Handbook of Essential Oils: Science, Technology, and Applications

Handbook of 

ESSENTIAL 

OILS 

Science, Technology, 

and Applications

ESSENTIAL 

OILS 

Edited by 

K. Hüsnü Can Başer 

Gerhard Buchbauer 

Handbook of 

Science, Technology, 

and Applications 

Boca Raton London New York 

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Library of Congress Cataloging-in-Publication Data 

Baser, K. H. C. (Kemal Hüsnü Can) 

Handbook of essential oils : science, technology, and applications / K. Hüsnü Can Baser, Gerhard 

Buchbauer. 

p. cm. 

Includes bibliographical references and index. 

ISBN 978-1-4200-6315-8 (hardcover : alk. paper) 

1. Essences and essential oils--Handbooks, manuals, etc. I. Buchbauer, Gerhard. II. Title. 

QD416.7.B37 2010 

661’.806--dc22 2009038020 

Visit the Taylor & Francis Web site at 

http://www.taylorandfrancis.com 

and the CRC Press Web site at 

http://www.crcpress.com

Contents 

Editors .......................................................................................................................................... ix 

Contributors .................................................................................................................................. xi 

Chapter 1 Introduction .............................................................................................................. 1 

K.Hüsnü Can Başer and Gerhard Buchbauer 

Chapter 2 History and Sources of Essential Oil Research ....................................................... 3 

Karl-Heinz Kubeczka 

Chapter 3 Sources of Essential Oils ........................................................................................ 39 

Chlodwig Franz and Johannes Novak 

Chapter 4 Production of Essential Oils ................................................................................... 83 

Erich Schmidt 

Chapter 5 Chemistry of Essential Oils ................................................................................. 121 

Charles Sell 

Chapter 6 Analysis of Essential Oils .................................................................................... 151 

Barbara d’Acampora Zellner, Paola Dugo, Giovanni Dugo, 

and Luigi Mondello 

Chapter 7 Safety Evaluation of Essential Oils: A Constituent-Based Approach .................. 185 

Timothy B. Adams and Sean V. Taylor 

Chapter 8 Metabolism of Terpenoids in Animal Models and Humans ................................ 209 

Walter Jäger 

Chapter 9 Biological Activities of Essential Oils .................................................................. 235 


v

vi 

Contents 

Chapter 10 Effects of Essential Oils in the Central Nervous System ..................................... 281 

10.1 Central Nervous System Effects of Essential Oils in 

Humans .................................................................................................................. 281 

Eva Heuberger 

10.2 Psychopharmacology of Essential Oils ..................................................... 297 

Domingos Sávio Nunes, Viviane de Moura Linck, Adriana Lourenço da Silva, 

Micheli Figueiró, and Elaine Elisabetsky 

Chapter 11 Phytotherapeutic Uses of Essential Oils ............................................................... 315 

Bob Harris 

Chapter 12 

In Vitro Antimicrobial Activities of Essential Oils Monographed in the 

European Pharmacopoeia 6th Edition ................................................................. 353 

Alexander Pauli and Heinz Schilcher 

Chapter 13 Aromatherapy with Essential Oils ....................................................................... 549 

Maria Lis-Balchin 

Chapter 14 

Chapter 15 

Biotransformation of Monoterpenoids by Microorganisms, Insects, 

and Mammals ....................................................................................................... 585 

Yoshiaki Noma and Yoshinori Asakawa 

Biotransformation of Sesquiterpenoids, Ionones, Damascones, Adamantanes, 

and Aromatic Compounds by Green Algae, Fungi, and Mammals ..................... 737 

Yoshinori Asakawa and Yoshiaki Noma 

Chapter 16 Industrial Uses of Essential Oils .......................................................................... 843 

W.S. Brud 

Chapter 17 

Encapsulation and Other Programmed Release Techniques for Essential Oils 

and Volatile Terpenes ........................................................................................... 855 

Jan Karlsen 

Chapter 18 Aroma-Vital Cuisine ............................................................................................ 863 

Maria M. Kettenring and Lara-M. Vucemilovic Geeganage 

Chapter 19 Essential Oils Used in Veterinary Medicine ........................................................ 881 

K. Hüsnü Can Başer and Chlodwig Franz

Contents 

vii 

Chapter 20 Trade of Essential Oils ......................................................................................... 895 

Hugo Bovill 

Chapter 21 Storage and Transport of Essential Oils ............................................................... 903 

Klaus-Dieter Protzen 

Chapter 22 

Recent EU Legislation on Flavors and Fragrances and Its Impact on 

Essential Oils ........................................................................................................ 917 

Jan C.R. Demyttenaere 

Index .......................................................................................................................................... 949

Editors 

K. Hüsnü Can Başer was born on July 15, 1949 in Çankırı, Turkey. He 

graduated from the Eskisehir I.T.I.A. School of Pharmacy with diploma 

number 1 in 1972 and became a research assistant in the pharmacognosy 

department of the same school. He did his PhD in pharmacognosy 

between 1974 and 1978 at Chelsea College of the University of London. 

Upon returning home, he worked as a lecturer in pharmacognosy at 

the school he had earlier graduated, and served as director of Eskisehir 

I.T.I.A. School of Chemical Engineering between 1978 and 1980. He 

was promoted to associate professorship in pharmacognosy in 1981. 

He served as dean of the faculty of pharmacy at Anadolu University 

(1993–2001), vice-dean of the faculty of pharmacy (1982–1993), head of 

the department of professional pharmaceutical sciences (1982–1993), 

head of the pharmacognosy section (1982–present), member of the 

University Board and Senate (1982–2001; 2007), and director of the Medicinal and Aromatic Plant 

and Drug Research Centre (TBAM) (1980–2002) in Anadolu University. 

During 1984–1994, he was appointed as the national project coordinator of Phase I and Phase II of 

the UNDP/UNIDO projects of the government of Turkey titled “Production of Pharmaceutical 

Materials from Medicinal and Aromatic Plants,” through which TBAM had been strengthened. 

He was promoted to full professorship in pharmacognosy in 1987. His major areas of research 

include essential oils, alkaloids, and biological, chemical, pharmacological, technological, and biological 

activity research into natural products. He is the 1995 Recipient of the Distinguished Service 

Medal of IFEAT (International Federation of Essential Oils and Aroma Trades) based in London, 

United Kingdom and the 2005 recipient of “Science Award” (Health Sciences) of the Scientific and 

Technological Research Council of Turkey (TUBITAK). He has published 537 papers in international 

refereed journals (378 in SCI journals), 105 papers in Turkish journals, and 134 papers in 

conference proceedings. 

For more information: http://www.khcbaser.com 

Gerhard Buchbauer was born in 1943 in Vienna, Austria. He studied 

pharmacy at the University of Vienna, from where he received his 

master’s degree (Mag.pharm.) in May 1966. In September 1966, he 

assumed the duties of university assistant at the Institute of Pharmaceutical 

Chemistry and received his doctorate (PhD) in pharmacy and 

philosophy in October 1971 with a thesis on synthetic fragrance compounds. 

Further scientific education was practised as post doc in the 

team of Professor C.H. Eugster at the Institute of Organic Chemistry, 

Univer sity of Zurich (1977–1978), followed by the habilitation (post 

doctoral lecture qualification) in Pharmaceutical Chemistry with the 

inaugural dissertation entitled “Synthesis of Analogies of Drugs and 

Fragrance Compounds with Contributions to Structure-Activity-Relationships” (1979) and 

appointment to the permanent staff of the University of Vienna, and head of the first department 

of the Institute of Pharmaceutical Chemistry. In November 1991, he was appointed as a full 

ix

x 

Editors 

professor of Pharmaceutical Chemistry, University of Vienna; in 2002, he was elected as head 

of this institute. He retired in October 2008. He is married since 1973 and was a father of a son 

since 1974. 

Among others, he is still a member of the permanent scientific committee of ISEO, a member of 

the scientific committee of Forum Cosmeticum (1990, 1996, 2002, and 2008), a member of editorial 

boards (e.g., Journal of Essential Oil Research, The International Journal of Essential Oil 

Therapeutics, Scientia Pharmaceutica, etc.), assistant editor of Flavour and Fragrance Journal, 

regional editor of Eurocosmetics, a member of many scientific societies, for example, Society of 

Austrian Chemists, head of its working group “Food Chemistry, Cosmetics, and Tensides” (2000– 

2004), Austrian Pharmaceutical Society, Austrian Phytochemical Society, vicehead of Austrian 

Society of Scientifi c Aromatherapy, and so on, technical advisor of IFEAT (1992–2008), and organizer 

of the 27th ISEO (September 2006, in Vienna) together with Professor Dr. Ch. Franz. 

Based on the sound interdisciplinary education of pharmacists, it was possible to establish almost 

completely neglected area of fragrance and flavor chemistry as a new research discipline within 

the pharmaceutical sciences. Our research team is the only one that conducts fragrance research in 

its entirety and covers synthesis, computer-aided fragrance design, analysis, and pharmaceutical/ 

medicinal aspects. Because of our efforts, it is possible to show and to prove that these small molecules 

possess more properties than merely emitting a good odor. Now, this “Viennese Centre of 

Flavour research” has gained a worldwide scientific reputation documented by more than 400 scientific 

publications, about 100 invited lectures, and about 200 contributions to symposia, meetings, 

and congresses, as short lectures and poster presentations.

Contributors 

Timothy B. Adams 

Flavor & Extract Manufacturers Association 

Washington, DC 

Yoshinori Asakawa 

Tokushima Bunri University 

Tokushima, Japan 

K. Hüsnü Can Başer 

Department of Pharmacognosy 

Anadolu University 

Eskisehir, Turkey 

Hugo Bovill 

Treatt PLC 

Bury St. Edmunds 

Suffolk, United Kingdom 

W. S. Brud 

Warsaw University of Technology and 

Pollena-Aroma Ltd 

Warsaw, Poland 


Department of Clinical Pharmacy and 

Diagnostics 

University of Vienna 

Vienna, Austria 

Jan C. R. Demyttenaere 

European Flavour and Fragrance Association 

(EFFA) 

Brussels, Belgium 

Giovanni Dugo 

Department of Drug-Chemical 

University of Messina 

Messina, Italy 

Paola Dugo 

Department of Food Science and the 

Environment 



Elaine Elisabetsky 

Laboratory of Ethnopharmacology 

The Federal University of 

Rio Grande do Sul 

Porto Alegre, Brazil 

Micheli Figueiró 


The Federal University of 

Rio Grande do Sul 


Chlodwig Franz 

Institute for Applied Botany and 

Pharmacognosy 

University of Veterinary Medicine Vienna 


Bob Harris 

SARL Essential Oil Consultants 

Provence, France 

Eva Heuberger 


Diagnostics 



Walter Jäger 


Diagnostics 



xi

xii 

Contributors 

Jan Karlsen 

Department of Pharmaceutics 

University of Oslo 

Oslo, Norway 

Maria M. Kettenring 

Neu-Isenburg, Germany 


Untere Steigstrasse 

Germany 

Viviane de Moura Linck 


The Federal University of Rio Grande do Sul 



South Bank University 

London, United Kingdom 

Luigi Mondello 




and 

Campus-Biomedical 

Rome, Italy 

Yoshiaki Noma 

Tokushima Bunri University 

Tokushima, Japan 

Johannes Novak 

Institute for Applied Botany and 

Pharmacognosy 

University of Veterinary Medicine Vienna 


Alexander Pauli 

ReviewScience 

Zirndorf, Germany 


Paul Kaders GmbH 

Hamburg, Germany 

Heinz Schilcher 

Immenstadt, Allgäu, Germany 

Erich Schmidt 

Nördlingen, Germany 


Kurt Kitzing GmbH 

Wallerstein, Germany 

Charles Sell 

Givaudan UK Ltd. 

Ashford, Kent, England 

Adriana Lourenço da Silva 


The Federal University of Rio Grande do Sul 


Sean V. Taylor 

Flavor & Extract Manufacturers Association 

Washington, DC 

Lara M. Vucemilovic 

Neu-Isenburg, Germany 

Barbara d’Acampora Zellner 




Domingos Sávio Nunes 

Departament of Chemistry 

State University of Ponta Grossa 

Ponta Grossa, Brazil

1 Introduction 

K. Hüsnü Can Başer and Gerhard Buchbauer 

Essential oils (EOs) are very interesting natural plant products and among other qualities they 

possess various biological properties. The term “biological” comprises all activities that these mixtures 

of volatile compounds (mainly mono- and sesquiterpenoids, benzenoids, phenylpropanoids, 

etc.) exert on humans, animals, and other plants. This book intends to make the reader acquainted 

with all aspects of EOs and their constituent aromachemicals ranging from chemistry, pharmaco logy, 

biological activity, production, and trade to uses, and regulatory aspects. After an overview of research 

and development activities on EOs with a historical perspective (Chapter 2), Chapter 3 “Sources of 

Essential Oils” gives an expert insight into vast sources of EOs. The chapter also touches upon agronomic 

aspects of EO-bearing plants. Traditional and modern production techniques of EOs are illustrated 

and discussed in Chapter 4. It is followed by two important chapters “Chemistry of Essential 

Oils” (Chapter 5) and “Analysis of Essential Oils” (Chapter 6) illustrating chemical diversity of EOs, 

and analytical techniques employed for the analyses of these highly complex mixtures of volatiles. 

They are followed by a cluster of articles on the biological properties of EOs, starting with “The 

Toxicology and Safety of Essential Oils: A Constituent-Based Approach” (Chapter 7). On account of 

the complexity of these natural products, the toxicological or biochemical testing of an EO will 

always be the sum of its constituents which either act in a synergistic or in an antagonistic way with 

one another. Therefore, the chemical characterization of the EO is very important for the understanding 

of its biological properties. The constituents of these natural mixtures upon being absorbed into 

the blood stream of humans or animals get metabolized and eliminated. This metabolic biotransformation 

leads mostly in two steps to products of high water solubility which enables the organism to 

get rid of these “xenobiotics” by renal elimination. This mechanism is thoroughly explained in 

Chapter 8, “Metabolism of Terpenoids in Animal Models and Humans.” In Chapter 9, “Biological 

Activities of Essential Oils,” “uncommon” biological activities of EOs are reviewed, such as anticancer 

properties, antinociceptive effects, antiviral activities, antiphlogistic properties, penetration 

enhancement activities, and antioxidative effects. The psychoactive, particularly stimulating, and 

sedative effects of fragrances as well as behavioral activities, elucidated, for example, by neurophysiological 

methods, are the topics of Chapter 10 (“Effects of Essential Oils in the Central Nervous 

Systemd”), Section 10.2. Here, the emphasis is put on the central nervous system and on psychopharmacology 

whereas Chapter 10, Section 10.1 mainly deals with reactions of the autonomic nervous 

system upon contact with EOs and/or their main constituents. The phytotherapeutic uses of EOs is 

another overview about scientific papers in peer-reviewed journals over the last 30 years, so to say the 

medical use of these natural plant products excluding aromatherapeutical treatments and single case 

studies (Chapter 11, “Phytotherapeutic Uses of Essential Oils”). Another contribution only deals with 

antimicrobial activities of those EOs that are monographed in the European Pharmacopoeia. In 

Chapter 12, “In Vitro Antimicrobial Activities of Essential Oils Monographed in the European 

Pharmacopoeia 6th Edition,” more than 81 tables show the importance of these valuable properties 

1

2 Handbook of Essential Oils 

of EOs. Aromatherapy with EOs covers the other side of the “classical” medical uses. “Aromatherapy 

with Essential Oils” (Chapter 13), is written by Maria Lis-Balchin, a known expert in this field and 

far from esoteric quackery. It completes the series of contributions dealing with the biological properties 

of EO regarded from various sides and standpoints. 

Chapters 14 and 15 by the world-renown experts Y. Asakawa and Y. Noma are concise treatises 

on the biotransformations of EO constituents. Enzymes in microorganisms and tissues metabolize 

EO constituents in similar ways by adding mainly oxygen function to molecules to render them 

water soluble to facilitate their metabolism. This is also seen as a means of detoxification for these 

organisms. Many interesting and valuable novel chemicals are biosynthesized by this way. These 

products are also considered as natural since the substrates are natural. 

Encapsulation is a technique widely utilized in pharmaceutical, chemical, food, and feed 

industries to render EOs more manageable in formulations. Classical and modern encapsulation 

techniques are explained in detail in Chapter 17, “Encapsulation and Other Programmed Release 

techniques for EOs and Volatile Terpenes.” 

EOs and aromachemicals are low-volume high-value products used in perfumery, cosmetics, 

feed, food, beverages, and pharmaceutical industries. Industrial uses of EOs are covered in an informative 

chapter from a historical perspective. 

“Aroma-Vital Cuisine” (Chapter 18) looks at the possibility to utilize EOs in the kitchen, where 

the pleasure of eating, the sensuality, and the enjoyment of lunching and dining of mostly processed 

food are stressed. Here, rather the holistic point of view and not too scientific way of understanding 

EOs is the topic, simply to show that these volatile natural plant products can add a lot of wellfeeling 

to their users. 

EOs are not only appealing to humans but also to animals. Applications of EOs as feed additives 

and for treating diseases in pets and farm animals are illustrated in Chapter 19, “Essential Oils Used 

in Veterinary Medicine,” that comprises a rare collection of information in this subject. 

The EO industry is highly complex and fragmented and the trade of EOs is rather conservative 

and highly specialized. EOs are produced and utilized in industrialized as well as in developing 

countries worldwide. Their trade situation in the world is summarized in “Trade of Essential Oils” 

(Chapter 20), authored by a world-renown expert Hugo Bovill. 

Storage and transport of EOs are crucial issues since they are highly sensitive to heat, moisture, 

and oxygen. Therefore, special precautions and strict regulations apply for their handling in storage 

and transport. “Storage and Transport of Essential Oils” (Chapter 21) will give the reader necessary 

guidelines to tackle this problem. 

Finally, the regulatory affairs of EOs are dealt with in Chapter 22 in order to give a better insight 

to those interested in legislative aspects. “Recent EU Legislation on Flavors and Fragrances and Its 

Impact on Essential Oils” comprises the most up-to-date regulations and legislative procedures 

applied on EOs in the European Union. 

This book is hoped to satisfy the needs of EO producers, traders, and users as well as researchers, 

academicians, and legislators who will find the most current information given by selected experts 

under one cover.

2 

History and Sources of 

Essential Oil Research 


CONTENTS 

2.1 First Systematic Investigations ............................................................................................ 3 

2.2 Research during the Last Half Century .............................................................................. 5 

2.2.1 Essential Oil Preparation Techniques ...................................................................... 5 

2.2.1.1 Industrial Processes .................................................................................. 5 

2.2.1.2 Laboratory-Scale Techniques ................................................................... 5 

2.2.1.3 Microsampling Techniques ...................................................................... 6 

2.2.2 Chromatographic Separation Techniques ............................................................... 11 

2.2.2.1 Thin-Layer Chromatography ................................................................... 12 

2.2.2.2 Gas Chromatography ............................................................................... 12 

2.2.2.3 Liquid Column Chromatography ............................................................. 18 

2.2.2.4 Supercritical Fluid Chromatography ........................................................ 20 

2.2.2.5 Countercurrent Chromatography ............................................................. 20 

2.2.3 Hyphenated Techniques ........................................................................................... 21 

2.2.3.1 Gas Chromatography-Mass Spectrometry ............................................... 21 

2.2.3.2 High-Resolution Gas Chromatography-Fourier Transform 

Infrared Spectroscopy .............................................................................. 23 

2.2.3.3 Gas Chromatography-Ultraviolet Spectroscopy ...................................... 23 

2.2.3.4 Gas Chromatography-Atomic Emission Spectroscopy ............................ 24 

2.2.3.5 Gas Chromatography-Isotope Ratio Mass Spectrometry ......................... 24 

2.2.3.6 High-Performance Liquid Chromatography-Gas Chromatography ............ 24 

2.2.3.7 HPLC-MS, HPLC-NMR Spectroscopy ................................................... 26 

2.2.3.8 Supercritical Fluid Extraction-Gas Chromatography .............................. 26 

2.2.3.9 Supercritical Fluid Chromatography-Gas Chromatography .................... 26 

2.2.3.10 Couplings of SFC-MS and SFC-FTIR Spectroscopy .............................. 27 

2.2.4 Identification of Multicomponent Samples without Previous Separation ............... 27 

2.2.4.1 UV Spectroscopy ..................................................................................... 27 

2.2.4.2 IR Spectroscopy ....................................................................................... 28 

2.2.4.3 Mass Spectrometry ................................................................................... 28 

2.2.4.4 

13 

C-NMR Spectroscopy ............................................................................ 28 

References .................................................................................................................................... 30 

2.1 FIRST SYSTEMATIC INVESTIGATIONS 

The first systematic investigations of constituents from essential oils may be attributed to the French 

chemist M. J. Dumas (1800–1884) who analyzed some hydrocarbons and oxygen as well as sulfurand 

nitrogen-containing constituents. He published his results in 1833. The French researcher 

3


M. Berthelot (1859) characterized several natural substances and their rearrangement products by 

optical rotation. However, the most important investigations have been performed by O. Wallach, an 

assistant of Kekule. He realized that several terpenes described under different names according to 

their botanical sources were often, in fact, chemically identical. He, therefore, tried to isolate the 

individual oil constituents and to study their basic properties. He employed together with his highly 

qualified coworkers Hesse, Gildemeister, Betram, Walbaum, Wienhaus, and others fractional distillation 

to separate essential oils and performed reactions with inorganic reagents to characterize the 

obtained individual fractions. The reagents he used were hydrochloric acid, oxides of nitrogen, 

bromine, and nitrosyl chloride—which was used for the first time by W. A. Tilden (1875)—by 

which frequently crystalline products have been obtained. 

At that time, hydrocarbons occurring in essential oils with the molecular formula C 10 H 16 were 

known, which had been named by Kekule terpenes because of their occurrence in turpentine oil. 

Constituents with the molecular formulas C 10 H 16 O and C 10 H 18 O were also known at that time under 

the generic name camphor and were obviously related to terpenes. The prototype of this group was 

camphor itself, which was known since antiquity. In 1891, Wallach characterized the terpenes 

pinene, camphene, limonene, dipentene, phellandrene, terpinolene, fenchene, and sylvestrene, which 

has later been recognized to be an artifact. 

During 1884–1914, Wallach wrote about 180 articles that are summarized in his book Terpene 

und Campher (Wallach, 1914) compiling all the knowledge on terpenes at that time and already in 

1887 he suggested that the terpenes must be constructed from isoprene units. In 1910, he was honored 

with the Nobel Prize for Chemistry “in recognition of his outstanding research in organic 

chemistry and especially in the field of alicyclic compounds.” 

In addition to Wallach, the German chemist A. von Baeyer, who also had been trained in Kekule’s 

laboratory, was one of the first chemists to become convinced of the achievements of structural 

chemistry and who developed and applied it to all of his work covering a broad scope of organic 

chemistry. Since 1893, he devoted considerable work to the structures and properties of cyclic 

terpenes (von Bayer, 1901). Besides his contributions to several dyes, the investigations of polyacetylenes, 

and so on, his contributions to theoretical chemistry including the strain theory of triple 

bonds and small carbon cycles have to be mentioned. In 1905, he was awarded the Nobel Prize for 

Chemistry “in recognition of his contributions to the development of Organic Chemistry and 

Industrial Chemistry, by his work on organic dyes and hydroaromatic compounds.” The frequently 

occurring acyclic monoterpenes geraniol, linalool, citral, and so on have been investigated by 

F. W. Semmler and the Russian chemist G. Wagner (1899), who recognized the importance of rearrangements 

for the elucidation of chemical constitution, especially the carbon-to-carbon migration 

of alkyl, aryl, or hydride ions, a type of reaction that was later generalized by H. Meerwein (1914) as 

Wagner–Meerwein rearrangement. 

More recent investigations of J. Read, W. Hückel, H. Schmidt, W. Treibs, and V. Prelog were 

mainly devoted to disentangle the stereochemical structures of menthols, carvomenthols, borneols, 

fenchols, and pinocampheols, as well as the related ketones (cf. Gildemeister and Hoffmann, 1956). 

A significant improvement in structure elucidation was the application of dehydrogenation of 

sesqui- and diterpenes with sulfur and later with selenium to give aromatic compounds as a major 

method, and the application of the isoprene rule to terpene chemistry, which have been very efficiently 

used by L. Ruzicka (1953) in Zurich, Switzerland. In 1939, he was honored in recognition 

of his outstanding investigations with the Nobel Prize in chemistry for his work on “polymethylenes 

and higher terpenes.” 

The structure of the frequently occurring bicyclic sesquiterpene ß-caryophyllene was for many 

years a matter of doubt. After numerous investigations W. Treibs (1952) has been able to isolate the 

crystalline caryophyllene epoxide from the autoxidation products of clove oil and F. Šorm et al. 

(1950) suggested caryophyllene to have a 4- and 9-membered ring on bases of infrared (IR) investigations. 

This suggestion was later confirmed by the English chemist D. H. R. Barton (Barton and 

Lindsay, 1951), who was awarded the Nobel Prize in Chemistry in 1969.

History and Sources of Essential Oil Research 5 

The application of ultraviolet (UV) spectroscopy in the elucidation of the structure of terpenes 

and other natural products was extensively used by R. B. Woodward in the early forties of the last 

century. On the basis of his large collection of empirical data, he developed a series of rules (later 

called the Woodward rules), which could be applied to finding out the structures of new natural 

substances by correlations between the position of UV maximum absorption and the substitution 

pattern of a diene or an a,b-unsaturated ketone (Woodward, 1941). He was awarded the Nobel Prize 

in Chemistry in 1965. However, it was not until the introduction of chromatographic separation 

methods and nuclear magnetic resonance (NMR) spectroscopy into organic chemistry, that a lot of 

further structures of terpenes were elucidated. The almost exponential growth in our knowledge in 

that field and other essential oil constituents is essentially due to the considerable advances in 

analytical methods in the course of the last half century. 

2.2 RESEARCH DURING THE LAST HALF CENTURY 

2.2.1 ESSENTIAL OIL PREPARATION TECHNIQUES 

2.2.1.1 Industrial Processes 

The vast majority of essential oils are produced from plant material in which they occur by different 

kinds of distillation or by cold pressing in the case of the peel oils from citrus fruits. 

In water- or hydrodistillation, the chopped plant material is submerged and in direct contact with 

boiling water. In steam distillation, the steam is produced in a boiler separate of the still and blown 

through a pipe into the bottom of the still, where the plant material rests on a perforated tray or in a 

basket for quick removal after exhaustive extraction. In addition to the aforementioned distillation at 

atmospheric pressure, high-pressure steam distillation is most often applied in European and 

American field stills and the applied increased temperature significantly reduces the time of distillation. 

The high-pressure steam-type distillation is often applied for peppermint, spearmint, lavandin, 

and the like. The condensed distillate, consisting of a mixture of water and oil, is usually separated 

in a so-called Florentine flask, a glass jar, or more recently in a receptacle made of stainless steel 

with one outlet near the base and another near the top. There the distillate separates into two layers 

from which the oil and the water can be separately withdrawn. Generally, the process of steam distillation 

is the most widely accepted method for the production of essential oils on a large scale. 

Expression or cold pressing is a process in which the oil glands within the peels of citrus fruits 

are mechanically crushed to release their content. There are several different processes used for the 

isolation of citrus oils; however, there are four major currently used processes. Those are Pellatrice 

and Sfumatrice—most often used in Italy—and the Brown Peel Shaver as well as the FMC extractor, 

which are used predominantly in North and South America. For more details see for example 

Lawrence 1995. All these processes lead to products that are not entirely volatile because they may 

contain coumarins, plant pigments, and so on; however, they are nevertheless acknowledged as 

essential oils by the International Organization for Standardization (ISO), the different pharmacopoeias, 

and so on. 

In contrast, extracts obtained by solvent extraction with different organic solvents, with liquid 

carbon dioxide or by supercritical fluid extraction (SFE) may not be considered as true essential oils; 

however, they possess most often aroma profiles that are almost identical to the raw material from 

which they have been extracted. They are therefore often used in the flavor and fragrance industry 

and in addition in food industry, if the chosen solvents are acceptable for food and do not leave any 

harmful residue in food products. 

2.2.1.2 Laboratory-Scale Techniques 

The following techniques are used mainly for trapping small amounts of volatiles from aromatic 

plants in research laboratories and partly for determination of the essential oil content in plant 

material. The most often used device is the circulatory distillation apparatus, basing on the


publication of Clevenger in 1928 and which has later found various modifications. One of those 

modified apparatus described by Cocking and Middleton (1935) has been introduced in the 

European Pharmacopoeia and several other pharmacopoeias. This device consists of a heated 

round-bottom flask into which the chopped plant material and water are placed and which is connected 

to a vertical condenser and a graduated tube, for the volumetric determination of the oil. 

At the bottom of the tube a three-way valve permits to direct the water back to the flask, since it 

is a continuous closed-circuit distillation device, and at the end of the distillation process to separate 

the essential oil from the water phase for further investigations. The length of distillation 

depends on the plant material to be investigated; however, it is usually fixed to 3–4 h. For the 

volumetric determination of the essential oil content in plants according to most of the pharmacopoeias, 

a certain amount of xylene—usually 0.5 mL—has to be placed over the water before 

running distillation to separate even small droplets of essential oil during distillation from the 

water. The volume of essential oil can be determined in the graduated tube after subtracting the 

volume of the applied xylene. 

Improved constructions with regard to the cooling system of the above-mentioned distillation 

apparatus have been published by Stahl (1953) and Sprecher (1963), and in publications of Kaiser 

et al. (1951) and Mechler et al. (1977), various apparatus used for the determination of essential oils 

in plant material are discussed and depicted. 

A further improvement was the development of a simultaneous distillation–solvent extraction 

device by Likens and Nickerson in 1964 (cf. Nickerson and Likens, 1966). The device permits continuous 

concentration of volatiles during hydrodistillation in one step using a closed-circuit distillation 

system. The water distillate is continuously extracted with a small amount of an organic and 

water-immiscible solvent. Although there are two versions described, one for high-density and one 

for low-density solvents, the high-density solvent version using dichloromethane is mostly applied 

in essential oil research. It has found numerous applications and several modified versions including 

different microdistillation devices have been described (e.g., Bicchi, 1987; Chaintreau, 2001). 

A sample preparation technique basing on Soxhlet extraction in a pressurized container using 

liquid carbon dioxide as extractant has been published by Jennings (1979). This device produces 

solvent-free extracts especially suitable for high-resolution gas chromatography (HRGC). As a less 

time-consuming alternative, the application of microwave-assisted extraction has been proposed by 

several researchers, for example by Craveiro et al. (1989), using a round-bottom flask containing the 

fresh plant material. This flask was placed into a microwave oven and passed by a flow of air. The 

oven was heated for 5 min and the obtained mixture of water and oil collected in a small and cooled 

flask. After extraction with dichloromethane the solution was submitted to gas chromatographymass 

spectrometry (GC-MS) analysis. The obtained analytical results have been compared with the 

results obtained by conventional distillation and exhibited no qualitative differences; however, the 

percentages of the individual components varied significantly. A different approach yielding 

solvent-free extracts from aromatic herbs by means of microwave heating has been presented by 

Lucchesi et al. (2004). The potential of the applied technique has been compared with conventional 

hydrodistillation showing substantially higher amounts of oxygenated compounds at the expense of 

monoterpene hydrocarbons. 

2.2.1.3 Microsampling Techniques 

2.2.1.3.1 Microdistillation 

Preparation of very small amounts of essential oils may be necessary if only very small amounts of 

plant material are available, and can be fundamental in chemotaxonomic investigations and control 

analysis but also for medicinal and spice plant breeding. In the past, numerous attempts have been 

made to minimize conventional distillation devices. As an example, the modified Marcusson device 

may be quoted (Bicchi et al., 1983) by which 0.2–3 g plant material suspended in 50 mL water can 

be distilled and collected in 100 μL analytical grade pentane or hexane. The analytical results 

proved to be identical with those obtained by conventional distillation.


Microversions of the distillation–extraction apparatus, described by Likens and Nickerson, have 

also been developed as well for high-density solvents (Godefroot et al., 1981), as for low-density 

solvents (Godefroot et al., 1982). The main advantage of these techniques is that no further enrichment 

by evaporation is required for subsequent gas chromatographic investigation. 

A different approach has been presented by Gießelmann et al. (1993) and Kubeczka et al. (1995). 

By means of a new developed microhydrodistillation device the volatile constituents of very small 

amounts of plant material have been separated. The microscale hydrodistillation of the sample is 

performed using a 20 mL crimp-cap glass vial with a Teflon ® -lined rubber septum contaning 10 mL 

water and 200–250 mg of the material to be investigated. This vial, which is placed in a heating 

block, is connected with a cooled receiver vial by a 0.32 mm I.D. fused silica capillary. By temperature-programmed 

heating of the sample vial, the water and the volatile constituents are vaporized 

and passed through the capillary into the cooled receiver vial. There, the volatiles as well as water 

are condensed and the essential oil collected in pentane for further analysis. The received analytical 

results have been compared to results from identical samples obtained by conventional hydrodistillation 

showing a good correlation of the qualitative and quantitative composition. Further applications 

with the commercially available Eppendorf MicroDistiller ® have been published in several 

papers, for example, by Briechle et al. (1997) and Baser et al. (2001). 

A simple device for rapid extraction of volatiles from natural plant drugs and the direct transfer 

of these substances to the starting point of a thin-layer chromatographic plate has been described by 

Stahl (1969a) and in his subsequent publications. A small amount of the sample (ca. 100 mg) is 

introduced into a glass cartridge with a conical tip together with 100 mg silica gel, containing 20% 

of water, and heated rapidly in a heating block for a short time at a preset temperature. The tip of the 

glass tube projects ca. 1 mm from the furnace and points to the starting point of the thin-layer plate, 

which is positioned 1 mm in front of the tip. Before introducing the glass tube it is sealed with a silicone 

rubber membrane. This simple technique has proven useful for many years in numerous investigations, 

especially in quality control, identification of plant drugs, and rapid screening of chemical 

races. In addition to the aforementioned microhydrodistillaion with the so-called TAS procedure 

(T = thermomicro and transfer; A = application; S = substance), several further applications, for 

example, in structure elucidation of isolated natural compounds such as zinc dust distillation, sulfur 

and selenium dehydrogenation, and catalytic dehydrogenation with palladium have been described 

in the microgram range (Stahl, 1976). 

2.2.1.3.2 Direct Sampling from Secretory Structures 

The investigation of the essential oils by direct sampling from secretory glands is of fundamental 

importance in studying the true essential oil composition of aromatic plants, since the usual applied 

techniques such as hydrodistillation and extraction are known to produce in some cases several 

artifacts. Therefore only direct sampling from secretory cavities and glandular trichomes and 

poperly performed successive analysis may furnish reliable results. One of the first investigations 

with a kind of direct sampling has been performed by Hefendehl (1966), who isolated the glandular 

hairs from the surfaces of Mentha piperita and Mentha aquatica leaves by means of a thin film of 

polyvinyl alcohol, which was removed after drying and extracted with diethyl ether. The composition 

of this product was in good agreement with the essential oils obtained by hydrodistillation. In 

contrast to these results, Malingré et al. (1969) observed some qualitative differences in course of 

their study on Mentha aquatica leaves after isolation of the essential oil from individual glandular 

hairs by means of a micromanipulator and a stereomicroscope. In the same year Amelunxen et al. 

(1969) published results on Mentha piperita, who separately isolated glandular hairs and glandular 

trichomes with glass capillaries. They found identical qualitative composition of the oil in both 

types of hairs, but differing concentrations of the individual components. Further studies have been 

performed by Henderson et al. (1970) on Pogostemon cablin leaves and by Fischer et al. (1987) on 

Majorana hortensis leaves. In the latter study, significant differences regarding the oil composition 

of the hydrodistilled oil and the oil extracted by means of glass capillaries from the trichomes


was observed. Their final conclusion was that the analysis of the respective essential oil is mainly 

an analysis of artifacts, formed during distillation, and the gas chromatographic analysis. Even if 

the investgations are performed very carefully and the successive GC has been performed by cold 

on-column injection to avoid thermal stress in the injection port, significant differences of the GC 

pattern of directly sampled oils versus the microdistilled samples have been observed in several 

cases (Bicchi et al., 1985). 

2.2.1.3.3 Headspace Techniques 

Headspace (HS) analysis has become one of the very frequently used sampling techniques in the 

investigation of aromatic plants, fragrances, and spices. It is a means of separating the volatiles from 

a liquid or solid prior to gas chromatographic analysis and is preferably used for samples that cannot 

be directly injected into a gas chromatograph. The applied techniques are usually classified according 

to the different sampling principles in static HS analysis and dynamic HS analysis. 

2.2.1.3.3.1 Static HS Methods In static HS analysis, the liquid or solid sample is placed into 

a vial, which is heated to a predetermined temperature after sealing. After the sample has reached 

equilibrium with its vapor (in equilibrium, the distribution of the analytes between the two phases 

depends on their partition coefficients at the preselected temperature, the time, and the pressure), an 

aliquot of the vapor phase can be withdrawn with a gas-tight syringe and subjected to gas chromatographic 

analysis. A simple method for the HS investigation of herbs and spices was described by 

Chialva et al. (1982), using a blender equipped with a special gas-tight valve. After grinding the herb 

and until thermodynamic equilibrium is reached, the HS sample can be withdrawn through the 

valve and injected into a gas chromatograph. Eight of the obtained capillary gas chromatograms are 

depicted in the paper of Chialva and compared with those of the respective essential oils exhibiting 

significant higher amounts of the more volatile oil constituents. However, one of the major problems 

with static HS analyses is the need for sample enrichment with regard to trace components. Therefore 

a concentration step such as cryogenic trapping, liquid absorption, or adsorption on a suitable solid 

has to be inserted for volatiles occurring only in small amounts. A versatile and often-used technique 

in the last decade is solid-phase microextraction (SPME) for sampling volatiles, which will be discussed 

in more detail in a separate paragraph. Since different other trapping procedures are a fundamental 

prerequisite for dynamic HS methods, they will be considered below. A comprehensive 

treatment of the theoretical basis of static HS analysis including numerous applications has been published 

by Kolb et al. (1997, 2006). 

2.2.1.3.3.2 Dynamic HS Methods The sensitivity of HS analysis can be improved considerably 

by stripping the volatiles from the material to be investigated with a stream of purified air or inert 

gas and trapping the released compounds. However, care has to be taken if grinded plant material 

has to be investigated, since disruption of tissues may initiate enzymatic reactions that may lead to 

formation of volatile artifacts. After stripping the plant material with gas in a closed vessel, the 

released volatile compounds are passed through a trap to collect and enrich the sample. This must 

be done because sample injection of fairly large sample volumes results in band broadening causing 

peak distortion and poor resolution. The following three techniques are advisable for collecting the 

highly diluted volatile sample according to Schaefer (1981) and Schreier (1984) with numerous 

references. 

Cryogenic trapping can be achieved by passing the gas containing the stripped volatiles through 

a cooled vessel or a capillary in which the volatile compounds are condensed (Kolb et al., 1986). 

The most convenient way for trapping the volatiles is to utilize part of the capillary column as a 

cryogenic trap. A simple device for cryofocusing of HS volatiles by using the first part of capillary 

column as a cryogenic trap has been shown in the aforementioned reference inclusive of a discussion 

of the theoretical background of cryogenic trapping. A similar on-column cold trapping device, suitable 

for extended period vapor sampling, has been published by Jennings (1981).


A different approach can be used if large volumes of stripped volatiles have to be trapped using 

collection in organic liquid phases. In this case, the volatiles distribute between the gas and the liquid 

and efficient collection will be achieved, if the distribution factor K is favorable for solving the 

stripped compounds in the liquid. A serious drawback, however, is the necessity to concentrate the 

obtained solution prior to GC with the risk to loose highly volatile compounds. This can be overcome 

if a short-packed GC column is used containing a solid support coated with a suitable liquid. 

Novak et al. (1965) have used Celite coated with 30% silicone elastomer E-301 and the absorbed 

compounds were introduced into a gas chromatograph after thermal desorption. Coating with 15% 

silicone rubber SE 30 has been successfully used by Kubeczka (1967) with a similar device and the 

application of a wall-coated tubing with methylsilicone oil SF 96 has been described by Teranishi 

et al. (1972). A different technique has been used by Bergström et al. (1973, 1980). They trapped the 

scent of flowers on Chromosorb ® W coated with 10% silicon high vacuum grease and filled a small 

portion of the sorbent containing the volatiles into a precolumn, which was placed in the splitless 

injection port of a gas chromatograph. There the volatiles were desorbed under heating and flushed 

onto the GC column. In 1987, Bichi et al. applied up to 50 cm pieces of thick-film fused silica 

capillaries coated with a 15 μm dimethylsilicone film for trapping the volatiles in the atmosphere 

surrounding living plants. The plants under investigation were placed in a glass bell into which the 

trapping capillary was introduced through a rubber septum while the other end of the capillary has 

been connected to pocket sampler. In order to trap even volatile monoterpene hydrocarbons, a capillary 

length of at least 50 cm and sample volume of maximum 100 mL has to be applied to avoid loss 

of components through breakthrough. The trapped compounds have been subsequently on-line 

thermally desorbed, cold trapped, and analyzed. Finally, a type of enfl eurage and especially designed 

for field experiments has been described by Joulain (1987) to trap the scents of freshly picked flowers. 

Around 100 g flowers were spread on the grid of a specially designed stainless steel device and 

passed by a stream of ambient air, supplied by an unheated portable air drier. The stripped volatiles 

are trapped on a layer of purified fat placed above the grid. After 2 h, the fat was collected and the 

volatiles recovered in the laboratory by means of vacuum distillation at low temperature. 

With a third often applied procedure the stripped volatiles from the HS of plant material and 

especially from flowers are passed through a tube filled with a solid adsorbent on which the volatile 

compounds are adsorbed. Common adsorbents most often used in investigations of plant volatiles 

are above all charcoal and different types of synthetic porous polymers. Activated charcoal is an 

adsorbent with a high adsorption capacity, thermal and chemical stability, and which is not deactivated 

by water, an important feature, if freshly collected plant material has to be investigated. The 

adsorbed volatiles can easily be recovered by elution with small amounts (10–50 μL) of carbon 

disulfide avoiding further concentration of the sample prior to GC analysis. The occasionally 

observed incomplete recovery of sample components after solvent extraction and artifact formation 

after thermal desorption have been largely solved by application of small amounts of special type of 

activated charcoal as described by Grob et al. (1976). Numerous applications have been described 

using this special type of activated charcoal, for example, by Kaiser (1993) in a great number of field 

experiments on the scent of orchids. In addition to charcoal the following synthetic porous polymers 

have been applied to collect volatile compounds from the HS from flowers and different other plant 

materials according to Schaefer (1981): Tenax ® GC, different Porapak ® types (e.g., Porapak ® P, Q, 

R, and T) as well as several Chromosorb ® types belonging to the 100 series. More recent developed 

adsorbents are the carbonaceous adsorbents such as Ambersorb ® , Carboxene ® , and Carbopak ® and 

their adsorbent properties lie between activated charcoal and the porous polymers. Especially the 

porous polymers have to be washed repeatedly, for example, with diethyl ether and conditioned 

before use in a stream of oxygen-free nitrogen at 200–280°C, depending on the sort of adsorbent. 

The trapped components can be recovered either by thermal desorption or by solvent elution and the 

recoveries can be different depending on the applied adsorbent (Cole, 1980). Another very important 

criterion for the selection of a suitable adsorbent for collecting HS samples is the breakthrough 

volume limiting the amount of gas passing through the trap.


A comprehensive review concerning HS gas chromatographic analysis of medicinal and aromatic 

plants and flowers with 137 references, covering the period from 1982 to 1988 has been published 

by Bicchi and Joulain in 1990, thoroughly describing and explaining the different 

methodological approaches and applications. Among other things most of the important contributions 

of the Finnish research group of Hiltunen and coworkers on the HS of medicinal plants and 

the optimization of the HS parameters have been cited in the mentioned review. 

2.2.1.3.4 Solid-Phase Microextraction 

SPME is an easy-to-handle sampling technique, initially developed for the determination of volatile 

organic compounds in environmental samples (Arthur et al., 1990), and has gained, in the last years, 

acceptance in numerous fields and has been applied to the analysis of a wide range of analytes in 

various matrices. Sample preparation is based on sorption of analytes from a sample onto a coated 

fused silica fiber which is mounted in a modified GC syringe. After introducing the coated fiber into 

a liquid or gaseous sample, the compounds to be analyzed are enriched according to their distribution 

coefficients and can be subsequently thermally desorbed from the coating after introducing the 

fiber into the hot injector of a gas chromatograph. The commercially available SPME device 

(Supelco Inc.) consists of a 1 cm length fused silica fiber of ca. 100 μm diameter coated on the outer 

surface with a stationary phase fixed to a stainless steel plunger and a holder that looks like a 

modified microliter syringe. The fiber can be drawn into the syringe needle to prevent damage. To 

use the device, the needle is pierced through the septum that seals the sample vial. Then, the plunger 

is depressed lowering the coated fiber into the liquid sample or the HS above the sample. After sorption 

of the sample, which takes some minutes, the fiber has to be drawn back into the needle and 

withdrawn from the sample vial. By the same procedure the fiber can be introduced into the gas 

chromatograph injector where the adsorbed substances are thermally desorbed and flushed by the 

carrier gas into the capillary GC column. 

SPME fibers can be coated with polymer liquid (e.g., polydimethylsiloxane, PDMS) or a mixed 

solid and liquid coating (e.g., Carboxen ® /PDMS). The selectivity and capacity of the fiber coating 

can be adjusted by changing the phase type or thickness of the coating on the fiber according to the 

properties of the compounds to be analyzed. Commercially available are coatings of 7, 30, and 

100 μm of PDMS, an 85 μm polyacrylate, and several mixed coatings for different polar components. 

The influence of fiber coatings on the recovery of plant volatiles was thoroughly investigated 

by Bicchi et al. (2000). Details concerning the theory of SPME, technology, its application, and 

specific topics have been described by Pawliszyn (1997) and references cited therein. A number of 

different applications of SPME in the field of essential oil analysis have been presented by Kubeczka 

(1997a). An overview on publications of the period 2000–2005 with regard to HS-SPME has been 

recently published by Belliardo et al. (2006) covering the analysis of volatiles from aromatic and 

medicinal plants, selection of the most effective fibers and sampling conditions, and discussing its 

advantages and limitations. The most comprehensive collection of references with regard to the different 

application of SPME can be obtained from Supelco on CD. 

2.2.1.3.5 Stir Bar Sorptive Extraction and Headspace Sorptive Extraction 

Despite the indisputable simplicity and rapidity of SPME, its applicability is limited by the small 

amount of sorbent on the needle (


stir bar has to be removed, introduced into a glass tube, and transferred to thermal desorption 

instrument. After desorption and cryofocusing within a cooled programmed temperature vaporization 

(PTV) injector, the volatiles were transferred onto the analytical GC column. Comparison of 

SPME and the above- mentioned stir bar sorptive extraction (SBSE) technique using identical phases 

for both techniques exhibited striking differences in the recoveries, which has been attributed to ca. 

100 times higher phase ratio in SBSE than in SPME. A comprehensive treatment of SBSE, discussion 

of the principle, the extraction procedure, and numerous applications was recently been published 

by David and Sandra (2007). 

A further approach for sorptive enrichment of volatiles from the HS of aqueous or solid samples 

has been described by Tienpont et al. (2000), referred to as headspace sorptive extraction (HSSE). 

This technique implies the sorption of volatiles into PDMS that is chemically bound on the surface 

of a glass rod support. The device consists of a ca. 5 cm length glass rod of 2 mm diameter and at 

the last centimeter of 1 mm diameter. This last part is covered with PDMS chemically bound to the 

glass surface. HS bars with 30, 50, and 100 mg PDMS are commercially available from Gerstel 

GmbH, Mühlheim, Germany. After thermal conditioning at 300°C for 2 h, the glass bar was introduced 

into the HS of a closed 20 mL HS vial containing the sample to be investigated. After sampling 

for 45 min, the bar was put into a glass tube for thermal desorption, which was performed with 

a TDS-2 thermodesorption unit (Gerstel). After desorption and cryofocusing within a PTV injector, 

the volatiles were transferred onto the analytical GC column. As a result, HSSE exceeded largely 

the sensitivity attainable with SPME. Several examples referring to the application of HSSE in HS 

analysis of aromatic and medicinal plants inclusive of details of the sampling procedure were 

described by Bicchi et al. (2000). 

2.2.2 CHROMATOGRAPHIC SEPARATION TECHNIQUES 

In the course of the last half century, a great number of techniques have been developed and applied 

to the analysis of essential oils. A part of them has been replaced nowadays by either more effective 

or easier-to-handle techniques, while other methods maintained their significance and have been 

permanently improved. Before going into detail, the analytical facilities in the sixties of the last 

century should be considered briefly. The methods available for the analysis of essential oils have 

been at that time (Table 2.1) thin-layer chromatography (TLC), various types of liquid column chromatography 

(LC), and already gas liquid chromatography (GC). In addition, several spectroscopic 

techniques such as UV and IR spectroscopy, MS, and 1 H-NMR spectroscopy have been available. 

TABLE 2.1 

Techniques Applied to the Analysis of Essential Oils 

Chromatographic Techniques 

Including Two- and Multidimensional Techniques 

TLC GC LC HPLC 

CCC 

SFC 

Spectroscopic and Spectrometric Techniques 

UV IR MS 1H-NMR 

13C-NMR NIR Raman 

Hyphenated Techniques 

GC-MS GC-UV HPLC-GC SFE-GC 

GC-FTIR GC-AES HPLC-MS SFC-GC 

GC-FTIR-MS GC-IRMS HPLC-NMR


In the following years, several additional techniques were developed and applied to essential oils 

analysis, including: high-performance liquid chromatography (HPLC); different kinds of countercurrent 

chromatography (CCC); supercritical fluid chromatography (SFC); including multidimensional 

coupling techniques, C-13 NMR, near infrared (NIR), and Raman spectroscopy; and a multitude 

of so-called hyphenated techniques, which means on-line couplings of chromatographic separation 

devices to spectrometers, yielding valuable structural information of the individual separated 

components made their identification feasible. 

2.2.2.1 Thin-Layer Chromatography 

TLC was one of the first chromatographic techniques and has been used for many years for the analysis 

of essential oils. This method provided valuable information compared to simple measurements of 

chemical and physical values and has therefore been adopted as a standard laboratory method for 

characterization of essential oils in numerous pharmacopoeias. Fundamentals of TLC have been 

described by Geiss (1987) and in a comprehensive handbook by Stahl (1969b), in which numerous 

applications and examples on investigations of secondary plant metabolites inclusive of essential oils 

are given. More recently, the third edition of the handbook of TLC from Shema and Fried (2003) 

appeared. Further approaches in TLC have been development of high-performance TLC (Kaiser, 1976), 

and the application of forced flow techniques such as overpressured layer chromatography (OPLC) and 

rotation planar chromatography (RPC) described by Tyihák et al. (1979) and Nyiredy (2003). 

In spite of its indisputable simplicity and rapidity, this technique is now largely obsolete for analyzing 

such complex mixtures like essential oils, due to its low resolution. However, for the rapid 

investigation of the essential oil pattern of chemical races or the differentiation of individual plant 

species, this method can still be successfully applied (Gaedcke and Steinhoff, 2000). In addition, 

silver nitrate and silver perchlorate impregnated layers have been used for the separation of olefinic 

compounds, especially sesquiterpene hydrocarbons (Prasad et al., 1947), and more recently for the 

isolation of individual sesquiterpenes (Saritas, 2000). 

2.2.2.2 Gas Chromatography 

However, the separation capability of GC exceeded all the other separation techniques, even if only 

packed columns have been used. The exiting evolution of this technique in the past can be impressively 

demonstrated with four examples of the gas chromatographic separation of the essential oil 

from rue (Kubeczka, 1981a), a medicinal and aromatic plant. This oil was separated by S. Bruno in 

1961 into eight constituents and represented one of the first gas chromatographic analyses of that 

essential oil. Only a few years later in 1964 separation of the same oil has been improved using a 

Perkin Elmer (PE) gas chromatograph equipped with a 2 m packed column and a thermal conductivity 

detector (TCD) operated under isothermal conditions yielding 20 separated constituents. 

A further improvement of the separation of the rue oil was obtained after the introduction of temperature 

programming of the column oven, yielding approximately 80 constituents. The last significant 

improvements were a result of the development of high-resolution capillary columns and the 

sensitive flame ionization detector (FID). By means of a 50 m glass capillary with 0.25 mm I.D., the 

rue oil could be separated into approximately 150 constituents, in 1981. However, the problems 

associated with the fragility of the glass capillaries and their cumbersome installation lessened the 

acknowledgment of this column types, despite their outstanding quality. This has changed since 

flexible fused silica capillaries became commercially available, which are nearly unbreakable in 

normal usage. In addition, by different cross-linking technologies, the problems associated with 

wall coating, especially with polar phases, have been overcome, so that all important types of 

stationary phases used in conventional GC have been commercially available. The most often used 

stationary phases for the analysis of essential oils have been, and are still today, the polar phases 

Carbowax 20M (DB-Wax, Supelcowax-10, HP-20M, Innowax, etc.), 14% cyanopropylphenyl–86% 

methyl polysiloxane (DB-1701, SPB-1701, HP-1701, OV-1701, etc.), and the nonpolar phases PDMS 

(DB-1, SPB-1, HP-1 and HP-1ms, CPSil-5 CB, OV-1, etc.), and 5% phenyl methyl polysiloxane


(DB-5, SPB-5, HP-5, CPSil-8 CB, OV-5, SE-54, etc.). Besides different column diameters of 0.53, 

0.32, 0.25, 0.10, and 0.05 mm I.D., a variety of film thicknesses can be purchased. Increasing column 

diameter and film thickness of stationary phase increases the sample capacity at the expense of separation 

efficiency. However, sample capacity has become important, particularly in trace analysis and 

with some hyphenated techniques such as gas chromatography-Fourier transform infrared (GC-FTIR), 

in which a higher sample capacity is necessary when compared to GC-MS. On the other hand, the 

application of a narrow bore column with 100 μm I.D. and a film coating of 0.2 μm have been shown 

to be highly efficient and theoretical plate numbers of approximately 250,000 were received with 

a 25 m capillary (Lancas et al., 1988). The most common detector in GC is the FID because of its 

high sensitivity toward organic compounds. The universal applicable TCD is nowadays used only for 

fixed-gas detection because of its very low sensitivity as compared to FID, and cannot be used in 

capillary GC. Nitrogen-containing compounds can be selectively detected with the aid of the selective 

nitrogen–phosphorus detector (NPD), and chlorinated compounds by the selective and very 

sensitive electron-capture detector (ECD), which is often used in the analysis of pesticides. 

Oxygen-containing compounds have been selectively detected with special O-FID analyzer even in 

very complex samples, which was primarily employed to the analysis of oxygenated compounds in 

gasoline, utilized as fuel-blending agents (Schneider et al., 1982). The oxygen selectivity of the FID 

is obtained by two on-line postcolumn reactions: First a cracking reaction forming carbon monoxide, 

which is reduced in a second reactor yielding equimolar quantities of methane, which can be 

sensitively detected by the FID. Since in total each oxygen atom is converted to one molecule methane, 

the FID response is proportional to the amount of oxygen in the respective molecule. Application 

of the O-FID to the analysis of essential oils has been presented by Kubeczka (1991). However, 

conventional GC using fused silica capillaries with different stationary phases, including chiral 

phases, and the sensitive FID, is up to now the prime technique for the analysis of essential oils. 

2.2.2.2.1 Fast and Ultrafast GC 

Due to the demand for faster GC separations in routine work in the field of GC of essential oils, the 

development of fast and ultrafast GC seems worthy to be mentioned. The various approaches for fast 

GC have been reviewed in 1999 (Cramers et al., 1999). The most effective way to speed up GC separation 

without loosing separation efficiency is to use shorter columns with narrow inner diameter 

and thinner coatings, higher carrier gas flow rates, and accelerated temperature ramps. In Figure 2.1 

the conventional and fast GC separation of lime oil is shown, indicating virtually the same separation 

efficiency in the fast GC and a reduction in time from approximately 60 to 13 min (Mondello 

et al., 2000). 

An ultrafast GC separation of the essential oil from lime with an outstanding reduction of time 

was recently achieved (Mondello et al., 2004) using a 5 m capillary with 50 μm I.D. and a film thickness 

of 0.05 μm operated with a high carrier gas velocity of 120 cm/min and an accelerated threestage 

temperature programme. The analysis of the essential oil was obtained in approximately 

90 sec, which equates to a speed gain of approximately 33 times in comparison with the conventional 

GC separation. However, such a separation cannot be performed with conventional GC 

instruments. In addition, the mass spectrometric identification of the separated components could 

only be achieved by coupling GC to a time-flight mass spectrometer. In Table 2.2 the separation 

parameters of conventional, fast, and ultrafast GC separation are given, indicating clearly the relatively 

low requirements for fast GC, while ultrafast separations can only be realized with modern 

GC instruments and need a significant higher employment. 

2.2.2.2.2 Chiral GC 

Besides fast and ultrafast GC separations, one of the most important developments in GC has been the 

introduction of enantioselective capillary columns in the past with high separation efficiency, so that a 

great number of chiral substances including many essential oil constituents could be separated and 

identified. The different approaches of gas chromatographic separation of chiral compounds are briefly


Monoterpenes Sesquiterpenes Coumarins 

and Psoralens 

0 

25 50 

Time (min) 

0 5 10 

FIGURE 2.1 Comparison of conventional and fast-GC separation of lime oil. (From Mondello, L., et al., 

2000. LC.GC Europe, 13: 495–502. With permission.) 

summarized in Table 2.3. In the mid-1960s, Gil-Av published results with chiral diamide stationary 

phases for gas chromatographic separation of chiral compounds, which interacted with the analytes by 

hydrogen bonding forces (Gil-Av et al., 1965). The ability to separate enantiomers using these phases 

was therefore limited to substrates with hydrogen bonding donor or acceptor functions. 

Diastereomeric association between chiral molecules and chiral transition metal complexes was 

first described by Schurig in 1977. Since hydrogen bonding interaction is not essential for chiral 

recognition in such a system, a number of compounds could be separated, but this method was limited 

by the nonsufficient thermal stability of the applied metal complexes. 

In 1988 König, as well as Schurig, described the use of cyclodextrin derivatives that act enantioselectively 

by host–guest interaction by partial intrusion of enantiomers into the cyclodextrin cavity. 

They are cyclic a-(1–4)-bounded glucose oligomers with 6-, 7-, or 8-glucose units, which can be 

prepared by enzymatic degradation of starch with specific cyclodextrin-glucanosyltransferases from 

different bacterial strains, yielding a-, b-, and g-cyclodextrins and are commercially available. Due 

to the significant lower reactivity of the 3-hydroxygroups of cyclodextrins, this position can be 

selectively acylated after alkylation of the 2- and 6-positions (Figure 2.2), yielding several nonpolar 

cyclodextrin derivatives, which are liquid or waxy at room temperature and which proved very useful 

for gas chromatographic applications. 

TABLE 2.2 

Conditions of Conventional, Fast-, and Ultrafast GC 

Conventional GC Fast GC Ultrafast GC 

Column 30 m 10 m 10–15 m 

0.25 mm I.D. 0.1 mm I.D. 0.1 mm I.D. 

0.25 μm film 0.1 μm film 0.1 μm film 

Temperature program 50–350°C 50–350°C 45–325°C 

3°C/min 14°C/min 45–200°C/min 

Carrier gas H 2 H 2 H 2 

u = 36 cm/s u = 57 cm/s u = 120 cm/s 

Sampling frequency 10 Hz 20–50 Hz 50–250 Hz


TABLE 2.3 

Different Approaches of Enantioselective GC 

1. Chiral diamide stationary phases (Gil-Av, 1965) 

Hydrogen bonding interaction 

2. Chiral transition metal complexation (Schurig, 1977) 

Complexation gas chromatography 

3. Cyclodextrin derivatives (König, Schurig, 1988) 

Host–guest interaction, inclusion gas chromatography 

König and coworkers reported their first results in 1988 with per-O-pentylated and selectively 3-Oacylated-2,6-di-O-pentylated 

a-, b-, and g-cyclodextrins, which are highly stable, soluble in nonpolar 

solvents, and which possess a high enantioselectivity toward many chiral compounds. In the following 

years a number of further cyclodextrin derivatives have been synthesized and tested by several groups, 

allowing the separation of a wide range of chiral compounds, especially due to the improved thermal 

stability (Table 2.4). With the application of 2,3-pentyl-6-methyl-b- and -g-cyclodextrin as stationary 

phases, all monoterpene hydrocarbons commonly occurring in essential oils could be separated (König 

et al., 1992). The reason for application of two different columns with complementary properties was 

that on one column not all enantiomers were satisfactorily resolved. Thus, the simultaneous use of these 

two columns provided a maximum of information and reliability in peak assignment. 

After successful application of enantioselective GC to the analysis of enantiomeric composition 

of monoterpenoids in many essential oils (e.g., Werkhoff et al., 1993; Bicchi et al., 1995; and references 

cited therein), the studies have been extended to the sesquiterpene fraction. Standard mixtures of 

known enantiomeric composition were prepared by isolation of individual enantiomers from numerous 

essential oils by preparative GC and by preparative enantioselective GC. A gas chromatographic 

separation of a series of isolated or prepared sesquiterpene hydrocarbon enantiomers, showing 

the separation of 12 commonly occurring sesquiterpene hydrocarbons on a 2,6-methyl-3-pentylb-cyclodextrin 

capillary column has been presented by König et al. (1995). Further investigations 

on sesquiterpenes have been published by König et al. (1994). However, due to the complexity of the 

sesquiterpene pattern in many essential oils, it is often impossible to perform directly an enantioselective 

analysis by coinjection with standard samples on a capillary column with a chiral stationary 

phase alone. Therefore, in many cases two-dimensional GC had to be performed. 

2.2.2.2.3 Two-Dimensional Gas Chromatography 

After preseparation of the oil on a nonchiral stationary phase, the peaks of interest have to be transferred 

to a second capillary column coated with a chiral phase, a technique usually referred to as 

“heart cutting.” In the simplest case, two GC capillaries with different selectivities are serially 

connected and the portion of unresolved components from the effluent of the first column is directed 

into a second column, for example, a capillary with a chiral coating. The basic arrangement used in 

two-dimensional gas chromatography (GC-GC) is shown in Figure 2.3. By means of a valve, the 

individual fractions of interest eluting from the first column are directed to the second, chiral 

column, while the rest of the sample may be discarded. With this heart-cutting technique many 

OR 6 

O 

O 

R 3 O 

FIGURE 2.2 a-Glucose unit of a cyclodextrin. 

R 2 O 

O


TABLE 2.4 

Important Cyclodextrin Derivatives 

Research Group Year Cyclodextrin Derivative 

Schurig and Novotny 1988 Per-O-methyl-b-CD 

König et al. 1988a Per-O-pentyl-(a,b,g)-CD 

König et al. 1988c 3-O-acetyl-2,6,-di-O-pentyl-(a,b,g)-CD 

König et al. 1989 3-O-butyryl-2,6-di-O-pentyl-(b,g)-CD 

König et al. 1990 6-O-methyl-2,3-di-O-pentyl-g-CD 

Köng et al. 1992 2,6-Di-O-methyl-3-O-pentyl-(b,g)-CD 

Dietrich et al. 1992 2,3-Di-O-acetyl-6-O-tert-butyl-dimethysilyl-b-CD 

Dietrich et al. 1992a 2,3-Di-O-methyl-6-O-tert-butyl-dimethylsilyl-(b,g)-CD 

Bicchi et al. 1996 2,3-Di-O-ethyl-6-O-tert-butyl-dimethylsilyl-(b,g)-CD 

Takahisa and Engel 2005 2,3-Di-O-methoxymethyl-6-O-tert-butyl-dimethylsilyl-b-CD 

Takahisa and Engel 2005a 2,3-Di-O-methoxymethyl-6-O-tert-butyl-dimethylsilyl-g-CD 

separations of chiral oil constituents have been performed in the past. As an example, the investigation 

of the chiral sesquiterpene hydrocarbon germacrene D shall be mentioned (Kubeczka, 1996), 

which was found to be a main constituent of the essential oil from the flowering herb from Solidago 

canadensis. The enantioselective investigation of the germacrene-D fraction from a GC run using a 

nonchiral DB-Wax capillary transferred to a 2,6-methyl-3-pentyl-b-cyclodextrin capillary exhibited 

the presence of both enantiomers. This is worthy to be mentioned, since in most of other germacrene 

D containing higher plants nearly exclusively the (−)-enantiomer can be found. 

The previously mentioned two-dimensional GC design, however, in which a valve is used to 

direct the portion of desired effluent from the first into the second column, has obviously several 

shortcomings: The sample comes into contact with the metal surface of the valve body, the pressure 

drop of both connected columns may be significant and the use of only one-column oven does not 

permit to adjust the temperature for both columns properly. Therefore, one of the best approaches 

Injector 

Det. 

Vent 

Det. 

Tee 

Valve 

Column 

Column 

FIGURE 2.3 Basic arrangement used in two-dimensional GC.


to overcome these limitations has been realized by a commercially available two-column 

oven instrument using a Deans-type pressure balancing interface between the two columns called a 

“live-T-connection” (Figure 2.4) providing considerable flexibility (Hener, 1990). By means of that 

instrument the enantiomeric composition of several essential oils has been investigated very successfully. 

As an example, the investigation of the essential oil from Lavandula angustifolia shall be 

mentioned (Kreis et al., 1992) showing the simultaneous stereoanalysis of a mixture of chiral compounds, 

which can be found in lavender oils, using the column combination Carbowax 20M as the 

precolumn and 2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl-b-cyclodextrin as the main column. 

All the unresolved enantiomeric pairs from the precolumn could be well separated after transferring 

them to the chiral main column in a single run. As a result, it was found that most of the characteristic 

and genuine chiral constituents of lavender oil exhibit a high enantiomeric purity. 

A different and inexpensive approach for transferring individual GC peaks onto a second column 

has been presented by Kubeczka (1997a), using an SPME device. The highly diluted organic vapor of 

a fraction eluting from a GC capillary in the carrier gas flow has been absorbed on a coated SPME 

fiber and introduced onto a second capillary. As could be demonstrated, no modification of the gas 

chromatograph had to be performed to realize that approach. The eluting fractions were sampled 

after shutting the valves of the air, of hydrogen and the make-up gas if applied. In order to minimize 

the volume of the detector to avoid dilution of the eluting fraction and to direct the gas flow to the 

fiber surface, a capillary glass tubing of 1.5 mm I.D. was inserted into the FID and fixed and tightened 

by an O-ring (Figure 2.5). At the beginning of peak elution, controlled only by time, a 100 μm 

PDMS fiber was introduced into the mounted glass capillary tubing and withdrawn at the end of peak 

elution. Afterward, the fiber within the needle was introduced into the injector of a second capillary 

column with a chiral stationary phase. Two examples concerning the investigation of bergamot oil 

have been shown. At first, the analysis of an authentic sample of bergamot oil, containing chiral 

linalool and the respective chiral actetate is carried out. Both components were cut separately and 

transferred to an enantioselective cyclodextrin Lipodex ® E capillary. The chromatograms clearly 

have shown that the authentic bergamot oil contains nearly exclusively the (−)-enantiomers of linalool 

and linalyl acetate, while the respective (+)-enantiomers could only be detected as traces. In contrast 

to the authentic sample, a commercial sample of bergamot oil, which was analyzed under the same 

Injector 

Nonchiral 

precolumn 

Δ p 

“live-T” 

Chiral main column 

ITD 

Oven I 

FID 

Detector precolumn 

Oven II 

: “Press-fit” connector 

FIGURE 2.4 Scheme of enantioselective multidimensional GC with “live-T” column switching. (From 

Hener, U. 1990. Chirale Aromastoffe—Beiträge zur Struktur, Wirkung und Analytik. Dissertation, Goethe- 

University of Frankfurt/Main, Germany. With permission.)


SPME-holder 

Inserted glass tubing 

O-ring 

Septum piercing needle 

Coated fused-silica fiber 

FID jet 

FIGURE 2.5 Cross section of an FID of an HP 5890 gas chromatograph with an inserted SPME fiber. (From 

Kubeczka, K.-H. 1997b. Essential Oil Symposium Proceedings, p. 145. With permission.) 

conditions, exhibited the presence of significant amounts of both enantiomers of linalool and linalyl 

acetate indicating a falsification by admixing the respective racemic alcohol and ester. 

2.2.2.2.4 Comprehensive Multidimensional Gas Chromatography 

One of the most powerful separation techniques that has been recently applied to the investigation 

of essential oils is the so-called comprehensive multidimensional gas chromatography (GC × GC). 

This technique is a true multidimensional gas chromatography (MDGC) since it combines two 

directly coupled columns and importantly is able to subject the entire sample to simultaneous twocolumn 

separation. Using that technique, the need to select heart cuts, as used in conventional 

MDGC, is no longer required. Since components now are retained in two different columns, the net 

capacity is the product of the capacities of the two applied columns increasing considerably the 

resolution of the total system. Details regarding that technique will be given in the chapter of Luigi 

Mondello. 

2.2.2.3 Liquid Column Chromatography 

The different types of LC have been mostly used in preparative or semipreparative scale for preseparation 

of essential oils or for isolation of individual oil constituents for structure elucidation 

with spectroscopic methods and were rarely used at that time as an analytical separation tool alone, 

because GC plays a central role in the study of essential oils. 

2.2.2.3.1 Preseparation of Essential Oils 

A different approach besides two-dimensional GC, which has often been used in the past to overcome 

peak overlapping in a single GC run of an essential oil has been preseparation of the oil with 

LC. The most common method of fractionation is the separation of hydrocarbons from the oxygenated 

terpenoids according to Miller et al. (1952), using silica gel as an adsorbent. After elution of the 

nonpolar components from the column with pentane or hexane, the more polar oxygen-containing 

constituents are eluted in order of increasing polarity after applying more and more polar eluents. 

A very simple and standardized fractionation in terms of speed and simplicity has been published 

by Kubeczka (1973) using dry-column chromatography. The procedure, which has been 

proved useful in numerous experiments for prefractionation of an essential oil, allows a preseparation 

into five fractions of increasing polarity. The preseparation of an essential oil into oxygenated 

constituents, monoterpene hydrocarbons, and sesquiterpene hydrocarbons, which is—depending on


the oil composition—sometimes of higher practical use, can be performed successfully using 

reversed-phase RP-18 HPLC (Schwanbeck et al., 1982). The HPLC was operated on a semipreparative 

scale by stepwise elution with methanol–water 82.5:17.5 (solvent A) and pure methanol 

(solvent B). The elution order of the investigated oil was according to decreasing polarity of the 

components and within the group of hydrocarbons to increasing molecular weight. Fraction 1 contained 

all oxygenated mono- and sesquiterpenoids, fraction 2 the monoterpene hydrocarbons, and 

fraction 3—eluted with pure methanol—the sesquiterpene hydrocarbons. A further alternative 

to the mentioned separation techniques is flash chromatography, initially developed by Still et al. 

(1978), and which has often been used as a rapid form of preparative LC based on a gas- or air 

pressure-driven short column chromatography. This technique, optimized for rapid separation of 

quantities typically in the range of 0.5–2.0 g uses dry-packed silica gel in an appropriate column. 

The separation of the sample generally takes only 5–10 min and can be performed with inexpensive 

laboratory equipment. However, impurities and active sites on dried silica gel were found to be 

responsible for isomerization of a number of oil constituents. After deactivation of the dried silica 

gel by adding 5% water, isomerization processes could be avoided (Scheffer et al., 1976). A different 

approach using HPLC on silica gel and isocratic elution with a ternary solvent system for the separation 

of essential oils has been published by Chamblee et al. (1985). In contrast to the aforementioned 

commonly used off-line pretreatment of a sample, the coupling of two or more chromatographic 

systems in an on-line mode offers advantages of ease of automation and usually of a shorter 

analysis time. 

2.2.2.3.2 High-Performance Liquid Column Chromatography 

The good separations obtained by GC have delayed the application of HPLC to the analysis of 

essential oils; however, HPLC analysis offers some advantages, if GC analysis of thermolabile compounds 

is difficult to achieve. Restricting factors for application of HPLC for analyses of terpenoids 

are the limitations inherent in the commonly available detectors and the relatively small range of k¢ 

values of liquid chromatographic systems. Since temperature is an important factor that controls k¢ 

values, separation of terpene hydrocarbons was performed at –15°C using a silica gel column and 

n-pentane as a mobile phase. Monitoring has been achieved with UV detection at 220 nm. Under 

these conditions, mixtures of commonly occurring mono- and sesquiterpene hydrocarbons could be 

well separated (Schwanbeck et al., 1979; Kubeczka, 1981b). However, the silica gel had to be deactivated 

by adding 4.8% water prior to separation to avoid irreversible adsorption or alteration of the 

sample. The investigation of different essential oils by HPLC already has been described in the 

seventies of the last century (e.g., Komae et al., 1975; Ross, 1976; Wulf et al., 1978; McKone, 1979; 

Scott and Kucera, 1979). In the last publication, the authors have used a rather long microbore 

packed column, which had several hundred thousand theoretical plates. Besides relatively expensive 

equipment, the HPLC chromatogram of an essential oil, separated on such a column could only be 

obtained at the expense of long analysis time. The mentioned separation needed about 20 h and may 

be only of little value in practical applications. 

More recent papers with regard to HPLC separation of essential oils were published, for example, 

by Debrunner et al. (1995), Bos et al. (1996), Frérot et al. (2004), and applications using silver 

ion-impregnated sorbents have been presented by Pettei et al. (1977), Morita et al. (1983), Friedel 

et al. (1987), and van Beek et al. (1994). The literature on the use and theory of silver complexation 

chromatography has been reviewed by van Beek et al. (1995). HPLC has also been used to separate 

thermally labile terpenoids at low temperature by Beyer et al. (1986), showing the temperature 

dependence of the separation efficiency. The investigation of an essential oil fraction from Cistus 

ladanifer using RP-18 reversed-phase HPLC at ambient temperature and an acetonitrile-water 

gradient was published by Strack et al. (1980). Comparison of the obtained HPLC chromatogram 

with the respective GC run exhibits a relatively good HPLC separation in the range of sesqui- and 

diterpenes, while the monoterpenes exhibited, as expected, a significant better resolution by GC. 

The enantiomeric separation of sesquiterpenes by HPLC with a chiral stationary phase has recently 

been shown by Nishii et al. (1997), using a Chiralcel ® OD column.


2.2.2.4 Supercritical Fluid Chromatography 

Supercritical fluids are highly compressed gases above their critical temperature and critical pressure 

point, representing a hybrid state between a liquid and a gas and which have physical properties 

intermediate between liquid and gas phases. The diffusion coefficient of a fluid is about two orders 

of magnitude larger and the viscosity is two orders of magnitude lower than the corresponding 

properties of a liquid. On the other hand, a supercritical fluid has a significant higher density than a 

gas. The commonly used carbon dioxide as a mobile phase, however, exhibits a low polarity 

(comparable to pentane or hexane), limiting the solubility of polar compounds, a problem that has 

been solved by adding small amounts of polar solvents, for example, methanol or ethanol, to increase 

mobile-phase polarity, thus permitting separations of more polar compounds (Chester et al., 1986). 

A further strength of SFC lies in the variety of detection systems that can be applied. The intermediate 

features of SFC between GC and LC can be profitable when used in a variety of detection 

systems, which can be classified in LC- and GC-like detectors. In the first case, measurement takes 

place directly in the supercritical medium or in the liquid phase, whereas GC-like detection proceeds 

after a decompression stage. 

Capillary SFC using carbon dioxide as mobile phase and a FID as detector has been applied to 

the analysis of several essential oils and seemed to give more reliable quantification than GC, 

especially for oxygenated compounds. However, the separation efficiency of GC for monoterpene 

hydrocarbons was, as expected, better than that of SFC. Manninen et al. (1990) published a 

comparison of a capillary GC versus a chromatogram obtained by capillary SFC from a linalool– 

methylchavicol basil oil chemotype exhibiting a fairly good separation by SFC. 

2.2.2.5 Countercurrent Chromatography 

CCC is according to Conway (1989) a form of liquid–liquid partition chromatography, in which 

centrifugal or gravitational forces are employed to maintain one liquid phase in a coil or train of 

chambers stationary, while a stream of a second, immiscible phase is passed through the system in 

contact with the stationary liquid phase. Retention of the individual components of the sample to be 

analyzed depends only on their partition coefficients and the volume ratio of the two applied liquid 

phases. Since there is no porous support, adsorption and catalytic effects encountered with solid 

supports are avoided. 

2.2.2.5.1 Droplet Countercurrent Chromatography (DCCC) 

One form of CCC, which has been sporadically applied to separate essential oils into fractions or in 

the ideal case into individual pure components, is DCCC. The device, which has been developed by 

Tanimura et al. (1970), consists of 300–600 glass tubes, which are connected to each other in series 

with Teflon ® tubing and filled with a stationary liquid. Separation is achieved by passing droplets of 

the mobile phase through the columns, thus distributing mixture components at different ratios 

leading to their separation. With the development of a water-free solvent system, separation of 

essential oils could be achieved (Becker et al., 1981, 1982). Along with the separation of essential 

oils, the method allows the concentration of minor components, since relatively large samples can 

be separated in one analytical run (Kubeczka, 1985). 

2.2.2.5.2 Rotation Locular Countercurrent Chromatography (RLCC) 

The RLCC apparatus (Rikakikai Co., Tokyo, Japan) consists of 16 concentrically arranged and 

serially connected glass tubes. These tubes are divided by Teflon ® disks with a small hole in the 

center, thus creating small compartments or locules. After filling the tubes with the stationary 

liquid, the tubes are inclined to a 30° angle from horizontal. In the ascending mode the lighter 

mobile phase is applied to the bottom of the first tube by a constant flow pump, displacing the 

stationary phase as its volume attains the level of the hole in the disk. The mobile phase passes 

through this hole and enters into the next compartment, where the process continues until the 

mobile phase emerges from the uppermost locule. Finally, the two phases fill approximately half


of each compartment. The dissolved essential oil subsequently introduced is subjected to a 

multistage partitioning process that leads to separation of the individual components. Whereas 

gravity contributes to the phase separation; rotation of the column assembly (60–80 rpm) produces 

circular stirring of the two liquids to promote partition. If the descending mode is selected 

for separation, the heavier mobile phase is applied at the top of each column by switching a valve. 

An overview on applications of RLCC in natural products isolation inclusive of a detailed 

description of the device and the selection of appropriate solvent systems has been presented by 

Snyder et al. (1984). 

Comparing RLCC to the aforementioned DCCC, one can particularly stress the superior flexibility 

of RLCC. While DCCC requires under all circumstances a two-phase system able to form 

droplets in the stationary phase, the choice of solvent systems with RLCC is nearly free. So the limitations 

of DCCC, when analyzing lipophilic samples, do not apply to RLCC. The separation of a 

mixture of terpenes has been presented by Kubeczka (1985). A different method, the high-speed 

centrifugal countercurrent chromatography (HSCCC) developed by Ito and coworkers in the mid of 

the sixties of the last century (Ito et al., 1966), has been applied to separate a variety of nonvolatile 

natural compounds; however, separation of volatiles has, strange to say, until now not seriously been 

evaluated. 

2.2.3 HYPHENATED TECHNIQUES 

2.2.3.1 Gas Chromatography-Mass Spectrometry 

The advantage on-line coupling of a chromatographic device to a spectrometer is that complex mixtures 

can be analyzed in detail by spectral interpretation of the separated individual components. 

The coupling of a gas chromatograph with a mass spectrometer is the most often used and a wellestablished 

technique for the analysis of essential oils, due to the development of easy-to-handle 

powerful systems concerning sensitivity, data acquisition and processing, and above all their relatively 

low cost. The very first application of a GC-MS coupling for the identification of essential oil 

constituents using a capillary column was already published by Buttery et al. (1963). In those times, 

mass spectra have been traced on UV recording paper with a five-element galvanometer and their 

evaluation was a considerable cumbersome task. 

This has changed after the introduction of computerized mass digitizers yielding the mass 

numbers and the relative mass intensities. The different kinds of GC-MS couplings available at the 

end of the seventies of the last century have been described in detail by ten Noever de Brauw 

(1979). In addition, different types of mass spectrometers have been applied in GC-MS investigations 

such as magnetic sector instruments, quadrupole mass spectrometers, ion-trap analyzers 

(e.g., ion-trap detector, ITD), and time-of-flight mass spectrometers, which are the fastest MS 

analyzers and therefore used for very fast GC-MS systems (e.g., in comprehensive multidimensional 

GC-MS). Surprisingly, a time-of-flight mass spectrometer was used in the very first description 

of a GC-MS investigation of an essential oil mentioned before. From the listed spectrometers, 

the magnetic sector and quadrupole instruments can also be used for selective ion monitoring 

(SIM), to improve sensitivity for the analysis of target compounds and for discrimination of 

overlapping GC peaks. 

The great majority of today’s GC-MS applications utilize one-dimensional capillary GC with 

quadrupole MS detection and electron ionization. Nevertheless, there are substantial numbers of 

applications using different types of mass spectrometers and ionization techniques. The proliferation 

of GC-MS applications is also a result of commercially available easy-to-handle dedicated 

mass spectral libraries (e.g., NIST/EPA/NIH 2005; WILEY Registry 2006; MassFinder 2007; and 

diverse printed versions such as Jennings and Shibamoto, 1980; Joulain and König, 1998; and 

Adams, 1989, 1995, 2007 inclusive of retention indices) providing identification of the separated 

compounds. However, this type of identification has the potential of producing some unreliable 

results, if no additional information is used, since some compounds, for example, the sesquiterpene


hydrocarbons a-cuprenene and b-himachalene, exhibit identical fragmentation pattern and only 

very small differences of their retention index values. This example demonstrates impressively that 

even a good library match and the additional use of retention data may lead in some cases to questionable 

results, and therefore require additional analytical data, for example, from NMR 

measurements. 

2.2.3.1.1 Gas Chromatography-Chemical Ionization-Mass Spectrometry and 

Gas Chromatography-Tandem Mass Spectrometry 

Although GC-electron impact (EI)-MS is a very useful tool for the analysis of essential oils, this technique 

can sometimes be not selective enough and requires more sophisticated techniques such as gas 

chromatography-chemical ionization-mass spectrometry (GC-CI-MS) and gas chromatographytandem 

mass spectrometry (GC-MS-MS). The application of CI-MS using different reactant gases 

is particularly useful, since many terpene alcohols and esters fail to show a molecular ion. The use 

of OH − as a reactant ion in negative CI-MS appeared to be an ideal solution to this problem. This 

technique yielded highly stable quasi-molecular-ions M–H, which are often the only ions in the 

obtained spectra of the above-mentioned compounds. As an example, the EI and CI spectra of 

isobornyl isovalerate—a constituent of valerian oil—shall be quoted (Bos et al., 1982). The respective 

EI mass spectrum shows only a very small molecular ion at 238. Therefore, the chemical ionization 

spectra of isobornyl acetate were performed exhibiting with isobutene as a reactant gas a 

[C 10 H 17 ] + -cation and in the negative CI-mode with OH - as a reactant gas two signals with the masses 

101, the isovalerate anion, and 237 the quasi-molecular-ion [M–H] − . Considering all these obtained 

data, the correct structure of the oil constituent could be deduced. The application of isobutane and 

ammonia as reactant gases has been presented by Schultze et al. (1992), who investigated sesquiterpene 

hydrocarbons by GC-CI-MS. Fundamental aspects of chemical ionization MS have been 

reviewed by Bruins (1987), discussing the different reactant gases applied in positive and negative 

ion chemical ionization and their applications in essential oil analysis. 

The utilization of GC-MS-MS to the analysis of a complex mixture will be shown in Figure 2.6. 

In the investigated vetiver oil (Cazaussus et al., 1988), one constituent, the nor-sesquiterpene ketone 

khusimone, has been identified by using GC-MS-MS in the collision-activated-dissociation mode. 

The molecular ion at m/z 204 exhibited a lot of daughter ions, but only one of them gave a daughter 

M +– 

Neutral loss of m/z 96 

204 

Fragment ion 

M +– 

×5 

108 

M +– 

Daughter ions 

of 

m/z 204 

30 

96 

108 (Main beam) 

(Main beam) 

Parent ions 

of m/z 108 

91 

119 

148 

161 

175 

189 

204 

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 m/z 

FIGURE 2.6 GC-EIMS-MS of khusimone of vetiver oil. (From Cazaussus, A., et al., 1988. Chromatographia, 

25: 865–869. With permission.)


ion at m/z 108, a fragment rarely occurring in sesquiterpene derivatives so that the presence of 

khusimone could be undoubtedly identified. 

2.2.3.2 High-Resolution Gas Chromatography-Fourier Transform 

Infrared Spectroscopy 

A further hyphenated technique, providing valuable analytical information is the on-line coupling of 

a gas chromatograph with a FTIR spectrometer. The capability of IR spectroscopy to provide discrimination 

between isomers makes the coupling of a gas chromatograph to an FTIR spectrometer 

suited as a complementary method to GC/MS for the analysis of complex mixtures like essential oils. 

The GC/FTIR device consists basically of a capillary gas chromatograph and an FTIR spectrometer 

including a dedicated computer and ancillary equipment. As each GC peak elutes from the GC column, 

it enters a heated IR measuring cell, the so-called light pipe, usually a gold-plated glass tube 

with IR transparent windows. There the spectrum is measured as an interferogram from which the 

familiar absorbance spectrum can be calculated by computerized Fourier transformation. After passing 

the light pipe, the effluent is directed back into the FID of the gas chromatograph. More detailed 

information on the experimental setup was given by Herres et al. (1986) and Herres (1987). 

In the latter publication, for example, the vapor-phase IR spectra of all the four isomers of 

pulegol and dihydrocarveol are shown, which have been extracted from a GC/FTIR run. These 

examples convincingly demonstrate the capability of distinguishing geometrical isomers with the 

aid of vapor-phase IR spectra, which cannot be achieved by their mass spectra. A broad application 

of GC-FTIR in the analysis of essential oils, however, is limited by the lack of sufficient vaporphase 

spectra of uncommon compounds, which are needed for reference use, since the spectra of 

isolated molecules in the vapor phase can be significantly different from the corresponding condensed-phase 

spectra. 

A different approach has been published by Reedy et al. in 1985, using a cryogenically freezing 

of the GC effluent admixed with an inert gas (usually argon) onto a rotating disk maintained at 

liquid He temperature to form a solid matrix trace. After the separation, reflection absorption spectra 

can be obtained from the deposited solid trace. A further technique published by Bourne et al. (1990) 

is the subambient trapping, whereby the GC effluent is cryogenically frozen onto a moving IR transparent 

window of zinc selenide (ZnSe). An advantage of the latter technique is that the unlike larger 

libraries of conventional IR spectra can be searched in contrast to the limited number of vaporphase 

spectra and those obtained by matrix isolation. A further advantage of both cryogenic techniques 

is the significant higher sensitivity, which exceeds the detection limits of a light pipe 

instrument by approximately two orders of magnitude. 

Comparing GC/FTIR and GC/MS, advantages and limitations of each technique become visible. 

The strength of IR lies—as discussed before—in distinguishing isomers, whereas identification of 

homologues can only be performed successfully by MS. The logical and most sophisticated way to 

overcome these limitations has been the development of a combined GC/FTIR/MS instrument, 

whereby simultaneously IR and mass spectra can be obtained. 

2.2.3.3 Gas Chromatography-Ultraviolet Spectroscopy 

The instrumental coupling of gas chromatograph with a rapid scanning UV spectrometer has been 

presented by Kubeczka et al. (1989). In this study, a UV-VIS diode-array spectrometer (Zeiss, 

Oberkochen, FRG) with an array of 512 diodes was used, which provided continuous monitoring in 

the range of 200–620 nm. By interfacing the spectrometer via fiber optics to a heated flow cell, 

which was connected by short heated capillaries to the GC column effluent, interferences of 

chromatographic resolution could be minimized. With the aid of this device, several terpene hydrocarbons 

have been investigated. In addition to displaying individual UV spectra, the available 

software rendered the analyst to define and to display individual window traces, three-dimensional 

plots and contour plots, which are valuable tools for discovering and deconvoluting gas chromatographic 

unresolved peaks.


2.2.3.4 Gas Chromatography-Atomic Emission Spectroscopy 

A device for the coupling of capillary gas chromatography with atomic emission spectroscopy 

(GC-AES) has been presented by Wylie et al. (1989). By means of this coupling 23 elements of a 

compound including all elements of organic substances separated by GC could be selectively 

detected providing the analyst not only with valuable information on the elemental composition of 

the individual components of a mixture, but also with the percentages of the elemental composition. 

The device incorporates a microwave-induced helium plasma at the outlet of the column coupled to 

an optical emission spectrometer. From the 15 most commonly occurring elements in organic compounds 

up to eight could be detected and measured simultaneously, for example, C, O, N, and S, 

which are of importance with respect to the analysis of essential oils. The examples given in the 

literature (e.g., Wylie et al., 1989; Bicchi et al., 1992; David et al., 1992; Jirovetz et al., 1992; Schultze, 

1993) indicate that the GC-AES coupling can provide the analyst with additional valuable information, 

which are to some extent complementary to the date obtained by GC-MS and GC-FTIR, making 

the respective library searches more reliable and more certain. 

However, the combined techniques GC-UV and GC-AES have not gained much importance in 

the field of essential oil research, since UV-spectra offer only low information and the coupling of 

a GC-AES, yielding the exact elemental composition of a component, can to some extent be obtained 

by precise mass measurement. Nevertheless, the on-line coupling GC-AES is still today efficiently 

used in environmental investigations. 

2.2.3.5 Gas Chromatography-Isotope Ratio Mass Spectrometry 

In addition to enantioselective capillary gas chromatography the on-line coupling of gas chromatography 

with isotope-ratio mass spectrometry (GC-IRMS) is an important technique in authentication 

of food flavors and essential oil constituents. The on-line combustion of effluents from capillary gas 

chromatographic separations to determine the isotopic compositions of individual components from 

complex mixtures was demonstrated by Matthews et al. (1978). On the basis of this work, the on-line 

interfacing of capillary GC with IRMS was later improved. With the commercially available 

GC-combustion IRMS device (GC-C-IRMS) measurements of the ratios of the stable isotopes 13 C/ 12 C 

have been accessible and respective investigations have been reported in several papers (e.g., Carle 

et al., 1990; Bernreuther et al., 1990; Braunsdorf et al., 1992, 1993; Frank et al., 1995; Mosandl et al., 

1997). A further improvement was the development of the GC-pyrolysis-IRMS (GC-P-IRMS) making 

measurements of 18 O/ 16 O ratios and later 2 H/ 1 H ratios feasible (Juchelka et al., 1998; Ruff et al., 

2000; Hör et al., 2001; Mosandl, 2004). Thus, the GC-P-IRMS device (Figure 2.7) appears today as 

one of the most sophisticated instruments for the appraisal of the genuineness of natural mixtures. 

2.2.3.6 High-Performance Liquid Chromatography-Gas Chromatography 

The on-line coupling of an HPLC device to a capillary gas chromatograph offers a number of 

advantages, above all higher column chromatographic efficiency, simple and rapid method development, 

simple cleanup of samples from complex matrices, and effective enrichment of the components 

of interest; additionally, the entire analytical procedure can easily be automated, thus increasing 

accuracy and reproducibility. The commercially available HPLC-GC coupling consists of an HPLC 

device that is connected with a capillary gas chromatograph via an interface allowing the transfer 

of HPLC fractions. Two different types of interfaces have been often used: The on-column interface 

is a modification of the on-column injector for GC; it is particularly suited for the transfer of fairly 

small fraction containing volatile constituents (Dugo et al., 1994; Mondello et al., 1994a, b, 1995). 

The second interface uses a sample loop and allows to transfer large sample volumes (up to 1 mL) 

containing components with limited volatilities. Figure 2.8 gives a schematic view of such an 

LC-GC instrument. In the shown position of the six-port valve, the desired fraction of the HPLC 

effluent is stored in the sample loop, while the carrier gas is passed through the GC columns. After 

switching the valve, the content of the sample loop is driven by the carrier gas into the large volume 

injector, vaporized and enters the precolumns, where the sample components are retained and most


MDGC Interface Mass spectrometer 

He 

Standard 

Ion 

H 2 

source 90º magnet 

Multicolumn 

switching system 

MCS 2 

Injector 

Monitor 

detector 

Pyrolysis 

reactor 

T=1450ºC 

Water 

separator 

He 

Open 

split 

CH 4 

2 3 

Faraday cups 


amplifiers 

Precolumn 

Transfer line 

heated 

Main column 

FIGURE 2.7 Scheme of an MDGC-C/P-IRMS device. (From Sewenig, S., et al., 2005. J. Agric. Chem., 53: 

838–844. With permission.) 

of the solvent vapor can be removed through the solvent vapor exit (SVE). After closing this valve 

and increasing the GC-oven temperature, the sample components are volatilized and separated in 

the main column reaching the detector. The main drawback of this technique, however, may be the 

loss of highly volatile compounds that are vented together with the solvent. As an example of an 

HPLC-GC investigation, the preseparation of lemon oil with gradient elution into four fractions is 

quoted (Munari et al., 1990). The respective gas chromatograms of the individual fractions exhibit 

good separation into hydrocarbons, esters, carbonyls, and alcohols, facilitating gas chromatographic 

separation and identification. Due to automation of all analytical steps involved, the manual 

Waste 

Carrier 

SVE 

LVI 

DE 

Sample 

HPLC 

column 

HPLC RG C1 C2 

FIGURE 2.8 Basic arrangement of an HPLC-GC device with a sample loop interface. RG: retention gap; 

C1: retaining column; C2: analytical column; LVI: large volume injector; and SVE: solvent vapor exit.


operations are significantly reduced and very good reproducibility was obtained. In three excellent 

review articles, the different kinds of HPLC-GC couplings are discussed in detail, describing their 

advantages and limitations with numerous references cited therein (Mondello et al., 1996, 1999; 

Dugo et al., 2003). 

2.2.3.7 HPLC-MS, HPLC-NMR Spectroscopy 

The on-line couplings of HPLC with MS and NMR spectroscopy are further important techniques 

combining high-performance separation with structurally informative spectroscopic techniques, 

but they are mainly applied to nonvolatile mixtures and shall not be discussed in more detail here, 

although they are very useful for investigating plant extracts. 

Some details concerning the different ionization techniques used in HPLC-MS have been presented 

among other things by Dugo et al. (2005). 

2.2.3.8 Supercritical Fluid Extraction-Gas Chromatography 

Although SFE is not a chromatographic technique, separation of mixtures can be obtained during 

the extraction process by varying the physical properties such as temperature and pressure to obtain 

fractions of different composition. Detailed reviews on the physical background of SFE and its 

application to natural products analysis inclusive of numerous applications have been published by 

Modey et al. (1995), and more recently by Pourmortazavi et al. (2007). The different types of 

couplings (off-line and on-line) have been presented by several authors. Houben et al. (1990) 

described an on-line coupling of SFE with capillary GC using a programmed temperature vaporizer 

as an interface. Similar approaches have been used by Blanch et al. (1994) in their investigations 

of rosemary leaves and by Ibanez et al. (1997) studying Spanish raspberries. In both the last two 

papers an off-line procedure was applied. A different device has been used by Hartonen et al. (1992) 

in a study of the essential oil of Thymus vulgaris using a cooled stainless steel capillary for trapping 

the volatiles connected via a six-port valve to the extraction vessel and the GC column. After 

sampling of the volatiles within the trap they have been quickly vaporized and flushed into the GC 

column by switching the walve. The recoveries of thyme components by SFE-GC were compared 

with those obtained from hydrodistilled thyme oil by GC exhibiting a good agreement. The SFE-GC 

analyses of several flavor and fragrance compounds of natural products by transferring the extracted 

compounds from a small SFE cell directly into a GC capillary has already been presented by 

Hawthorne et al. (1988). By inserting the extraction cell outlet restrictor (a 20 μm I.D. capillary) into 

the GC column through a standard on-column injection port, the volatiles were transferred and 

focused within the column at 40°C, followed by rapid heating to 70°C (30°C/min) and successive 

usual temperature programming. The suitability of that approach has been demonstrated with a 

variety of samples including rosemary, thyme, cinnamon, spruce needles, orange peel, and cedar 

wood. In a review article from Greibrokk, published in 1995, numerous applications of SFE 

connected on-line with gas chromatography and other techniques, the different instruments, and 

interfaces have been discussed, including the main parameters responsible for the quality of the 

obtained analytical results. In addition, the instrumental setups for SFE-LC and SFE-SFC couplings 

are given. 

2.2.3.9 Supercritical Fluid Chromatography-Gas Chromatography 

On-line coupling of SFC with gas chromatography has sporadically been used for the investigation 

of volatiles from aromatic herbs and spices. The requirements for instrumentation regarding the 

pumps, the restrictors, and the detectors are similar to those of SFE-GC. Additional parts of the 

device are the separation column and the injector, to introduce the sample into the mobile phase 

and successively into the column. The most common injector type in SFC is the high-pressure 

valve injector, similar to those used in HPLC. With this valve, the sample is loaded at ambient pressure 

into a sample loop of defined size and can be swept into the column after switching the valve 

to the injection position. The separation columns used in SFC may be either packed or open tubular


columns with their respective advantages and disadvantages. The latter mentioned open tubular 

columns for SFC can be compared with the respective GC columns; however, they must have 

smaller internal diameter. With regard to the detectors used in SFC, the FID is the most common 

applied detector, presuming that no organic modifiers have been admixed to the mobile phase. 

In that case, for example, a UV detector with a high-pressure flow cell has to be taken into 

consideration. 

In a paper, presented by Yamauchi et al. (1990), cold-pressed lemon-peel oil has been separated 

by semipreparative SFC into three fractions, namely hydrocarbons, aldehydes and alcohols, and 

esters together with other oil constituents. The obtained fractions were afterward analyzed by 

capillary GC. SFC has also often been combined with SFE prior to chromatographic separation in 

plant volatile oil analysis, since in both techniques the same solvents are used, facilitating an on-line 

coupling. SFE and on-line-coupled SFC have been applied to the analysis of turmeric, the rhizomes 

of Curcuma longa L., using modified carbon dioxide as the extractant, yielding fractionation of 

turmerones curcuminoids in a single run (Sanagi et al., 1993). A multidimensional SFC-GC system 

was developed by Yarita et al. (1994) to separate on-line the constituents of citrus essential oils by 

stepwise pressure programming. The eluting fractions were introduced into a split/splitless injector 

of a gas chromatograph and analyzed after cryofocusing prior to GC separation. An SFC-GC investigation 

of cloudberry seed oil extracted with supercritical carbon dioxide was described by 

Manninen et al. (1997), in which SFC was mainly used for the separation of the volatile constituents 

from the low-boiling compounds, such as triacylglycerols. The volatiles were collected in a trap 

column and refocused before being separated by GC. Finally, an on-line technique shall be mentioned 

by which the compounds eluting from the SFC column can be completely transferred to GC, 

but also for selective or multistep heart-cutting of various sample peaks as they elute from the SFC 

column (Levy et al., 2005). 

2.2.3.10 Couplings of SFC-MS and SFC-FTIR Spectroscopy 

Both coupling techniques such as SFC-MS and SFC-FTIR have nearly exclusively been used for the 

investigation of low-volatile more polar compounds. Arpino published in 1990 a comprehensive 

article on the different coupling techniques in SFC-MS, which have been presented up to 1990 

including 247 references. A short overview of applications using SFC combined with benchtop mass 

spectrometers was published by Ramsey and Raynor (1996). However, the only paper concerning 

the application of SFC-MS in essential oil research was published by Blum et al. (1997). With the 

aid of a newly developed interface and an injection technique using a retention gap, investigations 

of thyme extracts have been successfully performed. 

The application of SFC-FTIR spectroscopy for the analysis of volatile compounds has also rarely 

been reported. One publication found in the literature refers to the characterization of varietal 

differences in essential oil components of hops (Auerbach et al., 2000). In that paper, the IR spectra 

of the main constituents were taken as films deposited on AgCl disks and compared with spectra 

obtained after chromatographic separation in a flow cell with IR transparent windows, exhibiting 

a good correlation. 

2.2.4 IDENTIFICATION OF MULTICOMPONENT SAMPLES WITHOUT PREVIOUS SEPARATION 

In addition to chromatographic separation techniques including hyphenated techniques, several 

spectroscopic techniques have been applied to investigate the composition of essential oils without 

previous separation. 

2.2.4.1 UV Spectroscopy 

UV spectroscopy has only little significance for the direct analysis of essential oils due to the 

inability to provide uniform information on individual oil components. However, for testing the 

presence of furano-coumarins in various citrus oils, which can cause photodermatosis when applied


externally, UV spectroscopy is the method of choice. The presence of those components can be easily 

determined due to their characteristic UV absorption. In the European Pharmacopoeia for example, 

quality assessment of lemon oil, which has to be produced by cold pressing, is therefore 

performed by UV spectroscopy in order to exclude cheaper distilled oils. 

2.2.4.2 IR Spectroscopy 

Several attempts have also been made to obtain information about the composition of essential oils 

using IR spectroscopy. One of the first comprehensive investigations of essential oils was published 

by Bellanato and Hidalgo (1971) in the book Infrared Analysis of Essential Oils in which the IR 

spectra of approximately 200 essential oils and additionally of more than 50 pure reference components 

have been presented. However, the main disadvantage of this method is the low sensitivity 

and selectivity of the method in the case of mixtures with a large number of components and 

secondly the unsolvable problem when attempting to quantitatively measure individual component 

concentrations. 

New approaches to analyze essential oils by vibrational spectroscopy using attenuated reflection 

(ATR) IR spectroscopy and NIR-FT-Raman spectroscopy have recently been published by Baranska 

et al. (2005) and numerous papers cited therein. The main components of an essential oil can be 

identified by both spectroscopic techniques using the spectra of pure oil constituents as references. 

The spectroscopic analysis is based on characteristic key bands of the individual constituents and 

made it, for example, possible to discriminate the oil profiles of several eucalyptus species. As can 

be taken from this paper, valuable information can be obtained as a result of the combined application 

of ATR-IR and NIR-FT-Raman spectroscopy. Based on reference GC measurements, valuable 

calibration equations have been developed for numerous essential oil plants and related essential 

oils in order to quantify the amount of individual oil constituents applying different suitable chemometric 

algorithms. Main advantages of those techniques are their ability to control the quality of 

essential oils very fast and easily and above all, to quantify and analyze the main constituents of 

essential oils in situ, that means in living plant tissues without any isolation process, since both 

techniques are not destructive. 

2.2.4.3 Mass Spectrometry 

MS and proton NMR spectroscopy have mainly been used for structure elucidation of isolated 

compounds. However, there are some reports on mass spectrometric analyses of essential oils. One 

example has been presented by Grützmacher (1982). The depicted mass spectrum (Figure 2.9) of 

an essential oil exhibits some characteristic molecular ions of terpenoids with masses at m/z 136, 

148, 152, and 154. By the application of a double focusing mass spectrometer and special techniques 

analyzing the decay products of metastable ions, the components anethole, fenchone, 

borneol, and cineole could be identified, while the assignment of the mass 136 proved to be 

problematic. 

A different approach has been used by Schultze et al. (1986), investigating secondary metabolites 

in dried plant material by direct mass spectrometric measurement. The small samples (0.1–2 mg, 

depending on the kind of plant drug) were directly introduced into a mass spectrometer by means 

of a heatable direct probe. By heating the solid sample, stored in a small glass crucible, various 

substances are released depending on the applied temperature, and subsequently their mass spectra 

can be taken. With the aid of this technique, numerous medicinal plant drugs have been investigated 

and their main vaporizable components could be identified. 

2.2.4.4 

13 

C-NMR Spectroscopy 

13 

C-NMR spectroscopy is generally used for the elucidation of molecular structures of isolated 

chemical species. The application of 13 C-NMR spectroscopy to the investigation of complex 

mixtures is relatively rare. However, the application of 13 C-NMR spectroscopy to the analysis of


100% 

m/z 93 

50 

m/z 121 

m/z 136 m/z 152 

m/z 148 m/z 154 

50 100 150 

m/z 

FIGURE 2.9 EI-mass spectrum of an essential oil. (From Grützmacher, H.F. 1982. In Ätherische Öle 

Analytik, Physiologie, Zusammensetzung, K.H. Kubeczka (ed.), pp. 1–24. Stuttgart, New York: Georg Thieme 

Verlag. With permission.) 

essential oils and similar complex mixtures offers particular advantages, as have been shown in 

the past (Formaček and Kubeczka, 1979, 1982; Kubeczka, 2002), to confirm analytical 

results obtained by GC-MS and for solving certain problems encountered with nonvolatile mixture 

components or thermally unstable compounds, since analysis is performed at ambient 

temperature. 

The qualitative analysis of an essential oil is based on comparison of the oils spectrum, using 

broadband decoupling, with spectra of pure oil constituents which should be recorded under identical 

conditions regarding solvent, temperature, and so on to ensure that differences in the chemical 

shifts for individual 13 C-NMR lines of the mixture and of the reference substance are negligible. 

As an example, the identification of the main constituent of celery oil is shown (Figure 2.10). This 

constituent can be easily identified as limonene by the corresponding reference spectrum. Minor 

constituents give rise to less intensive signals that can be recognized after a vertical expansion of 

the spectrum. For recognition of those signals also a horizontal expansion of the spectrum is 

advantageous. 

The sensitivity of the 13 C-NMR technique is limited by diverse factors such as rotational sidebands, 

13 

C- 13 C-couplings, and so on, and at least by the accumulation time. For practical use, the concentration 

of 0.1% of a component in the entire mixture has to be seen as an interpretable limit. A very 

pretentious investigation has been presented by Kubeczka (1989). In the investigated essential oil, 

consisting of more than 80 constituents, approximately 1200 signals were counted after a horizontal 

and vertical expansion in the obtained broadband decoupled 13 C-NMR spectrum, which reflects 

impressively the complex composition of that oil. However, the analysis of such a complex mixture is 

made difficult by the immense density of individual lines, especially in the aliphatic region of the 

spectrum, making the assignments of lines to individual components ambiguous. Besides, qualitative 

analysis quantification of the individual sample components is accessible as described by Formaček 

and Kubeczka (1982a). After elimination of the 13 C-NMR signals of nonprotonated nuclei and calculation 

of average signal intensity per carbon atom as a measurement characteristic, it has been possible 

to obtain satisfactory results as shown by comparison with gas chromatographic analyses.


Limonene 

Celery oil 

170 

160 

150 

140 

130 

120 

110 

100 

90 

(ppm) 

80 

70 

60 

50 

40 

30 

20 

10 

FIGURE 2.10 Identification of limonene in celery oil by 13 C-NMR spectroscopy. 

During the last years, a number of articles have been published by Casanova and coworkers 

(e.g., Bradesi et al. (1996) and references cited therein). In addition, papers dealing with computer-aided 

identification of individual components of essential oils after 13 C-NMR measurements (e.g., Tomi 

et al., 1995), and investigations of chiral oil constituents by means of a chiral lanthanide shift 

reagent by carbon-13 NMR spectroscopy have been published (Ristorcelli et al., 1997). 

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3 

Sources of Essential Oils 

Chlodwig Franz and Johannes Novak 

CONTENTS 

3.1 “Essential Oil-Bearing Plants”: Attempt of a Definition ..................................................... 39 

3.2 Phytochemical Variation ..................................................................................................... 41 

3.2.1 Chemotaxonomy ...................................................................................................... 41 

3.2.2 Inter- and Intraspecific Variation ............................................................................. 42 

3.2.2.1 Lamiaceae (Labiatae) and Verbenaceae .................................................... 42 

3.2.2.2 Asteraceae (Compositae) ............................................................................ 46 

3.3 Identification of Source Materials ....................................................................................... 52 

3.4 Genetic and Protein Engineering ........................................................................................ 53 

3.5 Resources of Essential Oils: Wild Collection or Cultivation of Plants ............................... 54 

3.5.1 Wild Collection and Sustainability .......................................................................... 58 

3.5.2 Domestication and Systematic Cultivation .............................................................. 59 

3.5.3 Factors Influencing the Production and Quality of 

Essential Oil-Bearing Plants .................................................................................... 60 

3.5.3.1 Genetic Variation and Plant Breeding ....................................................... 61 

3.5.3.2 Plant Breeding and Intellectual Property Rights ....................................... 63 

3.5.3.3 Intraindividual Variation between Plant Parts and Depending on the 

Developmental Stage (Morpho- and Ontogenetic Variation) .................... 65 

3.5.3.4 Environmental Influences .......................................................................... 69 

3.5.3.5 Cultivation Measures, Contaminations, and Harvesting ........................... 69 

3.6 International Standards for Wild Collection and Cultivation ............................................. 72 

3.6.1 GA(C)P: Guidelines for Good Agricultural (and Collection) Practice of 

Medicinal and Aromatic Plants ............................................................................... 72 

3.6.2 ISSC-MAP: The International Standard on Sustainable Wild Collection 

of Medicinal and Aromatic Plants ........................................................................... 72 

3.6.3 FairWild ................................................................................................................... 73 

3.7 Conclusion ............................................................................................................................. 73 

References .................................................................................................................................... 73 

3.1 “ESSENTIAL OIL-BEARING PLANTS”: ATTEMPT OF A DEFINITION 

Essential oils are complex mixtures of volatile compounds produced by living organisms and 

isolated by physical means only (pressing and distillation) from a whole plant or plant part of known 

taxonomic origin. The respective main compounds are mainly derived from three biosynthetic pathways 

only, the mevalonate pathway leading to sesquiterpenes, the methyl-erithrytol-pathway leading 

to mono- and diterpenes, and the shikimic acid pathway en route to phenylpropenes. Nevertheless, 

there are an almost uncountable number of single substances and a tremendous variation in the 

composition of essential oils. Many of these volatile substances have diverse ecological functions. 

They can act as internal messengers, as defensive substances against herbivores or as volatiles 

39


directing not only natural enemies to these herbivores but also attracting pollinating insects to their 

host (Harrewijn et al., 2001). 

All plants possess principally the ability to produce volatile compounds, quite often, however, 

only in traces. “Essential oil plants” in particular are those plant species delivering an essential 

oil of commercial interest. Two principal circumstances determine a plant to be used as an essential 

oil plant: 

a. A unique blend of volatiles like the flower scents in rose (Rosa spp.), jasmine (Jasminum 

sambac), or tuberose (Polyanthes tuberosa). Such flowers produce and immediately emit 

the volatiles by the epidermal layers of their petals (Bergougnoux et al., 2007). Therefore 

the yield is even in intensive smelling flowers very low, and besides distillation special 

techniques, as an example, enfleurage has to be applied to recover the volatile fragrance 

compounds. 

b. Secretion and accumulation of volatiles in specialized anatomical structures. This leads to 

higher concentrations of the essential oil in the plant. Such anatomical storage structures 

for essential oils can be secretory idioblasts (secretory cells), cavities/ducts, or glandular 

trichomes (Fahn, 1979, 1988; colorfully documented by Svoboda et al., 2000). 

Secretory idioblasts are individual cells producing an essential oil in large quantities and retaining 

the oil within the cell like the essential oil idioblasts in the roots of Vetiveria zizanioides which 

occurs within the cortical layer and close to the endodermis (Bertea and Camusso, 2002). Similar 

structures containing essential oils are also formed in many flowers, for example, Rosa sp., Viola 

sp., or Jasminum sp. 

Cavities or ducts consist of extracellular storage space that originate either from schizogeny 

(created by the dissolution of the middle lamella between the duct initials and formation of an intercellular 

space) or by lysogeny (programmed death and dissolution of cells). In both cases, the peripheral 

cells are becoming epithelial cells highly active in synthesis and secretion of their products into 

the extracellular cavities (Pickard, 2008). Schizogenic oil ducts are characteristic for the Apiaceae 

family, for example, Carum carvi, Foeniculum vulgare, or Cuminum cyminum, but also for 

Hypericaceae or Pinaceae. Lysogenic cavities are found in Rutaceae (Citrus sp., Ruta graveolens), 

Myrtaceae (e.g., Syzygium aromaticum), and others. 

Secreting trichomes (glandular trichomes) can be divided into two main categories: peltate and 

capitate trichomes. Peltate glands consist of a basal epidermal cell, a neck-stalk cell and a secreting 

head of 4–16 cells with a large subcuticular space on the apex in which the secretion product is 

accumulated. The capitate trichomes possess only 1–4 secreting cells with only a small subcuticular 

space (Werker, 1993; Maleci Bini and Giuliani, 2006). Such structures are typical for Lamiaceae 

(the mint family), but also for Pelargonium sp. 

The monoterpene biosynthesis in different species of Lamiaceae, for example, sage (Salvia 

offi cinalis) and peppermint (Mentha × piperita), is restricted to a brief period early in leaf development 

(Croteau et al., 1981; Gershenzon et al., 2000). The monoterpene biosynthesis in peppermint 

reaches a maximum in 15-day-old leaves, only very low rates were observed in leaves 

younger than 12 days or older than 20 days. The monoterpene content of the peppermint leaves 

increased rapidly up to day 21, then leveled off, and kept stable for the remainder of the leaf life 

(Gershenzon et al., 2000). 

The composition of the essential oil often changes between different plant parts. Phytochemical 

polymorphism is often the case between different plant organs. In Origanum vulgare ssp. hirtum, 

a polymorphism within a plant could even be detected on a much lower level, namely between 

different oil glands of a leaf (Johnson et al., 2004). This form of polymorphism seems to be not 

frequently occurring, differences in the composition between oil glands is more often related to the 

age of the oil glands (Grassi et al., 2004; Johnson et al., 2004; Novak et al., 2006a; Schmiderer 

et al., 2008).

Sources of Essential Oils 41 

Such polymorphisms can also be found quite frequently when comparing the essential oil composition 

of individual plants of a distinct species (intraspecific variation, “chemotypes”) and is 

based on the plants genetical background. 

The differences in the complex composition of two essential oils of one kind may sometimes be 

difficult to assign to specific chemotypes or to differences arising in the consequence of the reactions 

of the plants to specific environmental conditions, for example, to different growing locations. 

In general, the differences due to genetical differences are much bigger than by different environmental 

conditions. However, many intraspecific polymorphisms are probably not yet detected or 

have been described only recently even for widely used essential oil crops like sage (Novak et al., 

2006b). 

3.2 PHYTOCHEMICAL VARIATION 

3.2.1 CHEMOTAXONOMY 

The ability to accumulate essential oils is not omnipresent in plants but scattered throughout the 

plant kingdom; in many cases, however, very frequent within—or a typical character of—certain 

plant families. From the taxonomical and systematic point of view, not the production of essential 

oils is the distinctive feature since this is a quite heterogeneous group of substances, but either the 

type of secretory containers (trichomes, oil glands, lysogenic cavities, or schizogenic oil ducts) or 

the biosynthetically specific group of substances, for example, mono- or sesquiterpenes, phenylpropenes, 

and so on; the more a substance is deduced in the biosynthetic pathway the more specific it 

is for certain taxa: monoterpenes are typical for the genus Mentha, but menthol is characteristic for 

Mentha piperita and Mentha arvensis ssp. piperascens only; sesquiterpenes are common in the 

Achillea–millefolium complex, but only Achillea roseo-alba (2¥) and Achillea collina (4¥) are able 

to produce matricine as precursor of (the artifact) chamazulene (Vetter et al., 1997). On the other 

hand, the phenylpropenoid eugenol, typical for cloves (Syzygium aromaticum, Myrtaceae) can also 

be found in large amounts in that distant species, for example, cinnamon (Cinnamomum zeylanicum, 

Lauraceae) or basil (Ocimum basilicum, Lamiaceae); as sources for anethole are known aniseed 

(Pimpinella anisum) and fennel (F. vulgare) both Apiaceae, but also star anise (Illicium verum, 

Illiciaceae), Clausena anisata (Rutaceae), Croton zetneri (Euphorbiaceae), or Tagetes lucida 

(Asteraceae). Finally, eucalyptol (1,8-cineole)—named after its occurrence in Eucalyptus sp. 

(Myrtaceae)—may also be a main compound of the essential oil of galangal (Alpinia offi cinarum, 

Zingiberaceae), bay laurel (Laurus nobilis, Lauraceae), Japan pepper (Zanthoxylum piperitum, 

Rutaceae), and a number of plants of the mint family, for example, sage (S. offi cinalis, Salvia fruticosa, 

Salvia lavandulifolia), rosemary (Rosmarinus offi cinalis), and mints (Mentha sp.). Taking the 

above facts into consideration, chemotaxonomically relevant are (therefore) common or distinct 

pathways, typical fingerprints, and either main compounds or very specific even minor or trace substances 

[e.g., d-3-carene to separate Citrus grandis from other Citrus sp. (Gonzalez et al., 2002)]. 

The plant families comprising species that yield a majority of the most economically important 

essential oils are not restricted to one specialized taxonomic group but are distributed among all 

plant classes: gymnosperms, for example, the families Cupressaceae (cedarwood, cedar leaf, juniper 

oil, etc.) and Pinaceae (pine and fir oils, etc.), as well as angiosperms, and among them within 

Magnoliopsida, Rosopsida, and Liliopsida. The most important families of dicots are Apiaceae 

(e.g., fennel, coriander, and other aromatic seed/root oils), Asteraceae or Compositae (chamomile, 

wormwood, tarragon oil, etc.), Geraniaceae (geranium oil), Illiciaceae (star anise oil), Lamiaceae 

(mint, patchouli, lavender, oregano, and many other herb oils), Lauraceae (litsea, camphor, cinnamon, 

sassafras oil, etc.), Myristicaceae (nutmeg and mace), Myrtaceae (myrtle, cloves, and allspice), 

Oleaceae (jasmine oil), Rosaceae (rose oil), and Santalaceae (sandalwood oil). In monocots 

(Liliopsida), it is substantially restricted to Acoraceae (calamus), Poaceae (vetiver and aromatic 

grass oils), and Zingiberaceae (e.g., ginger and cardamom).


Apart from the phytochemical group of substances typical for a taxon, the chemical outfit 

depends, furthermore, on the specific genotype, the stage of plant development—also influenced by 

environmental factors—and the plant part (see Section 3.2.1). Considering all these influences 

chemotaxonomic statements and conclusions have to be based on comparable material, grown and 

harvested under comparable circumstances. 

3.2.2 INTER- AND INTRASPECIFIC VARIATION 

Knowledge on biochemical systematics and the inheritance of phytochemical characters depends on 

extensive investigations of taxa (particularly species) and populations on single-plant basis, respectively, 

and several examples of genera show that the taxa do indeed display different patterns. 

3.2.2.1 Lamiaceae (Labiatae) and Verbenaceae 

The presumably largest genus among the Lamiaceae is sage (Salvia L). consisting of about 900 species 

widely distributed in the temperate, subtropical, and tropical regions all over the world with 

major centers of diversity in the Mediterranean, in Central Asia, the Altiplano from Mexico throughout 

Central and South America, and in southern Africa. Almost 400 species are used in traditional 

and modern medicine, as aromatic herbs or ornamentals worldwide; among them are S. offi cinalis, 

S. fruticosa, Salvia sclarea, Salvia divinorum, Salvia miltiorrhiza, and Salvia pomifera to name a 

few. Many applications are based on nonvolatile compounds, for example, diterpenes and polyphenolic 

acids. Regarding the essential oil, there are a vast number of mono- and sesquiterpenes found 

in sage but, in contrast to, for example, Ocimum sp. and Perilla sp. (also Lamiaceae), no phenylpropenes 

were detected. 

To understand species-specific differences within this genus, the Mediterranean S. offi cinalis 

complex (S. offi cinalis, S. fruticosa, and S. lavandulifolia) will be confronted with the Salvia stenophylla 

species complex (S. stenophylla, Salvia repens, and Salvia runcinata) indigenous to South 

Africa: In the S. offi cinalis group usually a- and b-thujones, 1,8-cineole, camphor, and in some 

cases linalool, b-pinene, limonene, or cis-sabinyl acetate are the prevailing substances, whereas in 

the S. stenophylla complex quite often sesquiterpenes, for example, caryophyllene or a-bisabolol, 

are main compounds. 

Based on taxonomical studies of Salvia spp. (Hedge, 1992; Skoula et al., 2000; Reales et al., 

2004) and a recent survey concerning the chemotaxonomy of S. stenophylla and its allies (Viljoen 

et al., 2006), Figure 3.1 shows the up-to-now-identified chemotypes within these taxa. Comparing 

the data of different publications the picture is, however, not as clear as demonstrated by six S. offi - 

cinalis origins in Figure 3.2 (Chalchat et al., 1998; Asllani, 2000). This might be due to the prevailing 

chemotype in a population, the variation between single plants, the time of sample collection, 

and the sample size. This is exemplarily shown by one S. offi cinalis population where the individuals 

varied in a-thujone, from 9% to 72%, b-thujone, from 2% to 24%; 1,8-cineole, from 4% to 18%; 

and camphor from 1% to 25%. The variation over three years and five harvests of one clone only 

ranged as follows: a-thujone 35–72%, b-thujone 1–7%, 1,8-cineole 8–15%, and camphor 1–18% 

(Bezzi, 1994; Bazina et al., 2002). But also all other (minor) compounds of the essential oil showed 

respective intraspecific variability (see, e.g., Giannouli and Kintzios, 2000). 

S. fruticosa was principally understood to contain 1,8-cineole as main compound but at best 

traces of thujones, as confirmed by Putievsky et al. (1986) and Kanias et al. (1998). In a comparative 

study of several origins, Máthé et al. (1996) identified, however, a population with atypically high 

b-thujone similar to S. offi cinalis. Doubts on if this origin could be true S. fruticosa or a spontaneous 

hybrid of both species were resolved by extensive investigations on the phytochemical and 

genetic diversity of S. fruticosa in Crete (Karousou et al., 1998; Skoula et al., 1999). There it was 

shown that all wild populations in western Crete consist of 1,8-cineole chemotypes only whereas in 

the eastern part of the island essential oils with up to 30% thujones, mainly b-thujone, could be 

observed. In Central Crete, finally, mixed populations were found. A cluster analysis based on


Chemotypes 

South African 

sage 

S. stenophylla S. repens S. runcinata 

α-bisabolol 

δ-3-carene 

limonene 

(E)-nerolidol 

γ-terpinene 

δ-3-carene 

ρ-cymene 

α-bisabolol 

(E)-nerolidol 

β-phellandrene 

β-caryophyllene 

ledol 

(E)-nerolidol 

α-bisabolol 

(E)-nerolidol 


α-pinene 

Chemotypes 

European sage 

S. officinalis S. lavandulifolia S. fruticosa 

α-pinene 

camphor 

β-thujone 

α-thujone 

camphor 

1,8-cineole 

β-thujone 

camphor 

β-pinene 

1,8-cineole 

caryophyllene 

limonene 

1,8-cineole 

camphor 

β-thujone 

1,8-cineole 

FIGURE 3.1 Chemotypes of some South African and European Salvia species. 

random amplification of polymorphic DNA (RAPD) patterns confirmed the genetic differences 

between the West- and East-Crete populations of S. fruticosa (Skoula et al., 1999). 

A rather interesting example of diversity is oregano, which counts to the commercially most 

valued spices worldwide. More than 60 plant species are used under this common name showing 

similar flavor profiles characterized mainly by cymyl-compounds, for example, carvacrol and thymol. 

With few exemptions the majority of oregano species belong to the Lamiaceae and Verbenaceae 

families with the main genera Origanum and Lippia (Table 3.1). In 1989, almost all of the estimated 

15,000 ton/yr dried oregano originated from wild collection; today, some 7000 ha of Origanum 

onites are cultivated in Turkey alone (Baser, 2002), O. onites as well as other Origanum species are 

cultivated in Greece, Israel, Italy, Morocco, and other countries. 

30 

25 

20 

15 

1,8-cineole 

α-thujone 

β-thujone 

10 

camphor 

5 

α-humulene 

0 

Albania 

Hungary 

Romania 

Czech 

Republic 

France 

Portugal 

FIGURE 3.2 Composition of the essential oil of six Salvia offi cinalis origins.


TABLE 3.1 

Species Used Commercially in the World as Oregano 

Family/Species 

Calamintha potosina Schaf. 

Coleus amboinicus Lour. (syn. C. aromaticus Benth) 

Labiatae 

Coleus aromaticus Benth. 

Hedeoma fl oribunda Standl. 

Hedeoma incona Torr. 

Hedeoma patens Jones 

Hyptis albida HBK. 

Hyptis americana (Aubl.) Urb. (H. gonocephala Gris.) 

Hyptis capitata Jacq. 

Hyptis pectinata Poit. 

Hyptis suaveolens (L.) Poit. 

Monarda austromontana Epling 

Ocimum basilicum L. 

Origanum compactum Benth. (syn. O. glandulosum Salzm, ex Benth.) 

Origanum dictamnus L. (Majorana dictamnus L.) 

Origanum elongatum (Bonent) Emberger et Maire 

Origanum fl oribundum Munby (O. cinereum Noe) 

Origanum grosii Pau et Font Quer ex letswaart 

Origanum majorana L. 

Origanum microphyllum (Benth) Vogel 

Origanum onites L. (syn. O. smyrneum L.) 

Origanum scabrum Boiss et Heldr. (syn. O. pulchrum Boiss et Heldr.) 

Origanum syriacum L. var. syriacum (syn. O. maru L.) 

Origanum vulgare L. subsp. gracile (Koch) letswaart (syn. O. gracile 

Koch, O. tyttanthum Gontscharov) 

Origanum vulgare ssp. hirtum (Link) letswaart (syn. O. hirtum Link) 

Origanum vulgare ssp. virens (Hoffmanns et Link) letswaart (syn. O. 

virens Hoffmanns et Link) 

Origanum vulgare ssp. viride (Boiss.) Hayek (syn. O. viride) Halacsy 

(syn. O. heracleoticum L.) 

Origanum vulgare L. subsp. vulgare (syn. Thymus origanum (L.) 

Kuntze) 

Origanum vulgare L. 

Poliomintha longifl ora Gray 

Salvia sp. 

Satureja thymbra L. 

Thymus capitatus (L.) Hoffmanns et Link (syn. Coridothymus 

capitatus (L.) Rchb.f.) 

Lantana citrosa (Small) Modenke 

Lantana glandulosissima Hayek 

Verbenaceae 

Lantana hirsuta Mart. et Gall. 

Lantana involucrata L. 

Lantana purpurea (Jacq.) Benth. & Hook. (syn. Lippia purpurea Jacq.) 

Lantana trifolia L. 

Commercial Name/s Found in Literature 

Oregano de la sierra, oregano, origanum 

Oregano, oregano brujo, oregano de Cartagena, 

oregano de Espana, oregano Frances 

Oregano de Espana, oregano, Origanum 

Oregano, Origanum 

Oregano 



Oregano 



Oregano, oregano cimarron, Origanum 








Oregano 


*Turkish oregano, oregano, Origanum 





Oregano, Origanum, oregano verde 

*Greek oregano, oregano, Origanum 


Oregano, orenga, Oregano de Espana 

Oregano 

Oregano 

Oregano cabruno, oregano, Origanum 

*Spanish oregano, oregano, Origanum 

Oregano xiu, oregano, Origanum 

Oregano xiu, oregano silvestre, oregano, 

Origanum 

Oreganillo del monte, oregano, Origanum 




continued


TABLE 3.1 (continued) 

Species Used Commercially in the World as Oregano 

Family/Species 

Lantana velutina Mart.&Gal. 

Lippia myriocephala Schlecht.&Cham. 

Lippia affi nis Schau. 

Lippia alba (Mill) N.E. Br. (syn. L. involucrata L.) 

Lippia berlandieri Schau. 

Lippia cordiostegia Benth. 

Lippia formosa T.S.Brandeg. 

Lippia geisseana (R.A.Phil.) Soler. 

Lippia graveolens HBK 

Lippia helleri Britton 

Lippia micromera Schau. 

Lippia micromera var. helleri (Britton) Moldenke 

Lippia origanoides HBK 

Lippia palmeri var. spicata Rose 

Lippia palmeri Wats. 

Lippia umbellata Cav. 

Lippia velutina Mart. et Galeotti 

Borreria sp. 

Scrophulariaceae 

Limnophila stolonifera (Blanco) Merr. 

Eryngium foetidum L. 

Rubiaceae 

Apiaceae 

Commercial Name/s Found in Literature 

Oregano xiu, oregano, Origanum 

Oreganillo 

Oregano 


Oregano 

Oreganillo, oregano montes, oregano, Origanum 



*Mexican oregano, oregano cimarron, oregano 

Oregano del pais, oregano, Origanum 

Oregano del pais, oregano, Origanum 

Oregano 

Oregano, origano del pais 

Oregano 


Oreganillo, oregano montes, oregano, Origanum 


Oreganos, oregano, Origanum 


Oregano de Cartagena, oregano, Origanum 

Asteraceae 

Coleosanthus veronicaefolius HBK 

Oregano del cerro, oregano del monte, oregano 

del campo 

Eupatorium macrophyllum L. (syn. Hebeclinium macrophyllum DC.) Oregano, Origanum 

*Oregano species with economic importance according to Lawrence (1984). 

In comparison with sage, the genus Origanum is much smaller and consists of 43 species and 18 

hybrids according to the actual classification (Skoula and Harborne, 2002) with main distribution 

areas around the Mediterranean. Some subspecies of O. vulgare only are also found in the temperate 

and arid zones of Eurasia up to China. Nevertheless, the genus is characterized by a large morphological 

and phytochemical diversity (Kokkini, 1996; Baser, 2002; Skoula and Harborne, 2002). 

The occurrence of several chemotypes is reported, for example, for commercially used Origanum 

species, from Turkey (Baser, 2002). In O. onites, two chemotypes are described, a carvacrol type 

and a linalool type. Additionally, a “mixed type” with both basic types mixed may occur. In Turkey, 

two chemotypes of Origanum majorana are known, one contains cis-sabinene hydrate as chemotypical 

lead compound and is used as marjoram in cooking (“marjoramy”), while the other one 

contains carvacrol in high amounts and is used to distil “oregano oil” in a commercial scale. 

Variability of chemotypes continues also within the “marjoramy” O. majorana. Novak et al. (2002) 

detected in cultivated marjoram accessions additionally to cis-sabinene hydrate the occurrence of 

polymorphism of cis-sabinene hydrate acetate. Since this chemotype did not influence the sensorial 

impression much, this chemotype was not eliminated in breeding, while an “off-flavor” chemotype 

would have been certainly eliminated in its cultivation history. In natural populations of O. majorana 

from Cyprus besides the “classical” cis-sabinene hydrate type, a chemotype with a-terpineol as


main compound was also detected (Novak et al., 2008). The two extreme “off-flavor” chemotypes 

in O. majorana, carvacrol-, and a-terpineol-chemotype are not to be found anywhere in cultivated 

marjoram, demonstrating one of the advantages of cultivation in delivering homogeneous qualities. 

The second “oregano” of commercial value—mainly used in the Americas—is “Mexican oregano” 

(Lippia graveolens HBK., Verbenaceae) endemic to California, Mexico, and throughout Central 

America (Fischer, 1998). Due to wild harvesting, only the few published data show essential oil contents 

largely ranging from 0.3% to 3.6%. The total number of up-to-now-identified essential oil compounds 

comprises almost 70 with the main constituents thymol (3.1–80.6%), carvacrol (0.5–71.2%), 

1,8-cineole (0.1–14%), and p-cymene (2.7–28.0%), followed by, for example, myrcene, g-terpinene, and 

the sesquiterpene caryophyllene (Lawrence, 1984, Dominguez et al., 1989, Uribe-Hernández et al., 

1992, Fischer et al., 1996; Vernin, 2001). 

In a comprehensive investigation of wild populations of L. graveolens collected from the hilly 

regions of Guatemala, three different essential oil chemotypes could be identified, a thymol, a carvacrol, 

and an absolutely irregular type (Fischer et al., 1996). Within the thymol type, contents of up 

to 85% thymol in the essential oil could be obtained and only traces of carvacrol. The irregular type 

has shown a very uncommon composition where no compound exceeds 10% of the oil, and also 

phenylpropenes, for example, eugenol and methyl eugenol, were present (Fischer et al., 1996; Fischer, 

1998). In Table 3.2, a comparison of recent data is given including Lippia alba commonly called 

“oregano” or “oregano del monte” although carvacrol and thymol are absent from the essential oil of 

this species. In Guatemala, two different chemotypes were found within L. alba: a myrcenone and a 

citral type (Fischer et al., 2004). Besides it, a linalool, a carvone, a camphor—1,8-cineole, and a 

limonene–piperitone chemotype have been described (Dellacassa et al., 1990; Pino et al., 1997; 

Frighetto et al., 1998; Senatore and Rigano, 2001). 

Chemical diversity is of special interest if on genus or species level both terpenes as well as phenylpropenes 

can be found in the essential oil. Most Lamiaceae preferentially accumulate mono- 

(and sesqui-)terpenes in their volatile oils but some genera produce oils also rich in phenylpropenes, 

among these Ocimum sp. and Perilla sp. 

The genus Ocimum comprises over 60 species, of which Ocimum gratissimum and O. basilicum 

are of high economic value. Biogenetic studies on the inheritance of Ocimum oil constituents were 

reported by Khosla et al. (1989) and an O. gratissimum strain named “Clocimum” containing 65% 

of eugenol in its oil was described by Bradu et al. (1989). A number of different chemotypes of basil 

(O. basilicum) has been identified and classified (Vernin, 1984; Marotti et al., 1996) containing up 

to 80% linalool, up to 21.5% 1,8-cineole, 0.3–33.0% eugenol, and also the presumably toxic compounds 

methyl chavicol (estragole) and methyl eugenol in concentrations close to 50% (Elementi 

et al., 2006; Macchia et al., 2006). 

Perilla frutescens can be classified in several chemotypes as well according to the main monoterpene 

components perillaldehyde, elsholtziaketone, or perillaketones, and on the other side phenylpropanoid 

types containing myristicin, dillapiole, or elemicin (Koezuka et al., 1986). A 

comprehensive presentation on the chemotypes and the inheritance of the mentioned compounds 

was given by this author in Hay and Waterman (1993). In the referred last two examples not only the 

sensorial but also the toxicological properties of the essential oil compounds are decisive for the 

(further) commercial use of the respective species’ biodiversity. 

Although the Labiatae family plays an outstanding role as regards the chemical polymorphism 

of essential oils, also in other essential oil containing plant families and genera a comparable phytochemical 

diversity can be observed. 

3.2.2.2 Asteraceae (Compositae) 

Only a limited number of genera of the Asteraceae are known as essential oil plants, among 

them Tagetes, Achillea, and Matricaria. The genus Tagetes comprises actually 55 species, all of 

them endemic to the American continents with the center of biodiversity between 30° northern 

and 30° southern latitude. One of the species largely used by the indigenous population is


TABLE 3.2 

Main Essential Oil Compounds of Lippia graveolens and L. alba According to Recent Data 

L. graveolens L. alba 

Fischer et al. 

(1996) 

Guatemala 

Senatore et al. 

(2001) 

Vernin et al. 

(2001) 

Fischer 

(1998) 

Guatemala 

Senatore 

et al. (2001) 

Lorenzo 

(2001) 

Compound Thymol-Type 

Carvacrol- 

Type 

Irregular 

Type Guatemala El-Salvador 

Myrcenone- 

Type 

Cineole- 

Type Guatemala Uruguay 

Myrcene 1.3 1.9 2.7 1.1 t 6.5 1.7 0.2 0.8 

p-Cymene 2.7 6.9 2.8 5.5 2.1 t t 0.7 n.d. 

1.8-Cineole 0.1 0.6 5.0 2.1 t t 22.8 14.2 1.3 

Limonene 0.2 0.3 1.5 0.8 t 1.0 3.2 43.6 2.9 

Linalool 0.7 1.4 3.8 0.3 t 4.0 2.4 1.2 55.3 

Myrcenon n.d. n.d. n.d. n.d. n.d. 54.6 3.2 n.d. n.d. 

Piperitone n.d. n.d. n.d. n.d. n.d. t t 30.6 n.d. 

Thymol 80.6 19.9 6.8 31.6 7.3 n.d. n.d. n.d. n.d. 

Carvacrol 1.3 45.2 1.1 0.8 71.2 n.d. n.d. n.d. n.d. 

b-Caryophyllene 2.8 3.5 8.7 4.6 9.2 2.6 1.2 1.0 9.0 

a-Humulene 1.9 2.3 5.7 3.0 5.0 0.7 t 0.6 0.9 

Caryophyll.-ox. 0.3 0.8 3.3 4.8 t 1.8 3.0 1.1 0.6 

Z-Dihydrocarvon/ 

n.d. n.d. n.d. n.d. n.d. 13.1 0.6 0.1 0.8 

Z-Ocimenone 

E-Dihydrocarvon n.d. n.d. n.d. n.d. n.d. 4.9 n.d. t 1.2 

n.d. = Not detectable; t = traces; main compounds in bold.


TABLE 3.3 

Main Compounds of the Essential Oil of Selected Tagetes lucida Types (in % of dm) 

Substance 

Anethole 

Type (2) 

Estragole 

Type (8) 

Methyleugenol 

Type (7) 

Nerolidol 

Type (5) 

Mixed 

Type 

Linalool 0.26 0.69 1.01 Tr. 3.68 

Estragole 11.57 78.02 8.68 3.23 24.28 

Anethole 73.56 0.75 0.52 Tr. 30.17 

Methyleugenol 1.75 5.50 79.80 17.76 17.09 

b-Caryophyllene 0.45 1.66 0.45 2.39 0.88 

Germacrene D 2.43 2.89 1.90 Tr. 5.41 

Methylisoeugenol 1.42 2.78 2.00 Tr. 3.88 

Nerolidol 0.35 0.32 0.31 40.52 1.24 

Spathulenol 0.10 0.16 0.12 Tr. 0.23 

Carophyllene oxide 0.05 0.27 0.45 10.34 0.53 

Location of origin in Guatemala: (2) Cabrican/Quetzaltenango, (5) La Fuente/Jalapa, (7) Joyabaj/El Quiche, (8) Sipacapa/ 

S.Marcos, Mixed Type: Taltimiche/San Marcos. 

Main compounds in bold. 

“pericon” (T. lucida Cav.), widely distributed over the highlands of Mexico and Central America 

(Stanley and Steyermark, 1976). In contrast to almost all other Tagetes species characterized by the 

content of tagetones, this species contains phenylpropenes and terpenes. A detailed study on its 

diversity in Guatemala resulted in the identification of several eco- and chemotypes (Table 3.3): 

anethol, methyl chavicol (estragol), methyl eugenol, and one sesquiterpene type producing higher 

amounts of nerolidol (Bicchi et al., 1997; Goehler, 2006). The distribution of the three main phenylpropenes 

in six populations is illustrated in Figure 3.3. In comparison with the plant materials 

investigated by Ciccio (2004) and Marotti et al. (2004) containing oils with 90–95% estragol, only 

the germplasm collection of Guatemaltekan provenances (Goehler, 2006) allows to select individuals 

with high anethol but low to very low estragol or methyleugenol content—or with interestingly 

high nerolidol content, as mentioned above. 

The genus Achillea is widely distributed over the northern hemisphere and consists of approximately 

120 species, of which the Achillea millefolium aggregate (yarrow) represents a polyploid 

complex of allogamous perennials (Saukel and Länger, 1992; Vetter and Franz, 1996). The different 

taxa of the recent classification (minor species and subspecies) are morphologically and chemically 

to a certain extent distinct and only the diploid taxa Achillea asplenifolia and A. roseo-alba as well 

60 

80 

70 

Anethole (%) 

50 

40 

30 

20 

10 

Estragole (%) 

60 

40 

20 

Methyleugenol (%) 

60 

50 

40 

30 

20 

10 

0 

1 

2 

3 

4 

7 

8 

0 

1 

2 

3 

4 

7 

8 

0 

1 

2 

3 

4 

7 

8 

Population 

Population 

Population 

FIGURE 3.3 Variability of anethole, methyl eugenol, and methyl chavicol (estragole) in the essential oil of 

six Tagetes lucida—populations from Guatemala.


TABLE 3.4 

Taxa within the Achillea-Millefolium-Group (Yarrow) 

Taxon 

Ploidy 

Level Main Compounds 

A. setacea W. et K. 2¥ Rupicoline 

A. aspleniifolia Vent. 2¥ (4¥) 7,8-Guajanolide 

Artabsin-derivatives 

3-Oxa-Guajanolide 

A. roseo-alba Ehrend. 2¥ Artabsin-derivatives 

3-Oxaguajanolide 

Matricinderivatives 

A. collina Becker 4¥ Artabsin-derivatives 

3-Oxaguajanolide 

Matricinderivatives 

Matricarinderivatives 

A. pratensis Saukel u. Länger 4¥ Eudesmanolides 

A. distans ssp. Distans W. et K. 6¥ Longipinenones 

A. distans ssp. Styriaca 4¥ 

A. tanacetifolia (stricta) W. et K. 6¥ 

A mill. ssp. sudetica 6¥ Guajanolidperoxide 

A. mill. ssp. Mill. L. 6¥ 

A. pannonica Scheele 8¥ (6¥) Germacrene 

Guajanolidperoxide 

Source: 

Franz et al., Unpublished. 

as the tetraploid A. collina and Achillea ceretanica are characterized by proazulens, for example, 

achillicin, whereas the other taxa, especially 6¥ and 8¥ contain eudesmanolides, longipinenes, germacranolides, 

and/or guajanolid peroxides, (Table 3.4). The intraspecific variation in the proazulene 

content ranged from traces up to 80%, other essential oil components of the azulenogenic 

species are, for example, a- and b-pinene, borneol, camphor, sabinene, or caryophyllene (Kastner 

et al., 1992). The frequency distribution of proazulene individuals among two populations is shown 

in Figure 3.4. 

Crossing experiments resulted in proazulene being a recessive character of di- and tetraploid 

Achillea sp. (Vetter et al., 1997) similar to chamomile (Franz, 1993a,b). Finally, according to 

3% 

1% 

1% 

Pro A 

12% 

Budakalaszi 210 

5% 

Pro-chamazulene 

1,8-cineole 

Linalool 


11% 

7% 

65% 

Pro-chamazulene 

1,8-cineole 

Linalool 

Borneol 


95% 

FIGURE 3.4 Frequency distribution of proazulene individuals among two Achillea sp. populations.


Steinlesberger et al. (2002) also a plant-to-plant variation in the enantiomers of, for example, a- and 

b-pinene as well as sabinene exists in yarrow oils, which makes it even more complicated to use 

phytochemical characters for taxonomical purposes. 

Differences in the essential oil content and composition of chamomile flowers (Matricaria 

recutita) have long been recognized due to the fact that the distilled oil is either dark blue, green, or 

yellow, depending on the prochamazulene content (matricin as prochamazulene in chamomile is 

transformed to the blue-colored artifact chamazulene during the distillation process). Recognizing 

also the great pharmacological potential of the bisabolols, a classification into the chemotypes 

(-)-a-bisabolol, (-)-a-bisabololoxide A, (-)-a-bisabololoxide B, (-)-a-bisabolonoxide (A), and 

(pro)chamazulene was made by Franz (1982, 1989a). Examining the geographical distribution 

revealed a regional differentiation, where an a-bisabolol—(pro) chamazulene population was identified 

on the Iberian peninsula, mixed populations containing chamazulene, bisabolol, and bisabololoxides 

A/B are most frequent in Central Europe, and prochamazulene—free bisabolonoxide 

populations are indigenous to southeast Europe and minor Asia. In the meantime, Wogiatzi et al. 

(1999) have shown for Greece and Taviani et al. (2002) for Italy a higher diversity of chamomile 

including a-bisabolol types. This classification of populations and chemotypes was extended by 

analyzing populations at the level of individual plants (Schröder, 1990) resulting in the respective 

frequency distributions (Figure 3.5). 

In addition, the range of essential oil components in the chemotypes of one Central European 

population is shown in Table 3.5 (Franz, 2000). 

Data on inter- and intraspecific variation of essential oils are countless and recent reviews are 

known for a number of genera published, for example, in the series “Medicinal and Aromatic Plants— 

Industrial Profiles” (Harwood publications, Taylor & Francis and CRC, Press, respectively). 

The generally observed quantitative and qualitative variation in essential oils draws the attention 

i.a. to appropriate random sampling for getting valid information on the chemical profile of a species 

or population. As concerns quantitative variations of a certain pattern or substance, Figure 3.6 shows 

2% 

2% 

H 29 

CH 29 

4% 

Menemen 

12% 

29% 

30% 

96% 

37% 

88% 

Bisabolol type 

Bisabolol oxide A type 

Bisabolol oxide B type 

Bisabolon oxide type 

Azulene type 

FIGURE 3.5 Frequency distribution of chemotypes in three varieties/populatios of chamomile [Matricaria 

recutita (L.) Rauschert].


TABLE 3.5 

Grouping within a European Spontaneous Chamomile, Figures in 

% of Terpenoids in the Essential Oil of the Flower Heads 

Chamazulen α-Bisabolol α-B.-Oxide A α-B.-Oxide B 

α-Bisabolol-Type 

Range 2.5–35.2 58.8–92.1 n.d.–1.0 n.d.–3.2 

Mean 23.2 68.8 n.d. n.d. 

α-Bisabololoxide A-Type 

Range 6.6–31.2 0.5–12.3 31.7–66.7 1.9–22.4 

Mean 21.3 2.1 53.9 11.8 

α-Bisabololoxide B-Type 

Range 7.6–24.2 0.8–6.5 1.6–4.8 61.6–80.5 

Mean 16.8 2.0 2.6 72.2 

Chamazulene-Type 

Range 76.3–79.2 5.8–8.3 n.d.–0.8 n.d.–2.6 

Mean 77.8 7.1 n.d. n.d. 

Source: Franz, Ch., 2000. Biodiversity and random sampling in essential oil plants. Lecture 

31st ISEO, Hamburg. 

Main compounds in bold. 

mg 

360 

Bisabolol 

340 

320 

300 

H 29 

280 

180 

160 

140 

120 

100 

CH 29 

80 

60 

40 

n plants (individuals) 

5 10 15 20 25 30 35 40 45 50 

FIGURE 3.6 (−)-a-Bisabolol-content (mg/100 g crude drug) in two chamomile (Matricaria recutita) populations: 

mean value in dependence of the number of individuals used for sampling.


80% 

70% 

60% 

50% 

40% 

30% 

1,8-cineole 

linalool 

trans-sabinene hydrate 

cis-sabinene hydrate 

α-terpineol 

α-terpinyl acetate 

20% 

10% 

0% 

All plants 

Linaloolchemotype 

1,8-Cineolechemotype 

Sabinene 

hydratechemotype 

α-Terpineolchemotype 

FIGURE 3.7 Mean values of the principal essential compounds of a Thymus vulgaris population (left) in 

comparison to the mean values of the chemotypes within the same population. 

exemplarily the bisabolol content of two chamomile populations depending on the number of individual 

plants used for sampling. At small numbers, the mean value oscillates strongly and only after 

at least 15–20 individuals the range of variation becomes acceptable. Quite different appears the situation 

at qualitative differences, that is, “either-or-variations” within populations or taxa, for example, 

carvacrol/thymol, a-/b-thujone/1,8-cineole/camphor, or monoterpenes/phenylpropenes. Any random 

sample may give nonspecific information only on the principal chemical profile of the respective 

population provided that the sample is representative. This depends on the number of chemotypes, 

their inheritance, and frequency distribution within the population, and generally speaking no less 

than 50 individuals are needed for that purpose, as it can be derived from the comparison of chemotypes 

in a Thymus vulgaris population (Figure 3.7). 

The overall high variation in essential oil compositions can be explained by the fact that quite 

different products might be generated by small changes in the synthase sequences only. On the other 

hand, different synthases may be able to produce the same substance in systematically distant taxa. 

The different origin of such substances can be identified by, for example, the 12 C: 13 C ratio (Mosandl, 

1993). “Hence, a simple quantitative analysis of the essential oil composition is not necessarily 

appropriate for estimating genetic proximity even in closely related taxa” (Bazina et al., 2002). 

3.3 IDENTIFICATION OF SOURCE MATERIALS 

As illustrated by the previous paragraph, one of the crucial points of using plants as sources for 

essential oils is their heterogeneity. A first prerequisite for reproducible compositions is therefore an 

unambiguous botanical identification and characterization of the starting material. The first approach 

is the classical taxonomical identification of plant materials based on macro- and micromorphological 

features of the plant. The identification is followed by phytochemical analysis that may contribute 

to species identification as well as to the determination of the quality of the essential oil. This 

approach is now complemented by DNA-based identification. 

DNA is a long polymer of nucleotides, the building units. One of four possible nitrogenous bases 

is part of each nucleotide and the sequence of the bases on the polymer strand is characteristic for


each living individual. Some regions of the DNA, however, are conserved on the species or family 

level and can be used to study the relationship of taxa (Taberlet et al., 1991; Wolfe and Liston, 1998). 

DNA sequences conserved within a taxon but different between taxa can therefore be used to identify 

a taxon (“DNA-barcoding”) (Hebert et al., 2003a; Kress et al., 2005). A DNA-barcoding consortium 

was founded in 2004 with the ambitious goal to build a barcode library for all eukaryotic 

life in the next 20 years (Ratnasingham and Hebert, 2007). New sequencing technologies (454, 

Solexa, SOLiD) enable a fast and representative analysis, but will be applied due to their high costs 

in the moment only in the next phase of DNA-barcoding (Frezal and Leblois, 2008). DNA-barcoding 

of animals has already become a routine task. DNA-barcoding of plants, however, are still not 

trivial and a scientific challenge (Pennisi, 2007). 

Besides sequence information-based approaches, multilocus DNA methods [RAPD, amplified 

fragment length polymorphism (AFLP), etc.] are complementing in resolving complicated taxa and 

can become a barcode for the identification of populations and cultivars (Weising et al., 2005). With 

multilocus DNA methods, it is furthermore possible to tag a specific feature of a plant of which the 

genetic basis is still unknown. This approach is called molecular markers (in sensu strictu) because 

they mark the occurrence of a specific trait like a chemotype or flower color. The gene regions visualized, 

for example, on an agarose gel is not the specific gene responsible for a trait but is located on the 

genome in the vicinity of this gene and therefore co-occurs with the trait and is absent when the trait 

is absent. An example for such an inexpensive and fast polymerase chain reaction (PCR)-system was 

developed by Bradbury et al. (2005) to distinguish fragrant from nonfragrant rice cultivars. If markers 

would be developed for chemotypes in essential oil plants, species identification by DNA and the 

determination of a chemotype could be performed in one step. 

Molecular biological methods to identify species are nowadays routinely used in feed- and foodstuffs 

to identify microbes, animals, and plants. Especially the discussion about traceability of genetically 

modified organisms (GMO’s) throughout the complete chain (“from the living organism to the 

super-market”) has sped up research in this area (Auer, 2003; Miraglia et al., 2004). One advantage 

of molecular biological methods is the possibility to be used in a number of processed materials like 

fatty oil (Pafundo et al., 2005) or even solvent extracts (Novak et al., 2007). The presence of minor 

amounts of DNA in an essential oil cannot be excluded a priori although distillation as separation 

technique would suggest the absence of DNA. However, small plant or DNA fragments could distill 

over or the essential oil could come in contact with plant material after distillation. 

3.4 GENETIC AND PROTEIN ENGINEERING 

Genetic engineering is defined as the direct manipulation of the genes of organisms by laboratory 

techniques, not to be confused with the indirect manipulation of genes in traditional (plant) breeding. 

Transgenic or genetically modifi ed organisms (GMOs) are organisms (bacteria, plants, etc.) 

that have been engineered with single or multiple genes (either from the same species or from a 

different species), using contemporary molecular biology techniques. These are organisms with 

improved characteristics, in plants, for example, with resistance or tolerance to biotic or abiotic 

stresses such as insects, disease, drought, salinity, and temperature. Another important goal in 

improving agricultural production conditions is to facilitate weed control by transformed plants 

resistant to broadband herbicides like glufosinate. Peppermint has been successfully transformed 

with the introduction of the bar gene, which encodes phosphinothricin acetyltransferase, an enzyme 

inactivating glufosinate-ammonium or the ammonium salt of glufosinate, phosphinothricin making 

the plant insensitive to the systemic, broad- spectrum herbicide Roundup (“Roundup Ready mint”) 

(Li et al., 2001). 

A first step in genetic engineering is the development and optimization of transformation (gene 

transfer) protocols for the target species. Such optimized protocols exist for essential oil plants such 

as lavandin (Lavandula × intermedia; Dronne et al., 1999), spike lavender (Lavandula latifolia; 

Nebauer et al., 2000), and peppermint (Mentha × piperita; Diemer et al., 1998; Niu et al., 2000).


TABLE 3.6 

Essential Oil Composition and Yield of Transgenic Peppermint Transformed with Genes 

Involved in Monoterpene Biosynthesis 

Gene Method Limonene Mentho-Furan Pulegone Menthone Menthol 

Oil Yield 

[lb/acre] 

WT — 1.7 4.3 2.1 20.5 44.5 97.8 

dxr overexpress 1.6 3.6 1.8 19.6 45.6 137.9 

Mfs antisense 1.7 1.2 0.4 22.7 45.2 109.7 

l-3-h cosuppress 74.7 0.4 0.1 4.1 3.0 99.6 

Source: Wildung, M.R. and R.B. Croteau, 2005. Transgenic Res., 14: 365–372. 

dxr = Deoxyxylulose phosphate reductoisomerase; l-3-h = limonene-3-hydroxylase; mfs = menthofuran synthase; WT = 

wild type. 

In spike lavender, an additional copy of the 1-deoxy-d-xylulose-5-phosphate synthase gene 

(DXS), the first enzymatic step in the methylerythritol phosphate (MEP) pathway leading to the 

precursors of monoterpenes, from Arabidopsis thaliana was introduced and led to an increase of 

the essential oil of the leaves of up to 360% and of the essential oil of flowers of up to 74% (Munoz- 

Bertomeu et al., 2006). 

In peppermint, many different steps to alter essential oil yield and composition were already 

targeted (reviewed by Wildung and Croteau 2005; Table 3.6). The overexpression of deoxyxylulose 

phosphate reductoisomerase (DXR), the second step in the MEP-pathway, increased the essential oil 

yield by approximately 40% tested under field conditions (Mahmoud and Croteau, 2001). The overexpression 

of geranyl diphosphate synthase (GPPS) leads to a similar increase of the essential oil. 

Menthofuran, an undesired compound, was downregulated by an antisense method (a method to 

influence or block the activity of a specific gene). Overexpression of the menthofuran antisense 

RNA was responsible for an improved oil quality by reducing both menthofuran and pulegone in 

one transformation step (Mahmoud and Croteau, 2003). The ability to produce a peppermint oil 

with a new composition was demonstrated by Mahmoud et al. (2004) by upregulating limonene by 

cosuppression of limonene-3-hydroxylase, the enzyme responsible for the transformation of 

(-)-limonene to (-)-trans-isopiperitenol en route to menthol. 

Protein engineering is the application of scientific methods (mathematical and laboratory methods) 

to develop useful or valuable proteins. There are two general strategies for protein engineering, 

random mutagenesis, and rational design. In rational design, detailed knowledge of the 

structure and function of the protein is necessary to make desired changes by site-directed mutagenesis, 

a technique already well developed. An impressive example of the rational design of 

monoterpene synthases was given by Kampranis et al. (2007) who converted a 1,8-cineole synthase 

from S. fruticosa into a synthase producing sabinene, the precursor of a- and b-thujone with 

a minimum number of substitutions. They went also a step further and converted this mono terpene 

synthase into a sesquiterpene synthase by substituting a single amino acid that enlarged the cavity 

of the active site enough to accommodate the larger precursor of the sesquiterpenes, farnesyl 

pyrophosphate (FPP). 

3.5 RESOURCES OF ESSENTIAL OILS: WILD COLLECTION 

OR CULTIVATION OF PLANTS 

The raw materials for producing essential oil are resourced either from collecting them in nature 

(“wild collection”) or from cultivating the plants (Table 3.7).


TABLE 3.7 

Important Essential Oil-Bearing Plants—Common and Botanical Names Incl. Family, 

Plant Parts Used, Raw Material Origin, and Trade Quantities of the Essential Oil 

Trade Name Species Plant Family 

Used Plant 

Part(s) 

Wild Collection/ 

Cultivation 

Trade 

Quantities a 

Ambrette seed Hibiscus abelmoschus L. Malvaceae Seed Cult LQ 

Amyris Amyris balsamifera L. Rutaceae Wood Wild LQ 

Angelica root Angelica archangelica L. Apiaceae Root Cult LQ 

Anise seed Pimpinella anisum L. Apiaceae Fruit Cult LQ 

Armoise Artemisia herba-alba Asso Asteraceae Herb Cult/wild LQ 

Asafoetida Ferula assa-foetida L. Apiaceae Resin Wild LQ 

Basil Ocimum basilicum L. Lamiaceae Herb Cult LQ 

Bay Pimenta racemosa Moore Myrtaceae Leaf Cult LQ 

Bergamot Citrus aurantium L. subsp. Rutaceae Fruit peel Cult MQ 

bergamia (Risso et Poit.) Engl. 

Birch tar Betula pendula Roth. (syn. Betulaceae Bark/wood Wild LQ 

Betula verrucosa Erhart. Betula 

alba sensu H.J.Coste. non L.) 

Buchu leaf Agathosma betulina (Bergius) Rutaceae Leaf Wild LQ 

Pillans. A. crenulata (L.) 

Pillans 

Cade Juniperus oxycedrus L. Cupressaceae Wood Wild LQ 

Cajuput Melaleuca leucandendron L. Myrtaceae Leaf Wild LQ 

Calamus Acorus calamus L. Araceae Rhizome Cult/wild LQ 

Camphor Cinnamomum camphora L. Lauraceae Wood Cult LQ 

(Sieb.) 

Cananga Cananga odorata Hook. f. et Annonaceae Flower Wild LQ 

Thoms. 

Caraway Carum carvi L. Apiaceae Seed Cult LQ 

Cardamom Elettaria cardamomum (L.) Zingiberaceae Seed Cult LQ 

Maton 

Carrot seed Daucus carota L. Apiaceae Seed Cult LQ 

Cascarilla Croton eluteria (L.) W.Wright Euphorbiaceae Bark Wild LQ 

Cedarwood, Cupressus funebris Endl. Cupressaceae Wood Wild MQ 

Chinese 

Cedarwood, Juniperus mexicana Schiede Cupressaceae Wood Wild MQ 

Texas 

Cedarwood, Juniperus virginiana L. Cupressaceae Wood Wild MQ 

Virginia 

Celery seed Apium graveolens L. Apiaceae Seed Cult LQ 

Chamomile Matricaria recutita L. Asteraceae Flower Cult LQ 

Chamomile, Anthemis nobilis L. Asteraceae Flower Cult LQ 

Roman 

Chenopodium Chenopodium ambrosioides (L.) Chenopodiaceae Seed Cult LQ 

Gray 

Cinnamon bark, Cinnamomum zeylanicum Nees Lauraceae Bark Cult LQ 

Ceylon 

Cinnamon bark, Cinnamomum cassia Blume Lauraceae Bark Cult LQ 

Chinese 

Cinnamon leaf Cinnamomum zeylanicum Nees Lauraceae Leaf Cult LQ 

continued






Used Plant 

Part(s) 


Cultivation 

Trade 

Quantities a 

Citronella, 

Ceylon 

Cymbopogon nardus (L.) 

W. Wats. 

Poaceae Leaf Cult HQ 

Citronella, Java Cymbopogon winterianus Jowitt. Poaceae Leaf Cult HQ 

Clary sage Salvia sclarea L. Lamiaceae Flowering Cult 

MQ 

herb 

Clove buds Syzygium aromaticum (L.) Myrtaceae Leaf/bud Cult LQ 

Merill et L.M. Perry 

Clove leaf Syzygium aromaticum (L.) Myrtaceae Leaf Cult HQ 

Merill et L.M. Perry 

Coriander Coriandrum sativum L. Apiaceae Fruit Cult LQ 

Cornmint Mentha canadensis L. (syn. Lamiaceae Leaf Cult HQ 

M. arvensis L. f. piperascens 

Malinv. ex Holmes; M. arvensis 

L. var. glabrata. M. haplocalyx 

Briq.; M. sachalinensis (Briq.) 

Kudo) 

Cumin Cuminum cyminum L. Apiaceae Fruit Cult LQ 

Cypress Cupressus sempervirens L. Cupressaeae Leaf/twig Wild LQ 

Davana Artemisia pallens Wall. Asteraceae Flowering Cult 

LQ 

herb 

Dill Anethum graveolens L. Apiaceae Herb/fruit Cult LQ 

Dill, India Anethum sowa Roxb. Apiaceae Fruit Cult LQ 

Elemi Canarium luzonicum Miq. Burseraceae Resin Wild LQ 

Eucalyptus Eucalyptus globulus Labill. Myrtaceae Leaf Cult/wild HQ 

Eucalyptus, Eucalyptus citriodora Hook. Myrtaceae Leaf Cult/wild HQ 

lemon-scented 

Fennel bitter Foeniculum vulgare Mill. subsp. Apiaceae Fruit Cult LQ 

vulgare var. vulgare 

Fennel sweet Foeniculum vulgare Mill. subsp. Apiaceae Fruit Cult LQ 

vulgare var. dulce 

Fir needle, Abies balsamea Mill. Pinaceae Leaf/twig Wild LQ 

Canadian 

Fir needle, Abies sibirica Ledeb. Pinaceae Leaf/twig Wild LQ 

Siberian 

Gaiac Guaiacum offi cinale L. Zygophyllaceae Resin Wild LQ 

Galbanum Ferula galbanifl ua Boiss. Apiaceae Resin Wild LQ 

F. rubricaulis Boiss. 

Garlic Allium sativum L. Alliaceae Bulb Cult LQ 

Geranium Pelargonium spp. Geraniaceae Leaf Cult MQ 

Ginger Zingiber offi cinale Roscoe Zingiberaceae Rhizome Cult LQ 

Gingergrass Cymbopogon martinii (Roxb.) Poaceae Leaf Cult/wild 

H. Wats var. sofi a Burk 

Grapefruit Citrus × paradisi Macfad. Rutaceae Fruit peel Cult LQ 

Guaiacwood Bulnesia sarmienti L. Zygophyllaceae Wood Wild MQ 

Gurjum Dipterocarpus spp. Dipterocarpaceae Resin Wild LQ 

Hop Humulus lupulus L. Cannabaceae Flower Cult LQ 

Hyssop Hyssopus offi cinalis L. Lamiaceae Leaf Cult LQ 

continued






Used Plant 

Part(s) 


Cultivation 

Trade 

Quantities a 

Juniper berry Juniperus communis L. Cupressaceae Fruit Wild LQ 

Laurel leaf Laurus nobilis L. Lauraceae Leaf Cult/wild LQ 

Lavandin Lavandula angustifolia Mill. × Lamiaceae Leaf Cult HQ 

L. latifolia Medik. 

Lavender Lavandula angustifolia Miller Lamiaceae Leaf Cult MQ 

Lavender, Spike Lavandula latifolia Medik. Lamiaceae Flower Cult LQ 

Lemon Citrus limon (L.) Burman fil. Rutaceae Fruit peel Cult HQ 

Lemongrass, 

Indian 

Lemongrass, West 

Indian 

Lime distilled 

Cymbopogon fl exuosus 

(Nees ex Steud.) H. Wats. 

Cymbopogon citratus (DC.) 

Stapf 

Citrus aurantiifolia (Christm. et 

Panz.) Swingle 

Poaceae Leaf Cult LQ 

Poaceae Leaf Cult LQ 

Rutaceae Fruit Cult HQ 

Litsea cubeba Litsea cubeba C.H. Persoon Lauraceae Fruit/leaf Cult MQ 

Lovage root Levisticum offi cinale Koch Apiaceae Root Cult LQ 

Mandarin Citrus reticulata Blanco Rutaceae Fruit peel Cult MQ 

Marjoram Origanum majorana L. Lamiaceae Herb Cult LQ 

Mugwort Artemisia vulgaris L. Asteraceae Herb Cult/wild LQ 

common 

Mugwort, Roman Artemisia pontica L. Asteraceae Herb Cult/wild LQ 

Myrtle Myrtus communis L. Myrtaceae Leaf Cult/wild LQ 

Neroli 

Citrus aurantium L. subsp. Rutaceae Flower Cult LQ 

aurantium 

Niaouli Melaleuca viridifl ora Myrtaceae Leaf Cult/wild LQ 

Nutmeg Myristica fragrans Houtt. Myristicaceae Seed Cult LQ 

Onion Allium cepa L. Alliaceae Bulb Cult LQ 

Orange Citrus sinensis (L.) Osbeck Rutaceae Fruit peel Cult HQ 

Orange bitter Citrus aurantium L. Rutaceae Fruit peel Cult LQ 

Oregano Origanum spp.. Thymbra spicata Lamiaceae Herb Cult/wild LQ 

L.. Coridothymus capitatus 

Rechb. fil.. Satureja spp. Lippia 

graveolens 

Palmarosa Cymbopogon martinii (Roxb.) Poaceae Leaf Cult LQ 

H. Wats var. motia Burk 

Parsley seed Petroselinum crispum (Mill.) Apiaceae Fruit Cult LQ 

Nym. ex A.W. Hill 

Patchouli Pogostemon cablin (Blanco) Lamiaceae Leaf Cult HQ 

Benth. 

Pennyroyal Mentha pulegium L. Lamiaceae Herb Cult LQ 

Pepper Piper nigrum L. Piperaceae Fruit Cult LQ 

Peppermint Mentha x piperita L. Lamiaceae Leaf Cult HQ 

Petitgrain Citrus aurantium L. subsp. Rutaceae Leaf Cult LQ 

aurantium 

Pimento leaf Pimenta dioica (L.) Merr. Myrtaceae Fruit Cult LQ 

Pine needle Pinus silvestris L.. P. nigra Arnold Pinaceae Leaf/twig Wild LQ 

Pine needle, Dwarf Pinus mugo Turra Pinaceae Leaf/twig Wild LQ 

Pine silvestris Pinus silvestris L. Pinaceae Leaf/twig Wild LQ 

continued






Used Plant 

Part(s) 


Cultivation 

Trade 

Quantities a 

Pine white Pinus palustris Mill. Pinaceae Leaf/twig Wild LQ 

Rose Rosa x damascena Miller Rosaceae Flower Cult LQ 

Rosemary Rosmarinus offi cinalis L. Lamiaceae Feaf Cult/wild LQ 

Rosewood Aniba rosaeodora Ducke Lauraceae Wood Wild LQ 

Rue Ruta graveolens L. Rutaceae Herb Cult LQ 

Sage, Dalmatian Salvia offi cinalis L. Lamiaceae Herb Cult/wild LQ 

Sage, Spanish Salvia lavandulifolia L. Lamiaceae Leaf Cult LQ 

Sage, three lobed 

(Greek. Turkish) 

Sandalwood, East 

Indian 

Sassafras, Brazilian 

(Ocotea 

cymbarum oil) 

Salvia fruticosa Mill. (syn. S. Lamiaceae Herb Cult/wild LQ 

triloba L.) 

Santalum album L. Santalaceae Wood Wild MQ 

Ocotea odorifera (Vell.) Rohwer 

[Ocotea pretiosa (Nees) Mez.] 

Lauraceae Wood Wild HQ 

Sassafras, Chinese Sassafras albidum (Nutt.) Nees. Lauraceae Root bark Wild HQ 

Savory 

Satureja hortensis L.. Satureja Lamiaceae Leaf Cult/wild LQ 

montana L. 

Spearmint, Native Mentha spicata L. Lamiaceae Leaf Cult MQ 

Spearmint, Scotch Mentha gracilis Sole Lamiaceae Leaf Cult HQ 

Star anise Illicium verum Hook fil. Illiciaceae Fruit Cult MQ 

Styrax Styrax offi cinalis L. Styracaceae Resin Wild LQ 

Tansy Tanacetum vulgare L. Asteraceae Flowering Cult/wild LQ 

herb 

Tarragon Artemisia dracunculus L. Asteraceae Herb Cult LQ 

Tea tree Melaleuca spp. Myrtaceae Leaf Cult LQ 

Thyme 

Thymus vulgaris L.. T. zygis Loefl. Lamiaceae Herb Cult LQ 

ex L. 

Valerian Valeriana offi cinalis L. Valerianaceae Root Cult LQ 

Vetiver Vetiveria zizanoides (L.) Nash Poaceae Root Cult MQ 

Wintergreen Gaultheria procumbens L. Ericaceae Leaf Wild LQ 

Wormwood Artemisia absinthium L. Asteraceae Herb Cult/wild LQ 

Ylang Ylang Cananga odorata Hook. f. et Thoms. Annonaceae Flower Cult MQ 

a 

HQ = High quantities (>1000 t/a); MQ = medium quantities (100–1000 t/a); LQ = low quantities (


• Some species are difficult to cultivate (slow growth rate and requirement of a special 

microclimate). 

• Market uncertainties or political circumstances do not allow investing in long-term 

cultivation. 

• The market is in favor of “ecological” or “natural” labeled wild collected material. 

Especially—but not only—in developing countries, parts of the rural population depend economically 

on gathering high-value plant material. Less than two decades ago, almost all oregano 

(crude drug as well as essential oil) worldwide came from wild collection (Padulosi, 1996) and even 

this well-known group of species (Origanum sp. and Lippia sp.) were counted under “neglected and 

underutilized crops.” 

Yarrow (Achillea millefolium s.l.), arnica, and even chamomile originate still partly from wild 

collection in Central and Eastern Europe, and despite several attempts to cultivate spikenard 

(Valeriana celtica), a tiny European mountain plant with a high content of patchouli alcohol, this 

species is still wild gathered in Austria and Italy (Novak et al., 1998, 2000). 

To regulate the sustainable use of biodiversity by avoiding overharvesting, genetic erosion, and 

habitat loss, international organizations such as IUCN (International Union for Conservation of 

Nature), WWF/TRAFFIC, and World Health Organization (WHO) have launched together the 

Convention on Biological Diversity (CBD, 2001), the Global Strategy for Plant Conservation (CBD, 

2002), and the Guidelines for the Sustainable Use of Biodiversity (CBD, 2004). These principles and 

recommendations address primarily the national and international policy level, but provide also the 

herbal industry and the collectors with specific guidance on sustainable sourcing practices (Leaman, 

2006). A standard for sustainable collection and use of medicinal and aromatic plants [the international 

standard on sustainable wild collection of medicinal and aromatic plants (ISSC-MAP)] was 

issued first in 2004 and its principles will be shown at the end of this chapter. This standard certifies 

wild-crafted plant material insofar as conservation and sustainability are concerned. Phytochemical 

quality cannot, however, be derived from it which is the reason for domestication and systematic 

cultivation of economically important essential oil plants. 

3.5.2 DOMESTICATION AND SYSTEMATIC CULTIVATION 

This offers a number of advantages over wild harvest for the production of essential oils: 

• Avoidance of admixtures and adulterations by reliable botanical identification. 

• Better control of the harvested volumes. 

• Selection of genotypes with desirable traits, especially quality. 

• Controlled influence on the history of the plant material and on postharvest handling. 

On the other side, it needs arable land and investments in starting material, maintenance, and 

harvest techniques. On the basis of a number of successful introductions of new crops a scheme and 

strategy of domestication was developed by this author (Table 3.8). 

Recent examples of successful domestication of essential oil-bearing plants are oregano (Ceylan 

et al., 1994; Kitiki 1996; Putievsky et al., 1996), Lippia sp. (Fischer, 1998), Hyptis suaveolens 

(Grassi, 2003), and T. lucida (Goehler, 2006). Domesticating a new species starts with studies at the 

natural habitat. The most important steps are the exact botanical identification and the detailed 

description of the growing site. National Herbaria are in general helpful in this stage. In the course 

of collecting seeds and plant material, a first phytochemical screening will be necessary to recognize 

chemotypes (Fischer et al., 1996; Goehler et al., 1997). The phytosanitary of wild populations 

should also be observed so as to be informed in advance on specific pests and diseases. The flower 

heads of wild Arnica montana, for instance, are often damaged by the larvae of Tephritis arnicae 

(Fritzsche et al., 2007).


TABLE 3.8 

Domestication Strategy for Plants of the Spontaneous Flora 

1. Studies at the natural habitat: botany, soil, climate, growing type, natural distribution and 

propagation, natural enemies, pests and diseases 

2. Collection of the wild grown plants and seeds: establishment of a germplasm collection, 

ex situ conservation, phytochemical investigation (screening) 

3. Plant propagation: vegetatively or by seeds, plantlet cultivation; (biotechnol.: in vitro 

propagation) 

4. Genetic improvement: variability, selection, breeding; phytochemical investigation, 

biotechnology (in vitro techniques) 

Æ GPS to exactly localize 

the place 

Æ Biotechnol./in vitro 

Æ Biotechnol./in vitro 

5. Cultivation treatments: growing site, fertilization, crop maintenance, cultivation techniques 

6. Phytosanitary problems: pests, diseases Æ Biotechnol./in vitro 

7. Duration of the cultivation: harvest, postharvest handling, phytochemical control of the 

crop produced 

Æ Technical processes, solar 

energy (new techniques) 

8. Economic evaluation and calculation Æ New techniques 

Source: Modified from Franz, Ch., 1993c. Plant Res. Dev., 37: 101–111; Franz, Ch., 1993d. Proc. 12th Int. Congr. of 

Essential Oils, Fragrances and Flavours, Vienna, pp. 27–44. 

The first phase of domestication results in a germplasm collection. In the next step, the appropriate 

propagation method has to be developed, which might be derived partly from observations at 

the natural habitat: while studying wild populations of T. lucida in Guatemala we found, besides 

appropriate seed set, also runners, which could be used for vegetative propagation of selected plants 

(Goehler et al., 1997). Wherever possible, propagation by seeds and direct sowing is however preferred 

due to economic reasons. 

The appropriate cultivation method depends on the plant type—annual or perennial, herb, vine, 

or tree—and on the agroecosystem into which the respective species should be introduced. In 

contrast to large-scale field production of herbal plants in temperate and Mediterranean zones, 

small-scale sustainable agroforesty and mixed cropping systems adapted to the environment have 

the preference in tropical regions (Schippmann et al., 2006). Parallel to the cultivation trials dealing 

with all topics from plant nutrition and maintenance to harvesting and postharvest handling, the 

evaluation of the genetic resources and the genetic improvement of the plant material must be started 

to avoid developing of a detailed cultivation scheme with an undesired chemotype. 

3.5.3 FACTORS INFLUENCING THE PRODUCTION AND QUALITY OF ESSENTIAL 

OIL-BEARING PLANTS 

Since plant material is the product of a predominantly biological process, prerequisite of its productivity 

is the knowledge on the factors influencing it, of which the most important ones are 

1. The already discussed intraspecific chemical polymorphism, derived from it the biosynthesis 

and inheritance of the chemical features, and as consequence selection and breeding 

of new cultivars. 

2. The intraindividual variation between the plant parts and depending on the developmental 

stages (“morpho- and ontogenetic variation”). 

3. The modification due to environmental conditions including infection pressure and 

immissions. 

4. Human influences by cultivation measures, for example, fertilizing, water supply, or pest 

management.


3.5.3.1 Genetic Variation and Plant Breeding 

Phenotypic variation in essential oils was detected very early because of their striking sensorial 

properties. Due to the high chemical diversity, a continuous selection of the desired chemotypes 

leads to rather homogenous and reproducible populations, as this is the case with the landraces 

and common varieties. But Murray and Reitsema (1954) stated already that “a plant breeding 

program requires a basic knowledge of the inheritance of at least the major essential oil compounds.” 

Such genetic studies have been performed over the last 50 years with a number of species 

especially of the mint family (e.g., Ocimum sp.: Sobti et al., 1978; Thymus vulgaris: Vernet, 

1976; Gouyon and Vernet, 1982; Perilla frutescens: Koezuka et al., 1986; Mentha sp.: Croteau, 

1991), of the Asteraceae/Compositae (Matricaria recutita: Horn et al., 1988; Massoud and Franz, 

1990), the genus (Eucalyptus: Brophy and Southwell, 2002; Doran, 2002), or the Vetiveria zizanioides 

(Akhila and Rani, 2002). 

The results achieved by inheritance studies have been partly applied in targeted breeding as 

shown exemplarily in Table 3.9. Apart from the essential oil content and composition there are 

also other targets to be observed when breeding essential oil plants, as particular morphological 

characters ensuring high and stable yields of the respective plant part, resistances to pest and 

diseases as well as abiotic stress, low nutritional requirements to save production costs, appropriate 

homogeneity, and suitability for technological processes at harvest and postharvest, especially 

readiness for distillation (Pank, 2007). In general, the following breeding methods are commonly 

used (Franz, 1999). 

3.5.3.1.1 Selection by Exploiting the Natural Variability 

Since many essential oil-bearing species are in the transitional phase from wild plants to systematic 

cultivation, appropriate breeding progress can be achieved by simple selection. Wild collections or 

accessions of germplasm collections are the basis, and good results were obtained, for example, 

with Origanum sp. (Putievsky et al., 1997) in limited time and at low expenses. 

Individual plants showing the desired phenotype will be selected and either generatively or vegetatively 

propagated (individual selection), or positive or negative mass selection techniques can be 

applied. Selection is traditionally the most common method of genetic improvement and the majority 

of varieties and cultivars of essential oil crops have this background. Due to the fact, however, 

that almost all of the respective plant species are allogamous, a recurrent selection is necessary to 

maintain the varietal traits, and this has especially to be considered if other varieties or wild populations 

of the same species are nearby and uncontrolled cross pollination may occur. 

The efficacy of selection has been shown by examples of many species, for instance, of the 

Lamiaceae family, starting from “Mitcham” peppermint and derived varieties (Lawrence, 2007), 

basil (Elementi et al., 2006), sage (Bezzi, 1994; Bernáth, 2000) to thyme (Rey, 1993). It is a wellknown 

method also in the breeding of caraway (Pank et al., 1996) and fennel (Desmarest, 1992) as 

well as of tropical and subtropical species such as palmarosa grass (Kulkarni, 1990), tea tree 

(Taylor, 1996), and eucalyptus (Doran, 2002). At perennial herbs, shrubs, and trees clone breeding, 

that is, the vegetative propagation of selected high-performance individual plants, is the method of 

choice, especially in sterile or not type-true hybrids, for example, peppermint (Mentha × piperita) 

or lavandin (Lavandula × hybrida). But this method is often applied also at sage (Bazina et al., 

2002), rosemary (Mulas et al., 2002), lemongrass (Kulkarni and Ramesh, 1992), pepper, cinnamon, 

and nutmeg (Nair, 1982), and many other species. 

3.5.3.1.2 Breeding with Extended Variability (Combination Breeding) 

If different desired characters are located in different individuals/genotypes of the same or a closely 

related crossable species, crossings are made followed by selection of the respective combination 

products. Artificial crossings are performed by transferring the paternal pollen to the stigma of the 

female (emasculated) or male sterile maternal flower. In the segregating progenies individuals with


TABLE 3.9 

Some Registered Cultivars of Essential Oil Plant 

Species Cultivar/Variety Country 

Year of 

Registration Breeding Method Specific Characters 

Achillea collina SPAK CH 1994 Crossing High in proazulene 

Angelica archangelica VS 2 FR 1996 Recurrent pedigree Essential oil index of roots: 180 

Foeniculum vulgare Fönicia HU 1998 Selection High anethole 

Lavandula offi cinalis Rapido FR 1999 Polycross High essential oil, 

high linalyl acetate 

Levisticum offi cinale Amor PL 2000 Selection High essential oil 

Matricaria recutita Mabamille DE 1995 Tetraploid High a-bisabolol 

Ciclo-1 IT 2000 Line breeding High chamazulene 

Lutea SK 1995 Tetraploid High a-bisabolol 

Melissa offi cinalis Ildikó HU 1998 Selection High essential oil, 

Citral A + B, linalool 

Landor CH 1994 Selection High essential oil 

Lemona DE 2001 Selection High essential oil, citral 

Mentha piperita Todd’s Mitcham USA 1972 Mutation Wilt resistant 

Kubanskaja RUS 1980ies Crossing and polyploid High essential oil, high menthol 

Mentha spicata MSH-20 DK 2000 Recurrent pedigree High menthol, good flavor 

Ocimum basilicum Greco IT 2000 Synthetic Flavor 

Perri ISR 1999 Cross-breeding Fusarium Resistant 

Cardinal ISR 2000 Cross-breeding 

Origanum syriacum Senköy TR 1992 Selection 5% essential oil, 

60% carvacrol 

Carmeli ISR 1999 Selection Carvacrol 

Tavor ISR 1999 Selection Thymol 

Origanum onites GR 2000 Selfing Carvacrol 

Origanum hirtum GR 2000 Selfing Carvacrol 

Vulkan DE 2002 Crossing Carvacrol 

Carva CH 2002 Crossing Carvacrol 

Darpman TR 1992 Selection 2.5% essential oil, 

55% carvacrol 

Origanum majorana Erfo DE 1997 Crossing High essential oil, 

(Majorana hortensis) Tetrata DE 1999 Ployploid Cis-sabinene-hydrate 

G 1 FR 1998 Polycross 

Salvia offi cinalis Moran ISR 1998 Crossing Herb yield 

Syn 1 IT 2004 Synthetic a-Thujone 

Thymus vulgaris Varico CH 1994 Selection Thymol/carvacrol 

T-16 DK 2000 Recurrent pedigree Thymol 

Virginia ISR 2000 Selection Herb yield 

the desired combination will be selected and bred to constancy, as exemplarily described for fennel 

and marjoram by Pank (2002). 

Hybrid breeding—common in large-scale agricultural crops, for example, maize—was introduced 

into essential oil plants over the last decade only. The advantage of hybrids on the one side 

is that the F 1 generation exceeds the parent lines in performance due to hybrid vigor and uniformity 

(“heterosis effect”) and on the other side it protects the plant breeder by segregating of the 

F 2 and following generations in heterogeneous low-value populations. But it needs as precondition


separate (inbred) parent lines of which one has to be male sterile and one male fertile with good 

combining ability. 

In addition, a male fertile “maintainer” line is needed to maintain the mother line. Few examples 

of F 1 hybrid breeding are known especially at Lamiaceae since male sterile individuals are 

found frequently in these species (Rey, 1994; Novak et al., 2002; Langbehn et al., 2002; Pank 

et al., 2002). 

Synthetic varieties are based on several (more than two) well-combining parental lines or clones 

which are grown together in a polycross scheme with open pollination for seed production. The 

uniformity and performance is not as high as at F 1 hybrids but the method is simpler and cheaper 

and the seed quality acceptable for crop production until the second or third generation. Synthetic 

cultivars are known for chamomile (Franz et al., 1985), arnica (Daniel and Bomme, 1991), marjoram 

(Franz and Novak, 1997), sage (Aiello et al., 2001), or caraway (Pank et al., 2007). 

3.5.3.1.3 Breeding with Artificially Generated New Variability 

Induced mutations by application of mutagenic chemicals or ionizing radiation open the possibility 

to find new trait expressions. Although quite often applied, such experiments are confronted with the 

disadvantages of undirected and incalculable results, and achieving a desired mutation is often like 

searching for a needle in a haystack. Nevertheless, remarkable achievements are several colchicineinduced 

polyploid varieties of peppermint (Murray, 1969; Lawrence, 2007), chamomile (Czabajska 

et al., 1978; Franz et al., 1983; Repčak et al., 1992), and lavender (Slavova et al., 2004). 

Further possibilities to obtain mutants are studies of the somaclonal variation of in vitro cultures 

since abiotic stress in cell and tissue cultures induces also mutagenesis. Finally, genetic engineering 

opens new fields and potentialities to generate new variability and to introduce new traits by gene 

transfer. Except research on biosynthetic pathways of interesting essential oil compounds genetic 

engineering, GMO’s and transgenic cultivars are until now without practical significance in essential 

oil crops and also not (yet) accepted by the consumer. 

As regards the different traits, besides morphological, technological, and yield characteristics as 

well as quantity and composition of the essential oil, also stress resistance and resistance to pests 

and diseases are highly relevant targets in breeding of essential oil plants. Well known in this 

respect are breeding efforts against mint rust (Puccinia menthae) and wilt (Verticillium dahliae) 

resulting in the peppermint varieties “Multimentha,” “Prilukskaja,” or “Todd’s Mitcham” (Murray 

and Todd, 1972; Pank, 2007; Lawrence, 2007), the development of Fusarium-wilt and Peronospora 

resistant cultivars of basil (Dudai, 2006; Minuto et al., 2006), or resistance breeding against Septoria 

petroselini in parsley and related species (Marthe and Scholze, 1996). An overview on this topic is 

given by Gabler (2002). 

3.5.3.2 Plant Breeding and Intellectual Property Rights 

Essential oil plants are biological, cultural, and technological resources. They can be found in nature 

gathered from the wild or developed through domestication and plant breeding. As long as the plant 

material is wild collected and traditionally used, it is part of the cultural heritage without any individual 

intellectual property and therefore not possible to protect, for example, by patents. Even 

finding a new plant or substance is a discovery in the “natural nature” and not an invention since a 

technical teaching is missing. Intellectual property, however, can be granted to new applications 

that involve an inventive step. Which consequences can be drawn from these facts for the development 

of novel essential oil plants and new selections or cultivars? 

Selection and genetic improvement of aromatic plants and essential oil crops is not only time 

consuming but also rather expensive due to the necessity of comprehensive phytochemical and possibly 

molecular biological investigations. In addition, with few exceptions (e.g., mints, lavender and 

lavandin, parsley but also Cymbopogon sp., black pepper, or cloves) the acreage per species is rather 

limited in comparison with conventional agricultural and horticultural crops. And finally, there are 

several “fashion crops” with market uncertainties concerning their longevity or half-life period,


respectively. The generally unfavorable cost: benefit ratio to be taken into consideration makes 

essential oil plant breeding economically risky and there is no incentive for plant breeders unless a 

sufficiently strong plant intellectual property right (IPR) exists. Questioning “which protection, 

which property right for which variety?” offers two options (Franz, 2001). 

3.5.3.2.1 Plant Variety Protection 

By conventional methods bred plant groupings that collectively are distinct from other known varieties 

and are uniform and stable following repeated reproduction can be protected by way of plant 

breeder’s rights. Basis is the International Convention for the Protection of New Varieties initially 

issued by UPOV (Union for the Protection of New Varieties of Plants) in 1961 and changed in 1991. 

A plant breeder’s right is a legal title granting its holder the exclusive right to produce reproductive 

material of his plant variety for commercial purposes and to sell this material within a particular 

territory for up to 30 years (trees and shrubs) or 25 years (all other plants). A further precondition is 

the “commercial novelty,” that is, it must not have been sold commercially prior to the filing date. 

Distinctness, uniformity, and stability (DUS) refer to morphological (leaf shape, flower color, etc.) 

or physiological (winter hardiness, disease resistance, etc.), but not phytochemical characteristics, 

for example, essential oil content or composition. Such “value for cultivation and use (VCU) characteristics” 

will not be examined and are therefore not protected by plant breeder’s rights (Franz, 

2001; Llewelyn, 2002; Van Overwalle, 2006). 

3.5.3.2.2 Patent Protection (Plant Patents) 

Generally speaking, patentable are inventions (not discoveries!) that are novel, involve an innovative 

step, and are susceptible to industrial application, including agriculture. Plant varieties or 

essentially biological processes for the production of plants are explicitly excluded from patenting. 

But other groupings of plants that fall neither under the term “variety” nor under “natural nature” 

are possible to be protected by patents. This is especially important for plant groupings with novel 

phytochemical composition or novel application combined with an inventive step, for example, 

genetical modification, a technologically new production method or a novel type of isolation 

(product by process protection). 

Especially for wild plants and essentially allogamous plants not fulfilling DUS for cultivated 

varieties (cultivars) and plants where the phytochemical characteristics are more important than the 

morphological ones, plant patents offer an interesting alternative to plant variety protection (PVP) 

(Table 3.10). 

TABLE 3.10 

Advantages and Disadvantages of PVP versus Patent Protection of Specialist 

Minor Crops (Medicinal and Aromatic Plants) 

PVP 

Beginning of protection: registration date 

Restricted to “varieties” 

Requirements: DUS = distinctness, uniformity, 

stability 

Free choice of characters to be used for DUS by 

PVO (Plant Variety Office) 

Phenotypical. Mainly morphological characters 

(phytochemicals of minor importance) 

Value for cultivation and use characteristics 

(VCU) not protected 

Patent 

Beginning of protection: application date 

“Varieties” not patentable, but any other grouping of plants 

Requirements: novelty, inventive step, industrial applicability (=NIA) 

Repeatability obligatory, product by process option 

“Essentially biological process” not patentable 

“Natural nature” not patentable 

Claims (e.g., phytochemical characters) depend on applicant 

Phytochemical characters and use/application (VCU) patentable


In conformity with the UPOV Convention of 1991 (http://www.upov.int/en/publications/ 

conventions/1991/content.htm) 

• A strong plant IPR is requested. 

• Chemical markers (e.g., secondary plant products) must be accepted as protectable 

characteristics. 

• Strong depending rights for essentially derived varieties are needed since it is easy to 

plagiarize such crops. 

• “Double protection” would be very useful (i.e., free decision by the breeder if PVR or 

patent protection is applied). 

• But also researchers exemption and breeders privilege with fair access to genotypes for 

further development is necessary. 

Strong protection does not hinder usage and development; it depends on a fair arrangement only 

(Le Buanec, 2001). 

3.5.3.3 Intraindividual Variation between Plant Parts and Depending on the 

Developmental Stage (Morpho- and Ontogenetic Variation) 

The formation of essential oils depends on the tissue differentiation (secretory cells and excretion 

cavities, as discussed above in the introduction to this chapter) and on the ontogenetic phase of the 

respective plant. The knowledge on these facts is necessary to harvest the correct plant parts at the 

right time. 

Regarding the differences between plant parts, it is known from cinnamon (Cinnamomum 

zeylanicum) that the root-, stem-, and leaf oils differ significantly (Wijesekera et al., 1974): only the 

stem bark contains an essential oil with up to 70% cinnamaldehyde, whereas the oil of the root bark 

consists mainly of camphor and linalool, and the leaves produce oils with eugenol as main compound. 

In contrast to it, eugenol forms with 70–90% the main compound in stem, leaf, and bud oils 

of cloves (Syzygium aromaticum) (Lawrence, 1978). This was recently confirmed by Srivastava 

et al. (2005) for clove oils from India and Madagascar, stating in addition that eugenyl acetate was 

found in buds up to 8% but in leaves between traces and 1.6% only. The second main substance 

in leaves as well as buds is b-caryophyllene with up to 20% of the essential oil. In Aframomum 

giganteum (Zingiberaceae), the rhizome essential oil consists of b-caryophyllene, its oxide, and 

derivatives mainly, whereas in the leaf oil terpentine-4-ol and pinocarvone form the principal 

components (Agnaniet et al., 2004). 

Essential oils of the Rutaceae family, especially citrus oils, are widely used as flavors and 

fragrances depending on the plant part and species: in lime leaves neral/geranial and nerol/geraniol 

are prevailing, whereas grapefruit leaf oil consists of sabinene and b-ocimene mainly. The peel of 

grapefruit contains almost limonene only and some myrcene, but lime peel oil shows a composition 

of b-pinene, g-terpinene, and limonene (Gancel et al., 2002). In Phellodendron sp., Lis et al. (2004, 

2006) found that in flower and fruit oils limonene and myrcene are dominating; in leaf oils, in contrast, 

a-farnesene, b-elemol, or b-ocimene, are prevailing. 

Differences in the essential oil composition between the plant parts of many Umbelliferae 

(Apiaceae) have exhaustively been studied by the group of Kubeczka, summarized by Kubeczka 

et al. (1982) and Kubeczka (1997). For instance, the comparison of the essential fruit oil of aniseed 

(Pimpinella anisum) with the oils of the herb and the root revealed significant differences (Kubeczka 

et al., 1986). Contrary to the fruit oil consisting of almost trans-anethole only (95%), the essential 

oil of the herb contains besides anethole, considerable amounts of sesquiterpene hydrocarbons, for 

example, germacrene D, b-bisabolene, and a-zingiberene. Also pseudoisoeugenyl-2-methylbutyrate 

and epoxi-pseudoisoeugenyl-2-methylbutyrate together form almost 20% main compounds of the 

herb oil, but only 8.5% in the root and 1% in the fruit oil. The root essential oil is characterized by


a high content of b-bisabolene, geijerene, and pregeijerene and contains only small amounts of 

trans-anethole (3.5%). Recently, Velasco-Neguerela et al. (2002) investigated the essential oil composition 

in the different plant parts of Pimpinella cumbrae from Canary Islands and found in all 

above-ground parts a-bisabolol as main compound besides of d-3-carene, limonene, and others, 

whereas the root oil contains mainly isokessane, geijerene, isogeijerene, dihydroagarofuran, and 

proazulenes—the latter is also found in Pimpinella nigra (Kubeczka et al., 1986). Pseudoisoeugenyl 

esters, known as chemosystematic characters of the genus Pimpinella, have been detected in small 

concentrations in all organs except leaves. 

Finally, Kurowska and Galazka (2006) compared the seed oils of root and leaf parsley cultivars 

marketed in Poland. Root parsley seeds contained an essential oil with high concentrations of apiole 

and some lower percentages of myristicin. In leaf parsley seeds, in contrast, the content of myristicin 

was in general higher than apiole, and a clear differentiation between flat leaved cultivars showing 

still higher concentrations of apiole and curled cultivars with only traces of apiole could be 

observed. Allyltetramethoxybenzene as the third marker was found in leaf parsley seeds up to 

12.8%, in root parsley seeds, however, in traces only. Much earlier, Franz and Glasl (1976) had published 

already similar results on parsley seed oils comparing them with the essential oil composition 

of the other plant parts (Figure 3.8). Leaf oils gave almost the same fingerprint than the seeds with 

high myristicin in curled leaves, some apiole in flat leaves, and higher apiole concentrations than 

myristicin in the leaves of root varieties. In all root samples, however, apiole dominated largely over 

myristicin. It is therefore possible to identify the parsley type by analyzing a small seed sample. 

As shown already by Figueiredo et al. (1997), in the major number of essential oil-bearing species 

the oil composition differs significantly between the plant parts, but there are also plant species—as 

mentioned before, for example, cloves—which form a rather similar oil composition in each plant 

organ. Detailed knowledge in this matter is needed to decide, for instance how exact the separation 

of plant parts has to be performed before further processing (e.g., distillation) or use. 

Ess. oil % in dm % in ess. oil Myristicin Apiole 

0.9 

0.6 

0.3 

75 

50 

25 

0.9 

0.6 

0.3 

75 

50 

25 

0.9 

0.6 

0.3 

75 

50 

25 

1 2 3 4 5 6 7 

FIGURE 3.8 Differences in the essential oil of fruits, leaves and roots of parsley cultivars (Petroselinum 

crispum (Mill.) Nyman); (left: ess. oil content, right: content of myristicin and apiole in the ess. oil. 1,2: flat 

leaved cv’s, 3–7 curled leaves cv’s, 7 root parsley).


Another topic to be taken into consideration is the developmental stage of the plant and the plant 

organs, since the formation of essential oils is phase dependent. In most cases, there is a significant 

increase of the essential oil production throughout the whole vegetative development. 

And especially in the generative phase between flower bud formation and full flowering, or until 

fruit or seed setting, remarkable changes in the oil yield and compositions can be observed. 

Obviously, a strong correlation is given between formation of secretory structures (oil glands, ducts, 

etc.) and essential oil biosynthesis, and different maturation stages, are associated with, for example, 

higher rates of cyclization or increase of oxygenated compounds (Figueiredo et al., 1997). 

Investigations on the ontogenesis of fennel (Foeniculum vulgare Mill.) revealed that the best time 

for picking fennel seeds is the phase of full ripeness due to the fact that the anethole content increases 

from


There is an extensive literature on ontogenesis and seasonal variation of Labiatae essential oils. 

Especially for this plant family, great differences are reported on the essential oil content and composition 

of young and mature leaves and the flowers may in addition influence the oil quality significantly. 

Usually, young leaves show higher essential oil contents per area unit compared to old leaves. 

But the highest oil yield is reached at the flowering period, which is the reason that most of the oils 

are produced from flowering plants. According to Werker et al. (1993) young basil (O. basilicum) 

leaves contained 0.55% essential oil while the content of mature leaves was only 0.13%. The same is 

also valid to a smaller extent for O. sanctum, where the essential oil decreases from young (0.54%) 

to senescing leaves (0.38%) (Dey and Choudhuri, 1983). Testing a number of basil cultivars mainly 

of the linalool chemotype, Macchia et al. (2006) found that only some of the cultivars produce methyl 

eugenol up to 8% in the vegetative stage. Linalool as main compound is increasing from the vegetative 

(10–50%) to the flowering (20–60%), and postflowering phase (25–80%), whereas the second 

important substance eugenol reaches its peak at the beginning of flowering (5–35%). According to the 

cultivars, different harvest dates are therefore recommended. In O. sanctum, the content of eugenol 

(60.3–52.2%) as well as of methyl eugenol (6.6–2.0%) is decreasing from young to senescent leaves 

and at the same time b-caryophyllene increases from 20.8% to 30.2% (Dey and Choudhuri, 1983). 

As regards oregano (O. vulgare ssp. hirtum), the early season preponderance of p-cymene over 

carvacrol was reversed as the season progressed and this pattern could also be observed at any time 

within the plant, from the latest leaves produced (low in cymene) to the earliest (high in cymene) 

(Johnson et al., 2004; Figure 3.9). Already Kokkini et al. (1996) had shown that oregano contains a 

higher proportion of p-cymene to carvacrol (or thymol) in spring and autumn, whereas carvacrol/ 

thymol prevails in the summer. This is explained by Dudai et al. (1992) as photoperiodic reaction: 

short days with high p-cymene, long days with low p-cymene production. But only young plants are 

capable of making this switch, whereas in older leaves the already produced and stored oil remains 

almost unchanged (Johnson et al., 2004). 

Presumably the most studied essential oil plant is peppermint (Mentha × piperita L.). Already in 

the 1950s Lemli (1955) stated that the proportion of menthol to menthone in peppermint leaves changes 

in the course of the development toward higher menthol contents. Lawrence (2007) has just recently 

shown that from immature plants via mature to senescent plants the content of menthol increases 

(34.8–39.9–48.2%) and correspondingly the menthone content decreases dramatically (26.8–17.4– 

4.7%). At the same time, also an increase of menthyl acetate from 8.5% to 23.3% of the oil could be 

observed. At full flowering, the peppermint herb oil contains only 36.8% menthol but 21.8% menthone, 

7.7% menthofuran, and almost 3% pulegone due to the fact that the flower oils are richer in 

% 

60 

50 

40 

30 

20 

10 

0 

Early March 

Late March 

Late May 

FIGURE 3.9 Average percentages and concentrations of p-cymene. g-terpinene and carvacrol at the different 

sampling dates of Origanum vulgare ssp. hirtum. Solid bars: p-cymene; diagonally hatched bars: g-terpinene; 

open bars: carvacrol.


menthone and pulegone and contain a high amount of menthofuran (Hefendehl, 1962). Corresponding 

differences have been found between young leaves rich in menthone and old leaves with high menthol 

and menthyl acetate content (Hoeltzel, 1964; Franz, 1972). The developmental stage depends, however, 

to a large extent from the environmental conditions, especially the day length. 

3.5.3.4 Environmental Influences 

Essential oil formation in the plants is highly dependent on climatic conditions, especially day 

length, irradiance, temperature, and water supply. Tropical species follow in their vegetation cycle 

the dry and rainy season; species of the temperate zones react more on day length, the more distant 

from the equator their natural distribution area is located. 

Peppermint as typical long day plant needs a minimum day length (hours of day light) to switch 

from the vegetative to the generative phase. This is followed by a change in the essential oil composition 

from menthone to menthol and menthyl acetate (Hoeltzel, 1964). Franz (1981) tested six peppermint 

clones at Munich/Germany and at the same time also at Izmir/Turkey. At the development 

stage “beginning of flowering,” all clones contained at the more northern site much more menthol 

than on the Mediterranean location, which was explained by a maximum day length in Munich of 

16 h 45 min, but in Izmir of 14 h 50 min only. Comparable day length reactions have been mentioned 

already for oregano (Kokkini et al., 1996; Dudai et al., 1992). Also marjoram (O. majorana L.) 

was influenced not only in flower formation by day length, but also in oil composition (Circella 

et al., 1995). At long day treatment the essential oil contained more cis-sabinene hydrate. Terpinene- 

4-ol prevailed under short day conditions. 

Franz et al. (1986) performed ecological experiments with chamomile, growing vegetatively 

propagated plants at three different sites, in South Finland, Middle Europe, and West Turkey. As 

regards the oil content, a correlation between flower formation, flowering period, and essential oil 

synthesis could be observed: the shorter the flowering phase, the less was the time available for oil 

formation, and thus the lower was the oil content. The composition of the essential oil, on the other 

hand, showed no qualitative change due to ecological or climatic factors confirming that chemotypes 

keep their typical pattern. In addition, Massoud and Franz (1990) investigated the 

genotype–environment interaction of a chamazulene–bisabolol chemotype. The frequency distributions 

of the essential oil content as well as the content on chamazulene and a-bisabolol have shown 

that the highest oil- and bisabolol content was reached in Egypt while under German climatic conditions 

chamazulene was higher. Similar results have been obtained by Letchamo and Marquard 

(1993). The relatively high heritability coefficients calculated for some essential oil components— 

informing whether a character is more influenced by genetic or other factors—confirm that the 

potential to produce a certain chemical pattern is genetically coded, but the gene expression will be 

induced or repressed by environmental factors also (Franz, 1993b,d). 

Other environmental factors, for instance soil properties, water stress, or temperature, are 

mainly influencing the productivity of the respective plant species and by this means the oil yield 

also, but have little effect on the essential oil formation and composition only (Figueiredo et al., 

1997; Salamon, 2007). 

3.5.3.5 Cultivation Measures, Contaminations, and Harvesting 

Essential oil-bearing plants comprise annual, biennial, or perennial herbs, shrubs, and trees, cultivated 

either in tropical or subtropical areas, in Mediterranean regions, in temperate, or even in arid 

zones. Surveys in this respect are given, for instance, by Chatterjee (2002) for India, by Carruba 

et al. (2002) for Mediterranean environments, and by Galambosi and Dragland (2002) for Nordic 

countries. Nevertheless, some examples should refer to some specific items. 

The cultivation method—if direct sowing or transplanting—and the timing influence the crop 

development and by that way also the quality of the product, as mentioned above. Vegetative propagation, 

necessary for peppermint due to its genetic background as interpecific hybrid, common in 

Cymbopogon sp. and useful to control the ratio between male and female trees in nutmeg (Myristica


fragrans), results in homogeneous plant populations and fields. A disadvantage could be the easier 

dispersion of pests and diseases, as known for “yellow rot” of lavandin (Lavandula × hybrida) 

(Fritzsche et al., 2007). Clonal propagation can be performed by leaf or stem cuttings (Goehler 

et al., 1997; Nicola et al., 2006; El-Keltawi and Abdel-Rahman, 2006) or in vitro (e.g., Figueiredo 

et al., 1997; Mendes and Romano, 1997), the latter method especially for mother plant propagation 

due to the high costs. In vitro essential oil production received increased attention in physiological 

experiments, but has up to now no practical significance. 

As regards plant nutrition and fertilizing, a numerous publications have shown its importance 

for plant growth, development, and biomass yield. The essential oil yield, obviously, depends on 

the plant biomass; the oil percentage is partly influenced by the plant vigor and metabolic activity. 

Optimal fertilizing and water supply results in better growth and oil content, for example, in 

marjoram, oregano, basil, or coriander (Menary, 1994), but also in delay of maturity, which causes 

quite often “immature” flavors. 

Franz (1972) investigated the influence of nitrogen and potassium on the essential oil formation 

of peppermint. He could show that higher nitrogen supply increased the biomass but retarded the 

plant development until flowering, whereas higher potassium supply forced the maturity. With 

increasing nitrogen, a higher oil percentage was observed with lower menthol and higher menthone 

content; potassium supply resulted in less oil with more menthol and menthyl acetate. Comparable 

results with Rosmarinus offi cinalis have been obtained by Martinetti et al. (2006), and Omidbaigi 

and Arjmandi (2002) have shown for Thymus vulgaris that nitrogen and phosphorus fertilization 

had significant effect on the herb yield and essential oil content, but did not change the thymol 

percentage. Also Java citronella (Cymbopogon winterianus Jowitt.) responded to nitrogen supply 

with higher herb and oil yields, but no influence on the geraniol content could be found (Munsi and 

Mukherjee, 1986). 

Extensive pot experiments with chamomile (Matricaria recutita) have also shown that high 

nitrogen and phosphorus nutrition levels resulted in a slightly increased essential oil content of the 

anthodia, but raising the potassium doses had a respective negative effect (Franz et al., 1983). With 

nitrogen the flower formation was in delay and lasted longer; with more potassium the flowering 

phase was reduced, which obviously influenced the period available for essential oil production. 

This was confirmed by respective 14 C-acetate labeling experiments (Franz, 1981). 

Almost no effect has been observed on the composition of the essential oil. Also a number of 

similar pot or field trials came to the same result, as summarized by Salamon (2007). 

Salinity and salt stress get an increasing importance in agriculture especially in subtropical and 

Mediterranean areas. Some essential oil plants, for example, Artemisia sp. and Matricaria recutita 

(chamomile) are relatively salt tolerant. Also thyme (Thymus vulgaris) showed a good tolerance to 

irrigation water salinity up to 2000 ppm, but exceeding concentrations caused severe damages 

(Massoud et al., 2002). Higher salinity reduced also the oil content, and an increase of p-cymene 

was observed. Recently, Aziz et al. (2008) investigated the influence of salt stress on growth and 

essential oil in several mint species. In all three mints, salinity reduced the growth severely from 

1.5 g/L onward; in peppermint, the menthone content raised and menthol went down to


plant material. More important in the production of essential oils are pests and diseases that cause 

damages to the plant material and sometimes alterations in the biosynthesis; but little is known in 

this respect. 

In contrast to organic production, where no use of pesticides is permitted, a small number of 

insecticides, fungicides, and herbicides are approved for conventional herb production. The number, 

however, is very restricted (end of 2008 several active substances lost registration at least in Europe), 

and limits for residues can be found in national law and international regulations, for example, the 

European Pharmacopoeia. For essential oils, mainly the lipophilic substances are of relevance since 

they can be enriched over the limits in the oil. 

Harvesting and the first steps of postharvest handling are the last part of the production chain 

of starting materials for essential oils. The harvest date is determined by the development stage 

or maturity of the plant or plant part, Harvesting techniques should keep the quality by avoiding 

adulterations, admixtures with undesired plant parts, or contaminations, which could cause 

“off-flavor” in the final product. There are many technical aids at disposal, from simple devices 

to large-scale harvesters, which will be considered carefully in Chapter 4. From the quality point 

of view, raising the temperature by fermentation should in general be avoided (except, in vanilla), 

and during the drying process further contamination with soil, dust, insects, or molds has to 

be avoided. 

Quality and safety of essential oil-bearing plants as raw materials for pharmaceutical products, 

flavors, and fragrances are of highest priority from the consumer point of view. To meet the respective 

demands, standards and safety as well as quality assurance measures are needed to ensure that 

the plants are produced with care, so that negative impacts during wild collection, cultivation, 

processing, and storage can be limited. To overcome these problems and to guarantee a steady, 

affordable and sustainable supply of essential oil plants of good quality (Figure 3.10), in recent 

years guidelines for good agricultural practices (GAP) and standards for Sustainable Wild 

Collection (ISSC) have been established at the national and international level. 

Cultivation 

Growing site, crop rotation, 

production technique 

and maintenance, 

fertilization, plant protection, 

spec. methods (e.g. organic 

production), harvest time and 

technique 

Starting material 

Species, 

variety, cultivar 

chemotype, 

resistances 

spec. characters 

(e.g. “usability value”) 

Stepwise 

quality 

control 

Safety 

Prevention 

against intoxications 

Storage 

Storehouse conditions, 

packaging 

Post harvest handling 

Transport from field, 

washing, drying, 

processing 

FIGURE 3.10 Main items of “good agricultural practices (GAP)” for medicinal and aromatic plants.


3.6 INTERNATIONAL STANDARDS FOR WILD COLLECTION 

AND CULTIVATION 

3.6.1 GA(C)P: GUIDELINES FOR GOOD AGRICULTURAL (AND COLLECTION) PRACTICE 

OF MEDICINAL AND AROMATIC PLANTS 

First initiatives for the elaboration of such guidelines trace back to a roundtable discussion in Angers, 

France in 1983, and intensified at an International Symposium in Novi Sad 1988 (Franz, 1989b). 

A first comprehensive paper was published by Pank et al. (1991) and in 1998 the European Herb 

Growers Association (EHGA/EUROPAM) released the first version (Máthé and Franz, 1999). The 

actual version can be downloaded from http://www.europam.net. 

In the following it was adopted and slightly modified by the European Agency for the Evaluation 

of Medicinal Products (EMEA), and finally as Guidelines on good agricultural and collection practices 

(GACP) by the WHO in 2003. 

All these guidelines follow almost the same concept dealing with the following topics: 

• Identification and authentication of the plant material, especially botanical identity and 

deposition of specimens. 

• Seeds and other propagation material, respecting the specific standards and certifications. 

• Cultivation, including site selection, climate, soil, fertilization, irrigation, crop maintenance, 

and plant protection with special regard to contaminations and residues. 

• Harvest, with specific attention to harvest time and conditions, equipment, damage, contaminations 

with (toxic) weeds and soil, transport, possible contact with any animals, and 

cleaning of all equipment and containers. 

• Primary processing, that is, washing, drying, distilling; cleanness of the buildings; according 

to the actual legal situation these processing steps including distillation—if performed 

by the farmer—is still part of GA(C)P; in all other cases, it is subjected to GMP (good 

manufacturing practice). 

• Packaging and labeling, including suitability of the material. 

• Storage and transportation, especially storage conditions, protection against pests and 

animals, fumigation, and transport facilities. 

• Equipment: material, design, construction, easy to clean. 

• Personnel and facilities, with special regard to education, hygiene, protection against 

allergens and other toxic compounds, welfare. 

In the case of wild collection the standard for sustainable collection should be applied (see 

Section 3.6.2). 

A very important topic is finally the documentation of all steps and measurements to be able to 

trace back the starting material, the exact location of the field, any treatment with agrochemicals, 

and the special circumstances during the cultivation period. Quality assurance is only possible if the 

traceability is given and the personnel is educated appropriately. Certification and auditing of the 

production of essential oil-bearing plants is not yet obligatory, but recommended and often requested 

by the customer. 

3.6.2 ISSC-MAP: THE INTERNATIONAL STANDARD ON SUSTAINABLE WILD COLLECTION 

OF MEDICINAL AND AROMATIC PLANTS 

ISSC-MAP is a joint initiative of the German Bundesamt für Naturschutz (BfN), WWF/TRAFFIC 

Germany, IUCN Canada, and IUCN Medicinal Plant Specialist Group (MPSG). ISSC-MAP intends 

to ensure the long-term survival of MAP populations in their habitats by setting principles and criteria 

for the management of MAP wild collection (Leaman, 2006; Medicinal Plant Specialist Group,


2007). The standard is not intended to address product storage, transport, and processing, or any 

issues of products, topics covered by the WHO Guidelines on GACP for Medicinal Plants (WHO, 

2003). ISSC-MAP includes legal and ethical requirements (legitimacy, customary rights, and transparency), 

resource assessment, management planning and monitoring, responsible collection, and 

collection, area practices and responsible business practices. One of the strengths of this standard is 

that resource management not only includes target MAP resources and their habitats but also social, 

cultural, and economic issues. 

3.6.3 FAIRWILD 

The FairWild standard (http://www.fairwild.org) was initiated by the Swiss Import Promotion 

Organization (SIPPO) and combines principles of FairTrade (Fairtrade Labelling Organizations 

International, FLO), international labor standards (International Labour Organization, ILO), and 

sustainability (ISSC-MAP). 

3.7 CONCLUSION 

This chapter has shown that a number of items concerning the plant raw material have to be taken 

into consideration when producing essential oils. A quality management has to be established tracing 

back to the authenticity of the starting material and ensuring that all known influences on the 

quality are taken into account and documented in an appropriate way. This is necessary to meet the 

increasing requirements of international standards and regulations. The review also shows that a 

high number of data and information exist, but sometimes without expected relevance due to the fact 

that the repeatability of the results is not given by a weak experimental design, an incorrect description 

of the plant material used, or an inappropriate sampling. On the other side, this opens the chance 

for many more research work in the field of essential oil-bearing plants. 

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4 

Production of Essential Oils 

Erich Schmidt 

CONTENTS 

4.1 Introduction ......................................................................................................................... 83 

4.1.1 General Remarks ..................................................................................................... 83 

4.1.2 Definition and History ............................................................................................. 85 

4.1.3 Production ................................................................................................................ 88 

4.1.4 Climate ..................................................................................................................... 89 

4.1.5 Soil Quality and Soil Preparation ............................................................................ 90 

4.1.6 Water Stress and Drought ........................................................................................ 90 

4.1.7 Insect Stress and Microorganisms ........................................................................... 90 

4.1.8 Location of Oil Cells ................................................................................................ 91 

4.1.9 Types of Biomass Used ............................................................................................ 91 

4.1.10 Timing of the Harvest .............................................................................................. 91 

4.1.11 Agricultural Crop Establishment ............................................................................. 92 

4.1.12 Propagation from Seed and Clones .......................................................................... 94 

4.1.13 Commercial Essential Oil Extraction Methods ....................................................... 95 

4.1.14 Expression ................................................................................................................ 95 

4.1.15 Steam Distillation .................................................................................................... 99 

4.1.16 Concluding Remarks ................................................................................................ 117 

Acknowledgments ........................................................................................................................ 118 

References .................................................................................................................................... 118 

4.1 INTRODUCTION 

4.1.1 GENERAL REMARKS 

Essential oils have become an integral part of everyday life. They are used in a great variety of 

ways: as food flavorings, as feed additives, as flavoring agents by the cigarette industry, and in the 

compounding of cosmetics and perfumes. Furthermore, they are used in air fresheners and deodorizers 

as well as in all branches of medicine such as in pharmacy, balneology, massage, and homeopathy. 

A more specialized area will be in the fields of aromatherapy and aromachology. In recent 

years, the importance of essential oils as biocides and insect repellents has led to a more detailed 

study of their antimicrobial potential. Essential oils are also good natural sources of substances with 

commercial potential as starting materials for chemical synthesis. 

Essential oils have been known to mankind for hundreds of years, even millenniums. Long 

before the fragrances themselves were used, the important action of the oils as remedies was recognized. 

Without the medical care as we enjoy in our time, self-healing was the only option to combat 

parasites or the suffering of the human body. Later on essential oils were used in the preparation of 

early cosmetics, powders, and soaps. As the industrial production of synthetic chemicals started and 

83


increased during the nineteenth century, the production of essential oils also increased owing to 

their importance to our way of life. 

The quantities of essential oils produced around the world vary widely. The annual output of 

some essential oils exceeds 35,000 tons while that of others may reach only a few kilograms. Some 

production figures, in metric tons, based on the year 2004 are shown in Table 4.1. 

Equally wide variations also occur in the monetary value of different essential oils. Prices range 

from $1.80/kg for orange oil to $120,000.00/kg for orris oil. The total annual value of the world 

market is of the order of several billions of USD. A large, but variable, labor force is involved in the 

production of essential oils. While in some cases, harvesting and oil production will require just a 

few workers, other cases will require manual harvesting and may require multiple working steps. 

Essential oil production from either wild-growing or from cultivated plants is possible almost anywhere, 

excluding the world’s coldest, permanently snow-covered regions. It is estimated that the 

global number of plant species is of the order of 300,000. About 10% of these contain essential oils 

and could be used as a source for their production. All continents possess their own characteristic 

flora with many odor-producing species. Occasionally, these plants may be confined to a particular 

geographical zone such as Santalum album to India and Timor in Indonesia, Pinus mugo to the 

European Alps, or Abies sibirica to the CIS [Commonwealth of Independent States (former 

Russia)]. For many countries, mainly in Africa and Asia, essential oil production is their main 

source of exports. Essential oil export figures for Indonesia, Sri Lanka, Vietnam, and even India 

are very high. 

Main producer countries are found in every continent. In Europe, the center of production is situated 

in the countries bordering the Mediterranean Sea: Italy, Spain, Portugal, France, Croatia, 

Albania, and Greece, as well as middle-eastern Israel, all of which produce essential oils in industrial 

quantities. Among Central European countries, Bulgaria, Romania, Hungary, and Ukraine 

should be mentioned. The huge Russian Federation spread over much of eastern Europe and northern 

Asia has not only nearly endless resources of wild-growing plants but also large areas of cultivated 

land. The Asian continent with its diversity of climates appears to be the most important 

TABLE 4.1 

Production Figures of Important Essential Oils (2008) 

Essential Oil 

Production in Metric 

Tons (2008) Main Production Countries 

Orange oils 51000 USA, Brasil, Argentina 

Cornmint oil 32000 India, China, Argentina 

Lemon oils 9200 Argentina, Italy, Spain 

Eucalyptus oils 4000 China, India, Australia, South Africa 

Peppermint oil 3300 India, USA, China 

Clove leaf oil 1800 Indonesia, Madagascar 

Citronella oil 1800 China, Sri Lanka 

Spearmint oils 1800 USA, China 

Cedarwood oils 1650 USA, China 

Litsea cubeba oil 1200 China 

Patchouli oil 1200 Indonesia, India 

Lavandin oil Grosso 1100 France 

Corymbia Citriodora 1000 China, Brazil, India, Vietnam 

Source: Perfumer & Flavorist, 2009. A preliminary report on the world production of some 

selected essential oils and countries, Vol. 34, January 2009.

Production of Essential Oils 85 

EO production worldwide quantities figures 2008 (partly esteemed) 

India; 25.8% 

Indonesia; 1.9% 

Hungary; 0.1% 

Marocco; 0.1% 

South Africa; 1.0% 

Vietnam; 0.1% 

Others; 10.0% 

USA; 16.8% 

France; 1.0% 

Argentina; 4.9% 

China; 9.0% 

Egypt; 0.1% 

Brasil; 28.6% 

Australia; 0.6% 

FIGURE 4.1 Production countries and essential oil production worldwide (2008). (Adapted from Perfumer 

& Flavorist, 2009. A preliminary report on the world production of some selected essential oils and countries, 

Vol. 34, January 2009.) 

producer of essential oils. China and India play a major role followed by Indonesia, Sri Lanka, and 

Vietnam. Many unique and unusual essential oils originate from the huge Australian continent and 

from neighboring New Zealand and New Caledonia. Major essential oil-producing countries in 

Africa include Morocco, Tunisia, Egypt, and Algeria with the Ivory Coast, South Africa, Ghana, 

Kenya, Tanzania, Uganda, and Ethiopia playing a minor role. The important spice-producing islands 

of Madagascar, the Comoros, Mayotte, and Réunion are situated along the eastern coast of the 

African continent. The American continent is also one of the biggest essential oil producers. The 

United States, Canada, and Mexico possess a wealth of natural aromatic plant material. In South 

America, essential oils are produced in Brazil, Argentina, Paraguay, Uruguay, Guatemala, and the 

island of Haiti. Apart from the above-mentioned major essential oil-producing countries there are 

many more, somewhat less important ones, such as Germany, Taiwan, Japan, Jamaica, and the 

Philippines. Figure 4.1 shows production countries and essential oil production worldwide (2008). 

Cultivation of aromatic plants shifted during the last two centuries. From 1850 to 1950, the centers 

of commercial cultivation of essential oil plants have been the Provence in France, Italy, Spain, 

and Portugal. With the increase of labor costs, this shifted to the Mediterranean regions of North 

Africa. As manual harvesting proved too expensive for European conditions, and following improvements 

in the design of harvesting machinery, only those crops that lend themselves to mechanical 

harvesting continued to be grown in Europe. By the early 1990s, even North Africa proved too 

expensive and the centers of cultivation moved to China and India. At the present time, manualhandling 

methods are tending to become too costly even in China and, thus India remains as today’s 

center for the cultivation of fragrant plant crops. 

4.1.2 DEFINITION AND HISTORY 

Not all odorous extracts of essential oil-bearing plants comply with the International Standards 

Organization (ISO) definition of an “Essential Oil.” An essential oil as defined by the ISO in document 

ISO 9235.2—aromatic natural raw materials—vocabulary is as follows. 

“Product obtained from vegetable raw material—either by distillation with water or steam 

or—from the epicarp of Citrus fruits by a mechanical process, or—by dry distillation” (ISO/DIS 9235.2,


1997, p. 2). Steam distillation can be carried out with or without added water in a still. By contrast, 

dry distillation of plant material is carried out without the addition of any water or steam to the still 

(ISO 9235, 1997). Note 2 in Section 3.1.1 of ISO/DIS 9235.2 is of importance. It states that “Essential 

oils may undergo physical treatments (e.g., re-distillation, aeration) which do not involve significant 

changes in their composition” (ISO/DIS 9235.2, 1997, p. 2). 

An alternative definition of essential oils, established by Professor Dr. Gerhard Buchbauer of the 

Institute of Pharmaceutical Chemistry, University of Vienna, includes the following suggestion: 

“Essential oils are more or less volatile substances with more or less odorous impact, produced 

either by steam distillation or dry distillation or by means of a mechanical treatment from one single 

species” (25th International Symposium on Essential Oils, 1994). This appears to suggest that 

mixing several different plant species within the production process is not allowed. As an example, 

the addition of lavandin plants to lavender plants will yield a natural essential oil but not a natural 

lavender essential oil. Likewise, wild-growing varieties of Thymus will not result in a thyme oil as 

different chemotypes will totally change the composition of the oil. It follows that blending of 

different chemotypes of the same botanical species is inadmissible as it will change the chemical 

composition and properties of the final product. However, in view of the global acceptance of some 

specific essential oils there will be exceptions. For example, Oil of Geranium ISO/DIS 4730, is 

obtained from Pelargonium ¥ ssp., for example, from hybrids of uncertain parentage rather than 

from a single botanical species (ISO/DIS 4731, 2005). It is a well established and important article 

of commerce and may, thus, be considered to be an acceptable exception. In reality it is impossible 

to define “one single species” as many essential oils being found on the market come from different 

plant species. Even in ISO drafts it is confirmed that various plants are allowed. There are several 

examples like rosewood oils, distilled from Aniba rosaedora and Aniba parvifl ora, two different 

plant species. The same happens with the oil of gum turpentine from China, where mainly Pinus 

massoniana will be used, beside other Pinus species. Eucalyptus provides another example: Oils 

produced in Portugal have been produced from hybrids such as Eucalyptus globulus subsp. globulus 

¥ Eucalyptus globulus subsp. bicostata and Eucalyptus globulus subsp. globulus ¥ Eucalyptus 

globulus subsp. Euca lyptus globulus subsp. pseudoglobulus. These subspecies were observed from 

various botanists as separate species. The Chinese eucalyptus oils coming from the Sichuan province 

are derived from Cinnamomum longipaniculatum. Oil of Melaleuca (terpinen-4-ol type) is 

produced from Melaleuca alternifolia and in smaller amounts also from Melaleuca linariifolia and 

Melaleuca dissitifl ora. For the future, this definition must be discussed on the level of ISO rules. 

Products obtained by other extraction methods, such as solvent extracts, including supercritical 

carbon dioxide extracts, concretes or pomades, and absolutes as well as resinoids, and oleoresins are 

not essential oils as they do not comply with the earlier mentioned definition. Likewise, products 

obtained by enzymic treatment of plant material do not meet the requirements of the definition of 

an essential oil. There exists, though, at least one exception that ought to be mentioned. The wellknown 

“essential oil” of wine yeast, an important flavor and fragrance ingredient, is derived from a 

microorganism and not from a plant. 

In many instances, the commercial terms used to describe perfumery products as essential oils 

are either wrong or misleading. So-called “artificial essential oils,” “nature-identical essential oils,” 

“reconstructed essential oils,” and in some cases even “essential oils complying with the constants 

of pharmacopoeias” are merely synthetic mixtures of perfumery ingredients and have nothing to do 

with pure and natural essential oils. 

Opinions differ as to the historical origins of essential oil production. According to some, China 

has been the cradle of hydrodistillation while others point to the Indus Culture (Levey, 1959; Zahn, 

1979). On the other hand, some reports also credit the Arabs as being the inventors of distillation. 

Some literature reports suggest that the earliest practical apparatus for water distillation has been 

dated from the Indus Culture of some 5000 years ago. However, no written documents have been 

found to substantiate these claims (Levey, 1955; Zahn, 1979). The earliest documented records of a 

method and apparatus of what appears to be a kind of distillation procedure were published by Levy


from the High Culture of Mesopotamia (Levey, 1959b). He described a kind of cooking pot from 

Tepe Gaure in northeastern Mesopotamia, which differed from the design of cooking pots of that 

period. It was made of brown clay, 53 cm in diameter and 48 cm high. Its special feature was a channel 

between the raised edges. The total volume of the pot was 37 L and that of the channel was 

2.1 L. As the pot was only half-filled when in use the process appears to represent a true distillation. 

While the Arabs appear to be, apart from the existence of the pot discovered in Mesopotamia, the 

inventors of hydrodistillation, we ought to go back 3000 years b.c. 

The archaeological museum of Texila in Pakistan has on exhibit a kind of distillation apparatus 

made of burnt clay. At first sight, it really has the appearance of a typical distillation apparatus but 

it is more likely that at that time it was used for the purification of water (Rovesti, 1977). Apart from 

that the assembly resembles an eighteenth century distillation plant (Figure 4.2). It was again Levy 

who demonstrated the importance of the distillation culture. Fire was known to be of greatest 

importance. Initial heating, the intensity of the heat, and its maintenance at a constant level right 

down to the cooling process were known to be important parameters. The creative ability to produce 

natural odors points to the fact that the art of distillation was a serious science in ancient 

Mesopotamia. While the art of distillation had been undergoing improvements right up to the eighth 

century, it was never mentioned in connection with essential oils, merely with its usefulness for 

alchemical or medicinal purposes (“Liber servitorius” of Albukasis). In brief, concentration and 

purification of alcohol appeared to be its main reason for being in existence, its “raison d’être” (Koll 

and Kowalczyk, 1957). 

The Mesopotamian art of distillation had been revived in ancient Egypt as well as being expanded 

by the expression of citrus oils. The ancient Egyptians improved these processes largely because 

of their uses in embalming. They also extracted, in addition to myrrh and storax, the exudates of 

certain East African coastal species of Boswellia, none of which are of course essential oils. The 

thirteenth century Arabian writer Ad-Dimaschki also provided a description of the distillation process, 

adding descriptions of the production of distilled rose water as well as of the earliest improved 

cooling systems. It should be understood that the products of these practices were not essential oils 

in the present accepted sense but merely fragrant distilled water extracts exhibiting the odor of the 

plant used. 

The next important step in the transfer of the practice of distillation to the Occident, from ancient 

Egypt to the northern hemisphere, was triggered by the crusades of the Middle Ages from the 

twelfth century onward. Hieronymus Brunschwyk listed in his treatise “The true art to distil” about 

FIGURE 4.2 Reconstruction of the distillation plant from Harappa.


25 essential oils produced at that time. Once again one should treat the expression “essential oils” 

with caution; it would be more accurate to refer to them as “fragrant alcohols” or “aromatic waters.” 

Improvements in the design of equipment led to an enrichment in the diversity of essential oils 

derived from starting materials such as cinnamon, sandalwood, and also sage and rosemary 

(Gildemeister and Hoffmann, 1931). 

The first evidence capable to discriminate between volatile oils and odorous fatty oils was provided 

in the sixteenth century. The availability of printed books facilitated “scientists” seeking 

guidance on the distillation of essential oils. While knowledge of the science of essential oils did not 

increase during the seventeenth century, the eighteenth century brought about only small progress 

in the design of equipment and in refinements of the techniques used. The beginning of the nineteenth 

century brought about progresses in chemistry, including wet analysis, and restarted again, 

chiefly in France, in an increased development of hydrodistillation methods. Notwithstanding the 

“industrial” production of lavender already in progress since the mid-eighteenth century, the real 

breakthrough occurred at the beginning of the following century. While until then the distillation 

plant was walled in, now the first moveable apparatus appeared. The “Alambique Vial Gattefossé” 

was easy to transport and placed near the fields. It resulted in improved product quality and reduced 

the length of transport. These stills were fired with wood or dried plant material. The first swiveling 

still pots had also been developed which facilitated the emptying of the still residues. These early 

stills had a capacity of about 50–100 kg of plant material. Later on their capacity increased to 

1000–1200 kg. At the same time, cooling methods were also improved. These improvements spread 

all over the northern hemisphere to Bulgaria, Turkey, Italy, Spain, Portugal, and even to northern 

Africa. The final chapter in the history of distillation of plant material came about with the invention 

of the “alembic à bain-marie,” technically speaking a double-walled distillation plant. Steam 

was not only passed through the biomass, but was also used to heat the wall of the still. This new 

method improved the speed of the distillation as well as the quality of the top notes of the essential 

oils thus produced. 

The history of the expression of essential oils from the epicarp of citrus fruits is not nearly as 

interesting as that of hydrodistillation. This can be attributed to the fact that these expressed 

fragrance concentrates were more readily available in antiquity as expression could be effected by 

implements made of wood or stone. The chief requirement for this method was manpower and that 

was available in unlimited amount. The growth of the industry led to the invention of new, and to 

the improvement of existing machinery, but this topic will be dealt with later on. 

4.1.3 PRODUCTION 

Before dealing with the basic principles of essential oil production it is important to be aware of the 

fact that the essential oil we have in our bottles or drums is not necessarily identical with what is 

present in the plant. It is wishful thinking, apart for some rare exceptions, to consider an “essential 

oil” to be the “soul” of the plant and thus an exact replica of what is present in the plant. Only 

expressed oils that have not come into contact with the fruit juice and that have been protected from 

aerial oxidation may meet the conditions of a true plant essential oil. The chemical composition of 

distilled essential oils is not the same as that of the contents of the oil cells present in the plant or 

with the odor of the plants growing in their natural environment. Headspace technology, a unique 

method allowing the capture of the volatile constituents of oil cells and thus providing additional 

information about the plant, has made it possible to detect the volatile components of the plant’s 

“aura.” One of the best examples is rose oil. A nonprofessional individual examining pure and natural 

rose oil on a plotter, even in dilution, will not recognize its plant source. The alteration caused 

by hydrodistillation is remarkable as plant material in contact with steam undergoes many chemical 

changes. Hot steam contains more energy than, for example, the surface of the still. Human skin 

that has come into contact with hot steam suffers tremendous injuries while short contact with a 

metal surface at 100°C results merely in a short burning sensation. Hot steam will decompose many


aldehydes and esters may be formed from acids generated during the vaporization of certain essential 

oil components. Some water soluble molecules may be lost by solution in the still water, thus 

altering the fragrance profile of the oil. 

Why do so many plants produce essential oils? Certainly neither to regale our nose with pleasant 

fragrances of rose or lavender, nor to heighten the taste (as taste is mostly related to odor) of 

ginger, basil, pepper, thyme, or oregano in our food! Nor to cure diseases of the human body or 

influence human behavior! Most essential oils contain compounds possessing antimicrobial 

properties, active against viruses, bacteria, and fungi. Often, different parts of the same plant, 

such as leaves, roots, flowers, and so on may contain volatile oils of different chemical composition. 

Even the height of a plant may play a role. For example, the volatile oil obtained from the 

gum of the trunk of Pinus pinaster at a height of 2 m will contain mainly pinenes and significant 

car-3-ene, while oil obtained from the gum collected at a height of 4 m will contain very little or 

no car-3-ene. The reason for this may be protection from deer that browse the bark during the 

winter months. Some essential oils may act not only as insect repellents but even prevent their 

reproduction. In many cases, it has been shown that plants attract insects that in turn assist in 

pollinating the plant. It has also been shown that some plants communicate through the agency 

of their essential oils. Sometimes essential oils are considered to be simply metabolic waste products! 

This may be so in the case of eucalypts as the oil cells present in the mature leaves of 

Eucalyptus species are completely isolated and embedded deeply within the leaf structure. In 

some cases essential oils act as germination inhibitors thus reducing competition by other plants 

(Porter, 2001). 

Essential oil yields vary widely and are difficult to predict. The highest oil yields are usually 

associated with balsams and similar resinous plant exudations, such as gurjun, copaiba, elemi, and 

Peru balsam, where they can reach 30–70%. Clove buds and nutmeg can yield between 15% and 

17% of essential oil while other examples worthy of mention are cardamom (about 8%), patchouli 

(3.5%) and fennel, star anise, caraway seed, and cumin seed (1–9%). Much lower oil yields are 

obtained with juniper berries, where 75 kg of berries are required to produce 1 kg of oil, sage (about 

0.15%), and other leaf oils such as geranium (also about 0.15%). 700 kg of rose petals will yield 1 kg 

of oil and 1000 kg of bitter orange flowers are required for the production of also just 1 kg of oil. 

The yields of expressed fruit peel oils, such as bergamot, orange, and lemon vary from 0.2% to 

about 0.5%. 

A number of important agronomic factors have to be considered before embarking on the production 

of essential oils, such as climate, soil type, influence of drought and water stress and stresses 

caused by insects and microorganisms, propagation (seed or clones), and cultivation practices. Other 

important factors include precise knowledge on which part of the biomass is to be used, location of 

the oil cells within the plant, timing of harvest, method of harvesting, storage, and preparation of 

the biomass prior to essential oil extraction. 

4.1.4 CLIMATE 

The most important variables include temperature, number of hours of sunshine, and frequency and 

magnitude of precipitations. Temperature has a profound effect on the yield and quality of the essential 

oils, as the following example of lavender will show. The last years in the Provence, too cold at 

the beginning of growth, were followed by very hot weather and a lack of water. As a result yields 

decreased by one-third. The relationship between temperature and humidity is an additional important 

parameter. Humidity coupled with elevated temperatures produces conditions favorable to the 

proliferation of insect parasites and, most importantly, microorganisms. This sometimes causes 

plants to increase the production of essential oil for their own protection. Letchamo have studied the 

relationship between temperature and concentration of daylight on the yield of essential oil and 

found that the quality of the oil was not influenced (Letchamo et al., 1994). Herbs and spices usually 

require greater amounts of sunlight. The duration of sunshine in the main areas of herb and spice


cultivation, such as the regions bordering the Mediterranean Sea, usually exceeds 8 h/day. In India, 

Indonesia and many parts of China this is well in excess of this figure and two or even three crops 

per year can be achieved. Protection against cooling and heavy winds may be required. Windbreaks 

provided by rows of trees or bushes and even stone walls are particularly common in southern 

Europe. In China the Litsea cubeba tree is used for the same purpose. In colder countries, the winter 

snow cover will protect perennials from frost damage. Short periods of frost with temperatures 

below −10°C will not be too detrimental to plant survival. However, long exposure to heavy frost at 

very low subzero temperatures will result in permanent damage to the plant ensuing from a lack of 

water supply. 

4.1.5 SOIL QUALITY AND SOIL PREPARATION 

Every friend of a good wine is aware of the influence of the soil on the grapes and finally on the 

quality of the wine. The same applies to essential oil-bearing plants. Some crops, such as lavender, 

thyme, oregano, and clary sage require meager but lime-rich soils. The Jura Chalk of the Haute 

Provence is destined to produce a good growth of lavender and is the very reason for the good quality 

and interesting top note of its oils by comparison with lavender oils of Bulgarian origin growing 

on different soil types (Meunier, 1985). Soil pH affects significantly oil yield and oil quality. 

Figueiredo et al. found that the pH value “strongly influences the solubility of certain elements in 

the soil. Iron, zinc, copper and manganese are less soluble in alkaline than in acidic soils because 

they precipitate as hydroxides at high pH values” (Figueiredo et al., 2005). It is essential that farmers 

determine the limits of the elemental profile of the soil. Furthermore, the spacing of plantings 

should ensure adequate supply with essential trace elements and nutrients. Selection of the optimum 

site coupled with a suitable climate plays an important role as they will provide a guarantee for 

optimum crop and essential oil quality. 

4.1.6 WATER STRESS AND DROUGHT 

It is well known to every gardener that lack of water, as well as too much water, can influence the 

growth of plants and even kill them. The tolerance of the biomass to soil moisture should be determined 

in order to identify the most appropriate site for the growing of the desired plant. Since fungal 

growth is caused by excess water, most plants require well-drained soils to prevent their roots 

from rotting and the plant from being damaged, thus adversely affecting essential oil production. 

Lack of water, for example, dryness, exerts a similar deleterious influence. Flowers are smaller than 

normal and yields drop. Extreme drought can kill the whole plant as its foliage dries closing down 

its entire metabolism. 

4.1.7 INSECT STRESS AND MICROORGANISMS 

Plants are living organisms capable of interacting with neighbor plants and warning them of any 

incipient danger from insect attack. These warning signals are the result of rapid changes occurring 

in their essential oil composition, which are then transferred to their neighbors who in turn transmit 

this information on to their neighbors forcing them to change their oil composition as well. In this 

way, the insect will come into contact with a chemically modified plant material, which may not suit 

its feeding habits thus obliging it to leave and look elsewhere. Microorganisms can also significantly 

change the essential oil composition as shown in the case of elderflower fragrance. Headspace gas 

chromatography coupled with mass spectroscopy (GC/MS) has shown that linalool, the main 

constituent of elderflowers, was transformed by a fungus present in the leaves, into linalool oxide. 

The larvae of Cécidomye (Thomasissiana lavandula) damage the lavender plant with a concomitant 

reduction of oil quality. Mycoplasmose and the fungus Armillaria mellex can affect the whole plantation 

and totally spoil the quality of the oil.


4.1.8 LOCATION OF OIL CELLS 

As already mentioned, the cells containing essential oils can be situated in various parts of the 

plant. Two different types of essential oil cells are known, superficial cells, for example, glandular 

hairs located on the surface of the plant, common in many herbs such as oregano, mint, lavender, 

and so on, and cells embedded in plant tissue, occurring as isolated cells containing the secretions 

(as in citrus fruit and eucalyptus leaves), or as layers of cells surrounding intercellular space (canals 

or secretory cavities), for example, resin canals of pine. Professor Dr. Johannes Novak (Institute of 

Applied Botany, Veterinary University, Vienna) has shown impressive pictures and pointed out that 

the chemical composition of essential oils contained in neighboring cells (oil glands) could be variable 

but that the typical composition of a particular essential oil was largely due to the averaging of 

the enormous number of individual cells present in the plant (Novak, 2005). It has been noted in a 

publication entitled “Physiological Aspects of Essential Oil Production” that individual oil glands 

do not always secrete the same type of compound and that the process of secretion can be different 

(Kamatou et al., 2006). Different approaches to distillation are dictated by the location of the oil 

glands. Preparation of the biomass to be distilled, temperature, and steam pressure affect the quality 

of the oil produced. 

4.1.9 TYPES OF BIOMASS USED 

Essential oils can occur in many different parts of the plant. They can be present in flowers (rose, 

lavender, magnolia, bitter orange, and blue chamomile) and leaves (cinnamon, patchouli, petitgrain, 

clove, perilla, and laurel); sometimes the whole aerial part of the plant is distilled (Melissa offi cinalis, 

basil, thyme, rosemary, marjoram, verbena, and peppermint). The so-called fruit oils are often 

extracted from seed, which forms part of the fruit, such as caraway, coriander, cardamom, pepper, 

dill, and pimento. Citrus oils are extracted from the epicarp of species of Citrus, such as lemon, 

lime, bergamot, grapefruit, bitter orange as well as sweet orange, mandarine, clementine, and tangerine. 

Fruit or perhaps more correctly berry oils are obtained from juniper and Schinus species. 

The well-known bark oils are obtained from birch, cascarilla, cassia, cinnamon, and massoia. Oil 

of mace is obtained from the aril, a fleshy cover of the seed of nutmeg (Myristica fragrans). Flower 

buds are used for the production of clove oil. Wood and bark exudations yield an important group 

of essential oils such as galbanum, incense, myrrh, mastix, and storax, to name but a few. The 

needles of conifers (leaves) are a source of an important group of essential oils derived from species 

of Abies, Pinus, and so on. Wood oils are derived mostly from species of Santalum (sandalwood), 

cedar, amyris, cade, rosewood, agarwood, and guaiac. Finally, roots and rhizomes are the source of 

oils of orris, valerian, calamus, and angelica. 

What happens when the plant is cut? Does it immediately start to die as happens in animals and 

humans? The water content of a plant ranges from 50% to over 80%. The cutting of a plant interrupts 

its supply of water and minerals. Its life-sustaining processes slow down and finally stop 

altogether. The production of enzymes stops, auto-oxidative processes start, including an increase 

in bacterial activity leading to rotting and molding. Color and organoleptic properties, such as fragrance, 

will also change usually to their detriment. As a consequence of this, unless controlled 

drying or preparation is acceptable options, treatment of the biomass has to be prompt. 

4.1.10 TIMING OF THE HARVEST 

The timing of the harvest of the herbal crop is one of the most important factors affecting the quality 

of the essential oil. It is a well-documented fact that the chemical composition changes throughout 

the life of the plant. Occasionally, it can be a matter of days during which the quality of the 

essential oil reaches its optimum. Knowledge of the precise time of the onset of flowering often has 

a great influence on the composition of the oil. The chemical changes occurring during the entire


life cycle of Vietnamese Artemisia vulgaris have shown that 1,8-cineole and b-pinene contents before 

flowering were below 10% and 1.2%, respectively, whereas at the end of flowering they reached 

values above 24% and 10.4% (Nguyen Thi Phuong Thao et al., 2004). These are very large variations 

indeed occurring during the plant’s short life span. In the case of the lavender life cycle, the ester 

value of the oil is the quality-determining factor. It varies within a wide range and influences the 

value of the oil. As a rule of thumb, it is held that its maximum value is reached at a time when about 

two-third of the lavender flowers have opened and thus, that harvesting should commence. In the 

past, growers knew exactly when to harvest the biomass. These days the use of a combination of 

microdistillation and gas chromatography (GC) techniques enables rapid testing of the quality of the 

oil and thus the determination of the optimum time for harvesting to start. Oil yields may in some 

cases be influenced by the time of harvesting. One of the best examples is rose oil. The petals should 

be collected in the morning between 6 a.m. and 9 a.m. With rising day temperatures the oil yield will 

diminish. In the case of oil glands embedded within the leaf structure, such as in the case of eucalypts 

and pines, oil yield and oil quality are largely unaffected by the time of harvesting. 

4.1.11 AGRICULTURAL CROP ESTABLISHMENT 

The first step is, in most cases, selection of plant seed which suits best the requirements of the product 

looked for. Preparation of seedbeds, growing from seed, growing and transplanting of seedlings, 

and so on should follow well-established agricultural practices. The spacing of rows has to be considered 

(Dey, 2007). For example, dill prefers wider row spacing than anise, coriander, or caraway 

(Novak, 2005). The time required before a crop can be obtained depends on the species used and 

can be very variable. Citronella and lemongrass may take 7–9 months from the time of planting 

before the first crop can be harvested while lavender and lavandin require up to three years. The 

most economical way to extract an essential oil is to transport the harvested biomass directly to the 

distillery. For some plants, this is the only practical option. Melissa offi cinalis (“lemon balm”) is 

very prone to drying out and thus to loss of oil yield. Some harvested plant material may require 

special treatment of the biomass before oil extraction, for example, grinding or chipping, breaking 

or cutting up into smaller fragments, and sometimes just drying. In some cases, fermentation of the 

biomass should precede oil extraction. Water contained within the plant material has been classified 

by Yanive and Palevitch as chemically, physicochemically, and mechanically bound water (Yanive 

and Palevitch, 1982). According to these authors only the mechanically bound water, which is 

located on the surface and the capillaries of plants, can be reduced. Drying can be achieved simply 

by spreading the biomass on the ground where wind movement effects the drying process. Drying 

can also be carried out by the use of appropriate drying equipment. Drying, too, can affect the quality 

of the essential oil. Until the middle of the 1980s cut lavender and lavandin have been dried in 

the field, (Figure 4.3) a process requiring about three days. The resulting oils exhibited the typical 

fine, floral odor; however, oil yields were inferior to yields obtained with fresh material. Compared 

with the present day procedure with container harvesting and immediate processing (the so-called 

“vert-broyé”) this quality of the oil is greener, harsher, and requires some time to harmonize. 

However, yields are better, and one step in the production process has been eliminated. Clary sage 

is a good example demonstrating the difference between oils distilled from fresh plant material on 

the one hand and dried plant material on the other. The chemical differences are clearly shown in 

Table 4.2. Apart from herbal biomass, fruits and seed may also have to be dried before distillation. 

These include pepper, coriander, cloves, and pimento berries, as well as certain roots such as vetiver, 

calamus, lovage, and orris. Clary sage is harvested at the beginning of summer but distilled only at 

the end of the harvesting season. 

Seeds and fruits of the families Apiaceae, Piperaceae, and Myristicaceae usually require grinding 

up prior to steam distillation. In many cases, the seed has to be dried before comminution takes 

place. Celery, coriander, dill, ambrette, fennel, and anise belong to the Apiaceae. All varieties of 

pepper belong to the Piperaceae while nutmeg belongs to the Myristicaceae. The finer the material


FIGURE 4.3 (See color insert following page 468.) Lavender drying on the field. 

is ground, the better will be the oil yield and, owing to shorter distillation times, also the quality of 

the oil. In order to reduce losses of volatiles by evaporation during the comminution of the seed or 

fruit, the grinding can also be carried out under water, preferably in a closed apparatus. Heartwood 

samples, such as those of Santalum album, Santalum spicatum, and Santalum austrocaledonicum 

TABLE 4.2 

Differences in the Composition of the Essential Oil of Clary Sage 

Manufactured Fresh and Dried 

Component “Vert Broyee” (%) Traditional (%) 

Myrcene 0.9–1.0 0.9–1.1 

Limonene 0.2–0.4 0.3–0.5 

Ocimene cis 0.3–0.5 0.4–0.6 

Ocimene trans 0.5–0.7 0.8–1.0 

Copaene alpha 0.5–0.7 1.4–1.6 

Linalool 13.0–24.0 6.5–13.5 

Linalyl acetate 56.0–70.5 62.0–78.0 

Caryophyllene beta 1.5–1.8 2.5–3.0 

Terpineol alpha 1.0–5.0 Max. 2.1 

Neryl acetate 0.6–0.8 0.7–1.0 

Germacrene d 1.1–7.5 1.5–12 

Geranyl acetate 1.4–1.7 2.2–2.5 

Geraniol 1.4–1.7 1.2–1.5 

Sclareol 0.4–1.8 0.6–2.8 

Minor changes 

Middle changes 

Big changes


have to be reduced to a very fine powder prior to steam distillation in order to achieve complete 

recovery of the essential oil. In some cases, coarse chipping of the wood is adequate for efficient 

essential oil extraction. This includes cedarwood, amyris, rosewood, birch, guaiac, linaloe, cade, 

cabreuva, and the like. 

Plant material containing small branches as well as foliage, which includes pine needles, has to 

be coarsely chopped up prior to steam distillation. Examples of such material are juniper branches, 

Melaleuca alternifolia, Corymbia citriodora, Pinus mugo, Pinus pinaster, Pinus sylvestris, Pinus 

nigra, Abies alba, Abies sibirica, and Abies grandis, as well as mint and peppermint. Present day 

mechanized harvesting methods automatically effect the chopping up of the biomass. This also 

reduces the volume of the biomass, thus increasing the quantity of material that can be packed into 

the still and making the process more economical. 

It appears that the time when the seed is sown influences both oil yield as well as essential oil 

composition, for example, whether it is sown in spring or autumn. Important factors affecting production 

of plant material are application of fertilizers, herbicides, and pesticides as well as the availability 

and kind of pollination agents. In many essential oil producing countries, no artificial 

nitrogen, potassium, or phosphorus fertilizers are used. Instead, both in Europe, as well as overseas, 

the biomass left over after steam distillation is spread in the fields as an organic fertilizer. Court 

et al. reported, from the field tests conducted with peppermint, that an increase in fertilizer affects 

plant oil yield. However, higher doses did not result in further increases in oil yield or in changes in 

the oil composition (Court et al., 1993). Herbicides and pesticides do not appear to influence either 

oil yield or oil composition. The accumulation of pesticide residues in essential oils has a negative 

influence on their quality and on their uses. The yield and quality of essential oils are also influenced 

by the timing and type of pollinating agent. If the flower is ready for pollination the intensity 

of its fragrance and the amount of volatiles present are at their maximum. If on the other hand the 

weather is too cold at the time of flowering, pollination will be adversely affected and transformation 

to fruit is unlikely to take place. Such an occurrence has a very significant effect on the plant’s 

metabolism and finally on its essential oil. Grapevine cultivators use the following trick to attract 

pollinators to their vines. A rose flower placed at the end of each grapevine row attracts pollinators 

who then also pollinate the unattractive flowers of the grapevine. 

4.1.12 PROPAGATION FROM SEED AND CLONES 

Plants can be grown from seed or propagated asexually by cloning. Lavender plants raised from seed 

are kept for one year in pots before transplanting into the field. It then takes another three years before 

the plantation yields enough flowers for commercial harvesting and steam distillation. Plants of any 

species raised from seed will exhibit wide genetic variations among the progeny, as exist between the 

members of any species propagated by sexual means, for example, humans. In the case of lavender 

(Lavandula angustifolia), the composition of the essential oil from individual plants varies from plant 

to plant or more precisely, from one genotype to the another. Improvement of the crop by selective 

breeding of those genotypes that yield the most desirable oil is a very slow process requiring years to 

accomplish. Charles Denny, who initiated the Tasmanian lavender industry in 1921, selected within 

11 years 487 genotypes from a source of 2500 genotypes of L. angustifolia for closer examination, 

narrowing them down to just 13 strains exhibiting large yields of superior oil. Finally, four of these 

genotypes were grown on a large scale and mixed together in what is called “comunelles.” The quality 

of the oils produced was fairly constant from year to year, both in their physicochemical properties as 

well as in their olfactory characteristics (Denny, 1995, private information to the author). 

Cloning is the preferred method for the replication of plants having particular, usually commercially 

desirable, characteristics. Clones are obtained from buds or cuttings of the same individual 

and the essential oils, for example, obtained from them are the same, or very similar to those of the 

parent. Cloning procedures are well established but may vary in their detail among different species. 

One important advantage of clones is that commercial harvesting may be possible after a shorter


time as compared with plantations grown from seed. One risk does exist though. If the mother plant 

is diseased all clones will also be affected and the plantation would have to be destroyed. 

No field of agriculture requires such a detailed and comprehensive knowledge of botany and soil 

science, as well as of breeding and propagation methods, harvesting methods, and so on as that of 

the cultivation of essential oil-bearing plants. The importance of this is evident from the very large 

amount of scientific research carried out in this field by universities as well as by industry. 

4.1.13 COMMERCIAL ESSENTIAL OIL EXTRACTION METHODS 

There are three methods in use. Expression is probably the oldest of these and is used almost exclusively 

for the production of Citrus oils. The second method, hydrodistillation or steam distillation, 

is the most commonly used one of the three methods, while dry distillation is used only rarely in 

some very special cases. 

4.1.14 EXPRESSION 

Cold expression, for example, expression at ambient temperature without the involvement of extraneous 

heat, was practiced long before humans discovered the process of distillation, probably because the 

necessary tools for it were readily available. Stones or wooden tools were well suited to breaking the oil 

cells and freeing their fragrant contents. This method was used almost exclusively for the production of 

Citrus peel oils. Citrus and the allied genus Fortunella belong to the large family Rutaceae. Citrus fruits 

used for the production of the oils are shown in Table 4.3. Citrus fruit cultivation is widely spread all over 

the world with a suitable climate. Oils with the largest production include orange, lemon, grapefruit, and 

mandarin. Taking world lemon production as an example, the most important lemon-growing areas in 

TABLE 4.3 

Important Essential Oil Production from Plants of the Rutaceae Family 

Botanical Term Expressed Distilled Used Plant Parts 

Citrus aurantifolia (Christm.) 

Swingle 

Citrus aurantium L., syn. Citrus 

amara Link, syn. Citrus bigaradia 

Loisel, syn. Citrus vulgaris Risso 

Citrus bergamia (Risso et Poit.), 

Citrus aurantium L. subsp. 

bergamia (Wight et Arnott) Engler 

Citrus hystrix DC., syn. 

Citrus torosa Blanco 

Lime oil Lime oil distilled Pericarp; fruit juice or 

crushed fruits 

Bitter orange oil 

Neroli oil, Bitter orange 

petitgrain oil 

Flower, pericarp, leaf, and 

twigs with sometimes 

little green fruits 

Bergamot oil Bergamot petitgrain oil Pericarp, leaf, and twigs 

with sometimes little 

green fruits 

Kaffir lime oil, Kaffir leaves oil Pericarp, leaves 

Combava 

Citrus latifolia Tanaka Lime oil Persian type Pericarp 

Citrus limon (L.) Burm. f. Lemon oil Lemon petitgrain oil Flower, pericarp, leaf, and 



Citrus reticulate Blanco syn. 

Citrus nobilis Andrews 

Citrus sinensis (L.) Osbeck, 

Citrus djalonis A. Chevalier 

Mandarin oil Mandarin petitgrain oil Flower, pericarp, leaf, and 



Sweet orange oil 

Pericarp 

Citrus × paradisi Macfad. Grapefruit oil Pericarp


Europe are situated in Italy and Spain with Cyprus and Greece being of much lesser importance. Nearly 

90% of all lemon fruit produced originates from Sicily where the exceptionally favorable climate enables 

an almost around the year production. There is a winter crop from September to April, a spring crop 

from February to May, and a summer crop from May to September. The Spanish harvest calendar is very 

similar. Other production areas in the northern hemisphere are in the United States, particularly in 

Florida, Arizona, and California, and in Mexico. In the southern hemisphere, large-scale lemon producers 

are Argentina, Uruguay, and Brazil. Lemon production is also being developed in South Africa, 

Ivory Coast, and Australia. China promises to become a huge producer of lemon in the future. 

The reason for extracting citrus oils from fruit peel using mechanical methods is the relative thermal 

instability of the aldehydes contained in them. Fatty, for example, aliphatic, aldehydes such as 

heptanal, octanal, nonanal, decanal, and dodecanal are readily oxidized by atmospheric oxygen, 

which gives rise to the formation of malodorous carboxylic acids. Likewise, terpenoid aldehydes such 

as neral, geranial, citronellal, and perilla aldehyde as well as the a- and b-sinensals are sensitive to 

oxidation. Hydrodistillation of citrus fruit yields poor quality oils owing to chemical reactions that can 

be attributed to heat and acid-initiated degradation of some of the unstable fruit volatiles. Furthermore, 

some of the terpenic hydrocarbons and esters contained in the peel oils are also sensitive to heat and 

oxygen. One exception to this does exist. Lime oil of commerce can be either cold pressed or steam 

distilled. The chemical composition of these two types of oil as well as their odors differs significantly 

from each other. The expressed citrus peel is normally treated with hot steam in order to recover any 

essential oil still left over in it. The products of this process, consisting mainly of limonene, are used 

in the solvent industry. The remaining peel and fruit flesh pulp is used as cattle feed. 

The oil cells of citrus fruit are situated just under the surface in the epicarp, also called flavedo, 

in the colored area of the fruit. Figure 4.4 is a cross section of the different parts of the fruit also 

showing the juice cells present in the fruit. An essential oil is also present in the juice cells. However, 

the amount of oil present in the juice cells is very much smaller than the amount present in the 

flavedo; also their composition differs from each other. 

Until the beginning of the twentieth century, industrial production of cold-pressed citrus oils was 

carried out manually. One has to visualize huge halls with hundreds of workers, men and women, 

seated on small chairs handling the fruit. First of all, the fruit had to be washed and cut into two 

halves. The pulp was then removed from the fruit using a sharp-edged spoon, called the “rastrello” 

and after the peel was soaked in warm water. The fruit peel was now manually turned inside out so 

that the epicarp was on the inside, squeezed by hand to break the oil glands and the oil soaked up 

with a sponge. The peel was now turned inside out once again and wiped with the sponge and the 

FIGURE 4.4 (See color insert following page 468.) Parts of a citrus fruit.


sponge squeezed into a terracotta bowl, the “concolina.” After decantation, the oil was collected in 

metal containers. This was an extremely laborious process characterized by substantial oil losses. A 

later improvement of the fruit peel expression process was the “scodella” method. The apparatus 

was a metallic hemisphere lined inside with small spikes, with a tube attached at its center. The fruit 

placed inside the hemisphere was rotated while being squeezed against the spikes thus breaking the 

oil cells. The oil emulsion, containing some of the wax coating the fruit, flowed into the central tube 

was collected, and the oil was subsequently separated by centrifugation. 

Neither of these methods, even when used simultaneously, was able to satisfy the increased 

demand for fruit peel oils at the start of the industrial era. The quantity of fruit processed could be 

increased but the extraction methods were time wasting and the oil yields too low. With the advent 

of the twentieth century the first industrial machinery was developed. Today the only systems of 

significance in use for the industrial production of peel oils can be classified into four categories: 

“sfumatrici” machines and “speciale sfumatrici,” “Pellatrici” machines, “FMC whole fruit process,” 

and “Brown oil extractors (BOEs)” (Arnodou, 1991). 

It is important to be aware of the fact that the individual oil glands within the epicarp are not 

connected to neighboring glands. The cell walls of these oil glands are very tough and it is believed 

that the oil they contain is either a metabolic waste product or a substance protecting the plant from 

being browsed by animals. 

The machines used in the “sfumatrici” methods consist in principle of two parts, a fixed part and 

a moveable part. The fruit is cut into two and the flesh is removed. In order to extract the oil, the 

citrus peel is gently squeezed, by moving it around between the two parts of the device, and rinsing 

off the squeezed-out oil with a jet of water. The oil readily separates from the liquid on standing and 

is collected by decantation. Since the epicarp may contain organic acids (citric acid, etc.), it is occasionally 

soaked in lime solution in order to neutralize the acids present. Greater concentrations of 

acid could alter the quality of the oil. Degradation of aldehydes is also an important consideration. 

In the “special sfumatrici” method, the peel is soaked in the lime solution for 24 h before pressing. 

By means of a metallic chain drawn by horizontal rollers with ribbed forms, the technical process 

is finished. The oils obtained by these methods may have to be “wintered,” for example, refrigerated 

in order to freeze out the peel waxes that are then filtered off. 

In the “Pellatrici” method the peel oil is removed during the first step and the fruit juice in a 

second step (Figure 4.5). In the first step, the fruit is fed through a slowly turning Archimedean 

screw-type valve. The screw is covered with numerous spikes that will bruise the oil cells in the epicarp 

and initiate the flow of oil. The oil is, once again, removed by means of a jet of water. The fruit 

FIGURE 4.5 (See color insert following page 468.) “Pellatrici method.” The spiked Archimedes screw 

with lemons, washed with water.


FIGURE 4.6 (See color insert following page 468.) “Brown” process. A battery of eight juice squeezers 

waiting for fruits. 

is finally carried to a fast-rotating, spiked, roller carpet where the remaining oil cells, located deeper 

within the epicarp, are bruised and their oil content recovered, thus resulting in maximum oil yield. 

The process involves centrifugation, filtration, and “wintering” as previously mentioned. 

The “Brown process” (Reeve, 2005) is used mainly in the United States and in South America, 

but less in Europe. The BOE (Figure 4.6) is somewhat similar to the machinery of the “Pellatrici” 

method. A device at the front end controls the quantity of fruit entering the machine. The machine 

itself consists of numerous pairs of spiked rollers turning in the same direction, as well as moving 

horizontally, thus reaching all oil cells. The spiked rollers as well as the fruit are submerged in water 

for easy transport. Any residual water and oil adhering to the fruit are removed by a special system 

of rollers and added to the oil emulsion generated on the first set of rollers. Any solid particles are 

then removed by passing it through a fine sieve. The emulsion is then centrifuged and the aqueous 

phase recycled. The BOE is manufactured in V4A steel to avoid contact with iron. 

The most frequently used type of extractor is the food machinery corporation (FMC)-in-Line. It is 

assumed that in the United States more than 50% of extractors are of the FMC type (Figure 4.7). 

Other large producer countries, such as Brazil and Argentina use exclusively FMC extractors. The 

FIGURE 4.7 (See color insert following page 468.) FMC extractor.


reason for this is the design of the machinery, as fruit juice and oil are produced in one step without 

the two coming into contact with each other. The process requires prior grading of the fruit as the cups 

used in this process are designed for different sizes of fruit. An optimum fruit size is important as 

bigger fruit would be over squeezed and some essential oil carried over into the juice making it bitter. 

On the other hand if the fruit were too small the yield of juice would be reduced. Different frame sizes 

allow treating 3, 5, or 8 fruits at the same time. This technique was revolutionary in its concept and 

works as follows: the fruit is carried to, and placed into, a fixed cup. Another cup, bearing a mirror 

image relationship to the fixed cup, is positioned exactly above it. Both cups are built of intermeshing 

jaws. The moveable cup is lowered toward the fixed cup thus enclosing the fruit. At the same time, a 

circular knife cuts a hole into the bottom of the fruit. When pressure is applied to the fruit the expressed 

juice will exit through the cut hole on to a mesh screen and be transported to the juice manifold while 

at the same time the oil is squeezed out of the surface of the peel. As before the oil is collected using 

a jet of water. The oil–water emulsion is then separated by centrifugation. 

An examination of the developments in the design of citrus fruit processing machinery shows 

quite clearly that the quality of the juice was more important than the quality of the oil, the only 

exception being oil of bergamot. Nevertheless, oil quality improved during the last decades and complies 

with the requirements of ISO Standards. The expressed pulp of the more valuable kind fruit is 

very often treated with high-pressure steam to recover additional amounts of colorless oils of variable 

composition. The kinds of fruit treated in this manner are bergamot, lemon, and mandarin. 

4.1.15 STEAM DISTILLATION 

Steam or water distillation is unquestionably the most frequently used method for the extraction of 

essential oil from plants. The already earlier mentioned history of steam distillation and the longstanding 

interest of mankind in extracting the fragrant and useful volatile constituents of plants 

testify to this. Distillation plants of varying design abound all over the world. While in some developing 

countries traditional and sometimes rather primitive methods are still being used (Figure 4.8), 

the essential oils produced are often of high quality. Industrialized countries employ technologically 

more evolved and complex equipment, computer aided with in-process analysis of the final 

product. Both of these very different ways of commercial essential oil production provide excellent 

quality oils. One depends on skill and experience, the other on superior technology and expensive 

equipment. It should be borne in mind that advice by an expert on distillation is a prerequisite for 

the production of superior quality oils. The term “distillation” is derived from the Latin “distillare” 

which means “trickling down.” In its simplest form distillation is defined as “evaporation and subsequent 

condensation of a liquid.” All liquids evaporate to a greater or lesser degree, even at room 

temperature. This is due to thermally induced molecular movements within the liquid resulting in 

some of the molecules being ejected into the airspace above them (diffusing into the air). As the 

temperature is increased these movements increase as well, resulting in more molecules being 

ejected, for example, in increased evaporation. The definition of an essential oil, ISO 9235, item 

3.1.1 is: “… product obtained from vegetable raw material—either by distillation with water or 

steam” and in item 3.1.2: “… obtained with or without added water in the still” (ISO/DIS 9235.2, 

1997, p. 2). This means that even “cooking” in the presence of water represents a method suitable 

for the production of essential oils. The release of the essential oil present in the oil glands (cells) 

of a plant is due to the bursting of the oil cell walls caused by the increased pressure of the heatinduced 

expansion of the oil cell contents. The steam flow acts as the carrier of the essential oil 

molecules. The basic principle of either water or steam distillation is a limit value of a liquid– 

liquid–vapor system. The theory of hydrodistillation is the following. Two nonmiscible liquids (in 

our case water and essential oil) A and B form two separate phases. The total vapor pressure of that 

system is equal to the sum of the partial vapor pressures of the two pure liquids: 

r = r A + r B (r is the total vapor pressure of the system).


FIGURE 4.8 Bush distillation device, opened. 

With complete nonmiscibility of both liquids, r is independent of the composition of the liquid 

phase. The boiling temperature of the mixture (T M ) lies below the boiling temperatures (T A and T B ) 

of liquids A and B. The proportionality between the quantity of each component and the pressure in 

the vapor phase is given in the formula 

N 

N 

oil 

water 

P 

= 

P 

oil 

water 

where N oil stands for the number of moles of the oil in the vapor phase and N water the number of 

moles of water in the vapor phase. It is nearly impossible to calculate the proportions as an essential 

oil is a multicomponent mixture of variable composition. 

The simplest method of essential oil extraction is by means of hydrodistillation, for example, by 

immersion of the biomass in boiling water. The plant material soaks up water during the boiling 

process and the oil contained in the oil cells diffuses through the cell walls by means of osmosis. 

Once the oil has diffused out of the oil cells, it is vaporized and carried away by the stream of 

steam. The volatility of the oil constituents is not influenced by the rate of vaporization but does 

depend on the degree of their solubility in water. As a result, the more water-soluble essential components 

will distil over before the more volatile but less water-soluble ones. The usefulness of 

hydrodiffusion can be demonstrated by reference to rose oil. It is well known that occasionally 

some of the essential oil constituents are not present as such in the plant but are artifacts of the 

extraction process. They can be products of either enzymic splitting or chemical degradation, 

occurring during the steam distillation, of high-molecular-weight and thus nonvolatile compounds 

present in the plants. These compounds are often glycosides. The main constituents of rose oil, 

citronellol, geraniol, and nerol are products of a fermentation that takes place during the waterdistillation 

process.


Hydrolysis of esters to alcohols and acids can occur during steam distillation. This can have 

serious implications in the case of ester-rich oils and special precautions have to be taken to prevent 

or at least to limit the extent of ester degradation. The most important examples of this are 

lavender or lavandin oils rich in linalyl acetate and cardamom oil rich in a-terpinyl acetate. 

Chamazulene, a blue bicyclic sesquiterpene, present in the steam-distilled oil of German chamomile, 

Chamomilla recutita (L.) Rauschert, flower heads is an artifact resulting from matricin by a 

complex series of chemical reactions: dehydrogenation, dehydration, and ester hydrolysis. As 

chamazulene is not a particularly stable compound, the deep blue color of the oil can change to 

green and even yellow on aging. 

The design of a water/steam distillation plant at its simplest, sometimes called “false bottom 

apparatus,” is as follows: a still pot (a mild steel drum or similar vessel) is fitted with a perforated 

metal plate or grate, fixed above the intended level of the water, and a lid with a goose neck outlet. 

The lid has to be equipped with a gasket or a water seal to prevent steam leaks. The steam outlet is 

attached to a condenser, for example, a serpentine placed in a drum containing cold water. An oil 

collector (Florentine flask) placed at the bottom end of the serpentine separates the oil from the 

distilled water (Figure 4.9). The whole assembly is fixed on a brick fireplace. A separate water inlet 

is often provided to compensate for water used up during the process. The biomass is placed inside 

the still pot above the perforated metal plate and sufficient biomass should be used to completely fill 

the still pot. The fuel used is firewood. This kind of distillation plant was extensively used at the end 

of the nineteenth century, mainly for field distillations. A disadvantage of this system was that in 

some cases excessive heat imparted a burnt smell to the oils. Furthermore, when the water level in 

the still dropped too much the, plant material could get scorched. Till today there is a necessity to 

clean the distillation vessel after two cycles with water to avoid burning notes in the essential oil. In 

any case, the quality of oils obtained in this type of apparatus was very variable and varied with 

each distillation. A huge improvement to this process was the introduction of steam generated externally. 

The early steam generators were very large and unwieldy and the distillation plant could no 

longer be transported in the field. The biomass had now to be transported to the distillation plant, 

unlike with the original type of distillation plant. Originally, the generator was fuelled with dry, 

FIGURE 4.9 Old distillation apparatus modernized by electric heating.


extracted biomass. Today gas or fuel oil is used. The delivery of steam can be carried out in various 

ways. Most commonly, the steam is led directly into the still through its bottom. Overheating is thus 

avoided and the biomass is heated rapidly. It also allows regulation of steam quantity and pressure 

and reduces distillation time and improves oil quality. In another method, the steam is injected in a 

spiraling motion. This method is more effective as the steam comes into contact with a greater surface 

of the biomass. The velocity of steam throughput and the duration of the distillation depend on 

the nature of the biomass. It can vary from 100 kg/h in the case of seed and fruits to 400 kg/h for 

clary sage. The duration of the distillation can vary from about 20 min for Lavender flowers (Denny, 

1995, personal communication) to 700 min for dried Angelica root. The values quoted are for a 4 m 3 

still pot (Omigbaigi, 2005). Specialists on distillation found as formula that distillation can be 

stopped when the ratio of oil to water coming from condenser will achieve 1:40. In all cases of 

hydrodistillation, the distillation water is recovered and reused for steam generation. In a cohobation, 

the aqueous phase of the distillate is continuously reintroduced into the still pot. In this 

method, any essential oil constituents emulsified or dissolved in the water are captured, thus increasing 

total oil yield. There is one important exception: In the case of rose oil the distillation water is 

collected and redistilled separately in a second step. The “floral water” contains increased amounts 

of b-phenylethyl alcohol, up to 15%, whereas its maximum permissible content in rose oil is 3%. 

The reason for this is its significant solubility in water, ca. 2%. 

The distillation of rose oil is an art in itself as not only quality but quantity as well play an important 

role. It takes two distillation cycles to produce between 200 and 280 g of rose oil. Jean-François Arnodou 

describes its manufacture as follows (Arnodou, 1991): The still pot is loaded with 400 kg of rose petals 

and 1600 L of water. The contents are heated until they boil and steam-distilled. Approximately, the 

quantity of flowers used is then distilled. That action will last about 2–3 h. Specially designed condensers 

are required in order to obtain a good quality. The condensing system comprises a tubular condenser 

followed by a second cooler to allow the oil to separate. The oil is collected in Florentine-type oil separators. 

About 300 L of the oil-saturated still waters are then redistilled in a separate still in order to 

recover most of the oil contained in them. Both oils are mixed together and constitute the rose oil of 

commerce. BIOLANDES described in 1991 the whole process, which uses a microprocessor to manage 

parameters such as pressure and temperature, regulated by servo-controlled pneumatic valves. 

A modern distillation plant consists of the biomass container (still pot), a cooling system (condenser), 

an oil separator, and a high-capacity steam generator. The kettle (still pot) looks like a cylindrical 

vertical storage tank with steam pipes located at the bottom of the still. Perforated sieve-like 

plates are often used to separate the plant charge and prevent compaction, thus allowing the steam 

unimpeded access to the biomass. The outlet for the oil-laden steam is usually incorporated into the 

design of the usually hemispherical, hinged still pot lid. The steam is then passed through the cooling 

system, either a plate heat exchanger or a surface heat exchanger, such as a cold-water condenser. The 

usually liquid condensate is separated into essential oil and distillation water in an appropriate oil 

separator such as a Florentine flask. The distillation water may, in some cases, be redistilled and any 

essential oil recovered dried and stored. Figure 4.10 shows a cross section of such a still. 

The following illustrations show different parts of an essential oil production plant. Figure 4.11 

shows a battery of four production units in the factory. Each still has a capacity of 3000–5000 L. Owing 

to their large size, the upper half of the stills is on the level as shown while the lower half is situated on 

the lower level. Figure 4.12 shows open stills and displays the steam/oil vapor outlets on the underside 

of the lids leading to the cooling units. On the right side of the illustration, one can see the perforated 

plate used to prevent clumping of the biomass. Several such perforated plates, up to 12, depending on 

the type of biomass, are used to prevent clumping. Spacers on the central upright control the optimum 

distance between these plates for improved steam penetration. Figure 4.13 shows the unloading of the 

still. Unloading is much faster than the loading process where the biomass is compacted either manually 

or by means of tractor wheel (Figure 4.14). This type of loading is called “open mouth” loading. 

Figure 4.15 shows the cooling unit. The cold water enters the tank equipped with a coil condenser. The 

cooling water is recycled so that no water is wasted. The two main types of industrially used condensers


Vapor + Oil 

Biomass 

Steam 

Separation bottoms 

Condenser 

Oil 

Back to steam generator 

Florentine flask 

Water 

FIGURE 4.10 Cross section of a hydrodistillation plant. 

are the following. The earliest was the coil condenser that consisted of a coiled tube fixed in an open 

vessel of cold water with cold water entering the tank from the bottom and leaving at the top. The oilrich 

steam is passed through the coils of the condenser from the top end. The second type of condenser 

is the pipe bundle condenser where the steam is passed through several vertical tubes immersed in a 

cold water tank. The tubes have on the inside walls horizontal protuberances that slow down the rate of 

the steam flow and thus result in more effective cooling. Figure 4.16 shows the inside of a Florentine 

flask where the oil is separated from the water. Most essential oils are lighter than water and thus float 

on top of the water. Some essential oils have a specific gravity >1, for example, they are heavier than 

water thus collecting at the bottom of the collection vessel. A modified design of the Florentine flask for 

such oils is shown in Figure 4.17. Figure 4.18 shows oil in the presence of turbid distillation water. The 

liquid phase is contaminated with biomass matter and the oil has to be filtered. The capacity of the still 

pot depends on the biomass. Weights vary from 150 to 650 kg/m 3 . Wilted and dried plants are much 

lighter than seeds and fruits or dried roots that can be very heavy. 

FIGURE 4.11 Battery of four distillation units.


FIGURE 4.12 Open kettle with opening for vapor and oil. 

A very special case is the production of the essential oils of Ylang-Ylang from the fresh flowers of 

Cananga odorata (Lam.) Hook. f. et Thomson forma genuina. The flowers are water distilled, stopped, 

and restarted again. In this manner, a total of four fractions is obtained. The chemical composition of 

the first fraction is characterized by a high concentration of p-cresol methylether, methyl benzoate, 

benzylacetate, linalool, and E-cinnamyl acetate. The second fraction contains less f those volatiles but 

an increased amount of geraniol, geranyl acetate, and b-caryophyllene. The third fraction contains 

higher boiling substances such as germacrene-D, (E,E)-a-farnesene, (E,E)-farnesole, benzyl benzoate, 

(E,E)-farnesyl acetate, and benzyl salicylate. Of course, smaller quantities of the lower boiling 

FIGURE 4.13 Unloading a kettle.


FIGURE 4.14 Loading a kettle and pressing by concreted tractor wheel. 

components are also present. This kind of fractionation has been practiced for a long time. At the same 

time, the whole oil, obtained by a single distillation is available as “Ylang complete.” This serves as 

an example of the importance the duration of the distillation can have on the quality of the oil. 

Raw materials occurring in the form of hard grains have to be comminuted, for example, ground 

up before water distillation. This is carried out in the presence of water, such as in a wet-grinding 

turbine, and the water is used later during the distillation. The stills themselves are equipped with 

blade stirrers ensuring thorough mixing and particularly dislodging oil particles or biomass articles 

sticking to the walls of the still, the consequence of which can be burning and burnt notes. Dry grinding 

is likely to result in a significant loss of volatiles. Pepper, coriander, cardamom, celery seed, and 

angelica seed as well as roots, cumin, caraway, and many other seeds and fruit are treated in this 

manner. The process used in all these cases is called “turbo distillation.” The ratio oil/condensate is 

FIGURE 4.15 Cooling unit.


FIGURE 4.16 Inner part of a Florentine flask. 

very low when this method is used and it is for that reason that turbo distillation uses a fractionating 

column to enrich the volatiles. This also assists in preventing small particles of biomass passing into 

the condenser and contaminating the oil. As in many other distillation and rectification units, 

cold traps are installed to capture any very volatile oil constituents that may be present. This waterdistillation 

procedure is also used for gums such as myrrh, olibanum, opopanax, and benzoin. 

Orris roots are also extracted by water distillation. However, in this case, the distillation has to 

be carried out under conditions of slightly elevated pressure. This is achieved by means of a reflux 

column filled with Raschig rings. This is important as the desired constituents, the irones, exhibit 

very high boiling points. It is noteworthy that in this case there is no cooling of the vapors, as not 

only the irones but also the long-chain hydrocarbons will immediately be transported to the top 

of the column. Figure 4.19 shows a Florentine flask with the condensed oil/water emerging at a 

temperature of nearly 98°C. Orris oil or orris butter (note that the term orris “concrete” is incorrect, 

as the process is not a solvent extraction) is one of the few essential oils that are, at least partly, solid 

at room temperature. Depending on its trans-anethole content rectified star anise oil is another 

example of this nature. 

Water 

Oil 

Oil 

Oil heavier than water 

Water 

Oil lighter than water 

FIGURE 4.17 Two varieties of Florentine flasks.


FIGURE 4.18 (See color insert following page 468.) Oil and muddy water in the Florentine flask. 

A relatively new technique that saves time in loading and unloading of the biomass is the “onsite” 

or “container” distillation. The technique is very simple as the container that is used to pick up 

the biomass and transport it to the distillery serves itself as the still pot. The first plant crops treated 

in this way were peppermint and mint, clary sage, lavandin grosso, L. angustifolia, Eucalyptus 

polybractea, and tea tree. In its simplest form, the mobile still assembly is composed of the following 

components: A tractor is coupled to an agricultural harvester that cuts the plant material and 

delivers it directly into the still pot (or vat) via a chute. The still pot (vat) is permanently fixed onto 

a trailer that is coupled to the harvester. Once the still pot is completely filled, it is towed by the 

tractor into the factory where it is uncoupled and attached to the steam supply and condenser and 

FIGURE 4.19 Orris distillation, Florentine flask at nearly 98°C.


distillation commences. Presupposition for a proper working of the container as vat is a perfect 

insulation. Every loss of steam and heat will guide to worse quality and diminished quantity. Lids 

will have to be placed properly and fixed by clamps. The tractor and harvester are attached to an 

empty still pot and the process is repeated. The design, shape, and size of the still pot as well as the 

type of agricultural harvester depend on the type of plant crop, the size of the plantation, the terrain, 

and so on. The extracted biomass can be used as mulch or, after drying, as fuel for the steam boiler. 

The unloading is automated using metal chains running over the tubes with steam valves. This 

method requires less manpower and thus reduces labor costs. Loading and unloading costs are 

minimal. It lends itself best to fresh biomass, lavender and lavandin, mallee eucalypts, tea tree, and 

so on. It may not be as useful for the harvesting and distillation of mint and peppermint as these 

crops have to be wilted before oil extraction. Figures 4.20 through 4.22 show the harvesting of 

mallee eucalyptus, containers in processing, and the whole site of container distillation. Figure 4.23 

gives a view into the interior of a container. 

Another interesting distillation method has been developed by the LBP Freising, Bavaria, 

Germany. The plant consists of two tubes, each 2 m long and 25 cm in diameter, open at the top. The 

tubes are attached vertically to a central axis that can be rotated. One tube is connected, hydraulically 

or mechanically, to the steam generator and on top to a condenser. During the distillation of 

the contents of the tube, which may take 25–40 min depending on the biomass, the other tube can 

be loaded. When the distillation of the first tube has been completed the tubes are rotated around 

the axis and distillation of the second tube commenced. The first distilled tube can now be emptied 

and reloaded. The only disadvantage of this type of apparatus is the small size. Only 8.5–21 kg of 

biomass can be treated. This system has been developed for farmers intending to produce small 

quantities of essential oil. The apparatus is transportable on a truck and will work satisfactorily 

provided a supply of power is available. 

Most commercially utilized essential oil distillation methods, excepting the mobile still on-site 

methods, suffer from high labor costs. Apart from harvesting the biomass, 3–4 laborers will be 

required to load and unload the distillation pots, regulating steam pressure and temperature, and so 

on. The loading and distribution of the biomass in the distillation vessel may not be homogeneous. 

This will adversely affect the steam flow through the biomass by channeling, for example, the steam 

passing through less compacted areas and thus not reaching other more compacted areas. This will 

result in lower oil yields and perhaps even alter the composition of the oil. In times of high energy 

FIGURE 4.20 Harvesting blue mallee with distillation container.


FIGURE 4.21 A battery of containers to be distilled, one opened to show the biomass. 

costs the need for consequent recovery has to be considered. Given the demand for greater quantities 

of essential oils, the question is how to achieve it and at the same time improve the quality of 

the oils. For this, several considerations have to be taken into account. The first is how to process 

large quantities of biomass in a given time? Manpower has to be decreased as it still is the most 

important factor affecting costs. The biomass as a whole has to be treated uniformly to ensure 

higher oil yields and more constant and thus better oil composition. How can energy costs and water 

requirements be reduced in an ecologically acceptable way? The answer to this was the development 

of continuous distillation during the last years of the twentieth century. Until then all distillation 

processes were discontinuous. Stills had to be loaded, the distillation stopped, and stills 

unloaded. The idea was to develop a process where the steam production was continuous with permanent 

unchanged parameters. This was achieved by the introduction of an endless screw that fed 

the plant material slowly into the still pot from the top and removed the exhausted plant material 

from the bottom at the same speed. The plant material moves against the flow of dry steam entering 

the still from the bottom. In this fashion, all of the biomass comes into contact with the steam ensuring 

optimum essential oil extraction. 

FIGURE 4.22 Distillation plant with container technique.


FIGURE 4.23 View inside the distillation container, showing the steam tubes and the metallic grid. 

The earliest of these methods is known as the “Padova System.” It consists of a still pot 6 m high 

and about 1.6 m in diameter (Arnodou, 1991). Its total volume is about 8 m 3 . The feeding of the still 

with the plant biomass as well as its subsequent removal is a continuous process. The plant material 

is delivered via a feed hopper situated at the top of the still. Before entering the still, it is compressed 

and cut by a rotating knife to ensure a more uniform size. Finally, a horizontal moving cone 

regulates the quantity of biomass entering the still. The biomass that enters the still moves in the 

opposite direction to that of the steam. The steam saturated with essential oil vapors is then passed 

into the cooling system. The exhausted plant material is simultaneously removed by means of an 

Archimedes screw. This type of plant was originally designed for the distillation of wine residues. 

A different system is provided by the distillation chimie fine (DCF) aroma process continuous distillation. 

Once again the plant material is delivered via a hopper to several interconnected tubes. 

These tubes are slightly inclined and connected to each other. The biomass is carried slowly through 

the tubes, by means of a worm screw, in a downward direction. Steam is injected at the end of the 

last tube and is directed upward in the opposite direction to that of the movement of the plant material. 

The essential oil-laden steam is deflected near the point of entrance of the biomass, into the 

condenser. The exhausted plant material is unloaded by another worm screw located near the point 

of the steam entrance to the system. 

Texarome, a big producer of cedarwood oil and related products holds a patent on another continuous 

distillation system. In contrast to other systems, the biomass is conveyed pneumatically 

within the system. It is a novel system spiked with new technology of that time. Texan cedarwood 

oil is produced from the whole tree, branches, roots, and stumps. Cedarwood used in Virginia uses 

exclusively branches, stumps, saw dust, and other waste for oil production; wood is used mainly 

for furniture making. The wood is passed through a chipper and then through a hammer mill. 

The dust is collected by means of a cyclone. Any coarse dust is reground to the desired size. The 

dust is now carried via a plug feeder to the first contactor where superheated steam in reverse flow


exhausts it in a first step and following that in a similar second step at the next contactor. The steam 

and oil vapors are carried into a condenser. The liquid distillate is then separated in Florentine 

flasks. This process does all transport entirely by pneumatic means. The recycling of cooling water 

and the use of the dried plant matter as a fuel contribute to environmental requirements (Arnodou, 

1991). In the 1990s, the BIOLANDES Company designed its own system of continuous distillation. 

The reason for this was BIOLANDES’ engagement in the forests of south-western France. 

Between Bordeaux and Biarritz exists the most important area of pine trees (Pinus pinaster Sol.) 

supplying the paper industry. Twigs and needles have been burnt or left to rot to assist with reforestation 

with new trees. These needles contain a fine essential oil very similar to that of the dwarf 

pine oil (Pinus mugo Turra.). Compared to other needle oils dwarf pine oil is very expensive and 

greatly appreciated. The oil was produced by a discontinuous distillation but as demand rose, new 

and improved methods were required. First of all, the collection of the branches had to be improved. 

A tractor equipped with a crude grinder and a ground wood storage box follows the wood and 

branch cutters and transports it to the nearby distillation unit where the biomass is exhausted via a 

continuous distillation process. In contrast to the earlier described methods the BIOLANDES 

continuous distillation process operates somewhat differently. The plant material is carried by 

mechanical means from the storage to the fine cutter and via an Archimedes screw to the top of the 

distillation pot. The plant material is now compressed by another vertical screw and transported 

into a chamber which is then hermetically closed on its back but opening at the front. Biomass is 

falling down allowing the countercurrent passage of hot steam through it. The steam is supplied 

through numerous nozzles. Endless screws at the bottom of the still continuously dispose of the 

exhausted biomass. Oil-laden steam is channeled from the top of the still into condenser and then 

the oil separator. 

It is well known that clary sage yields an essential oil on hydrodistillation. However, a very 

important component of this oil, sclareole, is usually recovered in only very small quantity when 

this method is used, the reason for this being its very high boiling point. Sclareole can be recovered 

in very high yield and quality by extraction with volatile solvents. Consequently, BIOLANDES has 

incorporated an extraction step in its system (Figure 4.24). Any waste biomass, whether of extracted or 

nonextracted material, is used for energy production or, mostly, for composting. The energy recovery 

management distinguishes this system from all other earlier described processes. In all of the latter, 

FIGURE 4.24 Scheme of the BIOLANDES continuous production unit. A: biomass; B: distillation vat; 

C: condenser; D: Florentine flask; E: extraction unit; F: solvent recovery; G: exhausted biomass.


large amounts of cold water are required to condense the essential oil-laden steam. This results in 

significant wastage of water as well as in latent energy losses. The BIOLANDES system recovers 

this latent heat. Hot water from the condenser is carried into an aerodynamic radiator. Air used as 

the transfer gas takes up the energy of the hot water, cooling it down, so that it can be recycled to 

the condenser. The hot air is then used to dry about one-third of the biomass waste that is used as 

an energy source for steam and even electricity production. In other words, this system is energetically 

self-sufficient. Furthermore, since it is fully automated, it results in constant quality products. 

A unit comprising two stills of 7.5 m 3 capacity can treat per hour 3 ton of pine needles, 1.5 ton of 

juniper branches, and 0.25 ton of cistus branches (Arnodou, 1991). The advantages are once 

again short processing time of large amounts of biomass, reduced labor costs, and near complete 

energy sufficiency. All operations are automated and water consumption is reduced to a minimum. 

The system can also operate under slight pressure thus improving the recovery of higher boiling 

oil constituents. 

The following is a controversial method for essential oil extraction by comparison with classical 

hydrodistillation methods. In this method, the steam enters the distillation chamber from the top 

passes through the biomass in the still pot (e.g., the distillation chamber) and percolates into the 

condenser located below it. Separation of the oil from the aqueous phase occurs in a battery of 

Florentine flasks. It is claimed that this method is very gentle and thus suitable for the treatment of 

sensitive plants. The biomass is held in the still chamber (e.g., still pot) on a grid that allows easy 

disposal of the spent plant matter at the completion of the distillation. The whole apparatus is relatively 

small, distillation times are reduced, and there is less chance of the oil being overheated. It 

appears that this method is fairly costly and thus likely to be used only for very high-priced 

biomass. 

Recently, microwave-assisted hydrodistillation methods have been developed, so far mainly in 

the laboratory or only for small-scale projects. Glass vessels filled with biomass, mainly herbs and 

fruits or seeds, are heated by microwave power. By controlling the temperature at the center of the 

vessel, dry heat conditions are established at about 100°C. As the plant material contains enough 

water, the volatiles are evaporated together with the steam solely generated by the microwave heat 

and can be collected in a suitably designed condenser/cooling system. In this case, changes in the 

composition of the oil will be less pronounced than in oil obtained by conventional hydrodistillation. 

This method has attracted interest owing to the mild heat to which the plant matter is exposed. 

Kosar reported improvements in the quality of microwave extracted fennel oil due to increases in 

the yields of its oxygenated components (Kosar et al., 2007). 

Very different products can result from the dry distillation of plant matter. ISO Standard 9235 

specifies in Section 3.1.4 that products of dry distillation, for example, “… obtained by distillation 

without added water or steam” are in fact essential oils (ISO/DIS 9235.2, 1997, p. 2). Dry distillation 

involves heating in the absence of aerial oxygen, normally in a closed vessel, preventing combustion. 

The plant material is thus decomposed to new chemical substances. Birch tar from the wood 

exsudate of Betula pendula Roth. and cade oil from the wood of Juniperus oxycedrus L. are manufactured 

in this way. Both oils contain phenols, some of which are recognized carcinogens. For this 

reason, the production of these two oils is no longer of any commercial importance, though very 

highly rectified and almost phenol-free cade oils do exist. 

Some essential oils require rectification. This involves redistillation of the crude oil in order to 

remove certain undesirable impurities, such as very small amounts of constituents of very low volatility, 

carried over during the steam or water distillation (such as high-molecular-weight phenols, 

leaf wax components, etc.) as well as small amounts of very volatile compounds exhibiting an undesirable 

odor, and thus affecting the top note of the oils, such as sulfur compounds (dimethyl sulfide 

present in crude peppermint oil), isovaleric aldehyde (present in E. globulus oil), and certain nitrogenous 

compounds (low-boiling amines, etc.). In some cases, rectification can also be used to enrich 

the essential oil in a particular component such as 1,8-cineole in low-grade eucalyptus oil. 

Rectification is usually carried out by redistillation under vacuum to avoid overheating and thus


partial decomposition of the oil’s constituents. It can also be carried out by steaming. Commonly 

rectified oils include eucalyptus, clove, mint, turpentine, peppermint, and patchouli. In the case of 

patchouli and clove oils, rectification improves their, often unacceptably, dark color. 

Fractionation of essential oils on a commercial scale is carried out in order to isolate fractions 

containing a particular compound in very major proportions and occasionally even individual 

essential oil constituents in a pure state. In order to achieve the required separation, fractionations 

are conducted under reduced pressure (e.g., under vacuum) to prevent thermal decomposition of 

the oil constituents, using efficient fractionating columns. A number of different types of fractionating 

columns are known but the one most commonly used in laboratory stills or small commercial 

stills is a glass or stainless steel column filled with Raschig rings. Raschig rings are short, 

narrow diameter, rings made of glass, or any other chemically inert material. Examples of compounds 

produced on a commercial scale are citral (a mixture of geranial and neral) from Litsea 

cubeba, 1,8-cineole from eucalyptus oil (mainly Eucalyptus polybractea and other cineole-rich 

species) as well as from Cinnamomum camphora oil, eugenol from clove leaf oil, a-pinene from 

turpentine, citronellal from citronella oil, linalool from Ho-oil, geraniol from palmarosa oil, and 

so on. A small-scale high vacuum plant used for citral production is shown in Figure 4.25. The 

reflux ratio, for example, the amount of distillate collected and the amount of distillate returned 

to the still, controls equilibrium conditions of the vapors near the top of the fractionation column, 

which are essential for good separation of the oil constituents. 

FIGURE 4.25 High vacuum rectification plant in small scale. The distillation assembly is composed of a 

distillation vessel (1) of glass, placed in an electric heating collar (2). The vessel is surmounted by a jacketed 

fractionation column (3), packed with glass spirals or Raschig rings, of such a height as to achieve maximum 

efficiency (e.g., have the maximum number of theoretical plates capable of being achieved for this type of 

apparatus). The reflux ratio is automatically regulated by a device (4), which also includes the head condenser 

(5), a glass tube leads the product to another condenser (6), from there to the both receivers (7). The vacuum 

pump unit is placed on the right (8).


Apart from employing fractional distillation, with or without the application of a vacuum, some 

essential oil constituents are also obtained on a commercial scale by freezing out from the essential 

oil, followed by centrifugation at below freezing point of the desired product. Examples are menthol 

from Mentha species (this is usually further purified by recrystallization from a suitable solvent, 

trans-anethole from anise oil, star anise oil, and particularly fennel seed oil and 1,8-cineole from 

cineole-rich eucalyptus oils. 

Most essential oils are complex mixtures of terpenic and sesquiterpenic hydrocarbons, and their 

oxygenated terpenoid and sesquiterpenoid derivatives (alcohols, aldehydes, ketones, esters, and 

occasionally carboxylic acids), as well as aromatic (benzenoid) compounds such as phenols, phenolic 

ethers, and aromatic esters. So-called “terpene-less” and “sesquiterpene-less essential oils are 

commonly used in the flavor industry. Many terpenes are bitter in taste and many, particularly the 

terpenic hydrocarbons, are poorly soluble or even completely insoluble in water–ethanol mixtures. 

Since the hydrocarbons rarely contribute anything of importance to their flavoring properties, their 

removal is a commercial necessity. They are removed by the so-called “washing process,” a method 

used mostly for the treatment of citrus oils. This process takes advantage of the different polarities 

of individual essential oil constituents. The essential oil is added to a carefully selected solvent (usually 

a water–ethanol solution) and the mixture partitioned by prolonged stirring. This removes some 

of the more polar oil constituents into the water–ethanol phase (e.g., the solvent phase). Since a 

single partitioning step is not sufficient to effect complete separation, the whole process has to be 

repeated several times. The water–ethanol fractions are combined and the solvent removed. The 

residue contains now very much reduced amounts of hydrocarbons but has been greatly enriched 

in the desired polar oxygenated flavor constituents, aldehydes such as octanal, nonanal, decanal, 

hexenal, geranial, and neral; alcohols such as nerol, geraniol, and terpinen-4-ol; oxides such as 

1,8-cineole and 1,4-cineole; as well as esters and sometimes carboxylic acids. Apart from water– 

ethanol mixtures, hexane or light petroleum fractions (sometimes called “petroleum ether”) have 

sometimes also been added as they will enhance the separation process. However, these are highly 

flammable liquids and care has to be taken in their use. 

“Folded” or “concentrated” oils are citrus oils from which some of the undesirable components 

(usually limonene) have been removed by high vacuum distillation. In order to avoid thermal degradation 

of the oil, temperatures have to be kept as low as possible. Occasionally, a solvent is used as 

a “towing” agent to keep the temperature low. 

Another, more complex, method for the concentration of citrus oils is a chromatographic separation 

using packed columns. This method allows a complete elimination of the unwanted hydrocarbons. 

This method, invented by Erich Ziegler, uses columns packed with either silica or aluminum 

oxide. The oil is introduced onto the column and the hydrocarbons eluted by means of a suitable 

nonpolar solvent of very low boiling point. The desirable polar citrus oil components are then 

washed out using a polar solvent (Ziegler, 1982). 

Yet another valuable flavor product of citrus fruits is the “essence oil.” The favored method for 

the transport of citrus juice is in the form of a frozen juice concentrate. The fruit juice is partly 

dehydrated by distilling off under vacuum the greater part of the water and frozen. Distilling off the 

water results in significant losses of the desirable volatiles responsible for the aroma of the fruit. 

These volatiles are captured in several cold traps and constitute the “aqueous essence” or “essence 

oil” that has the typical fruity and fresh fragrance, but slightly less aldehydic than that of the oil. 

This oil is used to enhance the flavor of the reconstituted juice obtained by thawing and dilution 

with water of the frozen concentrate. 

Producing essential oils today is, from a marketing point of view, a complex matter. As in the 

field of other finished products, the requirements of the buyer or producer of the consumer product 

must be fulfilled. The evaluation of commercial aspects of essential oil production is not an easy 

task and requires careful consideration. There is no sense in producing oils in oversupply. Areas of 

short supply, depending on climatic or political circumstances should be identified and acted 

upon. As in other industries global trends are an important tool and should continually be monitored.


For example, which are the essential oils that cannot be replaced by synthetic substitutes such as 

patchouli oil or blue chamomile oils? A solution to this problem can lie in the breeding of suitable 

plants. For example, a producer of a new kind “pastis,” the traditional aperitif of France, wants to 

introduce a new flavor with a rosy note in the fennel component of the flavor. This will require the 

study and identification of oil constituents with “rosy” notes and help biologists to create new botanical 

varieties by genetic crossing, for example, by genetic manipulation, of suitable target plant 

species. Any new lines will first be tested in the laboratory and then in field trials. Test distillations 

will be carried out and the chemical composition of the oils determined. Agronomists and farmers 

will be involved in all agricultural aspects of the projects: soil research and harvesting techniques. 

Variability of all physicochemical aspects of the new strains will be evaluated. At this point, the 

new types of essential oils will be presented to the client. If the client is satisfied with the quality 

of the oils, the first larger plantations shall be established and consumer market research will be 

initiated. If everything has gone to plan, that is all technical problems have been successfully 

resolved and the finished product has met with the approval of the consumers, large-scale production 

can begin. This example describes the current way of satisfying customer demands. 

Global demand for essential oils is on the increase. This also generates some serious problems 

for which immediate solutions may not easily be found. The first problem is the higher demand for 

certain essential oils by some of the world’s very major producers of cosmetics. They sometimes 

contract oil quantities that can be of the order of 70% of world production. This will not only raise 

the price but also restrict consumer access to certain products. From this arises another problem. 

Our market is to some extent a market of copycats. How can one formulate the fragrance of a competitor’s 

product without having access to the particular essential oil used by him, particularly as 

this oil may have other functions than just being a fragrance, such as, for example, certain physiological 

effects on both the body as well as the mind? Lavender oil from L. angustifolia is a calming 

agent as well as possessing anti-inflammatory activity. No similar or equivalent natural essential oil 

capable of replacing it is known. Another problem affecting the large global players is ensuring the 

continuing availability of raw material of the required quality needed to satisfy market demand. 

This is clearly an almost impossible demand as nobody can assure that climatic conditions required 

for optimum growth of a particular essential oil crop will remain unchanged. Another problem may 

be the farmer himself. Sometimes it may be financially more worthwhile for the farmer to cultivate 

other than essential oil plant crops. All these factors may have some detrimental effects on the availability 

of essential oils. Man’s responsibility for the continued health of the environment may also 

be one of the reasons for the disappearance of an essential oil from the market. Sandalwood (species 

of Santalum, but mainly Indian Santalum album) requires in some cases up to 100 years to regenerate 

to a point where they are large enough to be harvested. This and their uses in religious ceremonies 

have resulted in significant shortages of Indian oil. Owing to the large monetary value of Indian 

sandalwood oil, indiscriminate cutting of the wood has just about entirely eliminated it from native 

forests in Timor (Indonesia). Sandalwood oils of other origins are available, Santalum spicatum 

from western Australia, and Santalum austrocaledonicum from New Caledonia and Vanuatu. 

However, their wood oils differ somewhat in odor as well as in chemical composition from genuine 

Indian oils. 

Some essential oils are disappearing from the market owing to the hazardous components they 

contain and are, therefore, banned from most applications in cosmetics and detergents. These oil 

components, all of which are labeled as being carcinogenic, include safrole, asarone, methyleugenol, 

and elemicin. Plant diseases are another reason for essential oil shortages as they, too, can be 

affected by a multitude of diseases, some cancerous, which can completely destroy the total crop. 

For example, French lavender is known to suffer from a condition whereby a particular protein 

causes a decrease in the growth of the lavender plants. This process could only be slowed down by 

cultivation at higher altitudes. In the middle of the twentieth century lavender has been cultivated in 

the Rhône valley at an altitude of 120 m. Today lavender is growing only at altitudes around 800 m. 

The growing shoots of lavender plants are attacked by various pests, in particular the larvae of


Cécidomye (Thomasissiana lavandula) which, if unchecked, will defoliate the plants and kill them. 

Some microorganisms such as Mycoplasma and a fungus Armillaria mellex can cause serious 

damage to plantations. At the present time, the use of herbicides and pesticides is an unavoidable 

necessity. Wild-growing plants are equally prone to attack by insect pests and plant diseases. 

The progression from wild-growing plants to essential oil production is an environmental problem. 

In some developing countries, damage to the natural balance can be traced back to overexploitation 

of wild-growing plants. Some of these plants are protected worldwide and their collection, 

processing, and illegal trading are punishable by law. In some Asian countries, such as in Vietnam, 

collection from the wild is state controlled and limited to quantities of biomass accruing from 

natural regeneration. 

The state of technical development of the production in the developing countries is very variable 

and depends largely on the geographical zone they are located in. Areas of particular relevance are 

Asia, Africa, South America, and eastern Europe. As a rule, the poorer the country, the more 

traditional and less technologically sophisticated equipment is used. Generally, standards of the 

distillation apparatus are those of the 1980s. At that time, the distillation equipment was provided 

and installed by foreign aid programs with European and American know-how. Most of these units 

are still in existence and, owing to repairs and improvements by local people, in good working 

order. Occasionally, primitive equipment has been locally developed, particularly when the state did 

not provide any financial assistance. Initially, all mastery and expertise of distillation techniques 

came from Europe, mainly from France. Later on, that knowledge was acquired and transferred to 

their countries by local people who had studied in Europe. They are no longer dependent on foreign 

know-how and able to produce oils of constant quality. Conventional hydrodistillation is still the 

main essential oil extraction method used, one exception being hydrodiffusion often used in Central 

America, mainly Guatemala and El Salvador, and Brazil in South America. The construction of the 

equipment is carried out in the country itself and makes the producer independent from higherpriced 

imports. Steam is generated by oil-burning generators only in the vicinity of cities. In country 

areas, wood or dried spent biomass is used. As in all other essential oil-producing countries, the 

distillation plants are close to the cultivation areas. Wild-growing plants are collected, provided the 

infrastructure exists for their transport to the distillation plant. For certain specific products permanent 

fixed distillation plants are used. A forward leap in the technology will be only possible if 

sufficient investment funds became available in the future. Essential oil quantities produced in those 

countries are not small and important specialities such as citral-rich ginger oil from Ecuador play a 

role on the world market. It should be a compulsory requirement that developing countries treat 

their wild-growing plant resources with the utmost care. Harvesting has to be controlled to avoid 

their disappearance from the natural environment and quantities taken adjusted to the ability of the 

environment to spontaneously regenerate. On the other hand, cultivation will have to be handled 

with equal care. The avoidance of monoculture will prevent leaching the soil of its nutrients and 

guard the environment from possible insect propagation. Balanced agricultural practices will lead 

to a healthy environment and superior quality plants for the production of essential oils. 

The following are some pertinent remarks on the now prevailing views of “green culture” and 

“organically” grown plants for essential oil production. It is unjustified to suggest that such products 

are of better quality or greater activity. Comparisons of chemical analyses of “bio-oils,” for 

example, oils from “organically” grown plants, and commercially produced oils show absolutely no 

differences, qualitative or quantitative, between them. While the concept of pesticide- and fertilizerfree 

agriculture is desirable and should be supported, the huge worldwide consumption of essential 

oils could never be satisfied by bio-oils. 

Finally, some remarks as to the concept of honesty are attached to the production of natural 

essential oils. During the last 30 years or so, adulteration of essential oils could be found every day. 

During the early days, cheap fatty oils (e.g., peanut oil) were used to cut essential oils. Such adulterations 

were easily revealed by means of placing a drop of the oil on filter paper and allowing it to 

evaporate (Karg, 1981). While an unadulterated essential oil will evaporate completely or at worst


leave only a trace of nonvolatile residue, a greasy patch indicates the presence of a fatty adulterant. 

As synthetic components of essential oils became available around the turn of the twentieth century 

some lavender and lavandin oils have been adulterated by the addition of synthetic linalool and 

linalyl acetate to the stills before commencing the distillation of the plant material. With the advent 

of improved analytical methods, such as GC and GC/MS, techniques of adulterating essential oil 

were also refined. Lavender oil can again serve as an example. Oils distilled from mixtures of lavender 

and lavandin flowers mimicked the properties of genuine good-quality lavender oils. However, 

with the introduction of chiral GC techniques, such adulterations were easily identified and the 

genuineness of the oils guaranteed. This also allowed the verification of the enantiomeric distribution 

of monoterpenes, monoterpenoid alcohols, and esters present in essential oils. Nuclear magnetic 

resonance (NMR) is probably one of the best, but also one of the most expensive, methods available 

for the authentication of naturalness and will be cost effective only with large batch quantities or in 

the case of very expensive oils. In the future, two-dimensional GC (GC/GC) will provide the next 

step for the control of naturalness of essential oils. 

Another important aspect is the correct botanical source of the essential oil. This can perhaps 

best be discussed with reference to eucalyptus oil of the 1,8-cineole type. Originally, before commercial 

eucalyptus oil production commenced in Australia, eucalyptus oil was distilled mainly 

from E. globulus Labill. trees introduced into Europe [mainly Portugal and Spain (ISO Standard 

770)]. It should be noted that this species exists in several subspecies: E. globulus subsp. bicostata 

(Maiden, Blakely, & J. Simm.) Kirkpatr., E. globulus Labill. subsp. globulus., E. globulus subsp. 

pseudoglobulus (Naudin ex Maiden) Kirkpatr., and E. globulus subsp. maidenii (F. Muell.) Kirkpatr. 

It has been shown that the European oils were in fact mixed oils of some of these subspecies and of 

their hybrids (report by H. H. G. McKern of ISO/TC 54 meeting held in Portugal in 1966). The 

European Pharmacopoeia Monograph 0390 defines eucalyptus oil as the oil obtained from E. globulus 

Labill., Eucalyptus fruticetorum F. von Mueller Syn. Eucalyptus polybractea R. T. Baker (this 

is the correct botanical name), Eucalyptus smithii R. T. Baker, and other species of Eucalyptus rich 

in 1,8-cineole. The Council of Europe’s book Plants in cosmetics, Vol. 1, page 127 confuses the 

matter even further. It entitles the monograph as E. globulus Labill. et al. species, for example, 

includes any number of unnamed Eucalyptus species. The Pharmacopoeia of the Peoples Republic 

of China (English Version, Vol. 1) 1997 goes even further defining eucalyptus oil as the oil obtained 

from E. globulus Labill. and Cinnamomum camphora as well as from other plants of those two 

families. ISO Standard 3065—Oil of Australian eucalyptus, 80–85% cineole content, simply mentions 

that the oil is distilled from the appropriate species. The foregoing passage simply shows 

that Eucalyptus oil does not necessarily have to be distilled from a single species of Eucalyptus, 

for example, E. globulus, although suggesting that it is admissible to include 1,8-cineole-rich 

Cinna momum oils is incorrect and unrealistic. This kind of problem is not unusual or unique. For 

example, the so-called English lavender oil, considered by many to derive from L. angustifolia, is 

really, in the majority of cases, the hybrid lavandin (Denny, 1995, personal communication). 

Another pertinent point is how much twig and leaf material can be used in juniper berry oil? In 

Indonesia, it is common practice to space individual layers of patchouli leaves in the distillation 

vessel with twigs of the gurjun tree. Gurjun balsam present in the twigs contains an essential oil that 

contaminates the patchouli oil. Can this be considered to constitute an adulteration or simply a tool 

required for the production of the oil? 

4.1.16 CONCLUDING REMARKS 

As mentioned at the beginning, essential oils do have a future. In spite of regulatory limitations, dangerous 

substance regulations, and dermatological concerns as well as problems with pricing the world 

production of essentials oil will increase. Essential oils are used in a very large variety of fields. They 

are an integral constituent of fragrances used in perfumes and cosmetics of all kinds, skin softeners to 

shower gels and body lotions, and even to “aromatherapy horse care massage oils.” They are widely


used in the ever-expanding areas of aromatherapy or, better, aromachology. Very large quantities of 

natural essential oils are used by the food and flavor industries for the flavoring of smallgoods, fast 

foods, ice creams, beverages, both alcoholic as well as nonalcoholic soft drinks, and so on. Their 

medicinal properties have been known for many years and even centuries. Some possess antibacterial 

or antifungal activity while others may assist with the digestion of food. However, as they are multicomponent 

mixtures of somewhat variable composition, the medicinal use of whole oils has contracted 

somewhat, the reason being that single essential oil constituents were easier to test for effectiveness 

and eventual side effects. Despite all that, the use of essential oils is still “number one” on the natural 

healing scene. With rising health care and medicine costs, self-medication is on the increase and with 

it a corresponding increase in the consumption of essential oils. Parallel to this, the increase in various 

esoteric movements is giving rise to further demands for pure natural essential oils. 

In the field of agriculture, attempts are being made at the identification of ecologically more 

friendly natural biocides, including essential oils, to replace synthetic pesticides and herbicides. 

Essential oils are also used to improve the appetite of farm animals, leading to more rapid increases 

in body weight as well as to improved digestion. 

Finally, some very cheap essential oils or oil components such as limonene, 1,8-cineole, and the 

pinenes are useful as industrial solvents while phellandrene-rich eucalyptus oil fractions are marketed 

as industrial perfumes for detergents and the like. 

In conclusion, a “golden future” can be predicted for that useful natural product: the “Essential 

Oil”! 

ACKNOWLEDGMENTS 

The author thanks first of all Dr. Erich Lassak for his tremendous support, for so many details information, 

and also for some pictures. Klaus Dürbeck for some information about production of essential 

oils in development countries, Dr. Tilmann Miritz, Miritz Citrus Ingredients for Figures 4.4 

through 4.6, Bernhard Mirwald for Figure 4.9, and Tim Denny from Bridgestowe Estate, Lilydale, 

Tasmania for the Figures 4.20 through 4.23. 

REFERENCES 

25th International Symposium on Essential Oils, Aromatherapy Research: Studies on the Biological Effects of 

Fragrance Compounds and Essential Oils upon Inhalation, Grasse, France, 1994. 

Arnodou, J.F., 1991. The taste of nature; industrial methods of natural products extraction. Presented at a 

conference organized by the Royal Society of Chemistry in Canterbury, 16–19 July 1991. 

Court, W.A., R.C. Roy, R. Pocs, A.F. More, and P.H. White, 1993. Effects of harvest date on the yield and quality 

of the essential oil of peppermint. Can. J. Plant. Sci., 73: 815–824. 

Denny, T., 1995. Bridestowe estates. Tasmania, Private information to the author. 

Dey, D., Alberta government, Agriculture and Food. Available at http://www1.agric.gov.ab.ca/$department/ 

deptdocs.nsf/all/agdex122, July 2007. 

Figueiredo, A.C., J.G. Barroso, L.G. Pedro, and J.J.C. Scheffer, 2005. Physiological aspects of essential oil production. 

Plant Sci., 169(6): 1112–1117. 

Gildemeister, E., and F. Hoffmann, 1931. Die Ätherische Öle. Miltitz: Verlag Schimmel & Co. 

ISO/DIS 4731: 2005. Oil of Geranium. Geneva: International Standard Organisation. 

ISO/DIS 9235.2: 1997. Aromatic Natural Raw Materials—Vocabulary. Geneva: International Standard 

Organisation. 

Kamatou, G.P.P., R.L. van Zyl, S.F. van Vuuren, A.M. Viljoen, A.C. Figueiredo, J.G. Barroso, L.G. Pedro, and 

P.M. Tilney, 2006. Chemical composition, leaf trichome types and biological activities of the essential 

oils of four related salvia species indigenous to Southern Africa. J. Essent. Oil Res., 18 (Special edition): 

72–79. 

Karg, J.E., 1981. Das Geschäft mit ätherischen Ölen. Kosmetika, Aerosole, Riechstoffe, 54: 121–124. 

Koll, N. and W. Kowalczyk, 1957. Fachkunde der Parfümerie und Kosmetik. Leipzig: Fachbuchverlag.


Kosar, M., T. Özek, M. Kürkcüoglu, and K.H.C. Baser, 2007. Comparison of microwave-assisted hydrodistillation 

and hydrodistillation methods for the fruit essential oils of Foeniculum vulgare. J. Essent. Oil Res., 

19: 426–429. 

Letchamo, W., R. Marquard, J. Hölzl, and A. Gosselin, 1994. The selection of Thymus vulgaris cultivars to 

grow in Canada. Angewandte Botanik, 68: 83–88. 

Levey, M. 1955. Evidences of ancient distillation, sublimation and extraction in Mesopotamia. Centaurus, 

Freiburg, 4(1): 23–33. 

Levey, M. 1959. Chemistry and Chemical Technology in Ancient Mesopotamia. Amsterdam: Elsevier. 

Meunier, C., 1985. Lavandes & Lavandins. Aix-en-Provence: ÉDISUD. 

Nguyen, T.P.T., Nguyen, T.T., Tran, M.H., Tran, H.T., Muselli, A., Bighelli, A., Castola, V., and Casanova, J., 

2004. Artemisia vulgaris L. from Vietnam, chemical variability and composition of the oil along the 

vegetative life of the plant. J. Essent. Oil Res., 16: 358–361. 

Novak, J., 2005. Lecture held on the 35th Int. Symp. on Essential Oils, Giardini Naxos, Sicily. 

Omidbaigi, R., 2005. Processing of essential oil plants. In Processing, Analysis and Application of Essential 

Oils. Har Krishan Bhalla & Sons, Dehradun, India. 

Perfumer & Flavorist, 2009. A preliminary report on the world production of some selected essential oils and 

countries, Vol. 34, January 2009. 

Porter, N., 2001. Crop and Food Research. Crop & Foodwatch Research, Christchurch, No. 39, October. 

Reeve, D., 2005. A cultivated zest, Perf. Flav. 30 (3): 32–35. 

Rovesti, P., 1977. Die Destillation ist 5000 Jahre alt. Dragoco Rep., 3: 49–62. 

Yanive, Z., and D. Palevitch, 1982. Effect of drought on the secondary metabolites of medicinal and aromatic 

plants. In: Cultivation and Utilization of Medicinal Plants, C.V. Atal and B.M. Kapur (eds). CSIR Jammu 

Tawi, India. 

Zahn, J., 1979. Nichts neues mehr seit Babylon. Hamburg: Hoffmann und Campe. 

Ziegler, E., 1982. Die natürlichen und künstlichen Aromen, pp. 187–188. Heidelberg: Alfred Hüthig Verlag.

5 

Chemistry of Essential Oils 

Charles Sell 

CONTENTS 

5.1 Introduction ....................................................................................................................... 121 

5.2 Basic Biosynthetic Pathways ............................................................................................. 121 

5.3 Polyketides and Lipids ....................................................................................................... 123 

5.4 Shikimic Acid Derivatives ................................................................................................ 126 

5.5 Terpenoids ......................................................................................................................... 129 

5.5.1 Hemiterpenoids ..................................................................................................... 131 

5.5.2 Monoterpenoids ..................................................................................................... 131 

5.5.3 Sesquiterpenoids .................................................................................................... 135 

5.6 Synthesis of Essential Oil Components ............................................................................. 140 

References .................................................................................................................................. 149 


The term “essential oil” is a contraction of the original “quintessential oil.” This stems from the 

Aristotelian idea that matter is composed of four elements, namely, fire, air, earth, and water. The 

fifth element, or quintessence, was then considered to be spirit or life force. Distillation and evaporation 

were thought to be processes of removing the spirit from the plant and this is also reflected 

in our language since the term “spirits” is used to describe distilled alcoholic beverages such as 

brandy, whiskey, and eau de vie. The last of these again shows reference to the concept of removing 

the life force from the plant. Nowadays, of course, we know that, far from being spirit, essential oils 

are physical in nature and composed of complex mixtures of chemicals. One thing that we do see 

from the ancient concepts is that the chemical components of essential oils must be volatile since 

they are removed by distillation. In order to have boiling points low enough to enable distillation, 

and atmospheric pressure steam distillation in particular, the essential oil components need to have 

molecular weights below 300 Daltons (molecular mass relative to hydrogen = 1) and are usually 

fairly hydrophobic. Within these constraints, nature has provided an amazingly rich and diverse 

range of chemicals (Hay and Waterman, 1993; Lawrence, 1985) but there are patterns of molecular structure 

that give clues to how the molecules were constructed. These synthetic pathways have now been 

confirmed by experiment and will serve to provide a structure for the contents of this chapter. 

5.2 BASIC BIOSYNTHETIC PATHWAYS 

The chemicals produced by nature can be classified into two main groups. The primary metabolites 

are those that are universal across the plant and animal family and constitute the basic building 

blocks of life. The four subgroups of primary metabolites are proteins, carbohydrates, nucleic acids, 

and lipids. These families of chemicals contribute little to essential oils although some essential oil 

components are degradation products of one of these groups, lipids being the most significant. The 

121


secondary metabolites are those that occur in some species and not others and they are usually classified 

into terpenoids, shikimates, polyketides, and alkaloids. The most important as far as essential 

oils are concerned are the terpenoids and the shikimates are the second. There are a number of 

polyketides of importance in essential oils but very few alkaloids. Terpenoids, shikimates, and 

polyketides will therefore be the main focus of this chapter. 

The general scheme of biosynthetic reactions (Bu’Lock, 1965; Mann et al., 1994) is shown in 

Figure 5.1. Through photosynthesis, green plants convert carbon dioxide and water into glucose. 

Cleavage of glucose produces phosphoenolpyruvate (1), which is a key building block for the shikimate 

family of natural products. Decarboxylation of phosphoenolpyruvate gives the two-carbon unit 

of acetate and this is esterified with coenzyme-A to give acetyl CoA (2). Self-condensation of this 

species leads to the polyketides and lipids. Acetyl CoA is also a starting point for synthesis of 

mevalonic acid (3), which is the key starting material for the terpenoids. In all of these reactions, and 

indeed all the natural chemistry described in this chapter, Nature uses the same reactions that 

chemists do (Sell, 2003). However, nature’s reactions tend to be faster and more selective because of 

the catalysts it uses. These catalysts are called enzymes and they are globular proteins in which an 

active site holds the reacting species together. This molecular organization in the active site lowers 

the activation energy of the reaction and directs its stereochemical course (Lehninger, 1993; 

Matthews and van Holde, 1990). 

Many enzymes need cofactors as reagents or energy providers. Coenzyme-A has already been 

mentioned above. It is a thiol and is used to form thioesters with carboxylic acids. This has two 

effects on the acid in question. Firstly, the thiolate anion is a better leaving group than alkoxide and 

so the carbonyl carbon of the thioester is reactive toward nucleophiles. Secondly, the thioester group 

increases the acidity of the protons adjacent to the carbonyl group and therefore promotes the formation 

of the corresponding carbanions. In biosynthesis, a key role of adenosine triphosphate (ATP) 

is to make phosphate esters of alcohols (phosphorylation). One of the phosphate groups of ATP is 

added to the alcohol to give the corresponding phosphate ester and adenosine diphosphate (ADP). 

Another group of cofactors of importance to biosynthesis includes pairs such as NADP/NADPH, 

TPN/TPNH, and DPN/DPNH. These cofactors contain an N-alkylated pyridine ring. In each pair, 

one form comprises an N-alkylated pyridinium salt and the other the corresponding N-alkyl-1,4- 

dihydropyridine. The two forms in each pair are interconverted by gain or loss of a hydride anion 

and therefore constitute redox reagents. In all of the cofactors mentioned here, the reactive part of 

Carbon dioxide 

+ 

Water 

+ 

Sunlight 

Glucose 

Green plants 

Glycolysis 

OP 

CO 2 – 

Phosphoenolpyruvate 1 

O P = OPO 2– 3 

O OH 

HO 

OH 

OH 

Shikimic acid 4 

Lignans 

Coumarins 

Flavonoids 

Polyketides 

Lipids 

O 

O OH 

CoA 

HO 

Acetyl coenzyme A 

Mevalonic acid 

2 3 

OH 

Terpenoids 

FIGURE 5.1 General pattern of biosynthesis of secondary metabolites.

Chemistry of Essential Oils 123 

the molecule is only a small part of the whole. However, the bulk of the molecule has an important 

role in molecular recognition. The cofactor docks into the active site of the enzyme through recognition 

and this holds the cofactor in the optimum spatial configuration relative to the substrate. 

5.3 POLYKETIDES AND LIPIDS 

The simplest biosynthetic pathway to appreciate is that of the polyketides and lipids (Bu’Lock, 1965; 

Mann et al., 1994). The key reaction sequence is shown in Figure 5.2. Acetyl CoA (2) is carboxylated 

to give malonyl CoA (5) and the anion of this attacks the CoA ester of a fatty acid. Obviously, the 

fatty acid could be acetic acid, making this a second molecule of acetyl CoA. After decarboxylation, 

the product is a b-ketoester with a backbone that is two-carbon atoms longer than the first fatty acid. 

Since this is the route by which fatty acids are produced, it explains why fatty acids are mostly even 

numbered. If the process is repeated with this new acid as the feedstock, it can be seen that various 

poly-oxo-acids can be built up, each of which will have a carbonyl group on every alternate carbon 

atom, hence the name polyketides. Alternatively, the ketone function can be reduced to the corresponding 

alcohol, and then eliminated, and the double bond hydrogenated. This sequence of reactions 

gives a higher homologue of the starting fatty acid, containing two more carbon atoms in the 

chain. Long chain fatty acids, whether saturated or unsaturated, are the basis of the lipids. 

There are three main paths by which components of essential oils and other natural extracts are 

formed in this family of metabolites: condensation reactions of polyketides, degradation of lipids, 

and cyclization of arachidonic acid. 

Figure 5.3 shows how condensation of polyketides can lead to phenolic rings. Intramolecular 

aldol condensation of the tri-keto-octanoic acid and subsequent enolization leads to orsellinic acid 

(6). Polyketide phenols can be distinguished from the phenolic systems of the shikimates by the fact 

that the former usually retain evidence of oxygenation on alternate carbon atoms, either as acids, 

ketones, phenols, or as one end of a double bond. The most important natural products containing 

polyketide phenols are the extracts of oakmoss and treemoss (Evernia prunastrii). The most significant 

in odor terms is methyl 3-methylorsellinate (7) and ethyl everninate (8), which is usually also 

present in reasonable quantity. Atranol (9) and chloratranol (10) are minor components but they are 

skin sensitizers and so limit the usefulness of oakmoss and treemoss extracts, unless they are 

removed from them. Dimeric esters of orsellinic and everninic acids and analogues also exist in 

mosses. They are known as depsides and hydrolysis yields the monomers, thus increasing the odor 

of the sample. However, some depsides, such as atranorin (11), are allergens and thus contribute to 

safety issues with the extracts. 

O 

O 

–O 

CoA 

2 C 

2 5 

CoA 

R 

O 

CoA 

R 

O 

O 

CO 2 – 

CoA 

–CO 2 

O 

TPNH 

O 

–H 2 O 

OH 

O 

DPNH 

O 

O 

R 

CoA 

R 

CoA 

R 

CoA 

R 

CoA 

etc. 

etc. 

FIGURE 5.2 Polyketide and lipid biosynthesis.


O 

O 

O 

– 

O 

OH 

O 

O 

OH 

O 

O 

HO 

–H 2 O Enolise 

OH 

OH 

O O 

OH 

6 

O 

OH 

HO 

O 

HO 

HO 

O 

O O 

O 

OH O 

OH O 

OH 

OH 

7 8 9 10 

Cl 

O 

HO 

O 

OH 

O 

OH 

O 

O 

11 

FIGURE 5.3 Polyketide biosynthesis and oakmoss components. 

The major metabolic route for fatty acids involves b-oxidation and cleavage giving acetate and a 

fatty acid with two carbon atoms less than the starting acid, that is, the reverse of the biosynthesis 

reaction. However, other oxidation routes also exist and these give rise to new metabolites that were 

not on the biosynthetic pathway. For example, Figure 5.4 shows how allylic oxidation of a dienoic 

acid and subsequent cleavage can lead to the formation of an aldehyde. 

Allylic oxidation followed by lactonization rather than cleavage can, obviously, lead to lactones. 

Reduction of the acid function to the corresponding alcohols or aldehydes is also possible as are 

hydrogenation and elimination reactions. Thus a wide variety of aliphatic entities are made available. 

Some examples are shown in Figure 5.5 to illustrate the diversity that exists. The hydrocarbon 

(E,Z)-1,3,5-undecatriene (12) is an important contributor to the odor of galbanum. Simple aliphatic 

alcohols and ethers are found, the occurrence of 1-octanol (13) in olibanum and methyl hexyl ether 

(14) in lavender being examples. Aldehydes are often found as significant odor components of oils, 

for example, decanal (15) in orange oil and (E)-4-decenal (16) caraway and cardamom. The ketone 

2-nonanone (17) that occurs in rue and hexyl propionate (18), a component of lavender, is just one 

of a plethora of esters that are found. The isomeric lactones g-decalactone (19) and d-decalactone 

(20) are found in osmanthus (Essentials Oils Database, n.d.). Acetylenes also occur as essential oil 

components, often as polyacetylenes such as methyl deca-2-en-4,6,8-triynoate (21), which is a component 

of Artemisia vulgaris. 

[O] 

R R' R R' 

HO 

O 

H 

O 

R 

+ 

R' 

R 

R' 

FIGURE 5.4 Fragmentation of polyunsaturated fats to give aldehydes.


OH 

13 

12 

O 

14 

15 

17 

O 

O 

O 

O 

O 

16 

18 

O 

O 

O 

O 

19 20 

21 

O 

O 

FIGURE 5.5 Some lipid-derived components of essential oils. 

Arachidonic acid (22) is a polyunsaturated fatty acid that plays a special role as a synthetic intermediate 

in plants and animals (Mann et al., 1994). As shown in Figure 5.6, allylic oxidation at the 

11th carbon of the chain leads to the hydroperoxide (23). Further oxidation (at the 15th carbon) with 

two concomitant cyclization reactions gives the cyclic peroxide (24). This is a key intermediate for 

the biosynthesis of prostaglandins such as 6-ketoprostaglandin F 1a (25) and also for methyl jasmonate 

O 

O 

H 

OH 

OH 

HO 

O O O 

O 

22 

O 23 

O 

O 

OH 

HO 

O 

25 

OH 

O 

24 

HO 

HO 

O 

OH 

O 

O 

O 

26 

27 

O 

FIGURE 5.6 Biosynthesis of prostaglandins and jasmines.


(26). The latter is the methyl ester of jasmonic acid, a plant hormone, and is a significant odor component 

of jasmine, as is jasmone (27), a product of degradation of jasmonic acid. 

5.4 SHIKIMIC ACID DERIVATIVES 

Shikimic acid (4) is a key synthetic intermediate for plants since it is the key precursor for both the 

flavonoids and lignin (Bu’Lock, 1965; Mann et al., 1994). The flavonoids are important to plants as 

antioxidants, colors, protective agents against ultraviolet light, and the like, and lignin is a key component 

of the structural materials of plants, especially woody tissues. Shikimic acid is synthesized 

from phosphoenol pyruvate (1) and erythrose-4 phosphate (28), as shown in Figure 5.7, and thus its 

biosynthesis starts from the carbohydrate pathway. Its derivatives can usually be recognized by the 

characteristic shikimate pattern of a six-membered ring with either a one- or three-carbon substituent 

on position 1 and oxygenation in the third, and/or fourth, and/or fifth positions. However, the 

oxygen atoms of the final products are not those of the starting shikimate since these are lost initially 

and then replaced. 

Figure 5.8 shows some of the biosynthetic intermediates stemming from shikimic acid (4) and 

which are of importance in terms of generating materials volatile enough to be essential oil components. 

Elimination of one of the ring alcohols and reaction with phosphoenol pyruvate (1) gives 

chorismic acid (29) that can undergo an oxy-Cope reaction to give prephenic acid. (30) Decarboxylation 

and elimination of the ring alcohol now gives the phenylpropionic acid skeleton. Amination and 

reduction of the ketone function gives the essential amino acid phenylalanine (31) whereas reduction 

and elimination leads to cinnamic acid. (32) Ring hydroxylation of the latter gives the isomeric 

o- and p-coumaric acids, (33) and (34), respectively. Further hydroxylation gives caffeic acid (35) 

and methylation of this gives ferulic acid. (36) Oxidation of the methyl ether of the latter and subsequent 

cyclization gives methylenecaffeic acid (37). In shikimate biosynthesis, it is often possible to 

arrive at a given product by different sequences of the same reactions and the exact route used will 

depend on the genetic make-up of the plant. 

Aromatization of shikimic acid, without addition of the three additional carbon atoms from 

phosphoenolpyruvate, gives benzoic acid derivatives. Benzoic acid itself occurs in some oils and its 

esters are widespread. For example, methyl benzoate is found in tuberose, ylang ylang, and various 

lilies. Even more common are benzyl alcohol, benzaldehyde, and their derivatives (Arctander, 1960; 

Phosphoenol pyruvate 

1 

PO CO2 H 

OH 

HO 

O 

CO 2 H 

TPN + 

HO 

O 

CO 2 H 

HO 

O 

CO 2 H 

O 

OP OH 

Erythrose-4 phosphate 

28 

OH 

OP OH 

Heptulosonic acid 

monophosphate 

OP 

OH 

TPN 

OH 

H 

OH 

OH 

CO 2 H CO 2 H HO CO 2 H O CO 2 H 

TPNH 

HO 

OH 

OH 

Shikimic acid 

4 

O 

OH 

OH 

5-Dehydroshikimic acid 

O 

OH 

OH 

5-Dehydroquinic acid 

HO 

OH 

OH 

FIGURE 5.7 Biosynthesis of shikimic acid.


O 

OH 

O 

OH 

O 

OH 

O 

CO 2 H 

CO 2 H 

HO 

OH 

OH 

O 

4 

OH 29 

CO 2 H 

30 

OH 

31 

NH 2 

CO 2 H 

CO 2 H 

CO 2 H 

CO 2 H 

CO 2 H 

32 

O 

37 

O 

OH 

36 

O 

OH 

35 

OH 

OH 

34 

OH 

33 

CO 2 H 

FIGURE 5.8 Key intermediates from shikimic acid. 

Essential Oils Database, n.d.; Gildemeister and Hoffmann, 1956; Günther, 1948). Benzyl alcohol 

occurs in muguet, jasmine, and narcissus, for example, and its acetate is the major component of 

jasmine oils. The richest sources of benzaldehyde are almond and apricot kernels but it is also found 

in a wide range of flowers, including lilac, and other oils such as cassia and cinnamon. Hydroxylation 

or amination of benzoic acid leads to further series of natural products and some of the most significant, 

in terms of odors of essential oils, are shown in Figure 5.9. o-Hydroxybenzoic acid is known 

as salicylic acid (38) and both it and its esters are widely distributed in nature. For instance, methyl 

salicylate (39) is the major component (about 90% of the volatiles) of wintergreen and makes a significant 

contribution to the scents of tuberose and ylang ylang although only present at about 10% 

in the former and less than 1% in the latter. o-Aminobenzoic acid is known as anthranilic acid (40). 

Its methyl ester (41) has a very powerful odor and is found in such oils as genet, bitter orange flower, 

CO 2 H CO 2 H CO 2 H 

OH 

NH 2 

HO 

38 40 

43 

O 

O 

O 

O 

O 

OH 

39 41 

NH 2 

O 

44 

O 

O 

OH 

42 

N 

H 

O 

45 

FIGURE 5.9 Hydroxy- and aminobenzoic acid derivatives.


tuberose, and jasmine. Dimethyl anthranilate (42), in which both the nitrogen and acid functions 

have been methylated, occurs at low levels in citrus oils. p-Hydroxybenzoic acid has been found in 

vanilla and orris but much more common is the methyl ester of the corresponding aldehyde, commonly 

known as anisaldehyde (44). As the name suggests, the latter one is an important component 

of anise and it is also found in oils such as lilac and the smoke of agar wood. The corresponding 

alcohol, anisyl alcohol (45), and its esters are also widespread components of essential oils. 

Indole (46) and 2-phenylethanol (47) are both shikimate derivatives. Indole is particularly associated 

with jasmine. It usually occurs in jasmine absolute at a level of about 3–5% and makes a very 

significant odor contribution to it. However, it does occur in many other essential oils as well. 

2-Phenylethanol occurs widely in plants and is especially important for rose where it usually 

accounts for one-third to three-quarters of the oil. The structures of both are shown in Figure 5.10. 

Figure 5.10 also shows some of the commonest cinnamic acid-derived essential oil components. 

Cinnamic acid (32) itself has been found in, for example, cassia and styrax but its esters, particularly 

the methyl ester, are more frequently encountered. The corresponding aldehyde, cinnamaldehyde 

(48), is a key component of cinnamon and cassia and also occurs in some other oils. Cinnamyl 

alcohol (49) and its esters are more widely distributed, occurring in narcissus, lilac, and a variety 

of other oils. Lactonization of o-coumaric acid (33) gives coumarin (50). This is found in new 

mown hay to which it gives the characteristic odor. It is also important in the odor profile of lavender 

and related species and occurs in a number of other oils. Bergapten (51) is a more highly oxygenated 

and substituted coumarin. The commonest source is bergamot oil but it also occurs in 

other sources, such as lime and parsley. It is phototoxic and consequently constitutes a safety issue 

for oils containing it. 

Oxygenation in the p-position of cinnamic acid followed by methylation of the phenol and reduction 

of the acid to alcohol with subsequent elimination of the alcohol gives estragole (also known as 

methylchavicol (52) and anethole (53). Estragole is found in a variety of oils, mostly herb oils such as 

basil, tarragon, chervil, fennel, clary sage, anise, and rosemary. Anethole occurs in both the (E)- and 

(Z)-forms, the more thermodynamically stable (E)-isomer (shown in Figure 5.10) is the commoner, 

the (Z)-isomer is the more toxic of the two. Anethole is found in spices and herbs such as anise, fennel, 

lemon balm, coriander, and basil and also in flower oils such as ylang ylang and lavender. 

OH 

46 

N 

H 

47 

O 

OH 

48 

49 

O 

50 

O 

O 

O 

51 

O 

O 

O 

52 

O 

53 

FIGURE 5.10 Some shikimate essential oil components.


O 

O 

O 

O 

HO 

53 54 

HO 

HO 

55 

O 

O 

O 

O 

O 

56 

O 

57 O 

58 

O 

O 

O 

O 

O 

59 60 

O 

O 

61 

FIGURE 5.11 Ferulic acid derivatives. 

Reduction of the side chain of ferulic acid (36) leads to an important family of essential oil components, 

shown in Figure 5.11. The key material is eugenol (53), which is widespread in its occurrence. 

It is found in spices such as clove, cinnamon, and allspice, herbs such as bay and basil, and in 

flower oils including rose, jasmine, and carnation. Isoeugenol (54) is found in basil, cassia, clove, 

nutmeg, and ylang ylang. Oxidative cleavage of the side chain of shikimates to give benzaldehyde 

derivatives is common and often significant, as it is in this case, where the product is vanillin (55). 

Vanillin is the key odor component of vanilla and is therefore of considerable commercial importance. 

It also occurs in other sources such as jasmine, cabreuva, and the smoke of agar wood. The 

methyl ether of eugenol, methyleugenol (56), is very widespread in nature, which, since it is the subject 

of some toxicological safety issues, creates difficulties for the essential oils business. The oils 

of some Melaleuca species contain up to 98% methyleugenol and it is found in a wide range of species 

including pimento, bay, tarragon, basil, and rose. The isomer, methylisoeugenol (57), occurs as 

both (E)- and (Z)-isomers, the former being slightly commoner. Typical sources include calamus, 

citronella, and some narcissus species. Oxidative cleavage of the side chain in this set of substances 

produces veratraldehyde (58), a relatively rare natural product. Formation of the methylenedioxy 

ring, via methylenecaffeic acid (37), gives safrole (59), the major component of sassafras oil. The 

toxicity of safrole has led to a ban on the use of sassafras oil by the perfumery industry. Isosafrole 

(60) is found relatively infrequently in nature. The corresponding benzaldehyde derivative, heliotropin 

(61), also known as piperonal, is the major component of heliotrope. 

5.5 TERPENOIDS 

The terpenoids are, by far, the most important group of natural products as far as essential oils are 

concerned. Some authors, particularly in older literature, refer to them as terpenes but this term is 

nowadays restricted to the monoterpenoid hydrocarbons. They are defined as substances composed 

of isoprene (2-methylbutadiene) units. Isoprene (62) is not often found in essential oils and is not 

actually an intermediate in biosynthesis, but the 2-methylbutane skeleton is easily discernable in 

ter penoids. Figure 5.12 shows the structures of some terpenoids. In the case of geraniol (63), one end 

of one isoprene unit is joined to the end of another making a linear structure (2,6-dimethyloctane). In 

guaiol (64), there are three isoprene units joined together to make a molecule with two rings. It is easy 

to envisage how the three units were first joined together into a chain and then formation of bonds 

from one point in the chain to another produced the two rings. Similarly, two isoprene units were 

used to form the bicyclic structure of a-pinene (65). 

The direction of coupling of isoprene units is almost always in one direction, the so-called headto-tail 

coupling. This is shown in Figure 5.13. The branched end of the chain is referred to as the 

head of the molecule and the other as the tail.


OH 

62 

63 

64 

OH 

65 

FIGURE 5.12 Isoprene units in some common terpenoids. 

Head 

Head 

Tail 

Tail 

FIGURE 5.13 Head-to-tail coupling of two isoprene units. 

HO 

O 

OH 

Mn 2+ 

OH 

O P P O P P 

Mg 2+ 

3 66 67 

67 

66 

O 

P P 

H 

_ base 

O 

P P 

O 

P P 

O 

66 P P 

H 

_ base 

etc. 

68 

FIGURE 5.14 Coupling of C5 units in terpenoid biosynthesis. 

This pattern of coupling is explained by the biosynthesis of terpenoids (Bu’Lock, 1965; Croteau, 

1987; Mann et al., 1994). The key intermediate is mevalonic acid (3), which is made from three 

molecules of acetyl CoA (2). Phosphorylation of mevalonic acid followed by elimination of the 

tertiary alcohol and concomitant decarboxylation of the adjacent acid group gives isopentenyl pyrophosphate 

(66). This can be isomerized to give prenyl pyrophosphate (67). Coupling of these two 

5-carbon units gives a 10-carbon unit, geranyl pyrophosphate (68), as shown in Figure 5.14 and 

further additions of isopentenyl pyrophosphate (66) lead to 15-, 20-, 25-, and so on carbon units. 

It is clear from the mechanism shown in Figure 5.14 that terpenoid structures will always contain 

a multiple of five carbon atoms when they are first formed. The first terpenoids to be studied contained 

10 carbon atoms per molecule and were called monoterpenoids. This nomenclature has 

remained and so those with five carbon atoms are known as hemiterpenoids, those with 15, sesquiterpenoids, 

and those with 20, diterpenoids, and so on. In general, only the hemiterpenoids, monoterpenoids, 

and sesquiterpenoids are sufficiently volatile to be components of essential oils. Degradation 

products of higher terpenoids do occur in essential oils, so they will be included in this chapter.


5.5.1 HEMITERPENOIDS 

Many alcohols, aldehydes, and esters, with a 2-methylbutane skeleton, occur as minor components 

in essential oils. Not surprisingly, in view of the biosynthesis, the commonest oxidation pattern is 

that of prenol, that is, 3-methylbut-2-ene-1-ol. For example, the acetate of this alcohol occurs in 

ylang ylang and a number of other oils. However, oxidation has been observed at all positions. 

Esters such as prenyl acetate give fruity top notes to oils containing them and the corresponding 

thioesters contribute to the characteristic odor of galbanum. 

5.5.2 MONOTERPENOIDS 

Geranyl pyrophosphate (68) is the precursor for the monoterpenoids. Heterolysis of its carbon– 

oxygen bond gives the geranyl carbocation (69). In natural systems, this and other carbocations 

discussed in this chapter do not exist as free ions but rather as incipient carbocations held in enzyme 

active sites and essentially prompted into cation reactions by the approach of a suitable reagent. For 

the sake of simplicity, they will be referred to here as carbocations. The reactions are described in 

chemical terms but all are under enzymic control and the enzymes present in any given plant will 

determine the terpenoids it will produce. Thus essential oil composition can give information about 

the genetic make-up of the plant. A selection of some of the key biosynthetic routes to monoterpenoids 

(Devon and Scott, 1972) is shown in Figure 5.15. 

+ 

OH 

70 

69 

63 

O 

71 

73 

72 

77 

+ 

65 

+ 

76 

OH 

74 

75 

O 

OH 

+ 

+ 

O 

80 79 

78 

81 82 

FIGURE 5.15 Formation of monoterpenoid skeletons.


Reaction of the geranyl carbocation with water gives geraniol (63) that can subsequently be oxidized 

to citral (71). Loss of a proton from (69) gives myrcene (70) and this can be isomerized to 

other acyclic hydrocarbons. An intramolecular electrophilic addition reaction of (69) gives the 

monocyclic carbocation (72) that can eliminate a proton to give limonene (73) or add water to give 

a-terpineol (74). A second intramolecular addition gives the pinyl carbocation (75) that can lose a 

proton to give either a-pinene (65) or b-pinene (76). The pinyl carbocation (75) is also reachable 

directly from the menthyl carbocation (72). Carene (77), another bicyclic material, can be produced 

through similar reactions. Wagner–Meerwein rearrangement of the pinyl carbocation (75) gives the 

bornyl carbocation (78). Addition of water to this gives borneol (79) and this can be oxidized to 

camphor (80). An alternative Wagner–Meerwein rearrangement of (75) gives the fenchyl skeleton 

(81) from which fenchone (82) is derived. 

Some of the more commonly encountered monoterpenoid hydrocarbons (Arctander, 1960; 

Essential Oils Database, n.d.; Gildemeister and Hoffmann, 1956; Günther, 1948; Sell, 2007) are 

shown in Figure 5.16. Many of these can be formed by dehydration of alcohols and so their presence 

in essential oils could be as artifacts arising from the extraction process. Similarly, p-cymene (83) 

is one of the most stable materials of this class and can be formed from many of the others by appropriate 

cyclization and/or isomerization and/or oxidation reactions and so its presence in any essential 

oil could be as an artifact. 

Myrcene (70) is very widespread in nature. Some sources, such as hops, contain high levels and 

it is found in most of the common herbs and spices. All isomers of a-ocimene (84), b-ocimene (85), 

and allo-ocimene (86) are found in essential oils, the isomers of b-ocimene (85) being the most 

frequently encountered. Limonene (73) is present in many essential oils but the major occurrence is 

in the citrus oils that contain levels up to 90%. These oils contain the dextrorotatory (R)-enantiomer, 

and its antipode is much less common. Both a-phellandrene (87) and b-phellandrene (88) occur 

widely in essential oils. For example, (−)-a-phellandrene is found in Eucalyptus dives and (S)-(−)- 

b-phellandrene in the lodgepole pine, Pinus contorta. p-Cymene (83) has been identified in many 

essential oils and plant extracts and thyme and oregano oils are particularly rich in it. a-Pinene (65), 

70 84 85 86 

73 87 88 

83 

65 76 77 

89 

FIGURE 5.16 Some of the more common terpenoid hydrocarbons.


b-pinene (76), and 3-carene (77) are all major constituents of turpentine from a wide range of pines, 

spruces, and firs. The pinenes are often found in other oils, 3-carene less so. Like the pinenes, camphene 

(89) is widespread in nature. 

Simple hydrolysis of geranyl pyrophosphate gives geraniol, (E)-3,7-dimethylocta-2,6-dienol (63). 

This is often accompanied in nature by its geometric isomer, nerol (90). Synthetic material is usually 

a mixture of the two isomers and when interconversion is possible, the equilibrium mixture 

comprises about 60% geraniol (63) and 40% nerol (90). The name geraniol is often used to describe 

a mixture of geraniol and nerol. When specifying the geometry of these alcohols it is better to use 

the modern (E)/(Z) nomenclature as the terms cis and trans are somewhat ambiguous in this case 

and earlier literature is not consistent in their use. Both isomers occur in a wide range of essential 

oils, geraniol (63) being particularly widespread. The oil of Monarda fi stulosa contains over 90% 

geraniol (63) and the level in palmarosa is over 80%. Geranium contains about 50% and citronella 

and lemongrass each contain about 30%. The richest natural sources of nerol include rose, palmarosa, 

citronella, and davana although its level in these is usually only in the 10–15% range. Citronella 

and related species are used commercially as sources of geraniol but the price is much higher than 

that of synthetic material. Citronellol (91) is a dihydrogeraniol and occurs widely in nature in both 

enantiomeric forms. Rose, geranium, and citronella are the oils with the highest levels of citronellol. 

Geraniol, nerol, and citronellol, together with 2-phenylethanol, are known as the rose alcohols 

because of their occurrence in rose oils and also because they are the key materials responsible for 

the rose odor character. Esters (the acetates in particular) of all these alcohols are also commonly 

encountered in essential oils (Figure 5.17). 

Allylic hydrolysis of geranyl pyrophosphate produces linalool (92). Like geraniol, linalool occurs 

widely in nature. The richest source is Ho leaf, the oil of which can contain well over 90% linalool. 

Other rich sources include linaloe, rosewood, coriander, freesia, and honeysuckle. Its acetate is also 

frequently encountered and is a significant contributor to the odors of lavender and citrus leaf oils. 

Figure 5.18 shows a selection of cyclic monoterpenoid alcohols. a-Terpineol (74) is found in 

many essential oils as is its acetate. The isomeric terpinen-4-ol (93) is an important component of 

Ti tree oil but its acetate, surprisingly, is more widely occurring, being found in herbs such as marjoram 

and rosemary. l-Menthol (94) is found in various mints and is responsible for the cooling 

effect of oils containing it. There are eight stereoisomers of the menthol structure, l-menthol is the 

commonest in nature and also has the strongest cooling effect. The cooling effect makes menthol 

and mint oils valuable commodities, the two most important sources being cornmint (Mentha 

arvensis) and peppermint (Mentha piperita). Isopulegol (95) occurs in some species including 

Eucalyptus citriodora and citronella. Borneol (endo-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol) (79) 

and esters thereof, particularly the acetate, occur in many essential oils. Isoborneol (exo-1,7,7- 

trimethylbicyclo[2.2.1]heptan-2-ol) (96) is less common; however, isoborneol and its esters are 

found in quite a number of oils. Thymol (97), being a phenol, possesses antimicrobial properties, 

and oils, such as thyme and basil, which find appropriate use in herbal remedies. It is also found in 

various Ocimum and Monarda species. 

Three monoterpenoid ethers are shown in Figure 5.19. 1,8-Cineole (98), more commonly referred 

to simply as cineole, comprises up to 95% of the oil of Eucalyptus globulus and about 40–50% of 

OH 

OH 

OH 

OH 

63 90 91 92 

FIGURE 5.17 Key acyclic monoterpenoid alcohols.


OH 

OH 

OH 

OH 

74 93 94 95 

OH 

OH 

OH 

79 

96 

97 

FIGURE 5.18 Some cyclic monoterpenoid alcohol. 

Cajeput oil. It also can be found in an extensive range of other oils and often as a major component. 

It has antibacterial and decongestant properties and consequently, eucalyptus oil is used in various 

paramedical applications. Menthofuran (99) occurs in mint oils and contributes to the odor of peppermint. 

It is also found in several other oils. Rose oxide is found predominantly in rose and geranium 

oils. There are four isomers, the commonest being the laevorotatory enantiomer of cis-rose 

oxide (100). This is also the isomer with the lowest odor threshold of the four. 

The two most significant monoterpene aldehydes are citral (71) and its dihydro analogue citronellal 

(103), both of which are shown in Figure 5.20. The word citral is used to describe a mixture 

of the two geometric isomers geranial (101) and neral (102) without specifying their relative proportions. 

Citral occurs widely in nature, both isomers usually being present, the ratio between them 

usually being in the 40:60 to 60:40 range. Lemongrass contains 70–90% citral and the fruit of 

Litsea cubeba contains about 60–75%. Citral also occurs in Eucalyptus staigeriana, lemon balm, 

ginger, basil, rose, and citrus species. It is responsible for the characteristic smell of lemons although 

lemon oil usually contains only a few percent of it. Citronellal (103) also occurs widely in essential 

oils. Eucalyptus citriodora contains up to 85% citronellal and significant amounts are also found in 

some chemotypes of Litsea cubeba, citronella Swangi leaf oil, and Backhousia citriodora. Campholenic 

aldehyde (104) occurs in a limited range of species such as olibanum, styrax, and some eucalypts. 

Material produced from a-pinene (65) is important as an intermediate for synthesis. 

Figure 5.21 shows some of the commoner monoterpenoid ketones found in essential oils. Both 

enantiomers of carvone are found in nature, the (R)-(−)- (usually referred to as l-carvone) (105) 

being the commoner. This enantiomer provides the characteristic odor of spearmint (Mentha 

cardiaca, Mentha gracilis, Mentha spicata, and Mentha viridis), the oil of which usually contains 

55–75% of l-carvone. The (S)-(+)-enantiomer (106) is found in caraway at levels of 30–65% and 

O 

O 

O 

FIGURE 5.19 Some monoterpenoid ethers. 

98 99 100


O 

O 

O 

O 

101 102 103 104 

FIGURE 5.20 Some monoterpenoid aldehydes. 

in dill at 50–75%. Menthone is fairly common in essential oils particularly in the mints, pennyroyal, 

and sages but lower levels are also found in oils such as rose and geranium. The l-isomer is 

commoner than the d-isomer. Isomenthone is the cis-isomer and the two interconvert readily by 

epimerization. The equilibrium mixture comprises about 70% menthone and 30% isomenthone. 

The direction of rotation of plane-polarized light reverses on epimerization and therefore l-menthone 

(107) gives d-isomenthone (108). (+)-Pulegone (109) accounts for about 75% of the oil of 

pennyroyal and is also found in a variety of other oils. (−)-Piperitone (110) also occurs in a variety 

of oils, the richest source being Eucalyptus dives. Both pulegone and piperitone have strong minty 

odors. Camphor (80) occurs in many essential oils and in both enantiomeric forms. The richest 

source is the oil of camphor wood but it is also an important contributor to the odor of lavender and 

of herbs such as sage and rosemary. Fenchone (82) occurs widely, for example, in cedar leaf and 

lavender. Its laevorotatory enantiomer is an important contributor to the odor of fennel. 

5.5.3 SESQUITERPENOIDS 

By definition, sesquiterpenoids contain 15 carbon atoms. This results in their having lower volatilities 

and hence higher boiling points than monoterpenoids. Therefore, fewer of them (in percentage terms) 

contribute to the odor of essential oils but those that do often have low-odor thresholds and contribute 

significantly as end notes. They are also important as fixatives for more volatile components. 

Just as geraniol (63) is the precursor for all the monoterpenoids, farnesol (111) is the precursor 

for all the sesquiterpenoids. Its pyrophosphate is synthesized in nature by the addition of isopentenyl 

pyrophosphate (66) to geranyl pyrophosphate (68) as shown in Figure 5.14 and hydrolysis of that 

gives farnesol. Incipient heterolysis of the carbon–oxygen bond of the phosphate gives the nascent 

O 

O 

O 

O 

105 106 107 108 

O 

O 

O 

O 

109 110 

80 

82 

FIGURE 5.21 Some monoterpenoid ketones.


farnesyl carbocation (112) and this leads to the other sesquiterpenoids, just as the geranyl carbocation 

does to monoterpenoids. Starting from farnesyl pyrophosphate, the variety of possible cyclic 

structures is much greater than that from geranyl pyrophosphate because there are now three double 

bonds in the molecule. Similarly, there is also a greater scope for further structural variation resulting 

from rearrangements, oxidations, degradation, and so on (Devon and Scott, 1972). The geometry 

of the double bond in position 2 of farnesol is important in terms of determining the pathway 

used for subsequent cyclization reactions and so these are best discussed in two blocks. 

Figure 5.22 shows a tiny fraction of the biosynthetic pathways derived from (Z,E)-farnesyl pyrophosphate. 

Direct hydrolysis leads to acyclic sesquiterpenoids such as farnesol (111) and nerolidol 

(113). However, capture of the carbocation (112) by the double bond at position 6 gives a cyclic structure 

that of the bisabolane skeleton (114) and quenching of this with water gives bisabolol (115). A 

hydrogen shift in (114) leads to the isomeric carbocation (116) that still retains the bisabolane skeleton. 

Further cyclizations and rearrangements take the molecule through various skeletons, including 

those of the acorane (117) and cedrane (118) families, to the khusane family, illustrated by khusimol 

(119) in the figure. Obviously, a wide variety of materials can be generated along this route, an 

example being cedrol (120) formed by reaction of cation (118) with water. The bisabolyl carbocation 

(114) can also cyclize to the other double bonds in the molecule leading to, inter alia, the campherenane 

skeleton (121) and hence a-santalol (122) and b-santalol (123), or, via the cuparane (124) and 

chamigrane (125) skeletons, to compounds such as thujopsene (126). The carbocation function in 

128 

+ 

127 

+ 

112 

OH 

115 

+ 

+ 

124 

+ 

114 

121 

+ 

+ 

+ + 

129 

116 

HO 

122 123 

HO 

+ 

+ 

130 

125 

+ 

117 

OH 

+ 

+ 

119 

+ 

131 

126 

OH 

118 

+ 

120 

FIGURE 5.22 Some biosynthetic pathways from (Z,E)-farnesol.


(112) can also add to the double bond at the far end of the chain to give the cis-humulane skeleton 

(127). This species can cyclize back to the double bond at carbon 2 before losing a proton, thus 

giving caryophyllene (128). Another alternative is for a series of hydrogen shifts, cyclizations, 

and rearrangements to lead it through the himachalane (129) and longibornane (130) skeletons to 

longifolene (131). 

Figure 5.23 shows a few of the many possibilities for biosynthesis of sesquiterpenoids from 

(E,E)-farnesyl pyrophosphate. Cyclization of the cation (132) to C-11, followed by loss of a proton 

gives all trans- or a-humulene (133), whereas cyclization to the other end of the same double bond 

gives a carbocation (134) with the germacrane skeleton. This is an intermediate in the biosynthesis 

of odorous sesquiterpenes such as nootkatone (135) and a-vetivone (137). b-Vetivone (137) is synthesized 

through a route that also produces various alcohols, for example, (138) and (139), and an 

ether (140) that has the eudesmane skeleton. Rearrangement of the germacrane carbocation (134) 

leads to a carbocation (141) with the guaiane skeleton and this is an intermediate in the synthesis of 

guaiol (142). Carbocation (141) is also an intermediate in the biosynthesis of the a-patchoulane 

(143) and b-patchoulane (144) skeletons and of patchouli alcohol (145). 

O 

135 

+ 

132 133 

O 

136 

+ 

134 142 

OH 

+ 

+ 

138 

OH 

144 

141 

+ 

HO 

139 

O 

140 

143 

O 

OH 

137 

145 

FIGURE 5.23 Some biosynthetic pathways from (E,E)-farnesol.


All four isomers of farnesol (111) are found in nature and all have odors in the muguet and linden 

direction. The commonest is the (E,E)-isomer that occurs in, among others, cabreuva and ambrette 

seed while the (Z,E)-isomer has been found in jasmine and ylang, the (E,Z)-isomer in cabreuva, 

rose, and neroli, and the (Z,Z)-isomer in rose. Nerolidol (113) is the allylic isomer of farnesol and 

exists in four isomeric forms, namely, two enantiomers each of two geometric isomers. The 

(E)-isomer has been found in cabreuva, niaouli, and neroli oils among others and the (Z)-isomer in 

neroli, jasmine, ho leaf, and so on. Figure 5.24 shows the structures of farnesol and nerolidol with 

all of the double bonds in the trans-configuration. 

a-Bisabolol (115) is the simplest of the cyclic sesquiterpenoid alcohols. If farnesol is the sesquiterpenoid 

equivalent of geraniol and nerolidol of linalool, then a-bisabolol is the equivalent of 

a-terpineol. It has two chiral centers and therefore exists in four stereoisomeric forms, all of which 

occur in nature. The richest natural source is Myoporum crassifolium Forst., a shrub from New 

Caledonia, but a-bisabolol can be found in many other species including chamomile, lavender, and 

rosemary. It has a faint floral odor and anti-inflammatory properties and is responsible, at least in 

part, for the related medicinal properties of chamomile oil. 

The santalols (122) and (123) have more complex structures and are the principal components of 

sandalwood oil. Cedrol (120) is another complex alcohol but it is more widely occurring in nature 

than the santalols. It is found in a wide range of species, the most significant being trees of the 

Juniperus, Cupressus, and Thuja families. Cedrene (146) occurs alongside cedrol in cedarwood 

oils. Cedrol is dehydrated to cedrene in the presence of acid and so the latter can be an artifact of 

the former and the ratio of the two will often depend on the method of isolation. Thujopsene (126) 

also occurs in cedarwood oils, usually at a similar level to that of cedrol/cedrene, and it is found in 

various other oils also. Caryophyllene (128) and a-humulene (the all trans isomer) (133) are widespread 

in nature, cloves being the best-known source of the former and hops of the latter. The ring 

systems of these two materials are very strained making them quite reactive chemically and caryophyllene, 

extracted from clove oil as a by-product of eugenol production, is used as the starting 

material in the synthesis of several fragrance ingredients. Longifolene (131) also possesses a strained 

ring system. It is a component of Indian turpentine and is therefore readily available as a feedstock 

for fragrance ingredient manufacture. 

Guaicwood oil is the richest source of guaiol (142) and the isomeric bulnesol (147) but both are 

found in other oils, particularly guaiol that occurs in a wide variety of plants. Dehydration and 

dehydrogenation of these give guaiazulene (148), which is used as an anti-inflammatory agent. 

Guaiazulene is also accessible from a-gurjunene (149), the major component of gurjun balsam. 

Guaiazulene is blue in color as is the related olefin chamazulene (150). The latter occurs in a variety 

of oils but it is particularly important in chamomile to which it imparts the distinctive blue tint 

(Figure 5.25). 

OH 

OH 

111 113 

147 

OH 

FIGURE 5.24 Some sesquiterpenoid alcohols.


146 148 

FIGURE 5.25 Some sesquiterpenoid hydrocarbons. 

149 150 

Vetiver and patchouli are two oils of great importance in perfumery (Williams, 1996, 2004). Both 

contain complex mixtures of sesquiterpenoids, mostly with complex polycyclic structures (Sell, 

2003). The major components of vetiver oil are a-vetivone (136), b-vetivone (137), and khusimol 

(119), but the most important components as far as odor is concerned are minor constituents such as 

khusimone (151), zizanal (152), and methyl zizanoate (153). Nootkatone (154) is an isomer of 

a-vetivone and is an important odor component of grapefruit. Patchouli alcohol (145) is the major 

constituent of patchouli oil but, as is the case also with vetiver, minor components are more important 

for the odor profile. These include nor-patchoulenol (155) and nor-tetrapatchoulol (156) (Figure 5.26). 

The molecules of chamazulene (150), khusimone (151), nor-patchoulenol (155), and nor-tetrapatchoulol 

(156) each contain only 14 carbon atoms in place of the normal 15 of sesquiterpenoids. 

They are all degradation products of sesquiterpenoids. Degradation, either by enzymic action or 

from environmental chemical processes, can be an important factor in generating essential oil components. 

Carotenoids are a family of tetraterpenoids characterized by having a tail-to-tail fusion 

between two diterpenoid fragments. In the case of b-carotene (157), both ends of the chain have been 

cyclized to form cyclohexane rings. Degradation of the central part of the chain leads to a number of 

fragments that are found in essential oils and the two major families of such are the ionones and 

damascones. Both have the same carbon skeleton but in the ionones (Leffingwell & Associates, n.d.; 

H 

H 

H 

O 

O 

O 

O 

151 152 153 

HO 

HO 

154 

O 

155 156 

FIGURE 5.26 Components of vetiver, patchouli, and grapefruit.


157 

O 

O 

O 

O 

158 159 

163 

164 

O 

O 

O 

160 

161 

162 

O 

O 

O 

O 

O 

165 

166 167 168 

FIGURE 5.27 Carotenoid degradation products. 

Sell, 2003) the site of oxygenation is three carbon atoms away from the ring, in damascones oxygenation 

is found at the chain carbon next to the ring (Figure 5.27). 

The ionones occur naturally in a wide variety of flowers, fruits, and leaves, and are materials of 

major importance in perfumery (Arctander, 1960; Essential Oils Database, n.d.; Gildemeister and 

Hoffmann, 1956; Günther, 1948; Sell, 2007). About 57% of the volatile components of violet flowers 

are a- (158) and b-ionones (159) and both isomers occur widely in nature. The damascones are also 

found in a wide range of plants. They usually occur at a very low level but their very intense odors 

mean that they still make a significant contribution to the odors of oils containing them. The first to 

be isolated and characterized was b-damascenone (160), which was found at a level of 0.05% in the 

oil of the Damask rose. Both b-damascenone (160) and the a- (161) and b-isomers (162) have since 

been found in many different essential oils and extracts. In the cases of safranal (163) and cyclocitral 

(165), the side chain is degraded even further leaving only one of its carbon atoms attached to 

the cyclohexane ring. About 70% of the volatile component of saffron is safranal and it makes a 

significant contribution to its odor. Other volatile carotenoid degradation products that occur in 

essential oils and contribute to their odors include the theaspiranes (165), vitispiranes (166), edulans 

(167), and dihydroactindiolide (168). 

The similarity in structure between the ionones and the irones might lead to the belief that the 

latter are also carotenoid derived. However, this is not the case as the irones are formed by degradation 

of the triterpenoid iripallidal (169), which occurs in the rhizomes of the iris. The three isomers, 

a- (170), b- (171), and g-irone (172), are all found in iris and the first two in a limited number of 

other species (Figure 5.28). 

5.6 SYNTHESIS OF ESSENTIAL OIL COMPONENTS 

It would be impossible, in a volume of this size, to review all of the reported syntheses of essential 

oil components and so the following discussion will concentrate on some of the more commercially 

important synthetic routes to selected key substances. In the vast majority of cases, there is a balance 

between routes using plant extracts as feedstocks and those using petrochemicals. For some


HO 

OH 

169 

O 

OH 

O 

O 

O 

170 

FIGURE 5.28 Iripallidal and the irones. 

171 

172 

materials, plant-derived and petrochemical-derived equivalents might exist in economic competition 

while for others, one source is more competitive. The balance will vary over time and the 

market will respond accordingly. Sustainability of production routes is a complex issue and easy 

assumptions might be totally incorrect. Production and extraction of plant-derived feedstocks often 

requires considerable expenditure of energy in fertilizer production, harvesting, and processing and 

so it is quite possible that production of a material derived from a plant source would use more mineral 

oil than the equivalent derived from petrochemical feedstocks. 

Figure 5.29 shows some of the plant-derived feedstocks used in the synthesis of lipids and 

polyketides (Sell, 2006). Rapeseed oil provides erucic acid (173) that can be ozonolyzed to give 

brassylic acid (174) and heptanal (175), both useful building blocks. The latter can also be obtained, 

together with undecylenic acid (176), by pyrolysis of ricinoleic acid (177) that is available from 

Castor oil. Treatment of undecylenic acid (176) with acid leads to movement of the double bond 

along the chain and eventual cyclization to give g-undecalactone (178), which has been found in 

narcissus oils. Aldol condensation of heptanal (175) with cyclopentanone, followed by Bayer– 

Villiger oxidation, gives d-dodecalactone (179), identified in the headspace of tuberose. Such aldol 

reactions, followed by appropriate further conversions, are important in the commercial production 

of analogues of methyl jasmonate (26) and jasmone (27). 

Ethylene provides a good example of a petrochemical feedstock for the synthesis of lipids and 

polyketides. It can be oligomerized to provide a variety of alkenes into which functionalization can 

be introduced by hydration, oxidation, hydroformylation, and so on. Of course, telomerization can 

be used to provide functionalized materials directly. 

Eugenol (53) (e.g., clove oil) and safrole (59) (e.g., sassafras) are good examples of plant-derived 

feedstocks that are used in the synthesis of other shikimates. Methylation of eugenol produces methyleugenol 

(56) and this can be isomerized using acid or metal catalysts to give methylisoeugenol 

(57). Similarly, isomerization of eugenol gives isoeugenol (54) and oxidative cleavage of this, for 

example, by ozonolysis gives vanillin (55). This last sequence of reactions, when applied to safrole 

gives isosafrole (60) and heliotropin (61). All of these conversions are shown in Figure 5.30. 

Production of shikimates from petrochemicals for commercial use mostly involves straightforward 

chemistry (Arctander, 1969; Bauer and Panten, 2006; Däniker, 1987; Sell, 2006). Nowadays 

the major starting materials are benzene (180) and toluene (181), which are both available in bulk 

from petroleum fractions. Alkylation of benzene with propylene gives cumene (182), the hydroperoxide 

of which fragments to give phenol (183) and acetone. Phenol itself is an important molecular 

building block and further oxidation gives catechol (184). Syntheses using these last two materials 

will be discussed below. Alkylation of benzene with ethylene gives ethylbenzene, which is converted 

to styrene (185) via autoxidation, reduction, and elimination in a process known as styrene


OH 

Rapeseed oil 

173 

O 

OH 

O 3 

OH 

174 

O 

+ 

O 

O 

175 

Castor oil 

O 

H 

177 

OH 

O 

175 

O 

+ 

176 

OH 

O 

1) cyclopentanone/base 

2) H+ 

3) H2/cat. 

4) RCO 3 H 

O O 

FIGURE 5.29 Some natural feedstocks for synthesis of lipids and polyketides. 

179 

178 

H+ 

O 

O 

monomer/propylene oxide (SMPO) process. The epoxide (186) of styrene serves as an intermediate 

for 2-phenylethanol (47) and phenylacetaldehyde (187), both of which occur widely in essential oils. 

2-Phenylethanol is also available directly from benzene by Lewis acid catalyzed addition of ethylene 

oxide and as a by-product of the SMPO process. Currently, the volume available from the 

SMPO process provides most of the requirement. All of these processes are illustrated in 

Figure 5.31. 

O 

O 

O 

56 

O 

57 

O 

O 

O 

O 

HO 

53 

HO 

HO 

54 55 

O 

O 

O 

O 

O 

O 

O 

59 60 61 

FIGURE 5.30 Shikimates from eugenol and safrole.


OH 

OH 

O 2 

[O] 

OH 

182 

cat. 

183 184 

O 2 

180 

cat. 

cat. 

185 

O 

By-product 

[O] 

AlCl 3 

H + 

OH 

O 

O 

47 H / cat. 2 186 187 

FIGURE 5.31 Benzene as a feedstock for shikimates. 

Phenol (183) and related materials, such as guaiacol (188), were once isolated from coal tar 

but the bulk of their supply is currently produced from benzene via cumene as shown in 

Figure 5.31. The use of these intermediates to produce shikimates is shown in Figure 5.32. 

In principle, anethole (53) and estragole (methylchavicol) (52) are available from phenol, but in 

practice, the demand is met by extraction from turpentine. Carboxylation of phenol gives salicylic 

acid (38) and hence serves as a source for the various salicylate esters. Formylation of 

phenol by formaldehyde, in the presence of a suitable catalyst, has now replaced the Reimer– 

Tiemann reaction as a route to hydroxybenzaldehydes. The initial products are saligenin (189) 

and p-hydroxybenzyl alcohol (190), which can be oxidized to salicylaldehyde (191) and 

p-hydroxybenzaldehyde (192), respectively. Condensation of salicylaldehyde with acetic acid/ 

acetic anhydride gives coumarin (50) and O-alkylation of p-hydroxybenzaldehyde gives anisaldehyde 

(44). As mentioned earlier, oxidation of phenol provides a route to catechol (184) and 

guaiacol (188). The latter is a precursor for vanillin, and catechol also provides a route to 

heliotropin (61) via methylenedioxy benzene (193). 

Oxidation of toluene (181) with air or oxygen in the presence of a catalyst gives benzyl alcohol 

(194), benzaldehyde (195), or benzoic acid (196) depending on the chemistry employed. The 

demand for benzoic acid far exceeds that for the other two oxidation products and so such processes 

are usually designed to produce mostly benzoic acid with benzaldehyde as a minor product. 

For the fragrance industry, benzoic acid is the precursor for the various benzoates of interest while 

benzaldehyde, through aldol-type chemistry, serves as the key intermediate for cinnamate esters 

(such as methyl cinnamate (197)) and cinnamaldehyde (48). Reduction of the latter gives cinnamyl 

alcohol (49) and hence, through esterification, provides routes to all of the cinnamyl esters. 

Chlorination of toluene under radical conditions gives benzyl chloride (198). Hydrolysis of the 

chloride gives benzyl alcohol (194), which can, in principle, be esterified to give the various benzyl 

esters (199) of interest. However, these are more easily accessible directly from the chloride by 

reaction with the sodium salt of the corresponding carboxylic acid. All of these conversions are 

shown in Figure 5.33. 

Methyl anthranilate (41) is synthesized from either naphthalene (200) or o-xylene (201) as shown 

in Figure 5.34. Oxidation of either starting material produces phthalic acid (202). Conversion of this 

diacid to its imide, followed by the Hoffmann reaction, gives anthranilic acid and the methyl ester 

can then be obtained by reaction with methanol.


O 

53 

O 

52 

OH 

O 

OH 

183 

38 

OH 

188 

HO 

OH 

184 

189 

OH 

OH 

+ 

HO 

190 

OH 

O 

OH 

O 

O 

193 

191 

OH 

O 

HO 

192 

O 

HO 

O 

O 

O 

O 

O 

O 

O 

O 

O 

55 61 50 44 

FIGURE 5.32 Synthesis of shikimates from phenol. 

In volume terms, the terpenoids represent the largest group of natural and nature identical fragrance 

ingredients (Däniker, 1987; Sell, 2007). The key materials are the rose alcohols [geraniol 

(63)/nerol (90), linalool (23), and citronellol (91)], citronellal (103), and citral (71). Interconversion 

of these key intermediates is readily achieved by standard functional group manipulation. Materials 

in this family serve as starting points for the synthesis of a wide range of perfumery materials 

including esters of the rose alcohols. The ionones are prepared from citral by aldol condensation 

followed by cyclization of the intermediate y-ionones. 

The sources of the above key substances fall into three main categories: natural extracts, turpentine, 

and petrochemicals. The balance depends on economics and also on the product in question. 

For example, while about 10% of geraniol is sourced from natural extracts, it is only about 1% in the 

case of linalool. Natural grades of geraniol are obtained from the oils of citronella, geranium, and 

palmarosa (including the variants jamrosa and dhanrosa). Citronella is also used as a source of citronellal. 

Ho, rosewood, and linaloe were used as sources of linalool but conservation and economic 

factors have reduced these sources of supply very considerably. Similarly, citral was once extracted 

from Litsea cubeba but over-harvesting has resulted in loss of that source. 

Various other natural extracts are used as feedstocks for the production of terpenoids as 

shown in Figure 5.35. Two of the most significant ones are clary sage and the citrus oils (obtained 

as by-products of the fruit juice industry). After distillation of the oil from clary sage, sclareol (203) 

is extracted from the residue and this serves as a starting material for naphthofuran (204), known


181 

Cl 

RCO 2 Na 

O 

O 

R 

199 

[O] 

Cl 2 /R . or hn 

198 

H 2 O 

O OH O 

+ 

195 

194 

OH 

196 

O 

O 

O 

OH 

197 

48 

49 

FIGURE 5.33 Shikimates from toluene. 

under trade names such as Ambrofix, Ambrox, and Ambroxan. The conversion is shown in Figure 

5.35. Initially, sclareol is oxidized to sclareolide (205). This was once effected using oxidants such 

as permanganate and dichromate but nowadays, the largest commercial process uses a biotechnological 

oxidation. Sclareolide is then reduced using lithium aluminum hydride, borane, or similar 

reagents and the resulting diol is cyclized to the naphthofuran. d-Limonene (73) and valencene 

(206) are both extracted from citrus oils. Reaction of d-limonene with nitrosyl choride gives an 

adduct that is rearranged to the oxime of l-carvone and subsequent hydrolysis produces the free 

ketone (105). Selective oxidation of valencene gives nootkatone (135). 

Turpentine is obtained by tapping of pine trees and this product is known as gum turpentine. 

However, a much larger commercial source is the so-called crude sulfate turpentine (CST), which 

is obtained as a by-product of the Kraft paper process. The major components of turpentine are the 

two pinenes with a-pinene (65) predominating. Turpentine also serves as a source of p-cymene (83) 

and, as mentioned above, the shikimate anethole (53) (Zinkel and Russell, 1989). 

Figure 5.36 shows some of the major products manufactured from a-pinene (65) (Sell, 2003, 

2007). Acid-catalyzed hydration of a-pinene gives a-terpineol (74), which is the highest tonnage 

200 

OH 

O 

O 

O 

OH 

202 

41 

NH 2 

O 

201 

FIGURE 5.34 Synthesis of methyl anthranilate.


O 

OH 

OH 

O 

O 

H 

H 

H 

203 205 204 

73 105 

O 

206 

FIGURE 5.35 Partial synthesis of terpenoids from natural extracts. 

135 

O 

material of all those described here. Acid-catalyzed rearrangement of a-pinene gives camphene 

(89) and this, in turn, serves as a starting material for production of camphor (80). Hydrogenation 

of a-pinene gives pinane (207), which is oxidized to pinanol (208) using air as the oxidant. Pyrolysis 

of pinanol produces linalool (23) and this can be rearranged to geraniol (63). Hydrogenation of 

geraniol gives citronellol (91) whereas oxidation leads to citral (71). The major use of citral is not as 

74 89 

O 

OH 

80 

65 

OH 

OH 

OH 

207 208 

23 

63 

158 

O 

71 

O 

91 

OH 

FIGURE 5.36 Products from a-pinene.


OH 

76 

70 

63 

O 

94 

209 

OH 

FIGURE 5.37 Products from b-pinene. 

a material in its own right, but as a starting material for production of ionones, such as a-ionone 

(158) and vitamins A, E, and K. 

Some of the major products manufactured from b-pinene (76) are shown in Figure 5.37. Pyrolysis 

of b-pinene gives myrcene (70) and this can be “hydrated” (not in one step but in a multistage process) 

to give geraniol (63). The downstream products from geraniol are then the same as those 

described in the preceding paragraph and shown in Figure 5.36. Myrcene is also a starting point for 

d-citronellal (209), which is one of the major feedstocks for the production of l-menthol (94) as will 

be described below. 

Currently, there are two major routes to terpenoids that use petrochemical starting materials 

(Sell, 2003, 2007). The first to be developed is an improved version of a synthetic scheme demonstrated 

by Arens and van Dorp in 1948. The basic concept is to use two molecules of acetylene (210) 

and two of acetone (211) to build the structure of citral (71). The route, as it is currently practised, 

is shown in Figure 5.38. Addition of acetylene (210) to acetone (211) in the presence of base gives 

methylbutynol (212), which is hydrogenated, under Lindlar conditions, to methylbutenol (213). The 

second equivalent of acetone is introduced as the methyl ether of its enol form, that is, methoxypropene 

(214). This adds to methylbutenol and the resultant adduct undergoes a Claisen rearrangement 

210 

OH 

H 2 

/cat. 

OH 

O 

Base 

211 212 

213 

O 

214 

71 

O 

H + 

OH 

216 

210 

Base 

O 

215 

FIGURE 5.38 Citral from acetylene and acetone.


220 

H O 

OH 

218 

Pd 

OH 

217 

219 

1) Ag:SiO 2 

/O 2 

2) Pd 

221 

O 

O 

O 

O 

O 

71 

223 

222 

FIGURE 5.39 Citral from isobutylene and acetone. 

to give methylheptenone (215). Base catalyzed addition of the second acetylene to this gives dehydrolinalool 

(216), which can be rearranged under acidic conditions to give citral (71). Hydrogenation 

of dehydrolinalool under Lindlar conditions gives linalool (23) and thus opens up all the routes to 

other terpenoids as described above and illustrated in Figure 5.36. 

The other major route to citral is shown in Figure 5.39. This starts from isobutene (217) and 

formaldehyde (218). The ene reaction between these produces isoprenol (219). Isomerization of 

isoprenol over a palladium catalyst gives prenol (220) and aerial oxidation over a silver catalyst 

gives prenal (senecioaldehyde) (221). When heated together, these two add together to form the enol 

ether (222), which then undergoes a Claisen rearrangement to give the aldehyde (223). This latter 

molecule is perfectly set up (after rotation around the central bond) for a Cope rearrangement to give 

citral (71). Development chemists have always striven to produce economic processes with the highest 

overall yield possible thus minimizing the volume of waste and hence environmental impact. 

This synthesis is a very good example of the fruits of such work. The reaction scheme uses no 

reagents, other than oxygen, employs efficient catalysts, and produces only one by-product, water, 

which is environmentally benign. 

The synthesis of l-menthol (94) provides an interesting example of different routes operating in 

economic balance. The three production routes in current use are shown in Figure 5.40. The oldest 

N N O 

70 224 225 

209 

Mentha arvensis 

OH 

OH 

OH 

226 97 

227 

FIGURE 5.40 Competing routes to l-menthol. 

94 

OH


and simplest route is extraction from plants of the Mentha genus and Mentha arvensis (cornmint) in 

particular. This is achieved by freezing the oil to force the l-menthol to crystallize out. Diethylamine 

can be added to myrcene (70) in the presence of base and rearrangement of the resultant allyl amine 

(224) using the optically active catalyst ruthenium (S)-BINAP perchlorate gives the homochiral 

enamine (225). This can then be hydrolyzed to d-citronellol (209). The chiral center in this molecule 

ensures that, on acid catalyzed cyclization, the two new stereocentres formed possess the correct 

stereochemistry for conversion, by hydrogenation, to give l-menthol as the final product. Starting 

from the petrochemically sourced m-cresol (226), propenylation gives thymol (97), which can be 

hydrogenated to give a mixture of all eight stereoisomers of menthol (227). Fractional distillation of 

this mixture gives racemic menthol. Resolution was originally carried out by fractional crystallization, 

but recent advances include methods for the enzymic resolution of the racemate to give 

l-menthol. 

Estimation of the long-term sustainability of each of these routes is complex and the final outcome 

is far from certain. In terms of renewability of feedstocks, m-cresol might appear to be at a 

disadvantage against mint or turpentine. However, as the world’s population increases, use of agricultural 

land will come under pressure for food production, hence increasing pressure on mint 

cultivation and turpentine, hence, myrcene is a by-product of paper manufacture and is therefore 

vulnerable to trends in paper recycling and “the paperless office.” In terms of energy consumption, 

and hence current dependence on petrochemicals, the picture is also not as clear as might be imagined. 

Harvesting and processing of mint requires energy and, if the crop is grown in the same field 

over time, fertilizer is required and this is produced by the very energy-intensive Haber process. 

The energy required to turn trees in a forest into pulp at a sawmill is also significant and so turpentine 

supply will also be affected by energy prices. No doubt, the skills of process chemists will be 

of increasing importance as we strive to make the best use of natural resources and minimize 

energy consumption (Baser and Demirci, 2007). 

REFERENCES 

Arctander, S., 1960. Perfume and Flavour Materials of Natural Origin. Elizabeth, NJ: Steffen Arctander. 

(Currently available from Allured Publishing Corp.) 

Arctander, S., 1969. Perfume and Flavor Chemicals (Aroma Chemicals). Montclair, NJ: Steffen Arctander. 

(Currently available from Allured Publishing Corp.) 

Baser, K.H.C. and F. Demirci, 2007. Chemistry of Essential Oils, In Flavours and Fragrances: Chemistry, 

Bioprocessing and Sustainability, R.G. Berger (ed.), pp. 43–86, Berlin: Springer. 

Bauer, K. and J. Panten, 2006. Common Fragrance and Flavor Materials: Preparation, Properties and Uses. 

New York: Wiley-VCH. 

Bu’Lock, J.D., 1965. The Biosynthesis of Natural Products. New York: McGraw-Hill. 

Croteau, R., 1987. Biosynthesis and catabolism of monoterpenoids. Chem. Rev., 87: 929. 

Däniker, H.U., 1987. Flavors and Fragrances (Worldwide). Stamford: SRI International. 

Devon, T.K. and A.I. Scott, 1972. Handbook of Naturally Occurring Compounds, Vol. 2, The Terpenes. 

New York: Academic Press. 

Essential Oils Database, Boelens Aromachemical Information Systems, Huizen, The Netherlands. 

Gildemeister, E. and Fr. Hoffmann, 1956. Die Ätherischen ÖIe. Berlin: Akademie-Verlag. 

Günther, E., 1948. The Essential Oils. New York: D van Nostrand. 

Hay, R.K.M. and P.G. Waterman (eds), 1993. Volatile Oil Crops: Their Biology, Biochemistry and Production. 

London: Longman. 

Lawrence, B.M., 1985. A review of the world production of essential oils. Perfumer Flavorist, 10(5): 1. 

Leffingwell & Associates, http://www.leffingwell.com/ 

Lehninger, A.L., 1993. Principles of Biochemistry. New York: Worth. 

Mann, J., R.S. Davidson, J.B. Hobbs, D.V. Banthorpe, and J.B. Harbourne, 1994. Natural Products: Their 

Chemistry and Biological Signifi cance. London: Longman. 

Matthews C.K. and K.E. van Holde. 1990. Biochemistry. Redwood City: Benjamin/Cummings. 

Sell, C.S., 2003. A Fragrant Introduction to Terpenoid Chemistry. Cambridge: Royal Society of Chemistry.


Sell, C.S. (ed.), 2006. The Chemistry of Fragrances from Perfumer to Consumer, 2nd ed. Cambridge: Royal 

Society of Chemistry. 

Sell, C.S., 2007. Terpenoids. In the Kirk-Othmer Encyclopedia of Chemical Technology, 5th ed. New York: 

Wiley. 

Williams, D.G., 1996. The Chemistry of Essential Oils. Weymouth: Micelle Press. 

Williams, D.G., 2004. Perfumes of Yesterday. Weymouth: Micelle Press. 

Zinkel, D.F. and J. Russell (eds), 1989. Naval Stores. New York: Pulp Chemicals Association, Inc.

6 

Analysis of Essential Oils 

Barbara d’Acampora Zellner, Paola Dugo, Giovanni Dugo, 

and Luigi Mondello 

CONTENTS 

6.1 Introduction ....................................................................................................................... 151 

6.2 Classical Analytical Techniques ........................................................................................ 152 

6.3 Modern Analytical Techniques ......................................................................................... 156 

6.3.1 Use of GC and Linear Retention Indices in Essential Oils Analysis .................... 157 

6.3.2 Gas Chromatography-Mass Spectrometry ............................................................. 158 

6.3.3 Fast GC for Essential Oil Analysis ........................................................................ 159 

6.3.4 Gas Chromatography-Olfactometry for the Assessment of Odor-Active 

Components of Essential Oils ................................................................................ 162 

6.3.5 Gas Chromatographic Enantiomer Characterization of Essential Oils ................. 164 

6.3.6 LC and Liquid Chromatography Hyphenated to MS in the Analysis 

of Essential Oils ................................................................................................ 165 

6.3.7 Multidimensional Gas Chromatographic Techniques ........................................... 167 

6.3.8 Multidimensional Liquid Chromatographic Techniques ....................................... 174 

6.3.9 On-Line Coupled Liquid Chromatography-Gas Chromatography (LC-GC) ........ 176 

6.4 General Considerations on Essential Oil Analysis ............................................................ 177 

References .................................................................................................................................. 177 


The production of essential oils was industrialized in the first half of the nineteenth century, due to 

an increased demand for these matrices as perfume and flavor ingredients [1]. As a consequence the 

need to perform their systematic investigation also became unprecedented. It is interesting to point 

out that in the second edition of Parry’s monograph, published in 1908, about 90 essential oils were 

listed, and very little was known about their composition [2]. Further important contributions to the 

essential oil research field were made by Semmler [3], Gildemeister and Hoffmann [4], Finnemore [5], 

and Guenther [6]. Obviously, it is unfeasible to cite all the researchers involved in the progress of 

essential oil analysis. 

As widely acknowledged, the composition of essential oils is mainly represented by mono- and 

sesquiterpene hydrocarbons and their oxygenated (hydroxyl and carbonyl) derivatives, along with 

aliphatic aldehydes, alcohols, and esters. Terpenes can be considered as the most structurally varied 

class of plant natural products, derived from the repetitive fusion of branched five-carbon units 

(isoprene units) [7]. In this respect, analytical methods applied in the characterization of essential 

oils have to account for a great number of molecular species. Moreover, it is also of great importance 

to highlight that an essential oil chemical profile is closely related to the extraction procedure 

employed and, hence, the choice of an appropriate extraction method becomes crucial. On the basis 

151


of the properties of the plant material, the following extraction techniques can be applied: steam 

distillation (SD), possibly followed by rectification and fractionation, solvent extraction (SE), fractionation 

of solvent extracts, maceration, expression (cold pressing of citrus peels), enfl eurage, 

supercritical fluid extraction (SFE), pressurized-fluid extraction, simultaneous distillation– extraction 

(SDE), Soxhlet extraction, microwave-assisted hydrodistillation (MAHD), dynamic (DHS) and 

static (SHS) headspace (HS) techniques, solvent-assisted flavor evaporation (SAFE), solid-phase 

microextraction (SPME), and direct thermal desorption (DTD), among others. 

Apart from the great interest in performing systematic studies on essential oils, there is also the 

necessity to trace adulterations, mainly in economically important essential oils. As can be observed 

with almost all commercially available products, market changes occur rapidly, affecting individual 

plants, or industrial processes. In general, market competition, along with the limited interest of 

consumers with regard to essential oil quality, may induce producers to adulterate their commodities 

by the addition of products of lower value. Different types of adulterations can be encountered: 

(a) the simple addition of natural and/or synthetic compounds, with the aim of generating an oil 

characterized by specific quality values, such as density, optical rotation, residue percentage, ester 

value, and so on or (b) refined sophistications in the reconstitution and counterfeiting of commercially 

valuable oils. In the latter case, natural and/or synthetic compounds are added to enhance the 

market value of an oil, attempting to maintain the qualitative, or even quantitative, composition of 

natural essential oils, and making adulteration detection a troublesome task. Consequently, the 

exploitation of modern analytical methodologies, such as gas chromatography (GC) and related 

hyphenated techniques, is practically unavoidable. 

As a consequence of diffused illegal practice in the production of essential oils, there has been 

an enhanced request for legal standards of commercial purity, while essential oils were included as 

herbal drugs in pharmacopoeias [8–12], and also in a compendium denominated as Martindale: the 

complete drug reference (formerly named as Martindale’s: The Extra Pharmacopoeia) [13]. In view 

of the need for standardized methodologies, these pharmacopoeias commonly include the descriptions 

of several tests, processes, and apparatus. In addition, various international standard regulations 

have been introduced in which the characteristics of specific essential oils are described, and 

the botanical source and physicochemical requirements are reported. Such standardized information 

was created to facilitate the assessment of quality; for example, ISO 3761 (1997) specifies that 

for Brazilian rosewood essential oil (Aniba rosaeodora Ducke) an alcohol content in the 84–93% 

range, determined as linalool, is required [14]. Moreover, guidelines for the analysis of essential oils 

are also available; for example, for the measurement of the refractive index (ISO 280, 1198) and 

optical rotation (ISO 592, 1998), as also for GC analysis using capillary columns [ISO chromatography 

(ISO 8432, 1987)] [15]. The French Standards Association (Association Française de 

Normalisation—AFNOR) also develops norms and standard methods dedicated to the essential oil 

research field, with the aim of assessing quality in relation to specific physical, organoleptic, chemical, 

and chromatographic characteristics [16]. 

The present contribution provides an overview on the classical and modern analytical techniques 

commonly applied to characterize essential oils. Modern techniques will be focused on chromatographic 

analyses, including theoretical aspects and applications. 

6.2 CLASSICAL ANALYTICAL TECHNIQUES 

The thorough study of essential oils is based on the relationship between their physical and chemical 

properties, and is completed by the assessment of organoleptic qualities. The earliest analytical 

methods applied in the investigation of an essential oil were commonly focused on quality aspects, 

concerning mainly two properties, namely identity and purity [17]. 

The following techniques are commonly applied to assess an essential oil physical properties 

[6,17]: specific gravity (SG), which is the most frequently reported physicochemical property, and is 

a special case of relative density, [r] T(°C) , defined as the ratio of the densities of a given oil and of

Analysis of Essential Oils 153 

water when both are at identical temperatures. The attained value is characteristic for each essential 

oil and commonly ranges between 0.696 and 1.118 at 15°C [4]. In cases in which the determinations 

were made at different temperatures, conversion factors can be used to normalize data. 

The measurement of optical rotation, [a] D 

20 

, either dextrorotatory or laevorotatory, is also widely 

recognized. Optical activity is determined by using a polarimeter, with the angle of rotation depending 

on a series of parameters, such as oil nature, the length of the column through which the light 

passes, the applied wavelength, and the temperature. The degree and direction of rotation are of 

great importance for purity assessments, since they are related to the structures and the concentration 

of chiral molecules in the sample. Each optically active substance has its own specific rotation, 

as defined in Biot’s law: 

T 

a 

a = 

cl ◊ 

T l 

[] l 

, 

where a is the optical rotation at a temperature T expressed in °C, l is the optical path length in dm, 

l is the wavelength, and c is the concentration in g/100 mL. It is worthy of note that a standard 

100 mm tube is commonly used; in cases in which darker or lighter colored oils are analyzed, longer 

or shorter tubes are used, respectively, and the rotation should be extrapolated for a 100-mm-long 

tube. Moreover, prior to the measurement, the essential oil should be dried out with anhydrous 

sodium sulfate and filtered. 

The determination of the refractive index, [h] D 

20 

, also represents a characteristic physical constant 

of an oil, usually ranging from 1.450 to 1.590. This index is represented by the ratio of the sine of 

the angle of incidence (i) to the sine of the angle of refraction (e) of a beam of light passing from a 

less dense to a denser medium, such as from air to the essential oil: 

sini 

sine 

= 

N 

, 

n 

where N and n are, respectively, the indices of the more and the less dense medium. The Abbé-type 

refractometer, equipped with a monochromatic sodium light source, is recommended for routine 

essential oil analysis; the instrument is calibrated through the analysis of distilled water at 20°C, 

producing a refractive index of 1.3330. In cases in which the measurement is performed at a 

temperature above or below 20°C, a correction factor per degree must be added or subtracted, 

respectively [18]. 

A further procedure that can be applied for the purity assessment of essential oils is based on 

water solubility; the test, which reveals the presence of polar substances, such as alcohols, glycols 

and their esters, and glycerin acetates, is carried out as follows: the oil is added to a saturated solution 

of sodium chloride, which after homogenization is divided into two phases; the volume of the 

oil, which is the organic phase, should remain unaltered; volume reduction indicates the presence of 

water-soluble substances. On the other hand, the solubility, or immiscibility, of an essential oil in 

ethanol reveals much on its quality. Considering that essential oils are slightly soluble in water and 

are miscible with ethanol, it is simple to determine the number of volumes of water-diluted ethanol 

required for the complete solubility of one volume of oil; the analysis is carried out at 20°C, if the 

oil is liquid at this temperature. It must be emphasized that oils rich in oxygenated compounds 

are more readily soluble in dilute ethanol than those richer in hydrocarbons. Moreover, aged or 

improperly stored oils frequently present decreased solubility [6]. 

The investigation on the solubility of essential oils in other media is also widely accepted, such 

as the evaluation of the presence of water by means of a simple procedure: the addition of a volume 

of essential oil to an equal volume of carbon disulfide or chloroform; in case the oil is rich in oxygenated 

constituents it may contain dissolved water, generating turbidity. A further solubility test,


in which the oil is dissolved in an aqueous solution of potassium hydroxide, is applied to oils containing 

molecules with phenolic groups; finally, the incomplete dissolution of oils rich in aldehydes 

in a dilute bisulfite solution may denote the presence of impurities. 

The estimation of melting and congealing points, as well as the boiling range of essential oils, is 

also of great importance for identity and purity assessments. Melting point evaluations are a valuable 

modality to control essential oil purity, since a large number of molecules generally comprised 

in essential oils melt within a range of 0.5°C or, in the case of decomposition, over a narrow temperature 

range. On the other hand, the determination of the congealing point is usually applied in 

cases where the essential oil consists mainly of one molecule, such as the oil of cloves that contains 

about 90% of eugenol. In the latter case, such a test enables the evaluation of the percentage amount 

of the abundant compound. At congealing point, crystallization occurs accompanied by heat liberation, 

leading to a rapid increase in temperature which is then stabilized at the so-called congealing 

point. A further purity evaluation method is represented by the boiling range determination, through 

which the percentage of oil that distils below a certain temperature or within a temperature range is 

investigated. 

An additional test usually performed in essential oil analysis is the evaporation residue, in which 

the percentage of the oil that is not released at 100°C is determined. In the specific case of citrus 

oils, this test enables purity assessment, since a lower amount of residue in an expressed oil may 

indicate an addition of distilled volatile components to the oil; an increased residue amount reveals 

the possible presence of terpenes with higher molecular weights, through the addition of single 

compounds (or other essential oils), or of heavier oils, such as rosin oil, cheaper citrus oils, or by 

directly using the citrus oil residue. An example consists of the addition of lime oil to sophisticate 

lemon oils. In oxidized or polymerized oils the presence of less volatile compounds is common; in 

this case, a simple test may be carried out by applying a drop of oil on a piece of filter paper; if a 

transparent spot persists for a period of over 24 h, the oil is most probably degradated. Furthermore, 

the residue can be subjected to acid and saponification number analyses; for instance, the addition 

of rosin oil would increase the acid number since this oil, differently from other volatile oils, is 

characterized by the presence of complex acids. By definition, the acid number is the number of 

milligrams of potassium hydroxide required to neutralize the free acids contained in 1 g of an oil. 

This number is preserved in cases in which the essential oil has been carefully dried and stored in 

dark and airtight recipients. As commonly observed, the acid number increases along the aging 

process of an oil; oxidation of aldehydes and hydrolysis of esters trigger the increase of the acid 

number. 

Classical methodologies have been also widely applied to assess essential oil chemical properties 

[6,17], such as the determination of the presences of halogenated hydrocarbons and of heavy 

metals. The former investigation is exploited to reveal the presence of halogenated compounds, 

commonly added to the oils for adulteration purposes. Several tests have been developed for halogen 

detection, with the Beilstein method [19] the one most reported. In practice, a copper wire is 

cleaned and heated in a Bunsen burner flame to form a coating of copper (II) oxide. It is then 

dipped in the sample to be tested and once again heated in the flame. A positive test is indicated by 

a green flame caused by the formation of a copper halide. Attention is to be paid to positive 

or inconclusive results, since they may be induced by trace amounts of organic acids, nitrogencontaining 

compounds [6], or salts [20]. An alternative to the Beilstein method is the sodium 

fusion test, in which the oil is first mineralized, and in the case halogenated hydrocarbons are present, 

a residue of sodium halide is formed, which is soluble in nitric acid, and precipitates as the 

respective silver halide by the addition of a small amount of silver nitrate solution [17]. With regard 

to the detection of heavy metals, several tests are described to investigate and ensure the absence 

especially of copper and lead. One method is based on the extraction of the essential oil with a 

diluted hydrochloric acid solution, followed by the formation of an aqueous phase to which a 

buffered thioacetamide solution is added. The latter reagent leads to the formation of sulfite ions 

that are used in the detection of heavy metals.


The determination of esters derived from phthalic acid is also of great interest for the toxicity 

evaluation of an essential oil. Considering that esters commonly contained in essential oils are 

derived from monobasic acids, at first, saponification is carried out through the addition of an ethanolic 

potassium hydroxide solution. The formed potassium phthalate, which is not soluble in ethanol, 

generates a crystalline precipitate [17]. 

The use of qualitative information alone is not sufficient to correctly characterize an essential oil, 

and quantitative data are of extreme importance. Classical methods are generally focused on chemical 

groups and the assessment of quantitative information through titration is widely applied, for 

example, for the acidimetric determination of saponified terpene esters. Saponification can be performed 

with heat, and in the case readily saponified esters are to be investigated, in the cold, and 

afterward the alkali excess is titrated with aqueous hydrochloric acid; thereafter the ester number 

can be calculated. A further test is the determination of terpene alcohols by acetylating with acetic 

anhydride; part of the acetic anhydride is consumed in the reaction and can be quantified through 

titration of acetic acid with sodium hydroxide. The percentage of alcohol can then be calculated. 

The latter method is applied when the alcoholic constituents of an essential oil are not well known; 

in case these are established, the oil is saponified and the ester number of the acetylated oil is calculated 

and used to estimate the free alcohol content. 

Other chemical classes worthy of mention are aldehydes and ketones that may be investigated 

through different tests. The bisulfite method is recommended for essential oils rich in aldehydic 

compounds, as lemongrass, bitter almond, and cassia, while the neutral sulfite test is more suitable 

for ketone-rich oils, as spearmint, caraway, and dill oils. For essential oils presenting small amounts 

of aldehydes and ketones, the hydroxylamine method, or its modification, the Stillman–Reed method 

are the most indicated ones [20]. In the latter case, the aldehyde and ketone contents are determined 

through the addition of a neutralized hydroxylamine hydrochloride solution, and subsequent titration 

with standardized acid (the Stillman–Reed method) [21]; in the former analytical procedure, 

the aldehyde and ketone content is established through the addition of a hydroxylamine hydrochloride 

solution, followed by neutralization with the reaction products, that is, alkali of the hydrochloric 

acid. These methods may be applied in the determination of citral in citrus oils and carvone 

in caraway oil. With regard to the determination of phenols, such as eugenol in clove oil or thymol 

and carvacrol in thyme oil, the test is commonly made through the addition of potassium hydroxide 

solutions, forming water-soluble salts. It has to be pointed out that besides phenols, other constituents 

are soluble in alkali solutions and in water [6,20]. 

Essential oils are also often analyzed by means of chromatographic methods. In general, the 

principle of chromatography is based on the distribution of the constituents to be separated between 

two immiscible phases; one of these is a stationary bed (a stationary phase) with a large surface 

area, while the other is a mobile phase that percolates through the stationary bed in a definite direction 

[22]. Planar chromatography may be referred to as a classical method for essential oil analysis, 

being well represented by thin-layer chromatography (TLC) and paper chromatography (PC). In 

both techniques, the stationary phase is distributed as a thin layer on a flat support, in PC being selfsupporting, 

while in TLC coated on a glass, plastic, or metal surface; the mobile phase is allowed to 

ascend through the layer by capillary forces. TLC is a fast and inexpensive method for identifying 

substances and testing the purity of compounds, being widely used as a preliminary technique providing 

valuable information for subsequent analyses [23]. Separations in TLC involve the distribution 

of one or a mixture of substances between a stationary phase and a mobile phase. The stationary 

phase is a thin layer of adsorbent (usually silica gel or alumina) coated on a plate. The mobile phase 

is a developing solvent that travels up the stationary phase, carrying the samples with it. Components 

of the samples will separate on the stationary phase according to their stationary phase–mobile 

phase affinities [24]. In practice, a small quantity of the sample is applied near one edge of the plate 

and its position is marked with a pencil. The plate is then positioned in a developing chamber with 

one end immersed in the developing solvent, the mobile phase, avoiding the direct contact of the 

sample with the solvent. When the mobile phase reaches about two-third of the plate length, the


plate is removed, dried, the solvent front is traced, and the separated components are located. In 

some cases the spots are directly visible, but in others they must be visualized by using methods 

applicable to almost all organic samples, such as the use of a solution of iodine or sulfuric acid, both 

of which react with organic compounds yielding dark products. The use of an ultraviolet (UV) lamp 

is also advisable, especially if a substance that aids in the visualization of compounds is incorporated 

into the plate, as is the case of many commercially available TLC plates. Data interpretation 

is made through the calculation of the ratio of fronts (R f ) value for each spot, which is defined as 

R 

f 

= 

Z 

Z 

S 

St 

, 

where Z S is the distance from the starting point to the center of a specific spot, and Z St is the distance 

from the starting point to the solvent front [24,25]. A concise review on TLC has been made by 

Sherma [26]. 

The R f value is characteristic for any given compound on the same stationary phase using the 

identical mobile phase. Hence, known R f values can be compared to those of unknown substances 

to aid in their identification [24]. On the other hand, separations in PC involve the same principles 

as those in TLC, differing in the use of a high-quality filter paper as the stationary phase instead of 

a thin adsorbent layer, by the increased time requirements and poorer resolution. It is worthy to 

highlight that TLC has largely replaced PC in contemporary laboratory practice [22]. 

As is well known, essential oils can be characterized by their organoleptic properties, an assessment 

that involves human subjects as measuring tools. These procedures present an immediate 

problem, linked to the innate variability between individuals, not only as a result of their previous 

experiences and expectations, but also to their sensitivity [27]. In this respect, individuals are 

selected and screened for specific anosmia, as proposed by Friedrich et al. [28]. In the case no insensitivities 

are found, the panelists are introduced to two sensorial properties, quality, and intensity. 

Odor quality is described according to the odor families, while intensity is measured through the 

rating of a sensation based on an intensity interval scale. The assessment of an essential oil odor can 

be performed through its addition to filter paper strips and subsequent evaluation by the panelists. 

Considering that each volatile compound is characterized by a different volatility, the evaluation of 

the paper strip in different periods of time enables the classification of the odors in top, middle, and 

bottom notes [29]. In addition, the olfactive assessment during the determination of the evaporation 

residue is also of significance, since by-notes of low-boiling adulterants or contaminants may be 

detected as the oil vaporizes, and the odor of the final hot residue can reveal the addition of highboiling 

compounds. Olfactive analyses are also valuable after the determination of phenols in essential 

oils, by studying the nonphenolic portion [6]. 

It is noteworthy that the use of the earliest analytical techniques for the systematic study of essential 

oils, such as SG, relative density, optical activity, and refractive index, or melting, congealing, 

and boiling points determinations, are generally applied for the assessment of pure compounds, and 

may be extended to evaluate essential oils composed of a major compound. Classical methods cannot 

be used as stand-alone methods and need to be combined with modern analytical techniques, 

especially GC, for the assessment of essential oil genuineness. 

6.3 MODERN ANALYTICAL TECHNIQUES 

Most of the methods applied in the analysis of essential oils rely on chromatographic procedures, 

which enable component separation and identification. However, additional confirmatory evidence 

is required for reliable identification, avoiding equivocated characterizations. 

In the early stages of research in the essential oil field, attention was devoted to the development 

of methods in order to acquire deeper knowledge on the profiles of volatiles; however, this analytical


task was made troublesome due to the complexity of these real-world samples. Over the last decades, 

the aforementioned research area has benefited from the improvements in instrumental analytical 

chemistry, especially in the chromatographic area, and, nowadays, the number of known constituents 

has drastically increased. 

The primary objective in any chromatographic separation is always the complete resolution of 

the compounds of interest, in the minimum time. To achieve this task the most suitable analytical 

column (dimension and stationary phase type) has to be used, and adequate chromatographic parameters 

must be applied to limit peak enlargement phenomena. A good knowledge of chromatographic 

theory is, indeed, of great support for the method optimization process, as well as for the development 

of innovative techniques. 

In gas chromatographic analysis, the compounds to be analyzed are vaporized and eluted by the 

mobile gas phase, the carrier gas, through the column. The analytes are separated on the basis of 

their relative vapor pressures and affinities for the stationary bed. On the other hand, in liquid chromatographic 

analysis, the compounds are eluted by a liquid mobile phase consisting of a solvent or 

a mixture of solvents, the composition of which may vary during the analysis (gradient elution), and 

are separated according to their affinities for the stationary bed. In general, the volatile fraction of 

an essential oil is analyzed by GC, while the nonvolatile by liquid chromatography (LC). 

At the outlet of the chromatography column, the analytes emerge separated in time. The analytes 

are then detected and a signal is recorded generating a chromatogram, which is a signal versus time 

graphic, and ideally with peaks presenting a Gaussian distribution-curve shape. The peak area and 

height are a function of the amount of solute present and its width is a function of band spreading in 

the column [30], while retention time can be related to the solute’s identity. Hence, the information 

contained in the chromatogram can be used for qualitative and quantitative analysis. 

6.3.1 USE OF GC AND LINEAR RETENTION INDICES IN ESSENTIAL OILS ANALYSIS 

The analysis of essential oils by means of GC began in the 1950s, when professor Liberti [31] started 

analyzing citrus essential oils only a few years after James and Martin first described gas–liquid 

chromatography (GLC), commonly referred to as GC [32], a milestone in the evolution of instrumental 

chromatographic methods. 

After its introduction, GC developed at a phenomenal rate, growing from a simple research novelty 

to a highly sophisticated instrument. Moreover, the current-day requirements for high resolution 

and trace analysis are satisfied by modern column technology. In particular, inert, thermostable, 

and efficient open-tubular columns are available, along with associated selective detectors and 

injection methods, which allow on-column injection of liquid and thermally labile samples. The 

development of robust fused-silica columns, characterized by superior performances to that of glass 

columns, brings open-tubular GC columns within the scope of almost every analytical laboratory. 

At present, essential oil GC analyses are more frequently performed on capillary columns, 

which, after their introduction, rapidly replaced packed GC columns. In general, packed columns 

support larger sample size ranges, from 10 to 20 mL, and thus the dynamic range of the analysis can 

be enhanced. Trace-level components can be easily separated and quantified without preliminary 

fractionation or concentration. On the other hand, the use of packed columns leads to lower resolution 

due to the higher pressure drop per unit length. Packed columns need to be operated at higher 

column flow rates, since their low permeability requires high pressures to significantly improve 

resolution [33]. It is worthy of note that since the introduction of fused-silica capillary columns 

considerable progress has been made in column technology, a great number of papers regarding GC 

applications on essential oils have been published. 

The choice of the capillary column in an essential oil GC analysis is of great importance for the 

overall characterization of the matrix; the stationary phase chemical nature and film thickness, as 

well as the column length and internal diameter, are to be considered. In general, essential oil GC 

analyses are carried out on 25–50 m columns, with 0.20–0.32 mm internal diameters, and 0.25 mm


stationary phase film thickness. It must be noted that the degree of separation of two components on 

two distinct stationary phases can be drastically different. As is well known, nonpolar columns 

produce boiling-point separations, while on polar stationary phases compounds are resolved according 

to their polarity. Considering that essential oil components, such as terpenes and their oxygenated 

derivatives, frequently present similar boiling points, these elute in a narrow retention time 

range on a nonpolar column. In order to overcome this limit, the analytical method can be modified 

by applying a slower oven temperature rate to widen the elution range of the oil or by using a polar 

stationary phase, as oxygenated compounds are more retained than hydrocarbons. However, choosing 

different stationary phases may provide little improvement as resolution can be improved for a 

series of compounds but new coelutions can also be generated. 

Considering gas chromatographic analyses using flame ionization detector (FID), thermal conductivity 

detector (TCD), or other detectors which do not provide structural information of the 

analyzed molecules and retention data, more precisely retention indices, are used as the primary 

criterion for peak assignment. The retention index system was based on the fact that each analyte is 

referenced in terms of its position between the two n-paraffins that bracket its retention time. 

Furthermore, the index calculation is based on a linear interpolation of the carbon chain length of 

these bracketing paraffins. The most thoroughly studied, diffused, and accepted retention index 

calculation methods are based on the logarithmic-based equation developed by Kováts in 1958 [34], 

for isothermal conditions, and on the equation propounded by van den Dool and Kratz in 1963 [35], 

which does not use the logarithmic form and is used in the case of temperature-programming conditions. 

Values calculated using the latter approach are commonly denominated in literature as retention 

index (I), linear retention index (LRI), or programmed-temperature retention index (PTRI or 

I T ), while the ones derived from the former equation are usually referred to as Kováts index (KI). 

In general, retention index systems are based on the incremental structure–retention relationship, 

namely, that any regular increase in a series of chemical structures should provide a regular increase 

in the corresponding retention times. This means that the retention index concept is not restricted to 

the use of n-alkanes as standards. In practice, any homologous series presenting a linear relationship 

between the adjusted retention time, being logarithmic based or not, and the carbon number can 

be used. 

In the characterization of volatiles, the most commonly applied reference series is n-alkanes. 

However, the latter commonly present fluctuant behavior on polar stationary phases. In consideration 

of the fact that retention index values are correlated to retention mechanisms, alternative 

standard series of intermediate polarity have been introduced, such as 2-alkanones, alkyl ethers, 

alkyl halides, alkyl acetates, and alkanoic acid methyl esters [22]. Shibamoto [36] suggested the use 

of polar compounds series, such as ethyl esters, as an alternative. The most feasible choice, when 

analyzing volatiles, is to apply reference series as n-alkanes, fatty acid ethyl esters (FAEEs), or fatty 

acid methyl esters (FAMEs), employed according to the stationary phase to be used. 

Additionally, it is highly advisable to use two analytical columns coated with stationary phases 

of distinct polarities to obtain two retention index values and enhance confidence in assignments 

[37–39]. Identifications made on a single column can only be accepted if used in combination with 

spectroscopic detection systems. When n-alkanes are used, it is accepted that the reproducibility of 

retention indices between different laboratories are comprised within an acceptable range of ±5 units 

for methyl silicone stationary phases, and ±10 units for polyethylene glycol phases. A further aspect 

of great importance, which is frequently overseen, is the analytical reproducibility of retention 

indexes. Moreover, it is worthwhile to highlight that in practice it was found that the use of an initial 

isothermal hold in the GC oven temperature program does not provide additional resolution [40]. 

6.3.2 GAS CHROMATOGRAPHY-MASS SPECTROMETRY 

Mass spectrometry (MS) can be defined as the study of systems through the formation of gaseous 

ions, with or without fragmentation, which are then characterized by their mass-to-charge ratios (m/z)


and relative abundances [41]. The analyte may be ionized thermally, by an electric field or by impacting 

energetic electrons, ions, or photons. 

During the past decade, there has been a tremendous growth in popularity of mass spectrometers 

as a tool for both, routine analytical experiments and fundamental research. This is due to a number 

of features including relatively low cost, simplicity of design and extremely fast data acquisition 

rates. Although the sample is destroyed by the mass spectrometer, the technique is very sensitive 

and only low amounts of material are used in the analysis. 

In addition, the potential of combined gas chromatography-mass spectrometry (GC-MS) for 

determining volatile compounds, contained in very complex flavor and fragrance samples, is well 

known. The subsequent introduction of powerful data acquisition and processing systems, including 

automated library search techniques, ensured that the information content of the large quantities of 

data generated by GC-MS instruments was fully exploited. The most frequent and simple identification 

method in GC-MS consists of the comparison of the acquired unknown mass spectra with those 

contained in a reference MS library. 

A mass spectrometer produces an enormous amount of data, especially in combination with 

chromatographic sample inlets [42]. Over the years, many approaches for analysis of GC-MS data 

have been proposed using various algorithms, many of which are quite sophisticated, in efforts 

to detect, identify, and quantify all of the chromatographic peaks. Library search algorithms are 

commonly provided with mass spectrometer data systems with the purpose to assist in the identification 

of unknown compounds [43]. 

However, as is well known, compounds such as isomers, when analyzed by means of GC-MS, 

can be incorrectly identified; a drawback which is often observed in essential oil analysis. As is 

widely acknowledged, the composition of essential oils is mainly represented by terpenes, which 

generate very similar mass spectra; hence, a favorable match factor is not sufficient for identification 

and peak assignment becomes a difficult, if not impracticable, task (Figure 6.1). In order to increase 

the reliability of the analytical results and to address the qualitative determination of compositions 

of complex samples by GC-MS, retention indices can be an effective tool. The use of retention indices 

in conjunction with the structural information provided by GC-MS is widely accepted, and 

routinely used to confirm the identity of compounds. Besides, retention indices when incorporated 

to MS libraries can be applied as a filter, thus shortening the search routine for matching results, and 

enhancing the credibility of MS identification [44]. 

According to D. Joulain and W. A. König [45], provided data contained in mass spectral libraries 

have been recorded using authentic samples, it can be observed that the mass spectrum of a 

given sesquiterpene is usually sufficient to ensure its identification when associated with its retention 

index obtained on methyl silicone stationary phases. Indeed, for the aforecited class of compounds, 

there would be no need to use a polyethylene glycol phase, which could even lead to 

misinterpretations caused by possible changes in the retention behavior of sesquiterpene hydrocarbons 

as a result of column aging or deterioration. Moreover, according to the authors, attention 

should be paid to the retention index and the mass spectrum registration of each individual sesquiterpene, 

since many compounds with rather similar mass spectra elute in a narrow range; more 

than 160 compounds can elute within 100 retention index units on a methyl silicone-based column, 

for example, 1400–1500. 

6.3.3 FAST GC FOR ESSENTIAL OIL ANALYSIS 

Nowadays in daily routine work, apart from increased analytical sensitivity, demands are also made 

on the efficiency in terms of speed of the laboratory equipment. Regarding the rapidity of analysis, 

two aspects need to be considered: (i) the costs in terms of time required, for example, as is the case 

in quality control analysis, and (ii) the efficiency of the utilized analytical equipment. 

When compared to conventional GC, the primary objective of fast GC is to maintain sufficient 

resolving power in a shorter time, by using adequate columns and instrumentation in combination


(a) Inten.(×10,000) 

1.00 

93 

0.75 

0.50 

0.25 

0.00 

77 

91 

41 

53 65 69 80 

136 

105 

121 

40 50 60 70 80 90 100 110 120 130 m/z 

(b) Inten.(×10,000) 

1.00 

93 

0.75 

0.50 

0.25 

0.00 

53 65 41 

80 

107 121 

77 

91 

69 136 

40 50 60 70 80 90 100 110 120 130 m/z 

(c) Inten.(×10,000) 

1.00 

0.75 

0.50 

0.25 

0.00 

41 

53 

67 

79 

81 

93 

107 

121 

133136 147 

161 

50 75 100 125 150 175 200 

189 

204 

m/z 

(d) Inten.(×10,000) 

1.00 

121 

0.75 

0.50 

0.25 

0.00 

41 

93 

107 

67 79 

53 

81 133 161 

136 189 

147 

204 

50 75 100 125 150 175 200 m/z 

FIGURE 6.1 Representation of the similarity between mass spectra of monoterpenes: sabinene (a) and 

b-phellandrene (b); and sesquiterpenes: bicyclogermacrene (c) and germacrene B (d). 

with optimized run conditions to provide 3–10 times faster analysis times [46–48]. The technique 

can be accomplished by manipulating a number of analysis parameters, such as column length, 

column I.D., stationary phase, film thickness, carrier gas, linear velocity, oven temperature, and 

ramp rate. Fast GC is typically performed using short, 0.10 or 0.18 mm I.D. capillary columns with 

hydrogen carrier gas and rapid oven temperature ramp rates. In general, capillary gas chromatographic 

analysis may be divided into three groups, based solely on column internal diameter types; 

namely, as conventional GC when 0.25 mm I.D. columns are applied, fast GC using 0.10–0.18 mm 

I.D. columns, and ultrafast GC for columns with an I.D. of 0.05 mm or less. In addition, GC analyses 

times between 3 and 12 min can be defined as “fast,” between 1 and 3 min as “very fast,” and 

below 1 min as “ultrafast.” Fast GC requires instrumentation provided with high split ratio injection


systems because of low sample column capacities, increased inlet pressures, rapid oven heating 

rates, and fast electronics for detection and data collection [49]. 

The application of two methods, conventional (30 m ¥ 0.25 mm I.D., 0.25 mm d f column) and fast 

(10 m ¥ 0.10 mm I.D., 0.10 mm d f column), on five different citrus essential oils (bergamot, mandarin, 

lemon, bitter oranges, and sweet oranges) has been reported [49]. The fast method allowed the 

separation of almost the same compounds as the conventional analysis, while quantitative data 

showed good reproducibility. The effectiveness of the fast GC method, through the use of narrowbore 

columns, was demonstrated. An ultrafast GC lime essential oil analysis was also performed on 

a 5 m ¥ 50 mm capillary column with 0.05 mm stationary phase film thickness [50]. The total analysis 

time of this volatile essential oil was less than 90 s; a chromatogram is presented in Figure 6.2. 

Another technique, ultrafast module-GC (UFM-GC) with direct resistively heated narrow-bore 

columns, has been applied to the routine analysis of four essential oils of differing complexities; 

chamomile, peppermint, rosemary, and sage [51]. All essential oils were analyzed by conventional 

GC with columns of different lengths; namely, 5 and 25 m, with a 0.25 mm I.D., and by fast GC and 

UFM-GC with narrow-bore columns (5 m ¥ 0.1 mm I.D.). Column performances were evaluated 

and compared through the Grob test, separation numbers, and peak capacities. UFM-GC was successful 

in the qualitative and quantitative analysis of essential oils of different compositions with 

analysis times between 40 s and 2 min versus 20–60 min required by conventional GC. UFM-GC 

allows to drastically reduce the analysis time, although the very high column heating rates may lead 

to changes in selectivity compared to conventional GC, and that are more marked than those of 

classical fast GC. In a further work the same researchers [52] stated that in UFM-GC experiments 

the appropriate flow choice can compensate, in part, the loss of separation capability due to the heating 

rate increase. 

Besides the numerous fast GC application on citrus essential oils, other oils have also been 

subjected to analysis, such as rose oil by means of ultrafast GC [53] and very fast GC [54], both 

using narrow-bore columns. Rosemary and chamomile oils have been investigated by means of 

fast GC on two short conventional columns of distinct polarity (5 m ¥ 0.25 mm I.D.) [55]. The latter 

25,000 

2 4 5 6 9 11 

21 32 

20,000 

19 

27 

15,000 

22 

Intensity 

10,000 

1 

13 

26 

5000 

0 

16 

8 

14 

15 

3 7 10 12 17 18 20 

W h = 126 ms W h = 180 ms 

30 

23 

24 25 28 31 

29 

33 

W h = 192 ms 

35 

34 36 

0 12 24 36 48 60 72 

Seconds 

84 

FIGURE 6.2 Fast GC analysis of a lime essential oil on a 5 m ¥ 5 mm (0.05 mm film thickness) capillary 

column, applying fast temperature programming. The peak widths of three components are marked to provide 

an illustration of the high efficiency of the column, even under extreme operating conditions (for peak identification 

see on Ref. [50]). (From Mondello, L. et al., 2004. J. Sep. Sci., 27: 699–702. With permission.)


oil has also been analyzed through fast HS-SPME-GC on a narrow-bore column [56]. Fast and 

very fast GC analyses on narrow-bore columns have also been carried out on patchouli and peppermint 

oils [57]. 

6.3.4 GAS CHROMATOGRAPHY-OLFACTOMETRY FOR THE ASSESSMENT OF ODOR-ACTIVE 

COMPONENTS OF ESSENTIAL OILS 

The discriminatory capacity of the mammalian olfactory system is such that thousands of volatile 

chemicals are perceived as having distinct odors. It is accepted that the sensation of odor is triggered 

by highly complex mixtures of volatile molecules, mostly hydrophobic, and usually occurring in 

trace-level concentrations (ppm or ppb). These volatiles interact with odorant receptors of the olfactive 

epithelium located in the nasal cavity. Once the receptor is activated, a cascade of events is 

triggered to transform the chemical-structural information contained in the odorous stimulus into a 

membrane potential [58,59], which is projected to the olfactory bulb, and then transported to higher 

regions of the brain [60] where the translation occurs. 

It is known that only a small portion of the large number of volatiles occurring in a fragrant 

matrix contributes to its overall perceived odor [61,62]. Further, these molecules do not contribute 

equally to the overall flavor profile of a sample; hence, a large GC peak area, generated by a chemical 

detector does not necessarily correspond to high odor intensities, due to differences in intensity/ 

concentration relationships. 

The description of a gas chromatograph modified for the sniffing of its effluent to determine 

volatile odor activity was first published in 1964 by Fuller et al. [63]. In general, gas chromatograhyolfactometry 

(GC-O) is carried out on a standard GC that has been equipped with a sniffing port, 

also denominated olfactometry port or transfer line, in substitution of, or in addition to, the conventional 

detector. When a flame FID or a mass spectrometer is also used, the analytical column effluent 

is split and transferred to the conventional detector and to the human nose. GC-O was a 

breakthrough in analytical aroma research, enabling the differentiation of a multitude of volatiles, 

previously separated by GC, in odor-active and non-odor-active, related to their existing concentrations 

in the matrix under investigation. Moreover, it is a unique analytical technique that associates 

the resolution power of capillary GC with the selectivity and sensitivity of the human nose. 

GC-O systems are often used in addition to either a FID or a mass spectrometer. With regard to 

detectors, splitting column flow between the olfactory port and a mass spectral detector provides 

simultaneous identification of odor-active compounds. Another variation is to use an in-line, nondestructive 

detector such as a TCD [64] or a photoionization detector (PID) [65]. Especially when 

working with GC-O systems equipped with detectors that do not provide structural information, 

retention indexes are commonly associated to odor description supporting peak assignment. 

Over the last decades, GC-O has been extensively used in essential oil analysis in combination 

with sophisticated olfactometric methods; the latter were developed to collect and process GC-O 

data, and hence, to estimate the sensory contribution of a single odor-active compound. The odoractive 

compounds of essential oils extracted from citrus fruits (Citrus sp.), such as orange, lime, and 

lemon, were among the first character impact compounds identified by flavor chemists [66]. 

GC-O methods are commonly classified in four categories: dilution, time-intensity, detection 

frequency, and posterior intensity methods. Dilution analysis, the most applied method, is based on 

successive dilutions of an aroma extract until no odor is perceived by the panelists. This procedure, 

usually performed by a reduced number of assessors is mainly represented by CHARM (combined 

hedonic aroma response method) [67], developed by Acree and coworkers, and AEDA (aroma 

extraction dilution analysis), first presented by Ullrich and Grosch [68]. The former method has 

been applied to the investigation of two sweet orange oils from different varieties, one Florida 

Valencia and the other Brazilian Pera [69]. The intensities and qualities of their odor-active components 

were assessed. CHARM results indicated for both the oils that the most odor-active compounds 

are associated with the polar fraction compounds: straight chain aldehydes (C 8 –C 14 ), 

b-sinensal, and linalool presented the major CHARM responses. On the other hand, AEDA has


been used to investigate the odor-active compounds responsible for the characteristic odors of juzu 

oil (Citrus junos Sieb. ex Tanaka) [70] and dadai (Citrus aurantium L. var. cyathifera Y. Tanaka) 

[71] cold-pressed essential oils. 

Time-intensity methods, such as OSME (Greek word for odor), are based on the immediate 

recording of the intensity as a function of time by moving the cursor of a variable resistor [72]. An 

interesting application of the time-intensity approach was demonstrated for cold-pressed grapefruit 

oil [73], in which 38 odor-active compounds were detected and, among these, 22 were considered 

as aroma impact compounds. A comparison between the grapefruit oil GC chromatogram and the 

corresponding time-intensity aromagram for that sample is shown in Figure 6.3. 

A further approach, the detection frequency method [74,75], uses the number of evaluators 

detecting an odor-active compound in the GC effluent as a measure of its intensity. This GC-O 

method is performed with a panel composed of numerous and untrained evaluators; 8–10 assessors 

are a good agreement between low variation of the results and analysis time. It must be added that 

the results attained are not based on real intensities and are limited by the scale of measurement. An 

application of the detection frequency method was reported for the evaluation of leaf- and woodderived 

essential oils of Brazilian rosewood (Aniba rosaeodora Ducke) essential oils by means of 

enantioselective-GC-olfactometry (Es-GC-O) analyses [76]. 

Another GC-O technique, the posterior intensity method [77], proposes the measurement of a 

compound odor intensity, and its posterior scoring on a previously determined scale. This posterior 

registration of the perceived intensity may cause a considerable variance between assessors. The 

attained results may generally be well correlated with detection frequency method results, and to a 

lesser extent, with dilution methods. In the above-mentioned research performed on the essential 

oils of Brazilian rosewood, this method was also used to give complementary information on the 

intensity of the linalool enantiomers [76]. 

Other GC-O applications are also reported in literature using the so-called peak-to-odor impression 

correlation, the method in which the olfactive quality of an odor-active compound perceived 

by a panelist is described. The odor-active compounds of the essential oils of black pepper 

Response (mV) 

500 

400 

300 

200 

100 

0 

Limonene 

Octanal 

Nonanal 

Decanal 

Citronellal 

Linalool 

Octanol 

Terpenen-4-ol 

Neral 

2E-Decenal 

Dodecanal 

Geranial 

Nerol 

2E, 4E-Decadienal 

Geraniol 

β-Sinensal 

Nootkatone 

400 

800 

1200 

6 8 10 12 14 16 18 20 22 24 26 28 30 32 

Time (min) 

FIGURE 6.3 GC-FID chromatogram with some components identified by means of MS (top) and a time-intensity 

aromagram of grapefruit oil (bottom). The separation was performed on a polyethylene glycol column 

(30 m ¥ 0.32 mm I.D., 0.25 mm film thickness). (From Lin, J. and R.L. Rouseff, 2001. Flavour Fragr. J., 16: 

457–463. With permission.)


(Piper nigrum) and Ashanti pepper (Piper guineense) were assessed applying the aforecited correlation 

method [78]. The odor profile of the essential oils of leaves and flowers of Hyptis pectinata (L.) 

Poit. was also investigated by using the peak-to-odor impression correlation [79]. 

The choice of the GC-O method is of extreme importance for the correct characterization of a 

matrix, since the application of different methods to an identical real sample can distinctly select 

and rank the odor-active compounds according to their odor potency and/or intensity. Commonly, 

detection frequency and posterior intensity methods result in similar odor intensity/concentration 

relationships, while dilution analysis investigate and attribute odor potencies. 

6.3.5 GAS CHROMATOGRAPHIC ENANTIOMER CHARACTERIZATION OF ESSENTIAL OILS 

Capillary GC is currently the method of choice for enantiomer analysis of essential oils and enantioselective-GC 

(Es-GC) has become an essential tool for stereochemical analysis mainly after the 

introduction of cyclodextrin (CD) derivatives as chiral stationary phases (CSPs) in 1983 by Sybilska 

and Koscielski, at the University of Warsaw, for packed columns [80], and applied to capillary columns 

in the same decade [81,82]. Moreover, Nowotny et al. first proposed diluting CD derivatives 

in moderately polar polysiloxane (OV-1701) phases to provide them with good chromatographic 

properties and a wider range of operative temperatures [83]. 

The advantage on the application of Es-GC lies mainly in its high separation efficiency and sensitivity, 

simple detection, unusually high precision and reproducibility, as also the need for a small 

amount of sample. Moreover, its main use is related with the characterization of the enantiomeric 

composition and the determination of the enantiomeric excess (ee) and/or ratio (ER) of chiral 

research chemicals, intermediates, metabolites, flavors and fragrances, drugs, pesticides, fungicides, 

herbicides, pheromones, and so on. Information on ee or ER is of great importance to characterize 

natural flavor and fragrance materials, such as essential oils, since the obtained values are 

useful tools, or even “fingerprints,” for the determination of their quality, applied extraction technique, 

geographic origin, biogenesis, and also authenticity [84]. 

A great number of essential oils have already been investigated by means of Es-GC using distinct 

CSPs; unfortunately, a universal chiral selector with widespread potential for enantiomer separation 

is not available, and thus effective optical separation of all chiral compounds present in a matrix 

may be unachievable on a single chiral column. In 1997, Bicchi et al. [85] reported the use of columns 

that addressed particular chiral separations, noting that certain CSPs preferentially resolved 

certain enantiomers. Thus, a 2,3-di-O-ethyl-6-O-tert-butyldimethylsilyl-b-CD on polymethylphenylsiloxane 

(PS086) phase allowed the characterization of lavender and citrus oils containing linalyl 

oxides, linalool, linalyl acetate, borneol, bornyl acetate, a-terpineol, and cis- and trans-nerolidol. 

On the other hand, peppermint oil was better analyzed by using a 2,3-di-O-methyl-6-O-tertbutyldimethylsilyl-b-CD 

on PS086 phase, and especially for a- and b-pinene, limonene, menthone, 

isomenthone, menthol, isomenthol, pulegone, and methyl acetate. König [86] performed an exhaustive 

investigation of the stereochemical correlations of terpenoids, concluding that when using a 

heptakis (6-O-methyl-2,3-di-O-penthyl)-b-CD and octakis (6-O-methyl-2,3-di-O-penthyl)-g-CD 

in polysiloxane, the presence of both enantiomers of a single compound is common for monoterpenes, 

less common for sesquiterpenes, and never observed for diterpenes. 

Substantial improvements in chiral separations have been extensively published in the field of 

chromatography. At present, over 100 stationary phases with immobilized chiral selectors are available 

[22], presenting increased stability and extended lifetime. It can be affirmed that enantioselective 

chromatography has now reached a high degree of sophistication. To better characterize an 

essential oil, it is advisable to perform Es-GC analysis on at least two, or better three, columns 

coated with different CD derivatives. This procedure enables the separation of more than 85% of the 

racemates that commonly occur in these matrices [87], while the reversal of enantiomer elution 

order can take place in several cases. The analyst must be aware of some practical aspects prior to 

an Es-GC analysis: as is well accepted, variations in linear velocity can affect the separation of


enantiomeric pairs; resolution (R S ) can be improved by optimizing the gas linear velocity, a factor 

of high importance in cases of difficult enantiomer separation. Satisfactory resolution requires 

R S ≥ 1, and baseline resolution is obtained when R S ≥ 1.5 [88]. Resolution can be further improved 

by applying slow temperature ramp rates (1–2°C/min is frequently suggested). Moreover, according 

to the CSP used, the initial GC oven temperature can affect peak width; initial temperatures of 

35–40°C are recommended for the most column types. Furthermore, attention should be devoted to 

the column sample capacity, which varies with different compounds; overloading results in broad 

tailing peaks and reduced enantiomeric resolution. The troublesome separation and identification of 

enantiomers due to the fact that each chiral molecule splits into two chromatographic signals, for 

each existing stereochemical center is also worthy of note. As a consequence, the increase in complexity 

of certain regions of the chromatogram may lead to imprecise ee and/or ER values. In terms 

of retention time repeatability, and also reproducibility, it can be affirmed that good results are 

being achieved with commercially available chiral columns. 

The retention index calculation of optically active compounds can be considered as a troublesome 

issue due to complex inclusion complexation retention mechanisms on CD stationary phases; 

if a homologous series, such as the n-alkanes, is used, the hydrocarbons randomly occupy positions 

in the chiral cavities. As a consequence, n-alkanes can be considered as unsuitable for retention index 

determinations. Nevertheless, other reference series can be employed on CD stationary phases, such 

as linear chain FAMEs and FAEEs. However, retention indices are seldom reported for optically 

active compounds, and publications refer to retention times rather than indices. 

The innovations in Es-GC analysis have not only concerned the development and applications of 

distinct CSPs, but also the development of distinct enantioselective analytical techniques, such as 

Es-GC-mass spectrometry (Es-GC/MS), Es-GC-O, enantioselective multidimensional gas chromatography 

(Es-MDGC), Es-MDGC/MS, Es-GC hyphenated to isotopic ratio mass spectrometry 

(Es-GC/IRMS), Es-MDGC/IRMS, and so on. 

It is obvious that an enantioselective separation in combination with MS detection presents the 

additional advantage of qualitative information. Notwithstanding, a difficulty often encountered is 

that related to peak assignment, due to the similar fragmentation pattern of isomers. The reliability 

of Es-GC/MS results can be increased by using an effective tool, namely retention indices. It can be 

assumed that in the enantioselective recognition of optically active isomers in essential oils, mass 

spectra can be exploited to locate the two enantiomers in the chromatogram, and the LRI when 

possible, enables their identification [89]. In addition, the well-known property of odor activity recognized 

for several isomers can be assessed by means of Es-GC/MS/O and can represent an outstanding 

tool for precise enantiomer characterization (Figure 6.4). 

As demonstrated by Mosandl and his group [90], Es-GC-O is a valid tool for the simultaneous 

stereodifferentiation and olfactive evaluation of the volatile optically active components present in 

essential oils. It is worthwhile to point out that the preponderance of one of the enantiomers, defined 

by the enantiomeric excess, results in a characteristic aroma [91], and is of great importance for the 

olfactive characterization of the sample. 

6.3.6 LC AND LIQUID CHROMATOGRAPHY HYPHENATED TO MS IN THE ANALYSIS OF 

ESSENTIAL OILS 

Some natural complex matrices do not need sample preparation prior to GC analysis, for example, 

essential oils. The latter generally contain only volatile components, since their preparation is performed 

by SD. Citrus oils, extracted by cold-pressing machines, are an exception, containing more 

than 200 volatile and nonvolatile components. The volatile fraction represents 90–99% of the entire 

oil, and is represented by mono- and sesquiterpene hydrocarbons and their oxygenated derivatives, 

along with aliphatic aldehydes, alcohols, and esters; the nonvolatile fraction, constituting 1–10% of 

the oil, is represented mainly by hydrocarbons, fatty acids, sterols, carotenoids, waxes, and oxygen 

heterocyclic compounds (coumarins, psoralens, and polymethoxylated flavones—PMFs) [92].


Inten.(×10,000) 

1.00 41 

0.75 55 

0.50 

69 

81 

95 

(3S)-(–)-β-citronellol 

H 

HO 

0.25 

0.00 

40 

123 

109 

138 

156 

50 60 70 80 90 100 110 120 130 140 150 m/z 

floral, geranium-like odour 

LRI 1343 

Inten.(×10,000) 

1.00 41 

0.75 55 

0.50 

69 

81 

95 

(3R)-(+)-β-citronellol 

H 

OH 

0.25 

109 

123 

citronella oil like odour 

138 

156 

LRI 1346 

0.00 

40 50 60 70 80 90 100 110 120 130 140 150 m/z 

FIGURE 6.4 Representation of the mass spectra similarity of b-citronellol enantiomers. 

In some specific cases, the information attained by means of GC is not sufficient to characterize 

a citrus essential oil, and the analysis of the nonvolatile fraction can be required. Oxygen heterocyclic 

compounds, which are a distinct class of flavonoids, can have an important role in the identification 

of a cold-pressed oil and in the control of both quality and authenticity [92–95]. The analysis 

of these compounds is usually performed by means of LC, also referred to as high-performance 

liquid chromatography (HPLC), in normal (NP-HPLC) or reversed-phase (RP-HPLC) applications. 

The former method, commonly used when the analytes of interest are slightly polar, separates analytes 

based on polarity by using a polar stationary phase and a nonpolar mobile phase. The degree 

of adsorption on the polar stationary phase increases on the basis of analyte polarity, and the extension 

of this interaction has a great influence on the elution time. In general, the interaction strength 

is related to the nature of the analyte functional groups and to steric factors. On the other hand, 

RP-HPLC is based on the use of a nonpolar stationary phase and an aqueous, moderately polar 

mobile phase. Retention times are therefore shorter for polar molecules, which elute more readily. 

Moreover, retention times are increased by the addition of a polar solvent to the mobile phase, and 

decreased by the addition of a more hydrophobic solvent. 

The on-line coupling of two columns, namely, a m-Porasil (30 cm ¥ 3.9 mm I.D., with 10 mm 

particle size; Waters Corporation; Milford, USA) and a Zorbax silica (25 cm ¥ 4.6 mm I.D., with 

7 mm particle size; Phenomenex, Bologna, Italy), in the NP-HPLC analysis of bitter orange essential 

oils with UV detection has been reported. A large number of cold-pressed Italian and Spanish, commercial 

and laboratory-made oils, as also mixtures of bitter orange with sweet orange, lemon, lime, 

and grapefruit oils were analyzed [93]. A total of four coumarins [osthol (1), meranzin (5), isomeranzin 

(6), and meranzin hydrate (14)], three psoralens [bergapten (2), epoxybergamottin (3), and 

epoxybergamottin hydrate (13)], and four PMFs [tangeretin (8), heptamethoxyflavone (9), nobiletin 

(10), and tetra-O-methylscutellarein (11)] were identified. In addition, further three unidentified coumarins 

(peaks 4, 7, and 12) were detected. The bracketed numbers refer to those in Figure 6.5. In 

general, Italian essential oils exhibited a higher content of oxygen heterocyclic compounds than the 

Spanish oils. The use of NP and RP-HPLC with microbore columns and UV detection has also been 

reported for lemon and bergamot [96], bitter orange and grapefruit [97] essential oils. Orange and


1.60 

1.40 

8 

1.20 

1.00 

AU 

0.80 

0.60 

0.40 

0.20 

0.00 

i.s. 

3 

4 

5 

7 

9 

11 

10 12 13 14 

15 16 

17 

20.00 

Minutes 

40.00 

FIGURE 6.5 HPLC chromatogram of Italian genuine bitter orange oil. For peak identification, refer to the text 

(i.s.—internal standard). (From Dugo, P. et al., 1996. J. Agric. Food Chem., 44: 544–549. With permission.) 

mandarin essential oils have also been analyzed by NP and RP-HPLC, but with UV and spectrofluorimetric 

detection in series [98]. For the identification of chromatographic peaks of all the aforementioned 

oils, a preparative HPLC was used; the purified fractions were then analyzed by GC-MS 

and HPLC/MS. 

The oxygen heterocyclic compounds present in the nonvolatile residue of citrus essential oils 

has also been extensively investigated by means of high-performance liquid chromatography/ 

atmospheric pressure ionization mass spectrometry (HPLC/API/MS) [99]. The mass spectra 

obtained at different voltages of the “sample cone” have been used to build a library. Citrus essential 

oils have been analyzed with this system, using an optimized NP-HPLC method, and the mass 

spectra were compared with those of the laboratory-constructed library. This approach allowed the 

rapid identification and characterization of oxygen heterocyclic compounds of citrus oils, the detection 

of some minor components for the first time in some oils, and also the detection of authenticity 

and possible adulteration. 

Apart from citrus oils, other essential oils have also been analyzed by means of LC, such as the 

blackcurrant bud essential oil [100]. The latter was fractionated into hydrocarbons and oxygenated 

compounds and the two fractions were submitted to RP-HPLC analysis. Volatile carbonyls consist 

of some of the most important compounds for the blackcurrant flavor and, hence, were analyzed 

in detail. The carbonyls were converted into 2,4-dinitrophenylhydrazones and the mixture of 

2,4-dinitrophenylhydrazones was separated into derivatives of keto acids and monocarbonyl and 

dicarbonyl compounds. Each fraction was submitted to chromatographic investigation. 

6.3.7 MULTIDIMENSIONAL GAS CHROMATOGRAPHIC TECHNIQUES 

In spite of the considerable instrumental advances made, the detection of all the constituents of 

an essential oil is an extremely difficult task. For example, GC chromatograms relative to complex 

mixtures are characterized frequently by several overlapping compounds: Well-known examples 

are octanal and a-phellandrene, as well as limonene and 1,8-cineol, on 5% diphenyl–95% dimethylpolysiloxane 

stationary phases, while unsufficient resolution is observed between citronellol and 

nerol or geraniol and linalyl acetate. On the other hand, the overlapping of monoterpene alcohols


and esters with sesquiterpene hydrocarbons is frequently reported on polyethylene glycol stationary 

phases [101]. Hence, the direct identification of a component in such mixtures can be a cumbersome 

challenge. The use of multidimensional gas chromatography (MDGC) can be of great help in complex 

sample analysis. In MDGC, key fractions of a sample are selected from the first column 

and reinjected onto a second one, where ideally, they should be fully resolved. The instrumentation 

usually involves the use of a switching valve arrangement and two chromatographic columns of 

differing polarities, but generally of identical dimensions. Furthermore, when heart-cut operations 

are not carried out, the primary column elutes normally in the first dimension ( 1 D) GC system, 

while heart-cut fractions are chromatographically resolved on the secondary column [102]. The capillaries 

employed can be operated in either a single or two distinct GC ovens, with both GC systems 

commonly equipped with detectors. 

The use of MDGC has also been described in a wide range of enantioselective gas chromatographic 

applications [103], involving the use of chiral selectors as the stationary phase for the determination 

of ee and/or ER, as well as for adulteration assessments. 

A fully automated, multidimensional, double-oven GC-GC system has been presented by 

Mondello et al. The system is based on the use of mechanical valves that allow the automatic multiple 

transfers of different fractions from the precolumn to the analytical one, and the analysis of all 

transferred fractions during the same gas chromatographic run. A system of pneumatic valves emitted 

pressure variations in order to maintain constant retention times in the precolumn, even after 

numerous transfers. In addition, when the system was not applied in the multidimensional configuration, 

the two gas chromatographs could be operated independently. The system has been used for 

the determination of the enantiomeric distribution of monoterpene hydrocarbons and monoterpene 

alcohols in the essential oils of lime [104], mandarin [105], and lemon [106]. In Figure 6.6, the 

analysis of the latter oil is outlined to illustrate the technique: the chromatogram of the lemon oil 

obtained on an SE-52 column with the system in stand-by position is shown in Figure 6.6a, while 

the one attained with the system in the cut position is illustrated in Figure 6.6b. Figure 6.6c shows 

the chiral separation of the fractions transferred to the main analytical column. Well-resolved peaks 

of components present in large amounts, as also of the minor compounds, were attained through the 

partial transfer of the major concentration components. 

MDGC is a useful approach for the fractionation of compounds of particular interest in a specific 

sample, one of its major applicational areas is chiral analysis, using a conventional column as 1 D and 

a CSP capillary in the second dimension ( 2 D). Es-MDGC analysis has been used for the direct 

enantioselective evaluation of limonene in Rutaceae and Gramineae essential oils [107]. It is noteworthy 

that (4R)-(+)-limonene of high ee values were found in Rutaceae oils, as bergamot, orange, 

mandarin, lemon, or lime oils, while the (4S)-(-)-isomer was present in higher amounts in the 

Gramineae oils, such as citronella or lemongrass. The ratios of a-pinene and b-pinene enantiomers 

were also taken into consideration. 

The use of Es-MDGC using a primary polyethylene glycol stationary phase, and a secondary 

heptakis (2,3-di-O-acetyl-6-O-tert-butyldimethylsylil)-b-cyclodextrin, has been applied to rose 

oils. The technique proved to be highly efficient for the assessment of origin and quality control of 

economically important rose oil, using (3S)-(-)-citronellol, (2S,4R)-(-)-cis- and trans-rose oxides 

as markers [108]. Buchu leaf oil has also been assessed through Es-MDGC, and (1S)-menthone, 

isomenthone, (1S)-pulegone, (1S)-thiols, and (1S)-thiolacetates as enantiopure sulfur compounds 

[109]. A further application was reported on mint and peppermint essential oils, which contain 

(1R)-configured monoterpenoids [110]. Es-MDGC equipped with a 5% diphenyl–95% dimethylpolysiloxane 

and a 2,3-di-O-methyl-6-O-tert-butyldimethylsilyl-b-CD, as the 1 D and 2 D, respectively, 

enabled the simultaneous stereodifferentiation of menthone, neomenthol, isomenthone, 

menthol, neoisomenthol, and menthylacetate. 

The technique has also been successfully applied to the authenticity assessment of various 

commercially available rosemary oils [111]. The ER of a-pinene, camphene, b-pinene, limonene, 

borneol, terpinen-4-ol, a-terpineol, linalool, and camphor were measured; moreover, (1S)-(-)- 

borneol of high enantiomeric purity (higher than 90%) has been defined as a reliable indicator of


(a) 

Volts 

0.10 

0.08 

0.06 

0.04 

0.02 

Sabinene β-Pinene 

Limonene 

Linalool 

Terpinen-4-ol 

α-Terpineol 

0.10 

0.08 

0.06 

0.04 

0.02 

Volts 

0.00 

0 

25 

Minutes 

50 

0.00 

(b) 

0.10 

0.10 

0.08 

* 

* 

0.08 

0.06 

0.06 

Volts 

0.04 

* 

* 

0.04 

Volts 

0.02 

0.02 

* 

* 

0.00 

0 

Cut 1 Cut 2 Cut 3 Cut 4 Cut 5 

50 

0.00 

(c) 

Volts 

0.026 

0.022 

0.018 

0.014 

0.010 

0.006 

(+)-β-Pinene (–)-β-Pinene 

(+)-Sabinene (–)-Sabinene 

(–)-Limonene (+)-Limonene 

(–)-Linalol 

(+)-Linalol 

(+)-Terpinen-4-ol 

(–)-Terpinen-4-ol 

(–)-α-Terpineol 

(+)-α-Terpineol 

0.026 

0.022 

0.018 

0.014 

0.010 

0.006 

Volts 

0.002 

0.002 

15 20 25 30 35 40 45 50 55 

Minutes 

FIGURE 6.6 GC chromatogram of cold-pressed lemon oil obtained on an SE-52 column (a), GC chromatogram 

of cold-pressed lemon oil obtained on an SE-52 column with five heart-cuts (b), and GC-GC chiral 

chromatogram of the transferred components (c). (From Mondello, L. et al., 1999. J. High Res. Chromatogr., 

22: 350–356. With permission.)


genuine rosemary oils. A recently created high-performance MDGC system has been recently used 

in this specific field of essential oil research [111]. A conventional method and a fast MDGC method 

were developed and applied to the enantioselective analysis of rosemary oil. Prior to “heart-cutting,” 

a “stand-by” analysis was carried out in order to define the retention time cutting windows of the 

chiral components to be reanalyzed in the 2 D; retention time windows of eight peaks were defined. 

The eight peaks (camphene, b-pinene, sabinene, limonene, camphor, isoborneol, borneol, terpinen- 

4-ol, and a-terpineol) were then cut and transferred. It must be added that, in some cases, only a 

portion of the entire peak, that is, limonene, was diverted onto the secondary column. The fast 

MDGC method was applied on a twin set of 0.1 mm I.D. microbore columns with the objective of 

reproducing the conventional analytical result in a much shorter time (Figure 6.7). The overall runtime 

requested for the conventional analysis was of 43 min, while it was of 8.7 min for the fast 

MDGC application, with a speed gain of nearly a factor of five. 

MDGC heart-cutting methods, using valve and valveless flow switching interfaces, extend the 

separation power of capillary GC, although the 2 D analysis can only be restricted to a few regions 

UV(×10,000) 

2.5 

Chromatogram 

1 

4 

5 

7 

9 

E 

2.0 

1.5 

A 

2+3 

C 

8 

1.0 

0.5 

B 

D 

6 

0.0 

1.5 

2.0 

2.5 3.0 3.5 4.0 4.5 5.0 5.5 min 

UV(×10,000) 

Chromatogram 

(+)2 

(–)1 

4.0 (+)1 (–)2 

(+)5 

(–)5 

(–)7 

3.0 

2.0 

1.0 

0.0 

(+/–)3 

(–)4 

(+)4 

(+)6 

(–)6 

4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 min 

(+)7 

(–)8 

(+)8 

(–)9 

(+)9 

FIGURE 6.7 A 4.5 min chromatogram expansion relative to the fast MDGC rosemary analysis with the 

transfer system in stand-by position (top); tricyclene (peak A), a-phellandrene (peak B), unknown (peak C), 

a-terpinolene (peak D), and bornyl acetate (peak E). A 5 min 2 D chromatogram expansion relative to the fast 

MDGC rosemary oil application (bottom); the peak numbers refer to camphene (1), b-pinene (2), sabinene (3), 

limonene (4), camphor (5), isoborneol (6), borneol (7), terpinen-4-ol (8), and a-terpineol (9). (From Mondello, L. 

et al., 2006. J. Chromatogr. A, 1105: 11–16. With permission.)


of the chromatogram. In order to attain a complete two-dimensional characterization of a sample, a 

comprehensive approach, such as comprehensive two-dimensional gas chromatography (GC ¥ GC), 

has to be used. 

GC ¥ GC produces an orthogonal two-column separation, with the complete sample transfer 

achieved by means of a modulator; the latter entraps, refocuses, and releases fractions of the GC effluent 

from the 1 D, onto the 2 D column, in a continuous mode; this method enables an accurate screening 

of complex matrices, offering very high resolution and enhanced detection sensitivity [112,113]. The 

two columns must possess different separation mechanism, commonly a low polarity or nonpolar 

column is used in the 1 D, and a polar column is used as the fast 2 D column. Moreover, a two-dimensional 

separation can be defined as comprehensive if other two conditions are respected [114,115], 

namely, equal percentages (either 100% or less) of all sample components pass through both columns 

and eventually reach the detector; and the resolution obtained in the 1 D is essentially maintained. 

One of the first applications of GC ¥ GC to essential oils was performed by Dimandja et al. [116], 

who investigated the separation of peppermint (Mentha piperita) and spearmint (Mentha spicata) 

essential oil components. The latter oil is mainly characterized by four major components, that is, 

carvone, menthol, limonene, and menthone, while the former is mainly represented by menthol, 

menthone, menthylacetate, and eucalyptol. Both essential oils were considered as being of moderate 

complexity, containing


Injector 

Modulation 

device 

Detector 

Support guides 

CO 2 , N 2 supply and 

movement arm 

Cryogenic trap 

Retaining nut 

Capillary column 

FIGURE 6.9 Scheme of the LMCS located inside a GC oven. (From Marriott, P. et al., 2000. Flavour Fragr. J., 

15: 225–239. With permission.) 

cryotrap is presented; the columns are anchored with retaining nuts to the support frame so that the 

trap can move up and down along the column, its movement is controlled by a stepper motor. Liquid 

cryogen is supplied to the inlet of the trap, passing through a narrow restrictor and expanding to cool 

the body of the trap. A secondary, small flow of nitrogen passing through the center of the body prevents 

the build up of ice which would otherwise freeze the column to the trap [118]. Analysis were 

performed on a 5% phenyl–95% dimethylpolysiloxane 1 D column (25 m ¥ 0.25 mm I.D., 0.25 mm d f ) 

connected to a 50% phenyl–50% dimethylpolysiloxane 2 D column (0.8 m ¥ 0.1 mm I.D., 0.1 mm d f ), 

applying a modulation frequency of 4 s cycle. About 200 compounds could be detected by means of 

GC ¥ GC analysis. The authors reported that the GC-MS analysis of vetiver oil, with peak deconvolution, 

would still not have been sufficient for the identification of coeluting substances. 

French lavender (Lavandula angustifolia) and tea tree (Melaleuca alternifolia), essential oils 

were also submitted to GC ¥ GC analyses using a nonpolar (5% phenyl–95% dimethylpolysiloxane)– 

polar (polyethylene glycol) column set [119]. The work, developed using an LMCS, enabled the 

determination of elution patterns within the 2 D space useful for the correlation of component retention 

behavior with their chemical and structural properties. The GC ¥ GC approach provides higher 

sensitivity, greater peak resolution, and capacity, as well as an essential oil fingerprint pattern. The 

enhanced peak capacity and sensitivity of GC ¥ GC in the tea tree oil application can be observed 

in the conventional GC and untransformed GC ¥ GC chromatograms illustrated in Figure 6.10. 

Lavender essential oil has been further investigated by means of GC ¥ GC retrofitted with an 

LMCS and hyphenated to time-of-flight mass spectrometry (GC ¥ GC/TOFMS), thus generating a 

three-dimensional analytical approach [120]. The authors outlined that the vacuum effect on the 2 D 

column in GC ¥ GC/TOFMS may generate differing retention times with respect to an equivalent 

analysis performed on a GC ¥ GC system at ambient pressure conditions. Lavender essential oil was 

further investigated through the comparison of GC-MS and GC ¥ GC analyses [121], as illustrated 

in Figure 6.11. At least 203 components were counted in the 2 D separation space, which was characterized 

by a well-defined monoterpene hydrocarbon region, and a sesquiterpene hydrocarbon 

area. The oxygenated derivatives of both these groups generally elute closely after the main group 

in the 1 D, but due to their wide range of component polarities these are found to spread throughout 

a broader region of the 2 D plane. According to the authors, by using GC ¥ GC the detection of subtle


(a) 

80 

70 

60 

50 

40 

Response / pA 

30 

(b) 20 

80 

60 

(expanded scale) 

40 

20 

10 20 30 40 50 60 

Time / min 

FIGURE 6.10 Comparison of monodimensional GC and pulsed GC ¥ GC result for tea tree oil; both 

chromatograms are shown at identical sensitivity. (From Shellie, R. et al., 2000. J. High Resolution 

Chromatogr., 23: 554–560. With permission.) 

(a) 

(b) 

5.00 

D2 retention time (s) 

4.50 

4.00 

3.50 

3.00 

2.50 

2.00 

1.50 

1.00 

0.50 

M 

Y 

Z 

S 

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 

D1 retention time (min) 

FIGURE 6.11 Reconstructed gas chromatographic trace for a lavender essential oil (a), and the twodimensional 

separation space for the GC ¥ GC analysis of the same sample (b). The minor component Z 

overlaps completely from the major component Y in the 1 D; M: monoterpene hydrocarbons, S: sesquiterpene 

hydrocarbons. (From Shellie, R. et al., 2002. J. Chromatogr. A, 970: 225–234. With permission.)


differences in the analyses of lavender essential oils from different cultivars should be simplified, 

since it could be based on a 2 D pictorial representation of the volatile components. 

Further relevant essential oil investigations have been performed: the early works using FID, 

while the more recent ones using, preferably, a TOFMS as detector. Among the essential oils previously 

studied by means of GC ¥ GC are peppermint [122] and Australian sandalwood [123], with 

the latter also analyzed through GC ¥ GC/TOFMS in the same work. Essential oils derived from 

Thymbra spicata [124], Pistacia vera [125], hop [126], Teucrium chamaedrys [127], Rosa damascena 

[128], coriander [129], and Artemisia annua [130], as well as tobacco [131], have also been 

subjected to GC ¥ GC/TOFMS analyses. The references cited herein represent only a fraction of the 

studies performed by means of GC ¥ GC on essential oils. 

6.3.8 MULTIDIMENSIONAL LIQUID CHROMATOGRAPHIC TECHNIQUES 

HPLC has acquired a role of great importance in food analysis, as demonstrated by the wide variety 

of applications reported. Single LC column chromatographic processes have been widely applied 

for sample profile elucidation, providing satisfactory degrees of resolving power; however, whenever 

highly complex samples require analysis, a monodimensional HPLC system can prove to be 

inadequate. Moreover, peak overlapping may occur even in the case of relatively simple samples, 

containing components with similar properties. 

The basic principles of MDGC are also valid for multidimensional LC (MDLC). The most common 

use of MDLC separation is the pretreatment of a complex matrix in an off-line mode. The offline 

approach is very easy, but presents several disadvantages: it is time-consuming, operationally 

intensive, difficult to automate, and to reproduce. Moreover, sample contamination or formation of 

artifacts can occur. On the other hand, on-line MDLC, though requiring specific interfaces, offers the 

advantages of ease of automation and greater reproducibility in a shorter analysis time. In the on-line 

heart-cutting system, the two columns are connected by means of an interface, usually a switching 

valve, which allows the transfer of fractions of the first column effluent onto the second column. 

In contrast to comprehensive gas chromatography (GC ¥ GC), the number of comprehensive 

liquid chromatography (LC ¥ LC) applications reported in literature are much less. It can be affirmed 

that LC ¥ LC presents a greater flexibility when compared to GC ¥ GC since the mobile phase 

composition can be adjusted in order to obtain enhanced resolution [132]. Comprehensive HPLC 

systems, developed, and applied to the analysis of food matrixes, have employed the combination of 

either NP ¥ RP or RP ¥ RP separation modes. However, it is worthy of note that the two separation 

mechanisms exploited should be as orthogonal as possible, so that no or little correlation exists 

between the retention of compounds in both dimensions. 

A typical comprehensive two-dimensional HPLC separation is attained through the connection 

of two columns by means of an interface (usually a high-pressure switching valve), which entraps 

specific quantities of 1 D eluate, and directs it onto a secondary column. This means that the first 

column effluent is divided into “cuts” that are transferred continuously to the 2 D by the interface. 

The type of interface depends on the methods used, although multiport valve arrangements have 

been the most frequently employed. 

Various comprehensive HPLC systems have been developed and proven to be effective for the 

separation of complex sample components, and in the resolution of a number of practical problems. 

In fact, the very different selectivities of the various LC modes enable the analysis of complex mixtures 

with minimal sample preparation. However, comprehensive HPLC techniques are complicated 

by the operational aspects of switching effectively from one operation step to another, by data 

acquisition and interpretation issues. Therefore careful method optimization and several related 

practical aspects should be considered. 

In the most common approach, a microbore LC column in the first and a conventional column 

in the 2 D are used. In this case, an 8-, 10-, or 12-port valve equipped with two sample loops 

(or trapping columns) is used as an interface. A further approach foresees the use of a conventional


LC column in the first and two conventional columns in the 2 D. One or two valves that allow transfers 

from the first column to two parallel secondary columns (without the use of storage loops) are 

used as interface. 

One of the best examples of the application of comprehensive NPLC ¥ RPLC in essential oil 

analysis is represented by the analysis of oxygen heterocyclic components in cold-pressed lemon oil, 

by using a normal phase with a microbore silica column in the 1 D and a monolithic C18 column in 

the 2 D with a 10-port switching valve as interface [133]. In Figure 6.12, an NPLC ¥ RPLC separation 

of the oxygen heterocyclic fraction of a lemon oil sample is presented. Oxygen heterocyclic 

components (coumarins, psoralens, and PMFs) represent the main part of the nonvolatile fraction of 

cold-pressed citrus oils. Their structures and substituents have an important role in the characterization 

of these oils. Positive peak identification of these compounds was obtained by both the relative 

location of the peaks in the two-dimensional plane, which varied in relation to their chemical structure, 

and by characteristic UV spectra. In a later experiment, a similar setup was used for a citrus 

oil extract composed of lemon and orange oil [134]. The main difference with respect to the earlier 

published work [133] was the employment of a bonded-phase (diol) column in the 1 D. Under optimized 

LC conditions, the high degree of orthogonality between the NP and RP systems tested, 

resulted in increased 2 D peak capacity. 

A novel approach for the analysis of carotenoids, pigments mainly distributed in plant-derived 

foods, especially in orange and mandarin essential oils, has been recently developed by Dugo et al. 

[135,136]. In terms of structures, food carotenoids are polyene hydrocarbons, characterized by a C 40 

skeleton that derives from eight isoprene units. They present an extended conjugated double bond 

(DB) system that is responsible for the yellow, orange, or red colors in plants and are notable for their 

wide distribution, structural diversity, and various functions. Carotenoids are usually classified in 

two main groups: hydrocarbon carotenoids, known as carotenes (e.g., b-carotene and lycopene), and 

oxygenated carotenoids, known as xanthophylls (e.g., b-cryptoxanthin and lutein). The elucidation 

of carotenoid patterns is particularly challenging, because of the complex composition of carotenoids 

in natural matrices, their great structural diversity, and their extreme instability. An innovative comprehensive 

dual-gradient elution HPLC system was employed using an NPLC ¥ RPLC setup, composed 

of silica and C18 columns in the 1 D and 2 D, respectively. Free carotenoids in orange essential 

oil and juice (after saponification), were identified by combining the two-dimensional retention data 

1.000 

0.875 

0.750 

2 

5 

0.625 

4 

8 

0.500 

0.375 

0.250 

1 

3 

7 

9 

0.125 

6 

10 11 

0 5 10 15 20 25 30 35 40 45 

min 

FIGURE 6.12 Comprehensive NP (adsorption)-LC ¥ RP-LC separation of the oxygen heterocyclic fraction 

of a lemon oil sample (for peak identification see Ref. [133]). (From Dugo, P. et al., 2004. Anal. Chem., 73: 

2525–2530. With permission.)


t R (RP), sec 

120.0 

106.7 

93.4 

80.0 

66.7 

53.4 

40.0 

26.7 

13.4 

Hydrocarbons 

2 

1 

8 

7 

10 

13 

9 

Mono-ol 

mono-epoxides 

Mono-ols 

Di-ols 

17 

16 

12 

27 

21 

23 

19 

28 

20 

14 

15 

18 

22 

26 

41 

25 33 36 43 

32 

30 

40 

49 

42 53 

35 

38 

45 

31 44 52 

50 51 55 60 

59 

34 

Di-ol mono-epoxides 

Di-ol mono-epoxides 

54 

56 62 

57 

Di-ol di-epoxides 

63 

61 

64 

68 

70 

66 69 73 

UV at 450 nm 

Tri-ols + 

Tri-ol mono-epoxides 

75 

0 

8.5 

17.1 25.6 34.1 42.6 51.2 59.7 68.2 76.7 85.3 93.8 102.3 110.8 119.4 127.9 136.4 144.9 153.5 

t R (NP), min 

FIGURE 6.13 Contour plot of the comprehensive HPLC analyses of carotenoids present in sweet orange 

essential oil with peaks and compound classes indicated (for peak identification see Ref. 136). (From Dugo, P. 

et al., 2006. Anal. Chem., 78: 7743–7750. With permission.) 

with UV-visible spectra [136] obtained by using a photodiode array detection (DAD) detector 

(Figure 6.13). A recent study of the carotenoid fraction of a saponified mandarin oil has been 

performed by means of comprehensive LC, in which a 1 D microbore silica column was applied for 

the determination of free carotenoids, and a cyanopropyl column for the separation of esters; a monolithic 

column was used in the 2 D [135]. Detection was performed by connecting a DAD system in 

parallel with an MS detection system operated in the atmospheric pressure chemical ionization 

(APCI) positive-ion mode. Thus, the identification of free carotenoid and carotenoid esters was 

carried out by combining the information provided by the DAD and MS systems, and the peak positions 

in the two-dimensional chromatograms. 

6.3.9 ON-LINE COUPLED LIQUID CHROMATOGRAPHY-GAS CHROMATOGRAPHY (LC-GC) 

The analysis of very complex mixtures is often troublesome due to the variety of chemical classes to 

which the samples components belong to, and to their wide range of concentrations. As such, several 

compounds cannot be resolved by monodimensional GC. In this respect, less complex and more 

homogeneous mixtures can be attained by the fractionation of the matrix by means of LC prior to GC 

separation. The multidimensional LC-GC approach combines the selectivity of the LC separation 

with the high efficiency and sensitivity of GC separation, enabling the separation of compounds with 

similar physicochemical properties in samples characterized by a great number of chemical classes. 

For the highly volatile components, commonly present in essential oils, the most adequate transfer 

technique is partially concurrent eluent evaporation [137]. In the latter technique, proposed by Grob, a 

retention gap is installed, followed by a few meters of precolumn and the analytical capillary GC 

column, both with identical stationary phase, for the separation of the LC fractionated components. A 

vapor exit is placed between the precolumn and the analytical column, allowing partial evaporation of 

the solvent. Hence, column and detector overloading are avoided. This transfer technique can be applied 

to the analysis of GC components with a boiling point of at least 50°C higher than the solvent.


The composition of citrus essential oils has been greatly exploited by means of LC-GC, and the 

development of new methods for the study of single classes of components has been well reported. 

The aldehyde composition in sweet orange oil has been investigated [138], as also industrial citrus 

oil mono- and sesquiterpene hydrocarbons [139], and the enantiomeric distribution of monoterpene 

alcohols in lemon, mandarin, sweet orange, and bitter orange oils [140,141]. 

The hyphenation of LC-GC systems to mass spectrometric detectors has also been reported for 

the analyses of neroli [142], bitter and sweet oranges, lemon, and petitgrain mandarin oils [143]. It 

has to be highlighted that the preliminary LC separation, which reduces mutual component interference, 

greatly simplifies MS identification. 

6.4 GENERAL CONSIDERATIONS ON ESSENTIAL OIL ANALYSIS 

As evidenced by the numerous techniques described in the present contribution, chromatography, 

especially GC, has evolved into the dominant method for essential oil analysis. This is to be expected 

because the complexity of the samples must be unraveled by some type of separation, before the 

sample constituents can be measured and characterized; in this respect, GC provides the greatest 

resolving power for most of these volatile mixtures. 

In the past, a vast number of investigations have been carried out on essential oils, and many of 

these natural ingredients have been investigated following the introduction of GC-MS, which 

marked a real turning point in the study of volatile molecules. Es-GC also represented a landmark 

in the detection of adulterations, and in the cases where the latter technique could fail, gas chromatography 

correlated to isotope ratio mass spectrometry (GC-IRMS) by means of a combustion interface 

has proved to be a valuable method to evaluate the genuineness of natural product components. 

In addition, the introduction of GC-O was a breakthrough in analytical aroma research, enabling 

the differentiation of a multitude of volatiles in odor-active and non-odor-active, according to their 

existing concentrations in a matrix. The investigation of the nonvolatile fraction of essential oils, by 

means of LC and its related hyphenated techniques, contributed greatly toward the progress of 

the knowledge on essential oils. Many extraction techniques have also been developed, boosting the 

attained results. Moreover, the continuous demand for new synthetic compounds reproducing 

the sensations elicited by natural flavors triggered analytical investigations toward the attainment of 

information on scarcely known properties of well-known matrices. 

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7 

Safety Evaluation of Essential 

Oils: A Constituent-Based 

Approach 

Timothy B. Adams and Sean V. Taylor 

CONTENTS 

7.1 Introduction ....................................................................................................................... 186 

7.2 Constituent-Based Evaluation of an Essential Oil ............................................................. 187 

7.3 Scope of Essential Oils Used in Food ............................................................................... 187 

7.3.1 Plant Sources ......................................................................................................... 187 

7.3.2 Processing of Essential Oils for Flavor Functions ................................................ 188 

7.3.3 Chemical Composition and Congeneric Groups ................................................... 188 

7.3.4 Chemical Assay Requirements and Chemical Description of Essential Oil ......... 190 

7.3.4.1 Intake of the Essential Oil ....................................................................... 191 

7.3.4.2 Analytical Limits on Constituent Identification ...................................... 192 

7.3.4.3 Intake of Congeneric Groups .................................................................. 192 

7.4 Safety Considerations for Essential Oils, Constituents, and Congeneric Groups ............. 193 

7.4.1 Essential Oils ......................................................................................................... 193 

7.4.1.1 Safety of Essential Oils: Relationship to Food ....................................... 193 

7.4.2 Safety of Constituents and Congeneric Groups in Essential Oils ......................... 194 

7.5 The Guide and Example for the Safety Evaluation of Essential Oils ............................... 195 

7.5.1 Introduction ........................................................................................................... 195 

7.5.2 Elements of the Guide for the Safety Evaluation of the Essential Oil .................. 196 

7.5.2.1 Introduction ............................................................................................. 196 

7.5.2.2 Prioritization of Essential Oil According to Presence in Food ............... 196 

7.5.2.3 Organization of Chemical Data: Congeneric Groups and 

Classes of Toxicity .................................................................................. 197 

7.6 Summary ........................................................................................................................... 205 

References .................................................................................................................................. 205 

As a practical matter, the analytical requirements for the quantification and identification of chemical 

constituents are based on exposure to the essential oil from food and/or flavor use. With increased 

exposure there is a requirement for lower detection limits and therefore identification of a greater 

number of constituents. The flexibility of the guide is reflected in the fact that high intake of major 

congeneric groups of low toxicologic concern will be evaluated along with low intake of minor 

congeneric groups of significant toxicological concern. The guide also provides a comprehensive 

evaluation of all congeneric groups and constituents that account for the majority of the composition 

185


of the essential oil. The overall objective of the guide is to organize and prioritize the chemical 

constituents of an essential oil such that no significant risk associated with the intake of essential oil 

goes unevaluated. The guide is, however, not intended to be a rigid checklist and requires that expert 

judgment be applied to ensure that each essential oil is exhaustively evaluated. 


Based on their action on the human senses, plants and essential oils and extracts derived from them 

have functioned as sources of food, preservatives, medicines, symbolic articles in religious and 

social ceremonies, and remedies to modify behavior. In many cases, essential oils and extracts 

gained widespread acceptance as multifunctional agents due to their strong stimulation of the human 

gustatory (taste) and olfactory (smell) senses. Cinnamon oil exhibits a pleasing warm spicy aftertaste, 

characteristic spicy aroma, and preservative properties that made it attractive as a food flavoring 

and fragrance. Four millennia ago, cinnamon oil was the principal ingredient of a holy ointment 

mentioned in Exodus 32:22–26. Because of its perceived preservative properties, cinnamon and cinnamon 

oil were sought by Egyptians for embalming. According to Dioscorides (Dioscorides, first 

century ad), cinnamon was a breath freshener, would aid in digestion, counteract the bites of venomous 

beasts, reduce inflammation of the intestines and the kidneys, and act as a diuretic. Applied 

to the face, it was purported to remove undesirable spots. It is no wonder that in 1000 bc cinnamon 

was more expensive than gold. 

Based on histories of use of selected plants and plant products that strongly impact the senses, 

it is not unexpected that society would bestow powers to heal, cure diseases, and spur desirable 

emotions in the effort to improve the human condition, often with only a limited understanding or 

acknowledgment of the toxic effects associated with high doses of these plant products. The “natural” 

origin of these products and their long history of use by humans have, in part, mitigated concerns 

as to whether these products work or are safe under conditions of intended use (Arctander, 

1969). The adverse effects resulting from the human use of pennyroyal oil as an abortifacient or 

wild germander as a weight control agent are reminders that no substances is safe independent of 

considerations of dose. In the absence of information concerning efficacy and safety, recommendations 

for the quantity and quality of natural product to be consumed as a medicine remain 

ambiguous. However, when the intended use is as a flavor or fragrance that is subject to governmental 

regulation, effective and safe levels of use are defined by fundamental biological limits and 

careful risk assessment. 

Flavors and fragrances are complex mixtures that act directly on the gustatory and olfactory 

receptors in the mouth and nose leading to taste and aroma responses, respectively. Saturation of 

these receptors by the individual chemicals within the flavors and fragrances occurs at very low 

levels in animals. Hence, with few exceptions the effects of flavors and fragrances are self-limiting. 

The evolution of the human diet is tightly tied to the function of these receptors. Taste and aroma 

not only determine what we eat but often allow us to evaluate the quality of food and, in some cases, 

identify unwanted contaminants. The principle of self-limitation taken together with the long history 

of use of essential oils in food argues that these substances are safe under intended conditions of 

use. In the United States, the conclusion by the U.S. Food and Drug Administration (21 CFR Sec. 

182.10, 182.20, 482.40, and 182.50) that these oils are “generally recognized as safe” (GRAS) for 

their intended use was based, in large part, on these two considerations. In Europe and Asia, the 

presumption of “safe under conditions of use” has been bestowed on essential oils based on similar 

considerations. 

For other intended uses such as dietary supplements or direct food additives, a traditional toxicology 

approach has been used to demonstrate the safety of essential oils. This relies on performing 

toxicity tests on laboratory animals, assessing intake and intended use, and determining adequate 

margins of safety between daily intake by humans and toxic levels resulting from animal studies. 

Given the constantly changing marketplace and the consumer demand for new and interesting

Safety Evaluation of Essential Oils 187 

products, however, many new intended uses for natural products are required. The resources necessary 

to test all natural products for each intended use are simply not economical. For essential oils 

that are complex mixtures of chemicals, the traditional approach is effective only when specifications 

for the composition and purity are clearly defined and adequate quality controls are in place 

for the continued commercial use of the oil. In the absence of such specifications, the results of 

toxicity testing apply specifically and only to the article tested. Recent safety evaluation approaches 

(Schilter et al., 2003) suggest that a multifaceted decision-tree approach can be applied to prioritize 

natural products and the extent of data required to demonstrate safety under conditions of use. 

The latter approach offers many advantages, both economic as well as scientific, over more traditional 

approaches. Nevertheless, various levels of resource-intensive toxicity testing of an essential 

oil are required in this approach. 

7.2 CONSTITUENT-BASED EVALUATION OF AN ESSENTIAL OIL 

Because essential oils are mixtures of volatile organic substances of known chemical structure that 

react, either singly or in combination, with biomolecules (proteins, etc.) to produce biological 

responses, it should be possible to relate the intake of high doses of these substances to observed 

toxicity. No attempt has yet been made to evaluate the safety of a natural product based on its chemical 

composition and the variability of that composition for the intended use. The chemical constitution 

of a natural product is fundamental to understanding the product’s intended use and factors 

affecting its safety. Recent advances in analytical methodology have made intensive investigation of 

the chemical composition of a natural product economically feasible and even routine. Highthroughput 

instrumentation necessary to perform extensive qualitative and quantitative analysis of 

complex chemical mixtures and to evaluate the variation in the composition of the mixture is now 

a reality. In fact, analytical tools needed to chemically characterize these complex mixtures are 

becoming more cost effective, while the cost of traditional toxicology is becoming more cost intensive. 

Based on the wealth of existing chemical and biological data on the constituents of essential 

oils and similar data on essential oils themselves, it is possible to validate a constituent-based safety 

evaluation of an essential oil. 

As noted above, it is scientifically valid to evaluate the safety of a natural mixture based on its 

chemical composition. Fundamentally, it is the interaction between one or more molecules in the 

natural product and macromolecules (proteins, enzymes, etc.) that yield the biological response, 

regardless of whether it is a desired functional effect such as a pleasing taste, or a potential toxic 

effect such as liver necrosis. Many of the advertised beneficial properties of ephedra are based on 

the presence of the central nervous system stimulant ephedrine. So too, the gustatory and olfactory 

properties of coriander oil are, in part, based on the binding of the linalool, benzyl benzoate, and 

other molecules to the appropriate receptors. It is these molecular interactions of chemical constituents 

that ultimately determine conditions of use. 

7.3 SCOPE OF ESSENTIAL OILS USED IN FOOD 

7.3.1 PLANT SOURCES 

Essential oils, as products of distillation, are mixtures of mainly low-molecular-weight chemical 

substances. Sources of essential oils include components (e.g., pulp, bark, peel, leaf, berry, and 

blossom) of fruits, vegetables, spices, and other plants. Essential oils are prepared from foods and 

nonfood sources. Many of the approximately 100 essential oils used as flavoring substances in food 

are derived directly from food (i.e., lemon oil, basil oil, and cardamom oil); far fewer are extracts 

from plants not normally consumed as food (e.g., cedar leaf oil or balsam fir oil). 

Whereas an essential oil is typically obtained by steam distillation of the plant or plant part, an 

oleoresin is produced by extraction of the same with an appropriate organic solvent. The volatile


constituents of the plant isolated in the essential oil are primarily responsible for aroma and taste of 

the plant. Hence, borneol, bornyl acetate, camphor, and other volatile constituents in rosemary oil 

can provide a flavor intensity as potent as the mass of dried rosemary used to produce the oil. A few 

exceptions include cayenne pepper, black pepper, ginger, paprika, and sesame seeds that contain 

key nonvolatile flavor constituents (e.g., gingerol and zingerone in ginger). These nonvolatile constituents 

are often higher-molecular-weight hydrophilic substances that would be lost in the preparation 

of an essential oil but are present in the fixed oil of an oleoresin. For economic reasons, crude 

essential oils are often produced via distillation at the source of the plant raw material and subsequently 

further processed at modern flavor facilities. The methods of preparation of essential oils 

are reviewed in Chapter 4. 

7.3.2 PROCESSING OF ESSENTIAL OILS FOR FLAVOR FUNCTIONS 

Because essential oils are a product of nature, environmental and genetic factors will impact the 

chemical composition of the plant. Factors such as species and subspecies, geographical location, 

harvest time, plant part used, and method of isolation all affect the chemical composition of the 

crude material separated from the plant. The variability of the composition of the crude essential oil 

as isolated from nature has been the subject of much research and development since plant and oil 

yields are major economic factors in crop production. 

However, the crude essential oil that arrives at the flavor processing plant is not normally used as 

such. The crude oil is often subjected to a number of processes that are intended to increase purity 

and to produce a product with the intended flavor characteristics. Some essential oils may be distilled 

and cooled to remove natural waxes and improve clarity, while others are distilled more than 

once (i.e., rectified) to remove undesirable fractions or to increase the relative content of certain 

chemical constituents. Some oils are dry or vacuum distilled. Normally, at some point during processing, 

the essential oil is evaluated for its technical function as a flavor. This evaluation typically 

involves analysis [normally by gas chromatography (GC) or liquid chromatography] of the composition 

of the essential oil for chemical constituents that are markers for the desired technical flavor 

effect. For an essential oil such as cardamom oil, levels of target constituents such as terpinyl acetate, 

1,8-cineole, and limonene are markers for technical viability as a flavoring substance. Based 

on this initial assessment, the crude essential oil may be blended with other sources of the same oil 

or chemical constituents isolated from the oil to reach target ranges for key constituent markers that 

reflect flavor function. The mixture may then be further rectified by distillation. Each step of the 

process is driven by flavor function. Therefore, the chemical composition of product to be marketed 

may be significantly different from that of the crude oil. Also, the chemical composition of the processed 

essential oil is more consistent than that of the crude batches of oil isolated from various 

plant harvests. The range of concentrations for individual constituents and for groups of structurally 

related constituents in an essential oil are dictated, in large part, by the requirement that target levels 

of flavor-marker constituents must be maintained. 

7.3.3 CHEMICAL COMPOSITION AND CONGENERIC GROUPS 

In addition to the key chemical markers for the technical flavor effect, an essential oil found on the 

market will normally contain many other chemical constituents, some having little or no flavor 

function. However, the chemical constituents of essential oils are not infinite in structural variation. 

Because they are derived from higher plants, these constituents are formed via one of four or 

five major biosynthetic pathways: lipoxygenase oxidation of lipids, shikimic acid, isoprenoid (terpenoid), 

and photosynthetic pathways. In ripening vegetables, lipoxygenases oxidize polyunsaturated 

fatty acids, eventually yielding low-molecular-weight aldehydes (2-hexenal), alcohols 

(2,6-nonadienol), and esters, many exhibiting flavoring properties. Plant amino acids phenylalanine 

and tyrosine are formed via the shikimic acid pathway and can subsequently be deaminated,


oxidized, and reduced to yield important aromatic substances such as cinnamaldehyde and 

eugenol. The vast majority of constituents detected in commercially viable essential oils are terpenes 

[e.g., hydrocarbons (limonene), alcohols (menthol), aldehydes (citral), ketones (carvone), 

acids, and esters (geranyl acetate)] that are formed via the isoprene pathway (Roe and Field, 1965). 

Since all of these pathways operate in plants, albeit to different extents depending on the species, 

season, and growth environment, many of the same chemical constituents are present in a wide 

variety of essential oils. 

A consequence of having a limited number of plant biosynthetic pathways is that structural variation 

of chemical constituents in an essential oil is limited. Essential oils typically contain 5–10 

distinct chemical classes or congeneric groups. Some congeneric groups, such as aliphatic terpene 

hydrocarbons, contain upwards of 100 chemically identified constituents. In some essential oils, a 

single constituent (e.g., citral in lemongrass oil) or congeneric group of constituents (e.g., hydroxyallylbenzene 

derivatives; eugenol, eugenyl acetate, etc., in clove bud oil) comprise the majority of the 

mass of the essential oil. In others, no single congeneric group predominates. For instance, although 

eight congeneric groups comprise >98% of the composition of oil of mentha piperita (peppermint 

oil), >95% of the oil is accounted for by three chemical groups—(1) terpene aliphatic and aromatic 

hydrocarbons, (2) terpene alicyclic secondary alcohols, ketones, and related esters, and (3) terpene 

2-isopropylidene substituted cyclohexanone derivatives and related substances. 

The formation and members of a congeneric group are chosen based on a combination of structural 

features and known biochemical fate. Substances with a common carbon-skeletal structure 

and functional groups that participate in common pathways of metabolism are assigned to the same 

congeneric group. For instance, menthyl acetate hydrolyzes prior to absorption yield menthol, which 

is absorbed and is inconvertible with menthone in fluid compartments (e.g., the blood). Menthol is 

either conjugated with glucuronic acid and excreted in the urine or undergoes further hydroxylation 

mainly at C8 to yield a diol that is also excreted, either free or conjugated. Despite the fact that 

menthyl acetate is an ester, menthol is an alcohol, menthone a ketone, and 3,8-menthanediol a diol; 

they are structurally and metabolically related (Figure 7.1). Therefore are members of the same 

congeneric group. 

In the case of Mentha piperita, the three principal congeneric groups listed above have different 

metabolic options and possess different organ-specific toxic potential. The congeneric group of 

terpene aliphatic and aromatic hydrocarbons is represented mainly by limonene and myrcene. The 

second and most predominant congeneric group is the alicyclic secondary alcohols, ketones, and 

related esters that include d-menthol, menthone, isomenthone, and menthyl acetate. Although the 

O 

O 

OH 

Menthyl 

acetate 

Menthol 

OH 

OH 

O 

p-Menthane-3,8-diol 

Menthone 

FIGURE 7.1 Congeneric groups are formed by members sharing common structural and metabolic features, 

such as the group of 2-isopropylidene substituted cyclohexanone derivatives and related substances.


third congeneric group contains alicyclic ketones similar in structure to menthone, it is metabolically 

quite different in that it contains an exocyclic isopropylidene substituent that undergoes hydroxylation 

principally at the C9 position, followed by ring closure and dehydration to yield a heteroaromatic 

furan ring of increased toxic potential. In the absence of a C4–C8 double bond, neither 

menthone nor isomenthone can participate in this intoxication pathway. Hence they are assigned to 

a different congeneric group. 

The presence of a limited number of congeneric groups in an essential oil is critical to the 

organization of constituents and subsequent safety evaluation of the oil itself. Members of each 

congeneric group exhibit common structural features and participate in common pathways of pharmacokinetics 

and metabolism and exhibit similar toxicologic potential. If the mass of the essential 

oil (>95%) can be adequately characterized chemically and constituents assigned to well-defined 

congeneric groups, the safety evaluation of the essential oil can be reduced to (1) a safety evaluation 

of each of the congeneric groups comprising the essential oil; and (2) a “sum of the parts” evaluation 

of the all congeneric groups to account for any chemical or biological interactions between congeneric 

groups in the essential oil under conditions of intended use. Validation of such an approach 

lies in the stepwise comparison of the dose and toxic effects for each key congeneric group with 

similar equivalent doses and toxic effects exhibited by the entire essential oil. 

Potential interactions between congeneric groups can, to some extent, be analyzed by an in-depth 

comparison of the biochemical and toxicologic properties of different congeneric groups in the 

essential oil. For some representative essential oils that have been the subject of toxicology studies, 

a comparison of data for the congeneric groups in the essential oil with data on the essential oil itself 

(congeneric groups together) is a basis for analyzing the presence or absence of interactions. 

Therefore, the impact of interaction between congeneric groups is minimal if the levels of and endpoints 

for toxicity of congeneric groups (e.g., tertiary terpene alcohols) are similar to those of the 

essential oil (e.g., coriander oil). 

Since composition plays such a critical role in the evaluation, analytical identification requirements 

are also critical to the evaluation. Complete chemical characterization of the essential oil may 

be difficult or economically unfeasible based on the small volume of essential oil used as a flavor 

ingredient. In these few cases, mainly for low-volume essential oils, the unknown fraction may be 

appreciable and a large number of chemical constituents will not be identified. However, if the 

intake of the essential oil is low or significantly less than its intake from consumption of food 

(e.g., thyme) from which the essential oil is derived (e.g., thyme oil), there should be no significant 

concern for safety under conditions of intended use. For those cases in which chemical characterization 

of the essential oil is limited but the volume of intake is more significant, it may be necessary 

to perform additional analytical work to decrease the number of unidentified constituents or, in 

other cases, to perform selected toxicity studies on the essential oil itself. A principal goal of the 

safety evaluation of essential oils is that no congeneric groups that have significant human intakes 

should go unevaluated. 

7.3.4 CHEMICAL ASSAY REQUIREMENTS AND CHEMICAL DESCRIPTION OF ESSENTIAL OIL 

The safety evaluation of an essential oil involves specifying the biological origin, physical and 

chemical properties, and any other relevant identifying characteristics. An essential oil produced 

under good manufacturing practices (GMP) should be of an appropriate purity (quality), and chemical 

characterization should be complete enough to guarantee a sufficient basis for a thorough safety 

evaluation of the essential oil under conditions of intended use. Because the evaluation is based 

primarily on the actual chemical composition of the essential oil, full specifications used in a safety 

evaluation will necessarily include not only information on the origin of the essential oil (commercial 

botanical sources, geographical sources, plant parts used, degree of maturity, and methods of 

isolation) and physical properties (specific gravity, refractive index, optical rotation, solubility, etc.), 

but also chemical assays for a range of essential oils currently in commerce.


7.3.4.1 Intake of the Essential Oil 

Based on current analytical methodology, it is possible to identify literally hundreds of constituents 

in an essential oil and quantify the constituents to part per million levels. But is this necessary 

or desirable ? From a practical point of view, the level of analysis for constituents should be directly 

related to the level of exposure to the essential oil. The requirements to identify and quantify constituents 

for use of 2,000,000 kg of peppermint oil annually should be far greater than that for use 

of 2000 kg of coriander oil or 50 kg of myrrh oil annually. Also, there is a level at which exposure 

to each constituent is so low that there is no significant risk associated with intake of that substance. 

A conservative no-significant-risk-level of 1.5 mg/d (0.0015 mg/d or 0.000025 mg/kg/d) 

has been adopted by regulatory authorities as a level at which the human cancer risk is below 

one in one million (FDA, 2005). Therefore, if consumption of an essential oil results in an intake 

of a constituent that is


7.3.4.2 Analytical Limits on Constituent Identification 

As described above, the analytical requirements for detection and identification of the constituents 

of an essential oil are set by the intake of the oil and by the conservative assumption that 

constituents with intakes 0.15% would need to 

be chemically characterized and quantified: 

1.5 mg/d 

¥ 100 = 0.15 %. 

978 mg/d 

For the vast majority of essential oils, meeting these characterization requirements does not 

require exotic analytical techniques and the identification of the constituents is of a routine nature. 

However, what would the requirements be for very high-volume essential oils, such as orange oil, 

cold-pressed oil (567,000 kg), or peppermint oil (1,229,000 kg)? In these cases, a practical limit 

must be applied and can be justified based on the concept that the intake of these oils is widespread 

and far exceeds the 10% assumption of PCI ¥ 10. Based on current analytical capabilities, 0.10% or 

0.05% could be used as a reasonable limit of detection, with the lower level used for an essential oil 

that is known or suspected to contain constituents of higher toxic potential (e.g., methyl eugenol 

in basil). 

7.3.4.3 Intake of Congeneric Groups 

Once the analytical limits for identification of constituents have been met, it is key to evaluate the 

intake of each congeneric group from consumption of the essential oil. A range of concentration of 

each congeneric group is determined from multiple analyses of different lots of the essential oil used 

in flavorings. The intake of each congeneric group is determined from mean concentrations (%) of 

constituents recorded for each congeneric group. For instance, for peppermint oil the alicyclic secondary 

alcohol/ketone/related ester group may contain (−)-menthol, (−)-menthone, (−)-menthyl acetate, 

and isomenthone in mean concentrations of 43.0%, 20.3%, 4.4%, and 0.40%, respectively, with 

that congeneric group accounting for 68.1% of the oil. It should be emphasized that although members 

in a congeneric group may vary among the different lots of oil, the variation in concentration 

of congeneric groups in the oil is relatively small. 

Routinely, the daily PCI of the essential oil derived from the annual volumes is reported in industry 

surveys (NAS, 1965, 1970, 1975, 1982, 1987; Lucas et al., 1999, 2005; EFFA, 2005; JFFMA, 

2002). If a conservative estimate of intake of the essential oil is made using a volume- based approach 

such that a defined group of constituents and congeneric groups are set for each essential oil, target 

constituents can be monitored in an ongoing quality control program and the composition of the 

essential oil can become one of the key specifications linking the product that is distributed in the 

marketplace to the chemically based safety evaluation. 

Limited specifications for the chemical composition of some essential oils to be used as food 

flavorings are currently listed in the Food Chemicals Codex (FCC, 2008). For instance, the chemical 

assay for cinnamon oil is given as “not less than 80%, by volume, as total aldehydes.” Any 

specification developed related to this safety evaluation procedure should be consistent with 

already published specifications including FCC and ISO standards. However, based on chemical


analyses for the commercially available oil, the chemical specification or assay can and should be 

expanded to 

1. Specify the mean of concentrations for congeneric groups with confidence limits that constitute 

a sufficient number of commercial lots constituting the vast majority of the oil. 

2. Identify key constituents of intake >1.5 μg/d in these groups that can be used to efficiently 

monitor the quality of the oil placed into commerce over time. 

3. Provide information on trace constituents that may be of a safety concern. 

For example, given its most recent reported annual volume (649 kg), it is anticipated that a chemical 

specification for lemongrass oil would include (1) >97.6% of the composition chemically identified; 

(2) not more than 92% aliphatic terpene primary alcohols, aldehydes, acids, and related esters, 

typically measured as citral; and (3) not more than 15% aliphatic terpene hydrocarbons, typically 

measured as myrcene. The principal goal of a chemical specification is to provide sufficient chemical 

characterization to ensure safety of the essential oil from use as a flavoring. From an industry 

standpoint, the specification should be sufficiently descriptive as to allow timely quality control 

monitoring for constituents that are responsible for the technical flavor function. These constituents 

should also be representative of the major congeneric group or groups in the essential oil. Also, 

monitored constituents should include those that may be of a safety concern at sufficiently high 

levels of intake of the essential oil (e.g., pulegone). The scope of a specification should be sufficient 

to ensure safety in use, but not impose an unnecessary burden on industry to perform ongoing 

analyses for constituents unrelated to the safety or flavor of the essential oil. 

7.4 SAFETY CONSIDERATIONS FOR ESSENTIAL OILS, CONSTITUENTS, 

AND CONGENERIC GROUPS 

7.4.1 ESSENTIAL OILS 

7.4.1.1 Safety of Essential Oils: Relationship to Food 

The close relationship of natural flavor complexes to food itself has made it difficult to evaluate the 

safety and regulate the use of essential oils. In the United States, the Federal Food Drug and Cosmetic 

Act (FFDCA) recognizes that a different, lower standard of safety must apply to naturally occurring 

substances in food than applies to the same ingredient intentionally added to food. For a substance 

occurring naturally in food, the Act applies a realistic standard that the substance must “… not ordinarily 

render it [the food] injurious to health” (21 CFR 172.30). For added substances, a much higher standard 

applies. The food is considered to be adulterated if the added substance “… may render it [the food] 

injurious to health” (21 CFR 172.20). Essential oils used as flavoring substances occupy an intermediate 

position in that they are composed of naturally occurring substances, many of which are intentionally 

added to food as individual chemical substances. Because they are considered neither a direct food additive 

nor a food itself, no current standard can be easily applied to the safety evaluation of essential oils. 

The evaluation of the safety of essential oils that have a documented history of use in foods starts 

with the presumption that they are safe based on their long history of use over a wide range of human 

exposures without known adverse effects. With a high degree of confidence one may presume that 

essential oils derived from food are likely to be safe. Annual surveys of the use of flavoring substances 

in the United States (Lucas et al., 1999, 2005; NAS, 1965, 1970, 1975, 1981, 1987; 21 CFR 

172.510) in part, document the history of use of many essential oils. Conversely, confidence in the 

presumption of safety decreases for natural complexes that exhibit a significant change in the pattern 

of use or when novel natural complexes with unique flavor properties enter the food supply. Recent 

consumer trends that have changed the typical consumer diet have also changed the exposure levels 

to essential oils in a variety of ways. As one example, changes in the use of cinnamon oil in low-fat 

cinnamon pastries would alter intake for a specialized population of eaters. Secondly, increased


international trade has coupled with a reduction in cultural cuisine barriers, leading to the introduction 

of novel plants and plant extracts from previously remote geographical locations. Osmanthus absolute 

(FEMA No. 3750) and Jambu oleoresin (FEMA No. 3783) are examples of natural complexes 

recently used as flavoring substances that are derived from plants not indigenous to the United States 

and not commonly consumed as part of a Western diet. Furthermore, the consumption of some essential 

oils may not occur solely from intake as flavoring substances; rather, they may be regularly consumed 

as dietary supplements with advertised functional benefits. These impacts have brought 

renewed interest in the safety evaluation of essential oils. Although the safety evaluation of essential 

oil must still rely heavily on knowledge of the history of use, a flexible science-based approach would 

allow for rigorous safety evaluation of different uses for the same essential oil. 

7.4.2 SAFETY OF CONSTITUENTS AND CONGENERIC GROUPS IN ESSENTIAL OILS 

Of the many naturally occurring constituents so far identified in plants, there are none that pose, or 

reasonably might be expected to pose a significant risk to human health at current low levels of 

intake when used as flavoring substances. When consumed in higher quantities, normally for other 

functions, some plants do indeed exhibit toxicity. Historically, humans have used plants as poisons 

(e.g., hemlock) and many of the intended medicinal uses of plants (pennyroyal oil as an abortifacient) 

have produced undesirable toxic side effects. High levels of exposure to selected constituents 

in the plant or essential oil (i.e., pulegone in pennyroyal oil) have been associated with the observed 

toxicity. However, with regard to flavor use, experience through long-term use and the predominant 

self-limiting impact of flavorings on our senses have restricted the amount of a plant or plant part 

that we use in or on food. 

Extensive scientific data on the most commonly occurring major constituents in essential oils have 

not revealed any results that would give rise to safety concerns at low levels of exposure. Chronic 

studies have been performed on more 30 major chemical constituents (menthol, carvone, limonene, 

citral, cinnamaldehyde, benzaldehyde, benzyl acetate, 2-ethyl-1-hexanol, methyl anthranilate, geranyl 

acetate, furfural, eugneol, isoeugenol, etc.) found in many essential oils. The majority of these 

studies were hazard determinations that were sponsored by the National Toxicology Program (NTP) 

and they were normally performed at dose levels many orders of magnitude greater than the daily 

intakes of these constituents from consumption of the essential oil. Even at these high intake levels, 

the majority of the constituents show no carcinogenic potential (Smith et al., 2005a). In addition to 

dose/exposure, for some flavor ingredients the carcinogenic potential that was assessed in the study 

is related to several additional factors including the mode of administration, species and sex of the 

animal model, and target organ specificity. In the vast majority of studies, the carcinogenic effect 

occurs through a nongenotoxic mechanism in which tumors form secondary to preexisting highdose, 

chronic organ toxicity, typically to the liver or kidneys. Selected subgroups of structurally 

related substances (e.g., aldehydes and terpene hydrocarbons) are associated with a single-target 

organ and tumor type in a specific species and sex of rodent (i.e., male rat kidney tumors secondary 

to a-2u-globulin neoplasms with limonene in male rats) or using a single mode of administration (i.e., 

forestomach tumors that arise due to high doses of benzaldehyde and hexadienal given by gavage). 

Given their long history of use, it is unlikely that there are essential oils consumed by humans that 

contain constituents not yet studied that are weak nongenotoxic carcinogens at chronic high-dose levels. 

Even if there are such cases, because of the relatively low intake (Adams et al., 2005) as constituents of 

essential oils, these yet-to-be-discovered constituents would be many orders of magnitude less potent 

than similar levels of aflatoxins (found in peanut butter), the polycyclic heterocyclic amines (found in 

cooked foods), or the polynuclear aromatic hydrocarbons (also found in cooked foods). There is nothing 

to suggest that the major biosynthetic pathways available to higher plants are capable of producing substances 

such that low levels of exposure to the substance would result in a high level of toxicity or carcinogenicity. 

Thus, while the minor constituents should be considered, particularly in those plant 

families and genera known to contain constituents of concern, there is less need for caution than when 

dealing with xenobiotics, or with substances from origins other than those considered here.


The toxic and carcinogenic potentials exhibited by constituent chemicals in essential oils can 

largely be equated with the toxic potential of the congeneric group to which that chemical belongs. A 

comparison of the oral toxicity data (JECFA, 2004) for limonene, myrcene, pinene, and other members 

of the congeneric group of terpene hydrocarbons show similar low levels of toxicity with the same 

high-dose target organ endpoint (kidney). Likewise, dietary toxicity and carcinogenicity data (JECFA, 

2001) for cinnamyl alcohol, cinnamaldehyde, cinnamyl acetate, and other members of the congeneric 

group of 3-phenyl-1-propanol derivatives show similar toxic and carcinogenic endpoints. The safety 

data for the congeneric chemical groups that are found in vast majority of essential oils have been 

reviewed (Adams et al., 1996, 1997, 1998, 2004; Newberne et al., 1999; Smith et al., 2002a, 2002b; 

JECFA, 1997–2004). Available data for different representative members in each of these congeneric 

groups support the conclusion that the toxic and carcinogenic potential of individual constituents 

adequately represent similar potentials for the corresponding congeneric group. 

The second key factor in the determination of safety is the level of intake of the congeneric group 

from consumption of the essential oil. Intake of the congeneric group will, in turn, depend on the 

variability of the chemical composition of the essential oil in the marketplace and on the conditions 

of use. As discussed earlier, chemical analysis of the different batches of oil obtained from the same 

and different manufacturers will produce a range of concentrations for individual constituents in 

each congeneric group of the essential oil. The mean concentration values (%) for constituents are 

then summed for all members of the congeneric group. The total % determined for the congeneric 

group is multiplied by the estimated daily intake (PCI ¥ 10) of the essential oil to provide a conservative 

estimate of exposure to each congeneric group from consumption of the essential oil. 

In some essential oils, the intake of one constituent, and therefore, one congeneric group, may 

account for essentially all of the oil (e.g., linalool in coriander oil, citral in lemongrass oil, and benzaldehyde 

in bitter almond oil). In other oils, exposure to a variety of congeneric groups over a broad 

concentration range may occur. As noted earlier, cardamom oil is an example of such an essential 

oil. Ultimately, it is the relative intake and the toxic potential of each congeneric group that is the 

basis of the congeneric group-based safety evaluation. The combination of relative intake and toxic 

potential will prioritize congeneric groups for the safety evaluation. Hypothetically, a congeneric 

group of increased toxic potential that accounts for only 5% of the essential oil may be prioritized 

higher than a congeneric group of lower toxic potential accounting for 95%. 

The following guide and examples therein are intended to more fully illustrate the principles 

described above that are involved in the safety evaluation of essential oils. Fermentation products, 

process flavors, substances derived from fungi, microorganisms, or animals; and direct food additives 

are explicitly excluded. The guide is designed specifically for application to approximately 100 

essential oils that are currently in use as flavoring substances, and for any new essential oils that are 

anticipated to be marketed as flavoring substances. The guide is a tool to organize and prioritize the 

chemical constituents and congeneric groups in an essential oil in such a way as to allow a detailed 

analysis of their chemical and biological properties. This analysis as well as consideration of other 

relevant scientific data provides the basis for a safety evaluation of the essential oil under conditions 

of intended use. Validation of the approach is provided, in large part, by a detailed comparison of 

the doses and toxic effects exhibited by constituents of the congeneric group with the equivalent 

doses and effects provided by the essential oil. 

7.5 THE GUIDE AND EXAMPLE FOR THE SAFETY EVALUATION 

OF ESSENTIAL OILS 

7.5.1 INTRODUCTION 

The guide does not employ criteria commonly used for the safety evaluation of individual chemical 

substances. Instead, it is a procedure involving a comprehensive evaluation of the chemical and 

biological properties of the constituents and congeneric groups of an essential oil. Constituents 

in the oil that are of known structure are organized into congeneric groups that exhibit similar


metabolic and toxicologic properties. The congeneric groups are further classified according to levels 

(Structural Classes I, II, and III) of toxicologic concern using a decision-tree approach (Cramer et al., 

1978; Munro et al., 1996). Based on intake data for the essential oil and constituent concentrations, 

the congeneric groups are prioritized according to intake and toxicity potential. The procedure 

ultimately focuses on those congeneric groups that due to their structural features and intake may 

pose some significant risk from the consumption of the essential oil. Key elements used to evaluate 

congeneric groups include exposure, structural analogy, metabolism, and toxicology, which include 

toxicity, carcinogenicity, and genotoxic potential (Adams et al., 1996, 1997, 1998, 2004; Woods and 

Doull, 1991; Oser and Ford, 1991; Oser and Hall, 1977; Newberne et al., 1999; Smith et al., 2002a, 

2002b). Throughout the analysis of these data, it is essential that professional judgment and expertise 

be applied to complete the safety evaluation of the essential oil. As an example of how a typical 

evaluation process for an essential oil is carried out according to this guide, the safety evaluation for 

flavor use of cornmint oil (Mentha arvensis) is outlined in Section 7.5.3.2.1. 

7.5.2 ELEMENTS OF THE GUIDE FOR THE SAFETY EVALUATION OF THE ESSENTIAL OIL 

7.5.2.1 Introduction 

In Step 1 of the guide, the evaluation procedure estimates intake based on industry survey data for each 

essential oil. It then organizes the chemically identified constituents that have an intake >1.5 μg/d into 

congeneric groups that participate in common pathways of metabolism and exhibit similar toxic potential. 

In Steps 2 and 3, each identified chemical constituent is broadly classified according to toxic 

potential (Cramer et al., 1978) and then assigned to a congeneric group of structurally related substances 

that exhibit similar pathways of metabolism and toxicologic potential. 

Before the formal evaluation begins, it is necessary to specify the data (e.g., botanical, physical, 

and chemical) required to completely describe the product being evaluated. In order to effectively 

evaluate an essential oil, attempted complete analyses must be available for the product intended for 

the marketplace from a number of flavor manufacturers. Additional quality control data are useful, 

as they demonstrate consistency in the chemical composition of the product being marketed. A 

Technical Information paper drafted for the particular essential oil under consideration organizes 

and prioritizes these data for efficient sequential evaluation of the essential oil. 

In Steps 8 and 9, the safety of the essential oil is evaluated in the context of all congeneric groups 

and any other related data (e.g., data on the essential oil itself or for an essential oil of similar composition). 

The procedure organizes the extensive database of information on the essential oil constituents 

in order to efficiently evaluate the safety of the essential oil under conditions of use. It is 

important to stress, however, that the guide is not intended to be nor in practice operates as a rigid 

checklist. Each essential oil that undergoes evaluation is different, and different data will be available 

for each. The overriding objective of the guide and subsequent evaluation is to ensure that no 

significant portion of the essential oil should go unevaluated. 

7.5.2.2 Prioritization of Essential Oil According to Presence in Food 

In Step 1, essential oils are prioritized according to their presence or absence as components of 

commonly consumed foods. Many essential oils are isolated from plants that are commonly consumed 

as a food. Little or no safety concerns should exist for the intentional addition of the essential 

oil to the diet, particularly if intake of the oil from consumption of traditional foods (garlic) substantially 

exceeds intake as an intentionally added flavoring substance (garlic oil). In many ways, the 

first step applies the concept of “long history of safe use” to essential oils. That is, if exposure to the 

essential oil occurs predominantly from consumption of a normal diet a conclusion of safety is 

straightforward. Step 1 of the guide clearly places essential oils that are consumed as part of a 

traditional diet on a lower level of concern than those oils derived from plants that are either not part 

of the traditional diet or whose intake is not predominantly from the diet. The first step also mitigates


the need to perform comprehensive chemical analysis for essential oils in those cases where intake 

is low and occurs predominantly from consumption of food. An estimate of the intake of the essential 

oil is based on the most recent poundage available from flavor industry surveys and the assumption 

that the essential oil is consumed by only 10% of the population for an oil having a survey 

volume 50,000 kg/yr. 

In addition, the detection limit for constituents is determined based on the daily PCI of the 

essential oil. 

7.5.2.2.1 Cornmint Oil 

To illustrate the type of data considered in Step 1, consider cornmint oil. Cornmint oil is produced 

by the steam distillation of the flowering herb of Mentha arvensis. The crude oil contains upwards 

of 70% (−)-menthol, some of which is isolated by crystallization at low temperature. The resulting 

dementholized oil is cornmint oil. Although produced mainly in Brazil during the 1970s and 

1980s, cornmint oil is now produced predominantly in China and India. Cornmint has a more 

stringent taste compared to that of peppermint oil, Mentha piperita, but can be efficiently produced 

and is used as a more cost-effective substitute. Cornmint oil isolated from various crops undergoes 

subsequent “clean up,” further distillation, and blending to produce the finished commercial oil. 

Although there may be significant variability in the concentrations of individual constituents in 

different samples of crude essential oil, there is far less variability in the concentration of constituents 

and congeneric groups in the finished commercial oil. The volume of cornmint oil reported 

in the most recent U.S. poundage survey is 327,494 kg/yr (Gavin et al., 2008), which is approximately 

25% of the potential market of peppermint oil. Because cornmint oil is a high- volume 

essential oil, it is highly likely that the entire population consumes the annual reported volume, and 

therefore the daily PCI is calculated based on 100% of the population (280,000,000). This results 

in a daily PCI of approximately 3.2 mg/person/d (0.0533 mg/kg bw/d) of cornmint oil. 

9 

327,494 kg/yr ¥ 10 mg/kg 

6 

365 days/yr ¥ 280 ¥ 10 persons 

= 3204 mg/person/d. 

Based on the intake of cornmint oil (3204 μg/d), any constituent present at >0.047% would need to 

be chemically characterized and quantified: 

1.5 mg/d 

¥ 100 = 0.047 %. 

3204 mg/d 

7.5.2.3 Organization of Chemical Data: Congeneric Groups and Classes of Toxicity 

In Step 2, constituents are assigned to one of three structural classes (I, II, or III) based on toxic 

potential (Cramer et al., 1978). Class I substances contain structural features that suggest a low order 

of oral toxicity. Class II substances are clearly less innocuous than Class I substances, but do not 

contain structural features that provide a positive indication of toxicity. Class III substances contain 

structural features (e.g., an epoxide functional group, unsubstituted heteroaromatic derivatives) that 

permit no strong presumption of safety, and in some cases may even suggest significant toxicity. For 

instance, the simple aliphatic hydrocarbon, limonene, is assigned to Structural Class I while elemicin, 

which is an allyl-substituted benzene derivative with a reactive benzylic/allylic position, is 

assigned to Class III. Likewise, chemically unidentified constituents of the essential oil are automatically 

placed in Structural Class III, since no presumption of safety can be made.


The toxic potential of each of the three structural classes has been quantified (Munro et al., 1996). 

An extensive toxicity database has been compiled for substances in each structural class. The database 

covers a wide range of chemical structures, including food additives, naturally occurring substances, 

pesticides, drugs, antioxidants, industrial chemicals, flavors, and fragrances. Conservative noobservable-effect-levels 

(fifth percentile NOELs) have been determined for each class. These fifth percentile 

NOELs for each structural class are converted to human exposure thresholds levels by applying 

a 100-fold safety factor and correcting for mean bodyweight (60/100). The human exposure threshold 

levels are referred to as thresholds of toxicological concern (TTC). With regard to flavoring substances, 

the TTC are even more conservative, given that the vast majority of NOELs for flavoring substances are 

above the 90th percentile. These conservative TTC have since been adopted by the WHO and 

Commission of the European Communities for use in the evaluation of chemically identified flavoring 

agents by Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the European Food 

Safety Authority (EFSA) (JECFA, 1997; EC, 1999). 

Step 3 is a key step in the guide. It organizes the chemical constituents into congeneric groups 

that exhibit common chemical and biological properties. Based on the well-recognized biochemical 

pathways operating in plants, essentially all of the volatile constituents found in essential oils, 

extracts, and oleoresins belong to well-recognized congeneric groups. Recent reports (Maarse et al., 

1992, 1994, 2000; Nijssen et al., 2003) of the identification of new naturally occurring constituents 

indicate that newly identified substances fall into existing congeneric groups. The Expert Panel, 

JECFA, and the European Communities (EC) have acknowledged that individual chemical substances 

can be evaluated in the context of their respective congeneric group (Smith et al., 2005; 

JECFA, 1997; EC, 1999). The congeneric group approach provides the basis for understanding the 

relationship between the biochemical fate of members of a chemical group and their toxicologic 

potential. Within this framework, the objective is to continuously build a more complete understanding 

of the absorption, distribution, metabolism, and excretion of members of the congeneric 

group and their potential to cause systemic toxicity. Within the guidelines, the structural class of 

each congeneric group is assigned based on the highest structural class of any member of the group. 

Therefore, if an essential oil contained a group of furanone derivatives that were variously assigned 

to structural classes II and III, then in the evaluation of the oil the congeneric group would, in a 

conservative manner, be assigned to Class III. 

The types and numbers of congeneric groups in a safety evaluation program are, by no means, 

static. As new scientific data and information become available, some congeneric groups are combined 

while others are subdivided. This has been the case for the group of alicyclic secondary 

alcohols and ketones that were the subject of a comprehensive scientific literature review (SLR) in 

1975 (FEMA, 1975). Over the last two decades, experimental data have become available indicating 

that a few members of this group exhibit biochemical fate and toxicologic potential inconsistent 

with that for other members of the same group. These inconsistencies, almost without exception, 

arise at high-dose levels that are irrelevant to the safety evaluation of low levels of exposure to flavor 

use of the substance. However, given the importance of the congeneric group approach in the safety 

assessment program, it is critical to resolve these inconsistencies. Additional metabolic and toxicologic 

studies may be required to distinguish the factors that determine these differences. Often the 

effect of dose and a unique structural feature results in utilization of a metabolic activation pathway 

not utilized by other members of a congeneric group. Currently, evaluating bodies including JECFA, 

EFSA, and the FEMA Expert Panel have classified flavoring substances into the same congeneric 

groups for the purpose of safety evaluation. 

Step 4 incorporates the available analytical data for constituents in the essential oil into the congeneric 

group approach. First, the percentages of the individual constituents that comprise each 

congeneric group are summed. The highest determined percentages for each constituent are used to 

calculate a total amount for each congeneric group, since this provides the highest possible amount of 

each congeneric group within the oil. Based on that high percentage and the estimated daily PCI of the 

essential oil, the daily PCI of the congeneric group from the essential oil is estimated.


In Steps 5, 6, and 7, each congeneric group in the essential oil is evaluated for safety in use. In 

Step 5, an evaluation of the metabolism and disposition is performed to determine, under current 

conditions of intake, whether the group of congeneric constituents is metabolized by wellestablished 

detoxication pathways to yield innocuous products. That is, such pathways exist for the congeneric 

group of constituents in an essential oil and safety concerns will arise only if intake of the congeneric 

group is sufficient to saturate these pathways potentially leading to toxicity. If a significant 

intoxication pathway exists (e.g., pulegone), this should be reflected in a higher decision-tree class 

and lower TTC threshold. At Step 6 of the procedure, the intake of the congeneric group relative to 

the respective TTC for one of the three structural classes (1800 μg/d for Class I; 540 μg/d for Class 

II; 90 μg/d for Class III; see Table 7.1) is evaluated. If the intake of the congeneric group is less than 

the threshold for the respective structural class, the intake of the congeneric group presents no significant 

safety concerns. The group passes the first phase of the evaluation and is then referred to 

Step 8, the step in which the safety of the congeneric group is evaluated in the context of all congeneric 

groups in the essential oil. 

If, at Step 5, no sufficient metabolic data exist to establish safe excretion of the product, or if 

activation pathways have been identified for a particular congeneric group, then the group moves to 

Step 7 and toxicity data are required to establish safe use under current conditions of intake. There 

are examples where low levels of xenobiotic substances can be metabolized to reactive substances. 

In the event that reactive metabolites are formed at low levels of intake of naturally occurring substances, 

a detailed analysis of dose-dependent toxicity data must be performed. Also, if the intake 

of the congeneric group is greater than the human exposure threshold (suggesting metabolic saturation 

may occur), then toxicity data are also required. If, at Step 7, a database of relevant toxicological 

data for a representative member or members of the congeneric group indicates that a sufficient 

margin of safety exists for the intake of the congeneric group, the members of that congeneric group 

are concluded to be safe under conditions of use of the essential oil. The congeneric group then 

moves to Step 8. 

TABLE 7.1 

Structural Class Definitions and Their Human Intake Thresholds 

Class 

I 

II 

III 

Description 

Structure and related data suggest a low order of toxicity. 

If combined with low human exposure, they should enjoy 

an extremely low priority for investigation. The criteria 

for adequate evidence of safety would also be minimal. 

Greater exposures would require proportionately higher 

priority for more exhaustive study 

Intermediate substances. They are less clearly innocuous 

than those of Class I, but do not offer the basis either of 

the positive indication of toxicity or of the lack of 

knowledge characteristic of those in Class III 

Permit no strong initial presumptions of safety, or that 

may even suggest significant toxicity. They thus deserve 

the highest priority for investigation. Particularly, when 

per capita intake is high of a significant subsection of the 

population has a high intake, the implied hazard would 

then require the most extensive evidence for safety-in-use 

Fifth Percentile 

NOEL (mg/kg/d) 

Human Exposure 

Threshold (TTC) a (µg/d) 

3.0 1800 

0.91 540 

0.15 90 

a 

The human exposure threshold was calculated by multiplying the fifth percentile NOEL by 60 (assuming an individual 

weighs 60 kg) and dividing by a safety factor of 100.


In the event that insufficient data are available to evaluate a congeneric group at Step 7, or the 

currently available data result in margins of safety that are not sufficient, the essential oil cannot be 

further evaluated by this guide and must be set aside for further considerations. 

Use of the guide requires scientific judgment at each step of the sequence. For instance, if a congeneric 

group that accounted for 20% of a high-volume essential oil was previously evaluated and 

found to be safe under intended conditions of use, the same congeneric group found at less than 2% 

of a low-volume essential oil does not need to be further evaluated. 

Step 8 considers additivity or synergistic interactions between individual substances and between 

the different congeneric groups in the essential oil. As for all other toxicological concerns, the level of 

exposure to congeneric groups is relevant to whether additive or synergistic effects present a significant 

health hazard. The vast majority of essential oils are used in food in extremely low concentrations, 

which therefore results in very low intake levels of the different congeneric groups within that oil. 

Moreover, major representative constituents of each congeneric group have been tested individually 

and pose no toxicological threat even at dose levels that are orders of magnitude greater than normal 

levels of intake of essential oils from use in traditional foods. Based on the results of toxicity studies 

both on major constituents of different congeneric groups in the essential oil and on the essential oil 

itself, it can be concluded that the toxic potential of these major constituents is representative of that of 

the oil itself, indicating the likely absence of additivity and synergistic interaction. In general, the margin 

of safety is so wide and the possibility of additivity or synergistic interaction so remote that combined 

exposure to the different congeneric groups and the unknowns are considered of no health 

concern, even if expert judgment cannot fully rule out additivity or synergism. However, case-by-case 

considerations are appropriate. Where possible combined effects might be considered to have toxicological 

relevance, additional data may be needed for an adequate safety evaluation of the essential oil. 

Additivity of toxicologic effect or synergistic interaction is a conservative default assumption 

that may be applied whenever the available metabolic data do not clearly suggest otherwise. The 

extensive database of metabolic information on congeneric groups (JECFA, 1997–2004) that are 

found in essential oils suggests that the potential for additive effects and synergistic interactions 

among congeneric groups in essential oils is extremely low. Although additivity of effect is the 

approach recommended by National Academy of Sciences (NAS)/National Research Council (NRC) 

committees (NRC, 1988, 1994) and regulatory agencies (EPA, 1988), the Presidential Commission 

of Risk Assessment and Risk Management recommended (Presidential Commission, 1996, p. 68) 

that “For risk assessments involving multiple chemical exposures at low concentrations, without 

information on mechanisms, risks should be added. If the chemicals act through separate mechanisms, 

their attendant risks should not be added but should be considered separately.” Thus, the 

risks of chemicals that act through different mechanisms, that act on different target systems, or that 

are toxicologically dissimilar in some other way should be considered to be independent of each 

other. The congeneric groups in essential oils are therefore considered separately. 

Further, the majority of individual constituents that comprise essential oils are themselves used as 

flavoring substances that pose no toxicological threat at doses that are magnitudes greater than their 

level of intake from the essential oil. Rulis (1987) reported that “The overwhelming majority of additives 

present a high likelihood of having safety assurance margins in excess of 10 5 .” He points out that 

this is particularly true for additives used in the USA at less than 100,000 lb/yr. Because more than 

90% of all flavoring ingredients are used at


(+)-isomenthone, (−)-menthyl acetate, and other related substances. Samples of triple-distilled 

commercial cornmint oil may contain up to 95% of this congeneric group. The biochemical and 

biological fate of this group of substances has been previously reviewed (Adams et al., 1996; 

JECFA, 1999). Key data on metabolism, toxicity, and carcinogenicity are cited below in order to 

complete the evaluation. Although constituents in this group are effectively detoxicated via conjugation 

of the corresponding alcohol or w-oxidation followed by conjugation and excretion 

(Yamaguchi et al., 1994; Madyastha and Srivatsan, 1988; Williams, 1940), the intake of the congeneric 

group (3044 μg/person/d or 3.04 mg/person/d; see Table 7.2) is higher than the exposure 

threshold of 540 μg/person/d or 0.540 mg/person/d for Structural Class II. Therefore, toxicity data 

are required for this congeneric group. In both short- and long-term studies (Madsen et al., 1986; 

JEFCA, 2000a), menthol, menthone, and other members of the group exhibit no-observable- adverseeffect-levels 

(NOAELs) at least 1000 times the daily PCI (“eaters only”) (3.04 mg/person/d or 

0.05 mg/kg bw/d) of this congeneric group resulting from intake of the essential oil. For members 

of this group, numerous in vitro and in vivo genotoxicity assays are consistently negative (Heck 

et al., 1989; Sasaki et al., 1989; Muller, 1993; Florin et al., 1980; Rivedal et al., 2000; Zamith et al., 

1993; NTP Draft, 2003). Therefore, the intake of this congeneric group from consumption of 

Mentha arvensis is not a safety concern. 

Although it is a constituent of cornmint oil and is also a terpene alicyclic ketone structurally 

related to the above congeneric group, pulegone exhibits a unique structure (i.e., 2-isopropylidenecyclohexanone) 

that participates in a well-recognized intoxication pathway (Figure 7.2) 

(McClanahan et al., 1989; Thomassen et al., 1992; Adams et al., 1996; Chen et al., 2001) that 

leads to the formation of menthofuran. This metabolite subsequently oxidizes and the ring opens 

to yield a highly reactive 2-ene-1,4-dicarbonyl intermediate that reacts readily with proteins 

resulting in hepatotoxicity at intake levels at least two orders of magnitude less than no observable 

effect levels for structurally related alicyclic ketones and secondary alcohols (menthone, 

carvone, and menthol). Therefore, pulegone and its metabolite (menthofuran), which account for 

2% of the composition of cornmint oil is a 

congeneric group of terpene hydrocarbons [(+) and (−)-pinene, (+) limonene, etc.]. Although these 

may contribute up to 8% of the oil, upon multiple redistillations during processing the hydrocarbon 

content can be significantly reduced (


TABLE 7.2 

Safety Evaluation of Cornmint Oil, Mentha arvensis a 

Steps 2 and 3: Constituent 

Identification/Congeneric 

Grouping/Structural Class 

Assignment 

Step 4: Oil Composition 

by Congeneric Group, 

Resulting Intake Steps 5,6,7,8: Metabolism/Toxicology Data/Evaluation 

Congeneric Group 

Assignment 

Structural 

Class (TTC) 

(µg/Person/d) 

High % From 

Multiple 

Commercial 

Samples 

Intake 

(µg/ 

Person/d) Metabolism Pathways 

Intake of Congeneric Group 

or Total of Unidentified 

Constituents Group < TTC 

for Class? 

Relevant Toxicity 

Data if Intake of 

Group >TTC 

Secondary alicyclic 

saturated and 

unsaturated 

alcohol/ketone/ 

ketal/ester (e.g., 

menthol, menthone, 

isomenthone, 

menthyl acetate) 

Aliphatic terpene 

hydrocarbon (e.g., 

limonene, pinene) 

II (540) 95 3044 1. Glucuronic acid conjugation of 

the alcohol followed by excretion 

in the urine 

2. w-Oxidation of the side chain 

substituents to yield various 

polyols and hydroxy acids 

and excreted as glucuronic acid 

conjugates 

I (1800) 8 256 1. w-Oxidation to yield polar 

hydroxy and carboxy metabolites 

excreted as glucuronic acid 

conjugates 

No, 3044 μg/person/d >540 μg/ 

person/d 

Yes, 256 μg /person/d,


2-Isopropylidene 

cyclohexanone and 

metabolites 

(e.g., pulegone) 

III (90) 2 64 1. Reduction to yield menthone or 

isomenthone, followed by 

hydroxylation of ring or side 

chain positions and then 

conjugation with glucuronic acid 

2. Conjugation with glutathione in 

a Michael-type addition leading to 

mercapturic acid conjugates that 

are excreted or further 

hydroxylated and excreted 

3. Hydroxylation catalyzed by 

cytochrome P-450 to yield a series 

of ring- and side chainhydroxylated 

pulegone 

metabolites, one of which is a 

reactive 2-ene-1,4-dicarbonyl 

derivative. This intermediate is 

known to form protein adducts 

leading to enhanced toxicity 

(Austin et al., 1988) 

Note: In Steps 2 and 3, individual constituents and their assignment to structural classes are not shown. 

a 

Based on daily PCI of 3204 μg/person/d for cornmint oil, as determined in Step 1. 

Yes, 64 μg/person/d


H 

H 

H 

Hydrolysis Reduction Isomerization 

H 

OAc 

OH 

O 

O 

Isopulegyl 

acetate 

Isopulegol 

Isopulegone 

Pulegone 

9-Hydroxylation 

Glucuronic 

acid 

conjugate 

H 

OH 

9-Hydroxyisopulegone 

H 

O 

Menthofuran 

FIGURE 7.2 Metabolism of isopulegone, pulegone, and isopulegyl acetate. 

Finally, the essential oil itself is evaluated in the context of the combined intake of all congeneric 

groups and any other related data in Step 8. Interestingly, members of the terpene alicyclic secondary 

alcohols, ketones, and related esters, multiple members of the monoterpene hydrocarbons, and 

peppermint oil itself show a common nephrotoxic effect recognized as a-2u-globulin nephropathy. 

The microscopic evidence of histopathology of the kidneys for male rats in the mint oil study is 

consistent with the presence of a-2u-globulin nephropathy. In addition, a standard immunoassay for 

detecting the presence of a-2u-globulin was performed on kidney sections from male and female 

rats in the mint oil study (Serota, 1990). Results of the assay confirmed the presence of a-2uglobulin 

nephropathy in male rats (Swenberg and Schoonhoven, 2002). This effect is found only in 

males rats and is not relevant to the human health assessment of cornmint oil. Other toxic interactions 

between congeneric groups are expected to be minimal given that the NOELs for the 

congeneric groups and those for finished mint oils are on the same order of magnitude. 

Based on the above assessment and the application of the scientific judgment, cornmint oil is 

concluded to be “GRAS” under conditions of intended use as a flavoring substance. Given the 

criteria used in the evaluation, recommended specifications should include the following chemical 

assay: 

1. Less than 95% alicyclic secondary alcohols, ketones, and related esters, typically measured 

as (−)-menthol. 

2. Less than 2% 2-isopropylidenecyclohexanones and their metabolites, measured as 

(−)-pulegone. 

3. Less than 10% monoterpene hydrocarbons, typically measured as limonene.


7.6 SUMMARY 

The safety evaluation of an essential oil is performed in the context of all available data for congeneric 

groups of identified constituents and the group of unidentified constituents, data on the 

essential oil or a related essential oil, and any potential interactions that may occur in the essential 

oil when consumed as a flavoring substance. 

The guide provides a chemically based approach to the safety evaluation of an essential oil. The 

approach depends on a thorough quantitative analysis of the chemical constituents in the essential 

oil intended for commerce. The chemical constituents are then assigned to well-defined congeneric 

groups that are established based on extensive biochemical and toxicologic information, and this 

is evaluated in the context of intake of the congeneric group resulting from consumption of the 

essential oil. The intake of unidentified constituents considers the consumption of the essential 

oil as a food, a highly conservative toxicologic threshold, and toxicity data on the essential oil 

or an essential oil of similar chemical composition. The flexibility of the guide is reflected in the 

fact that high intake of major congeneric groups of low toxicologic concern will be evaluated 

along with low intake of minor congeneric groups of significant toxicological concern (i.e., higher 

structural class). The guide also provides a comprehensive evaluation of all congeneric groups 

and constituents that account for the majority of the composition of the essential oil. The overall 

objective of the guide is to organize and prioritize the chemical constituents of an essential oil 

in order that no reasonably possible significant risk associated with the intake of essential oil goes 

unevaluated. 

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8 

Metabolism of Terpenoids in 

Animal Models and Humans 

Walter Jäger 

CONTENTS 

8.1 Introduction ....................................................................................................................... 209 

8.2 Metabolism of Monoterpenes ............................................................................................ 210 

8.2.1 Camphene .............................................................................................................. 210 

8.2.2 Camphor ................................................................................................................ 210 

8.2.3 Carvacrol ............................................................................................................... 212 

8.2.4 Carvone ................................................................................................................. 213 

8.2.5 1,4-Cineole ............................................................................................................ 213 

8.2.6 1,8-Cineole ............................................................................................................ 214 

8.2.7 Citral ...................................................................................................................... 216 

8.2.8 Citronellal .............................................................................................................. 217 

8.2.9 Fenchone ................................................................................................................ 217 

8.2.10 Geraniol ................................................................................................................. 218 

8.2.11 Limonene ............................................................................................................... 218 

8.2.12 Linalool .................................................................................................................. 219 

8.2.13 Linalyl Acetate ...................................................................................................... 221 

8.2.14 Menthol .................................................................................................................. 221 

8.2.15 Myrcene ................................................................................................................. 223 

8.2.16 Pinene .................................................................................................................... 223 

8.2.17 Pulegone ................................................................................................................ 225 

8.2.18 a-Terpineol ............................................................................................................ 225 

8.2.19 a- and b-Thujone ................................................................................................... 225 

8.2.20 Thymol .................................................................................................................. 226 

8.3 Metabolism of Sesquiterpenes .......................................................................................... 227 

8.3.1 Caryophyllene ........................................................................................................ 227 

8.3.2 Farnesol ................................................................................................................. 227 

8.3.3 Longifolene ............................................................................................................ 229 

8.3.4 Patchouli Alcohol .................................................................................................. 230 

References .................................................................................................................................. 232 


Terpenoids are main constituents of plant-derived essential oils. Because of their pleasant odor or 

flavor they are widely used in the food, fragrance, and pharmaceutical industry. Furthermore, in 

traditional medicine, terpenoids are also well known for their analeptic, antibacterial, antifungal, 

209


antitumor, and sedative activities. Although large amounts are used in the industry, the knowledge 

about their biotransformation in humans is still scarce. Yet, metabolism of terpenoids can lead to the 

formation of new biotransformation products with unique structures and often different flavor and 

biological activities compared to the parent compounds. 

All terpenoids easily enter the human body by oral absorption, penetration through the skin, or 

inhalation very often leading to measurable blood concentrations. A number of different enzymes, 

however, readily metabolize these compounds to more water-soluble molecules. Although nearly 

every tissue has the ability to metabolize drugs, the liver is the most important organ of drug 

biotransformation. In general, metabolic biotransformation occurs at two major categories called 

Phase I and Phase II reactions (Spatzenegger and Jäger, 1995). Phase I concerns mostly cytochrome 

P450 (CYP)-mediated oxidation as well as reduction and hydrolysis. Phase II is a further step where 

a Phase I product is completely transformed to high water solubility. This is done by attaching 

already highly water-soluble endogenous entities such as sugars (glucuronic acids) or salts (sulfates) 

to the Phase I intermediate and forming a Phase II final product. It is not always necessary for a 

compound to undergo both Phases I and II; indeed for many terpenoids one or the other is enough 

to eliminate these volatile plant constituents. 

In the following concise review, special emphasis will be put on metabolism of selected monoand 

sesquiterpenoids not only in animal and in vitro models but also in humans. 

8.2 METABOLISM OF MONOTERPENES 

8.2.1 CAMPHENE 

Camphene is found in many plants at high concentrations, especially in the essential oil of the leaves 

and flowers of Thymus vulgaris. Based on expectorant, spasmolytic, and antimicrobial properties, 

camphene-containing remedies are successfully used in the treatment against cough and infections 

of the respiratory tract. The far greatest amount of camphene, however, is used in the liquor industry 

(Wichtel, 2002). Data about the in vivo metabolism of camphene are scarce as there is only one 

publication demonstrating various biotransformation products in the urine of rabbits after its oral 

administration. As shown in Figure 8.1, camphene is metabolized into two diasteromeric glycols 

(camphene-2,10-glycols). Their formation obviously involves two isomeric epoxide intermediates, 

which are hydrated by epoxide hydrolase. Further metabolites, namely 6-hydroxycamphene, 

7-hydroxycamphene, 3-hydroxytricyclene, and 10-hydroxytricyclene, were apparently formed 

through the nonclassical cation intermediate (shown in brackets), structures of which were identified 

by IR, UV NMR, mass spectrometry, and chemical degradation (Ishida et al., 1979). So far, there 

are no studies available about the biotransformation of camphene in human liver microsomes or in 

human subjects. 

8.2.2 CAMPHOR 

Camphor, a bicyclic monoterpene, is extracted from the woods of Cinnamomum camphora, a tree 

located in Southeast Asia and North America. Furthermore, it is also one of the major constituents 

of the essential oil of common sage (Salvia offi cinalis). Solid camphor forms white, fatty crystals 

with intensive camphoraceous odor and is used commercially as a moth repellent and preservative 

in pharmaceuticals and cosmetics (Wichtel, 2002). In dogs, rabbits, and rats, camphor is extensively 

metabolized whereas the major hydroxylation products of d- and l-camphor were 5-endo-and 

5-exo-hydroxycamphor. A small amount was also identified as 3-endo-hydroxycamphor (Figure 8.2). 

Both 3- and 5-bornane groups can be further reduced to 2,5-bornanedione. Minor biotransformation 

steps also involve the reduction of camphor to borneol and isoborneol. Interestingly, all hydroxylated 

camphor metabolites are further conjugated in a Phase II reaction with glucuronic acid

Metabolism of Terpenoids in Animal Models and Humans 211 

Camphene 

Rabbit 

in vivo 

O 

+ 

O 

Rabbit 

in vivo 

OH 

10-Hydroxytricyclene 

+ 

OH 

OH 

Camphene-2,10-glycol 

OH 

OH 

Camphene-2,10-glycol 

HO 

HO 

HO 

7-Hydroxycamphene 6-exo-Hydroxycamphene 3-Hydroxytricyclene 

FIGURE 8.1 Urinary excretion of camphene metabolites in rabbits. (Adapted from Ishida, T. et al., 1979. 

J. Pharm. Sci., 68: 928–930; Jahrmann, R., 2007. Metabolismus von Monterpenen und Sesquiterpenen in 

Mensch und Säugetier: Bedeutung für die pharmazeutische Praxis. MPharm. diploma thesis, University of 

Vienna, Austria.) 

O 

Human, rat, rabbit, dog 

in vitro 

Rabbit 

in vivo 

Camphor 

Rabbit 

in vitro/in vivo 

HO 

O 

+ 

3-endo-Hydroxycamphor 

not in vitro human 

O 

OH 

H 

+ 

5-endo-Hydroxycamphor 

not in vitro human 

O 

H 

OH 

5-exo-Hydroxycamphor 

not in vivo rabbit 

HO 

H 

H 

HO 

O 

O 

Borneol 

Isoborneol 

O 

2,3-Bornanedione 

FIGURE 8.2 Metabolisms of camphor in dogs, rabbits, and rats. (Adapted from Leibmann, K.C. and E. 

Ortiz, 1972. Drug Metab. Dispos., 1: 543–551; Jahrmann, R., 2007. Metabolismus von Monterpenen und 

Sesquiterpenen in Mensch und Säugetier: Bedeutung für die pharmazeutische Praxis. MPharm. diploma 

thesis, University of Vienna, Austria; Gyoubu, K. and M. Miyazawa, 2007. Biol. Pharm. Bull., 30: 230–233.) 

O 

2,5-Bornanedione


(Leibmann and Ortiz, 1972; Gyoubu and Miyazawa, 2007). Based on animal data, camphor should 

be also extensively metabolized in humans. Indeed, a very recent study using human liver microsomes 

could also show hydroxylation to 5-exo-hydroxycamphor. The formation of other metabolites 

namely borneol, isoborneol, and various glucuronides are therefore also suggested in the blood and 

urine of human volunteers after oral administration of camphor. 

8.2.3 CARVACROL 

Carvacrol is a monoterpenic alcohol which is found in high concentrations (3–5%) in the essential 

oil of Thymus vulgaris, a plant widely distributed in Middle and South Europe. Carvacrol is reportedly 

used as a flavor additive in a number of foods and beverages (Wichtel, 2002). Screening the 

literature for its metabolism, only one study could be found investigating biotransformation product 

in rat urine after oral application. In rat, only small amounts of unchanged carvacrol were excreted 

after 24 h. Based on the sample preparation protocol using b-glucuronidase and sulfatase before GC 

analysis, carvacrol might also be excreted as their glucuronide and sulfate, respectively. An interesting 

feature of this study is the demonstration that both of the aliphatic groups present undergo 

extensive meta bolism. Noteworthy is also the fact that aromatic hydroxylation to 2,3-dihydroxy-pcymene 

is only a minor important pathway for carvacrol. Further oxidation of 2-hydroxymethyl-5- 

(1-methylethyl)-phenol may also take place leading to the monocarboxylic acid metabolite, whose 

chemical structure was identified as 2-hydroxymethyl-4-(1-methyl)benzoic acid (Austgulen et al., 

1987) (Figure 8.3). There currently are not any studies available in the literature about carvacrol 

biotransformation in humans. 

OH 

Rat 

in vivo 

OH 

Rat 

in vivo 

OH 

OH 

2,3-Dihydroxy-p-cymene 

Rat 

in vivo 

OH 

Carvacrol 

Rat 

in vivo 

OH 

2-(3-Hydroxy-4-methylphenyl)propan-2-ol 

CH 2 OH 

OH 

CH 2 OH 


2-Hydroxymethyl-5-(1-methylethyl)phenol 

OH 

CH 2 OH 

OH 

COOH 

OH 

COOH 

2-(3-Hydroxy-4-methylphenyl)propionic acid 

2-Hydroxymethyl-4-(1-methylethyl)benzoic acid 

CH 2 OH 

2-(4-Hydroxymethyl-3-hydroxyphenyl)propan-1-ol 

FIGURE 8.3 Metabolism and urinary excretion of carvacrol in rats. (Adapted from Austgulen, L.T. et al., 1987. 

Pharmacol. Toxicol., 61: 98–102; Jahrmann, R., 2007. Metabolismus von Monterpenen und Sesquiterpenen in 


Vienna, Austria.)


8.2.4 CARVONE 

The (R)-(-)- and (S)-(+)-enantiomers of monoterpene ketone carvone are found in various plants. 

While (S)-(+)-carvone is the main constituent of the essential oil of caraway (Carum carvi), the oil 

of spearmint leaves (Mentha spicata var. crispa) contains about 50% of (R)-(-)-carvone besides 

other terpenes (Wichtel, 2002). Both enantiomers are not only different in odor and taste, but they 

also have different use in the food, fragrance, and pharmaceutical industries. Because of minty odor 

and taste, large amounts of (R)-(-)-carvone are frequently added to toothpastes, mouth washes, and 

chewing gums. (S)-(+)-carvone possesses the typical caraway aroma and is therefore mainly used 

as a taste enhancer in the food and fragrance industry. Due to its spasmolytic effect, (S)-(+)-carvone 

is also used as stomachic and carminative in many pharmaceutical formulations. Furthermore, in 

combination with other essential oils, (S)-(+)- and (R)-(-)-carvone are applied in aromatherapy 

massage treatments for nervous tension and several skin disorders (Jäger et al., 2001). After separate 

topical applications of (R)-(-)- and (S)-(+)-carvone, both enantiomers are rapidly absorbed resulting 

in significantly higher maximal plasma concentrations (C max ) and areas under the blood concentrations 

time curves (AUC) for (S)-(+)- compared to (R)-(-)-carvone (88.0 versus 23.9 ng/mL plasma 

and 5420 versus 1611 ng/mL min, respectively). As demonstrated in Figure 8.4, analysis of controland 

ß-glucuronidase pretreated urine samples only revealed stereoselective metabolism of 

(R)-(-)-carvone but not of (S)-(+)-carvone to (4R,6S)-(-)-carveol and (4R,6S)-(-)-carveol 

glucuronide indicating that stereoselectivity in Phases I and II metabolism has significant effects on 

(R)-(-)- and (S)-(+)-carvone pharmacokinetics (Jäger et al., 2000) (Figure 8.4). 

The metabolites in plasma for both enantiomers in plasma were below detection limit. Contrary 

to the study of Jäger et al. (2000), however, a recent study of Engel could not demonstrate any differences 

in the formation of metabolites after peroral application of (R)-(-)- and (S)-(+)-carvone 

(1 mg/kg body weight) to human volunteers. This may be due to the separation of biotransformation 

products on a nonchiral gas chromatography column. As shown in Figure 8.5, besides carveol, also 

dihydrocarveol, carvonic acid (possibly via 10-hydroxycarvone formation), dihydrocarvonic acid, 

and uroterpenolone could be identified in the urine samples (Engel, 2001). 

8.2.5 1,4-CINEOLE 

1,4-Cineole, a monoterpene cyclic ether, is known to be a major flavor constituent of lime (Citrus 

aurantiifolia) and Eucalyptus polybractea has been used for many years as a fragrance and flavoring 

agent (Miyazawa et al., 2001a). Although there are no in vivo data about the metabolism of 

1,4- cineole in humans, recent in vitro and in vivo animal studies demonstrated extensive biotransformation 

of this monoterpene strongly suggesting biotransformation in the human body too. After 

oral application to rabbits, four neutral and one acidic metabolite could be isolated from urine 

namely 9-hydroxy-1,4-cineole, 3,8-dihydroxy-1,4-cineole, 8,9-dihydroxy-1,4-cineole, 1,4-cineole-8- 

en-9-ol, and 1,4-cineole-9-carboxylic acid (Asakawa et al., 1988). Using rat and human liver 

O 

OH 

O-glu 

H 

H 

H 

L-Carvone 4L,6D-Carveol 4L,6D-Carveol-glucuronic acid 

FIGURE 8.4 Metabolic pathway of (R)-(-)-carvone in healthy subjects. (Adapted from Jäger, W. et al., 2000. 

J. Pharm. Pharmacol., 52: 191–197; Jahrmann, R., 2007. Metabolismus von Monterpenen und Sesquiterpenen 

in Mensch und Säugetier: Bedeutung für die pharmazeutische Praxis. MPharm. diploma thesis, University of 

Vienna, Austria.).


OH 

OH 

Dihydrocarveol 

Carveol 

Human 

in vivo 

Human 

in vivo 

Carveol-glucuronid 

O 

Human 

in vivo 

O 

Human 

in vivo 

O 

OH 

10-Hydroxycarvone 

Carvone 

Human 

in vivo 

HO 

OH 

Uroterpenolone 

Human 

in vivo 

O 

O 

O 

O 

OH 

Carvonic acid 

OH 

Dihydrocarvonic acid 

FIGURE 8.5 Proposed metabolic pathway of (R)-(-)- and (S)-(+)-carvone in healthy volunteers. (Adapted 

from Engel, W., 2001. J. Agric. Food Chem., 49: 4069–4075; Jahrmann, R., 2007. Metabolismus von 

Monterpenen und Sesquiterpenen in Mensch und Säugetier: Bedeutung für die pharmazeutische Praxis. 

MPharm. diploma thesis, University of Vienna, Austria.) 

microsomes, however, only 1,4-cineole 2 hydroxylation could be observed indicating species-related 

differences in 1,4-cineole metabolism (Miyazawa et al., 2001a) (Figure 8.6). 

8.2.6 1,8-CINEOLE 

1,8-Cineole, a monoterpene cyclic ether which is also named eucalyptol, is widely distributed in 

plants and is found in high concentrations in the essential oil of Eucalyptus polybractea. It is extensively 

used in cosmetics, for cough treatment, muscular pain, neurosis, rheumatism, asthma, and 

urinary stones (Wichtel, 2002). Using rat liver microsomes, 1,8-cineole is predominantly converted 

to 3-hydroxy-1,8-cineole, followed by 2- and then 9-hydroxycineole (Miyazawa et al., 2001b). As 

seen in Figure 8.7, in human liver microsomes, however, only the 2-hydroxy- and 3-hydroxy products


Rabbit 

in vivo 

O 

OH 

O 

HO 

OH 

3,8-Dihydroxy-1,4-cineole 

O 

1,4-Cineole 

Human, rat 

in vitro 

O 

Rabbit 

in vivo 

OH 

O 

OH 

9-Hydroxy-1,4-cineole 

O 

OH 

OH 

8,9-Dihydroxy-1,4-cineole 

O 

COOH 

1,4-Cineole-9-carboxylic acid 

O 

OH 

1,4-Cineole-8-en-9-ol 

2-exo-Hydroxy-1,4-cineole 

FIGURE 8.6 Proposed metabolism of 1,4-cineole in rabbits and in rat and human liver microsomes. 

(Adapted from Asakawa, Y., M. Toyota, and T. Ishida, 1988. Xenobiotica, 18: 1129–1134; Miyazawa, M. et al., 

2001a. Xenobiotica, 31: 713–723; Jahrmann, R., 2007. Metabolismus von Monterpenen und Sesquiterpenen 



Human 

O 

Human 

O 

in vivo/vitro 

in vivo/in vitro 

HO 

Rat 

Rat 

HO 

in vitro 

in vitro 

2-Hydroxy-1,8-cineole 1,8-Cineole 3-Hydroxy-1,8-cineole 

Rat 

in vitro 

CH 2 OH 

O 

9-Hydroxy-1,8-cineole 

FIGURE 8.7 Proposed metabolism of 1,8-cineole in vitro (rat and human liver microsomes) and in vivo 

(rabbits and humans). (Adapted from Miyazawa, M. et al., 2001b. Drug Metab. Dispos., 29: 200–205; Jahrmann, 

R., 2007. Metabolismus von Monterpenen und Sesquiterpenen in Mensch und Säugetier: Bedeutung für die 

pharmazeutische Praxis. MPharm. diploma thesis, University of Vienna, Austria.)


catalyzed by the isoenzmye CYP3A4 were seen (Miyazawa et al., 2001b). Both metabolites could 

also be identified in the urine of three human volunteers after oral administration of a cold medication 

containing 1,8-cineole (Miyazawa et al., 2001b). Both metabolites can therefore be used as 

urinary markers for the intake of 1,8-cineole in humans. 

8.2.7 CITRAL 

Both natural and synthetic citral are composed of an isomeric mixture of geranial (E-3,7-dimethyl- 

2,6-octadienal) and neral (Z-3,7-dimethyl-2,6-octadienal). In the isomeric mixtures, geranial is 

usually the predominant isomer. It occurs naturally in essential oils of citrus fruits (i.e., up to 5% 

in lemon oil) and in a variety of herbs and plants such as Melissa offi cinalis, lemongrass (70–80%), 

and eucalyptus (Wichtel, 2002). Because of its intense lemon aroma and flavor, citral has been 

used extensively in the food, cosmetic, and detergent industries since the early 1900s (Boyer and 

Petersen, 1991). Studies in rats have shown that citral is rapidly metabolized to several acids and 

a biliary glucuronide and excreted, with urine (48–63%) as the major route of elimination of 

citral, followed by expired air (8–17%), and feces (7–16%). As demonstrated in Figure 8.8, seven 

urinary metabolites were isolated and identified: 3-hydroxy-3,7-dimethyl-6-octenedioic acid, 

CHO 

CHO 

Rat 

in vivo 

CHO 

CHO 

CHO 

CHO 

OH 

OH 

(E) (Z) (E) (Z) 

(E) 

CHO 

(Z) 

CHO 

Citral 

Rat 

in vivo 

8-Hydroxy-3,7-dimethyl-2,6-octadienal 

3,7-Dimethyl-2,6-octadienedial 

COOH 

COOH 

COOH 

COOH 

(E) 

(Z) 

E-3,8-Dimethyl-2,6-octadienedioic acid 

COOH 

COOH 

(E) 

(Z) 

3,7-Dimethyl-2,6-octadienedioic acid 

OH 

OH 

OH 

COOH 

COOH 

COOH 

COOH 

OH 

(E) 

(Z) 

OH 

COOH 

(E) 

COOH 

(Z) 

3,8-Dihydroxy-3,7-dimethyl-6-octenoic 

acid 

3-Hydroxy-3,7-dimethyl-6-octenedioic 

acid 

3,7-Dimethyl-6-octenedioic 

acid 

FIGURE 8.8 Proposed metabolism of citral in rats. (Adapted from Diliberto, J.J. et al., 1990. Drug Metab. 

Dispos., 18: 886–875; Jahrmann, R., 2007. Metabolismus von Monterpenen und Sesquiterpenen in Mensch und 

Säugetier: Bedeutung für die pharmazeutische Praxis. MPharm. diploma thesis, University of Vienna, Austria.)


3,8-dihydroxy-3,7-dimethyl-6-octenedioic acid 3,9-dihydroxy-3,7-dimethyl-6-octenedioic acid, 

E- and Z-3,7-dimethyl-2,6-octenedioic acid, 3,7-dimethyl-6-octenedioic acid, and E-3,7-dimethyl- 

2,6-octenedioic acid (Diliberto et al., 1990). There currently are not any in vitro or in vivo data 

available about the metabolism of citral in humans. However, based on the rat study mentioned 

above, extensive biotransformation of citral is highly suggested. 

8.2.8 CITRONELLAL 

Citronellal is a monocyclic monoterpene with highest concentrations in the essential oil of 

Melissa offi cinalis (1–20%). Although citronellal is not commonly used in food and flavor industry, 

it is a main constituent in many pharmaceutical preparations as a mild sedative or stomachicum 

(Wichtel, 2002). Although extensively used in patients, data about its metabolism are scarce. Only 

one study described biotransformation of citronellal in rabbits. Ishida et al. could isolate three 

neutral metabolites of (+)-citronellal in the urine of rabbits namely (-)-trans-menthane-3,8-diol, 

(+)-cis menthane-3,8-diol, and (-)-isopulegol (Figure 8.9). An additional acidic metabolite (6E)-3, 

7-dimethyl-6-octene-1,8-dioic acid was formed as the result of regioselective oxidation of the aldehyde 

and dimethyl allyl groups (Ishida et al., 1989). Based on animal data, metabolism of citronellal 

is also expected in humans. 

8.2.9 FENCHONE 

Fenchone, a bicyclic monoterpene, is widely distributed in plants with highest concentrations in the 

essential oil of Foeniculum vulgare. Fenchone has camphoraceous fragrance and is used as a food 

flavor and in perfumes (Wichtel, 2002). A recent study (Miyazawa and Gyoubu, 2006) investigated 

the biotransformation of fenchone in human liver microsomes demonstrating the formation of 6-exohydroxyfenchone, 

6-endo-hydroxyfenchone, and 10-hydroxyfenchone (Figure 8.10). There currently 

are not any data about metabolism of this compound in human volunteers. However, based on the 

CHO 

(+) Citronellal 

Rabbit (urine and gastric juice) 

CO 2 H 

OH 

+ + + 

OH 

OH 

OH 

OH 

CO 2 H 

(–)-trans-menthane-3,8-diol (+)-cis-Menthane-3,8-diol (–)-Isopulegone (6E)-3,7-Dimetyl-6-octene-1,8-dioic acid 

FIGURE 8.9 Proposed metabolism of citronellal in rabbits. (Adapted from Ishida, T. et al., 1989. Xenobiotica, 

19: 843–855; Jahrmann, R., 2007. Metabolismus von Monterpenen und Sesquiterpenen in Mensch und Säugetier: 

Bedeutung für die pharmazeutische Praxis. MPharm. diploma thesis, University of Vienna, Austria.)


Human 

in vivo 

HO 

H 

O 

+ 

H 

HO 

O 

Human 

in vivo 

6-exo-Hydroxyfenchone 

6-endo-Hydroxyfenchone 

O 

(+)-Fenchone 

HO 

O 

10-Hydroxyfenchone 

FIGURE 8.10 Proposed metabolism of fenchone in human liver microsomes. (Adapted from Miyazawa, M. 

and K. Gyoubu, 2006. Biol. Pharm. Bull., 29: 2354–2358; Jahrmann, R., 2007. Metabolismus von Monterpenen 

und Sesquiterpenen in Mensch und Säugetier: Bedeutung für die pharmazeutische Praxis. MPharm. diploma 

thesis, University of Vienna, Austria.) 

in vitro experiments using human liver microsomes, identical biotransformation products should also 

be found in blood or urine samples of humans after dietary intake of fenchone-containing products. 

8.2.10 GERANIOL 

Geraniol is a monoterpene alcohol with pronounced concentrations in the essential oil of Cymbopogon 

winteranus Jowitt (12–25%). It is also found in small quantities in rose, palmarosa, citronella, geranium, 

lemon, and many other essential oils. It has a rose-like odor and is commonly used in perfumes 

and in the flavor industry (Chadha and Madyastha, 1984). In a study of Chadha and Madyastha, 

several metabolites could be identified in rat urine after oral administration (Chadha and Madyastha, 

1984). Geraniol can be either metabolized to 8-hydroxygeraniol and via 8-carboxygeraniol to 3,7- 

dimethyl-2,6-octenedioic acid (Hildebrandt’s acid) or directly oxidized to geranic acid and 3- 

hydroxycitronelic acid (Figure 8.11). Formation of 8-hydroxygeraniol and 8-carboxygeraniol are due 

to selective oxidation of the C-8 in geraniol. The 8-hydroxylation of geraniol also occurs in higher 

plants where it is the first step in the biosynthesis of indole alkaloids. 

8.2.11 LIMONENE 

The monocyclic monoterpene (+)- and (-)-limonene enantiomers have been shown to be present in 

orange peel (Citrus aurantium L. sp. aurantium) and other plants and are extensively used as fragrances 

in household products and components of artificial essential oils. The (+)-limonene isomeric 

form is more abundantly present in plants than the racemic mixture and the (-)-limonene isomeric 

form (Wichtel, 2002). It has previously been shown that (+) limonene has chemopreventive activities 

in experimental animal models including rats and mice (Crowell et al., 1992). Because of the greater 

importance of (+)-limonene in the food and fragrance industry, only its metabolism and not that of 

(-)-limonene is described below. Several research groups have successfully described the biotransformation 

of (+)-limonene in vitro (rat and human liver microsomes) and in vivo (rat, mice, guinea


OH 

Rat 

in vivo 

Rat 

in vivo 

OH 

Rabbit 

in vitro 

Geraniol 

COOH 

OH 

8-Hydroxygeraniol 

Geranic acid 

Rat 

in vivo 

OH 

COOH 

OH 

COOH 

COOH 

COOH 

8-Carboxygeraniol 

Hildebrandt’s acid 

3-Hydroxycitronelic acid 

FIGURE 8.11 Proposed metabolism of geraniol in rats. (Adapted from Chadha, A. and M.K. Madyastha, 

1984. Xenobiotica, 14: 365–374; Jahrmann, R., 2007. Metabolismus von Monterpenen und Sesquiterpenen in 



pigs, dogs, rabbits, human volunteers, and patients). As shown in Figure 8.12, (+)-limonene is 

extensively biotransformed to several metabolites whereas in humans the main biotransformation 

products are perillyl alcohol; perillic acid; p-mentha-1,8-dien-carboxylic acid (an isomer of perillic 

acid); cis-dihydroperillic acid; trans-dihydroperillic acid; limonene; 1,2-diol; limonene-10-ol; 

limonene-8,9-diol; several glucuronides of perillic acid; dihydroperillic acid; and limonene-10-ol 

(Crowell et al., 1992; Miyazawa et al., 2002; Shimada et al., 2002). 

8.2.12 LINALOOL 

Linalool can be obtained naturally by fractional distillation and subsequent rectification from oils of 

the Cajenne rosewood, Brazil rosewood, Mexican linaloe, and coriander seed. The far highest concentration 

of linalool is found in the essential oil of Ocimum basilicum (up to 75%). Pure linalool possesses 

a fresh, clean, mild, light floral odor with a slight citrus impression and is used in large quantities 

in soap and detergent products (Wichtel, 2002). Although linalool is used in large quantities in the 

fragrance industry, there are no data available about its biotransformation in humans. In rat, however, 

linalool is metabolized by cytochrome P450 (CYP) isoenzymes to dihydrolinalool and tetrahydrolinalool 

and to 8-hydroxylinalool, which is further oxidized to 8-carboxylinalool (Figure 8.13). CYPderived 

metabolites are then converted to glucuronide conjugates (Chadha and Madyastha, 1984).


CH 2 OH 

HO 

Rabbit, O 

guinea pig, dog 

in vitro 

OH 

OH 

Perillyl alcohol 

trans-Carveol 

Carvone 

Limonene-1,2-diol 

Human, rabbit, 


in vitro 

Human, rabbit, 


in vitro 

Human 

in vivo 

Rat 

in vitro 

HO 

OH 

O 

Human 

in vivo 

1,2-Glycol 

1,2-Epoxid 

Rat 

in vitro 

OH 

OH 

Human 

in vivo 

Limonene-8,9-diol 

(Uroterpenol) 

Rat 

in vitro 

OH 

O-Glu 

Limonene-8,9-diolglucuronide 

OH OH 

8,9-Glycol 

O 

8,9-Epoxid 

Human 

in vivo 

Rat 

in vitro 

Rat 

in vitro 

Human, rat 

in vivo 

D-Limonene 

Rat 

in vitro 

CH 2 OH 

Human 

in vivo 

COOH 

OH 

Limonene-10-ol 

Human 

in vivo 

COO-Glu 

O-Glu 

Limonene-10-olglucuronide 

HOOC 

Isomer of perillic acid 

(p-Mentha-1,8-dien-carboxylic acid) 

Perillyl alcohol 

Perillic acid 

Human, rat 

in vivo 

Rat 

in vitro 

Perillic acid-glucuronide 

COO-Glu 

+ 

COO-Glu 

Human 

in vivo 

COOH 

+ 

COOH 

cis-Dihydroperillic acidglucuronide 

trans-Dihydroperillic acidglucuronide 

cis-Dihydroperillic acid 

trans-Dihydroperillic acid 

FIGURE 8.12 Proposed metabolism of (+)-limonene in rats, rabbits, guinea pigs, dogs, and humans. 

(Adapted from Crowell, P.L. et al., 1992. Cancer Chemother. Pharmacol., 31: 205–212; Miyazawa, M. et al., 

2002. Drug Metab. Dispos., 30: 602–607; Shimada, T. et al., 2002. Drug Metab. Pharmacokinetics, 17: 

507–515; Jahrmann, R., 2007. Metabolismus von Monterpenen und Sesquiterpenen in Mensch und Säugetier: 

Bedeutung für die pharmazeutische Praxis. MPharm. diploma thesis, University of Vienna, Austria.)


O-Glu 

Linalool-glucuronic acid 

Rat 

in vivo 

OH 

OH 

Rat 

in vivo 

OH 

Rat 

in vivo 

OH 

OH 

OH 

OH 

O 

8-Carboxylinalool 8-Hydroxylinalool 

Linalool 

Dihydrolinalool Tetrahydrolinalool 

FIGURE 8.13 Proposed metabolism of linalool in rats. (Adapted from Chadha, A. and M.K. Madyastha, 

1984. Xenobiotica, 14: 365–374; Jahrmann, R., 2007. Metabolismus von Monterpenen und Sesquiterpenen 



8.2.13 LINALYL ACETATE 

Linalyl acetate is a fragrance ingredient used in many fragrance compounds. It may be found in 

fragrances used in decorative cosmetics, fine fragrances, shampoos, and toilet soaps, as well as in 

noncosmetic products such as household cleaners and detergents. 

Linalyl acetate can be found in many plants, however, it is in the highest concentration in the essential 

oil of Citrus aurantium spp. aurantium (Wichtel, 2002). As an ester, linalyl acetate is hydrolyzed 

in vivo by carboxylesterases or esterases to linalool (Figure 8.14), which is then further metabolized 

to numerous oxidized biotransformation products (see metabolism of linalool) (Bickers et al., 2003). 

8.2.14 MENTHOL 

Menthol is a major component of various mint oils. The plant oil, often referred to as peppermint oil 

(from Mentha piperita) or cornmint oil (from Mentha arvensis), is readily extracted from the plant 

by steam distillation (Wichtel, 2002). It has a pleasant typical minty odor and taste, and is widely 

O 

OH 

O 

Rat 

in vitro 

Linalyl acetate 

Linalool 

FIGURE 8.14 Proposed metabolism of linalyl acetate in rats. (Adapted from Bickers, D. et al., 2003. Food 

Chem. Toxicol., 41: 919–942; Jahrmann, R., 2007. Metabolismus von Monterpenen und Sesquiterpenen in 


Vienna, Austria.)


used to flavor foods and oral pharmaceutical preparations ranging from common cold medications 

to toothpastes (Mühlbauer et al., 2003). After oral administration to human volunteers, menthol is 

rapidly metabolized and only menthol glucuronide could be measured in plasma or urine. 

Interestingly, unconjugated menthol was only detected after a transdermal application. In rats, however, 

hydroxylation at the C-7 methyl group and at C-8 and C-9 of the isopropyl moiety form a series 

a mono- and dihydroxymenthols and carboxylic acids, some of which are excreted in part as 

glucuronic acid conjugates. Additional metabolites are mono- and or dihydroxylated menthol derivatives 

(Figure 8.15). Similar to humans, the main metabolite in rats was again menthol glucuronide 

(Madyastha and Srivatsan, 1988b; Gelal et al., 1999; Spichinger et al., 2004). 

OH 

COOH 

OH 

OH 

OH 

COOH 

3-Hydroxy-p-menthane-9-carboxylic 

acid 

Rat 

in vivo 

OH 

OH 


Human/rat 

in vivo 

Glucuronide 

Rat 


Rat 


Menthol 

Rat 

in vivo 

Glucuronide 

OH 


Rat 

in vivo 

Rat 

in vitro/vivo 

OH 

OH 


Rat 

in vivo 

3-Hydroxy-p-menthane-7-carboxylic 

acid 

Rat 

in vivo 

OH 

OH 

COOH 

OH 

OH 

3,8-Dihydroxy-7-carboxylic 

acid 

OH 

p-Menthane-3,7,8-triol 

COOH 

COOH 

OH 

OH 

O 

O 

3,8-Dihydroxy-p-menthane-7-carboxylic acid 

3,8-Oxy-p-menthane-7-carboxylic acid 

FIGURE 8.15 Proposed metabolism of menthol in rats and humans. (Adapted from Madyastha, K.M. and 

V. Srivatsan, 1988a. Environ. Contam. Toxicol., 41: 17–25; Gelal, A. et al., 1999. Clin. Pharmacol. Ther., 66: 

128–235; Spichinger, M. et al., 2004. J. Chromatogr. B, 799: 111–117; Jahrmann, R., 2007. Metabolismus 

von Monterpenen und Sesquiterpenen in Mensch und Säugetier: Bedeutung für die pharmazeutische Praxis. 

MPharm. diploma thesis, University of Vienna, Austria.)


8.2.15 MYRCENE 

Myrcene is the major constituent of the essential oil of hop (Humulus lupulus), which is used in the 

manufacture of alcoholic beverages. Hop is also frequently used in many pharmaceutical preparations 

as a mild sedative in the treatment of insomnia (Wichtel, 2002). After oral application, Ishida 

and coworkers could identify several metabolites in the urine of rabbits whereby the formation of 

the two glycols may be due to the hydration of the corresponding epoxides formed as intermediates 

(Ishida et al., 1981). The formation of uroterpenol may proceed through limonene, which was clearly 

derived from myrcene in the acidic conditions of rabbit stomachs (Figure 8.16). 

8.2.16 PINENE 

There are two structural isoforms found in nature: a- and b-pinene. As the name suggests, both isoforms 

are important constituents of pine resin. Interestingly, a-pinene is more common in European 

pines, whereas b-pinene is more common in North America. They are also found in the resin of many 

O 

OH 

OH 

O 

OH 

OH 

Rabbit 

in vivo 

Myrcene-3(10)-glycol 

3-Hydroxymyrcene-10-carboxylic acid 

O 

Rabbit 

in vivo 

O 

OH 

OH 

OH 

OH 

Myrcene 

Myrcene-1,2-glycol 

2-Hydroxymyrcene-1-carboxylic acid 

Rabbit 

in vivo 

OH 

CH 2 OH 

Limonene 

Uroterpenol 

FIGURE 8.16 Proposed metabolism of myrcene in rabbits. (Adapted from Ishida, T. et al., 1981. J. Pharm. 

Sci., 70: 406–415; Jahrmann, R., 2007. Metabolismus von Monterpenen und Sesquiterpenen in Mensch und 

Säugetier: Bedeutung für die pharmazeutische Praxis. MPharm. diploma thesis, University of Vienna, Austria.)


other conifers, and more widely in many plants. One of the highest concentrations of a- and b-pinene 

is in the essential oil of the fruit of juniper (Juniperis communis) with a total content of over 80% of 

these isoforms. Furthermore, a-pinene is also found in the essential oil of rosemary (Rosmarinus 

offi cinalis) and the racemic mixture in eucalyptus oil. In the industry, a- and b-pinene are used in the 

production of alcoholic beverages like gin (Wichtel, 2002). As shown in Figure 8.17, metabolism of 

CH 2 OH 

COOH 

Rabbit 

in vivo 

+ 

Rabbit 

in vivo 

OH 

α-Pinene 

trans-Verbenol 

Myrtenol 

Myrtenic acid 

Human 

in vivo 

Human 

in vivo 

CH 2 OH 

OH 

cis-Verbenol 

trans-Pinocarveol 

10-Pinanol 

Human 

in vivo 

Rabbit 

in vivo 

Rabbit 

in vivo 

O 

β-Pinene 

Rabbit 

in vivo 

CH 2 OH 

OH 

α-Terpineol 

FIGURE 8.17 Proposed metabolism of a- and b-pinene in rabbits and humans. (Adapted from Ishida, T. 

et al., 1981. J. Pharm. Sci., 70: 406–415; Eriksson, K. and J.O. Levin, 1996. J. Chromatogr. B, 677: 85–98; 

Jahrmann, R., 2007. Metabolismus von Monterpenen und Sesquiterpenen in Mensch und Säugetier: Bedeutung 

für die pharmazeutische Praxis. MPharm. diploma thesis, University of Vienna, Austria.) 

OH 

1-p-Menthene-7,8-diol


a- and b-pinene in humans leads to the formation of trans- and cisverbenol, respectively. Recent 

data analyzing the human urine after occupational exposure of sawing fumes also suggest that cisand 

trans-verbenol are being further hydroxylated to diols. The main urinary metabolite of a-pinene 

in rabbits is trans-verbenol; the minor biotransformation products are myrtenol and myrtenic acid. 

The main urinary metabolites of b-pinene, in rabbits, however, is cis-verbenol indicating stereoselective 

hydroxylation (Ishida et al., 1981; Eriksson and Levin, 1996). 

8.2.17 PULEGONE 

(R)-(+)-Pulegone is a monoterpene ketone present in essential oils from many mint species. Two 

mints, Hedeoma pulegoides and Mentha pulegium, both commonly called pennyroyal, contain 

essential oils, which are chiefly, pulegone (Madyastha and Raj, 1992). (S)-(-) Pulegone is found 

rarely in essential oil. Pennyroyal oil has been used as a flavoring agent in food and beverages, as 

well as a component in fragrance products and flea repellents. Pennyroyal herb has also been used 

for the purpose of inducing menstruation and abortion. In higher doses, however, penny royal oil 

may have resulted in central nervous system toxicity, gastritis, hepatic and renal failure, pulmonary 

toxicity, and death. The content of pulegone was found to be greater than 80% of the terpenes 

in pennyroyal oils that were obtained from health food stores, and was found to be both 

hepatotoxic and pneumotoxic in mice (Engel, 2003). Based on the observed toxicity in animal 

models and humans, several studies were performed in order to investigate the metabolism of 

(R)-(+)-pulegone. At nontoxic concentrations, pulegone is oxidized selectively at the 10-position, 

forming 10-hydroxypulegone. Alternatively, it may be reduced to menthone, which has been 

detected in trace levels in urine samples. It might be possible that pulegone is also reduced at the 

carbonyl group first; however, no trace of pulegol was found in the urine samples. Consequently, 

pulegol is either reduced very efficiently to menthol or rearranged to 3-p-menthen-8-ol (Engel, 

2003) (Figure 8.18). 

8.2.18 α-TERPINEOL 

a-Terpineol, a monocyclic monoterpene tertiary alcohol, which has been isolated from a variety of 

a-terpineol, was found in the essential sources such as cajuput oil, pine oil, and petit grain oil. But 

the far highest concentration (up to 30%) of a-terpineol was found in the essential oil of the tee tree 

(Melaleuca alternifolia). Based on its pleasant odor similar to lilac, a-terpineol is widely used in the 

manufacture of perfumes, cosmetics, soaps, and antiseptic agents (Madyastha and Srivatsan, 1988a; 

Wichtel, 2002). After oral administration to rats (600 mg/kg body weight), a-terpineol is metabolized 

to p-menthane-1,2,8-triol probably formed from the epoxide intermediate. Notably, allylic 

methyl oxidation and the reduction of the 1,2-double bound are the major routes for the biotransformation 

of a-terpineol in rat (Figure 8.19). Although allylic oxidation of C-1 methyl seems to be the 

major pathway, the alcohol p-ment-1-ene-7,8-diol could not be isolated from the urine samples. 

Probably, this compound is accumulated and is readily further oxidized to oleuropeic acid 

(Madyastha and Srivatsan, 1988a). 

8.2.19 α- AND β-THUJONE 

a-Thujone and b-thujone are bicyclic monoterpenes that differ in the stereochemistry of the C-4 

methyl group. The isomer ratio depends on the plant source, with high content of a-thujone in cedar 

leaf oil and b-thujone in wormwood oil. They are also common constituents in herbal medicines, 

essential oils, foods, flavorings, and beverages. a-Thujone is perhaps best known as the active 

ingredient of the alcoholic beverage absinthe, which was a very popular European drink in the 

1800s (Wichtel, 2002). In vivo rat and mouse models conformed to the in vitro data using rat, mice, 

and human liver microsomes where a- and b-thujone are extensively metabolized to six hydroxythujones 

and three dehydrothujones (Figure 8.20). The dehydro derivatives are possibly formed by


Human 

in vivo 

O 

O 

O 

Pulegone 

Human 

in vivo 

Human 

in vivo 

OH 

10-Hydroxypulegone 


OH O O 

Pulegol 

Menthone 

OH 

8-Hydroxymenthone 

OH 

OH 

O 

O 

OH 

3-p-Menthen-8-ol Menthol 1-Hydroxymenthone Piperitone 

FIGURE 8.18 Proposed metabolism of pulegone in humans. (Adapted from Engel, W., 2003. J. Agric. Food 

Chem., 51: 6589–6597; Jahrmann, R., 2007. Metabolismus von Monterpenen und Sesquiterpenen in Mensch 

und Säugetier: Bedeutung für die pharmazeutische Praxis. MPharm. diploma thesis, University of Vienna, 

Austria.) 

hydroxylation of methyl substituents at position 8 or 10 followed by dehydration. In Phase II 

reactions, several metabolites are further conjugated with glucuronic acid (Ishida et al., 1989; 

Höld et al., 2000, 2001). Based on the in vitro and in vivo data, biotransformation should also be 

pronounced in humans after the intake of a- and b-thujone. 

8.2.20 THYMOL 

Natural sources of thymol are the bee balms (Monarda fi stulosa and Monarda didyma). Larger 

quantities of thymol exist in the essential oil of thyme (Thymus vulgaris) with concentrations up to 

70%. Thymol is the primary ingredient in modern commercial mouthwash formulae as it demonstrates 

antiseptic properties. This may explain the use of thyme in herbal medicine to treat mouth 

and throat infections (Wichtel, 2002). It is noteworthy that thymol is also used as flavor additive in 

a number of foods and beverages. Although after oral application to rats, large quantities were 

excreted unchanged or as their glucuronide and sulfate conjugates; extensive oxidation of the methyl 

and isopropyl groups also occurred (Austgulen et al., 1987). This resulted in the formation of derivatives 

of benzyl alcohol and 2-phenylpropanol and their corresponding carboxylic acids. Ring 

hydroxy lation was only a minor reaction (Figure 8.21).


O 

OH 

OH 

Rat 

in vitro 

OH 

OH 

p-Menthane-1,2,8-triol 

OH 

Rat 

in vitro 

CH 2 OH 

COOH 

COOH 

α-Terpineol 

OH 

OH 

OH 

p-Menth-1-ene-7,8-diol 

Oleuropeic acid 

Dihydrooleuropeic acid 

FIGURE 8.19 Proposed metabolism of a-terpineol in rats. (Adapted from Madyastha, K.M. and V. Srivatsan, 

1988a. Environ. Contam. Toxicol., 41: 17–25; Jahrmann, R., 2007. Metabolismus von Monterpenen und 

Sesquiterpenen in Mensch und Säugetier: Bedeutung für die pharmazeutische Praxis. MPharm. diploma thesis, 

University of Vienna, Austria.) 

8.3 METABOLISM OF SESQUITERPENES 

8.3.1 CARYOPHYLLENE 

(-)-b-Caryophyllene is the main sesquiterpene of hops and is being used as a cosmetic additive in 

soaps and fragrances (Wichtel, 2002). Storage of fresh hops under aerobic conditions leads to rapid 

oxidation. The main product of this reaction (-)-caryophyllene-5,6-oxide markedly affects the 

quality of beer. In herbal medicine, (-)-b-caryophyllene is also responsible for the mild sedative 

properties of hops. Furthermore, it also demonstrates cytotoxicity against breast cancer cells in vitro 

(Asakawa et al., 1986; DeBarber et al., 2004). The biotransformation of (-)-b-caryophyllene in 

rabbits yielded the main metabolite [10S-(-)-14-hydroxycaryophyllene-5,6-oxide] and the minor 

biotransformation product cayrophyllene-5,6-oxide-2,12-diol. The formation of the minor metabolites 

is easily explained via the diepoxide intermediate. Thus, it is suggested that regioselective 

hydroxylation of the epoxide occurred (Asakawa et al., 1981, 1986). Whether (-)-b-caryophyllene 

also shows the same metabolic pathway in human is not known yet. However, based on in vivo data 

from rabbits, extensive biotransformation in humans is highly suggested after oral administration 


8.3.2 FARNESOL 

Farnesol is a natural organic compound, which is a sesquiterpene alcohol present in many essential 

oil such as citronella, neroli, cyclamen, lemon grass, rose, and musk. Interestingly, it is also produced 

in humans where it acts on numerous nuclear receptors and has received considerable attention 

due to its apparent anticancer properties. It is used in perfumery to emphasize the odors of 

sweet floral perfumes (DeBarber et al., 2004). In vitro studies using recombinant drug metabolizing


Glucuronide Glucuronide Glucuronide 

Glucuronide 

O 

O 

HO 

O 

OH 

O 

OH 

OH 

2-Hydroxy-α-thujon 

2-Hydroxy-β-thujon 4-Hydroxy-α-thujon 4-Hydroxy-β-thujon 

Human, rat, mouse 

in vitro 

Rat, mouse 

in vivo 


in vitro 

Rat, mouse 

in vivo 


in vitro 

Rat, mouse 

in vivo 


in vitro 

Rat, mouse 

in vivo 

OH 

Rabbit, 

in vivo 

O 

Rat, mouse 

in vivo 

O 

Mouse, 

in vitro/ in vivo 

Thujol (R) 

Neothujol (S) 

OH 


8-Hydroxy-β-thujon 

Human, 

rat, mouse 

in vitro 

α-Thujon 

β-Thujon 

Rat, mouse 

in vivo 

Human, 

rat, mouse 

in vitro 

OH 

O 

O 


in vitro 

Rat, mouse 

in vivo 


in vitro 

Rat, mouse 

in vivo Rat, mouse 

in vivo 

Human, 

rat, mouse 

O 

in vitro 



O 

Glucuronide 

7,8-Dehydro-α-thujon 

7,8-Dehydro-β-thujon 

OH 



4,10-Dehydro-α-thujon 

Sabinon 

FIGURE 8.20 Proposed in vitro and in vivo metabolism of a- and b-thujone in rats. (Adapted from Ishida, T. 

et al., 1989. Xenobiotica, 19: 843–855; Höld, K.M. et al., 2000. Environ. Chem. Toxicol., 97: 3826–3831; Höld, 

K.M. et al., 2001. Chem. Res. Toxicol., 14: 589–595; Jahrmann, R., 2007. Metabolismus von Monterpenen 

und Sesquiterpenen in Mensch und Säugetier: Bedeutung für die pharmazeutische Praxis. MPharm. diploma 

thesis, University of Vienna, Austria.)


HO 

OH 

OH 

2,5-Dihydroxy-p-cymene 

Thymol 

OH 

OH 

OH 

OH 


5-Hydroxymethyl-2-(1-methylethyl)phenol 

OH 

COOH 

OH 

OH 

OH 

COOH 

3-Hydroxy-4-(1-methylethyl)bezoic acid 

2-(2-Hydroxy-4-methylphenyl)propionic acid 

OH 

2-(4-Hydroxymethyl-2-hydroxyphenyl)propan-1-ol 

FIGURE 8.21 Proposed metabolism of thymol in rats. (Adapted from Austgulen, L.T. et al., 1987. Pharmacol. 

Toxicol., 61: 98–102; Jahrmann, R., 2007. Metabolismus von Monterpenen und Sesquiterpenen in Mensch 

und Säugetier: Bedeutung für die pharmazeutische Praxis. MPharm. diploma thesis, University of Vienna, 

Austria.) 

enzymes and human liver microsomes have shown that cytochrome P450 isoenzymes participate in 

the metabolism of farnesol to 12-hydroxyfarnesol (Figure 8.23). Subsequently, farnesol and 

12-hydroxyfarnesol are glucuronidated to farnesyl glucuronide and 12-hydroxyfarnesyl glucuronide, 

respecively (DeBarber et al., 2004; Staines et al., 2004). 

8.3.3 LONGIFOLENE 

Longifolene is the common chemical name of a naturally occurring, oily liquid hydrocarbon, which 

is found primarily in certain pine resins especially in that of Pinus longifolia, a tree that gives longifolene 

its name. Besides pines, longifolene is also a main constituent of clove (Wichtel, 2002). Based 

on its pleasant odor, longifolene is used in the food industry. In rabbits, longifolene is metabolized 

as follows: attack on the exo-methylene group from the endo-face to form its epoxide followed by 

isomerization of the epoxide to a stable endo-aldehyde. Then rapid cytochrome P450-catalyzed 

hydroxylation of this endo-aldehyde occurs (Asakawa et al., 1986) (Figure 8.24).


H 

H 

(–)-Caryophyllene 

Rabbit 

in vivo 

Rabbit 

in vivo 

O 

H 

H 

(–)-Caryophyllene-5,6-oxide 

O 

H 

H 

OH 

OH 

HO H 

H 

HO H O 

H 

Caryophyllene-5,6-oxide-2,12-diol 

(10S)-(–)-14-Hydroxycaryophyllene-5,6-oxide 

FIGURE 8.22 Proposed metabolism of b-caryophyllene in rabbits. (Adapted from Asakawa, Y. et al., 

1981. J. Pharm. Sci., 70: 710–711; Asakawa, Y. et al., 1986. Xenobiotica, 16: 753–767; Jahrmann, R., 2007. 

Metabolismus von Monterpenen und Sesquiterpenen in Mensch und Säugetier: Bedeutung für die pharmazeutische 

Praxis. MPharm. diploma thesis, University of Vienna, Austria.) 

8.3.4 PATCHOULI ALCOHOL 

Patchouli alcohol is the major active ingredient and the most odor-intensive component of patchouli 

oil, the volatile oil of Pogostemon cablin and Pogostemon patchouli. Patchouli oil is widely used in 

the cosmetic and oral hygiene industries to scent perfumes and flavor toothpaste (Bang et al., 1975). 

Modern research has also demonstrated various pharmacological activities of this oil including 

antiemetic, antibacterial, and antifungal properties. In rabbits and dogs, patchouli alcohol is 

hydroxylated at the C-15 yielding a diol that is subsequently oxidized to a hydroxyl acid. After 

decarboxylation and oxidation, the 3,4-unsaturated norpatchoulen-1-ol is formed, which also has a 

characteristic odor (Figure 8.25). All these urinary metabolites are also found as glucuronides, 

explaining their excellent water solubility (Bang et al., 1975; Ishida, 2005).


HO 

O 

O 

HO 

COOH 

OH 

OH 

Human 

in vitro 

Hydroxyfarnesyl-glucuronide 

OH 

Human, 

rabbit, rat 

in vitro 

HO 

OH 

Farnesol 

Human 

in vitro 

Hydroxyfarnesol 

O 

O 

HO 

COOH 

OH 

OH 

Farnesyl-glucuronide 

FIGURE 8.23 Proposed metabolism of farnesol in human liver microsomes. (Adapted from DeBarber, A.E. 

et al., 2004. Biochim. Biophys. Acta, 1682: 18–27; Staines, A.G. et al., 2004. Biochem. J., 384: 637–645; 


für die pharmazeutische Praxis. MPharm. diploma thesis, University of Vienna, Austria.) 

Rabbit 

in vivo 

O 

Longifolene 

HO 

CHO 

CHO 

14-Hydroxyisolongifolaldehyde 

Isolongifolaldehyde 

FIGURE 8.24 Proposed metabolism of (+)-longifolene in rabbits. (Adapted from Asakawa, Y. et al., 1986. 

Xenobiotica, 16: 753–767; Jahrmann, R., 2007. Metabolismus von Monterpenen und Sesquiterpenen in 


Vienna, Austria.)


HO 

Rabbit 

in vivo 

HO 

HO 

HO 

OH 

COOH 

Patchouli alcohol 

Yielding diol 

Hydroxyoxid acid 

3,4-Unsaturated norpatchoulen-1-ol 

FIGURE 8.25 Proposed metabolism of patchouli alcohol in rabbits and dogs. (Adapted from Bang, L. et al., 

1975. Tetrahedron Lett. 26: 2211–2214; Ishida, T., 2005. Chem. Biodivers., 2: 569–590; Jahrmann, R., 2007. 

Metabolismus von Monterpenen und Sesquiterpenen in Mensch und Säugetier: Bedeutung für die pharmazeutische 

Praxis. MPharm. diploma thesis, University of Vienna, Austria.) 

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9 

Biological Activities 

of Essential Oils 


CONTENTS 

9.1 Introduction ........................................................................................................................ 235 

9.1.1 Anticancer Properties ............................................................................................. 236 

9.1.2 Antinociceptive Effects .......................................................................................... 239 

9.1.3 Antiviral Activities ................................................................................................. 244 

9.1.4 Antiphlogistic Activity ........................................................................................... 246 

9.1.5 Penetration Enhancement ....................................................................................... 254 

9.1.6 Antioxidative Properties ......................................................................................... 256 

9.1.6.1 Reactive Oxygen Species ......................................................................... 256 

9.1.6.2 Antioxidants ............................................................................................. 256 

9.1.7 Test Methods ........................................................................................................... 257 

9.1.7.1 Free Radical Scavenging Assay ............................................................... 257 

9.1.7.2 b-Carotene Bleaching Test ....................................................................... 257 

9.1.7.3 Deoxyribose Assay ................................................................................... 258 

9.1.7.4 TBA Test .................................................................................................. 258 

9.1.7.5 Xanthine–Xanthine Oxidase Assay ......................................................... 258 

9.1.7.6 Linoleic Acid Assay ................................................................................. 258 

References ................................................................................................................................... 273 


The term “biological” in this context comprises all properties, from for example “abdomen” to 

“zymase,” which any natural product may possess. And these can be attributed to the whole animated 

nature, to all living organisms, namely plants, animals, and especially humans. However, 

only the effects of essential oils (EOs) on human beings is the topic of the present chapter. Excluded 

therefore, are all “botanical” activities, for example, plant care and interplant communication such 

as the prevention of germination of seeds of a potentially rivalry plant by emitting an EO, or the 

“cry for help” of a plant when it is attacked by pests and the “victim” volatilizes a fragrance which 

itself attracts enemies of these varmints. Neither are covered animal messengers, so-called pheromones, 

as well as EOs as veterinary therapeutics in animal care and feed. All these properties go 

beyond the scope and frame of this treatise and would rather fill a separate volume. 

Therefore, the subject matter of this chapter is the therapeutic uses of EOs and/or single fragrance 

compounds in human medicine and care. However, even this field seems to be too extensive, 

so that cosmetic uses and repellents are excluded, too. Since a chapter on the pharmacological properties 

of EOs is already contained in this volume (see Chapter 10), only those pharmacological activities 

235


which are of secondary (but not less important!) interest to the traditional pharmacologist will be 

discussed in this chapter. In particular, these are properties which do not directly aim at the central 

or autonomic nervous systems and for which the molecular mechanisms are only of minor relevance, 

for example, antioxidative effects, anticancer properties, penetration enhancing activities, 

and so on. 

In general, the prominent literature databases have been searched and the literature mainly back 

to the year 2000 is discussed in this chapter. Readers interested in earlier studies are referred to 

three reviews by the author, which were published some years ago (Buchbauer, 2002, 2004, 2007). 

However, where it seemed necessary also earlier investigations have been included here and topics 

are discussed shortly which were not dealt with in these earlier compilations. Nevertheless, in a few 

cases some overlapping with related chapters may occur in the present book. 

9.1.1 ANTICANCER PROPERTIES* 

A very promising field of treatment with EOs is their application against tumors. Especially since 

the 1990s the anticancer properties of EOs and/or their main constituents and/or metabolites have 

gained more and more interest, inasmuch as such a “natural” therapy is accepted all over the world 

by the patients. One of the most prominent compounds in that sense is either d-limonene, the main 

constituent of the EO of sweet orange peel oil (Citrus sinensis, Rutaceae) as well as of other citrus 

fruit peel oils, or perillyl alcohol, the most important metabolite of this monoterpene hydrocarbon. 

Perillyl alcohol has been developed as a clinical candidate at the National Cancer Institute because 

of its greater potency than limonene, which may enable potentially effective systemic concentrations 

of the active principles to be achieved at considerably lower doses (Phillips et al., 1995). 

Perillyl alcohol is effective as an inhibitor of farnesyl transferase. In the early developmental stages 

of mouse lung carcinogenesis the ras-protein undergoes a series of modifications, and farnesylation 

at the cysteine is one of these, which leads to the anchoring of ras-p-21-gen to the plasma membrane 

in its biologically active state. Perillyl alcohol administered to test mice showed a 22% reduction in 

tumor incidence and a 58% reduction in tumor multiplicity (Lantry et al., 1997). Perillyl alcohol 

reduced the growth of hamster pancreatic tumors (>50% of the controls), or even led to a complete 

regression (16%). Thus, perillyl alcohol may be an effective chemotherapeutic agent for human 

pancreatic cancer (Löw-Baselli et al., 2000; Stark et al., 1995). Perillyl alcohol also inhibited significantly 

the incidence (percentage of animals with tumors) and multiplicity (tumor/animals) of 

invasive adenocarcinomas of the colon and exhibited increased apoptosis of the tumor cells. 

Scientists from the Purdue University report that the rate of apoptosis is over sixfold higher in perillyl 

alcohol-treated pancreatic adenocarcinoma cells than in untreated cells, and that the effect of 

perillyl alcohol on pancreatic tumor cells is significantly greater than its effect on nonmalignant 

pancreatic ductal cells (Stayrock et al., 1997). Moreover, this monoterpene alcohol-induced increase 

in apoptosis in all of the pancreatic tumor cells is associated with a 2–8-fold increase in the expression 

of a proapoptotic protein which preferentially stimulates the apoptosis in malignant cells. 

Perillyl alcohol is also effective in reducing liver tumor growth. Two weeks after diethyl nitrosamine 

exposure was discontinued, the animals were divided into perillyl alcohol-treated and 

untreated groups. The mean liver tumor weight for the perillyl alcohol-treated rats of perillyl alcohol 

treatment was 10-fold less than that for the untreated animals (Mills et al., 1995). A newer study 

found that this monoterpene alcohol potentially attenuates ferric-nitrilo-acetate-induced oxidative 

damage and tumor promotional events (Jahangir et al., 2007). Monoterpenes such as d-limonene 

and perillyl alcohol, as well as other terpene alcohols, such as geraniol, carveol, farnesol, nerolidol, 

b-citronellol, linalool, and menthol, showed inhibitory activities on induced neoplasia of the large 

bowl and duodenum. Nerolidol, especially, has an impact on the protein prenylation and is able to 

reduce the adenomas in rats fed with these compounds to an extent of about 82% compared to the 

* Adorjan, M., 2007. Part of her master thesis, University of Vienna.

Biological Activities of Essential Oils 237 

controls (Wattenberg, 1991). Geraniol prevents the growth of cultured tumor cells, especially those 

of rat hepatomas and melanomas (Yu et al., 1995). Dietary geraniol increased the 50% survival time 

of mice significantly and even 20% of the animals remained free of tumors when fed a geraniolcontaining 

diet 14 days before an intraperitoneal transfer of the tumor cells (Shoff et al., 1991). 

Similar studies indicate that the colon tumors of animals fed with perillyl alcohol exhibited increased 

apoptosis as compared to those fed the control diet (Reddy et al., 1997). Therefore, consumption of 

diets containing fruits and vegetables rich in monoterpenes, such as d-limonene, reduces the risk of 

developing cancer of the colon, mammary gland, liver, pancreas, and lung (Crowell, 1999). 

In the following, the anticancer activity of some EOs published since 2000 up to now will be 

discussed. In these papers several different cell lines were used to determine the anticancer activity 

of the EOs tested: A-549 (human lung carcinoma cell line), B16F10 (mouse cell line), CO25 (N-ras 

transformed mouse myoblast cell line), DLD-1 (human colon adenocarcinoma cell line), Hep-2 

(human laryngeal cancer cell line), HL-60 (human promyelocytic leukemia cell line), J774 (mouse 

monocytic cell line), K562 (human erythroleucemic cell line), nuclear-factor-k-B (human mouth 

epidermal carcinoma cell line), M14 WT (human melanoma cell line), Neuro-2a (mouse neuroblastoma 

cell line), P388 (murine leukemia cell line), SP2/0 (mouse plasmocytoma cell line), and then 

Caco-2, K562, MCF-7, PC-3, M4BEU, ACHN, Bel-7402, Hep G2, HeLa, and CT-26 (different 

human cancer cell lines). 

El Tantawy et al. (2000) investigated the EO of Senecio mikanioides O. (cape ivy, Asteraceae), 

grown in Egypt. The main components of the oil of the plant’s aerial parts were a-pinene (23%) and 

b-myrcene (11.3%), whereas dehydroaromadendrene (31.8%) and camphene (19.7%) were the major 

compounds in the underground organs, analyzed by gas chromatography/mass spectrometry 

(GC-MS). Both EOs had a potent cytotoxic activity against the growth of certain human cell lines 

in vitro. Another study dealt with the EO of Nigella sativa (black cumin, Ranunculaceae) seeds and 

its main constituent, thymoquinone (Badary et al., 2000). This substance was tested against fibrosarcomas 

induced by 20-methylcholanthrene (MC) in Swiss albino mice in vivo and in vitro. The 

mice got 0.01% thymoquinone in drinking water 1 week before and thereafter MC treatment. At the 

end of the experiment there was a significant inhibition of MC-induced fibrosarcoma compared to 

MC alone (tumor incidence 43%, less MC-induced mortality). In comparison to the control group, 

in the liver of MC-induced tumor-bearing mice a reduction in hepatic lipid peroxides, an increase in 

glutathione content and enzyme activities of glutathione S-transferase (GST) and quinone transferase 

(QT) are observed. Furthermore, the in vitro tests showed an inhibition of the survival of the 

tumor cells. These data indicate that thymoquinone could be a powerful chemopreventive agent 

against MC-induced fibrosarcoma tumors, probably because of its interference with DNA synthesis. 

Some years later, Ali and Blunden (2003) published a review about the seeds of black cumin, which 

is used in folk medicine. The EO and its major constituent thymoquinone were found to have antineoplastic 

activity and to be protective against nephrotoxicity and hepatotoxicity induced by diseases 

or chemicals. 

The anticancerogenic effect of the EO of the Melissa offi cinalis L. (Lamiaceae) was investigated 

by Allahverdiyev et al. (2001) using cell cultures of Hep-2 cells derived from human laryngeal 

cancer. The activity of the EO was examined by morphologic changes and by flow cytometry, 

compared to methotrexate (MTX, an antagonist of folic acid) and etoposid, a partial synthetically 

obtainable glycoside of podophyllotoxin. The EO was able to terminate the cells of the G1 and S 

phases, whereas MTX was active in the S and G2 phases and etoposid in the G1 phase. Their findings 

showed that the essential balm oil possesses anticancerogenic effects because MTX blocks the 

transfer of one-carbon fragments by its affinity to dihydrofolic acid reductase, which leads to an 

obstruction of the nucleic acid synthesis. De Sousa et al. (2004) found in an in vitro assay that the 

EO of this plant was very effective against various human cancer cell lines (A549, MCF-7, Caco-2, 

HL-60, K562) and mouse cell lines (B16F10). 

Legault et al. (2003) performed a study about the antitumor activity of balsam fir oil (Abies 

balsamea, Pinaceae) using the tumor cell lines MCF-7, PC-3, A-549, DLD-1, M4BEU, and CT-26.


Balsam fir oil was active against all tumor cell lines with GI 50 values ranging from 0.76 to 1.7 mg/ mL. 

After GC-MS analysis among the monoterpenes found (about 96%) a-humulene proved itself to be 

responsible for cytotoxicity (GI 50 55 mM). Both the EO and a-humulene induced a dose- and timedependent 

decrease in cellular glutathione (GSH) content and an increase in reactive oxygen species 

(ROS) production. 

Zeytinoglu et al. (2003) studied the effects of carvacrol, one of the main compounds in the EO 

of oregano (obtained from the Lamiaceae Origanum onites L.) on the DNA synthesis of N-rastransformed 

mouse myoblast cells CO25. This monoterpenic phenol was able to inhibit the DNA 

synthesis in the growth medium and ras-activating medium, which contained dexamethason. The 

authors concluded that carvacrol may find application in cancer therapy because of its growth inhibition 

of myoblast cells even after activation of mutated N-ras-oncogene. Also, Ipek et al. (2003) 

investigated the thymol-isomer carvacrol using the in vitro sister-chromatid-exchange (SCE) assay 

on human peripheral blood lymphocytes. The inhibitory effect of carvacrol was checked in the presence 

of mitomycin C (MMC) in the same assay. The formation of SCE was not increased by any 

dose of carvacrol, while it decreased the rate of SCE induced by MMC. These findings demonstrate 

that carvacrol shows a significant antigenotoxic activity in mammalian cells, indicating its usage as 

an antigenotoxic agent. 

The fresh leaf the EO of the Moraceae Streblus asper Lour. comprising the major compounds 

phytol (45.1%), a-farnesene (6.4%), trans-farnesyl acetate (5.8%), caryophyllene (4.9%), and 

trans,trans-a-farnesene (2.0%) was tested against mouse lymphocytic leukemia cells (P388) 

and showed a significant anticancer activity (ED 50 30 mg/mL) (Phutdhawong et al., 2004). Also 

another EO, this time from the fruits of the Anonaceae Xylopia aethiopica (Ethiopian pepper), 

a plant grown in Nigeria, showed in a concentration of 5 mg/mL a cytotoxic effect in the carcinoma 

cell line (Hep-2) (Asekun and Adeniyi, 2004). Last but not least, also terpinen-4-ol, the 

major component of the tea tree oil (TTO) (Melaleuca alternifolia, Myrtaceae), was investigated 

by Calcabrini et al. (2004) as to its anticancer effects in human melanoma M14 WT cells and 

their drug-resistant counterparts, M14 adriamycin-resistant cells. TTO as well as terpinen-4-ol 

were able to impair the growth of human M14 melanoma cells, whereupon the effect was stronger 

on their resistant variants, which express high levels of P-glycoprotein in the plasma membrane, 

overcoming resistance to caspase-dependent apoptosis exerted by P-glycoprotein-positive 

tumor cells. 

Some other EOs from “prominent” plants were investigated if they could be used in cancer 

therapy. One of these plants is the Asteraceae Chrysanthemum boreale Makino whose EO was 

studied on the apoptosis of KB cells by Cha et al. (2005). Different cytotoxic effects hallmarking 

apoptosis (DNA fragmentation, apoptotic body formation, and sub-G1 DNA content) proceeded 

dose dependently. The caspase-3 activity was induced rapidly and transiently by treatment with an 

apoptosis-inducing concentration of the EO. Eugenol isolated from clove oil (Eugenia caryophyllata, 

Myrtaceae) was investigated by Yoo et al. (2005) using human promyelocytic leukemia cells 

(HL-60) and might be a potent agent in cancer therapy. After treatment with eugenol the HL-60 

cells showed hallmarks of apoptosis such as DNA fragmentation and formation of DNA ladders in 

agarose gel electrophoresis. Apoptotic cell death was induced via generation of ROS, inducing a 

mitochondrial permeability transition, reducing antiapoptotic protein bcl-2 level and inducing cytochrome 

C release to the cytosol. In traditional medicine very prominent is the bog myrtle Myrica 

gale L. (Myricaceae), a native plant in Canada as well as in Scotland. GC-MS analysis of the leaf 

EO revealed 53 components with myrcene, limonene, a-phellandrene, and b-caryophyllene as the 

major compounds. In the 60-min fraction of this oil the caryophyllene oxide content was higher 

(9.9%) than in the 30-min fraction (3.5%). The anticancer activity of these extracts was tested in 

human lung carcinoma cell line A-549 and human colon adenocarcinoma cell line DLD-1. 

The 60-min fraction showed a higher anticancer activity against both cell types than the 30-min 

fraction. The higher cell growth inhibition induced by the 60-min fraction could be caused by the 

accumulation of sesquiterpenes (Sylvestre et al., 2005).


Some Thai medicinal plants were checked for their antiproliferative activity on human mouth 

epidermal carcinoma (KB) and murine leukemia (P388) cell lines by Manosroi et al. (2006) using 

the MTX assay. From 17 Thai plants the Myrtaceae Psidium guajava L. (common guava) leaf oil 

showed the highest antiproliferative activity in KB cell line, which is 4.37 times more potent than 

vincristine, a well-known mitosis inhibitor. In P388 cells the Lamiaceae Ocimum basilicum L. 

(basil) oil had the highest effect, which is 12.7 times less potent than the thymin antagonist 

5-fluorouracil. 

In another study, the EO of the leaves of the Euphorbiaceae Croton fl avens L. (yellow balsam) 

from Guadeloupe, a native plant from the Caribbean area, was analyzed by Sylvestre et al. (2006) 

and as main components viridiflorene (12.2%), germacrone (5.3%), (E)-g-bisabolene (5.3%), and 

b-caryophyllene (4.9%) ascertained. The EO was found to be active against human lung carcinoma 

cell line A-549 and human colon adenocarcinoma cell line DLD-1. Three of the 47 components 

of the EO, namely a-cadinol, b-elemene, and a-humulene, showed also a cytotoxic activity 

against tumor cell lines. Yu et al. (2007) tested the EO of the rhizome of the Aristolochiaceae 

Aristolochia mollissima for its cytotoxicity on four human cancer cell lines (ACHN, Bel-7402, 

Hep G2, HeLa). The rhizome oil possessed a significantly greater cytotoxic effect on these cell 

lines than the oil from the aerial plant. 

The success of chemotherapeutic agents is often hindered by the development of drug resistance, 

with multidrug-resistant phenotypes reported in a number of tumors. In a recent study of an Italian 

research team, the effects of the monoterpene alcohol linalool on the growth of two human breast 

adenocarcinoma cell lines were investigated, both as a single agent and in combination with doxorubicin. 

Linalool inhibited only moderately cell proliferation; however, in subtoxic concentrations 

potentiates doxorubicin-induced cytotoxicity and proapoptotic effects in both cell lines, MCF7 WT 

and MCF7 AdrR. The results of the Italian author group suggest that linalool improves the therapeutic 

index in the management of breast cancer, especially multidrug resistance (MDR) tumors 

(Ravizza et al., 2008). 

The EO of Cyperus rotundus (Cyperaceae) contains cyperene, a-cyperone, isolongifolen-5-one, 

rotundene, and cyperorotundene as principal constituents. An in vitro cytotoxicity assay indicated 

that this oil was very effective against L1210 leukemia cells, which correlates with significantly 

increased apoptotic DNA fragmentation (Kilani et al., 2008). 

Finally, Yan et al. (2008) reported on the cytotoxic activity of the EO and extracts of Lynderia 

strychnifolia (Lauraceae), a plant which is widely used in traditional Chinese medicine. Three 

human cancer cell lines (A549, HeLa, and Hep G2) were examined by in vitro assays. The strongest 

cytotoxicity on the cancer cells showed the leaf oil with 50% inhibitory concentration (IC 50 ) values 

ranging between 22 and 24 mg/mL after 24 h of treatment. The EO of the leaves and also of the roots 

exhibited greater cytotoxicity than ethanol extracts. 

9.1.2 ANTINOCICEPTIVE EFFECTS* 

Although it has been mentioned in the introduction that in this book the comprehensive term 

“biological properties” does not include activities affecting the central and the peripheral nervous 

system, nevertheless for this subchapter a small exception had to be made, because the antinociceptive 

system belongs to the central nervous system. The function of antinociception is to aggravate 

the forwarding of pain impulses, which alleviates the sensation of pain. It is assumed that the socalled 

nociceptors are nerve endings responsible for nociception. They are sensory receptors that 

send signals, which cause the perception of pain in response to a potentially damaging stimulus. 

When the nociceptors are activated, they can trigger a reflex. Due to this system it can be explained 

why pains in a stress situation (e.g., caused by an injury after a traffic accident) are not noted 

in the first instance, but later after the decay of the tension. To test this pain-relieving capacity, 



experimentally generated pains in animal experiments were performed (Hunnius, 2007). The animal 

experiments that were mostly used are as follows: 

1. Formalin test: A small volume of formalin is injected in the hind paw of a rat or a mouse 

and the pain-related behavior (paw lifting, paw licking) is observed. There are two phases 

of the test. In the first phase (10 min) the immediate reaction, that reflects the activation of 

peripheral nociceptors, is measured. The second phase (60 min) reflects a spinal hypersensibilization 

(Pharmacon, 2007). 

2. Acetic acid-induced writhing test: Acetic acid is administered intraperitoneally to the test 

animals and the number of writhings is registered. 

3. Tail-fl ick test: The tail is irritated by a thermal stimulus and the movement of the tail is 

monitored. 

4. Hot-plate test: The test animal is put on a heated surface (“a hot plate”) and the thermal 

pain reflexes are recorded. 

5. Carrageenin edema test: The test animals get carrageenin injected and the volume of the 

paw is measured. 

6. Dextran edema test: The test animals get dextran injected and the paw volume is 

measured. 

7. PBQ-induced abdominal constriction test: The test animals get injected intraperitoneally 

a solution of p-benzoquinone and the number of abdominal contractions is recorded. 

The antinociceptive effects of N. sativa (black cumin, Ranunculaceae) oil and its major component 

thymoquinone were investigated by Abdel-Fattah et al. (2000). After oral administration of 

doses ranging from 50 to 400 mg/kg the nociceptive response was suppressed in the hot-plate test, 

tail-pinch test, acetic acid-induced writhing test, and in the early phase of formalin test. There was 

also an inhibition of nociceptive response in the late phase of the formalin test after systemic administration 

and i.p. injection of thymoquinone. The s.c. injection of naloxone (1 mg/kg) significantly 

blocked the antinociceptive effect of N. sativa oil and thymoquinone in the early phase of the formalin 

test. In N. sativa oil- and thymoquinone-tolerant mice the antinociceptive effect of morphine was 

significantly reduced, but not vice versa. These findings indicate that N. sativa oil as well as thymoquinone 

induce an antinociceptive effect by means of an indirect activation of m1- and m-opioid 

receptor subtypes. 

The EO from the leaves of Cymbopogon citratus (lemongrass, Poaceae) showed in the acetic 

acid-induced writhing test a strong inhibition dose dependently. Also in the formalin test the EO 

could cause an inhibition especially in the second phase of the test (100% at 200 mg/kg i.p.). 

Otherwise the opioid antagonist naloxone reversed the central antinociception, suggesting that the 

EO of Cymbopogon citratus plays a role in central and peripheral levels (Viana et al., 2000). 

Abdon et al. (2002) studied the antinociceptive effect of the EO of Croton nepetaefolius Baill. 

(Euphorbiaceae), an aromatic plant distributed in the northeast of Brazil and used in folk medicine 

as sedative, orexigen, and antispasmodic medicine, on male Swiss mice using the acetic acidinduced 

writhings, the hot-plate test and the formalin test. The EO was administered orally. 

Writhings were reduced effectively at the highest dose tested. In hot-plate test latency was assessed 

at all times of observation. In formalin test a significant reduction of paw licking could be noticed 

in the second phase of the test at 100 mg/kg and in both phases at 300 mg/kg. The analgesic effect 

of morphine was reversed significantly by pretreatment with naloxone in both phases in the formalin 

test. 

The antinociceptive effect of Satureja hortensis L. (summer savory, Lamiaceae) extracts and EO, 

a medicinal plant used in Iranian folk medicine as stomachic, muscle, and bone pain deliver was 

assessed by Hajhashemi et al. (2002). The hydroalcoholic extract as well as the polyphenolic fraction 

and EO of the aerial parts of the herb were screened for their antinociceptive activity in the light 

tail-flick test, as well as in the formalin and also in the acetic acid-induced writhing test. While


there was no significant result in the light tail-flick test the EO decreased the number of writhings 

induced by acetic acid compared to the control at the highest doses given. In the formalin test the 

hydroalcoholic extract, then the polyphenolic fraction and the EO showed an antinociceptive activity, 

which could not be reversed by pretreatment with naloxone or caffeine. These findings demonstrate 

that this effect caused by Satureja hortensis L. is not mediated by opioidergic or adenosine 

receptors. 

Another Satureja species of the Lamiaceae family, namely Satureja thymbra L., was investigated 

by Karabay-Yavasoglu et al. (2006). The antinociceptive activity of the EO was assessed in 

mice by the formalin test and in rats by the light tail-flick test and the hot-plate test. An antinociceptive 

effect could only be detected in the hot-plate test during the early phase and the late phase. In 

the tail-flick test the EO did not produce any significant antinociceptive effect. Nevertheless the 

authors concluded that the EO of Satureja thymbra may have an analgesic activity in mice and rats. 

A screening of the leaf EO of the Lauraceae Laurus nobilis L. (sweet bay) for antinociception in 

mice and rats was made by Sayyah et al. (2003). They reported a significant analgesic effect in tail 

flick and formalin tests, which was comparable to reference analgesics such as morphine and 

piroxicam. 

In another study the antinociceptive effects of Teucrium polium L., a wild-growing Iranian plant 

belonging to the Lamiaceae, were investigated by Abdollahi et al. (2003). The total extract and the 

EO significantly inhibited pain-related behavior in the acetic acid-induced writhing test compared 

to the control. 

The hydroalcoholic extract, the polyphenolic fraction, and the EO of the Lamiaceae Zataria 

multifl ora Boiss. (Zataria), a plant used in traditional medicine for pain therapy and several gastrointestinal 

diseases, were checked by Jaffary et al. (2004) using writhing, tail flick, and formalin test 

in mice and rats. As main components in the EO linalool, linalyl acetate, and p-cymene could be 

detected. In the writhing test the EO and the hydroalcoholic extract were able to decrease the pain 

reflexes significantly (p < .05, n = 6). Both EO and hydroalcoholic extract were effective in tail-flick 

test (p < .05, p < .01, n = 6), whereas oral administration did not show any effect, which indicates an 

inactivation or extensively metabolization in liver or in gastrointestinal sections (see Figure 9.1). 

Furthermore, antinociception was indicated in both phases of formalin test (p < .01, n = 6). Due to 

the overall activity in these tests scientists concluded that Zataria multifl ora has a clear central and 

peripheral antinociceptive activity. 

The antinociceptive effects of Lavandula hybrida Reverchon “Grosso” (Lamiaceae) EO and its 

main components linalool and linalyl acetate were examined by Barocelli et al. (2004). The number 

of acetic acid-induced writhings was significantly decreased after oral administration of 100 mg/kg or 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

15 min 

30 min 

45 min 

60 min 

75 min 

90 min 

105 min 

120 min 

Control 

5 mg/kg 

Essential 

oil 

900 mg/kg 

p.o. 

500 mg/kg 

i.p 

Morphine 

5 mg/kg 

FIGURE 9.1 Antinociceptive activity of Zataria multifl ora in tail-flick test in rats.


inhalation of lavender EO for 60 min. After pretreatment with naloxone, atropine, and mecamylamine 

the postinhalative analgesia in hot-plate test was suppressed, which indicates an involvement both 

of the opioidergic and of the cholinergic system. 

In another study the involvement of adenosine A1 and A2A receptors in (−)-linalool-induced 

antinociception was found by Peana et al. (2006b). The authors also mentioned that they have 

already shown the antinociceptive effect of (−)-linalool in recent studies in different animal 

models. The antinociceptive and antihyperalgesic effects were ascribed to the stimulation of 

opioidergic, cholinergic, and dopaminergic systems as well as to the interaction with K-channels, 

the local anesthetic activity, the negative modulation of glutamate transmission, and the blockade 

of N-methyl-d-aspartic acid (NMDA) receptors (Peana et al., 2006a). In the present study 

the authors used 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a selective A1 receptor antagonist, 

and 3,7-dimethyl-1-propargylxanthine (DMPX), a selective A2A receptor antagonist, to 

measure the depression of the antinociceptive effect of (−)-linalool in the hot-plate test in mice. 

The decrease of the antinociceptive effect of linalool was significantly for both DPCPX (0.1 mg/ 

kg i.p.) and DMPX (0.1 mg/kg i.p.) at the highest doses tested. These results indicate that the 

antinociceptive effects of (−)-linalool, the natural occurring enantiomer in the EOs of lavender, 

are, at least partially, mediated by adenosine A1 and A2A. The authors also examined the role 

of nitric oxide (NO) and prostaglandin E2 (PGE2) using lipopolysaccharide (LPS)-induced 

responses in macrophage cell line J774.A1. The nitrite accumulation in the culture medium was 

significantly inhibited after exposure of LPS-stimulated cells to (−)-linalool, whereas the LPSstimulated 

increase of inducible nitric oxide synthetase (iNOS) expression was not inhibited at 

all. On the other hand, (−)-linalool had no effect on the release of PGE2 and on the increase of 

inducible cyclooxygenase-2 (COX-2) expression. These findings demonstrate that the reduction 

of NO production is, at least partially, responsible for the molecular mechanism of (−)-linalool 

antinociceptive effect, supposably through cholinergic and glutamatergic activities. Coming 

back to an earlier study of this author group (Peana et al., 2003), the influence of opioidergic and 

cholinergic systems in (−)-linalool-induced antinociception was examined. In acid-induced 

writhing test a significant reduction of writhings could be shown at doses ranging from 25 to 

75 mg/kg. Whereupon the effect was completely inverted by naloxone, an opioid receptor antagonist, 

and by atropine, an unselective muscarinic receptor antagonist. In hot-plate test only the 

dose of 100 mg/kg was of significance. Moreover (−)-linalool showed a dose-dependent increase 

of motility effects, which excludes the participation of any sedative effect. The conclusion of this 

study is that opioidergic and cholinergic system play an important role in (−)-linalool-induced 

antinociception. 

In a recent published study, the contribution of the glutamergic system in the antinociception 

elicited by (−)-linalool in mice was investigated (Batista et al., 2008). This monoterpene alcohol 

administered intraperitoneally, or orally, or intrathecally inhibited dose dependently glutamateinduced 

nociception in mice. Furthermore, (−)-linalool reduced significantly the biting response 

caused by intrathecal injection of glutamate when this alcohol was given i.p. This antinociception is 

possible due to mechanisms operated by ionotropic glutamate receptors, namely AMPA, NMDA, 

and kainite. 

In a further study De Araujo et al. (2005) screened the EO of Alpinia zerumbet (Pers.) Burtt. 

et Smith (shell ginger, Zingiberaceae), an aromatic plant native to the tropical and subtropical 

regions of the world and used in folk medicine for various diseases, including hypertension. In 

the acetic acid-induced writhing test the oral administration was effective, in the hot-plate test 

the EO increased the remedy time and also paw licking could be reduced significantly in the 

second phase of formalin test at 100 mg/kg. At 300 mg/kg a decrease was noticed in both phases 

of the test. After pretreatment with naloxone i.p. the analgesia was reversed significantly, completely 

for the first phase and partially for the second phase of the test. Therefore, also this EO 

shows a dose-dependent antinociceptive effect, which supposably includes the participation of 

opiate receptors.


Lino et al. (2005) used the EO of Ocimum micranthum Willd. (Lamiaceae) from Northeastern 

Brazil to study its antinociceptive activity in the hot plate and in the acetic acid-induced writhing 

test. Upon administration of low doses the EO could inhibit the number of writhings up to 79%. The 

antinociceptive effect was not influenced by pretreatment with naloxone. In the formalin test pawlicking 

time decreased to 61%. Also in this case the pretreatment with naloxone could not reverse 

the antinociceptive effect, confirming that there is no involvement of the opioid system. However, 

an involvement of the NO system was suggested because of the reverse of the antinociception by 

l-arginine in the second phase of the formalin test. 

Santos et al. (2005) tested the antinociceptive activity of leaf EO of the Euphorbiaceae Croton 

sonderianus in mice using chemical and thermal methods. After i.p. injection of acetic acid, formalin, 

and capsaicin the EO could provoke an inhibition of nociception. On the other hand, there was 

no evidence for effectivity against thermal nociception in the hot-plate test; however, the acetic acidinduced 

writhing and the capsaicin-induced hind-paw licking could be reduced more effectively. 

The antinociceptive effect in both capsaicin and formalin test was significantly antagonized by 

glibenclamide. These findings indicate that glibenclamide-sensitive KATP + -channels are involved 

in the antinociceptive effect of Croton sonderianus EO. 

The same author studied also the antinociceptive properties in animal experiments after oral 

administration of 1,8-cineole, a terpenoid oxide in many EOs. By pretreatment of mice with naloxone 

the antinociceptive effect of this bicyclic ether was not inverted in the formalin test (Santos 

et al., 2000). 

Methyleugenol, a prominent fragrance substance because of its allergenic potential, was isolated 

from Asiasari radix (Aristolochiaceae) and its antinociceptive effect on formalin-induced hyperalgesia 

in mice investigated by Yano et al. (2006). The oral administration of methyleugenol suppressed 

the duration of licking and biting in the second phase of the test in the same way as diclofenac, 

a nonsteroidal anti-inflammatory drug. Furthermore, the substance could decrease pain-related 

behaviors induced by intrathecal injection of NMDA, whereas diclofenac did not influence this 

behavior. All these antinociceptive effects of methyleugenol were depressed by bicuculline, a 

g-aminobutyric acid (A) antagonist, whereas COX-1 and -2 activity was not affected. The conclusion 

of this study was that methyleugenol is a potent inhibitor of NMDA-receptor-mediated hyperalgesia 

via GABA(A) receptors. 

Iscan et al. (2006) analyzed the EO of the Asteraceae Achillea schischkinii Sosn. and Achillea 

aleppica DC. ssp. aleppica by GC and GC-MS and found as main component in both oils 1,8-cineole 

(32.5% and 26.1%). For testing the antinociceptive effect, male Swiss albino mice were used for the 

p-benzoquinone-induced abdominal constriction test. The number of abdominal contractions 

(writhing moments) was counted for 15 min, whereupon the antinociceptive activity was illustrated 

as percentage change from writhing controls. As reference drug Aspirin ® at doses of 100 and 

200 mg/kg was used. The EO of Achillea aleppica ssp. aleppica, which was also rich in bisabolol 

and its derivates, could induce a significant antinociception by reducing the number of writhes. In 

comparison with acetylsalicylic acid the active component of Achillea aleppica ssp. aleppica was 

not as potent as the drug. There are a number of isolated components from both EOs, which cause 

an antinociceptive effect. In particular, (−)-linalool has been closely investigated. The molecular 

mechanisms of the antinociceptive effect are different. They can be mediated by adenosine A1 and 

A2A or NMDA receptors, or the reduction of the NO production can play an important role. Also 

some can be glibenclamid-sensitive KATP + -channel dependent or influenced by the opioidergic or 

cholinergic system. 

Finally, also the antinociceptive and anti-inflammatory effects of the EO from Eremanthus 

erythropappus (Asteraceae) leaves were reported by a Brazilian author group. The EO proved to be 

significantly antinociceptive in the acetic acid-induced writhing test in mice, as well in the formalin 

test, and also in both phases of the paw-licking test, and in the hot-plate test. The exudate volume 

after intrapleural injection of carrageenan was significantly reduced as well as the leukocyte mobilization 

by administration of this oil 4 h before the start of the study (Sousa et al., 2008).


9.1.3 ANTIVIRAL ACTIVITIES* 

Besides the manifold, well-documented, and, in human and veterinary medicine, very often used 

antimicrobial and antifungal properties of nearly all EOs (see Chapter 12), this group of natural 

compounds also possesses distinct antiviral properties. Viruses are submicroscopic particles (ranging 

from 20 to 300 nm) that can infect cells of a biological organism. They replicate themselves 

only by infecting a host cell and cannot reproduce on their own. Unlike living organisms, viruses 

do not respond to changes in their environment (Hunnius, 2007). EOs are able to suppress the 

viruses in different ways. They can inhibit their replication or they can prevent their spread from 

cell to cell. In the following the antiviral activity of some EOs against different viruses such as 

Herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2), pseudorabies virus (PrV), influenza 

virus A3, Junin virus (JUNV), and dengue virus type 2 (DEN-2) will be discussed. In 1999, 

Benencia et al. (1999) published their results on the antiviral activity of sandalwood oil (Santalum 

album, Santalaceae) against Herpes simplex virus type 1 and type 2. The authors found that the 

EO inhibited the replication of the viruses. HSV-1 was more influenced than HSV-2 dose 

dependently. 

De Logu et al. (2000) investigated the inactivation of HSV-1 and HSV-2 and the prevention of 

cell-to-cell virus spread by the EO of the Asteraceae Santolina insularis. The plaque-reduction 

assay showed an IC 50 values of 0.88 mg/mL for HSV-1 and 0.7 mg/mL for HSV-2, respectively, 

whereas another test on Vero cells showed a cytotoxic concentration (CC 50 ) of 112 mg/mL, which 

leads to a CC 50 /IC 50 ratio of 127 for HSV-1 and 160 for HSV-2. These findings indicate that the 

antiviral effect of the EO was caused by direct virucidal effects. There was no antiviral activity 

detected in a postattachment assay. Due to attachment assays it was shown that virus adsorption was 

not reduced. Additionally, the reduction of plaque formation assay indicated that the EO reduced 

cell-to-cell transmission of both HSV-1 and HSV-2. 

Another study was made on the antiviral activity of Australian TTO and eucalyptus oil against 

Herpes simplex virus in cell culture by Schnitzler et al. (2001). The authors used a standard neutral 

red dye uptake assay to evaluate the cytotoxic effects of TTO and eucalyptus oil and found a moderate 

toxicity for RC-37 cells of both oils approaching 50% (TC 50 ) at very low concentrations. In the 

plaque-reduction assay an IC 50 for plaque formation of 0.0009% HSV-1 and 0.0008% HSV-2 for 

TTO and of 0.009% (HSV-1) and 0.008% (HSV-2) for eucalyptus oil was determined. In a viral 

suspension test a very strong virucidal activity against HSV-1 and HSV-2 could be shown. TTO 

reduced plaque formation by 98.2% for HSV-1 and by 93.0% for HSV-2 at noncytotoxic concentrations, 

respectively, whereas eucalyptus oil reduced virus titers by 57.9% for HSV-1 and 75.4% for 

HSV-2 at noncytotoxic concentrations. Additionally, the authors investigated the mode of antiviral 

action of both EOs. After pretreatment of the virus prior to adsorption plaque formation was clearly 

inhibited. The findings show that both oils affect the virus before or during adsorption, but not after 

penetration into the host cell. The authors suggest the application of both oils as antiviral agents in 

recurrent herpes infections, although the active components are yet unknown. 

Farag et al. (2004) examined the chemical and biological properties of the EOs of different 

Melaleuca species (teatree, TTO, Myrtaceae). The authors used the EOs of the fresh leaves of 

Melaleuca ericifolia, Melaleuca leucadendron (weepin teatree), Melaleuca armillaris, and 

Melaleuca styphelioides. Methyl eugenol (96.8%) was the main compound of the EO of Melaleuca 

ericifolia, whereas 1,8-cineole (64.3%) was the major constituent of Melaleuca leucadendron. The 

EO of Melaleuca armillaris was rich in 1,8-cineole (33.9%) and of terpinen-4-ol (18.8%). The main 

constituents of Melaleuca styphelioides were caryophyllene oxide (43.8%) followed by (−)spathulenol 

(9.6%). The highest virucidal effect of the EOs against HSV-1 in African green monkey kidney 

cells (Vero) by plaque reduction was caused by the volatile oil of Melaleuca armillaris (up to 99%), 

followed by that of Melaleuca leucadendron (92%) and Melaleuca ericifolia (91.5%). 



Primo et al. (2001) examined in vitro the antiviral activity of the EO from the Lamiaceae 

Minthostachys verticillata (Griseb.) Epling against HSV-1 and PrV using the viral plaque-reduction 

assay. The EO influences HSV-1 and PrV multiplication, an activity which is attributed to the main 

constituents of the EO, namely menthone (39.5%) and especially pulegone (44.6%). The therapeutic 

index values attained 10.0 and 9.5 for HSV-1 and PrV, respectively. 

In a chicken embryo hemagglutination valence-reduction test, Yan et al. (2002) investigated 

the inhibition of influenza virus A3 by the Lamiaceae Mosla chinensis EO. The authors assessed the 

activity of the EO for treatment against pneumonia in experiments with mice and found that the 

cytopathic effect (CPE) caused by influenza virus A3 was reduced in Vero cells by this EO. 

The hemagglutination valence was reduced from 1:1280 to 1:20 and 1:160 at concentrations of 500, 

250, and 50 mg/mL in 9-day-old chicken, respectively. At a dosage of 100 mg/g/d, the therapeutic 

treatment of mice against pneumonia was successful. 

The virucidal effect of the EO of the Lamiaceae Mentha piperita against HSV-1 and HSV-2 was 

tested in vitro on RC-37 cells using a plaque-reduction assay (Schuhmacher et al., 2003). The EO 

showed high virucidal effects against HSV-1 and HSV-2. At concentrations that did not produce a 

cytotoxic effect plaque formation was significantly inhibited by 82% for HSV-1 and 92% for HSV-2, 

respectively. A reduction of more than 90% for both herpes viruses could be achieved at higher 

concentrations of peppermint oil. The authors also demonstrated that the antiviral effect depended 

on time. After 3 h of incubation of HSV with the EO an antiviral activity of about 99% was shown. 

To investigate the mechanism of antiviral action, peppermint oil was added at different times to the 

cells or viruses during infection. When HSV was pretreated with the EO before adsorption, both 

HSV types were significantly reduced. These findings demonstrate that Mentha piperita oil influences 

the virus before adsorption, but not after penetration into the host cell. The EO also reduces 

plaque formation of an acyclovir-resistant strain of HSV-1 significantly by 99%. This oil might be 

useful for topical application as a virucidal agent in recurrent infection, considering its lipophilic 

properties, which enables it to penetrate the skin. 

Another study as to the inhibiory effect of some EOs on HSV-1 replication in vitro was carried 

out by Minami et al. (2003). The best results were achieved by lemongrass, which inhibited the viral 

replication completely even at a concentration of 0.1%. 

Furthermore, Garcia et al. (2003) studied the virucidal activity against HSV-1, JUNV, and DEN-2 

of eight different EOs obtained from plants of San Luis Province, Argentinia. The EOs of Lippia 

junelliana and Lippia turbinata (Verbenaceae) exhibited the highest virucidal effect against JUNV 

at virucidal concentrations (VC 50 ) values from 14 to 20 ppm, whereas the EOs of Aloysia gratissima 

(whitebrush, Verbenaceae), Heterotheca latifolia (camphorweed, Asteraceae), and Tessaria 

absinthioides (tessaria, Asteraceae) reduced JUNV from 52 to 90 ppm. The virucidal activity 

depended on time and temperature. The EOs of Aloysia gratissima, Artemisia douglasiana (mugwort, 

Asteraceae), Eupatorium patens (Asteraceae), and Tessaria absinthioides inhibited HSV-1 in 

the range of 65–125 ppm. A discernible effect on DEN-2 infectivity could only be produced by 

Artemisia douglasiana and Eupatorium patens with VC 50 values of 60 and 150 ppm, respectively. 

The antiviral activity of the EO of the Lamiaceae Melissa offi cinalis L. against HSV-2 was examined 

by Allahverdiyev et al. (2004). The effect of the essential oil on HSV-2 replication in Hep-2 cells 

was tested in five different concentrations (25, 50, 100, 150, and 200 mg/mL). Up to a concentration of 

100 mg/mL Melissa offi cinalis oil did not cause any toxic effect to Hep-2 cells, but it was slightly 

toxic at concentrations over 100 mg/mL. At nontoxic concentrations the replication of HSV-2 was 

reduced. Recently, Schnitzler et al. (2008) confirmed these findings: The lipophilic nature of the EO 

of lemon balm helps to affect the virus before adsorption thus exerting a direct antiviral effect. After 

the pene tration of the herpes virus into the host cell there was no affection recorded anymore. 

Yang et al. (2005) studied the anti-influenza virus activities of the volatile oil from the roots of 

the Asclepiadaceae Cynanchum stauntonii and found that the volatile oil caused an antiviral effect 

against influenza virus in vitro and also in in vivo experiments and was able to prevent the number 

of deaths induced by the virus in a dose-dependent manner.


Another study was carried out on the liposomal incorporation of Artemisia arborescens L. 

(powis castle, Asteraceae) EO and its in vitro antiviral activity by Sinico et al. (2005). The antiviral 

effect was tested against HSV-1 by a quantitative tetrazolium-based colorimetric method. The 

authors found that the EO can be incorporated in good amounts in vesicular dispersions and that 

these vesicle dispersions were stable for at least 6 months. During this period neither oil leakage nor 

vesicle size alteration occurred and even after a year of storage oil retention was still good, but 

vesicle fusion was present. The best antiviral results were observed when vesicles were made with 

P90H (= hydrogenated (P90H) soy phosphatidyl-choline). 

An evaluation of antiviral properties of various EOs from South American plants was carried out 

by Duschatzky et al. (2005). The authors assessed the cytotoxicity and in vitro inhibitory activity of 

the EOs against HSV-1, DENV-2, and JUNV by a virucidal test. The best results were observed 

with the EOs of Heterothalamus alienus (Asteraceae) and Buddleja cordobensis (Scrophulariaceae) 

against JUNV, with virucidal concentration 50% (VC 50 ) values of 44.2 and 39.0 ppm and therapeutic 

indices (cytotoxicity to virucidal action ratio) of 3.3 and 4.0, respectively. The oils caused the 

inhibitory effect interacting directly with the virions. 

Reichling et al. (2005) investigated the virucidal activity of a b-triketone-rich EO of the 

Myrtaceae Leptospermum scoparium (manuka oil) against HSV-1 and HSV-2 in vitro on RC-37 

cells (monkey kidney cells) using a plaque-reduction assay. The addition of the oil to the cells or 

viruses at different times during the infection cycle made it possible to determine the mode of the 

antiviral action. After pretreatment with manuka oil 1 h before cell infection both virus types were 

significantly inhibited. At concentrations that were not cytotoxic, the plaque formation reduction 

reached levels of 99.5% for HSV-1 and 98.9% for HSV-2. The IC 50 of the EO for virus plaque formation 

was 0.0001% V/V (= 0.96 mg/mL) for HSV-1 and 0.00006% V/V (= 0.58 mg/mL) for HSV-2. 

When the host cells were pretreated before viral infection, plaque formation could not be influenced. 

After the virus penetrated the host cells only the replication of HSV-1 particle was significantly 

reduced to about 41% by manuka oil. 

A phytochemical analysis and in vitro evaluation of the biological activity against HSV-1 of 

Cedrus libani A. Rich. (cedar of libanon, Pinaceae) was made by Loizzo et al. (2008a). The authors 

identified the active constituents for the in vitro antiviral activity against HSV-1 and evaluated the 

cytotoxic effects in Vero cells. The IC 50 values of cones and leaves extract were 0.50 and 0.66 mg/ mL, 

respectively, without provoking a cytotoxic effect, whereas the EO showed a comparable activity 

with an IC 50 value of 0.44 mg/mL. In another study the author group found that the EO of Laurus 

nobilis (Lauraceae) and Thuja orientalis (Cupressaceae) were very effective against SARScoronavirus 

(IC 50 : 120 ± 1.2 mg/mL, resp. 130 ± 0.4 mg/mL) and against Herpes simplex virus type 

1 (IC 50 : 60 + 0.5 mg/mL, resp. > 1000 mg/mL) (Loizzo et al., 2008b). 

Ryabchenko et al. (2007, 2008) presented a study that dealt with antitumor, antiviral, and cytotoxic 

effects of some single fragrance compounds. The antiviral properties were investigated in an 

in vitro plaque formation test in 3T6 cells against mouse polyoma virus. Natural and synthetic nerolidol 

showed the highest inhibitory activity, followed by trans,trans-farnesol and longifolene. 

9.1.4 ANTIPHLOGISTIC ACTIVITY* 

Processes by which the body reacts to injuries or infections are called inflammations. There are 

several inflammatory mediators such as the tumor necrosis factor-a (TNF-a); interleukin (IL)-1b, 

IL-8, IL-10; and the PGE2. In the following the inhibitory effects of some EOs on the expression of 

these inflammatory mediators and on other reasons for inflammations will be shown. Shinde et al. 

(1999) performed studies on the anti-inflammatory and analgesic activity of the Pinaceae Cedrus 

deodara (Roxb.) Loud. (deodar cedar, Pinaceae) wood oil. They examined the volatile oil obtained 

by steam distillation of the wood of this Cedrus species for its anti-inflammatory and analgesic 



effect at doses of 50 and 100 mg/kg body weight and observed a significant inhibition of carrageenin-induced 

rat paw edema. At doses of 100 mg/kg body weight both exudative–proliferative 

and chronic phases of inflammation in adjuvant arthritic rats were reduced. In acetic acid-induced 

writhing and also in hot-plate test both tested doses exhibited an analgesic effect in mice. 

The anti-inflammatory-related activity of EOs from the leaves and resin of species of Protium 

(Burseraceae), which are commonly used in folk medicine, was evaluated by Siani et al. (1999). The 

resin oil contains mainly monoterpenes and phenylpropanoids: a-terpinolene (22%), p-cymene 

(11%), p-cimen-8-ol (11%), limonene (5%), and dillapiol (16%), whereas the leaves dominantly comprise 

sesquiterpenes. The authors tested the resin of Protium heptaphyllum (PHP) and the leaves of 

Protium strumosum (PS), Protium grandifolium (PG), Protium lewellyni (PL), and Protium 

hebetatum (PHT) for their anti-inflammatory effect using a mouse pleurisy model induced by 

zymosan and LPS. In addition, they screened the plants for their NO production from stimulated 

macrophages and for the proliferation of neoplastic cell lines: Neuro-2a (mouse neuroblastoma), 

SP2/0 (mouse plasmocytoma), and J774 (mouse monocytic cell line). After administration of 

100 mg/kg p.o. 1 h before stimulation with zymosan, an inhibition of protein extravasation could be 

observed with the oils from PHP, PS, and PL, whereas total or different leukocyte counts could not 

be reduced. Also the neutrophilic accumulation could be decreased by the oils from PG, PL, and 

PHT, while PHP and especially PL lead to a reduction of LPS-induced eosinophilic accumulation 

in mouse pleural cavity. PHT also showed the ability to inhibit the mononuclear accumulation. 

The NO production from stimulated mouse macrophages could be changed by in vitro treatment 

with the EOs. A reduction of the LPS-induced NO production of 74% was achieved by PHP and of 

46% by PS. On the contrary, PL caused an increase of 49% in NO production. Concerning the cellline 

proliferation, Neuro-2a was affected in the range of 60–100%, SP2/0 of 65–95%, and J774 of 

70–90%. As to the suggestion of the authors, these EOs could be used as efficient pharmacological 

tools. 

Another study was made on the anti-inflammatory effect in rodents of the EO of the Euphorbiaceae 

Croton cajucara Benth. (Sacaca, Euphorbiaceae) by Bighetti et al. (1999). At a dose of 

100 mg/kg the EO exerted an anti-inflammatory effect in animal models of acute (carrageenininduced 

paw edema in mice) and chronic (cotton pellet granuloma) inflammation. Compared with 

the negative control a dose-dependent reduction of carrageenin-induced edema was achieved. This 

EO also reduced chronic inflammation by 38%, whereas diclofenac only achieved an inhibition of 

36%. The migration of neutrophils into the peritoneal cavity could not be inhibited by the EO. The 

anti-inflammatory effect seemed to be related to the inhibition of COX. 

Santos et al. (2000) investigated the anti-inflammatory activity of 1,8-cineole, a terpenoid oxide 

present in many plant EOs. Inflammation could be reduced in some animal models, that is, paw 

edema induced by carrageenin and cotton pellet-induced granuloma. This effect was caused at an 

oral dose range of 100–400 mg/kg. The authors suggest a potentially beneficial use in therapy as an 

anti-inflammatory and analgesic agent. 

The anti-inflammatory effect of the EO of the Myrtaceae Melaleuca alternifolia (TTO) was 

evaluated by Hart et al. (2000). The authors tested the ability of TTO to inhibit the production of 

inflammatory mediators such as the TNF-a, IL-1b, IL-8, IL-10, and the PGE2 by LPS-activated 

human peripheral blood monocytes. A toxic effect on monocytes was achieved at a concentration of 

0.016% vol/vol by TTO emulsified by sonication in a glass tube into culture medium containing 

10% fetal calf serum (FCS). In addition, a significant suppression of LPS-induced production of 

TNF-a, IL-1b and IL-10 (by approximately 50%), and PGE2 (by approximately 30%) after 40 h 

could be observed with the water-soluble components of TTO at a concentration equivalent to 

0.125%. The main constituents of this EO were terpinen-4-ol (42%), a-terpineol (3%), and 1,8-cineole 

(2%). When tested individually, only terpinen-4-ol inhibited the production of the inflammatory 

mediators after 40 h. 

The mechanisms involved in the anti-inflammatory action of inhaled TTO in mice were investigated 

by Golab et al. (2007). The authors used sexually mature, 6–8-week-old, C57BI10 × CBA/H


(F1) male mice and divided them into two groups. One group was injected i.p. with zymosan to 

induce peritoneal inflammation and the other simultaneously with antalarmin, a CRH-1 receptor 

antagonist, to block hypothalamic–pituitary–adrenal (HPA) axis function. After 24 h of injection 

the mice were killed by CO 2 asphyxia, and the peritoneal leukocytes (PTLs) isolated and counted. 

Additionally, the levels of ROS and COX activity were detected in PTLs by fluorometric and colorimetric 

assays, respectively. The result was that TTO inhalation led to a strong anti-inflammatory 

effect on the immune system stimulated by zymosan injection, whereas PTL number, ROS level, 

and COX activity in mice without inflammation were not affected. The HPA axis was shown to play 

an important role in the anti-inflammatory effect of TTO and antalarmin was observed to abolish 

the influence of inhaled TTO on PTL number and their ROS expression in mice with experimental 

peritonitis. In mice without inflammation these parameters were not affected. 

A further study was made on the anti-inflammatory activity of linalool and linalyl acetate constituents 

of many EOs by Peana et al. (2002). The authors evaluated the anti-inflammatory effect of 

(−)-linalool, that is, the naturally occurring enantiomer, and its racemate form, present in various 

amounts in distilled or extracted EOs. Due to the fact that in linalool-containing oils there is also 

linalyl acetate present, this monoterpene ester was also tested for its anti-inflammatory activity. 

Both the pure enantiomer and its racemate caused a reduction of edema after systemic administration 

in carrageenin-induced rat paw edema test. Better results could be observed with the pure 

enantiomer, which elicited a delayed and more prolonged effect at a dose of 25 mg/kg, whereas the 

efficiency of the racemate form lasted only for 1 h after carrageenin administration. At higher doses, 

there were no differences between the (−) enantiomer and the racemate and there could be achieved 

no increase of the effect with increasing the dose. Equimolar doses of linalyl acetate on local edema 

did not provoke the same effect as the corresponding alcohol. These results demonstrate a typical 

prodrug behavior of linaly acetate. 

Salasia et al. (2002) examined the anti-inflammatory effect of cinnamyl tiglate contained in the 

volatile oil of kunyit (Curcuma domestica Val.; Zingiberaceae) on carrageenin-induced inflammation 

in Wistar albino rats (Rattus norvegicus). Cinnamyl tiglate was found in the second fraction of 

the volatile oil at a concentration of 63.6%. After induction of an inflammation in rats by injection 

of 1% carrageenin and administration of cinnamyl tiglate orally at various doses (control group 

treated with aspirin) and the EO a plethysmograph measured the degree of inflammation: At a dose 

of 17.6% of the volatile oil of kunyit/kg body weight the highest anti-inflammatory effect could be 

observed (p ≤ 0.01), followed by the effect of a dose of 4.4%/kg body weight (p ≤ 0.05). At lower 

doses the inflammation could not be reduced (p ≥ 0.05). 

The in vitro anti-inflammatory activity of the EO from the Caryophyllaceae Ligularia fi scheri var. 

spiciformis (ligularia) in murine macrophage RAW 264.7 cells was evaluated by Kim et al. (2002). 

They examined the effects of the EOs isolated from various plants on LPS-induced release of NO, 

PGE2, and TNF-a by the macrophage RAW 264.7 cells. The EO of Ligularia fi scheri var. spiciformis 

achieved the best results among the tested oils inhibiting significantly the LPS-induced generation of 

NO, PGE2, and TNF-a in RAW 264.7 cells. Additionally, the EO reduced the expression of iNOS and 

COX-2 enzyme in a dose-dependent manner. Therefore, the mechanism of the anti-inflammatory 

effect of this EO is the suppression of the release of iNOS, COX-2 expression, and TNF-a. 

The in vitro anti-inflammatory activity of the EO of the Asteraceae Chrysanthemum sibiricum 

in murine macrophage RAW 264.7 cells was evaluated by Lee et al. (2003). The aim was to study 

the effect not only on the formation of NO, PGE2, and TNF-a, but also on iNOS and COX-2 in 

LPS-induced murine macrophage RAW 264.7 cells. The EO had a similar effect on both enzymes 

and the inhibitory effects were concentration dependent. Additionally, the volatile oil also furnished 

a reduction of the formation of TNF-a. 

An evaluation of the anti-inflammatory activity of the EOs from the Asteraceae Porophyllum 

ruderale (PR) (yerba porosa) and Conyza bonariensis (CB) (asthmaweed) in a mouse model of 

pleurisy induced by zymosan and LPS was made by Souza et al. (2003). The activity of the main 

compounds of each oil, b-mycrene (in PR), and limonene (in CB) in the LPS-induced pleurisy


model as well as the immunoregulatory activity was examined by measurement of the inhibition of 

NO and production of the cytokines, g-interferon, and IL-4. After oral administration of the oils, a 

reduction of the LPS-induced inflammation including cell migration could be observed. A similar 

effect could be provoked with the use of limonene alone. Pure b-mycrene and limonene were also 

able to reduce the production of NO at not cytotoxic doses. In addition, b-myrcene and limonene 

also inhibited significantly g-interferon. 

The anti-inflammatory effect of the leaf EO of the Lauraceae Laurus nobilis Linn. (sweet bay) in 

mice and rats was investigated by Sayyah et al. (2003). A dose-dependent anti-inflammatory effect 

could be observed in the formalin-induced edema test, which could be compared with the effect of 

nonsteroid anti-inflammatory drugs such as piroxicam. 

Silva et al. (2003) examined the anti-inflammatory effect of the EO of three species of the 

Myrtaceae Eucalyptus citriodora (EC, lemon eucalyptus), Eucalyptus tereticornis (ET, forest red 

gum), and Eucalyptus globulus (EG, blue gum eucalyptus), from which many species are used in 

Brazilian folk medicine to treat various diseases such as cold, flu, fever, and bronchial infections. 

An inhibition of rat paw edema induced by carrageenan and dextran, neutrophil migration into rat 

peritoneal cavities induced by carrageenan and vascular permeability induced by carrageenan and 

histamine could be observed. But there were no consistent results obtained for parameters as activity 

and dose–response relationship, which demonstrates the complex nature of the oil and the assays 

used. However, these findings provide support for the traditional use of Eucalyptus in Brazilian folk 

medicine and further investigations should be made in order to develop possibly new classes of antiinflammatory 

drugs from components of the EOs of the Eucalyptus species. 

The anti-inflammatory activity of the EOs of Ocimum gratissimum (African brasil, Lamiaceae), 

Eucalyptus citriodora (lemon eucalyptus, Myrtaceae), and Cymbopogon giganteus (Poaceae) was 

evaluated by Sahouo et al. (2003). The authors tested the inhibitory effect of the three plants in vitro 

on soybean lipoxygenase L-1 and COX function of prostaglandin H synthetase. The two enzymes 

play an important role in the production of inflammatory mediators. The EO of Eucalyptus citriodora 

evidently suppressed L-1 with an IC 50 value of 72 mg/mL. Both enzymes were inhibited by only one 

EO that of Ocimum gratissimum with IC 50 values of 125 mg/mL for COX function of PGHS and 

144 mg/mL for L-1, respectively, whereas the oils of Eucalyptus citriodora and Cymbopogon 

giganteus did not affect the COX. 

Lourens et al. (2004) evaluated the in vitro biological activity and chemical composition of the 

EOs of four indigenous South African Helichrysum species (Asteraceae), such as Helichrysum 

dasyanthum, Helichrysum felinum (strawberry everlasting), Helichrysum excisum, and Helichrysum 

petiolare (licorice plant). An interesting anti-inflammatory effect could be observed in the lipooxygenase-5 

assay at doses between 25 and 32 mg/mL. Analysis of the chemical composition showed 

that the EOs comprise mainly monoterpenes such as a-pinene, 1,8-cineole, and p-cymene, only the 

oil of Helichrysum felinum was dominated by sesquiterpenes in low concentrations with b-caryophyllene 

as main compound on top. 

The composition and in vitro anti-inflammatory activity of the EO of South African Vitex species 

(Verbenaceae), such as Vitex pooara (waterberg poora-berry), Vitex rehmanni (pipe-stem tree), 

Vitex obovata ssp. obovata (hairy fingerleaf), V. obovata ssp. wilmsii, and Vitex zeyheri (silver pipestem 

tree), were analyzed by Nyiligira et al. (2004). After determination of the composition of the 

EOs by GC-MS their in vitro anti-inflammatory activity was investigated in a 5-lipoxygenase assay. 

All EOs effectively suppressed 5-lipoxygenase, which plays an important role in the inflammatory 

cascade. The best results were achieved by V. pooara with an IC 50 value of 25 ppm. The use of the 

EO data matrix presents chemotaxonomic evidence, which supports infrageneric placement of 

V. pooara in subgenus Vitex, whereas the other four species are placed in subgenus Holmskiodiopsis. 

Ganapaty et al. (2004) examined the composition and anti-inflammatory activity of the 

Geraniaceae Pelargonium graveolens (rose geranium) EO. Citronellol, geranyl acetate, geraniol, 

citronellyl formate, and linalool were identified in the leaf oil by GC-MS. A significant antiinflammatory 

effect could be observed in the carrageenan-induced rat paw edema test.


Another study was made on the in vitro anti-inflammatory activity of paeonol from the EO of the 

Paeoniaceae Paeonia moutan (tree peony) and its derivate methylpaeonol by Park et al. (2005). The 

authors isolated paeonol (2-hydroxy-5-methoxyacetophenone) by silica gel column chromatography 

and methylated it by dimethylsulfate to yield methylpaeonol (2,5-di-O-methylacetophenone). A suppression 

of the NO formation in LPS-induced macrophage RAW 264.7 cells was observed with both 

compounds in nitrite assay. Additionally, a reduction of iNOS-synthase and COX-2 formation was 

achieved in the Western blotting assay. These findings demonstrate that paeonol is partly responsible 

for the anti-inflammatory effect of Paeonia moutan and that synthesized derivates are promising 

candidates for new anti-inflammatory agents. 

The anti-inflammatory effect of the EO from the leaves of indigenous Cinnamomum osmophloeum 

Kaneh. (camphor tree, Lauraceae) was studied by Chao et al. (2005). Twenty-one components, 

among which the monoterpenes 1,8-cineole (17%) and santolina triene (14.2%) and the sesquiterpenes 

spathulenol (15.7%) and caryophyllene oxide (11.2%), were analyzed as main constituents. In 

the anti-inflammatory assay it was found that the EO exerted a high capacity to suppress pro-IL-1b 

protein expression induced by LPS-treated J774A.1 murine macrophage at dosages of 60 mg/mL. 

Additionally, IL-1b and IL-6 production was reduced at the same dose. The TNF-a production 

could not be influenced by this dose of the EO. 

A further study was carried out on the anti-inflammatory activity of the EO from Casaeria sylvestris 

Sw. (wild coffee, Flacourtiaceae) by Esteves et al. (2005). The EO having a total yield of 2.5% showed 

a LD 50 of 1100 mg/kg in mouse. Its composition was analyzed by GC and mainly sesquiterpenes, such 

as caryophyllene, thujopsene, a-humulene, b-acoradiene, germacrene-D, bicyclogermacrene, calamenene, 

germacrene B, spathulenol, and globulol identified as main compounds. After oral administration 

of this EO to rats, a reduction by 36% in carrageenan-induced edema was achieved in the rat assay 

(p < 0.05, Student’s t-test). In rat paw edema dextran-induced and vascular permeability assay using 

histamine, no significant result could be observed. Additionally, the writhing test using acetic acid 

demonstrated an inhibition of writhes with the EO by 58% and with indomethacin by 56%. 

Ramos et al. (2006) investigated the anti-inflammatory activity of EOs from five different 

Myrtaceae species, Eugenia brasiliensis (grumichama), Eugenia involucrate, Eugenia jambolana, 

Psidium guajava (guava), and Psidium widgrenianum. The oils were obtained by steam distillation 

and analyzed by GC-MS and the correlation of retention indexes. In Eugenia brasiliensis, 

Eugenia involucrate, and Psidium guajava mainly sesquiterpenes could be identified, whereas 

monoterpenes dominated in Psidium widgrenianum and Eugenia jambolana. Afterwards the 

volatile compounds in zymosan and LPS-induced inflammatory models were tested. In zymosaninduced 

pleurisy, no reduction of leukocyte accumulation or protein leakage could be observed 

after p.o. administration of up to 100 mg/kg. Eugenia jambolana suppressed the total leukocyte 

(up to 56%) and eosinophil (up to 74%) migration in LPS-induced pleurisy, but the response did 

not correlate with the dose. Psidium widgrenianum only inhibited eosinophil migration (up to 

70%) and both Eugenia jambolana and Psidium widgrenianum were also tested in vitro for 

their inhibitory effect on the production of NO. A potent suppression was achieved by Eugenia 

jambolana (up to 100%) in a dose-dependent manner, whereas only a moderate effect could 

be observed with Psidium widgrenianum (51%) test at concentrations below cytotoxic activity 

(25 mg/well). 

The anti-inflammatory activity of the Myrtaceae Eugenia caryophyllata (clove) EO was evaluated 

in an animal model by Ozturk et al. (2005). The chemical composition of this EO by GC 

yielded b-caryophyllene (44.7%), eugenol (44.2%), a-humulene (3.5%), eugenyl acetate (1.3%), and 

a-copaene (1.0%) as main constituents. Then the volumes of the right hind paws of rats were measured 

with a plethysmometer in eight groups: physiological serum, ethylalcohol, indomethacin 

(3 mg/kg), etodolac (50 mg/kg), cardamom (0.05 ml/kg), EC-I (0.025 ml/kg), EC-II (0.050 ml/kg), 

EC-III (0.100 ml/kg), and EC-IV (0.200 ml/kg). Afterwards the drugs were injected i.p. and 

g-carrageenan s.c. into the plantar regions and the difference of the volumes determined after 3 h. 

A reduction of the inflammation by 95.7% could be observed with indomethacin, to a lesser extent


by the other seven groups (Table 9.1). The result of the study was that the EO of Eugenia caryophyllata 

exerted a remarkable anti-inflammatory effect. 

Alitonou et al. (2006) investigated the EO of Poaceae Cymbopogon giganteus, widely used 

in traditional medicine against several diseases, from Benin for its potential use as an anti-inflammatory 

agent. The analysis of this EO furnished trans-p-1(7),8-menthadien-2-ol (22.3%), cis-p-1- 

(7),8-menthadien-2-ol (19.9%), trans-p-2,8-menthadien-1-ol (14.3%), and cis-p-2,8-menthadien-1-ol 

(10.1%) as main components. Additionally, it was found that the leaf EO suppressed the 5-lipoxygenase 

in vitro. Moreover, the antiradical scavenging activity was measured by the 1,1-diphenyl-2- 

picrylhydrazyl (DPPH) method (as to this property see Section 9.1.6). 

A further study was carried out on the anti-inflammatory effect of the EO of Iranian black cumin 

seeds (BCS) (N. sativa L.; Ranunculaceae) by Hajhashemi et al. (2004). p-Cymene (37.3%) and 

thymoquinone (13.7%) were found to be the main compounds. For the detection of the anti-inflammatory 

activity, carrageenan-induced paw edema test in rats was used and also the croton oilinduced 

ear edema in mice. After oral administration of this EO at various doses no significant 

anti-inflammatory effect could be observed in the carrageenan test, whereas i.p. injection of the 

same doses significantly reduced carrageenan-induced paw edema. At doses of 10 and 20 mL/ear, 

BCS-EO also caused a reduction of a croton oil-induced edema. An anti-inflammatory effect could 

be observed after both systemic and local administration and thymoquinone seemed to play an 

important role in this pharmacological effect. 

The downregulation of the leukotriene biosynthesis by thymoquinone, the active compound of 

the EO of the Ranunculaceae N. sativa, and its influence on airway inflammation in a mouse model 

was examined by El Gazzar et al. (2006). Bronchial asthma is often caused by chronic airway 

inflammation and leukotrienes are potent inflammatory mediators. Their levels arose in the air passages 

when an allergen challenge has been going on. The authors sensitize mice and challenged 

them with ovalbumin (OVA) antigen, which led to an increase of leukotrien B4 and C4, Th2 cytokines, 

and eosinophils in bronchoalveolar lavage (BAL) fluid. Additionally, lung tissue eosinophilia 

and nose goblet cells were remarkably elevated. After administration of thymoquinone before OVA 

challenge, 5-lipoxygenase expression by lung cells was suppressed and therefore the levels of LTB4 

and LTC4 were reduced. A reduction of Th2 cytokines and BAL fluid and lung tissue eosinophilia, 

all parameters of airway inflammation, could also be observed. These findings demonstrate the 

anti-inflammatory activity of thymoquinone in experimental asthma. 

Also El Mezayen et al. (2006) studied the effect of thymoquinone, the major constituent of the 

EO of N. sativa seeds, on COX expression and prostaglandin production in a mouse model of allergic 

airway inflammation. Prostaglandins play an important role in modulating the inflammatory 

responses in a number of conditions, including allergic airway inflammation. They are formed 

through arachidonic acid metabolism by COX-1 and -2 in response to various stimuli. The authors 

sensitized mice and challenged them through the air passage with OVA, which caused a significant 

TABLE 9.1 

Reduction of Inflammation by the EO of Eugenia caryophyllata 

(EC), Indomethacin, and Etodolac in Animal Model 

Reduction of Inflammation (%) 

Indomethacin (3 mg/kg) 95.7 

Etodolac (50 mg/kg) 43.4 

EC-I (0.025 mL/kg) 46.5 

EC-II (0.050 mL/kg) 90.2 

EC-III (0.100 mL/kg) 66.9 

EC-IV (0.200 mL/kg) 82.8


elevation of the PGD2 and PGE2 expression in the airways. Additionally, inflammatory nose cells 

and Th2 cytokine levels in the BAL fluid, lung airway eosinophilia, and goblet cell hyperplasia were 

raised and the COX-2-protein expression in the lung was induced. After i.p. injection of thymoquinone 

for 5 days before the first OVA challenge a significant decrease in Th2 cytokines, lung eosinophilia, 

and goblet cell hyperplasia could be observed, which was caused by the suppression of 

COX-2 protein expression and a reduction of the PGD2 production. Thymoquinone also slightly 

inhibited the COX-1 expression and the production of PGE2. This time the results demonstrated the 

anti-inflammatory effect of thymoquinone during the allergic response in the lung caused by the 

suppression of PGD2 synthesis and Th2-driven immune response. 

The effect of thymoquinone from the volatile oil of black cumin on rheumatoid arthritis in rat 

models was investigated by Tekeoglu et al. (2006). Arthritis was induced in rats by Freund’s incomplete 

adjuvant and the rats were divided into five groups: controls 0.9% NaCl (n = 7), 2.5 mg/kg 

thymoquinone (n = 7), 5 mg/kg thymoquinone (n = 7), Bacilli Chalmette Guerin (BCG) 6 × 105 CFU 

(n = 7), and MTX 0.3 mg/kg (n = 7). The level of inflammation was characterized by radiological 

and visual signs on the claw and by TNF-a and IL-1b expression and the results of the different 

groups were compared. Thymoquinone reduced adjuvant-induced arthritis in rats, which was confirmed 

clinically and radiologically. 

Biochemical and histopathological evidences for beneficial effects of the EO of the Lamiaceae 

Satureja khuzestanica Jamzad, an endemic Iranian plant, on the mouse model of inflammatory 

bowel diseases were found by Ghazanfari et al. (2006). The EO was tested on the experimental 

mouse model of inflammatory bowel disease, which is acetic acid-induced colitis and used prednisolone 

as control. For best results, also biochemical, macro- and microscopic examinations of the 

colon were performed. In acetic acid-treated mice a significant increase of lipid peroxidation could 

be observed compared to the control group, which was significantly restored by treatment with the 

EO and prednisolone. The EO decreased the lipid peroxidation up to 42.8% dose dependently, 

whereas prednisolone caused a decrease of 33.3%. Also a significant increase of the myeloperoxidase 

activity could be observed compared to the control group in acetic acid-treated mice, which 

was also significantly restored by treatment with the EO and prednisolone. The EO lowered the 

myeloperoxidase activity by 25% and 50% on average, whereas that of the control group was 

decreased by 53%. In addition, the EO- and prednisolone-treated groups exhibited significantly 

lower score values of macro- and microscopic characters after comparison to the acetic acid-treated 

group. These findings demonstrate that the beneficial effect of Satureja khuzestanica Jamzad EO 

could be compared to that of prednisolone. The antioxidant, antimicrobial, anti-inflammatory, and 

antispasmodic properties of the EO may be responsible for the protection of animals against experimentally 

induced inflammatory bowel diseases. 

The biological activity and the composition of the EOs of 17 indigenous Agathosma (Rutaceae) 

species were examined by Viljoen et al. (2006a) in order to validate their traditional use. The analysis 

of the EOs furnished 322 different components. The anti-inflammatory activity was detected 

with the 5-lipoxygenase assay and all oils inhibited inflammations in vitro with Agathosma collina 

achieving the best results (IC 50 value of about 25 mg/mL). These results show that the EOs of the 

different Agathosma species strongly suppress the 5-lipoxgenase. 

The chemical composition and biological activity of the EOs of four related Salvia species 

(Lamiaceae) also indigenous to South Africa was evaluated by Kamatou et al. (2006). The authors 

isolated the EOs from fresh aerial parts by hydrodistillation and analyzed the chemical composition 

by GC-MS. The differences between the different species were rather quantitative. Forty-three 

components were identified accounting for 78% of Salvia africana-caerulea, 78% of Salvia 

africana-lutea, 96% of Salvia chamelaeagnea, and 81% of Salvia lanceolata total EO. Salvia 

africana-caerulea and Salvia lanceolata mainly contained oxygenated sesquiterpenes (59% and 

48%, respectively), whereas Salvia chamelaeagnea was dominated by oxygen-containing monoterpenes 

(43%) and Salvia africana-lutea by monoterpene hydrocarbons (36%). The anti-inflammatory 

effect was tested with the 5-lipoxygenase method.


Viljoen et al. (2006b) studied the chemical composition and in vitro biological activities of 

seven Namibian species of Eriocephalus L. (Asteraceae). The EOs of Eriocephalus ericoides ssp. 

ericoides (samples 1 and 2), Eriocephalus merxmuelleri, Eriocephalus scariosus, Eriocephalus 

dinteri, Eriocephalus luederitzianus, Eriocephalus klinghardtensis, and Eriocephalus pinnatus were 

analyzed by GC-MS. Eriocephalus ericoides ssp. ericoides (sample 1), Eriocephalus merxmuelleri, 

and Eriocephalus scariosus contained high levels of 1,8-cineole and camphor and Eriocephalus 

scariosus was also rich in santolina alcohol (14.8%). Most camphor was found in Eriocephalus dinteri 

(38.4%), whereas the major compound of Eriocephalus ericoides ssp. ericoides (sample 2) was linalool 

(10.4%). The composition of Eriocephalus luederitzianus and Eriocephalus klinghardtensis was 

similar, both containing high levels of a-pinene, b-pinene, p-cymene, and g-terpinene. Eriocephalus 

luederitzianus additionally was rich in a-longipinene (10.3%) and b-caryophyllene (13.3%). 

Eriocephalus pinnatus was different from the other taxa containing mainly isoamyl 2-methyl butyrate 

(7.9%) and isoamyl valerate (6.5%). The anti-inflammatory activity was evaluated using the 5-lipoxygenase 

enzyme and Eriocephalus dinteri achieved best results (IC 50 : 35 mg/mL). 

Wang and Zhu (2006) studied the anti-inflammatory effect of ginger oil (Zingiber offi cinale, 

Zingiberaceae) with the model of mouse auricle edema induced by xylene and rat paw edema 

induced by egg white for acute inflammation and the granuloma hyperplasia model in mouse caused 

by filter paper for chronic inflammation. Additionally, the influence of ginger oil on delayed-type 

hypersensitivity (DHT) induced by 2,4-dinitrochlorobenzene (DNCB) in mice was observed. The 

authors found that ginger oil significantly inhibited both mouse auricle edema and rat paw edema 

and it also reduced the mouse granuloma hyperplasia and DHT. 

The anti-inflammatory activity of Carlina acanthifolia (acanthus-leaved thistle, Asteraceae) root 

EO was evaluated by Dordevic et al. (2007). In traditional medicine the root of the plant is used for 

the treatment of a variety of diseases concerning stomach and skin. The anti-inflammatory activity 

was tested in the carrageenan-induced rat paw edema assay and the oil inhibited the edema in all 

applied concentrations. The effect could be compared to indomethacin, which was used as control. 

Another study was made on the anti-inflammatory effect of the EO and the active compounds of 

the Boraginaceae Cordia verbenacea (black sage) by Passos et al. (2007). It was found that the carrageenan-induced 

rat paw edema, the myeloperoxidase activity, and the mouse edema elicited by 

carrageenan, bradykinin, substance P, histamine, and the platelet-activating factor could be inhibited 

after systemic (p.o.) administration of 300–600 mg/kg EO. It also suppressed carrageenan-evoked 

exudation, the neutrophil influx to the rat pleura and the neutrophil migration into carrageenanstimulated 

mouse air pouches. Additionally, a reduction of edema caused by Apis mellifera venomous 

OVA in sensitized rats and OVA-evoked allergic pleurisy could be observed. TNF-a was 

significantly inhibited in carrageenan-treated rat paws by the EO, whereas the IL-1b expression was 

not influenced. No affection was caused of neither the PGE2 formation after intrapleural injection of 

carrageenan, nor of the COX-1 or COX-2 activities in vitro. Both sesquiterpenes, a-humulene and 

trans-caryophyllene (50 mg/kg p.o.) obtained from the EO, lead to a remarkable reduction of the 

carrageenan-induced mice paw edema. All in all, this study demonstrated the anti-inflammatory 

effect of the EO of Cordia verbenacea and its active compounds. The possible mechanism of this 

effect might be caused by the interaction with the TNF-a production. The authors suggest that Cordia 

verbenacea EO could represent new therapeutic options for the treatment of inflammatory diseases. 

The topical anti-inflammatory effect of the leaf EO of the Verbenaceae Lippia sidoides Cham. 

was studied by Monteiro et al. (2007). In northwestern Brazil, the plant is widely used in the social 

medicine program “Live Pharmacies” as a general antiseptic because of its strong activity against 

many microorganisms. After topical application of 1 and 10 mg/ear, in 45.9% and 35.3%, a significant 

reduction (p < 0.05) of the acute ear edema induced by 12-tetradecanoylphorbol 13-acetate 

(TPA) could be observed. 

The anti-inflammatory effects of the EO from Eremanthus erythropappus leaves (Asteraceae) 

have already been discussed in the chapter dealing with antinociception (Sousa et al., 2008). 

An interesting study concerning the healing of Helicobacter pylori-associated gastritis by the


volatile oil of “Amomum” (several Amomum species, which are used in similar manner to cardamon 

(Elettaria cardamomum), Zingiberaceae) was published recently by a Chinese author group. The 

effects of this EO on the expressions of mastocarcinoma-related peptide and platelet-activating factor 

was assessed and its potential mechanism discussed. The mechanism of this volatile oil for its 

antigastritis activity could be the influence on the decrease of the expression of the platelet-activating 

factor and thus regulating the hydrophobicity of the gastric membrane. About 88% of the patients 

with a proven Helicobacter pylori infection were treated and showed a higher healing rate compared 

to the control group having received a traditional “Western” tertiary medicinal treatment 

(Huang et al., 2008). 

9.1.5 PENETRATION ENHANCEMENT* 

A number of EOs are able to improve the penetration of various drugs through living membranes, 

for example, the skin. The improvement of the penetration can be achieved by the interaction of the 

EOs with liquid crystals of skin lipids. In the following, the penetration enhancing effect of some 

EOs will be discussed. For determination of the penetration enhancing effect different experimental 

setups were used: Valia–Chien horizontal diffusion cells, Keshary–Chien diffusion cells, and Franz 

diffusion cells. Furthermore, the scientists using polarizing microscopy, differential scanning calorimetry 

(DSC), x-ray diffraction, and high performance liquid chromatography (HPLC) to detect the 

penetration through the skin. 

The interaction of eucalyptus oil with liquid crystals of skin lipids was proved by Abdullah et al. 

(1999) using polarizing microscopy, DSC, and x-ray diffraction. Crystal 1 (matrix 1) consisted of 

five fatty acids of stratum corneum, crystal 2 (matrix 2) consisted of cholesterol together with five 

fatty acids. Dispersion and swelling of the lamellar structure were observed after application of 

small amounts of eucalyptus oil, whereas large amounts resulted in their breakage and disappearance. 

The EO did not promote the formation of any other structures. This interaction seems to be 

the explanation for the increase of permeation of drugs through stratum corneum in the presence of 

eucalyptus oil and similar penetration enhancers. 

Li et al. (2001) investigated the effects of eucalyptus oil on percutaneous penetration and absorption 

of a clobetasol propionate cream using vertical diffusion cells. The in vitro penetration of the cream 

containing 0.05% clobetasol propionate through mouse abdominal skin was detected at 2, 4, 6, 8, 10, 

and 24 h (cumulative amount Q, mg/g) and at steady state (J, mg/cm2/h). The quantity of clobetasol 

propionate within the whole stria of skin after 24 h (D, mg/g) was measured too. Eucalyptus oil was 

able to increase Q and J, whereas D was not influenced in that way, which indicates that eucalyptus oil 

would increase clobetasol propionate percutaneous absorption and cause unwanted side effects. 

Cinnamon oil, eugenia oil, and galangal oil have been studied for their potency as percutaneous 

penetration enhancers for benzoic acid (Shen et al., 2001). Valia–Chien horizontal diffusion cell and 

HPLC were used to detect benzoic acid penetration through skin. 

Skin penetration of benzoic acid was significantly enhanced by all three volatile oils. In combination 

with ethanol and propylene glycol the amount of benzoic acid was increased, but the permeability 

coefficients were decreased. In conclusion, cinnamon oil, eugenia oil, and galangal oil might 

be used as percutaneous penetration enhancers for benzoic acid. 

Monti et al. (2002) tried to examine the effects of six terpene-containing EOs on permeation of 

estradiol through hairless mouse skin. Therefore, in vitro tests with cajeput, cardamom, melissa, 

myrte, niaouli, and orange oil (all 10% wt/wt concentration in propylene glycol) have been carried 

out. Niaouli oil was found as the best permeation promoter for estradiol. Tests with its single main 

components 1,8-cineole, a-pinene, a-terpineol, and d-limonene (all 10% wt/wt concentration in 

propylene glycol) showed that the whole niaouli oil was a better activity promoter than the single 

compounds. These data demonstrate complex terpene mixtures to be potent transdermal penetration 

enhancers for moderately lipophilic drugs like estradiol. 



Different terpene-containing EOs have been investigated for their enhancing effect in the percutaneous 

absorption of trazodone hydrochloride through mouse epidermis (Das et al., 2006). Fennel 

oil, eucalyptus oil, citronella oil, and mentha oil were applied on the skin membrane in the transdermal 

device, as a pretreatment or both using Keshary–Chien diffusion cells and constantly stirring 

saline phosphate buffer of pH 7.4 at 37 ± 1°C as receptor phase. Pretreatment of the skin with EOs 

increased the flux values of trazodone hydrochloride compared with the values obtained when the 

same EOs were included in the transdermal devices. The percutaneous penetration flux was 

increased with skin permeation by 10% EOs in the following order: fennel oil > eucalyptus 

oil > citronella oil > mentha oil. The quantity of trazodone hydrochloride kept in the skin was very 

similar for all EOs and much higher than in control group. 

An in vitro study about Australian TTO was made by Reichling et al. (2006). The aim of the 

study was to investigate the penetration enhancement of terpinen-4-ol, the main compound of TTO, 

using Franz diffusion cells with heat seperated human epidermis and infinite dosing conditions. 

The three semisolid preparations with 5% TTO showed the following flux values: semisolid oil-inwater 

(O/W) emulsion (0.067 mL/cm2/h) > white petrolatum (0.051 mL/cm2/h) > ambiphilic cream 

(0.022 mL/cm2/h). Because of the lower content of terpinen-4-ol, the flux values were significantly 

reduced compared to native TTO (0.26 mL/cm2/h). The papp values for native TTO 

(1.62 ± 0.12 × 10 −7 cm/s) and ambiphilic cream were comparable (2.74 ± 0.06 × 10 −7 cm/s), whereas 

with white petrolatum (6.36 ± 0.21 × 10 −7 cm/s) and semisolid O/W emulsion (8.41 ± 0.15 × 10 −7 cm/s) 

higher values indicated a penetration enhancement. Between permea tion and liberation there was 

no relationship observed (Table 9.2). The stratum corneum absorption and retention of linalool and 

terpinen-4-ol was investigated by Cal and Krzyzaniak (2006). Both monoterpenes were applied to 

eight human subjects as oily solution or as carbomeric hydrogel. The stratum corneum absorption 

after application of carbomeric hydrogel was better than that achieved with an oily solution. Two 

mechanisms of elimination from the stratum corneum were observed: evaporation from the outer 

layer and drainage of the stratum corneum reservoir via penetration into dermis. The retention of 

the monoterpenes in the stratum corneum during the elimination phase was a steady state between 

6 and 13 mg/cm2. 

Also linalool alone, one of the most prominent monoterpene alcohols, is used in many dermal 

preparations as penetration enhancer. In a series of in vitro studies it was shown that linalool 

enhanced its own penetration (Cal and Sznitowska, 2003) as well as the absorption of other therapeutics, 

such as haloperidol (Vaddi et al., 2002a, 2002b), metoperidol (Komuru et al., 1999), propanolol 

hydrochloride (Kunta et al., 1997), and transcutol (Ceschel et al., 2000). Cal (2005) showed in 

another in vitro study the influence of linalool on the absorption and elimination kinetics and was 

able to prove that this monoterpene alcohol furnished the highest absorption ratio compared to an 

oily solution or an O/W emulsion. 

Wang, L.H. et al. (2008) reported on enhancer effects on human skin penetration of aminophylline 

from cream formulations and investigated for this report four EOs, namely rosemary, ylang, 

lilac, and peppermint oil compared with three plant oils, the liquid wax jojoba oil, and the fatty oils 

TABLE 9.2 

Flux Values and Papp Values of the Three Semisolid Preparations 

of Australian TTO 5% 

Flux Values (μL/cm²/h) 

Papp Values (cm/s) 

Native TTO 0.26 1.62 ± 0.12 × 10 −7 

Ambiphilic cream 0.022 2.74 ± 0.06 × 10 −7 

Semisolid O/W emulsion 0.067 8.41 ± 0.15 × 10 −7 

White petrolatum 0.051 6.36 ± 0.21 × 10 −7


of corn germs and olives. In this study the EOs were less effective in their penetration enhancement 

than the three plant oils. The effect of penetration enhancers on permeation kinetics of nitrendipine 

through two different skin models was evaluated by Mittal et al. (2008) and also this author group 

found that the used EOs (thyme oil, palmarosa oil, petit grain oil, and basil oil) were inferior in their 

penetration enhancement effects to oleic acid but superior to a lot of other common permeation 

enhancers, such as sodium lauryl sulfate, myristic acid, lauric acid, Tween 80, or Span 80. In contrast 

to these two reports Jain et al. (2008) found that basil oil is a promising penetration enhancer 

for improved drug delivery of labetolol. The effect of clove oil on the transdermal delivery of ibuprofen 

in the rabbit in vitro and in vivo methods was investigated by Shen et al. (2007). The in vitro 

results indicated a significant penetration enhancement effect of the clove oil whereas the in vivo 

results showed a weaker enhancement. The good transdermal delivery of ibuprofen from the essential 

clove oil could be attributed to the principal constituents eugenol and acetyl eugenol. 

9.1.6 ANTIOXIDATIVE PROPERTIES* 

Free radicals are aggressive, unstable, and highly reactive atoms or compounds because of their 

single electron. They attack other molecules to reach a steady stage, thereby changing their properties 

and making disorders inside possible. Free radicals result from products from different metabolic 

activities. A large number occur because of smog, nitrogen oxides, ozone, cigarette smoke, and 

toxic heavy metal. Also chemicals such as organic solvents, halogenated hydrocarbons, pesticides, 

and cytostatic drugs cause a high number of free radicals. If a lot of energy is built or has to be supplied, 

such as sporty high-performance, extreme endurance sports, sunbathing, solarium, exposure, 

x-rays, UV radiation, pyrexia, or infections, they are also massively produced (Schehl and Schroth, 

2004). Preferred for attack are nucleic acids of the DNA and RNA, proteins, and especially polyunsaturated 

fatty acids of the membrane lipids. To protect the body’s own structure from damages, 

all aerobe living cells use enzymatic and nonenzymatic mechanisms. Scavangers are able to yield 

electrons and so they dispose free radicals. Also, enzymes such as superoxide dismutase (SOD), 

catalase, and glutathione peroxidase are very important in protection mechanisms (Eckert et al., 

2006). Oxidative and antioxidative processes should keep the balance. If the balance is in benefit for 

the oxidative processes, it is called “oxidative stress.” 

9.1.6.1 Reactive Oxygen Species 

Basically, oxygen radicals (Table 9.3) are built by one-electron reduction in the context of autoxidation 

of cell-mediated compounds. The superoxide radical arises from autoxidation of hydroquinone, 

flavine, hemoglobin, glutathione, and other mercaptans and also from UV light, x-rays, or gamma 

rays. Two-electron reduction yields hydrogen peroxide. The very reactive hydroxyl radical is built 

by three-electron reduction, which is mostly catalyzed by metal ion (Löffler and Petrides, 1998). A 

lot of other oxygen radicals are accumulated by secondary reactions because they damage the 

biomolecules, ROS participate in different diseases, mainly cancer, aging, diabetes mellitus, and 

atherosclerosis. 

9.1.6.2 Antioxidants 

Antioxidants are substances that are able to protect organisms from oxidative stress. A distinction 

is drawn between three types of antioxidants: enzymatic antioxidants, nonenzymatic antioxidants, 

and repair enzymes. 

Well-known naturally occurring antioxidants are vitamin C (ascorbic acid), which are contained 

in many citrus fruits, or on the other hand members of vitamin E family, which appear for example 

in nuts and sunflower seeds. Also b-carotene and lycopine, which also belong to the family of carotenoides, 

are further examples of natural antioxidants. On the other hand, there are many synthetic 

* Scheurecker, M., 2007. Part of her master thesis, University of Vienna.


TABLE 9.3 

Reactive Oxygen Species 

Species 

Name 

O2 -• Superoxide radical 

HO 

• 

2 Perhydroxyl 

H 2 O 2 

Hydrogen peroxide 

HO • Hydroxyl radical 

RO • R-oxyl radical 

ROO • R-dioxyl radical 

substances such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and the 

water-soluble vitamin E derivative troxol (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) 

noted for their antioxidative activity. Different studies suspect the synthetic antioxidants to cause 

different diseases, so there has been much interest in the antioxidative activity of naturally occurring 

substances (Salehi et al., 2005). 

To the group of enzymatic antioxidants belongs the manganese- or zinc-containing SOD, the 

selenium-containing glutathione peroxidase, and the iron-containing catalase. Their capacity is 

addicted to adequate trace elements and minerals (Schehl et al., 2004). Nonenzymatic antioxidants 

have to be admitted with food or substitution, for example, a-tocopherol, l-ascorbic acid, b-carotene, 

and secondary ingredients of plants. Repair enzymes delete damaged molecules and substitute them. 

9.1.7 TEST METHODS 

9.1.7.1 Free Radical Scavenging Assay 

This spectrophotometric assay uses the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) as 

a reagent (Yadegarinia et al., 2006). The model of scavenging stable DPPH-free radicals can be used 

to evaluate the antioxidative activities in a relatively short time (Conforti et al., 2006). The samples 

are able to reduce the stable free DPPH radical to 1,1-diphenyl-2-picrylhydrazyl that is yellow 

colored. The hydrogen or electron donation abilities of the samples are measured by means of the 

decrease of the absorbance resulting in a color change from purple to yellow (Gutierrez et al., 2006). 

Another procedure can be applied for an EO. A dilution of the EO in toluene is applied onto a thinlayer 

chromatography (TLC) plate and toluene-ethyl-acetate is used as a developer (Sökmen et al., 

2004a). The plates are sprayed with 0.4 mM DPPH in methanol. The active compounds were 

detected as yellow spots on a purple background. Only those compounds, which changed the color 

within 30 min, are taken as a positive result. 

9.1.7.2 β-Carotene Bleaching Test 

The lipid peroxidation inhibitory activities of EOs are assessed by the b-carotene bleaching 

tests (Yadegarinia et al., 2006). In this method, the ability to minimize the coupled oxidation of 

b-carotene and linoleic acid is measured with a photospectrometer. The reaction with radicals shows 

a change in this orange color. The b-carotene bleaching test shows better results than the DPPH 

assay because it is more specialized in lipophilic compounds. The test is important in the food 

industry because the test medium is an emulsion, which is near to the situation in food, therefore 

allowable alternatives to synthetic antioxidants can be found. An only qualitative assertion uses the 

TLC procedure. A sample of the EOs is applied onto a TLC plate and is sprayed with b-carotene 

and linoleic acid. Afterwards, the plate is abandoned to the daylight for 45 min. Zones with constant 

yellow colors show an antioxidative activity of the component (Guerrini et al., 2006).


9.1.7.3 Deoxyribose Assay 

This assay is used for determining the scavenging activity on the hydroxyl radical. The pure EO is 

applied in different concentrations (Dordević et al., 2006). The competition between deoxyribose 

and the sample about hydroxyl radicals that are engendered by an Fe 3+ /EDTA/H 2 O 2 system is measured. 

The radicals were formed to attack the deoxyribose and they are detected by their ability to 

degrade 2-deoxy-2-ribose into fragments. These degradation products generate with 2-thiobarbituric 

acid (TBA) at a low pH and upon heating pink chromogens. The TBA-reactive substances could 

be determined spectrophotometrically at 532 nm. So the damage of 2-deoxy-2-ribose by the radicals 

is detected with the aid of the TBA assay. 

9.1.7.4 TBA Test 

This assay is basically used to appoint the lipid oxidation. It is an older test for receiving the oxidation 

status of fats spectrophotometrically. Thereby the aldehydes, which are generated by the autoxidation 

of unsaturated fatty acids, are converted into red or yellow colorimeters with TBA. But it is 

also used for the determination of the potency of antioxidants with thiobarbituric acid reactive substances 

(TBARS). Thereby the antioxidant activity is measured by the inhibition of the lipid oxidation. 

It concerns the spectrophotometric detection of malonic aldehyde, one of the secondary lipid 

peroxidation products, which generates a pink pigment with TBA (Ruberto et al., 2000; Varda-Ünlü 

et al., 2003). 

9.1.7.5 Xanthine–Xanthine Oxidase Assay 

Superoxide radicals are produced by a xanthine–xanthine oxidase system. Xanthine is able to generate 

O 2 

• 

and H 2 O 2 by using xanthine oxidase as a substrate. The superoxide radicals are able to 

reduce the yellow colored nitro triazolium blue (NTB) to the blue formazan, which is used for monitoring 

the reaction. The superoxide anions are measured spectrophotometrically. This test method 

was developed to explore the reaction between antioxidants and O 2• . So the inhibition of the superoxide 

reductase is a mark for the ability of antioxidative activity. 

9.1.7.6 Linoleic Acid Assay 

This test system was developed to determine the ability of substances to inhibit the generation of 

hydroxy peroxides at the early stages of the oxidation of linoleic acid, as well as for its inhibitory 

potential after the formation of secondary oxidized products such as aldehydes, ketones, or hydrocarbons 

(Jirovetz et al., 2006). Either the oxidation of linoleic acid is monitored by measuring the values 

of conjugated dienes or TBARS spectrophotometrically or hydroperoxy-octadeca-dienoic acid isomers 

(HOPES) generated during the oxidation are measured ( Marongiu et al., 2004). 

The biological features of Mentha piperita L. (peppermint, Lamiaceae) oil and Myrtus communis 

L. (myrtle, Myrtaceae) oil from Iran were studied by Yadegarinia et al. (2006). One of the main 

constituents of the EO of Myrtus communis is 1,8-cineole (also called eucalyptol), which showed 

the most powerful DPPH radical scavenging activity. Myrtle oil contains about 18% 1,8-cineole and 

showed a scavenging activity of 3.5% upon reduction of the DPPH radical to the neutral DPPH-H 

form. Better results yielded the b-carotene bleaching test. Also the EO of Mentha aquatica (water 

mint) contains up to 14% 1,8-cineole and proved itself in the DPPH assay to be an acceptably radical 

scavenger and the most active compound in the oil. With Mentha communis oil comprising 21.5% 

limonene, an antioxidative activity was assessed with about 43% in contrast to the reference oil of 

Thymus porlock (Lamiaceae) having an efficiency of 77% (Mimica-Dukic et al., 2003). Also a-terpinene, 

a constituent of the EO of Mentha piperita (about 20%), showed in both test procedures a 

scavenging activity lower than that of the standard found with the EO of Thymus porlock, but 

similar to the efficiency of the synthetic antioxidant trolox (23.5% versus 28.3%). Another main 

compound of this oil is b-caryophyllene with about 8%. Mentha piperita oil attained a scavenging 

power of 23.5% and is also able to inhibit the lipid oxidation, which was determined in the b-carotene 

bleaching test. The results of the standard and the EO were correlated with the results from the


DPHH assay. The peppermint oil shows a lipid peroxidation inhibition of 50% compared with 77% 

of the standard. 

Sesuvium portulacastrum (sea purslane, Ficoidaceae) was collected in the northern, western, and 

central part of Zimbabwe to determine the chemical activity of the essential leaf oils, which showed 

besides an antibacterial and antifungal also a significant antioxidative activity (Magwa et al., 2006). 

Sea purslane is used by the traditional healers in Southern Africa to treat various infections and 

kidney problems. The secondary metabolites from this plant species have a great potential as substitutes 

for synthetic raw materials in food, perfumery, cosmetic, and pharmaceutical industries. 

Thus, the composition and the biological activities of the EO of this Ficoidaceae were studied. One 

of the major chemical compounds is 1,8-cineole (6.8%). The antioxidative testing was carried out by 

a modified b-carotene bleaching test. The background of these test methods is that b-carotene is a 

yellow antioxidant, which becomes colorless when it encounters light or oxygen. With the attendance 

of another antioxidant, which is not so sensitive to light or oxygen, the yellow color of the 

b-carotene could continue for some time. Retention of the yellow areas around the test compounds 

shows an antioxidative activity. In comparison to the positive control (ascorbic acid) with a diameter 

of 27 mm, the EO showed a 15.9-mm zone of color retention. Mainly responsible to the antioxidative 

activity besides this bicyclic ether is the content of 2-b-pinene (13.6%), a-pinene (14%), 

limonene (6.4%), and ocimene, a-terpinolene, and camphene, which also belong to the group of 

main compounds of the EO of Sesuvium portulacastrum. 

Thymus (thyme, Lamiaceae) is a very big genus, to which more than 300 evergreen species 

belong. All of them are well-known aromatic and medical plants and also the oil of different species 

are used against various diseases. So, many species of this genus were tested for their biological 

activities inclusive the antioxidative properties of the EOs and their constituents. The EOs of Thymus 

caespititius (Azoricus thyme, Lamiaceae), Thymus camphoratus (camphor thyme), and Thymus 

mastichina (mastic thyme) show in different investigations an antioxidative activity, which is comparable 

to the action of a-tocopherol, a well-known naturally occurring antioxidant (Miguel et al., 

2003). One reason for this capacity could be that the EO of all three species contains a high concentration 

of 1,8-cineole. Because species of the genus Thymus (thyme) are widely used as medicinal 

plants and spices, the antioxidative activity of the EO of Thymus pectinatus comprising up to 16% 

g-terpinene was determined by multifarious test systems. To detect the hydroxyl scavenging activity 

a deoxyribose assay was used, a DPPH assay was arranged, and the inhibition of lipid peroxidation 

formation by a TBA assay was measured. Fifty percent of the free DPPH radicals were scavenged 

by the EO, which is a stronger antioxidative activity than that of the used standards (BHT, curcumin, 

and ascorbic acid). Furthermore, the EO showed an inhibitory effect in the deoxyribose assay, and 

comparable results emanated from the TBA assay, where the oil had an IC 50 value similar to the 

activity of BHT. Finally, also the EO of Thymus zygis (sauce thyme) which contains among other 

monoterpenes p-cymene showed a dose-dependent antioxidative activity (Dorman and Deans, 2004). 

Furthermore, the above described species, Thymus pectinatus, is characterized by a very high content 

of the phenolic compound thymol. Therefore, two fractions of this EO, characterized by a high 

content of thymol (95.5% and 80.7%, respectively), were studied using four different test methods to 

assess their antioxidative activity. Fifty percent of the free radicals in the test with DPPH were scavenged 

by 0.36 ± 0.10 mg/mL of the EO. This is a stronger inhibition as the controls afford. Then 

the deoxyribose assay was used to identify the inhibition of the degradation of hydroxyl radicals by 

the EO and its main compounds. The investigation showed that thymol has an IC 50 value of 

0.90 ± 0.05 mg/mL and the EO an IC 50 value of 1.40 ± 0.03 mg/mL. To prove the inhibition of the 

lipid peroxidation, also the TBA method was used and afforded an IC 50 value of 9.50 ± 0.02 mg/mL 

for the EO. Also in this case the EO showed a better protection than the control substances BHT and 

curcumin, so to say the two major compounds, thymol and carvacrol, show a very strong antioxidative 

activity. Then the EO of another thyme species with a thymol content of about 31%, namely 

Thymus eigii, was studied by the DPPH and the b-carotene bleaching test (Tepe et al., 2004). For 

the rapid screening DPPH on TLC was applied. Compendiously after the plate had been sprayed


three spots appeared, which were identified as thymol, carvacrol, and a-terpineol. Thus, an antioxidative 

activity of this EO was established as well. Also in the volatile extract of Thymus vulgaris 

(common thyme), thymol dominates with 72%. In a test system, where the inhibition of hexane 

oxidation is used to determine the antioxidative activity, thymol shows one of the strongest activities. 

In a concentration of 10 mg/mL, the capacity to decrease the oxidation is equal to the activity 

of BHT and a-tocopherol. These two, well-known, antioxidants inhibit the hexane oxidation up to 

89% and 99% in a concentration of 5 mg/mL over 30 days. Also carvacrol, an isomer of thymol and 

existing in a concentration of about 6% in this oil, is able to hinder the oxidation of hexane by 

95–99% at 5 mg/mL over a period of 30 days. This is comparable with the activity of BHT and 

a-tocopherol (Randonic et al., 2003; Sacchetti et al., 2004; Faleiro et al., 2005). These findings 

could be confirmed by a very recent study of Chizzola et al. (2008). The antioxidative activity 

depends on the concentration of the phenolic constituents thymol and/or carvacrol. 

Thymol as well as carvacrol protects low-density lipoprotein (LDL) from oxidation (Pearson 

et al., 1997), and this antioxidative activity is dependent on the concentration: below 1.25 mM no 

effect was detected, whereas at a concentration between 2.5 and 5.0 mM thymol offers a very strong 

antioxidative capacity. Finally, the EOs of 15 different wild grown Thymus species show the ability 

to delay lard becoming rancid, because of the inhibition of lipid oxidation. The value of generated 

peroxides was measured to detect the antioxidative activity. Therefore, variable concentrations of 

the EO were added to lard and each mixture stored at 60ºC. In regular time intervals samples were 

taken and the peroxide amount was determined. As standards BHT, BHA, and thymol were used. 

A “thymol- and a thymol/carvacrol group” were able to keep the peroxide value low for a period of 

7 days. The EOs of Thymus serpyllus (creeping thyme) and Thymus spathulifolius comprise high 

contents of carvacrol (58% and 30% respectively) and exhibit both an antioxidative activity near to 

BHT. Another species is Thymus capitata (Corido thyme), with an amount of 79% carvacrol, and 

shows an antioxidative activity assessed by the TBA assay in variable concentrations. There is no 

difference at the concentration of 1000 mg/L between the capacity of the oil and the control BHT, 

but the antioxidative properties are better than BHA and a-tocopherol. In a micellar model system, 

where the decrease of just generated conjugated dienes is used to determine the antioxidative ability, 

Thymus capitata oil showed an antioxidative index of 96% and proved a good protective activity in 

the primary lipid oxidation. The oil is better effective against lipid oxidation and in higher concentrations 

better than BHA and a-tocopherol. The addition of a radical inducer reduced the antioxidative 

activity of the EO. Carvacrol is capable to prevent up to 96% the primary step of lipid oxidation. 

Also Thymus pectinatus EO contains carvacrol as one of the main compound and shows a strong 

antioxidative activity. Fifty percent inhibition of lipid peroxidation (detected by the TBA assay) was 

attained at a concentration of 5.2 mg/mL carvacrol (Vardar-Ünlü et al., 2003; Miguel et al., 2003; 

Hazzit et al., 2006). 

The EO of Ziziphora clinopodioides ssp. rigida (blue mint bush) was isolated by hydrodistillation 

of the dried aerial parts, which was collected during the anthesis. The main compounds are 

thymol and 1,8-cineole with a content of 8% and 2.7%, respectively. Different extracts were tested by 

the DPPH assay to determine the antioxidative activity and showed that the free radical scavenging 

activity of the menthol extract was superior to all other extracts. Polar extracts exhibited stronger 

antioxidant activity than nonpolar extracts (Salehi et al., 2005). 

Many species of the genus Artemisia (wormwood, Asteraceae) are used as spices, for alcoholic 

drinks and also in the folk and traditional medicine. The chemical compounds and the antioxidative 

activity of the EOs isolated from the aerial parts of Artemisia absinthium (vermouth), Artemisia 

santonicum (sea wormwood), and Artemisia spicigera (sluggish wormwood) were investigated 

(Kordali et al., 2005). The analysis of the EO of Artemisia santonicum and Artemisia spicigera 

showed two main components, namely 1,8-cineole and camphor. In addition, it is noticed that 

the EO of these two species contain no thujone derivates in contrast to Artemisia absinthium. 

Earlier studies have also shown that 1,8-cineole and camphor are main components of the EO of 

some Artemisia species. The antioxidative activity of the EO of Artemisia santonicum and


Artemisia spicigera was analyzed by the thiocyanate method and a high antioxidative activity 

was found. With the thiocyanate test and the DPPH radical scavenging assay, the high antioxidative 

activity of Artemisia santonicum EO is ensured. On account of their adequate antioxidative activity, 

Artemisia santonicum and Artemisia spicigera could possibly be used in the liqueur-making industry, 

because they do not include thujone derivates. The antioxidative activity of the EO from 

another wormwood species, namely Artemisia molinieri (mugwort), was investigated by chemoluminescence. 

The main compounds of this EO are a-terpinene with 36.4% and then 1,4-cineole 

(


extraction, had a better activity in the assay than the EOs. These test scores are an important argument 

for the use of some plants of this genus in the traditional medicine (Grassmann et al., 2000). 

Also the EO of the fresh fruits by Xylopia aethiopica (Meleguetta pepper, Negro pepper, and 

African pepper tree, Anonaceae) is characterized by the occurrence of 1,8-cineole and b-pinene. 

But also EOs from the leaves (containing about 17% b-pinene and even 24.5% germacrene D), 

barks, stems, and roots are known and examined for their composition and antioxidative activities. 

The antioxidative activity of the EO from different parts of the plant was determined by the free 

radical scavenging assay with DPPH and the xanthine–xanthine assay. All tested samples were 

found to interact with the stable free radical DPPH in a time-dependent manner (Karioti et al., 

2004). The highest radical scavenging in the DPPH assay was measured with the EO of the fresh 

fruits (comprising about 9% germacrene D) with an interaction of 85.6%. Generally, all the EOs of 

the different parts possess the ability to scavenge free radicals. Also the xanthine–xanthine assay 

showed that the capability to reduce the superoxide radical is given. 

Plants of the genus Melaleuca (Tea tree, Myrtaceae) are rich in volatile oils. On this account, 

different species were investigated for their biological activity as well as for their antioxidative 

capacity. A GC-MS analysis revealed that 1,8-cineole is the major compound of the EO extracted 

from Melaleuca armillaris (33.9%). For the study 50 male albino rats were treated differently. 

Besides the control group the rats received multiple doses of the EO 3 times a week for 1 month. To 

evaluate the antioxidative activity the following estimations were carried out: SOD, vitamin C, catalase, 

glutathione, and lipid peroxides (Farag et al., 2004). An alternation of the antioxidant status 

induced by CCl 4 , as free radical inducer, before and after the administration of the EO was studied 

as well. The EO of Melaleuca armillaris increased the value of vitamin E and vitamin C. The same 

effect was given by the level of SOD and lipid peroxide compared to the control group. Only the 

level of catalase was decreased by the treatment of Melaleuca oil. TTO from Melaleuca alternifolia 

(Australian tea tree) is known for its wide spectrum of biological activities, therefore the antioxidative 

properties of TTO was assessed by various methods, such as the DPPH radical scavenging or 

the hexanal/hexanoic acid assay (Hyun-Jin et al., 2004). The crude EOs, as well as the most active 

fractions (fractions 5 and 6 after silica gel open column chromatography and C 18 -HPLC), were 

used for the investigation. The three major compounds in these fractions were g-terpinene (20.6%), 

a-terpinene (9.6%), and a-terpineol. A correlation between the scavenging activity and the concentration 

was emerged in the DPPH assay. At a concentration of 10 mM, the activity of a-terpinene 

with more than 80% was near to the scavenging capacity of BHT. Eighty-two percent activity was 

determined for a concentration of 180 mM which is also close to the synthetic antioxidant (85%). It 

was shown that a-terpinene had the strongest scavenging effect in this test system compared to the 

other two compounds. The second test system, which was performed, was the hexanal/hexanoic 

acid assay. In lower concentrations (30 and 90 mM) a-terpinene exhibited a strong inhibitory 

activity at first but it decreased extremely fast. The inhibitory effect increased with the arising 

concentration. a-Terpinene (180 mM) as well as g-terpinene had a blocking action of 65% over the 

30 days. It should be noticed that the antioxidative activity of BHT was stronger compared to any 

isolated compound of the TTO at lower concentrations. 

Vitamin E is a natural antioxidant, which occurs in the plasma red cells and tissues, disarms the 

free radicals, and anticipates the peroxidation of polyunsaturated fatty acids and phospholipids. 

Also vitamin C is one of the naturally occurring antioxidants, which actually increase the efficiency 

of vitamin E to avoid the lipid peroxidation. SOD protects the cells of hydrogen peroxide anion free 

radicals, furnishes the decrease of the catalase which decomposes H 2 O 2 , thus yielding a reduction 

of the oxidative process. This research shows that the EO of Melaleuca armillaris, which has 

1,8-cineole as the major chemical compound, can be used as a suppressor for free radicals and is 

able to avoid damages caused by oxidative stress generated by chemical or physical factors. 

Myrtol standardized and eucalyptus oil, which both contain about 70–80% 1,8-cineole, were 

examined in a Fenton system which is a very sensitive indicator for ROS such as a-keto-g-methiolbutyric 

acid (KMB) or 1-aminocylopropane-1-carboxylic acid (ACC). OH radicals are generated by


the reaction between hydrogen peroxide and Fe 2+ (Grassmann et al., 2000). The KMB is transformed 

into ethene, carbon dioxide, formiate, and dimethyldisulfide. The ethene can be detected in 

very low quantities by GC. Both oils prevent KMB before destruction and so no ethene can be 

detected. 

The EOs of citrus fruits (Rutaceae)—obtained upon pressing the peels—are also called “agrumen 

oils” and are characterized normally by a high content of the monoterpene hydrocarbon 

d-limonene which even after boiling the fruits remains in the peel in a substantial quantity. So, 

citrus fruits are a very interesting source for the occurrence of antioxidants. Twenty-six citrus fruits 

and their flavor compounds were investigated for an antioxidative activity by the thiocyanate test. 

The EO of Citrus sinesis Osbeck var. Sanguinea Tanaka form a Tarocco (Tarocco orange) which 

contains as major component limonene (84.5%) shows a very high antioxidative activity of about 

more than 90%. Also the oil of Citrus aurantium Linn. var. cyathifera Y. Tanaka (Dadai) exerts an 

antioxidative activity up to 19% but it was slightly weaker than that of the standard troxol, a watersoluble 

vitamin E derivative. Also all the citrus EOs show an antioxidative activity against linoleic 

acid peroxidation (Song et al., 2001). The scavenging activity of the authentic compounds was evaluated 

in the DPPH test and the activity from limonene was assessed from 8.8% to 16.5%, whereas 

g-terpinene showed that in this test a noticeable radical scavenging effect for g-terpinene was 84.7%, 

which is 3.5 times stronger as that of trolox. The results of the thiocyanate test reveal that the EOs 

and their flavor components can be used in the food industry to protect aliments of oxidation and to 

avoid lipid peroxidation. Correlating results are afforded by another investigation of 24 different 

citrus species and their authentic compounds. g-Terpinene showed also in the linoleic acid assay a 

very strong antioxidative activity, which manifests itself in a better peroxide value than trolox. The 

EOs obtained from Citrus yuko Hort. ex Tanaka (Yuko) and Citrus limon Brum. f. cv. Eureka 

(Lisbon lemon) contain an appreciable content of g-terpinene with 18.6% and 8.8%, respectively. 

Both citrus species offered a strong antioxidative activity of more than 90% due to the high amount 

of g-terpinene in the oils (Choi et al., 2000). Ao et al. (2008) studied whether EOs from Rutaceae 

can effectively scavenge singlet oxygen upon irradiation, using electron spin resonance and found 

that the investigated 12 oils (eight of them obtained by conventional expression, four by steam distillation) 

enhanced singlet oxygen production, whereby the content of limonene is made responsible 

for this result. However, two expressed oils and three oils obtained by steam distillation showed 

singlet oxygen scavening activity. 

The EO of Cuminum cyminum (cumin, Apiaceae) consists of 21.5% limonene which is able to 

abate the concentration of the DPPH free radical, although its efficiency is a bit lower than that of 

trolox. Since the EO is also able to decrease the lipid peroxidation, a b-carotene bleaching test was 

arranged. This assay furnished better results than the DPPH free radical scavenging test. The possibility 

for that could be the higher specificity of this test method for lipophilic compounds. a-Pinene 

is another major constituent of this oil and contributes as well to the antioxidative activity. The 

implication of these investigations is the fact that the EO of cumin is competent enough to neutralize 

free radicals and to protect unsaturated fatty acids of oxidation (Gachkar et al., 2007). 

Limonene is also one of the major compounds of the EO of Ocotea bofo Kunth (Lauraceae, “anis 

de arbol” in Ecuador, “moena rosa” in northern Peru or “pau de quiabo” in Brazil), (~5.0%), which 

was tested for its antioxidative activity by three different methods. The DPPH assay furnished a 

scavenging activity that was higher than that of the synthetic reference trolox, but lower than the 

activity of the natural and commercial EO reference Thymus vulgaris, whereas the b-carotene 

bleaching test showed that the EO is comparable to standards in the inhibition of oxidation. Another 

method was carried out to assess the antioxidative activity using a specific test kit of photochemistry. 

A light emission curve was recorded over 130 s, using inhibition as the parameter to evaluate the 

antioxidant potential (Guerrini et al., 2006). The activity was calculated by the integral under the 

curve and showed that Ocotea bofo EO here has a comparable scavenging activity to trolox. Besides, 

also the aromatic monoterpene p-cymene (about 5% in the EO) contributes to the antioxidative 

activity (Table 9.4).


TABLE 9.4 

Antioxidative Activity of Ocotea bofo EO Performed by DPPH and 

β-Carotene Bleaching Assays 

Inhibition % 

Sample 

DPPH 

β-Carotene 

Bleaching Test 

Photochemiluminescence 

(mmol of trolox/L) 

Ocotea bofo EO 64.23 ± 0.03 75.82 ± 0.04 3.14 ± 0.02 

Thymus vulgaris EO 75.64 ± 0.04 90.94 ± 0.05 0.34 ± 0.06 

BHA 84.35 ± 0.04 86.74 ± 0.04 

A-tocopherol 4.28 ± 0.5 

Trolox 94.44 ± 0.05 84.60 ± 0.04 3.94 ± 0.06 

Salvia (belonging to the family of Lamiaceae) EO is used in folk medicine all over the world and 

many studies were carried out to assess its constitution and biological activity. Three different species 

were collected in the southern part of Africa: Salvia stenophylla (blue mountain sage), Salvia 

repens (creeping sage), and Salvia runcinata (African sage) (Kamatou et al., 2005). Limonene is 

one of the major compounds of the EO from Salvia repens with 9.8%. Interestingly, in the other two 

species this monoterpene was absent. The antioxidant behavior of the EO and the phenolic composition 

of Rosmarinus offi cinalis (Lamiaceae) and Salvia fruticosa M., both collected in an island of 

the Ionian Sea (Greece) were investigated. The principal component of the EO was 1,8-cineole and 

flavonoids that of the methanolic extracts. The phenolic content correlates with the antioxidant 

activity (Papageorgiou et al., 2008). 

To determine the radical scavenging capacity, a modified DPPH test was used where the test 

tubes were analyzed with HPLC. Different concentrations were plated out in a 96-well plate with 

control wells containing dimethyl sulfoxide (DMSO). The decolorization was investigated by measuring 

the absorbance at 560 nm. Vitamin C provided as a positive control. The LC 50 value of Salvia 

repens EO (comprising about 22% b-caryophyllene) rests with more than 100.0 mg/mL or Salvia 

multicaulis which showed an IC 50 value of 17.8 mg/mL, two examples for a low scavenging activity 

(Erdemoglu et al., 2006). But the reason for this result could be that in this method a stable free radical 

is used while in other investigations unstable radicals were applied and there a better antioxidative 

activity was adopted. 

The EO of Crithmum maritimum (= Cachrys maritima, Apiaceae, rock samphire) comprises 

limonene and g-terpinene with an amount of 22.3% and 22.9%, respectively, as the major components. 

Two different test methods (TBA assay and a micellar model system where the antioxidative 

activity in different stages of the oxidative process of the lipid matrix was monitored) were used. 

Both assays explain the very high activity of this EO. In the TBA assay BHT and a-tocopherol were 

used as positive standards and the oil showed a better capacity than those substances. Comparable 

results were obtained by the micellar method system where the EO acts as a protector of the oxidation 

of linoleic acid and inhibits the formation of conjugated dienes (Ruberto et al., 2000). The 

modification of LDL by an oxidative process for instance can lead to atherosclerosis. Natural antioxidants 

such as b-carotene, ascorbic acid, a-tocopherol, EOs, and so on are able to protect LDL 

against this oxidative modification. g-Terpinene proved itself to be the strongest inhibitor of all used 

authentic compounds for the formation of TBARS in the Cu 2+ -induced lipid oxidation system 

(Grassmann et al., 2003). So, the addition of g-terpinene to food can possibly stop the oxidative 

modification of LDL and reduce the atherosclerosis risk. 

a-Pinene and a-terpineol are two of the main compounds of the EO from Juniperus procera 

(African juniper, Cupressaceae). The oil was studied for its antioxidative activity, because the aerial 

parts of this plant are used in traditional medicine against different diseases and ailments, for example,


ulcers, headaches, stomach disorders, intestinal worms, rheumatic pains, liver diseases, as an 

emmenagogue, and to heal wounds. To determine the antioxidative activity in vitro test methods 

were used: DPPH assay, deoxyribose assay, and the assay for nonenzymatic lipid peroxidation. The 

EO showed an IC 50 value of 14.9 mL/mL in the DPPH assay, a 50% inhibition in the deoxyribose 

assay at 0.4 mL/mL, and an IC 50 value of 0.20 mL/mL by inhibition of the lipid peroxidation. On the 

other hand, pure a-pinene showed an IC 50 value of 0.78 mL/mL and 50% of the lipid peroxidation 

were inhibited by 0.51 mL/mL. To determine the inhibition of nonenzymatic lipid peroxidation 

bovine liposomes, FeCl 3 and ascorbic acid were used. The generated aldehydes form pink compounds 

with TBA and were detected by measuring the absorbance at 532 nm. 

Eleven different EOs were investigated for their antioxidative activities. The oils extracted from 

Eucalyptus globulus (Eucalyptus, Myrtaceae), Pinus radiata (Monterey Pine, Pinaceae), Piper 

crassinervium (Piperaceae), and Psidium guajava (Guayava, Myrtaceae) contain 20%, 21.9%, 10%, 

and 29.5% a-pinene, respectively. Three different test methods were used to evaluate the antioxidative 

activities of these EOs: DPPH assay, b-carotene bleaching test, and photoluminescence. 

In the DPPH assay the EO from Piper crassinervium expressed an activity of 43.0 ± 0.30%, which 

was lower than that of the reference Thymus vulgaris EO, but comparable with the activity of trolox, 

whereas the other oils were almost ineffective. In the b-carotene bleaching test similar data were 

obtained. The EOs of Eucalyptus globulus and Piper crassinervium showed results between 66% 

and 49% inhibition. The photoluminescence test method is very rapid on the photo-induced autoxidation 

inhibition of luminol by antioxidants mediated from the radical anion superoxide (Sacchetti 

et al., 2005). With this method only the EO of Piper crassinervium showed an antioxidative activity, 

whereas the other oils were almost ineffective. 

One of the main compounds of the eucalyptus oil besides 1,8-cineole are the monoterpene hydrocarbons 

a-pinene (10–12%), p-cymene, and a-terpinene, and the monoterpene alcohol linalool. 

This oil is used to treat diseases of the respiratory tract in which ROS play an important role, so the 

antioxidative activity of eucalyptus oil was of interest. The results obtained by assessing this activity 

were compared with those of myrtle standardized oil of Myrtus communis (Myrtaceae), which 

is also used to combat infections of the respiratory tract. The antioxidative activity was determined 

by the Fenton test where the OH ∑ -radicals are built from H 2 O 2 , whereas Fe 2+ acts as the electron 

donator. The ROS reacts with the sensitive indicators KMB and ACC, which release a measurable 

signal (Grassmann et al., 2000). After an incubation time of 30 min at 37ºC, the generated ethene 

can be quantified by GC, because KMB dissociates into ethene, carbon dioxide, formiate, and 

dimethylsulfide. Both, the eucalyptus oil and the myrtle standardized oil were able to inhibit the 

generation of ethene in dependence on the concentration of the oils. In an in vitro system the EO of 

Eucalyptus globulus was investigated for its property to inhibit the autoxidation of linoleic acid and 

compared with the activity of BHT and showed very good protection for linoleic acid with an IC 50 

value of 7 mg. In another test the inhibition of the nonenzymatic lipid peroxidation was investigated. 

A solution of bovine brain extract and various concentrations of linalool were submitted to a TBA 

assay: Linalool caused a 50% inhibition at a concentration of 0.67 mL/mL. 

The EO, obtained from the leaves of Origanum syriacum L. (Syrian oregano, Lamiaceae), is a 

very popular Arab spice which is used as a herbal (traditional) medicament, flavor, fragrance, and 

for aromatherapy in form of bath, massage, steam inhalation, and vaporization (Alma et al., 2003), 

was investigated with regard to the antioxidative activity of its chemical compounds, for example, 

g-terpinene, p-cymene, and b-caryophyllene (about 28%, 16%, and 13% respectively) using the thiocyanate 

method, DPPH radical scavenging activity, and the reducing power. The latter property was 

determined in a concentration from 100 to 500 mg/L and compared with the reducing power of 

ascorbic acid. The EO was able to increase the absorbance but the reducing power is lower than that 

of the used standard. It should be noticed that the power of the EO increased with a higher concentration. 

Also in the DPPH assay the standard BHT showed a better radical scavenging than the EO. 

In addition, 500 mg/L EO scavenged only 17% DPPH. In comparison to that, BHT showed an activity 

of 82% in a concentration of 100 mg/L, again dependent on the concentration. The thiocyanate


test was carried out with concentrations of 20, 40, and 60 mg/L of the EO. Also here the activity 

increased with a higher concentration. The antioxidative activity was comparable to that of BHT. 

Even at low concentrations (20 mg/L) a high antioxidative activity was found. At least also the 

reducing power of the EO using potassium ferricyanide was assessed by measuring the absorbance 

at 700 nm; however, the EO attained only a reducing power of 0.77, compared with that of ascorbic 

acid with a value of 0.96. Because an increasing absorbance means a stronger reducing power, it was 

shown that in this case only a low reducing capacity could be recorded, as in the method with 

DPPH. The EO of Origanum fl oribundum comprises only 8.4% thymol, but about 30% of carvacrol. 

This oil was able to decrease the generation of TBARS in the TBA assay just as well as BHT, BHA, 

and a-tocopherol, in the absence and presence of a radical inducer. The DPPH radical activity of 

oregano EO in a menthol extract exists, because it is able to reduce the stable free radical DPPH 

with values of IC 50 ranging from 378 to 826 mg/L, however inferior to the capacity of BHA (Hazzit 

et al., 2006). Similar results also furnished the carvacrol-rich oil from Origanum glandulosum 

(Algerian oregano) and from Origanum acutidens (hops oregano). This EO was tested with the radical 

scavenging method using DPPH and the b-carotene/linoleic assay. In both test systems the oil 

exhibits an antioxidative activity. As shown in other investigations, the activity in the b-carotene/ 

linoleic assay was higher than that in the DPPH assay and is comparable with the ability of BHT, 

which was used as a positive control. 

Also the species oregano (Origanum vulgare) is characterized by a high content of thymol (33%) 

and carvacrol, as was shown by a GC-MS analysis of (oregano) EO. The property to inhibit the 

generation of malonic aldehyde in the first stage of lipid oxidation is determined by TBA test 

method. And the micellar model system is used to measure the decrease of conjugated dienes formed 

by linoleic acid spectrophotometrically at 234 nm. In a concentration of 640–800 mg/L the EO 

shows a protective activity, which is superior to the standard substances BHA and a-tocopherol but 

equal to BHT. When the amount of the EO is increased to 1000 mg/L, the antioxidative capacity is 

better than that of BHT (Faleiro et al., 2005). The meat of poultry is very sensitive for oxidative 

deterioration because the meat is rich in polyunsaturated fatty acids. Turkeys are more sensitive 

than chicken because they cannot store a-tocopherol in their tissues to the same extent. A study was 

carried out if a diet with the EO of Origanum vulgare ssp. hirtum (oregano) is able to degrade the 

susceptibility to lipid oxidation. Thirty 10-week-old female turkeys were divided into five groups. 

In the control group the animals were fed with a standard diet. The other turkeys were also fed with 

the same bush but containing different concentrations of oregano EO and oregano herbs: 5 g oregano 

herb/kg, 10 g oregano herb/kg, 100 mg oregano EO, and 200 mg EO. After 4 weeks all the turkeys 

were slaughtered and worked up. The breasts were tight, minced, and stored at 4°C over 9 days. At 

days 0, 3, 6, and 9 the concentration of malonic aldehyde was measured spectrophotometrically 

using the TBA assay. At each stage the control group showed the highest content of malonic aldehyde. 

On the third day the amount of malonic aldehyde increased in every group. The group with 

100 mg EO addition showed a lower concentration than the control group but a higher one as the 

group with 200 mg EO. On the sixth day the content of malonic aldehyde also increased and similar 

to the previous sample the group with 200 mg EO had the lowest content. Thus, it is shown that a 

diet with oregano EO can delay the deterioration induced by lipid oxidation (Botsoglou et al., 2003; 

Florou-Paneri et al., 2005). Responsible for this result is carvacrol, which is one of the main compounds 

of the oil and exerts also in other studies a strong antioxidative activity. In conclusion, these 

results prove that the EO of Origanum vulgare possesses protective properties in the first and second 

step of lipid peroxidation and can be used to replace synthetic antioxidants in the food industry 

or other areas. Another species, namely Origanum majorana L. from Albania, showed in the DPPH 

test a better antiradical activity than the phenolic compound thymol. This EO exhibited a scavenging 

effect on the hydroxyl radical OH as well and finally was also capable of antioxidant activity in 

a linoleic acid emulsion system where at a concentration of 0.05% it inhibited conjugated dienes 

formation by 50% and the generation of linoleic acid secondary oxidized products by about 80% 

(Schmidt et al., 2008).


One of the main compounds with about 23% found in the volatile oil from Trachyspermum ammi 

(ajwain, ajowan, omum, Apiaceae), which is a very popular aromatic plant in India and used for 

flavoring food as well as in the Ayurvedic medicine, is g-terpinene. Another main compound of the 

ajowan oil is p-cymene (about 31%), which contributes to the antispamodic and carminative properties 

as well. To determine the antioxidative activity of the EO a wide range of test methods were 

used. To simulate the different stages of lipid peroxidation three different test methods were exerted: 

the determination of peroxide values, a TBA assay, and the linoleic acid assay. The antioxidative 

activity of the oil and of an acetone extract was compared to that of BHT, BHA, and of a control: 

a sample with crude linseed oil. It could be shown that the oil was a better inhibitor than the synthetic 

antioxidants (Singh et al., 2004). Due to the fact that all results of the different test methods 

correlate, it can be concluded that at a concentration of 200 ppm the inhibitor activity can be put into 

the following order: acetone extract > EO > BHA > BHT > control. 

Terpinolene was identified as one of the main compounds (about 6%) in the EO from Curcuma 

longa (turmeric, Zingiberaceae). In the course of an investigation of 11 different EOs also turmeric 

oil was determined for its antioxidative activity. The EO of Curcuma longa showed a noteworthy 

scavenging activity of about 62%, an antioxidative activity twice of that of trolox but only marginally 

lower than that of reference oil from Thymus vulgaris. Also the b-carotene bleaching test 

furnished comparable results, namely an inhibition activity of 72% versus 91% of Thymus vulgaris 

oil and 87% of BHA. This prevention of oxidation was also shown by the photoluminescence test 

method that is based on the photo-induced autoxidation inhibition of luminol by antioxidants mediated 

from the radical anion superoxide (Sacchetti et al., 2004). The EO of Curcuma longa showed 

a noticeable inhibition activity of about 28 mmol trolox/L. 

The protective effect of terpinolene with reference to the lipoproteins of human blood and compared 

to that of the well-known antioxidative substances, such as a-tocopherol and b-carotene, was 

investigated. The oxidative modification of LDL, which was obtained from the blood of healthy 

volunteers, can be detected with the use of the increasing absorbance at 234 nm. An elongation of 

the time until rapid extinction (lag-phase) exhibits an antioxidative activity. The longer the lag-phase 

lasts, the better is the antioxidative capacity. Similar to other test systems with other EOs, the antioxidative 

capacity is dependent on the concentration. In that case a higher concentration of terpinolene 

means that the LDL particles are better loaded with terpinolene molecules. The result of this 

investigation proved that the protective and thus the antioxidative activity of terpinolene is only a bit 

weaker than that of the most common antioxidant a-tocopherol. 

In the course of the determination of the antioxidative activity of the EOs obtained from 34 different 

citrus species and the main compounds of the oils, among them terpinolene, were investigated 

for their radical scavenging activity in a DPPH test system. Terpinolene offers a scavenging power 

of 87%, which was much higher than the antioxidative activity of the standard trolox. Terpinolene 

also showed a relative lipid peroxidation rate of 18%, which is an indication of a superior antioxidative 

activity, because the relative lipid peroxidation rate offered by the well-known and often used 

antioxidant a-tocopherol was about 30%. 

The leaves, flowers, and stems of Satureja hortensis (summer savory, Lamiaceae), a common 

plant widely spread in Turkey, are used as tea or as addition to foods on account of the aroma and 

the flavor. As a medical plant it is known for its antispasmodic, antidiarrheal, antioxidant, sedative, 

and antimicrobial properties. Also this EO was investigated for its antioxidative properties. The 

GC-MS analysis showed that besides 9% p-cymene, carvacrol and thymol are the main compounds 

of about 22 constituents of the oil. They occur at a ratio of approximately 1:1, which is representative 

for the genus Satureja, namely 29% of thymol and 27% of carvacrol. In a linoleic acid test system 

the EO showed an inhibition activity of 95%, this is an indicator for a strong antioxidative activity 

because the control BHT attained an inhibition of 96% (Güllüce et al., 2003). Thymol is one of the 

main components of the EO from Satureja montana L., ssp. montana (savory) and also one of the 

glycosidically bound volatile aglycones that were found. The EO with 45% thymol shows a very 

strong antioxidative capacity that was a bit lower than the standards, BHT, and a-tocopherol. The


activity of the isolated glycosides is similar to that of the EO. Identification of the volatile aglycone 

shows a value of 2.5% thymol (Randonic et al., 2003). To evaluate the antioxidative properties of 

ingredients, EO and glycosides, the b-carotene bleaching test was chosen. The DPPH assay yields a 

weaker result than the b-carotene bleaching test, in which the EO had an efficiency of inhibition 

about 95%. Compared to the inhibition from BHT of 96%, this result shows a high antioxidative 

activity of the EO, due to the high content of thymol and carvacrol. 

Since Nigella sativa (black cumin, devil in the bush, fennel flower, Ranunculaceae) seeds are 

used for the treatment of inflammations it was reasonable to investigate the ability of the volatile oil 

to act as a radical scavenger (Burits and Bucar, 2000). Experiments have shown that Nigella oil and 

the main compounds are able to inhibit in liposomes the nonenzymatic lipid peroxidation. Besides 

carvacrol (6–12%) thymoquinone, p-cymene is one of the major constituents of the oil (7–15%). In 

the free radical DPPH test the EO showed to be a very weak scavenger for radicals (IC 50 value: 

460.0 mg/ml). Totally contrary results were furnished by the TBA assay: the volatile oil of Nigella 

seeds exhibited a very strong inhibition capacity, namely at a concentration of 0.0011 mg/ml already 

50% of the lipid peroxidation could be stopped. 

As to the next oil a scientific confusion must be mentioned: the common name black caraway 

belongs to the seeds of Nigella sativa (Ranunculaceae) and not to the Apiaceae plant Carum. 

Therefore, Carum nigrum is a wrong botanical name, even if the seeds of N. sativa are black and 

resemble to the seeds of Carum carvi. The confusion that has been created by the authors of the 

articles (Singh et al., 2006) by using possibly local justified terms, but nevertheless wrong botanical 

names still persists. 

The EO of Carum nigrum (black caraway) comprising, for example, thymol (~19%), b-caryophyllene 

(~8%), and germacrene D (~21%), and its oleoresin were able to scavenge free radicals in the 

DPPH assay with an effect of 41–71% and 50–80%, respectively. This can be compared with the 

efficiency of BHT and BHA. However, this activity could only be observed using a high concentration 

of the EO and oleoresin not in lower ones. A good antioxidative activity was assessed also by the 

other tests: the linoleic acid assay exhibited that the EO and oleoresin are able to decrease the rate of 

peroxide during the incubation time. The deoxyribose assay proved that both EO and oleoresin are 

able to prevent the formation of hydrogen peroxides dependently of the concentration. Also the 

chelating effect with iron was screened and the absorbance of the mixture determined at 485 nm 

(Singh et al., 2006). A mixture of this EO, the oleoresin, and crude mustard oil were studied to assess 

the generated peroxides as well as the TBA value was measured in order to determine secondary 

products of the oxidation. This mixture showed a clear antioxidative activity, better than the control, 

and in the linoleic system were also able to keep the amount of the peroxides, formed by the oxidation 

of linoleic acid, very low in comparison to the standard antioxidants BHA and BHT. Finally, the 

rather moderate chelating capacity could—nevertheless—be beneficial for the food industry because 

ferrous ions are the most effective pro-oxidants in food systems (Singh et al., 2006). 

The EO of Monarda citriodora var. citriodora (lemon bee balm, Lamiaceae) contains about 10% 

p-cymene. The oil was determined for its antioxidative activity in two different in vitro test systems. 

In one the oxidation of lipids was induced by Fe 2+ and in the other assay AAPH was used. Oxygen 

in the presence of iron(II) generates superoxide anion radicals. In the aqueous phase AAPH undergoes 

steady-state decomposition into carbon-centered free radicals (Dorman and Deans, 2004). The 

oil of Monarda citriodora was active oil at concentrations of 50 and 100 ppm. In the test system 

where the radicals were built from AAPH, the oil of Monarda citriodora showed a concentrationdependent 

pattern of antioxidative activity. Monarda citriodora showed a better activity at 10 ppm 

than the EO of Thymus zygis and an equal inhibition power like Origanum vulgare but the activity 

was lower than that of the standards BHT and BHA. The EO obtained from the stem with leaves and 

the flowers of Monarda didyma L. (golden balm or honey balm, Lamiaceae) contains p-cymene, 

10.5% in the stem with leaves and 9.7% in the flowers. The EO was studied by two different test 

methods to determine its antioxidative activity. The EO showed a good free radical scavenging


activity in the DPPH assay. Also the properties to inhibit the lipid peroxidation in the 5-lipogenase 

test system furnished a strong inhibition that is similar to BHT. 

In some Mediterranean countries Thymbra spicata (spiked thyme, Lamiaceae) is applied as spice 

for different meals and as herbal tea. Two of the main compounds of the EO from this plant are 

carvacrol (86%) and thymol (4%). The investigation of different natural antioxidants becomes more 

and more interesting in order to attain more safety in the food industry. The lipid oxidation may be 

one of the reasons for different changes in the quality of meat and thus the addition of Thymus 

spicata EO has a positive influence on its quality. As material of examination served the Turkish 

meal sucuk, which is prepared of lamb, lamb tail fat, beef, salt, sugar, clean dry garlic spices, and 

olive oil. During ripening on the days 2, 4, 6, 8, 10, 13, and 15, a sample was taken and different 

parameters were determined, among them was also TBARS. They were detected spectrophotometrically 

by measuring the absorbance at 538 nm. During the first 8 days the TBARS increased 

from 0.18 to 1.14 mg/kg. The addition of Thymus spicata EO reduced the value of TBARS more 

than BHT. This result shows that the EO from Thymus spicata exerts a safety effect on the quality 

of the meat and thus can be used as a natural antioxidant in the food industry. The EO of Thymbra 

capitata (conehead thyme, Lamiaceae) collected in Portugal contains 68% carvacrol. Both the oil 

and carvacrol were tested for their antioxidative activity together with sunflower oil via the assessment 

of liberated iodine and its titration with sodium thiosulfate solution in the presence of starch 

as an indicator (Miguel et al., 2003). The antioxidative capacity was determined by the peroxide 

value of the samples, which were taken continuously during a period when the test solution was 

stored at 60°C. The EO and carvacrol exhibit approximately the same antioxidative activity during 

a period of 36 days. 

It is known that free radicals induce deterioration of food because they start the chain reaction of 

the oxidation of polyunsaturated fatty acids. Zataria multifl ora (Zataria, Lamiaceae) is used for 

flavoring yoghurt and as a medical plant, and therefore aroused the interest to study also the biological 

activities of its EO. Using the ammonium thiocyanate method this oil exhibits a strong antioxidative 

activity, as could be shown in an experiment. To determine the inhibition of oxidation, 

ammonium thiocyanate and ferrous chloride were added and the absorbance was measured spectrophotometrically 

at 500 nm. BHT was used for the positive control. Thymol with about 38% the main 

compound of this EO shows a very strong capacity to avoid the lipid peroxidation up to 80%, which 

is close to the inhibition of BHT (97.8%). The high content of thymol, carvacrol (38%), and g-terpinene 

is the reason for the excellent antioxidative activity. In the DPPH assay the results were less 

convincing. The EO of Zataria multifl ora is more liable to prevent lipid peroxidation than scavenging 

free radicals. The value of conjugated dienes is also decreased by the oil. 

Linalool is one of the main compounds (up to 15%) of the EO obtained from Rosmarinus offi cinalis 

L. (rosemary, Lamiaceae). The EO was tested using two different methods: radical scavenging 

with the DPPH assay and the b-carotene bleaching test. The oil was able to reduce the stable free 

radical DPPH and showed a slightly weaker scavenging activity than the standard trolox. But the 

efficiency of rosemary oil was clearly weaker than that of the reference oil Thymus porlock. By the 

b-carotene bleaching test it could be shown that this EO has the ability to prevent the lipid peroxidation 

with a capacity close to the used standards. 

The genus Ocimum contains various species and the EOs are used as an appendage in food, 

cosmetics, and toiletries. Ocimum basilicum (sweet basil, Lamiaceae) is used fresh or dried as a 

food spice nearly all over the world. The antioxidative activities of different Ocimum species were 

studied in order to assess the potential to substitute synthetic antioxidants. Linalool and eugenol 

(~12%) are the main compounds in the diverse oils. In the HPLC-based xanthine–xanthine assay, 

the EO of Ocimum basilicum var. purpurascens (dark opal basil) contains linalool, eugenol, and 

b-caryophyllene as main compounds and shows a very strong antioxidative capacity with an IC 50 

value of 1.84 mL (Salles-Trevisan et al., 2006). Linalool as a pure substance yielded the same test 

results. In the DPPH assay linalool showed a bit weaker activity than in the xanthine–xanthine test


method. Ocimum micranthum (least basil) is original in the South and Central American tropics 

and is used in these territories as a culinary and medical plant. The EO comprises eugenol (up to 

51%) as main compound. In the DPPH assay the EO was able to scavenge about 77% of the free 

DPPH radicals, which is 3 times stronger than that of trolox. The efficiency was also better than 

those of Ocimum basilicum (basil) commercial EO and only slightly weaker than those of Thymus 

vulgaris (thyme) commercial EO. These two EOs were used as a standard because of their known 

antioxidative activity (Sacchetti et al., 2004). The b-carotene bleaching test proved that the EO 

inhibited the lipid peroxidation up to 93% after an incubation time of 60 min. The antioxidative 

activity determined in this test is better than the activity of BHA. In a special test using the photochemoluminescence 

method the EO of Ocimum micranthum attained a 10 times better antioxidative 

activity as Ocimum basilicum and Thymus vulgaris commercial EO. These data are of 

significance because the results of this assay correlate easily with the therapeutic, nutritional, and 

cosmetic potential of a given antioxidant and the capability to quench O 2 

•− 

is useful to describe the 

related capacity to counteract ROS-induced damages to the body (Sacchetti et al., 2004). Furthermore, 

the antioxidative activities of the EOs obtained from Thymus vulgaris and Ocimum basilicum were 

determined by the aldehyde/carboxylic acid assay. Various amounts of the EOs were added to a 

dichloromethane solution of hexanal-containing undecane as a GC internal standard and the antioxidant-standard 

substances BHT and a-tocopherol (Lee et al., 2005). After 5 days the concentration 

of hexane was determined. All samples showed good antioxidative properties as well as pure 

eugenol furnishing an inhibition of the hexanal oxidation by 32%. 

Linalool (~12%), limonene (~18%), and a-terpineol (~2%) are the main volatile compounds in an 

infusion prepared of the green leaves from Illex paraguariensis (mate, Aquifoliaceae). In order to 

determine the protective activity against the oxidation of lipids, the infusion was submitted to the 

ferric thiocyanate test. It could be seen that the infusion of green mate shows similar antioxidative 

activity as the synthetic antioxidant BHT (Bastos-Markowizc et al.,2006). 

The leaves and barks of Cinnamomum zeylanicum Blume syn. Cinnamomum verum (cinnamon, 

Lauraceae) are widely used as spice, flavoring agent in foods, and in various applications in 

medicine (Schmidt et al., 2006). The leaf oil is very rich in eugenol (up to 75%). To investigate the 

antioxidative activity five different methods were used: scavenging effect on DPPH, detection of 

the hydroxyl radicals by deoxyribose assay, evaluation of the antioxidant activity in the linoleic 

acid model system, determination of conjugated dienes formation, and determination of the 

TBARS. In the DPPH radical scavenging assay the EO showed an inhibition of 94% at a concentration 

of 8.0 mg/mL. To reach a radical scavenging activity of 89% a concentration of 20.0 mg/mL 

was necessary, which is comparable to the efficiency of the standard compounds BHT or BHA. 

The EO also exhibits a very strong inhibition of the hydroxyl radicals in the deoxyribose assay. 

The oil prevented 90% at 0.1 mg/mL and eugenol causes a blocking of 71% at the same concentration. 

Quercetin, which was used in this method as a positive control, showed a weaker efficiency. 

In a modified deoxyribose assay, in which FeCl 3 is added to the sample, both the EO and eugenol 

showed an antioxidative activity. The EO and eugenol were able to chelate the Fe 3+ ions and so the 

degradation of deoxyribose was prevented. To assess the oxidation of linoleic acid the determination 

of the conjugated diene content and TBARS were used. Cinnamon EO is able to inhibit the 

generation of conjugated dienes. In a concentration of 0.01% the formation of conjugated dienes 

is avoided (up to 57%) similar to the efficiency of BHT (~59%). On the day 5 of linoleic acid 

storage, malonic aldehyde was detected with TBARS. The cinnamon oil showed an inhibitory 

action of 76% at a concentration of 0.01% oil compared with the 76% of BHT at the same 

concentration. 

Another Lauraceae is Laurus nobilis (laurel). The EO obtained from the leaves of wild grown 

shrubs is characterized by a very high content of eugenol. The biological activities, especially the 

antioxidative properties, of the extract were studied in different in vitro test methods. The scavenging 

capacity in the DPPH assay yielded an IC 50 value of 0.113 mg/mL. Also the b-carotene bleaching 

test of the nonpolar fractions was able to protect the lipids from oxidation. After an incubation


time of 60 min an IC 50 value of 1 mg/mL was calculated. The last applied method to determine the 

antioxidative activity used liposomes obtained from bovine brain extract. This test offers a high 

antioxidative activity with an IC 50 value of 115.0 mg/mL. The high content of eugenol renders it possible 

to use Laurus nobilis leaf extract as a natural antioxidant. Similar results of the antioxidative 

power were found using the FRAP assay and the DPPH assay of the EO obtained from Laurus 

nobilis leaves collected in Dalmatia (Conforti et al., 2003). In the first test system the change in 

absorbance at 593 nm owing to the formation of blue-colored Fe III tripyridyltriazine from a colorless 

Fe III form by the action of electron-donating antioxidants was measured. The sample was incubated 

at 37ºC and over a defined period the ferric reducing antioxidant power (FRAP) values were determined. 

Using the DPPH assay the EO of Laurus nobilis exhibited an antioxidative activity of nearly 

90% at a concentration of 20 g/L, which is near to the activity of BHT with 91%. 

Many aromatic herbs are used as an aroma additive in foodstuffs and in fat-containing food 

systems to prevent or delay some chemical deteriorations occurring during the storage (Politeo 

et al., 2006). For the investigation of the antioxidative activities of the EO of Syzygium aromaticum 

(cloves, Myrtaceae), three variable tests methods were employed: TBA assay, radical scavenging 

with DPPH, and the determination of FRAP. In all three assays it was found that the cloves 

EO—probably on account of its high content of eugenol (up to 91%)—shows a noticeable antioxidative 

activity. The determination of the FRAP assay measures the aforementioned change in absorbance 

at 593 nm owing to the formation of blue-colored Fe III tripyridyltriazine (Politeo et al., 2006). 

In the radical scavenging test with DPPH an inhibition capacity of 91% at 5.0 mg/mL was found for 

the EO. Pure eugenol, BHT, and BHA needed a concentration of 20 mg/mL to arrive at the same 

result. In the deoxyribose assay the EO furnished a hydroxyl radical scavenging of about 94% at 

0.2 mg/mL. The inhibitory effect of eugenol was 91% at a concentration of 0.6 mg/ml. Quercetin, the 

positive control, showed an inhibition of 77.8% at 20.0 mg/mL. The capture of OH • by cloves oil is 

attributed to the hydrogen-donating ability of the phenolic compound eugenol, which is found in a 

high concentration in the EO (Jirovetz et al., 2006). In a linoleic acid model system the inhibition of 

generated hydroxy peroxides in the early stages of the oxidation of linoleic acid and the secondary 

oxidized products were detected by two different indicators: The adoption of conjugated dienes and 

TBARS. At a concentration of 0.005% the cloves oil outbid the activity of BHT. The capacity of the 

EO was about 74% compared to 59% achieved by BHT at 0.001%. The same results were obtained 

by the determination of TBARS. Here the EO’s activity was also equal to BHT. 

Melissa offi cinalis L. (lemon balm, Lamiaceae) is a well-known herb and is also used as a medicinal 

plant for the treatment of different diseases such as headache, gastrointestinal disorders, nervousness, 

and rheumatism. The EO is well known for its antibacterial and antifungal properties, so 

it was investigated for its antioxidative activity too (Mimica-Dukic et al., 2004). The analyses of the 

chemical composition of the EO showed that b-caryophyllene is one of the main compounds (~4.6%) 

besides geranial, neral, citronellal, and linalool. The free radical scavenging capacity was determined 

by the DPPH assay, the protection of lipid peroxidation was investigated with the TBA assay, 

and the scavenging activity of the oil for hydroxyl radicals was measured with the deoxyribose 

assay, also a rapid screening for the scavenging compounds was made. Lemon balm scavenged in 

the DPPH test 50% of the radicals at a concentration of 7.58 mg/mL compared with the standard 

BHT, which attained an IC 50 value of 5.37 mg/mL. At a concentration of 2.13 mg/mL the highest 

inhibition (about 60%) of generated hydroxyl radicals in the deoxyribose assay was found compared 

with BHT as a positive control (~19%). Strong antioxidative activity of the EO was also determined 

in the TBA test system, where the inhibition of the lipid peroxidation was determined. Sixty-seven 

percent inhibition was caused by 2.13 mg/mL EO, in comparison with 37% of BHT. A rapid screening 

for the scavenging capacity with DPPH on a TLC plate exhibited that caryophyllene is one of 

the most active compounds. 

Croton urucurana (Euphorbiaceae) is known as “dragon’s blood” and is used in traditional medicine 

because of its wound and ulcer healing, antidiarrheic, anticancer, anti-inflammatory, antioxidant, 

and antirheumatic properties. The antioxidative fractions of the EO obtained were determined with


a rapid screening using the DPPH assay on TLC. The main compound of the most active fraction was 

a-bisabolol with 38.3%. The isolated fraction exhibited a 50% radical scavenging activity at a concentration 

of 1.05 mg/mL. This is a lower antioxidative activity than that of BHT. The EO of Croton 

urucurana showed an activity in the DPPH assay with an IC 50 value of 3.21 mg/mL (Simionatto 

et al., 2007). 

The EO of Elionurus elegans Kunth. (African pasture grass, Poaceae) was investigated for its 

biological activities. GC/MS analyses showed that the oil contains a-bisabolol (1.6% in the roots and 

1.2% in the aerial parts). None of the other compounds exhibited in individual test systems antioxidative 

activities with the exception of limonene and, which were available only in low amounts. The 

antioxidant activity of the EO was tested with the chemiluminescence method using a luminometer, 

where the chemiluminescence intensity of the reaction mixture containing the EO or a standard 

(a-tocopherol), AAPH, and luminol was measured. The IC 50 value for the EO obtained from the 

aerial parts amounted to 30% and the 50% inhibition rate of the roots EO to 46%. 

Teucrium marum (mint plant, Lamiaceae) is used in the traditional medicine for its antibacterial, 

anti-inflammatory, and antipyretic activities. The antioxidative activity of the EO, which contains 

about 15% b-bisabolene, was investigated in three different test systems. The inhibition of lipid 

peroxide formation and of superoxide radicals and the radical scavenging activity with DPPH were 

tested. The oil exhibited a scavenging power in the DPPH assay (IC 50 value 13.13 mg/mL), which is 

similar to the well-known antioxidants BHT, ascorbic acid, and trolox. Using the xanthine–xanthine 

assay, in which the inhibition of superoxide radicals is tested, the EO showed a better scavenging 

activity for the superoxide radicals than BHT (IC 50 value 0.161 mg/mL against 2.35 g/mL); however, 

the efficiency of ascorbic acid and trolox was higher with IC 50 values of 0.007 and 0.006 mg/mL, 

respectively. The inhibition of lipid peroxidation was determined with the 5-lipidoxygenase 

test, where the formation of the hydroxy peroxides was measured spectrophotometrically at 235 nm. 

The activities of the EO and trolox were similar with IC 50 values of 12.48 and 11.88 mg/mL, respectively. 

The inhibition power was better than ascorbic acid with an IC 50 value of 18.63 mg/mL. 

The antioxidative activity of BHT was higher than the activity of the EO with an IC 50 value of 

3.86 mg/mL (Ricci et al., 2005). 

In the course of the investigation of the antioxidative activity of EOs obtained from three 

different citrus species (Rutaceae), b-bisabolene was also determined for its antioxidative property. 

The LDL oxidation was measured spectrophotometrically at 234 nm by the formation of 

TBARS. The results were expressed as nmol malonic dialdehyde/mg of protein. In the test system, 

b-bisabolene inhibited only TBARS formation from the AAPH-induced oxidation of LDL 

(Takahashi et al., 2003). 

Several naturally occurring EOs containing for example carvacrol, anethole, perillaldehyde, cinnamaldehyde, 

linalool, and p-cymene were investigated for their effectiveness in their antioxidant 

activities and simultaneously also as to their ability in reducing a decay in fruit tissues. The tested 

EOs show positive effects on enhancing anthocyanins and antioxidative activity of fruits (Wang, 

C.Y. et al., 2008). In another study, the EO from black currant buds (Ribes nigrum L., Grossulariaceae) 

was analyzed by GC-MS and GC/-O and was tested for its radical scavenging activity, which—and 

this was the outcome of the present study—varied within a broad range, for example, from 43% 

to 79% in the DPPH reaction system (Dvaranauskaite et al., 2008). The antioxidant and antimicrobial 

properties of the rhizome EO of four different Hedychium species (Zingiberaceae) were investigated 

by Joshi et al. (2008). The rhizome EOs from all Hedychium species tested exhibited 

moderate to good Fe 2+ chelating activity, whereas especially Hedychium spicatum also showed a 

complete different DPPH radical scavenging profile than the samples from the other species. Finally, 

a very strong superoxide anion scavenging and an excellent DPPH-scavenging activity besides a 

strong hypolipidemic property possessed a methanol fractionate of the mountain celery seed EO 

(Cryptotaenia japonica Hassk, Apiaceae). The principal constituents of this fraction after a successive 

gel column adsorption were g-selinene, 2-methylpropanal, and (Z)-9-octadecenamide (Cheng 

et al., 2008).


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10 

Effects of Essential Oils in the 

Central Nervous System 

CONTENTS 

10.1 Central Nervous System Effects of Essential Oils in Humans ....................................... 281 

Eva Heuberger 

10.1.1 Introduction ........................................................................................................ 281 

10.1.2 Activation and Arousal: Definition and Neuroanatomical Considerations ....... 284 

10.1.3 Influence of EOs and Fragrances on Brain Potentials Indicative of Arousal .... 285 

10.1.3.1 Spontaneous Electroencephalogram Activity ................................... 285 

10.1.3.2 Contingent Negative Variation .......................................................... 289 

10.1.4 Effects of EOs and Fragrances on Selected Basic 

and Higher Cognitive Functions ........................................................................ 290 

10.1.4.1 Alertness and Attention ..................................................................... 290 

10.1.4.2 Learning and Memory ....................................................................... 293 

10.1.4.3 Other Cognitive Tasks ....................................................................... 295 

10.1.5 Conclusions ........................................................................................................ 296 

10.2 Psychopharmacology of Essential Oils ........................................................................... 297 



10.2.1 Aromatic Plants Used in Traditional Medical Systems 

as Sedatives or Stimulants ................................................................................. 297 

10.2.2 Effects of EOs in Animal Models ...................................................................... 299 

10.2.2.1 Effects of Individual Components ..................................................... 301 

10.2.2.2 Effects of Inhaled EOs ...................................................................... 301 

10.2.3 Mechanism of Action Underlying Psychopharmacological Effects of EOs ...... 302 

References .................................................................................................................................. 306 

10.1 CENTRAL NERVOUS SYSTEM EFFECTS OF ESSENTIAL OILS IN HUMANS 

Eva Heuberger 

10.1.1 INTRODUCTION 

A number of attempts have been made to unravel the effects of natural essential oils (EOs) and 

fragrances on the human central nervous system (CNS). Among these attempts two major lines of 

research have been followed to identify psychoactive, particularly stimulating and sedative, effects 

of fragrances. On the one hand, researchers have investigated the influence of EOs and fragrances 

on brain potentials, which are indicative of the arousal state of the human organism by means of 

281


neurophysiological methods. On the other hand, behavioral studies have elucidated the effects of 

EOs and fragrances on basic and higher cognitive functions, such as alertness and attention, learning 

and memory, or problem solving. The scope of the following section, although not claiming to 

be complete, is to give an overview about the current knowledge in these fields. Much of the research 

reviewed has been carried out in healthy populations and only recently investigators have started to 

focus on clinical aspects of the administration of fragrances and EOs. However, since this topic is 

covered in another chapter of this volume, it is omitted here in the interest of space. 

Olfaction differs from other senses in several ways. First, in humans and many other mammals, 

the information received by peripheral olfactory receptor cells is mainly processed in brain areas 

located ipsilaterally to the stimulated side of the body, whereas in the other sensory systems, it is 

transferred to the contralateral hemisphere. Second, in contrast to the other sensory systems, olfactory 

information reaches a number of cortical areas without being relayed in the thalamus (Kandel 

et al., 1991; Zilles and Rehkämpfer, 1998; Wiesmann et al., 2001) (Figure 10.1). Owing to this missing 

thalamic control as well as to the fact that the olfactory system presents anatomical connections 

and overlaps with brain areas involved in emotional processing, such as the amygdala, hippocampus, 

and prefrontal cortex of the limbic system (Reiman et al., 1997; Davidson and Irwin, 1999; 

Phan et al., 2002; Bermpohl et al., 2006), the effects of odorants on the organisms are supposedly 

exerted not only via pharmacological but also via psychological mechanisms. In humans and probably 

also in other mammals, psychological factors may be based on certain stimulus features, such 

los 

mos 

olf 

OB 

tos 

OT 

AON 

Tu 

L 

OpT 

PIR-FR 

PIR-TP 

AM 

EA 

G 

MB 

CP 

FIGURE 10.1 Macroscopic view of the human ventral forebrain and medial temporal lobes, depicting the 

olfactory tract, its primary projections, and surrounding nonolfactory structures. The right medial temporal 

lobe has been resected horizontally through the mid-portion of the amygdala (AM) to expose the olfactory cortex. 

AON, anterior olfactory nucleus; CP, cerebral peduncle; EA, entorhinal area; G, gyrus ambiens; L, limen 

insula; los, lateral olfactory sulcus; MB, mammillary body; mos, medial olfactory sulcus; olf, olfactory sulcus; 

PIR-FR, frontal piriform cortex; OB, olfactory bulb; OpT, optic tract; OT, olfactory tract; tos, transverse 

olfactory sulcus; Tu, olfactory tubercle; PIR-TP, temporal piriform cortex. Figure prepared with the help of 

Dr. Eileen H. Bigio, Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, 

Illinois. (From Gottfried, J.A. and D.A. Zald, 2005. Brain Res. Rev., 50: 287–304. With permission.)

Effects of Essential Oils in the Central Nervous System 283 

as odor valence (Baron and Thomley, 1994), on semantic cues, for example, memories and experiences 

associated with a particular odor, as well as on placebo effects related to the expectation of 

certain effects (Jellinek, 1997). None of the latter mechanisms is substance, that is, odorant, specific 

but their effectiveness depends on cognitive mediation and control. 

Many odorants stimulate not only the olfactory system via the first cranial nerve (N. olfactorius) 

but also the trigeminal system via the fifth cranial nerve (N. trigeminus), which enervates the nasal 

mucosa. The trigeminal system is part of the body’s somatosensory system and mediates mechanical 

and temperature-related sensations, such as itching and burning or warmth and cooling sensations. 

Trigeminal information reaches the brain via the trigeminal ganglion and the ventral posterior 

nucleus of the thalamus. The primary cortical projection area of the somatosensory system is the 

contralateral postcentral gyrus of the parietal lobe (Zilles and Rehkämpfer, 1998). The reticular 

formation in the brain stem, which is part of the reticular activating system (RAS) (Figure 10.2), 

receives collaterals from the trigeminal system. Thus, trigeminal stimuli have direct effects on 

arousal. Utilizing this direct connection, highly potent trigeminal stimulants, such as ammonia and 

menthol, have been used in the past in smelling salts to awaken people who fainted. 

It has been shown in experimental animals that, due to their lipophilic properties, fragrances 

not only penetrate the skin (Hotchkiss, 1998) but also the blood–brain barrier (Buchbauer et al., 

1993). Also, odorants have been found to bind to several types of brain receptors (Aoshima 

and Hamamoto, 1999; Elisabetsky et al., 1999; Okugawa et al., 2000), and it has been suggested 

Hypothalamus 

Basal 

ganglia 

Cortex 

Thalamus 

Midbrain 

Raphé Nuclei 

Amygdala 

Pons 

Medulla 

Cerebellum 

Locus ceruleus 

Cell 

bodies 

Axon 

terminals 

Noradrenergic neurons 

Serotonergic neurons 

FIGURE 10.2 Schematic of the RAS with noradrenergic and serotonergic connections. (From Grilly, D.M., 

2002. Drugs & Human Behavior. Boston: Allyn & Bacon. With permission.)


that these odorant–receptor interactions are responsible for psychoactive effects of fragrances in 

experimental animals. With regard to these findings, it is important to note that Heuberger et al. 

(2001) have observed differential effects of fragrances as a function of chirality. It seems likely that 

such differences in effectiveness are related to enantiomeric selectivity of receptor proteins. However, 

the question whether effects of fragrances on human arousal and cognition rely on a similar psychopharmacological 

mechanism remains to be answered. 

10.1.2 ACTIVATION AND AROUSAL: DEFINITION AND NEUROANATOMICAL CONSIDERATIONS 

Activation, or arousal, refers to the ability of an organism to adapt to internal and external challenges 

(Schandry, 1989). Activation is an elementary process that serves in the preparation for overt 

activity. Nevertheless, it does not necessarily result in overt behavior (Duffy, 1972). Activation varies 

in degree and can be described along a continuum from deep sleep to overexcitement. Early 

theoretical accounts of activation have emphasized physiological responses as the sole measurable 

correlate of arousal. Current models, however, consider physiological, cognitive, and emotional 

activity as observable consequences of activation processes. It has been shown that arousal processes 

within each of these three systems, that is, physiological, cognitive, and emotional, can occur 

to varying degrees so that the response of one system need not be correlated linearly to that of the 

other systems (Baltissen and Heimann, 1995). 

It has long been established that the RAS, which comprises the reticular formation with its sensory 

afferents and widespread hypothalamic, thalamic, and cortical projections, plays a crucial role 

in the control of both phasic and tonic activation processes (Becker-Carus, 1981; Schandry, 1989). 

Pribram and McGuinness (1975) distinguish three separate but interacting neural networks in the 

control of activation (Figure 10.3). The arousal network involves amygdalar and related frontal 

cortical structures and regulates phasic physiological responses to novel incoming information. The 

activation network centers on the basal ganglia of the forebrain and controls the tonic physiological 

readiness to respond. Finally, the effort network, which comprises hippocampal circuits, coordinates 

the arousal and activation networks. Noradrenergic projections from the locus ceruleus, which 

is located within the dorsal wall of the rostral pons, are particularly important in the regulation of 

OFC 

HI 

CC 

SMP 

AM 

S 

CS 

MDT 

ADT 

LDT 

AH 

PH 

SR 

RF 

T 

SC 

SC 

FIGURE 10.3 Control of activation processes. OFC, orbitofrontal cortex; AM, amygdala; MDT, medial 

dorsal thalamus; AH, anterior hypothalamus; RF, reticular formation; SC, spinal cord; HI, hippocampus; CC, 

cingulate cortex; S, septum; ADT, anterior dorsal thalamus; PH, posterior hypothalamus; SMP, sensory-motor 

projections; CS, corpus striatum; LDT, lateral dorsal thalamus; SR, subthalamic regions; T, tectum. Left, 

structures of the arousal network; middle, structures of the effort network; right, structures of the activation 

network. (Adapted from Pribram, K. H. and D. McGuinness, 1975. Psychol. Rev., 82(2): 116–149.)


circadian alertness, the sleep–wake rhythm, and the sustenance of alertness (alerting) (Pedersen 

et al., 1998; Aston-Jones et al., 2001). On the other hand, tonic alertness seems to be dependent on 

cholinergic (Baxter and Chiba, 1999; Gill et al., 2000) frontal and inferior parietal thalamic structures 

of the right hemisphere (Sturm et al., 1999). Other networks that are involved in the control of 

arousal and attentional functions are found in posterior parts of the brain, for example, the parietal 

cortex, superior colliculi, and posterior-lateral thalamus, as well as in anterior regions, for example, 

the cingular and prefrontal cortices (Posner and Petersen, 1990; Paus, 2001). 

10.1.3 INFLUENCE OF EOS AND FRAGRANCES ON BRAIN POTENTIALS INDICATIVE OF AROUSAL 

10.1.3.1 Spontaneous Electroencephalogram Activity 

Recordings of spontaneous electroencephalogram (EEG) activity during the administration of EOs 

and fragrances have widely been used to assess stimulant and sedative effects of these substances. 

Particular attention has been paid to changes within the a and b bands, sometimes also the q band 

of the EEG in response to olfactory stimulation since these bands are thought to be most indicative 

of central arousal processes. Alpha waves are slow brain waves within a frequency range of 8–13 Hz 

and amplitudes between 5 and 100 mV, which typically occur over posterior areas of the brain in an 

awake but relaxed state, especially with closed eyes. The a rhythm disappears immediately when 

subjects open their eyes and when cognitive activity is required, for example, when external stimuli 

are processed or tasks are solved. This phenomenon is often referred to as a block or desynchronization. 

Simultaneously with the a block, faster brain waves occur, such as b waves with smaller 

amplitudes (2–20 mV) and frequencies between 14 and 30 Hz. The b rhythm, which is most evident 

frontally, is characteristic of alertness, attention, and arousal. In contrast, q waves are very slow 

brain waves occurring in fronto-temporal areas with amplitudes between 5 and 100 mV in the frequency 

range between 4 and 7 Hz. Although the q rhythm is most commonly associated with 

drowsiness and light sleep, some researchers found q activity to correlate with memory processes 

(Grunwald et al., 1999; Hoedlmoser et al., 2007) and creativity (Razumnikova, 2007). Other authors 

found correlations between q activity and ratings of anxiety and tension (Lorig and Schwartz, 1988). 

With regard to animal olfaction, it has been proposed that the q rhythm generated by the hippocampus 

is concomitant to sniffing and allows for encoding and integration of olfactory information with 

other cognitive and motor processes (Kepecs et al., 2006). 

A large number of measures can be derived from recordings of the spontaneous EEG. Time (index) 

or voltage (power)-based rates of typical frequency bands, as well as ratios between certain frequency 

bands (e.g., between the a and b band or the q and b band) within a selected time interval are most 

commonly used to quantify EEG patterns. Period analysis quantifies the number of waves that occur 

in the various frequency bands within a distinct time interval of the EEG record and is supposed to be 

more sensitive to task-related changes than spectral analysis (Lorig, 1989). Other parameters that 

describe the covariation of a given signal at different electrodes are coherence and neural synchrony. 

These measures inform on the functional link between brain areas (Oken et al., 2006). 

Generally speaking, the pattern of the spontaneous EEG varies with the arousal level of the CNS. 

Thus, different states of consciousness, such as sleep, wakefulness, or meditation, can be distinguished 

by their characteristic EEG patterns. For instance, an increase in central activation is typically 

characterized by a decrease in a and an increase in b activity (Schandry, 1989). More precisely, 

a decline of a and b power together with a decrease of a index and an increase of b and q activity 

have been observed under arousing conditions, that is, in a mental calculation and a psychosocial 

stress paradigm (Walschburger, 1976). Also when subjects are maximally attentive, frequencies in 

the a band are attenuated and activity in the b and even higher frequency bands can be observed. 

Fatigue and performance decrements in situations requiring high levels of attention are often associated 

with increases in q and decreases in b activity (Oken et al., 2006). On the other hand, drowsiness 

and the onset of sleep are characterized by an increase in slow and a decrease in fast EEG 

waves. However, high activity in the a band, particularly in the range between 7 and 10 Hz, is not


indicative of low arousal states of the brain, such as relaxation, drowsiness, and the onset of sleep, 

but rather seems to be a component of selective neural inhibition processes that are necessary for a 

number of cognitive processes, such as perception, attention, and memory (Miller and O’Callaghan, 

2006; Palva and Palva, 2007). 

Because changes of spontaneous EEG activity accompany a wide range of cognitive as well as 

emotional brain processes and “EEG measurements […] do not tell investigators what the brain is 

doing” (Lorig 1989, p. 93), it is somewhat naïve to interpret changes that are induced by the application 

of an odorant as a result of a single and specific process, particularly when no other correlates 

of the process of interest are assessed. Nevertheless, this is exactly the approach that has been 

taken by many researchers to identify stimulant or relaxing effects of odors. The simplest, but in 

terms of interpretation of the results probably most problematic setup for such experiments is the 

comparison of spontaneous EEG activity in response to odorants with a no-odor baseline. Using 

this design, Sugano (1992) observed increased EEG a activity after inhalation of a-pinene (1), 

1,8-cineole (2), lavender, sandalwood, musk, and eucalyptus odors. Considering that traditional 

aromatherapy discriminates these fragrances by their psychoactive effects, for example, lavender 

is assigned relaxing properties while eucalyptus is supposedly stimulant (Valnet, 1990); these findings 

are at least rather curious. Also Ishikawa et al. (2002) recorded the spontaneous EEG in 

13 Japanese subjects while drinking either lemon juice with a supplement of lemon odor or lemon 

juice without it. It was shown that a power was enhanced by supplementation with lemon odor, an 

indicative of increased relaxation. Again, with regard to aromatherapeutical accounts of the lemon 

EO this result is somewhat counterintuitive, even more so as in the same study the juice supplemented 

with lemon odor increased spontaneous locomotion in experimental animals. Haneyama 

and Kagatani (2007) tested a fragrant spray made from extracts of Chinese spikenard roots 

[Nardostachys chinensis Batalin (Valerianaceae)] in butylene glycol and found increased a activity 

in subjects under stress. This finding was interpreted by the authors as demonstrating a sedative 

effect of the extract. However, it is unknown how stress was induced in the subjects and how it was 

measured. Similarly, Ishiyama (2000) concluded from measurements of the frequency fluctuation 

patterns of the a band that smelling a blend of terpene compounds, typically found in forests, 

induced feelings of refreshment and relaxation in human subjects without proper description of 

how these feelings were assessed. 

Inconclusive findings as those described above are not unexpected with such simple experimental 

designs as there are several problems associated with this kind of experiments. First, a no-odor 

baseline is often inappropriate as it does not control for cognitive activity of the subject. For instance, 

subjects might be puzzled by the fact that they do not smell anything eventually focusing attention 

to the search for an odor. This may lead to quite high arousal levels rather than the intended resting 

brain state. This was the case with Lorig and Schwarzt (1988), who tested changes of spontaneous 

EEG in response to spiced apple, eucalyptus, and lavender fragrances diluted in an odorless base: 

contrary to the authors’ expectations it was found that a activity in the no-odor condition was less 

than that during odor presentation. 

Considering the various mechanisms outlined by Jellinek (1997) by which fragrances influence 

human arousal and behavior, another inherent problem of such simple designs is that little is 

known about how subjects process stimulus-related information, for example, the pleasantness or 

intensity of an odorant, and whether or not higher cognitive processes related to the odorant are 

initiated by the stimulation. For instance, subjects might be able to identify and label some odors 

but might fail to do so with others; similarly, some odors might trigger the recall of associated 

memories while others might not. In order to assess psychoactive effects of EOs and fragrances, it 

seems necessary to control for these factors, for instance by assessing additional variables that 

inform on the subject’s perception of primary and secondary stimulus features and that correlate 

to the subject’s cognitive or emotional arousal state. In the above-mentioned study, Lorig and 

Schwarzt (1988) collected ratings of intensity and pleasantness of the tested fragrances as well as 

subjective ratings of a number of affective states in addition to the EEG recordings. Analysis of the


amount of EEG q activity revealed that the spiced apple odor produced more relaxation than 

lavender and eucalyptus; the analysis of the secondary variables suggested that this relaxing effect 

was correlated with subjective estimates of anxiety and tension. As to the EEG patterns, similar 

results were observed when subjects imagined food odors and practiced relaxation techniques. 

Thus, the authors conclude that the relaxing effect of spiced apple was probably related to its association 

with food. These cognitive influences also seem to be a plausible explanation for the 

increase in a power by lemon odor in the Ishikawa et al. (2002) study. Other studies related to food 

odors were conducted by Kaneda et al. (2005, 2006). These authors investigated the influence of 

smelling beer flavors on the frequency fluctuation of a waves in frontal areas of the brain. The 

results showed relaxing effects of the aroma of hop extracts as well as of linalool (3). In addition, 

in the right hemisphere these fluctuations were correlated with subjective estimates of arousal and 

with the intensity of the hop aroma. Lee et al. (1994) also found evidence for differential EEG patterns 

as a function of odor intensity for citrus, lavender, and a floral odor. A 10-min exposure to 

the weaker intensity of the citrus fragrance in comparison to lavender odor increased the rate of 

occipital a. Moreover, there was a general trend for citrus to be rated as more comfortable than 

other fragrances. In contrast, the higher intensity of the floral fragrance increased the rate of 

occipital b more than the lavender odor. 

Several authors have shown influences of odor pleasantness and familiarity on changes of the 

spontaneous EEG. For instance, Kaetsu et al. (1994) reported that pleasant odors increased the a 

activity, while unpleasant ones decreased it. In a study on the effects of lavender and jasmine odor 

on electrical brain activity (Yagyu, 1994), it was shown that changes in the a, b, and q bands in 

response to these fragrances were similar when subjects rated them as pleasant, while lavender and 

jasmine odor led to distinct patterns when they were rated as unpleasant. Increases of a activity in 

response to pleasant odors might be explained by altered breathing patterns since it has been demonstrated 

that pleasant odors induce deeper inhalations and exhalations than unpleasant odors, and 

that this form of breathing by itself increases the activity in the a band (Lorig, 2000). Masago et al. 

(2000) tested the effects of lavender, chamomile, sandalwood, and eugenol (4) fragrances on ongoing 

EEG activity and self-ratings of comfort and found a significant positive correlation between the 

degree of comfort and the odorants’ potency to decrease the a activity in parietal and posterior 

temporal regions. In relation to the previously described investigations, this finding is rather difficult 

to explain, although it differs from the other studies in that it differentiated between electrode 

sites rather than reporting merely global changes in electrical brain activity. Therefore, this result 

suggests that topographical differences in electrical brain activity induced by fragrances may be 

important and need further investigation. In fact, differences in hemispheric localization of spontaneous 

EEG activity in response to pleasant and unpleasant fragrances seem to be quite consistent. 

While pleasant odors induced higher activation in left frontal brain regions, unpleasant ones led to 

bilateral and widespread activation (Kim and Watanuki, 2003) or no differences were observed 

when an unpleasant odor (valerian) was compared to a no-odor control condition (Kline et al., 

2000). Another interesting finding in the study of Kim and Watanuki (2003) was that EEG activity 

in response to the tested fragrances was observed when subjects were at rest but vanished after 

performing a mental task. The importance of distinguishing EEG activity arising from different 

areas of the brain is highlighted by an investigation by Van Toller et al. (1993). These authors 

recorded a wave activity at 28 sites of the scalp immediately after the exposure to a number of fragrances 

covering a range of different odor types and hedonic tones at iso-intense concentrations; the 

odorants had to be rated in terms of pleasantness, familiarity, and intensity. It was shown that in 

posterior regions of the brain changes in a activity in response to these odors compared to an odorless 

blank were organized in distinct topographical maps. Moreover, a activity in a set of electrodes 

at frontal and temporal sites correlated with the psychometric ratings of the fragrances. 

As to influences of the familiarity of or experience with fragrances, Kawano (2001) reported 

that the odors of lemon, lavender, patchouli, marjoram, rosemary, and sandalwood increased a 

activity over occipital electrode sites in subjects to whom these fragrances were well known. On the


one hand, lag times between frontal and occipital a phase were shorter in subjects less experienced 

with the fragrances, indicating that these subjects were concentrating more on smelling—and probably 

identifying—the odorants. These findings were confirmed in an investigation comparing professional 

perfume researchers, perfume salespersons, and general workers (Min et al., 2003). This 

study showed that measures of cortico-cortical connectivity, that is, the averaged cross mutual 

information content, in odor processing were more pronounced in frontal areas with perfume 

researchers, whereas with perfume salespersons and general workers a larger network of posterior 

temporal, parietal, and frontal regions was activated. These results could result from a greater 

involvement of orbitofrontal cortex neurons in perfume researchers, who exhibit high sophistication 

in discriminating and identifying odors. Moreover, it was shown that the value of the averaged cross 

mutual information content was inversely related to preference in perfume researchers and perfume 

salespersons, but not in general workers. 

As pointed out above, the administration of EO and fragrances to naïve subjects can lead to 

cognitive processes that are unknown to the investigator, and sometimes even the subject, but that 

nevertheless affect spontaneous EEG activity. Some researchers have sought to solve this problem 

by engaging subjects in a secondary task while the influence of the odorant of interest is assessed. 

This procedure does not only draw the subject’s attention away from the odor stimulus, but also 

provides the desired information about his/her arousal state. Another benefit of such experimental 

designs is that the task may control for the subject’s arousal state if a certain amount of attention is 

required to perform it. Measurement of changes of a and q activity in the presence or absence of 

1,8-cineole (2), methyl jasmonate (5), and trans-jasmin lactone (6) in subjects who performed a 

simple visual task showed that the increase in slow wave activity was attenuated by 1,8-cineole (2) 

and methyl jasmonate (5), while augmented by trans-jasmin lactone (6) (Nakagawa et al. 1992). 

At least in the case of 1,8-cineole (2) these findings are supported by results from experimental 

animals and humans, indicating activating effects of this odorant (Kovar et al., 1987; Nasel et al., 

1994; Bensafi et al., 2002). An investigation of the effects of lemon odor EEG a, b, and q activity 

during the administration of lemon odor showed that the odor reduced power in the lower a range 

while it increased power in the higher a, lower b, and lower q bands (Krizhanovs’kii et al., 2004). 

These findings of increased arousal were in agreement with better performance in a cognitive task. 

In addition, it was shown that inhalation of the lemon fragrance was most effective during rest and 

in the first minutes of the cognitive task, but wore off after less than 10 min. In several experiments, 

the group of Sugawara demonstrated complex interactions between electrical brain activity induced 

by the exposure to fragrances, sensory profiling, and various types of tasks (Sugawara et al., 2000; 

Satoh and Sugawara, 2003). In one study they showed that the odor of peppermint, in contrast to 

basil, was rated less favorable on a number of descriptors and reduced the magnitude of b waves 

after as compared to before performance of a cognitive task. In a similar investigation, it was shown 

that the sensory evaluation as well as changes in spontaneous EEG activity in response to the odors 

of the linalool enantiomers differed as a function of the molecular structure and the kind of task. 

For instance, R-(-)-linalool (7) was rated more favorable and led to larger decreases of b activity 

after listening to natural sounds than before. In contrast, after as compared to before cognitive effort 

R-(-)-linalool (7) was rated as less favorable and tended to increase b power. A similar pattern was 

found for RS-(±)-linalool (3), whereas for S-(+)-linalool (8) the pattern was different, particularly 

with regard to EEG activity. 

In the study of Yagyu (1994), the effects of lavender and jasmine fragrances on the performance 

in a critical flicker fusion and an auditory reaction time task were assessed in addition to 

changes of the ongoing EEG. It was demonstrated that in contrast to the EEG findings, lavender 

decreased performance in both tasks independent of its hedonic evaluation. Jasmine, however, 

had no effect on task performance. The EEG changes in response to these odorants might well 

explain their effects on performance: lavender induced decreases of activity in the b band, which 

is associated with states of low attention regardless of being rated as pleasant or unpleasant; 

jasmine, on the other hand, increased EEG b activity when it was judged unpleasant but lowered


b activity when judged pleasant, so that overall its effect on performance levelled out. The effects 

of lavender and rosemary fragrances on electrical brain activity, mood states, and math computations 

were investigated by Diego, Field, and co-workers (Diego et al., 1998; Field et al., 2005). 

These investigations showed that the exposure to lavender increased b power, elevated feelings 

of relaxation, reduced feelings of depression, and improved both speed and accuracy in the cognitive 

task. In contrast, rosemary odor decreased frontal a and b power, decreased feelings of 

anxiety, increased feelings of relaxation and alertness, and increased speed in the math computations. 

The EEG results were interpreted as indicating increased drowsiness in the lavender group 

and increased alertness in the rosemary group; however, the behavioral data showed performance 

improvement and similar mood ratings in both groups. These findings suggest that different 

electrophysiological arousal patterns may still be associated with similar behavioral arousal patterns 

emphasizing the importance of collecting additional endpoints to evaluate psychoactive 

effects of EO and fragrances. 

10.1.3.2 Contingent Negative Variation 

The contingent negative variation (CNV) is a slow, negative event-related brain potential, which is 

generated when an imperative stimulus is preceded by a warning stimulus and reflects expectancy 

and preparation (Walter et al., 1964). The amplitude of the CNV is correlated to attention and 

arousal (Tecce, 1972). Since changes of the magnitude and latency of CNV components have long 

been associated with the effects of psychoactive drugs (Kopell et al., 1974; Ashton et al., 1977), 

measurement of the CNV has also been used to evaluate psychostimulant and sedating effects of 

EOs and fragrances. In a pioneering investigation, Torii et al. (1988) measured CNV magnitude 

changes evoked by a variety of EOs, such as jasmine, lavender, and rose oil, in male subjects. CNV 

was recorded at the frontal, central, and parietal sites after the presentation of an odorous or blank 

stimulus in the context of a cued reaction time paradigm. In addition, physiological markers of 

arousal, that is, skin potential level and heart rate, were simultaneously measured. Results showed 

that at frontal sites the amplitude of the early negative shift of the CNV was significantly altered 

after the presentation of odor stimuli, and that these changes were mostly congruent with stimulating 

and sedative properties reported for the tested oils in the traditional aromatherapy literature. In 

contrast to other psychoactive substances, such as caffeine or benzodiazepines, presentation of the 

EOs neither affected physiological parameters nor reaction times. The authors concluded that the 

EOs tested influenced brain waves “almost exclusively” while having no effects on other indicators 

of arousal. 

Subsequently, CNV recordings have been used by a number of researchers on a variety of EOs 

and fragrances to establish effects of odors on the human brain along the activation–relaxation 

continuum. For instance, Sugano (1992) in the aforementioned study demonstrated that a-pinene 

(1), sandalwood, and lavender odor increased the magnitude of the CNV in healthy young adults, 

whereas eucalyptus reduced it. It is interesting to note, however, that all of these odors—despite 

their differential influence on the CNV—increased spontaneous a activity in the same experiment. 

An increase of CNV magnitude was also observed with the EO from pine needles (Manley, 1993), 

which was interpreted as having a stimulating effect. Aoki (1996) investigated the influence of 

odors from several coniferous woods, that is, hinoki [Chamaecyparis obtusa (Siebold & Zucc.) 

Endl. (Cupressaceae)], sugi [Cryptomeria japonica D.Don (Cupressaceae)], akamatsu [Pinus densifl 

ora Siebold & Zucc. (Pinaceae)], hiba [Thujopsis dolabrata var. hondai Siebold & Zucc. 

(Cupressaceae)], Alaska cedar [Chamaecyparis nootkatensis (D.Don) Spach (Cupressaceae)], 

Douglas fir [Pseudotsuga manziesii (Mirbel) Franco (Pinaceae)], and Western red cedar [Thuja 

plicata Donn (Cupressaceae)], on the CNV and found conflicting effects: the amplitude of the early 

CNV component at central sites was decreased by these wood odors, and the a/b-wave ratio of the 

EEG increased. Moreover, the decrease of CNV magnitude was correlated with the amount of 

a-pinene (1) in the tested wood odors. Also Sawada et al. (2000) measured changes of the early 

component of the CNV in response to stimulation with terpenes found in the EO of woods and


leaves. These authors noticed a reduction of the CNV magnitude after the administration of 

a-pinene (1), D-3-carene (9), and bornyl acetate (10). However, a more recent investigation (Hiruma 

et al., 2002) showed that hiba [Thujopsis dolabrata Siebold & Zucc. (Cupressaceae)] odor increased 

the CNV magnitude at frontal and central sites and shortened reaction times to the imperative 

stimulus in female subjects. These authors thus concluded that the odor of hiba heightened the 

arousal level of the CNS. 

Although the CNV is believed to be largely independent of individual differences, such as age, 

sex, or race (Manley, 1997), there seem to be cognitive influences that must not be neglected when 

interpreting the effects of odor stimuli on the CNV. Lorig and Roberts (1990) repeated the study by 

Torii et al., (1988) and investigated cognitive factors by introducing a manipulation of their subjects 

as an additional variable into the paradigm. In this experiment subjects were exposed to the original 

two odors, that is, lavender and jasmine, referred to as odor A and odor B, respectively, as well as to 

a mixture of the two fragrances. However, in half of the trials in which the mixture was administered 

subjects were led to believe that they received a low concentration of odor A, whereas in the 

other half of trials they thought that they would be exposed to a low concentration of odor B. In none 

of the four conditions were the subjects given the correct odor names. As in the Torii et al. study, 

lavender reduced the amplitude of the CNV whereas jasmine increased it. When the mixture was 

administered, however, the CNV magnitude decreased when subjects believed to receive a low 

concentration of lavender, but increased when they thought they were inhaling a low concentration 

of jasmine. This means that the alteration of the CNV amplitude was not solely related to the substance 

that had been administered but also related to the expectation of the subjects. Another point 

made by Lorig and Roberts (1990) is that in their study self-report data indicated that lavender was 

actually rated as more arousing than jasmine. Since low CNV amplitudes are not only associated 

with low arousal but also with high arousal in the context of distraction (Travis and Tecce, 1998), 

the lavender odor might in fact have led to higher arousal levels than jasmine even though the CNV 

magnitude was smaller with lavender. Other authors have noted that CNV changes might not only 

reflect effects of odor stimuli but also the anticipation, expectancy, and the emotional state of the 

subjects who are exposed to these odorants (Hiruma et al., 2005). The involvement of these and 

other cognitive factors might well explain why the findings of CNV changes in response to odorants 

are rather inconsistent. 

10.1.4 EFFECTS OF EOS AND FRAGRANCES ON SELECTED BASIC 

AND HIGHER COGNITIVE FUNCTIONS 

Psychoactive effects of odorants at the cognitive level have been explored in humans using a large 

number of methods. A variety of testing procedures ranging from simple alertness or mathematical 

tasks to tests that assess higher cognitive functions, such as memory or creativity, have been 

employed to study stimulant or relaxing/sedating effects of EOs and fragrances. Nevertheless, the 

efficiency of odorants is commonly defined by changes in performance in such tasks as a function 

of the exposure to fragrances. 

10.1.4.1 Alertness and Attention 

A number of studies are available on the influence of fragrances on attentional functions. The integrity 

and the level of the processing efficiency of the attentional systems is a fundamental prerequisite 

of all higher cognitive functions. Attentional functions can be divided into four categories: alertness, 

selective and divided attention, and vigilance (Posner and Rafal, 1987; Keller and Groemminger, 

1993; Sturm, 1997). Alertness is the most basic form of attention and is intrinsically dependent on 

the general level of arousal. Selective attention describes the ability to focus on relevant stimulus 

information while nonrelevant features are neglected; divided attention describes the ability to concomitantly 

process several stimuli from different sensory modalities. Vigilance refers to the sustenance 

of attention over longer periods of time. Since the critical stimuli typically occur only rarely


in time, vigilance can be seen as a counterforce against increasing fatigue in boring situations, which 

is crucial in everyday life, in situations such as long-distance driving (particularly at night), working 

in assembly lines, or monitoring a radar screen (e.g., in air traffic control). 

In a pioneering study, Warm et al. (1991) investigated the influence of peppermint and muguet 

odors on human visual vigilance. Peppermint, which was rated as stimulant, was expected to 

increase the task performance, whereas muguet, rated as relaxant, was expected to impair it. After 

intermittent inhalation, none of these fragrances increased processing speed in the task, but subjects 

in both odorant conditions detected more targets than a control group receiving unscented air. 

On the other hand, neither fragrance influenced subjective mood or judgments of workload. Gould 

and Martin (2001) studied the effects of bergamot and peppermint EOs on human sustained attention, 

where again peppermint was expected to improve performance, whereas bergamot, characterized 

as relaxing by an independent sample of subjects, was expected to have a deteriorating 

effect on vigilance performance. However, only bergamot had a significant influence in the anticipated 

direction, that is, subjects in this condition detected fewer targets than subjects in the peppermint 

or a no-odor control condition, which was probably related to subjects’ expectation of a 

relaxing effect. 

The influence of the inhalation of a number of EOs, which were expected to have activating 

effects on performance in an alertness task, was assessed by Ilmberger et al. (2001). Contrary to the 

authors’ hypotheses, the results suggested that these EOs, when compared to an odorless control did 

not increase the speed of information processing. Even more unexpected, motor learning was 

impaired in the groups that received EOs; this effect is likely a consequence of distraction induced 

by the strong odor stimuli, an explanation that was supported by the reaction times that tended to be 

higher in the EO-treated groups than in the corresponding control groups. Alternatively, these 

authors argued that a ceiling effect might be responsible for the observed effects. Given that healthy 

subjects with intact attentional systems already perform at optimal levels of information processing 

in such basic tasks, it seems likely that activating EOs cannot enhance performance any further. 

Similarly, performance of healthy subjects may be too robust to be influenced by deactivating fragrances. 

This hypothesis has been supported by investigations on the EO of peppermint (Ho and 

Spence, 2005), lavender, and rosemary (Moss et al., 2003). These fragrances rather affected performance 

in difficult tasks or in tasks testing higher cognitive functions than in simple ones testing 

basic functions. Another interesting finding of the study of Ilmberger et al. was that changes in 

performance were correlated with subjective ratings of characteristic odor properties, particularly 

with pleasantness and efficiency. Similar results were obtained in another study for the EO of peppermint 

(Sullivan et al., 1998), which showed that in a vigilance task subjects benefited most from 

the effects of this fragrance when they experienced the task as quite difficult and thought that the 

EO had a stimulant effect. In addition, the study of Ilmberger and co-workers clearly demonstrated 

effects of expectation, that is, a placebo effect, as correlations between individual task performance 

and odor ratings were not only revealed in the EO groups but also in the no-odor control groups. The 

effects of a pleasant and an unpleasant blend of fragrances on selective attention was studied by 

Gilbert et al. (1997). No influence of either fragrance blend was found on attentional performance, 

but the authors observed a sex-specific effect of suggesting the presence of ambient odors. In the 

presence of a pleasant or no odor in the testing room, male subjects performed better when they 

were led to believe that no odor was present. Female subjects, however, performed better when they 

thought that they were exposed to an odorant under the same conditions. No such interaction was 

found in the unpleasant fragrance condition. These data again emphasize that, in addition to hedonic 

preferences, expectation of an effect may crucially influence the effects of odorants on human 

performance and that these factors may affect women and men differently. 

Millot et al. (2002) evaluated the influence of pleasant (lavender oil) and unpleasant (pyridine, 

11) ambient odors on performance in a visual or auditory alertness task, and in a divided attention 

task. The results showed that in the alertness task, irrespective of the tested modality, both fragrances 

independent of their hedonic valence improved performance by shortening reaction times compared


to an unscented control condition. However, none of the odorants exerted any influence on performance 

in the selective attention task in which subjects had to attend to auditory stimuli while neglecting 

visual ones. The authors conclude that pleasant odors enhance task performance by decreasing 

subjective feelings of stress, that is, by reducing overarousal, while unpleasant fragrances increase 

activation from suboptimal to optimal levels, thus having the same beneficial effects on cognitive 

performance. With this explanation the authors, however, presume that subjects in their experimental 

groups started from dissimilar arousal levels which seems rather unlikely given that subjects were 

assigned to these groups at random. Moreover, cognitive performance should be affected by alterations 

of the arousal level more readily with increasing task difficulty. Thus, the interpretation given 

by the authors does not thoroughly explain why reaction times were influenced by the odorants in 

the simple alertness task but not in the more sophisticated selective attention task. 

Degel and Köster (1999) exposed healthy subjects to either lavender, jasmine, or no fragrance 

with subjects being unaware of the odorants. Subjects had to perform a mathematical test, a letter 

counting (i.e., selective attention) task, and a creativity test. The authors expected a negative effect 

on performance of lavender and a positive effect of jasmine. The results, however, showed that lavender 

decreased the error rate in the selective attention task, whereas jasmine increased the number 

of errors in the mathematical test. Ratings of odor valence collected after testing demonstrated that 

lavender was judged more pleasant than jasmine, independent of which odor had been presented 

during the testing. Although subjects did not know that a fragrance had been administered, implicit 

evaluation of odor pleasantness has probably influenced their performance. This relation is supported 

by the fact that subjects who were not able to correctly identify the odors preferentially 

associated pictures of the room they had been tested in with the odor that had been present during 

the testing. Improvement of performance as a result of the inhalation of lavender EO has also been 

reported in another investigation (Sakamoto et al., 2005). In this study, subjects were exposed to 

lavender, jasmine or no aroma during phases of rest in-between sessions in which they completed a 

visual vigilance task involving tracking of a moving target. In the penultimate of five sessions, when 

fatigue was highest and arousal lowest as estimated from the decrement in performance between 

sessions of the control group, tracking speed increased and tracking error decreased in the lavender 

group when compared to the no-aroma group. Jasmine had no effect on task performance. The 

authors argued that lavender aroma may have decreased arousal during the resting period and hence 

helped to achieve optimal levels for the following task period. Since no secondary variables indicative 

of arousal or of subjective evaluation of aroma quality were assessed in this investigation, no 

inferences can be made on the mechanisms underlying the observed effects. Diego et al. (1998) in 

the aforementioned investigation studied the influence of lavender and rosemary EOs after a 3-min 

inhalation period on a mathematical task. In contrast to the authors’ expectations, both odorants 

positively affected performance by increasing calculation speed, although only lavender improved 

calculation accuracy. In addition, subjects in both fragrance groups reported to be more relaxed. 

Those in the lavender group had less depressed mood, whereas those in the rosemary group felt 

more alert and had lower state anxiety scores. These findings were interpreted as indicating overarousal 

caused by rosemary EO, which led to an increase of calculation speed at the cost of accuracy. 

In contrast, lavender EO seemed to have reduced the subjects’ arousal level and thus led to 

better performance than rosemary EO. However, since subjects in both fragrance groups felt more 

relaxed—but obviously only the lavender group benefited from this increase in relaxation—this is 

a somewhat unsatisfying explanation for the observed results. 

Evidence for the influence of physico-chemical odorant properties on visual information processing 

was supplied by Michael et al. (2005). These authors found that the exposure to both allyl 

isothiocyanate (AIC, 12), a mixed olfactory/trigeminal stimulus, and 2-phenyl ethyl alcohol 

(2-PEA, 13), a pure olfactory stimulant, impaired performance in a highly demanding visual attention 

task involving reaction to a target stimulus when a distractor appeared at different intervals 

after presentation of the target, although different mechanisms were responsible for these effects. In 

trials without a distractor, only 2-PEA (13) significantly increased the reaction times of healthy


subjects; in trials with a distractor, subjects reacted more slowly in both odor conditions as compared 

to the no-odor control condition. However, AIC impaired performance, independent of the 

interval between distractor and target, whereas 2-PEA (13) only had a negative effect when the 

interval between target and distractor was short. While 2-PEA (13) seemed to have led to performance 

decrements by decreasing subjects’ arousal level, AIC as a strong trigeminal irritant seemed 

to have affected the shift of attention toward the distractor stimuli, so that they were considered 

more important than in the other conditions. 

Differences in effectiveness of fragrances as a function of the route of administration were 

explored by Heuberger et al. (2008). In several experiments, these authors investigated the influence 

of two monoterpenes, that is, 1,8-cineole (2) and (±)-linalool (3), on performance in a visual sustained 

attention task after 20 min inhalation and dermal application, respectively. 1,8-cineole (2) 

was expected to induce activation and improve task performance whereas (±)-linalool (3) was considered 

sedating/relaxing thus impairing performance. Since one of the aims of the study was to 

assess fragrance effects that were not mediated by stimulation of the olfactory system, inhalation of 

the odorants was prevented in the dermal application conditions. In each condition, subjects rated 

their mood and well-being. In addition, ratings of odor pleasantness, intensity, and effectiveness 

were assessed in the inhalation conditions. In regard to performance on the vigilance task, the results 

showed no difference between the fragrance groups compared to a control group, which had received 

odorless air. However, 1,8-cineole (2) increased feelings of relaxation and calmness whereas 

(±)- linalool (3) led to increased vigor and mood. In addition, individual performance was correlated 

to the pleasantness of the odor and of expectations of its effect. In contrast, in the dermal application 

conditions, subjects having received 1,8-cineole (2) performed faster than those having received 

(±)-linalool (3). These findings were interpreted as indicating the involvement of different mechanisms 

after inhalation and nonolfactory administration of fragrances. It seems that psychological 

effects are predominant when fragrances are applied by means of inhalation, that is, when the sense 

of smell is stimulated. On the other hand, pharmacological effects of odorants that might be overridden 

when fragrances are inhaled are evident when processing of odor information is prevented. 

10.1.4.2 Learning and Memory 

Effects of EOs and fragrances on memory functions and learning have less frequently been explored 

than influences on more basic cognitive functions. While learning can briefly be defined as “a process 

through which experience produces a lasting change in behavior or mental processes” (Zimbardo 

et al., 2003, p. 206), memory is a cognitive system composed of three separate subsystems or stages 

that cooperate closely to encode, store, and retrieve information. Sensory memory constitutes the 

first of the three memory stages and is responsible for briefly retaining sensory information. The 

second stage, working memory, transitorily preserves recent events and experiences. Long-term 

memory, the third subsystem, has the highest capacity of all stages and stores information based on 

meaning associated with the information (Zimbardo et al., 2003). A basic form of learning, which 

has been identified as a potent mediator of fragrance effects in humans (Jellinek, 1997), is conditioning, 

that is, the (conscious or unconscious) association of a stimulus with a specific response or 

behavior. For instance, Epple and Herz (1999) demonstrated that children who were exposed to an 

odorant during the performance of an insolvable task performed worse on other, solvable tasks 

when the same odorant was presented again. In contrast, no such impairment was observed when no 

odor or a different odor was presented. These results were interpreted as demonstrating negative 

olfactory conditioning. Along the same lines, Chu (2008) was able to show positive olfactory conditioning. 

In his study, children successfully performed a cognitive task, which they believed was 

insolvable in the presence of an ambient odor. When they were re-exposed to the same odorant, 

performance on other tasks improved significantly in comparison to another group of children who 

received a different odor. Since the children in Chu’s investigation were described in school reports 

as underachieving and lacking self-confidence, one might speculate that only children with these 

specific attributes benefit the influence of fragrances. This seems to be confirmed by the study of


Kerl (1997), who found that, in general, ambient odors of lavender and jasmine did not improve 

memory functions in school children. However, lavender tended to increase performance in the 

memory task in children with high anxiety levels, which may have been consequent to the stressrelieving 

properties of lavender. On the other hand, jasmine impaired performance in lethargic 

children and this impairment in performance was negatively correlated with the children’s rating of 

the odor. This result might indicate that lethargic children were distracted by the presence of an 

odorant they liked. 

Several studies examined the influence of EOs on a number of memory-related variables in 

adults (Moss et al., 2003, 2008; Tildesley et al., 2005). These authors reported that lavender reduced 

the quality of memory and rosemary increased it, while both EOs reduced the speed of memory 

when compared to a no-odor control condition. At the same time, rosemary increased alertness in 

comparison to both the control and the lavender group, but exposure to the odorants led to higher 

contentedness than no scent. Similarly, peppermint enhanced memory quality while ylang-ylang 

impaired it and reduced processing speed. Ratings of mood showed that peppermint increased alertness 

while ylang-ylang decreased it and increased subjective calmness. In a third experiment, oral 

administration of Spanish sage [Salvia lavandulifolia Vahl (Lamiaceae)] improved both quality and 

speed of memory, and increased subjective ratings of alertness, calmness, and contentedness. The 

beneficial influence of an odorant being present in the learning phase on successive retrieval of 

information was shown by Morgan (1996). In this study, subjects were exposed or not exposed to a 

fragrance during the encoding of words unrelated to odor. Recall of the learned material was tested 

in three unannounced sessions 15 min apart, as well as 5 days after the learning phase. The results 

showed that performance in those groups that had not been exposed to an odorant in the learning 

phase declined continuously over time whereas it remained stable in those groups that had learned 

with ambient odor present. In addition, subjects who had learned under odor exposure performed 

significantly better when the odor was present during recall than those who had not received an 

odorant during the learning phase. These findings show that odorants in the encoding phase may 

serve as cues for later recall of the stored information. Recently, similar results have been reported 

in subjects who were presented with odorants while asleep (Rasch et al., 2007) demonstrating that 

fragrances may prompt memory consolidation during sleep. According to a study by Walla et al. 

(2002), it seems to be crucial whether an odorant in the encoding phase of a mnemonic task is consciously 

perceived and processed. These authors found differences in brain activation in a word 

recognition task as a function of conscious versus unconscious olfactory processing in the encoding 

phase. In other words, when odorants were presented during the learning phase and consciously 

perceived word recognition was more likely negatively affected than when the odor was not consciously 

processed. In addition, the same group of researchers demonstrated that word recognition 

performance was significantly poorer when the odorants were presented simultaneously with the 

words as opposed to continuously during the encoding phase, and when semantic (deep) as opposed 

to nonsemantic (shallow) encoding was required. These effects can be explained by a competition 

of processing resources in brain areas engaged in both language and odor processing (Walla et al., 

2003). Similar results were also observed in an experiment involving the encoding of faces with and 

without odorants present in the learning phase (Walla et al. 2003). Again, recognition accuracy was 

impaired when an odor was simultaneously presented during encoding. 

As discussed above, subjective experience of valence seems to modulate the influence of 

fragrances on cognition. For instance, Danuser et al. (2003) found no effects of pleasant olfactory 

stimuli on short-term memory whereas unpleasant odorants reduced the performance of healthy 

subjects, probably by distracting them. Habel et al. (2007) studied the effect of neutral and unpleasant 

olfactory stimulation on the performance of a working memory task and found that malodors 

significantly deteriorated working memory in only about half of the subjects. It was also shown that 

subjects in the affected group differed significantly in brain activation patterns from those in the 

unaffected group, that is, the latter showing stronger activation in fronto-parieto-cerebellar networks 

associated with working memory. In contrast, subjects whose performance was impaired by


the unpleasant odor showed greater activation in areas associated with emotional processing, such 

as the temporal and medial frontal cortex. The authors concluded that individual differences exist 

for the influence of fragrances on working memory and that unaffected subjects were better able to 

counteract the detrimental effect of unpleasant odor stimuli. 

Leppanen and Hietanen (2003) tested the recognition speed of happy and disgusted facial expressions 

when pleasant or unpleasant odorants were presented during the recognition task. This study 

showed that pleasant olfactory stimuli had no particular influence on the speed of recognition of 

emotional facial expressions, that is, happy faces were recognized faster than disgusted faces. This 

result was also observed when no odorant was administered. In the unpleasant condition, however, 

the advantage for recognizing happy faces disappeared. These findings were interpreted as evidence 

for the modulation of emotion-related brain structures that form the perceptual representation of 

facial expressions by unpleasant odorants. Walla et al. (2005) supplied evidence that performance 

in a face recognition task was only affected when conscious odor processing took place In this 

study, two olfactory stimuli, that is, 2-PEA (13) and dihydrogen sulfide (H 2 S), a trigeminal stimulus, 

that is, carbon dioxide (CO 2 ), and no odor were presented briefly and simultaneously to the presentation 

of faces. The results showed that the pure odorants irrespective of their valence improved 

recognition performance whereas CO 2 decreased it, and only CO 2 , which is associated with painful 

sensations, was processed consciously by the participants of this investigation. 

With regard to the content of memory, some researchers have claimed a special relationship 

between autobiographical, that is, personally meaningful episodic, memories and fragrances. As a 

result of this special link, it has been observed that memories evoked by olfactory cues are often 

older, more vivid, more detailed, and more affectively toned than those cued by other sensory 

stimuli (Chu and Downes, 2002; Goddard et al., 2005; Willander and Larsson, 2007). This phenomenon 

has been explained by the peculiar neuroanatomical connection of the memory systems with 

the emotional systems. Evidence for this hypothesis has for instance been supplied by the group of 

Herz (Herz and Cupchik, 1995; Herz, 2004; Herz et al., 2004), who demonstrated that presentation 

of odorants resulted in more emotional memories than presentation of the same cue in auditory or 

visual form. The authors also showed that if the odor cue was hedonically congruent with the item 

that had to be remembered, memory for associated emotional experience improved. Moreover, 

personally salient fragrance cues were associated with higher functional activity in emotion-related 

brain regions, such as the amygdala and the hippocampus. 

10.1.4.3 Other Cognitive Tasks 

The study of Degel and Köster (1999) described earlier showed that under certain conditions odorants 

may influence attentional performance even without subjects being aware of their presence. 

According to an investigation by Holland et al. (2005) the unnoticed presence of odorants may also 

affect everyday behavior and higher cognitive functions. The authors reported that subliminal concentrations 

of a citrus-scented cleaning product increased identification of cleaning-related words in 

a lexical decision task. Moreover, subjects exposed to the subliminal odor listed cleaning-related 

activities more frequently when asked to describe planned activities during the day and kept their 

environment tidier during an eating task. 

The effects of pleasant suprathreshold fragrances on other higher cognitive functions were, for 

instance, investigated by Baron (1990). In this study, subjects were exposed to pleasant or neutral 

ambient odors while solving a clerical coding task and negotiating about monetary issues with a 

fellow participant. Before performing these tasks subjects indicated self-set goals and self-efficacy. 

Following the tasks subjects rated the experimental rooms in terms of pleasantness and comfort, 

as well as their mood. In addition, they were asked which conflict management strategies they 

would adopt in the future. Although neither fragrance had a direct effect on performance in the 

clerical task, subjects in the pleasant odor condition set higher goals and adopted a more efficient 

strategy in the task than those in the neutral odor condition. In addition, male subjects in the 

pleasant odor condition rated themselves as more efficient than those in the neutral odor condition.


In the negotiation task, subjects in the pleasant odor condition set higher monetary goals and made 

more concessions than those in the neutral odor condition. Moreover, subjects in the pleasant odor 

condition were in better mood and reported planning to handle future conflicts less often through 

confrontation and avoidance. Thus, this study showed that pleasant ambient fragrances offer a 

potential to create a more comfortable work environment and diminish aggressive behavior in 

situations involving competition. Gilbert et al. (1997) examined the effect of a pleasant and an 

unpleasant blend of fragrances on the same clerical coding task but were not able to show any 

influence of either fragrance on task performance. However, subjects exposed to unpleasant odorants 

believed that these odorants had negative effects on their performance in simple and difficult 

mathematical and verbal tasks (Knasko, 1993). 

Finally, Ludvigson and Rottman (1989), studying the influence of lavender and clove EOs in 

ambient air in comparison to a no-odor control condition, found that lavender impaired performance 

in a mathematical reasoning task, while clove was devoid of effects. However, the lavender 

effect was only observed in the first of two sessions held one week apart. Also, subjects in the 

lavender condition rated the experimental conditions more favorably, while clove odor decreased 

subjects’ willingness to return to the second session. Moreover, subjects who were exposed to an 

odor in one of the two sessions were generally less willing to return and had worse mood than subjects 

who never received an odor. To further complicate things, these odorant by session interactions 

were related to personality factors in a highly complex manner. 

10.1.5 CONCLUSIONS 

The presented review of the literature on the effects of EOs and fragrances on human arousal and 

cognition demonstrates that coherent findings are quite scarce and that we are still far from painting 

a detailed picture of which effects can be achieved by administering a particular EO and how these 

effects are precisely exerted. One reason for the inconsistency in results may be that in most studies 

in humans clear associations between constituents of EOs and observed effects are missing. While 

exact specifications about the origin, composition, and concentration of the tested oils would be 

necessary to establish clear pharmacological profiles and dose response curves for specific oils, in 

many investigations no such details are given. This renders attempts to compare the results from 

different studies and to generalize findings rather difficult. 

Another aspect that clearly contributes to inconclusive findings is the involvement of a variety of 

mechanisms of action in the effects of EOs on human arousal and cognition. In a very valuable 

review on the assessment of olfactory processes with electrophysiological techniques, Lorig (2000) 

points out that EEG changes induced by odorants have to be interpreted with great care, since other 

direct odor effects may be responsible for changes, particularly when relaxing effects reflected by 

the induction of slow-wave activity are concerned. Physiological processes, such as altered breathing 

patterns in response to pleasant versus unpleasant odors, cognitive factors, for example, as a consequence 

of expectancy or the processing of secondary stimulus features, or an inappropriate baseline 

condition can lead to changes of the EEG pattern, which are quite unrelated to any psychoactive 

effect of the tested odorant. Well-designed paradigms are thus necessary to control for cognitive 

influences that might mask substance-specific effects of fragrances. Also, while EEG and other 

electrophysiological techniques are highly efficient to elucidate fragrance effects on the CNS in the 

time domain, we know only little about spatial aspects of such effects. Brain imaging techniques, 

such as functional magnetic resonance imaging (fMRI), will prove valuable to address this issue. 

Using fMRI, preliminary results from the author’s group (Friedl et al., 2007) have shown that, after 

prolonged exposure, fragrances alter neuronal activity in distinct regions of the brain in a timedependent 

manner and that this influence is sex specific. 

When evaluating psychoactive effects of EOs and fragrances on human cognitive functions, the 

results should be interpreted just as cautiously as those of electrophysiological studies as similar 

confounding factors, ranging from influences of stimulus-related features, for example, pleasantness,


to expectation of fragrance effects and even personality traits, may be influencing the observed 

outcome. In regard to higher cognitive functioning, such as language or emotional processing, conscious 

as opposed to sub- or unconscious processing of odor information seems to differentially 

affect performance due to differences in the utilization of shared neuronal resources. Again, it 

seems worthwhile to measure additional parameters that are indicative of (subjective) stimulus 

information processing and emotional arousal if hypotheses are being built on direct (pharmacological) 

and cognitively mediated (psychological) odor effects on human behavior. Moreover, comparisons 

of different forms of application that involve or exclude stimulation of the olfactory system, 

such as inhalative versus noninhalative, dermal administration, have proven useful in the distinction 

of pharmacological from psychological mechanisms and will serve to enlarge our understanding of 

psychoactive effects of EO and fragrances in humans. 

10.2 PSYCHOPHARMACOLOGY OF ESSENTIAL OILS 



The use of aromas goes back to ancient times, as implied by the nearly 200 references in the Bible 

relating the use of aromas for “mental, spiritual, and physical healing” (Perry and Perry, 2006). It is 

currently accepted that aromas and some of its individual components may in fact possess pharmacological 

and/or psychological properties, and in many instances the overall effect is likely to result 

from a combination of both. The following sections review the psychopharmacology of EOs and/or 

its individual components, as well as its mechanisms of action. 

10.2.1 AROMATIC PLANTS USED IN TRADITIONAL MEDICAL SYSTEMS 

AS SEDATIVES OR STIMULANTS 

EOs are generally products of rather complex compositions used contemporaneously in aromatherapy, 

and for centuries as aromatic medicinal plant species in traditional systems of medicine. 

Aromatic formulas are used for the treatment of a variety of illnesses, including those that affect the 

CNS (Almeida et al., 2004). Volatile compounds presenting sedative or stimulatory properties have 

been and continue to be identified in EOs from aromatic medicinal species spread across different 

families and genera. The majority of these substances have small structures with less than 12 carbons 

and present low polarity chemical functions, being therefore quite volatile. Since most natural 

EOs are formed by complex mixtures, their bioactivity(ies) are obviously dependent on the contribution 

of their various components. Therefore, studies failing to characterize at least the main components 

of the EO studied are not discussed in this chapter. 

Several Citrus EOs contain high proportions of limonene (14) as its major component. Orange 

peels are used as sedative in several countries, and EOs obtained from Citrus aurantium L. (Rutaceae) 

fruit peels can contain as much as 97.8% of limonene (14). The anxiolytic and sedative properties of 

Citrus EO suggested by traditional uses have been assessed in mice (Carvalho-Freitas and Costa, 

2002; Pultrini et al., 2006) and also shown in a clinical setting (Lehrner et al., 2000). The relaxant 

effects observed in female patients in a dental office were produced with a Citrus sinensis (L.) 

Osbeck (Rutaceae) EO composed of 88.1% limonene (14) and 3.77% myrcene (15). 

The common and large variability in the composition of natural EOs poses difficulties for the 

evaluation and the safe and effective use of aromatic medicinal plants. Genetic variations led to the 

occurrence of chemotypes, as in the case of Lippia alba (P. Mill.) N.E. Br. ex Britt. & Wilson 

(Verbenaceae). Analyses revealed three monoterpenic chemotypes characterized by the prevalence 

of myrcene (15) and citral (16) in chemotype I, limonene (14) and citral (16) in chemotype II, and 

limonene (14) and carvone (17) in chemotype III (Matos, 1996). This species is known in Brazil as 

“cidreira”; the aromatic tea from its leaves is traditionally used as a tranquilizer being one of the


most widely known home-made remedies. Pharmacological assays showed anxiolytic (Vale et al., 

1999) and anticonvulsant (Viana et al., 2000) effects of EO samples from all three chemotypes. 

Anticonvulsive and sedative effects in mice were also demonstrated for the three isolated principal 

constituents of Lippia alba oils: limonene (14), myrcene (15), and citral (16) (Vale et al., 2002). 

Various Ocimum species are used traditionally for sedative and anticonvulsive purposes, and its 

EOs seem to play an important role for these properties. However, besides the normal variability in 

the composition of the EOs, the occurrence of chemotypes seems to be generalized in this genus 

(Grayer et al., 1996; Vieira et al., 2001). The comparison of EO samples from different accessions 

of Ocimum basilicum L. (Lamiaceae) pointed to the occurrence of linalool (3) and methylchavicol 

(18) types, and it has been suggested that such data should be taken into account for an intraspecific 

classification of the taxon (Grayer et al., 1996). An EO of O. basilicum L. of the linalool-type containing 

44.18% linalool (3), 13.65% 1,8-cineole (2), and 8.59% eugenol (4) as main constituents 

presented anticonvulsant and hypnotic activities (Ismail, 2006). There are two varieties of Ocimum 

gratissimum L. (Lamiaceae) (O. gratissimum L. var. gratissimum and O. gratissimum var. macrophyllum 

Briq.) that form a polymorphic complex very difficult to differentiate by morphological 

traits (Vieira et al., 2001). It was clearly demonstrated that the genetic variations of O. gratissimum 

led to three different chemotypes (Vieira et al., 2001): eugenol (4), thymol (19), and geraniol (20). 

EOs obtained from O. gratissimum of the eugenol-type collected during the 4 year seasons contained 

eugenol (4) (44.89–56.10%) and 1,8-cineole (2) (16.83–33.67%) as main components, and 

presented sedative and anticonvulsant activities slightly altered by the composition of each sample 

(Freire et al., 2006). A dose-dependent sedative effect was observed in mice and rats (Orafidiya et 

al., 2004) treated with O. gratissimum thymol-type EO containing 47.0% thymol (19), 16.2% p-cymene 

(21), and 6.2% a-terpinene (22) as major constituents. 

Among Amazonian traditional communities, a widespread recipe that includes Cissus sicyoides 

L. (Vitaceae), Aeolanthus suaveolens Mart. ex Spreng. (Lamiaceae), Ruta graveolens L. (Rutaceae), 

and Sesamum indicum L. (Pedaliaceae) was identified as the most frequently indicated for the management 

of epilepsy-like symptoms. The results of pharmacological studies on the traditional preparations 

and the EO obtained from Aeolanthus suaveolens and its principal components led to the 

suggestion that the volatile lactones could be interesting target compounds in the search for new 

anticonvulsant agents (de Souza et al., 1997). Linalool (3) was identified as one of the principal 

active components of the Aeolanthus suaveolens EO (Elisabetsky et al., 1995b), and proved to play 

a key role in the central activities of the traditional preparation (Elisabetsky et al., 1995b, 1999; 

Brum et al., 2001a, b). (R)-(–)-Linalool (7) is recognized today as the sedative and calming component 

of numerous traditional and commercial preparations or their isolated natural EOs (Sugawara 

et al., 1998; Heuberger et al., 2004; Kuroda et al., 2005; Shaw et al., 2007). Epinepetalactone (23) is 

a volatile apolar compound and major component in the EO from Nepeta sibthorpii Benth. 

(Lamiaceae) responsible for the EO anticonvulsant activity (Galati et al., 2004). 

Nature continues to reveal its inventiveness in combining different monoterpenes and arylpropenoids 

to achieve special nuances regarding central activities. An EO obtained from Artemisia 

dracunculus L. (Asteraceae) containing 21.1% trans-anethole (24), 20.6% a-trans-ocimene (25), 

12.4% limonene (14), 5.1% a-pinene (1), 4.8% allo-ocimene (26), and 2.2% methyleugenol (27) 

shows anticonvulsant activity likely to be assigned to a combination of these various monoterpenes 

(Sayyah et al., 2004). b-Asarone (28) and its isomers are recognized as important sedative and 

anticonvulsive active components as in, for example, drugs based on Acorus species in which the 

total proportion of isomers can reach 90% (Koo et al., 2003; Mukherjee et al., 2007). In the EO 

from Acorus tatarinowii Schott (Acoraceae) rhizome, traditionally used for epilepsy, the combination 

of monoterpenes and arylpropenoids was found to be ~25% 1,8-cineole (2), ~12% linalool (3) 

and ~10% b-asarone (28), and isomers (Ye et al., 2006). 

Traditional Chinese medicine (TCM) is known to use especially complex preparations, more 

often than not composed of several plant materials including therefore a number of active principles 

pertaining to different chemical classes. The TCM prescription SuHeXiang Wan combines different


proportions of as much as 15 crude drugs and is used orally for the treatment of seizure, infantile 

convulsion, and other conditions affecting the CNS (Koo et al., 2004). The EO obtained by hexane 

extraction at room temperature from a SuHeXiang Wan composed of nine crude drugs proved to 

have a relatively simple composition: 21.4% borneol (29), 33.3% isoborneol (30), 5.9% eugenol (4), 

and other minor components (Koo et al., 2004). The inhalation of this volatile mixture delayed the 

appearance of pentylenetetrazole (PTZ)-induced convulsions suggesting GABAergic modulation 

(Koo et al., 2004). 

Other monoterpenoid or arylpropenoid derivatives identified as the active components of traditional 

sedatives include: methyleugenol (27) (Norte et al., 2005), isopulegol (31) (Silva et al., 2007), 

and a-terpineol (32) (de Sousa et al., 2007). It is worth mentioning that the monoterpene thujone 

(33), the dangerous principle of the ancient Absinthii herba, Artemisia absinthium L. (Asteraceae), 

induces marked central stimulatory effects, especially when used in the form of liqueur. Frequent 

and excessive use of this drug can cause intoxicated states accompanied by clonic convulsions 

among other serious consequences (Bielenberg, 2007). 

Only a few volatile sesquiterpenes presenting important central activities are currently known. 

b-Eudesmol (34) was found to be one of the volatile active principles of the Chinese medicinal herb 

Atractylodes lancea DC. (Asteraceae) with alleged antagonist properties useful in intoxication by 

anticholinesterase agents of the organophosphorous type (Chiou et al., 1997). Experimental data 

show that b-eudesmol (34) prevents convulsions and lethality induced by electroshock but not those 

induced by PTZ or picrotoxin (Chiou et al., 1995). With a very similar chemical structure, a- eudesmol 

(35) protects the development of postischemic brain injury in rats by blocking v-Aga-IVA-sensitive 

Ca 2+ channels (Asakura et al., 2000). 

The sesquiterpenes caryophyllene oxide (36) and b-selinene (=b-eudesmene) (37) isolated from 

the hexane extract from leaves of Psidium guajava var. minor Mattos (Myrtaceae) potentiated 

pentobarbital sleep and increased the latency for PTZ-induced convulsions in mice; blockade of 

extracellular Ca 2+ was observed in isolated guinea-pig ileum with the hexane extract and its fractions 

containing both sesquiterpenes (Meckes et al., 1997). The similarity between the chemical structures 

of the sesquiterpenes (34), (35), and (37) is noteworthy and could indicate relevant characteristic 

patterns required for central activity. 

10.2.2 EFFECTS OF EOS IN ANIMAL MODELS 

Pure compounds isolated from aromas and complex EOs have been proved to induce a variety of 

effects on human and other mammalian species. Biological properties such as antispasmodic 

(Carvalho-Freitas et al., 2002) or other autonomic nervous system-related activities (Haze et al., 

2002; Sadraei et al., 2003) are outside the scope of this chapter. Central effects have been extensively 

documented, and fall more often than not into the sedative (Buchbauer et al., 1991, 1993; Elisabetsky 

et al., 1995a; Lehrner et al., 2000), anxiolytic (Diego et al., 1998; Cooke and Ernst, 2000; Lehrner 

et al., 2000; Carvalho-Freitas et al., 2002), antidepressant (Komori et al., 1995a,b), and hypnotic 

(Diego et al., 1998) categories. As earlier stated, it is likely that the overall effects of EOs in humans 

results from a combination of physiological (in this case psychopharmacological) and psychological 

effects. Even when accepting the limitations of the validity of animal models in regard to assessing 

the effects of drugs on complex human emotional states, in the case of EOs the usefulness of experiments 

with rodent models is largely limited to clarify the physiological part of the potential effects in 

humans. Again, the reproducibility of the data compiled in this section would be highly dependent 

on the composition of EOs, unfortunately not always adequately reported. 

Relevant to the data that follows, it has been shown that individual components of EOs administered 

orally, by means of intraperitoneal or subcutaneous injections, dermally, or by inhalation do 

reach and adequately cross the blood brain barrier (Kovar et al., 1987; Jirovetz et al., 1990; Buchbauer 

et al., 1993; Fujiwara et al., 1998; Moreira et al., 2001; Perry et al., 2002). The question of whether 

psychopharmacological effects in animals are dependent on olfactory functions is surprisingly not


yet entirely clarified. Cedrol (from pine EO) was shown to be sedative in normal rats and rats made 

anosmic with zinc sulfate (Kagawa et al., 2003). In contrast, a mix of chamomile and lavender oils 

reduced pentobarbital-induced sleeping time in normal but not anosmic rats and mice (Kagawa 

et al., 2003). 

Rose and lavender oil, administrated intraperitoneally (i.p.) to mice, showed anticonflict effects 

(Umezu, 1999; Umezu et al., 2002), suggesting anxiolytic properties. Unequivocal anxiolytic properties 

were also demonstrated for lavender EO (i.p., gerbils) with the elevated plus maze test (Bradley 

et al., 2007a). Chen et al. (2004) reported that Angelica sinensis (Oliv.) Diels (Apiaceae) EO (orally 

administrated, mice) also has anxiolytic effect in the elevated plus maze, the light/dark model, and 

the stress-induced hyperthermia paradigms. 

The dry rhizomes of Acorus gramineus Soland (Acoraceae) are officially listed in the Korean 

pharmacopoeia for sedative, digestive, analgesic, diuretic, and antifungal effects (Koo et al., 2003). 

Various CNS effects have been characterized for Acorus gramineus EO, including antagonism of 

PTZ-induced convulsion, potentiation of pentobarbital-induced sleeping time, sedation, and 

decreased spontaneous locomotion with the water and methanol extracts (mice, i.p.) (Vohora et al., 

1990; Liao et al., 1998). Unexpectedly, given that sedative drugs usually impair cognition in animals, 

the oral administration of Acorus gramineus rhizoma EO, with eugenol (4) as the principal 

component, improved cognitive function in aged rats and mice; based on brain amine analyses, the 

authors suggest that such effects may be related to increased norepinephrine, dopamine, and serotonin 

relative levels, and to decreased activity of brain acetylcholinesterase (Zhang et al., 2007). 

Cymbopogon winterianus Jowitt (Poaceae), popularly known as “citronella” and “java grass,” is 

an important EO yielding aromatic grass, mostly cultivated in India and Brazil. Cymbopogon winterianus 

EO is rich in citronellal (38), geraniol (39), and citronellol (40) (Cassel and Vargas 2006), 

and has demonstrated anticonvulsant effects (i.p., mice) (Quintans-Júnior et al., 2007). Pharmacological 

studies with Cymbopogon citratus Stapf EO [presenting high percentage of citral (16) in 

its composition] revealed anxiolytic, hypnotic, and anticonvulsant properties when orally administrated 

to mice (Blanco et al., 2007). 

The EO of Eugenia caryophyllata Thunb (Myrtaceae), used in Iranian traditional medicine, 

exhibits anticonvulsant activity against tonic seizures induced by maximal electroshock (MES) (i.p., 

mice) (Pourgholami et al., 1999a). The leaf EO of Laurus nobilis L. (Lauraceae), which has been 

used as an antiepileptic remedy in Iranian traditional medicine, demonstrated more anti convulsant 

activity against experimental seizures induced by PTZ (i.p., in mice) than MES-induced seizures. 

Components responsible for this effect may include methyleugenol, eugenol, and pinene present in 

the EO (Sayyah et al., 2002). In the same manner, EO obtained from fruits of Pimpinella anisum S.G. 

Gmel. (Umbelliferae) demonstrated anticonvulsant activity against seizures induced by PTZ or MES 

in mice (i.p.) (Pourgholami et al., 1999b). As mentioned earlier, since no chemical analysis is reported 

for the studied EOs one can only speculate on the components that may be responsible for the activities 

observed with E. caryophyllata, Laurus nobilis, and Pimpinella anisum. 

Various Thymus species of the Lamiaceae family (including Thymus fallax Fisch. & Mey., 

Thymus kotschyanus Boiss. & Hohen., Thymus pubescens Boiss. & Kotschy ex Celak, Thymus 

vulgaris M. Bieb.), which are widely distributed as aromatic and medicinal plants in many regions 

of Iran, have shown CNS effects (Duke et al., 2002). Pharmacological studies in mice (i.p.) with 

Thymus fallax, Thymus kotschyanus, and Thymus pubescens containing, respectively, 30.2% of 

carvacrol (41), 18.7% of pulegone (42), and 32.1% of carvacrol (41), demonstrated that the Thymus 

fallax EO has more antidepressant activity than Thymus kotschyanus and Thymus pubescens during 

the forced swimming test (Morteza-Semnani et al., 2007). These results illustrate that minor 

components of EOs can modify the activity of main components, reaffirming the importance of 

chemically characterizing EOs in order to understand its overall bioactivity. 

An EO fraction obtained from powdered seeds of Licaria puchury-major (Mart.) Kosterm. 

(Lauraceae) containing 51.3% of safrol (43), 3.3% of eugenol (4), and 2.9% of methyleugenol (27) 

reduced locomotion and anesthetized mice, as well as affording protection against electroshockinduced 

convulsions (Carlini et al., 1983).


10.2.2.1 Effects of Individual Components 

Section 10.2 listed the effects of several EO components found in traditionally used species, including 

a-pinene (1), eugenol (4), limonene (14), myrcene (15), citral (16), epi-nepetalactone (23), transanethole 

(24), a-trans-ocimene (25), allo-ocimene (26), methyleugenol (27), borneol (29), isoborneol 

(30), citronellal (38), geraniol (39), and citronellol (40); anticonvulsive, muscle relaxants, anxiolityc 

and/or hypnotic properties were observed with these compounds when given i.p. to mice (Vale et al., 

1999, 2002; Galati et al., 2004; Koo et al., 2004; Sayyah et al., 2004; Cassel and Vargas, 2006; 

Blanco et al., 2007; Quintans-Júnior et al., 2007). 

Linalool (3) is a monoterpene commonly found as a major volatile component of EOs in several 

aromatic plant species, such as Lavandula angustifolia Mill (Lamiaceae), Rosa damascena Mill. 

(Rosaceae), Citrus bergamia Risso (Rutaceae), Melissa offi cinalis L. (Lamiaceae), Rosmarinus 

offi cinalis L. (Lamiaceae), Cymbopogon citratus DC ex Nees (Poaceae), and Mentha piperita L. 

(Lamiaceae). Interestingly, many linalool-producing species are traditionally used as sedative, analgesic, 

hypnotic, or anxiolytic remedies in traditional medicine and some as well in aromatherapy 

(Elisabetsky et al., 1995a). 

As mentioned above, Aeolanthus suaveolens Mar. ex Spreng. (Lamiaceae) is used as an anticonvulsant 

through the Brazilian Amazon. The EO obtained from Aeolanthus suaveolens and its main 

component linalool (3) proved to be anticonvulsant against several types of experimental convulsions, 

including those induced by PTZ and transcorneal electroshock (Elisabetsky et al., 1995a), 

intracerebraly injected quinolinic acid, and i.p. NMDA (Elisabetsky et al., 1999). Moreover, psychopharmacological 

evaluation of linalool (3) showed dose-dependent marked sedative effects, 

including hypnotic, hypothermic, increased sleeping time, and decreased spontaneous locomotion 

in mice (i.p.) (Elisabetsky et al., 1995a; Linck et al., 2008). Decreased motor activity was also 

reported in mice by Buchbauer’s group (Buchbauer et al., 1991). Indicating anxiolityc properties 

linalool (3) was reported to have anticonflict effects (mice, i.p.) in the Geller and Vogel tests, and 

similar findings were reported for lavender oil (Umezu, 2006). Analgesic properties were observed 

against chemical (rats, p.o.; Barocelli et al., 2004) (mice, s.c.; Peana et al., 2004a, 2004b) and thermal 

(mice, s.c., Peana et al., 2003) nociceptive stimuli. Since anti-inflammatory activity (rats, s.c.; 

Peana et al., 2002) has also been reported, it is not clear if linalool-induced analgesia is of central 

origin. Nevertheless, these experimental data are relevant to clinical studies indicating that aromatherapy 

with lavender can reduce the demand for opioids during the immediate postoperative 

period (Kim et al., 2007), and deserve further investigation. Linalool local anesthetic effects were 

observed in vivo by the conjunctival reflex test, and in vitro by phrenic nerve-hemidiaphragm preparation 

(Ghelardini et al., 1999). 

10.2.2.2 Effects of Inhaled EOs 

Despite the wide and growing use of aromatherapy in the treatment of a diversity of ailments, including 

those of central origin (Perry and Perry, 2006), and the alleged effects of incenses and other means 

of ambient aromas, experimental data on psychopharmacological properties of inhaled EOs is surprisingly 

scarce. Moreover, few of the studies control for inhalation flow and it is difficult to estimate 

the actual concentration of whatever is being inhaled. However, evidences that EOs and its components 

are absorbed by inhalation are available. After mice were exposed to a cage loaded with 27 mg 

of linalool (3) for 30, 60, or 90 min, plasma linalool concentrations of, respectively, 1.0, 2.7, and 

3.0 ng/mL were found (Buchbauer et al., 1991); these exposure schedules resulted in an exposuredependent 

decrease in locomotion. Moreover, detectable plasma concentrations at the nanogram level 

were reported for as many as 40 different aromatic compounds after 60 min of inhalation (Buchbauer 

et al., 1993). Therefore, despite the lack of precise measures of inhaled and/or absorbed quantities, it 

is arguable that animal studies with inhaled aromas are nevertheless informative. 

Inhalation of citrus-based aromas [Citrus sinensis (L.) Osbeck (Rutaceae)] or fragrances were 

found to restore stress-induced immunosuppression (Shibata et al., 1990) and antidepressant-like 

effects in rats (2 mL/min EO in the air flux) (Komori et al., 1995a). A clinical study with depressed


patients revealed that it was possible to reduce the needed antidepressants’ doses by inhaling a 

mixture of citrus oils; moreover, inhalation of the oil by itself was antidepressive and normalized 

neuroendocrine hormone levels (cortisol and dopamine) in depressive patients (Komori et al., 1995b). 

Relevant to these findings, inhaled lemon oil [Citrus limonum Risso (Rutaceae)] has been shown to 

increase the turnover of dopamine and serotonin after inhalation in mice (Komiya et al., 2006). 

Anxiolytic proprieties were observed in rats with the plus maze model after 7 min rose oil inhalation; 

this is the only report of inhaled effects in animals after a short period of inhalation, although 

the procedure of leaving four cotton balls embedded with 2 mL of EO lacks standardization (Almeida 

et al., 2004). As mentioned above, inhaled cedrol was shown to be sedative in rats (Kagawa et al., 

2003), and the inhalation of a volatile mixture from the TCM SuHeXiang Wan composed of 21.4% 

borneol (29), 33.3% isoborneol (30), 5.9% eugenol (4), and other minor components delayed the 

appearance of PTZ-induced convulsions suggesting GABAergic modulation (Koo et al., 2004). 

Inhaled lavender oil [0–1–2 mL, 25% of linalool (3) and 46% of linalyl acetate (44)] demonstrated 

anxiolytic effects in rats, as shown by decreased peripheral movement and defecation in an open 

field, after at least 30 min of inhalation (Shaw et al., 2007). Similar effects were observed with 

inhaled lavender containing 38.47% of linalool (3) and 43.98% of linalyl acetate (44) in gerbils, 

showing increased exploratory behavior in the elevated plus maze test after 1 or 14 days inhalation 

(Bradley et al., 2007a). In agreement with studies with inhaled lavender EO, anxiolytic activity was 

observed in mice after inhaling (±)-linalool (3) at 1% or 3% inhaled for 60 min using the light/dark 

and an immobilization-induced stress paradigms; moreover, after the same inhalation procedure 

isolated mice exhibited decreased aggression toward an intruder, and increased social interaction 

was also observed (da Silva et al., 2008). 

Finally, antinociceptive (mice) and gastroprotective effects (rats) of orally given or inhaled 

(60 min in a camera saturated with 2.4 μL/L) Lavandula hybrida Rev. (Lamiaceae) EO and its 

principal constituents linalool (3) and linalyl acetate (44) have also been reported (Barocelli et al., 

2004). 

10.2.3 MECHANISM OF ACTION UNDERLYING PSYCHOPHARMACOLOGICAL EFFECTS OF EOS 

The mechanisms of action underlying the effects of fragrances as complex mixtures as found in 

natura, or even for isolated components are far from been clarified, but seem to differ among 

different fragrances (Komiya et al., 2006). If the overall pharmacological effects of an EO depend 

on the contribution of its various components, the mechanism of action of a complex mixture is 

far more complex than a simple sum of each component’s physiological consequence. Interaction 

among the various substances present in EOs can modify each other’s pharmacodynamic and 

pharmacokinetic properties; nevertheless, studying the pharmacodynamic basis of isolated components 

is helpful for a comprehensive understanding of the basis of the physiological and psychopharmacological 

effects of EOs. 

Rats treated (i.p.) with the isolated components of lemon EO, such as R-(+)-limonene (14), 

S-(−)-limonene (45), and citral, did not show the cold stress-induced elevation in norepinephrine 

and dopamine (Fukumoto et al., 2008); the authors suggested that these effects are related to 

changes in monoamine release in rat brain slices induced by these compounds (Fukumoto et al., 

2003). Complementary to these findings, inhaled lemon oil (cage with a cotton ball with 1 mL, 

90 min) has also been reported to increase the turnover of dopamine and serotonin in mice (Komiya 

et al., 2006). 

Inhalation (2 g of fragrance/day, 2 × 3 h/day, for 7, 14, or 30 days at home cages) of Acorus 

gramineus Solander (Acoraceae) EO inhibited the activity of g-aminobutyric acid (GABA) transaminase 

(an enzyme critical for metabolizing GABA at synapses), thereby significantly increasing 

GABA levels; a decrease in glutamate levels was also reported in this study (Koo et al., 2003). Both 

of these alterations in the inhibitory and excitatory neurotransmitter systems are compatible with 

and relevant to the sedative and anticonvulsant effects mentioned earlier. Neuroprotective effects on


cultured neurons were also reported for Acorus gramineus EO, apparently attained through the 

blockade of NMDA receptors given that this oil inhibits [ 3 H]-MK801 binding (Cho et al., 2001). 

Other examples of EO actions on amino acid neurotransmitters are available. Morrone et al. 

(2007) using in vivo microdialysis showed that bergamot EO (i.p.) increased the levels of aspartate, 

glycine, taurine, GABA, and glutamate in a Ca 2+ -dependent manner in rat hippocampus; the authors 

suggested that these same effects may be relevant for other monoterpenes affecting the CNS. 

Inhibition of glutamatemediated neurotransmission is in part responsible for the mechanisms of 

action underlying the above-mentioned anticonvulsant effects of linalool (3). Neurochemical assays 

reveal that (±)-linalool (3) acts as a competitive antagonist of l-[ 3 H]-glutamate binding (Elisabetsky 

et al., 1999), and also shows a dose-dependent noncompetitive inhibition of [ 3 H]-MK801 binding 

(IC 50 =2.97 mM) indicating antagonism of NMDA glutamate receptors (Brum et al., 2001a). 

(±)-Linalool (3) also decreases the potassium-stimulated glutamate release and uptake in mice cortical 

synaptosomes, without affecting basal glutamate release (Brum et al., 2001b). This neurochemical 

profile explains, for instance, the linalool-induced delay in NMDA-induced convulsions 

and blockade of quinolinic acid-induced convulsions (Elisabetsky et al., 1999). Eugenol (4) was also 

found to inhibit excitotoxic neuronal effects induced by NMDA, apparently involving modulation 

of NMDA glutamate receptor, and inhibition of Ca 2+ uptake (Wie et al., 1997; Won et al., 1998). 

Because (±)-linalool (3) is also able to protect against PTZ- and picrotoxin-induced convulsions 

(Elisabetsky et al., 1999), a GABAergic modulation could also be in place. Nevertheless, (±)-linalool 

(3) did not alter [ 3 H]-muscinol binding (Brum, 2001a) suggesting that, if existing at all, linalool 

modulation of the GABAergic system is not mediated by GABA A receptors. 

Potentially relevant to the pharmacology profile of linalool (3), patch-clamp techniques demonstrated 

that the monoterpene (no isomer specified) suppressed the Ca 2+ current in rat sensory neurons 

and in rat cerebellar Purkinje cells (Narusuye et al., 2005). Based on their study with lavender EO, 

Aoshima and Hamamoto (1999) suggested that the GABAergic transmission may be of relevance for 

the mechanism of action of other monoterpenes, such as a-pinene (1), eugenol (4), citronellal (38), citronellol 

(40), and hinokitiol (46) (Aoshima and Hamamoto, 1999). Additionally, the antinociceptive 

activity of R-(−)-linalool (7) seems to involve several receptors, including opioids, cholinergic M 2 , dopamine 

D 2, adenosine A 1 and A 2A , as well as changes in K + channels (Peana et al., 2003, 2004a, 2006). 

Relevant to studies on cognition, Salvia lavandulifolia Vahl (Lamiaceae) EO and isolated 

monoterpene constituents were shown to inhibit brain acetylcholinesterase and to present antioxidant 

properties (Perry et al., 2001, 2002). 

Overall, its seems reasonable to argue that the modulation of glutamate and GABA neurotransmitter 

systems are likely to be the critical mechanisms responsible for the sedative, anxiolytic, and 

anticonvulsant proprieties of linalool and EOs containing linalool (3) in significant proportions. 

CHEMICAL STRUCTURES OF MENTIONED CNS ACTIVE COMPOUNDS 

O 

a-Pinene (1) 

1,8-Cineole (2) 

HO 

MeO 

Linalool (3) 

HO 

Eugenol (4)


O 

O 

O 

O 

O 

Methyl jasmonate (5) 

trans-Jasminlactone (6) 

HO 

HO 

H 3 C 

(R)-(-)-linalool (7) (S)-(+)-linalool (8) 

H 3 C 

D-3-Carene (9) 

O 

Bornyl acetate (10) 

O 

NCS 

N 

Pyridine (11) 

Allyl isothiocyanate (12) 

OH 

2-Phenyl ethyl alcohol (13) Limonene (14) 

O 

Myrcene (15) 

Citral (16) 

O 

MeO 

Methylchavicol (18) 

Carvone (17) 

OH 

Geraniol (20) 

OH 

Thymol (19)


p-Cymene (21) a-Terpinene (22) 

O 

O 

MeO 

trans-Anethole (24) 

Epi-nepetalactone (23) 

trans-Ocimene (25) allo-Ocimene (26) 

MeO 

MeO 

MeO 

MeO 

Methyl eugenol (27) 

b-Asarone (28) 

OMe 

H 

OH 

Borneol (29) 

OH 

H 

Isoborneol (30) 

OH 

OH 

a-Terpineol (32) 

Isopulegol (31) 

O 

OH 

Thujone (33) 

b-Eudesmol (34)


OH 

O 

a-Eudesmol (35) 

Caryophyllene oxide (36) 

Citronellal (38) 

O 

b-Eudesmene (37) 

Geraniol (39) 

OH 

Citronellol (40) 

OH 

HO 

O 

Carvacrol (41) 

O 

Pulegone (42) 

CH 3 COO 

O 

Linalyl acetate (44) 

Safrol (43) 

O 

S-(-)-limonene (45) 

HO 

Hinokitiol (46) 

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11 

Phytotherapeutic Uses of 

Essential Oils 

Bob Harris 

CONTENTS 

11.1 Introduction ..................................................................................................................... 316 

11.2 Acaricidal activity ........................................................................................................... 316 

11.3 Anticarcinogenic ............................................................................................................. 317 

11.4 Antimicrobial .................................................................................................................. 317 

11.4.1 Antibacterial .................................................................................................... 317 

11.4.1.1 Methicillin-Resistant Staphylococcus aureus ............................... 318 

11.4.2 Antifungal ........................................................................................................ 318 

11.4.3 Antiviral .......................................................................................................... 319 

11.4.4 Microbes of the Oral Cavity ............................................................................ 320 

11.4.4.1 Activity of Listerine against Plaque and/or Gingivitis ................... 321 

11.4.4.2 Antiviral Listerine ........................................................................... 321 

11.4.4.3 Activity of Essential Oils ................................................................ 321 

11.4.5 Controlling Microflora in Atopic Dermatitis .................................................. 323 

11.4.6 Odor Management for Fungating Wounds ...................................................... 323 

11.5 Dissolution of Hepatic and Renal Stones ........................................................................ 323 

11.5.1 Gall and Biliary Tract Stones .......................................................................... 323 

11.5.2 Renal Stones .................................................................................................... 325 

11.6 Functional Dyspepsia ...................................................................................................... 325 

11.7 Gastroesophageal reflux .................................................................................................. 326 

11.8 Hyperlipoproteinemia ..................................................................................................... 326 

11.9 Irritable Bowel Syndrome ............................................................................................... 327 

11.10 Medical Examinations .................................................................................................... 328 

11.11 Nausea ............................................................................................................................. 329 

11.12 Pain Relief ....................................................................................................................... 329 

11.12.1 Dysmenorrhea .................................................................................................. 330 

11.12.2 Headache ......................................................................................................... 330 

11.12.3 Infantile Colic .................................................................................................. 331 

11.12.4 Joint Physiotherapy .......................................................................................... 331 

11.12.5 Nipple Pain ...................................................................................................... 331 

11.12.6 Osteoarthritis ................................................................................................... 331 

11.12.7 Postherpetic Neuralgia .................................................................................... 331 

11.12.8 Postoperative Pain ........................................................................................... 332 

11.12.9 Prostatitis ......................................................................................................... 332 

11.12.10 Pruritis ............................................................................................................. 332 

11.13 Pediculicidal Activity ...................................................................................................... 332 

11.14 Recurrent Aphthous Stomatitis ....................................................................................... 333 

315


11.15 Respiratory Tract ............................................................................................................. 333 

11.15.1 Menthol ............................................................................................................ 333 

11.15.1.1 Antitussive ....................................................................................... 334 

11.15.1.2 Nasal Decongestant ......................................................................... 334 

11.15.1.3 Inhibition of Respiratory Drive 

and Respiratory Comfort ................................................................ 335 

11.15.1.4 Bronchodilation and Airway Hyperresponsiveness ........................ 335 

11.15.1.5 Summary ......................................................................................... 336 

11.15.2 1,8-Cineole ...................................................................................................... 336 

11.15.2.1 Antimicrobial .................................................................................. 336 

11.15.2.2 Antitussive ....................................................................................... 337 

11.15.2.3 Bronchodilation ............................................................................... 337 

11.15.2.4 Mucolytic and Mucociliary Effects ................................................ 337 

11.15.2.5 Anti-Inflammatory Activity ............................................................ 338 

11.15.2.6 Pulmonary Function ........................................................................ 339 

11.15.2.7 Summary ......................................................................................... 341 

11.15.3 Treatment with Blends Containing both Menthol and 1,8-Cineole ................ 341 

11.16 Allergic Rhinitis .............................................................................................................. 342 

11.17 Snoring ............................................................................................................................ 342 

11.18 Swallowing Dysfunction ................................................................................................. 342 

11.19 Conclusion ....................................................................................................................... 343 

References .................................................................................................................................. 343 


For many, the term “aromatherapy” originally became associated with the concept of the holistic 

use of essential oils to promote health and well-being. As time has progressed and the psychophysiological 

effects of essential oils have been explored further, their uses to reduce anxiety and aid 

sedation have also become associated with the term. This is especially so since the therapy has 

moved into the field of nursing, where such activities are of obvious benefit to patients in a hospital 

environment. More importantly, the practice of aromatherapy (in English-speaking countries) is 

firmly linked to the inhalation of small doses of essential oils and their application to the skin in 

high dilution as part of an aromatherapy massage. 

This chapter is concerned with the medical use of essential oils, given to the patient by all routes 

of administration to treat specific conditions and in comparably concentrated amounts. Studies that 

use essential oils in an aromatherapy-like manner, for example, to treat anxiety by essential oil massage, 

are therefore excluded here. 

Of the literature published in peer-reviewed journals over the last 30 years, only a small percentage 

concerns the administration of essential oils or their components to humans in order to treat 

disease processes. These reports are listed below in alphabetical order of their activity. The exception 

is the section on the respiratory tract, where the many activities of the two principal components 

(menthol and 1,8-cineole) are discussed and related to respiratory pathologies. 

All of the references cited are from peer-reviewed publications; a minority is open to debate 

regarding methodology and/or interpretation of results, but this is not the purpose of this compilation. 

Reports of individual case studies have been omitted. 

11.2 ACARICIDAL ACTIVITY 

A number of essential oils have been found to have effective acaricidal activity against infections in 

the animal world. Recent examples include Origanum onites against cattle ticks (Coskun et al.,

Phytotherapeutic Uses of Essential Oils 317 

2008) and Cinnamomum zeylanicum against rabbit mange mites (Fichi et al., 2007). In comparison 

to veterinary research, there have been few investigations into human acaricidal infections. 

The scabies mite, Sarcoptes scabiei var. hominis, is becoming increasingly resistant to existing 

acaricidal compounds such as lindane, benzyl benzoate, permethrin, and oral ivermectin. The 

potential use of a 5% Melaleuca alternifolia essential oil solution to treat scabies infections was 

investigated in vitro. It was found to be highly effective at reducing mite survival times and the main 

active component was terpinen-4-ol. However, the in vivo effectiveness was only tested on one 

individual, in combination with benzyl benzoate and ivermectin (Walton et al., 2004). 

A double-blind, randomized, parallel group study was used to compare the effects of 25% w/w 

benzyl benzoate emulsion with 20% w/w Lippia multifl ora essential oil emulsion in the treatment 

of scabies infection in 105 patients. Applied daily, the cure rates for the oil emulsion were 50%, 

80%, and 80% for 3, 5, and 7 days, respectively, compared to 30%, 60%, and 70% for the benzyl 

benzoate emulsion. There were also less adverse reactions to the oil emulsion, leading it to be 

considered as an additional formulation for the treatment of scabies (Oladimeji et al., 2005). 

Although not an infection, the lethal activity of essential oils toward the house dust mite 

(Dermatophagoides farina and Dermatophagoides pteronyssinus) is important as these mites are a 

major cause of respiratory allergies and an etiologic agent in the sensitization and triggering of 

asthma in children. Numerous studies have been conducted, including the successful inclusion of 

Eucalyptus globulus in blanket washing solutions (Tovey and McDonald, 1997), the high acaricidal 

activity of clove, rosemary, eucalyptus, and caraway (El-Zemity et al., 2006), and of tea tree and 

lavender (Williamson et al., 2007). 

11.3 ANTICARCINOGENIC 

Despite the popularity of in vitro experimentation concerning the cellular mechanisms of carcinogenic 

prevention by essential oil components (mainly by inducing apoptosis), there is no evidence 

that the direct administration of essential oils can cure cancer. There is evidence to suggest that the 

mevalonate pathway of cancer cells is sensitive to the inhibitory actions of dietary plant isoprenoids 

(e.g., Elson and Yu, 1994; Duncan et al., 2005). Animal testing has shown that some components 

can cause a significant reduction in the incidence of chemically induced cancers when administered 

before and during induction (e.g., Reddy et al., 1997; Uedo et al., 1999). 

Phase II clinical trials have all involved perillyl alcohol. Results demonstrated that despite 

preclinical evidence, there appeared to be no anticarcinogenic activity in cases of advanced ovarian 

cancer (Bailey et al., 2002), metastatic colorectal cancer (Meadows et al., 2002), and metastatic 

breast cancer (Bailey et al., 2008). Only one trial has demonstrated antitumor activity as evidenced 

by a reduction of tumor size in patients with recurrent malignant gliomas (Orlando da 

Fonseca et al., 2008). 

11.4 ANTIMICROBIAL 

Considering that the majority of essential oil research is directed toward antimicrobial activity, 

there is a surprising lack of corresponding in vivo human trials. This is disappointing since the topical 

and systemic application of essential oils to treat infection is a widespread practice among therapists 

with (apparently) good results. 

11.4.1 ANTIBACTERIAL 

Antibiotics that affect Propionibacterium acnes are a standard treatment for acne but antibiotic 

resistance is becoming prevalent. A preliminary study of 126 patients showed that topical 2% essential 

oil of Ocimum gratissimum (thymol chemotype) in a hydrophilic cream base was more effective 

than 10% benzyl peroxide lotion at reducing the number of lesions when applied twice daily for 

4 weeks (Orafidiya et al., 2002).


In a randomized, single-blind, parallel-group-controlled trial, the same group examined the 

effects of the addition of aloe vera gel at varying concentrations to the Ocimum gratissimum cream 

and compared its activity with 1% clindamycin phosphate. In the 84 patients with significant acne, 

it was found that increasing the aloe gel content improved efficacy; the essential oil preparations 

formulated with undiluted or 50% aloe gels were more effective at reducing lesions than the reference 

product. The aloe vera gels alone had minimal activity (Orafidiya et al., 2004). 

A later report judged the efficacy of a 5% Melaleuca alternifolia gel in the amelioration of mild 

to moderate acne, since a previous study (Raman et al., 1995) had demonstrated the effectiveness 

of tea tree oil components against Propionibacterium acnes. The randomized, double-blind, 

placebo-controlled trial used 60 patients who were given the tea tree oil gel or the gel alone twice 

daily for 45 days. The total acne lesion count was significantly reduced by 43.64% and the acne 

severity index was significantly reduced by 40.49% after the tea tree oil treatment, as compared to 

the placebo scores of 12.03% and 7.04%, respectively (Enshaieh et al., 2007). 

11.4.1.1 Methicillin-Resistant Staphylococcus aureus 

A number of papers have demonstrated the in vitro effects of various essential oils against methicillinresistant 

Staphylococcus aureus (MRSA); for example, Lippia origanoides (Dos Santos et al., 2004), 

Backhousia citriodora (Hayes and Markovic, 2002), Mentha piperita, Mentha arvensis, and Mentha 

spicata (Imai et al., 2001), and Melaleuca alternifolia (Carson et al., 1995). There have been no trials 

involving the use of essential oils to combat active MRSA infections, although there have been two 

studies involving the use of tea tree oil as a topical decolonization agent for MRSA carriers. 

A pilot study compared the use of 2% mupirocin nasal ointment and triclosan body wash (routine 

care) with 4% Melaleuca alternifolia essential oil nasal ointment and 5% tea tree oil body wash in 

30 MRSA patients. The interventions lasted for a minimum of 3 days and screening for MRSA was 

undertaken at 48 and 96 h post-treatment from sites previously colonized by the bacteria. There was 

no correlation between length of treatment and outcome in either group. Of the tea tree oil group, 

33% were initially cleared of MRSA carriage while 20% remained chronically infected at the end 

of the treatment; this was in comparison with routine care group of 13% and 53%, respectively. The 

trial was too small to provide significant results (Caelli et al., 2000). 

A randomized, controlled trial compared the use of a standard regime for MRSA decolonization 

with Melaleuca alternifolia essential oil. The 5-day study involved 236 patients. The standard treatment 

group was given 2% mupirocin nasal ointment thrice daily, 4% chlorhexidine gluconate soap 

as a body wash once daily, and 1% silver sulfadiazine cream for skin lesions, wounds, and leg ulcers 

once daily. The tea tree oil group received 10% essential oil cream thrice daily to the nostrils and to 

specific skin sites and 5% essential oil body wash at least once daily. In the tea tree oil group, 41% 

were cleared of MRSA as compared to 49% using the standard regime; this was not a significant 

difference. Tea tree oil cream was significantly less effective at clearing nasal carriage than mupirocin 

(47% compared to 78%), but was more effective at clearing superficial sites than chlorhexidine 

or silver sulfadiazine (Dryden et al., 2004). 

11.4.2 ANTIFUNGAL 

The essential oil of Citrus aurantium var. amara was used to treat 60 patients with tinea corporis, 

cruris, or pedis. One group received a 25% bitter orange (BO) oil emulsion thrice daily, a second 

group was treated with 20% bitter orange oil in alcohol (BOa) thrice daily, and a third group used 

undiluted BO oil once daily. The trial lasted for 4 weeks and clinical and mycological examinations 

were performed every week until cure, which was defined as an elimination of signs and symptoms. 

In the BO group, 80% of patients were cured in 1–2 weeks and the rest within 2–3 weeks. By using 

BOa, 50% of patients were cured in 1–2 weeks, 30% in 2–3 weeks, and 20% in 3–4 weeks. With the 

undiluted essential oil, 25% of patients did not continue treatment, 33.3% were cured in 1 week, 

60% in 1–2 weeks, and 6.7% in 2–3 weeks (Ramadan et al., 1996).


A double-blind, randomized, placebo-controlled trial investigated the efficacy of 2% butenafine 

hydrochloride cream with added 5% Melaleuca alternifolia essential oil in 60 patients with 

toenail onychomycosis. After 16 weeks, 80% of patients in the treatment group were cured, as 

opposed to none in the control group (Syed et al., 1999). However, butenafine hydrochloride is 

a potent antimycotic in itself and the results were not compared with this product when 

used alone. 

After an initial in vitro study, which showed that the essential oil of Eucalyptus paucifl ora had a 

strong fungicidal activity against Epidermophyton fl occosum, Microsporum canis, Microsporum 

nanum, Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton 

tonsurans, and Trichophyton violaceum, an in vivo trial was commenced. Fifty patients with confirmed 

dermatophytosis were treated with 1% v/v essential oil twice daily for 3 weeks. At the end of 

the treatment, a cure was demonstrated in 60% of patients with the remaining 40% showing significant 

improvement (Shahi et al., 2000). 

On the surmise that infection with Pityrosporum ovale is a major contributing factor to dandruff 

and that anti-Pityrosporum drugs such as nystatin were proven effective treatments, the use of 5% 

Melaleuca alternifolia essential oil was investigated. In this randomized, single-blind, parallel-group 

study tea tree oil shampoo or placebo shampoo was used daily for 4 weeks by 126 patients with mild 

to moderate dandruff. In the treatment group, the dandruff severity score showed an improvement of 

41%, as compared to 11% in the placebo group. The area involvement and total severity scores also 

demonstrated a statistically significant improvement, as did itchiness and greasiness. Scaliness was 

not greatly affected. The condition resolved for one patient in each group and so ongoing application 

of tea tree oil shampoo was recommended for dandruff control (Satchell et al., 2002a). 

For inclusion in a randomized, double-blind, controlled trial, 158 patients with the clinical features 

of intertriginous tinea pedis and confirmed dermatophyte infection were recruited. They were 

administered 25% or 50% Melaleuca alternifolia essential oil (in an ethanol and polyethylene glycol 

vehicle) or the vehicle alone, twice daily for 4 weeks. There was an improvement in the clinical 

severity score, falling by 68% and 66% in the 25% and 50% tea tree oil groups, in comparison with 

41% for the placebo. There was an effective cure in the 25% and 50% tea tree oil and placebo groups 

of 48%, 50%, and 13%, respectively. The essential oil was less effective than standard topical treatments 

(Satchell et al., 2002b). 

The anticandida properties of Zataria multifl ora essential oil and its active components (thymol, 

carvacrol, and eugenol) were demonstrated in vitro by Mahmoudabadi et al. (2006). A randomized, 

clinical trial was conducted using 86 patients with acute vaginal candidiasis. They were treated with 

a cream containing 0.1% Zataria multifl ora essential oil or 1% clotrimazole once daily for 7 days. 

Statistically significant decreases in vulvar pruritis (80.9%), vaginal pruritis (65.5%), vaginal 

burning (73.95), urinary burning (100%), and vaginal secretions (90%) were obtained by the essential 

oil treatment as compared to the clotrimazole treatment of 73.91%, 56.7%, 82.1%, 100%, and 

70%, respectively. In addition, the Zataria multifl ora cream reduced erythema and satellite vulvar 

lesions in 100% of patients, vaginal edema in 100%, vaginal edema in 83.3%, and vulvo-vaginal 

excoriation and fissures in 92%. The corresponding results for clotrimazole were 100%, 100%, 76%, 

and 88%. In terms of overall efficacy, the rates of improvement were 90% and 74.8% for the Zataria 

multifl ora and clotrimazole groups, respectively. Use of the cream alone provided no significant 

changes (Khosravi et al., 2008). 

11.4.3 ANTIVIRAL 

The in vitro studies that have been conducted so far indicate that many essential oils possess antiviral 

properties, but they affect only enveloped viruses and only when they are in the free state, that is, 

before the virus is attached to, or has entered the host cell (e.g., Schnitzler et al., 2008). This is in 

contrast to the majority of synthetic antiviral agents, which either bar the complete penetration of 

viral particles into the host cell or interfere with viral replication once the virus is inside the cell.


A randomized, investigator-blinded, placebo-controlled trial used 6% Melaleuca alternifolia 

essential oil gel to treat recurrent herpes labialis. It was applied five times daily and continued until 

re-epithelialization occurred and the polymerase chain reaction (PCR) for Herpes simplex virus 

was negative for two consecutive days. The median time to re-epithelialization after treatment with 

tea tree oil was 9 days as compared to 12.5 days with the placebo, which is similar to reductions 

caused by other topical therapies. The median duration of PCR positivity was the same for both 

groups (6 days) although the viral titers appeared slightly lower in the oil group on days 3 and 4. 

None of the differences reached statistical significance, probably due to the small group size 

(Carson et al., 2001). 

Children below 5 years were enrolled in a randomized trial to test a 10% v/v solution of the 

essential oil of Backhousia citriodora against molluscum contagiosum (caused by Molluscipoxvirus). 

Of the 31 patients, 16 were assigned to the treatment group and the rest to the control of 

olive oil. The solutions were applied directly to the papules once daily at bedtime for 21 days or 

until the lesions had resolved. In the essential oil group, five children had a total resolution of 

lesions and four had reductions of greater than 90% at the end of 21 days. In contrast, none of the 

control group had any resolution or reduction of lesions by the end of the study period (Burke 

et al., 2004). 

A study was conducted on 60 patients who were chronic carriers of hepatitis B or C. The essential 

oils of Cinnamomum camphora ct 1,8-cineole, Daucus carota, Ledum groelandicum, Laurus 

nobilis, Helichrysum italicum, Thymus vulgaris ct thujanol, and Melaleuca quinquenervia were 

used orally in various combinations. They were used as a monotherapy or as a complement to allopathic 

treatment. The objectives of treatment were normalization of transaminase levels, reduction 

of viral load, and stabilization or regression of fibrosis. There was an improvement of 100%, when 

patients with hepatitis C were given bitherapy with essential oils. With essential oil monotherapy, 

improvements were noted in 64% of patients with hepatitis C and there were two cures of hepatitis B 

(Giraud-Robert, 2005). 

11.4.4 MICROBES OF THE ORAL CAVITY 

The activities of essential oils against disease-producing microbes in the oral cavity have been 

documented separately because there are numerous reports of relevance. The easy administration of 

essential oils in mouthrinses, gargles, and toothpastes, and the success of such commercial preparations, 

has no doubt led to the popularity of this research. 

The in vitro activities of essential oils against the oral microflora are well documented and these 

include effects on cariogenic and periodontopathic bacteria. One example is the in vitro activity of 

Leptospermum scoparium, Melaleuca alternifolia, Eucalyptus radiata, Lavandula offi cinalis, and 

Rosmarinus offi cinalis against Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, 

Fusobacterium nucleatum, and Streptococcus mutans. The essential oils inhibited all of the test 

bacteria, acting bactericidally except for Lavandula offi cinalis. In addition, significant adhesioninhibiting 

activity was shown against Streptococcus mutans by all essential oils and against 

Porphyromonas gingivalis by tea tree and manuka (Takarada et al., 2004). 

There have been at least six in vivo studies concerning the activity of individual essential oils 

against the microflora of the oral cavity. In addition, a review of the literature fi nds a surprising 

number of in vivo papers that detail the activities of “an essential oil mouthrinse.” Closer examination 

reveals that the essential oil mouthrinse is the commercial product, Listerine. 

Although Listerine contains 21% or 26% alcohol (depending on the exact product), a 6-month 

study has shown that it contributes nothing to the efficaciousness of the mouthrinse (Lamster 

et al., 1983). The active ingredients are 1,8-cineole (0.092%), menthol (0.042%), methyl salicylate 

(0.06%), and thymol (0.64%). For this reason, a small random selection of such papers is 

included below.


11.4.4.1 Activity of Listerine against Plaque and/or Gingivitis 

An observer-blind, 4-day plaque regrowth, crossover study compared the use of Listerine ® with a 

triclosan mouthrinse and two placebo controls in 32 volunteers. All normal hygiene procedures were 

suspended except for the rinses. The triclosan product produced a 45% reduction in plaque area and 

a 12% reduction in plaque index against its placebo, in comparison with 52% and 17%, respectively, 

for the essential oil rinse. The latter was thus deemed more effective (Moran et al., 1997). 

A similar protocol was used to compare the effects of Listerine against an amine fluoride/stannous 

fluoride-containing mouthrinse (Meridol ® ) and a 0.1% chlorhexidine mouthrinse (Chlorhexamed ® ) 

in inhibiting the development of supragingival plaque. On day 5 of each treatment, the results from 

23 volunteers were evaluated. In comparison with their placebos, the median plaque reductions 

were 12.2%, 23%, and 38.2% for the fluoride, essential oil, and chlorhexidine rinses, respectively. 

The latter two results were statistically significant (Riep et al., 1999). 

After the assessment for the presence of gingivitis and target pathogens (Porphyromonas 

gingivalis, Fusobacterium nucleatum, and Veillonella sp.) and total anaerobes, 37 patients undertook 

a twice daily mouthrinse with Listerine for 14 days. After a washout period, the study was 

conducted again using a flavored hydroalcoholic placebo. The results of this randomized, doubleblind, 

crossover study showed that the essential oil rinse significantly lowered the number of all 

target pathogens by 66.3–79.2%, as compared to the control (Fine et al., 2007). 

The effect of adding Listerine mouthrinse to a standard oral hygiene regime in 50 orthodontic 

patients was examined. The control group brushed and flossed twice daily, whereas the test group 

also used the mouthrinse twice daily. Measurements of bleeding, gingival, and plaque indices were 

conducted at 3 and 6 months. All three indices were significantly lowered in the test group as compared 

to the control at both time intervals (Tufekci et al., 2008). 

The same fixed combination of essential oils that is found in Listerine mouthrinse has been incorporated 

into a dentifrice. Such a dentifrice was used in a 6-month double-blind study to determine its 

effect on the microbial composition of dental plaque as compared to an identical dentifrice without 

essential oils. Supragingival plaque and saliva samples were collected at baseline and their microbial 

content characterized, after which the study was conducted for 6 months. The essential oil dentifrice did 

not significantly alter the microbial flora and opportunistic pathogens did not emerge, nor was there any 

sign of developing resistance to the essential oils in tested bacterial species (Charles et al., 2000). 

The same dentifrice was examined for antiplaque and antigingivitis properties in a blinded, randomized, 

controlled trial. Before treatment, 200 patients were assessed using a plaque index, a 

modified gingival index (GI), and a bleeding index. The dentifrice was used for 6 months, after 

which another assessment was made. It was found that the essential oil dentifrice had a statistically 

significant lower whole-mouth and interproximal plaque index (18.3% and 18.1%), mean GI (16.2% 

and 15.5%), and mean bleeding index (40.5% and 46.9%), as compared to the control. It was therefore 

proven to be an effective antiplaque and antigingivitis agent (Coelho et al., 2000). 

11.4.4.2 Antiviral Listerine 

A trial was conducted to examine whether a mouthrinse could decrease the risk of viral crosscontamination 

from oral fluids during dental procedures. Forty patients with a perioral outbreak of 

recurrent herpes labialis were given a 30-s mouthrinse with either water or Listerine. Salivary 

samples were taken at baseline, immediately following the rinse and 30 min after the rinse and 

evaluated for the viral titer. Infectious virions were reduced immediately to zero postrinse and there 

was a continued significant reduction 30 min postrinse. The reduction by the control was not significant 

(Meiller et al., 2005). 

11.4.4.3 Activity of Essential Oils 

The antibacterial activity of the essential oil of Lippia multifl ora was first examined in vitro for 

antimicrobial activity against ATCC strains and clinical isolates of the buccal flora. A significant


activity was found, with an MBC of 1/1400 for streptococci and staphylococci, 1/800 for enterobacteria 

and neisseria, and 1/600 for candida. A mouthwash was prepared with the essential oil at a 

1/500 dilution and this was used in two clinical trials. 

The buccodental conditions of 26 French children were documented by measuring the percentage 

of dental surface free of plaque, gum inflammation, and the papillary bleeding index (PBI). 

After 7 days of rinsing with the mouthwash for 2 min, the test group was found to have a reduction 

of dental plaque in 69% of cases and a drop in PBI with a clear improvement of gum inflammation 

in all cases. The second trial was conducted in the Cote d’Ivoire with 60 adult patients with a variety 

of conditions. After using the mouthwash after every meal for 5 days, it was found that candidiasis 

had disappeared in most cases, gingivitis was resolved in all patients, and 77% of dental abscesses 

had resorbed (Pélissier et al., 1994). 

Fluconazole-refractory oropharyngeal candidiasis is a common condition in HIV patients. Twelve 

such patients were treated with 15 mL of a Melaleuca alternifolia oral solution (Breath-Away) four 

times daily for 2 weeks, in a single center, open-label clinical trial. The solution was swished in the 

mouth for 30–60 s and then expelled, with no rinsing for at least 30 min. Clinical assessment was 

carried out on days 7 and 14 and also on days 28 and 42 of the follow-up. Two patients were clinically 

cured and six were improved after the therapy; four remained unchanged and one deteriorated. 

The overall clinical response rate was thus 67% and was considered as a possible alternative antifungal 

treatment in such cases (Jandourek et al., 1998). 

A clinical pilot study compared the effect of 0.34% Melaleuca alternifolia essential oil 

solution with 0.1% chlorhexidine on supragingival plaque formation and vitality. Eight subjects 

participated, with a 10-day washout period between each treatment regime of 1 week. The plaque 

area was calculated using a stain and plaque vitality was estimated using a fluorescence 

technique. Neither of these parameters was reduced by the tea tree oil treatment (Arweiler et al., 

2000). 

A gel containing 2.5% Melaleuca alternifolia essential oil was used in a double-blind, 

longitudinal noncrossover trial and compared with a chlorhexidine gel positive control and a placebo 

gel in the treatment of plaque and chronic gingivitis. The gels were applied as a dentifrice 

twice daily by 49 subjects for 8 weeks and the treatment was assessed using a gingival index (GI), 

a PBI, and a plaque staining score. The tea tree group showed a significant reduction in PBI and 

GI scores, although plaque scores were not reduced. It was apparent that the tea tree gel decreased 

the level of gingival inflammation more than the positive or negative controls (Soukoulis and 

Hirsch, 2004). 

A mouthcare solution consisting of an essential oil mixture of Melaleuca alternifolia, Mentha 

piperita, and Citrus limon in a 2:1:2 ratio diluted in water to a 0.125% solution was used to treat oral 

malodor in 32 intensive care unit patients, 13 of whom were ventilated. The solution was used to 

clean the teeth, tongue, and oral cavity twice daily. The level of malodor was assessed by a nurse 

using a visual analogue scale, and volatile sulfur compounds (VSC) were measured via a probe in 

the mouth, before, 5 and 60 min after treatment. On the second day, the procedure was repeated 

using benzydamine hydrochloride (BH), which is normally used for oral hygiene, instead of essential 

oil solution. The perception of oral malodor was significantly lowered after the essential oil 

treatment but not after the BH treatment. There was a decrease in VSC levels at 60 min for both 

treatment groups, but not after 5 min for the oil mixture. The results suggested that just one session 

with the essential oil mixture could improve oral malodor and VSC in intensive care patients (Hur 

et al., 2007). 

The essential oil of Lippia sidoides (rich in thymol and carvacrol) was used in a double-blind, 

randomized, parallel-armed study against gingival inflammation and bacterial plaque. Fifty-five 

patients used a 1% essential oil solution as a mouthrinse twice daily for 7 days and the results were 

compared with a positive control, 0.12% chlorhexidine. Clinical assessment demonstrated decreased 

plaque index and gingival bleeding scores as compared to the baseline, with no significant difference 

between test and control. The essential oil of Lippia sidoides was considered a safe and effective 

treatment (Botelho et al., 2007).


11.4.5 CONTROLLING MICROFLORA IN ATOPIC DERMATITIS 

Rarely found on healthy skin, Staphylococcus aureus is usually present in dry skin and is one of the 

factors that can worsen atopic dermatitis. Toxins and enzymes deriving from this bacteria cause 

skin damage and form a biofilm from fibrin and glycocalyx, which aids adhesion to the skin and 

resistance to antibiotics. An initial in vitro study found that a mixture of xylitol (a sugar alcohol) and 

farnesol was an effective agent against Staphylococcus aureus; xylitol inhibited the formation 

of glycocalyx whereas farnesol dissolved fibrin and suppressed Staphylococcus aureus growth 

without affecting Staphylococcus epidermidis (Masako et al., 2005a). 

The same mixture of xylitol and farnesol was used in a double-blind, randomized, placebocontrolled 

study of 17 patients with mild to moderate atopic dermatitis on their arms. A skin-care 

cream containing 0.02% farnesol and 5% xylitol or the cream alone was applied to either the left or 

the right arms for 7 days. The ratio of Staphylococcus aureus to other aerobic skin microflora was 

significantly decreased in the test group compared to placebo, from 74% to 41%, while the numbers 

of coagulase-negative staphylococci increased. In addition, skin conductance (indicating hydration 

of skin surface) significantly increased at the test cream sites compared to before application and to 

the placebo (Masako et al., 2005b). 

11.4.6 ODOR MANAGEMENT FOR FUNGATING WOUNDS 

Fungating wounds may be caused by primary skin carcinomas, underlying tumors or via spread 

from other tissues. The malodor associated with such necrosis is caused by the presence of aerobic 

and anaerobic bacteria. The wounds rarely heal and require constant palliative treatment, leading to 

social isolation of the patients and poor quality of life. 

Smell reduction with essential oils was first reported by Warnke et al. (2004) in 25 malodorous 

patients with inoperable squamous cell carcinoma of the head and neck. A commercial product 

containing eucalyptus, grapefruit, and tea tree essential oils (Megabac ® ) was applied topically to the 

wounds twice daily. Normal medication apart from Betadine disinfection was continued. The smell 

disappeared completely within 2–3 days and signs of superinfection and pus secretion were reduced 

in the necrotic areas. 

Megabac has also been used in a small pilot study (10 patients) to treat gangrenous areas, being 

applied via spray thrice daily until granulation tissue formed. The treatment was then continued 

onto newly formed split skin grafts. All wounds healed within 8 weeks and no concurrent antibiotics 

were used (Sherry et al., 2003). 

Use of essential oils to reduce the smell of fungating wounds in 13 palliative care patients was 

detailed by another group the following year. Lavandula angustifolia, Melaleuca alternifolia, and 

Pogostemon cablin essential oils were used alone or in combinations at 2.5–5% concentrations in a 

cream base. The treatments were effective (Mercier and Knevitt, 2005). 

A further study was conducted with 30 patients suffering incurable head and neck cancers with 

malodorous necrotic ulcers. A custom-made product (Klonemax ® ) containing eucalyptus, tea tree, 

lemongrass, lemon, clove, and thyme essential oils was applied topically (5 mL) twice daily. All 

patients had a complete resolution of the malodor; in addition to the antibacterial activity, an 

anti-inflammatory effect was also noted (Warnke et al., 2006). 

The use of essential oils to treat malodorous wounds in cancer patients is becoming widespread 

in many palliative care units although no formal clinical trials have been conducted as yet. 

11.5 DISSOLUTION OF HEPATIC AND RENAL STONES 

11.5.1 GALL AND BILIARY TRACT STONES 

Rowachol and Rowatinex are two commercial products that have been marketed for many years and 

are based on essential oil components. They are sometimes thought of as being the same product but


TABLE 11.1 

Composition of Rowachol and Rowatinex 

Declared by the Manufacturers 

Component Rowachol Rowatinex 

a-pinene 20.0 37.0 

b-pinene 5.0 9.0 

Camphene 8.0 22.0 

1,8-cineole 3.0 4.0 

Fenchone — 6.0 

Menthone 9.0 — 

Borneol 8.0 15.0 

Menthol 48.0 — 

anethole — 6.0 

Source: Sybilska, D. and M. Asztemborska, 2002. J. Biochem. 

Biophys. Methods., 54: 187–195. 

in fact they are different. The compositions have changed slightly over the years and the most 

recently disclosed are shown in Table 11.1. 

Rowachol has been in use for over 50 years for the dissolution of gallstones and biliary tract stones. 

There have been many published works on its effects and at least one double-blind trial (Lamy, 1967). 

It has been stated that although the dissolution rate of Rowachol is not impressive, it is still much 

greater than Rowatinex and could occur spontaneously (Doran and Bell, 1979). It has been employed 

alone as a useful therapy for common duct stones (Ellis and Bell, 1981) although improved results were 

demonstrated when Rowachol was used in conjunction with bile acid therapy (Ellis et al., 1981). 

Rowachol has been shown to inhibit hepatic cholesterol synthesis mediated by a decreased 

hepatic S-3-hydroxy-3-methylgutaryl-CoA reductase activity (Middleton and Hui, 1982); the 

components mostly responsible for this activity were menthol and 1,8-cineole, with pinene and 

camphene having no significant effect (Clegg et al., 1980). A reduction in cholesterol crystal formation 

in the bile of gallstone patients has been demonstrated in a small trial using Rowachol 

(von Bergmann, 1987). 

Two early uncontrolled trials reported that Rowachol significantly increased plasma high-density 

lipoprotein (HDL) cholesterol when administered to patients with low HDL cholesterol; a twofold 

increase was found in 10 subjects after 6 weeks of treatment (Hordinsky and Hordinsky, 1979), 

while a progressive increase in HDL of 14 subjects was noted, >100% after 6 months (Bell 

et al., 1980). This was interesting as low plasma concentrations of HDLs are associated with an 

elevated risk of coronary heart disease. However, a double-blind, placebo-controlled trial that 

administered six capsules of Rowachol daily for 24 weeks to 19 men found that there were no significant 

HDL-elevating effects of the treatment (Cooke et al., 1998). It is currently thought that 

monoterpenes have no HDL-elevating potential that is useful for disease prevention. 

In vitro, a solution of 97% d-limonene was found to be 100-fold better at solubilizing cholesterol 

than sodium cholate. A small trial followed with 15 patients, whereby 20 ml of the d-limonene 

preparation was introduced into the gallbladder via a catheter on alternate days for up to 48 days. 

The treatment was successful in 13 patients with gallstone dissolution sometimes occurring after 

three infusions. Side effects included vomiting and diarrhea (Igimi et al., 1976). 

A further study was conducted by Igimi et al. (1991) using the same technique with 200 patients. 

Treatments lasted from 3 weeks to 4 months. Complete or partial dissolution of gallstones was achieved 

in 141 patients, with complete disappearance of stones in 48% of cases. Epigastric pain was experienced 

by 168 patients and 121 suffered nausea and vomiting. Further trials have not been conducted.


11.5.2 RENAL STONES 

While Rowachol is used as a measure against gallstones and biliary tract stones, Rowatinex is 

used in the treatment of renal stones. The first double-blind, randomized trial was conducted by 

Mukamel et al. (1987) on 40 patients with acute renal colic. In the Rowatinex group, there was a 

significantly higher expulsion rate of stones ≥3 mm in diameter in comparison with the placebo 

(61% and 28%, respectively). There was also a higher overall success rate in terms of spontaneous 

stone expulsion and/or disappearance of ureteral dilatation in the treatment group compared to 

placebo (78–52%), but the difference was not statistically significant. 

A second double-blind, randomized trial was conducted on 87 patients with ureterolithiasis. 

Four Rowatinex capsules were prescribed four times a day, the average treatment time being 

two weeks. The overall stone expulsion rate was significantly higher in the Rowatinex group as 

compared to placebo; 81% and 51%, respectively. Mild to moderate gastrointestinal disturbances 

were noted in seven patients. It was concluded that the early treatment of ureteral stones 

with Rowatinex was preferable before more aggressive measures were considered (Engelstein 

et al., 1992). 

Rowatinex has also been used with success in the removal of residual stone fragments after extracorporeal 

shock wave lithotriptsy, a situation that occurs in up to 72% of patients when given this 

therapy. With 50 patients, it was found that Rowatinex decreased the number of calculi debris, 

reducing the number of late complications and further interventions. By day 28, 82% of patients 

were free of calculi whereas this situation is normally reached after 3 months without Rowatinex 

treatment (Siller et al., 1998). 

A minor study examined the use of Rowatinex in the management of childhood urolithiasis. Six 

children aged from 4 months to 5 years were administered varying doses of the preparation from 

10 days to 12 weeks. All patients became stone-free with no side effects, although a definite conclusion 

as to the efficacy of treatment could not be established due to the small patient number involved 

(Al-Mosawi, 2005). 

A comparison of the effects of an a-blocker (tamsulosin) and Rowatinex for the spontaneous 

expulsion of ureter stones and pain control was undertaken using 192 patients. They were divided 

into three groups: analgesics only, Rowatinex with analgesics, and tamsulosin with analgesics. For 

ureter stones less than 4 mm in diameter, their excretion was accelerated by both Rowatinex and 

tamsulosin. The use of these two treatments also decreased the amount of analgesics required and 

it was concluded that they should be considered as adjuvant regimes (Bak et al., 2007). 

11.6 FUNCTIONAL DYSPEPSIA 

Several essential oils have been used in the treatment of functional (nonulcer) dyspepsia. All of the 

published trials have concerned the commercial preparation known as Enteroplant ® , an entericcoated 

capsule containing 90 mg of Mentha ¥ piperita, and 50 mg of Carum carvi essential oils. 

The combination of peppermint and caraway essential oils has been shown to act locally in the 

gut as an antispasmodic (Micklefield et al., 2000, 2003) and to have a relaxing effect on the gallbladder 

(Goerg and Spilker, 2003). The antispasmodic effect of peppermint is well documented and 

that of caraway essential oil has also been demonstrated (Reiter and Brandt, 1985). The latter alone 

has also been shown to inhibit gallbladder contractions in healthy volunteers, increasing gallbladder 

volume by 90% (Goerg and Spilker, 1996). 

One of the first studies involved 45 patients in a double-blind, placebo-controlled multicenter trial 

with the administration of Enteroplant thrice daily for 4 weeks. It was found to be superior to placebo 

with regard to pain frequency, severity, efficacy, and medical prognosis. Clinical Global Impressions 

were improved for 94.5% of patients using the essential oil combination (May et al., 1996). 

The activity of Enteroplant (twice daily) was compared with that of cisapride (30 mg daily), a 

serotonin 5-HT 4 agonist that stimulates upper gastrointestinal tract motility, over a 4-week period.


This double-blind, randomized trial found that both products had comparable efficacy in terms of 

pain severity and frequency, Dyspeptic Discomfort Score, and Clinical Global Impressions (Madisch 

et al., 1999). 

Another double-blind, randomized trial administered either Enteroplant or placebo twice daily 

for 28 days. Pain intensity and pressure, heaviness, and fullness were reduced in the test group by 

40% and 43% as compared to 22% for both in the placebo group, respectively. In addition, Clinical 

Global Impressions were improved by 67% for the peppermint/caraway combination whereas the 

placebo scored 21% (May et al., 2000). 

Holtmann et al. (2001) were the first to investigate the effect of Enteroplant (twice daily) on disease-specific 

quality of life as measured by the Nepean Dyspepsia Index. All scores were significantly 

improved compared to the placebo. In 2002, the same team also demonstrated that patients 

suffering with severe pain or severe discomfort both responded significantly better in comparison 

with the placebo. 

Approximately 50% of patients suffering from functional dyspepsia are infected with Helicobacter 

pylori (Freidman, 1998). The Helicobacter status of 96 patients and the efficacy of Enteroplant were 

compared by May et al. (2003). They found that patients with Helicobacter pylori infection demonstrated 

a substantially better treatment response than those who were not infected. However, a previous 

study found no efficacy differences between infected and noninfected functional dyspepsia 

patients (Madisch et al., 2000) and so the effect of the presence of the bacterium on Enteroplant 

treatment has yet to be elucidated. 

A short review of the literature concluded that treatment with the fixed peppermint/caraway 

essential oil combination had demonstrated significant efficacy in placebo-controlled trials, had 

good tolerability and safety, and could thus be considered for the long-term management of functional 

dyspepsia patients (Holtmann et al.,2003). 

11.7 GASTROESOPHAGEAL REFLUX 

d-Limonene has been found to be effective in the treatment of gastroesophageal reflux disorder. 

Nineteen patients took one capsule of 1000 mg d-limonene every day and rated their symptoms 

using a severity/frequency index. After 2 days, 32% of patients had significant relief and by day 14, 

89% of patients had complete relief of symptoms (Wilkins, 2002). 

A double-blind, placebo-controlled trial was conducted with 13 patients who were administered 

one 1000 mg capsule of d-limonene daily or on alternate days. By day 14, 86% of patients were 

asymptomatic compared to 29% in the placebo group (Wilkins, 2002). 

The mechanism of action of d-limonene has not been fully elucidated in this regard but it is 

thought that it may coat the mucosal lining and offer protection against gastric acid and/or promote 

healthy peristalsis. 

11.8 HYPERLIPOPROTEINEMIA 

Girosital is a Bulgarian encapsulated product consisting of rose essential oil (68 mg) and vitamin A 

in sunflower vegetable oil. Initial animal studies found that rose oil administered at 0.01 

and 0.05 mL/ kg had a hepatoprotective effect against ethanol. Dystrophy and lipid infiltration were 

lowered and glycogen tended to complete recovery, suggesting a beneficial effect of rose oil on lipid 

metabolism (Kirov et al., 1988a). 

Girosital was administered to 33 men with long-standing alcohol abuse, twice daily for 3 months. 

It significantly reduced serum triglycerides and low-density lipoprotein and increased the level of 

HDL-cholesterin; it was particularly effective for the treatment of hyperlipoproteinemia types IIb 

and IV. Liver lesions relating to alcohol intoxication improved and subjective complaints such as 

dyspeptic symptoms and pain were reduced (Konstantinova et al., 1988).


The hypolipidemic effect of Girosital was again studied by giving a capsule once daily for 

20 days in 35 patients with hyperlipoproteinemia. In type IIa hyperlipoproteinemia cases, the total 

lipids were reduced by 23.91% and the total cholesterol by 10.64%. For type IIb patients, the total 

lipid reduction was 15.93%, triglycerides fell by 25.45%, and cholesterol by 14.06%; in type IV cases 

the reductions were 33.83%, 25.33%, and 36%, respectively. Girosital was more effective in comparison 

with the treatment with bezalipe and clofibrate (Stankusheva, 1988). 

Thirty-two patients with hyperlipoproteinemia and arterial hypertension were administered one 

Girosital capsule twice daily for 110 days. A marked reduction in hyperlipoproteinemia was demonstrated 

in all patients. The hypocholesterolemic effect manifested first in type IIa patients after 

20 days, and later in type IIb cases. Reduction of serum triglycerides in type IIb began 50 days after 

the commencement of treatment (Kirov et al., 1988b). 

A further study (Mechkov et al., 1988) examined the effect of Girosital capsules twice daily for 

110 days in 30 patients with cholelithiasis, liver steatosis, and hyperlipoproteinemia. Total cholesterol 

decreased after 20 days of treatment although it tended to rise slightly later in the test period. 

The triglycerides were most affected in hyperlipoproteinemia types IIb and IV. The b-lipoprotein 

values were not altered by the treatment. 

11.9 IRRITABLE BOWEL SYNDROME 

The essential oil of Mentha ¥ piperita has been used for many years as a natural carminative of the 

gastrointestinal tract. This effect is principally due to the antispasmodic activity of menthol, which 

acts as a calcium channel antagonist of the intestinal smooth muscle (Taylor et al., 1984, 1985). 

Secondary effects include a reduction of gastrointestinal foam by peppermint oil (Harries et al., 

1978) and a choleretic activity that is attributed to menthol (Rangelov et al., 1988). The reduction of 

intestinal hydrogen production caused by bacterial overgrowth has also been demonstrated in 

patients by enteric-coated peppermint oil (Logan and Beaulne, 2002). 

The first clinical trial of peppermint for the treatment of irritable bowel syndrome was conducted 

by Rees et al. (1979). They prescribed 0.2 mL of peppermint oil in enteric-coated capsules (1–2 

capsules depending on symptom severity) thrice daily. Patient assessment considered the oil to 

be superior to the placebo in relieving abdominal symptoms. 

Since then, a further 15 double-blind and two open trials have been conducted; examples of these 

can be seen in Table 11.2. 

Eight studies used the commercial preparation known as Colpermin ® and two used Mintoil ® , the 

capsules of which contain 187 and 225 mg of peppermint oil, respectively. The other studies used 

enteric-coated capsules usually containing 0.2 mL of the essential oil. 

The latest trial (Cappello et al., 2007) used a randomized, double-blind, placebo-controlled 

design to test the efficacy of two capsules of Mintoil twice daily for 4 weeks. The symptoms evaluated 

before the treatment and at 4 and 8 weeks post-treatment were abdominal bloating, pain or 

discomfort, diarrhea, constipation, incomplete or urgency of defecation, and the passage of gas or 

mucus. The frequency and intensity of these symptoms was used to calculate the total irritable 

bowel syndrome symptoms score. At 4 weeks, 75% of patients in the peppermint oil group demonstrated 

a >50% reduction of the symptoms score as compared to 38% in the placebo group. At 4 and 

8 weeks in the peppermint oil group compared to that before the treatment, there was a statistically 

significant reduction of the total irritable bowel syndrome symptoms score whereas there was no 

change with the placebo. 

A critical review and meta-analysis of the use of peppermint oil for irritable bowel syndrome was 

published by Pittler and Ernst (1998). They examined five double-blind, placebo-controlled trials; 

there was a significant difference between peppermint oil and placebo in three cases and no significant 

difference in two cases. It was concluded that although a beneficial effect of peppermint oil was 

demonstrated, its role in treatment was not established.


TABLE 11.2 

Examples of Clinical Trials of Peppermint Oil in the Treatment of Irritable 

Bowel Syndrome 

Patients Treatment Outcome Reference 

18 0.2–0.4 mL thrice daily 

for 3 weeks 

29 0.2–0.4 mL thrice daily 

for 2 weeks 

25 0.2 mL thrice daily 

for 2 weeks 

35 One Colpermin thrice daily 

for 24 weeks 

110 One Colpermin 3–4 times 

daily for 2 weeks 

42 1–2 Colpermin thrice daily 

for 2 weeks 

178 Two Mintoil thrice daily 

for 3 months 

57 Two Mintoil twice daily 

for 4 weeks 

Superior to placebo in relieving Rees et al. (1979) 

abdominal symptoms 

Superior to placebo in relieving Dew et al. (1984) 

abdominal symptoms 

No significant change in symptoms Lawson et al. (1988) 

as compared to placebo 

Effective in relieving symptoms Shaw et al. (1991) 

Significant improvement in 

symptoms as compared to placebo 

75% of children had reduced pain 

severity 

Significant improvement in 

gastroenteric symptoms as 

compared to placebo (97% 

versus 33%, respectively) 

Significant reduction in overall 

symptom score 

Liu et al. (1997) 

Kline et al. (2001) 

Capanni et al. (2005) 

Cappello et al. (2007) 

A review of 16 trials was conducted by Grigoleit and Grigoleit (2005). They concluded that there 

was reasonable evidence that the administration of enteric-coated peppermint oil (180–200 mg) 

thrice daily was an effective treatment for irritable bowel syndrome when compared to placebo or 

the antispasmodic drugs investigated (mebeverine, hyoscyamine, and alverine citrate). 

A comparison between two commercial delayed release peppermint oil preparations found that 

there were differences in the pharmacokinetics in relation to bioavailability times and release site. 

A capsule that is more effective in delivering the peppermint oil to the distal small intestine and 

ascending colon would be more beneficial in the treatment of irritable bowel syndrome (White 

et al., 1987). It has also been suggested that the conflicting results in some trials may be due to 

the inclusion of patients suffering from lactose intolerance, syndrome of small intestinal bacterial 

overgrowth, and celiac disease, all of which have symptoms similar to irritable bowel disease 

(Cappello et al., 2007). 

11.10 MEDICAL EXAMINATIONS 

Although not employed in a treatment context, the antispasmodic activity of peppermint essential 

oil has been used to facilitate examinations of the upper and lower gastrointestinal tract. A few 

examples are highlighted below. 

Peppermint oil has also been used during double-contrast barium enemas. The study comprised 

383 patients in four groups, two being no-treatment and Buscopan groups. The preparation, consisting 

of 8 mL of essential oil, 0.2 mL of Tween 80 in 100 mL water, was administered in 30 mL 

quantities via the enema tube or mixed in with the barium meal. Peppermint oil had the same spasmolytic 

effect as systemic Buscopan in the transverse and descending colon and a stronger effect in 

the cecum and ascending colon. Both methods of peppermint oil administration were equally effective 

(Asao et al., 2003).


Orally administered peppermint oil was used in a randomized trial in 430 patients undergoing a 

double-contrast barium meal examination, without other antispasmodics. A reduction in spasms of 

the esophagus, lower stomach, and duodenal bulb was found, along with an inhibition of barium 

flow to the distal duodenum and an improvement of diagnostic quality (Shigeaki et al., 2006). 

During endoscopic retrograde cholangiopancreatography, Buscopan or glucagon is used to 

inhibit duodenal motility but produce adverse effects. Various concentrations of peppermint oil 

were introduced into the upper gastrointestinal tract of 40 patients undergoing the procedure. 

Duodenal relaxation was obtained with 20 mL of 1.6% peppermint oil solution and the procedure 

was performed successfully in 91.4% of patients. The inhibitory effect of peppermint oil appeared 

to be identical to that of glucagon, but without side effects (Yamamoto et al., 2006). 

11.11 NAUSEA 

A small study examined a variety of aromatherapy treatments to 25 patients suffering from nausea 

in a hospice and palliative care facility. Patients were offered the essential oils of Foeniculum vulgare 

var. dulce, Chaemomelum nobile, and Mentha ¥ piperita, either singly or in blends, depending 

on individual preferences. Delivery methods included abdominal compress or massage, personal air 

spritzer or scentball diffuser. Only 32% of patients reported no response to the treatments and they 

had all just finished heavy courses of chemotherapy. Using a visual-numeric analogue scale, the 

remainder of patients experienced an improvement in their nausea symptoms when using the aromatherapy 

interventions. All patients were also taking antiemetic drugs and so the essential oils 

were regarded as successful complements to standard medications (Gilligan, 2005). 

A 6-month trial investigated the effect of inhaled 5% Zingiber offi cinale essential oil in the prevention 

of postoperative nausea and vomiting (PONV). All patients were at a high risk for PONV and 

all used similar combinations of prophylactic intravenous antiemetics. The test group had the essential 

oil applied to the volar aspects of both wrists via a rollerball immediately prior to surgery. In the 

recovery room, patients were questioned as to their feelings of nausea. Any patient who felt that they 

required further medication was considered a “failure.” Prevention of PONV by ginger essential oil 

was effective in 80% of cases, as measured by no complaint of nausea during the recovery period. In 

those patients who did not receive the essential oil, 50% experienced nausea (Geiger, 2005). 

Another experiment used essential oils to prevent PONV, but they were applied after surgery if 

the patient complained of nausea. An undiluted mixture of Zingiber offi cinale, Elettaria cardamomum, 

and Artemisia dracunculus essential oils in equal parts was applied with light friction to 

the sternocleidomastoid area and carotid-jugular axis of the neck. Of the 73 cases treated, 50 had 

a positive response, that is, a complete block of nausea and vomiting within 30 min. It was found 

that the best response (75%) was with patients who had received a single analgesic/anesthetic 

(de Pradier, 2006). 

The use of essential oils to alleviate motion sickness has also been investigated. A blend of 

Zingiber offi cinale, Lavandula angustifolia, Mentha spicata, and Mentha ¥ piperita essential oils 

in an inhalation dispenser (QueaseEase) was given to 55 ocean boat passengers with a history of 

motion sickness. The oil blend was inhaled as needed during the trip and queasiness was assessed 

using a linear analogue scale. The product was more effective than the placebo in lowering sensations 

of nausea when the seas were roughest, but was not significant at other times (Post-White and 

Nichols, 2007). 

11.12 PAIN RELIEF 

There follows a number of differing conditions that have been treated with essential oils with varying 

biological activities, such as antispasmodic, anti-inflammatory, and so on. They all share a 

common effect, that of pain relief.


11.12.1 DYSMENORRHEA 

The seeds of Foeniculum vulgare have been used in traditional remedies for the treatment of 

dysmenorrhea, an action attributed to the antispasmodic effect of the essential oil. An in vitro experiment 

demonstrated that fennel essential oil inhibited oxytocin- and prostaglandin E 2 (PGE 2 )- 

induced contractions of isolated uterus; the former was considered to have a similar activity to 

diclofenac, a nonsteroidal anti-inflammatory drug. The overall mechanism of action is still unknown 

(Ostad et al., 2001). 

A randomized, double-blind crossover study examined the effect of oral fennel essential oil at 

1% or 2% concentration as compared to placebo for the treatment of 60 women with mild to moderate 

dysmenorrhea. Up to 1 mL of the solution was taken as required for the pain at intervals of not 

less than 4 h. In the treatment groups, the severity of the pain was significantly decreased; the 

efficacy of the 2% fennel oil was 67.4%, which was comparable to the efficacy of nonsteroidal antiinflammatory 

drugs (Khorshidi et al., 2003). 

Thirty patients with moderate to severe dysmenorrhea took part in a study to compare the activity 

of mefenamic acid with the essential oil of Foeniculum vulgare var. dulce. The evaluation was 

carried out during the first 5 days of three consecutive menstrual cycles. In the first cycle, no intervention 

was given (control); during the second cycle, 250 mg of mefenamic acid 6 hourly was 

prescribed; and in the third, 25 drops of a 2% solution of fennel essential oil were given 4 hourly. 

A self-scoring linear analogue technique was used to determine effect and potency. Both interventions 

effectively relieved menstrual pain as compared to the control. Mefenamic acid was more 

potent on the second and third days, but the result was not statistically significant. It was concluded 

that fennel essential oil was a safe and effective remedy but was probably less effective than 

mefenamic acid at the dosage used (Jahromi et al., 2003). 

A third study used aromatherapy massage for the relief of the symptoms of dysmenorrhea in 

67 students. The essential oils of Lavandula offi cinalis, Salvia sclarea, and Rosa centifolia (2:1:1 ratio) 

were diluted to 3% in 5 mL of almond oil and applied in a 15-min abdominal massage daily, 1 week 

before the start of menstruation, and stopping on the first day of menstruation. The control group 

received no treatment and the placebo group received massage with almond oil only. The results 

showed a significant improvement of dysmenorrhea as assessed by a verbal multidimensional scoring 

system for the essential oil group compared to the other two groups (Han et al., 2006). 

11.12.2 HEADACHE 

The effect of peppermint and eucalyptus essential oils on the neurophysiological, psychological, 

and experimental algesimetric parameters of headache mechanisms were investigated using a 

double-blind, placebo-controlled trial with 32 healthy subjects. Measurements included sensitivity 

to mechanical, thermal, and ischemically induced pain. Four preparations consisting of varying 

amounts of peppermint and/or eucalyptus oils in ethanol were applied to the forehead and temples. 

Eucalyptus alone had no effect on the parameters studied. A combination of both oils (10% peppermint 

and 5% eucalyptus) increased cognitive performance and had a muscle-relaxing and 

mentally relaxing effect, but did not influence pain sensitivity. Peppermint alone (10%) had a significant 

analgesic effect with reduction in sensitivity to headache. It was shown to exert significant 

effects on the pathophysiological mechanisms of clinical headache syndromes (Göbel et al., 

1995a). 

A second study used the same essential oils when investigating the skin perfusion of the head in 

healthy subjects and migraine patients. In the former, capillary flow was increased by 225% 

in comparison with baseline by peppermint oil, while eucalyptus decreased the flow by 16%. In 

migraine patients, neither essential oil had any effect. It was suggested that the absence of capillary 

vasodilation (normally caused by menthol) was due to impaired calcium channel function in 

migraine patients (Göbel et al., 1995b).


11.12.3 INFANTILE COLIC 

Since animal studies had demonstrated that the essential oil of Foeniculum vulgare reduced intestinal 

spasm and increased the motility of the small intestine, it was used in a double-blind, randomized, 

placebo-controlled trial in the treatment of infantile colic. The 125 infants were all 2–12 weeks 

of age and those in the treatment group received a water emulsion of 0.1% fennel essential oil 

and 0.4% polysorbate (5–20 mL) up to four times a day. The dose was estimated to provide about 

12 mg/kg/day of fennel essential oil. The control group received the polysorbate only. The treatment 

provided a significant improvement of colic, eliminating symptoms in 65% of infants as compared 

to 23.7% for the control. No side effects were noted (Alexandrovich et al., 2003). 

11.12.4 JOINT PHYSIOTHERAPY 

Six sports physiotherapists treated 30 patients suffering from knee or ankle pathologies of traumatic 

or surgical origin. Two commercial products were used simultaneously, Dermasport ® and Solution 

Cryo ® . The former was a gel consisting of the essential oils of Betula alba, Melaleuca leucadendron, 

Cinnamomum camphora, Syzygium aromaticum, Eucalyptus globulus, and Gaultheria 

procumbens. Solution Cryo contained the same essential oils minus Gaultheria procumbens but 

with the addition of Chamaemelum nobile, Citrus limon, and Cupressus sempervirens. Both products 

were at an overall concentration of 6%. Thirty minutes after application a net reduction in 

movement pain and joint circumference was demonstrated, along with an increase in articular flexion 

and extension of both joints in all patients (Le Faou et al., 2005). 

11.12.5 NIPPLE PAIN 

Nipple cracks and pain are a common cause of breastfeeding cessation. In a randomized trial 196 

primiparous women were studied during the first 2 weeks postpartum. The test group applied peppermint 

water (essential oil in water, concentration not given) to the nipple and areola after each 

breastfeed while the control group applied expressed breast milk. The overall nipple crack rate at 

the end of the period in the peppermint group was 7% as compared to 23% for the control. Only 2% 

of peppermint group experienced severe nipple pain in contrast to 23% of the control, with 93% and 

71% experiencing no pain, respectively (Melli et al., 2007). 

11.12.6 OSTEOARTHRITIS 

A blend of Zingiber offi cinale (1%) and Citrus sinensis (0.5%) essential oils was used in an experimental 

double-blind study using 59 patients with moderate to severe knee pain caused by osteoarthritis. 

The treatment group received six massage sessions over a 3-week period; the placebo 

received the same massage sessions but without the essential oils and the control had no intervention. 

Assessment of pain intensity, stiffness, and physical functioning was carried out at baseline 

and at post 1 and 4 weeks. There were improvements in pain and function for the intervention group 

in comparison with the placebo and control at post 1 week but this was not sustained to week 4. The 

treatment was suggested for the relief of short-term knee pain (Yip et al., 2008). 

11.12.7 POSTHERPETIC NEURALGIA 

A double-blind crossover study examined the effect of the essential oil of Pelargonium spp. on moderate 

to severe postherpetic pain in 30 subjects. They were assigned to groups receiving 100, 50, or 

10% geranium essential oil (in mineral oil), mineral oil placebo, or capsaicin control. Pain relief was 

measured using a visual analogue scale from 0 to 60 min after treatment. Mean values for the time 

integral of spontaneous pain reduction was 21.3, 12.7, and 8.0 for the 100%, 50%, and 10% geranium


oils and evoked pain-reduction values were 15.8, 7.7, and 5.9, respectively. Both evoked and spontaneous 

pains were thus significantly reduced in a dose-dependent manner (Greenway et al., 2003). 

The result is interesting because topical capsaicin cream (one of the standard treatments for this 

condition) relieves pain gradually over 2 weeks, while the essential oil acted within minutes. 

Geranium essential oil applied cutaneously in animal studies has suppressed cellular inflammation 

and neutrophil accumulation in inflammatory sites (Maruyama et al., 2006) but postherpetic 

neuralgia normally occurs after the inflammation has subsided. One of the main components of the 

essential oil, geraniol, and the minor components of geranial, nerol, and neral, have been shown to 

interact with the transient receptor potential channel, TRPV1, as does capsaicin (Stotz et al., 2008). 

This sensory inhibition may explain the efficacy of topical geranium oil. 

11.12.8 POSTOPERATIVE PAIN 

A randomized, placebo-controlled clinical trial was conducted to determine whether the inhalation 

of lavender essential oil could reduce opioid requirements after laparoscopic adjustable gastric 

banding. In the postanesthesia care unit, 54 patients were given either lavender (two drops of a 2% 

dilution) or nonscented oil in a face mask. It was found that patients in the lavender group required 

significantly less morphine postoperatively than the placebo group (2.38 and 4.26 mg, respectively). 

Moreover, significantly more patients in the placebo group required analgesics in comparison with 

the lavender group; 82% compared to 46% (Kim et al., 2007). 

A similar study in the previous year with 50 patients who had undergone breast biopsy surgery 

had found that lavender essential oil had no significant effect on postoperative pain or analgesic 

requirements. However, a significantly higher satisfaction with pain control was noted by patients in 

the lavender group (Kim et al., 2006). 

11.12.9 PROSTATITIS 

One study has evaluated the use of Rowatinex for the treatment of chronic prostatitis/chronic 

pelvic pain syndrome, the rationale being based on the known anti-inflammatory properties of the 

product. A 6-week, randomized single-blind trial compared the use of Rowatinex 200 mg thrice 

daily with ibuprofen 600 mg thrice daily in 50 patients. Efficacy was measured by the National 

Institutes of Health (NIH)-Chronic Prostatitis Symptom Index (NIH-CPSI) that was completed by 

the patients on four occasions. The decrease in the NIH-CPSI was significant in both groups at the 

end of treatment and a 25% improvement in the total score was superior in the Rowatinex group 

(68%) compared to the ibuprofen group (40%). Although the symptomatic response was significant, 

no patients became asymptomatic (Lee et al., 2006). 

11.12.10 PRURITIS 

Pruritis is one of the most common complications of patients undergoing hemodialysis. Thirteen such 

patients were given an arm massage with lavender and tea tree essential oils (5% dilution in sweet 

almond and jojoba oil) thrice a week for 4 weeks. A control group received no intervention. Pruritis 

score, pruritis-related biochemical markers, skin pH, and skin hydration were measured before and 

after the study. There was a significant decrease in the pruritis score and blood urea nitrogen level for 

the test group. The control group showed a decreased skin hydration between pre- and post-test whereas 

for the essential oil group it was significantly increased (Ro et al., 2002). The lack of a massage only 

group in the study meant that the effects could not be definitely associated with the essential oils. 

11.13 PEDICULICIDAL ACTIVITY 

The activity of essential oils against the human head louse, Pediculus humanus capitis, has 

been investigated in a number of reports. Numerous essential oils have been found to exhibit


pediculicidal activity in vitro, with common oils such as Eucalyptus globulus, Origanum marjorana, 

Rosmarinus offi cinalis, and Elettaria cardamomum being comparable to, or more effective 

than d-phenothrin and pyrethrum (Yang et al., 2004). Melaleuca alternifolia and Lavandula 

angustifolia have also been found to be highly effective pediculicidal agents (Willimason 

et al., 2007). 

Despite the availability of positive in vitro results, only one trial involving application to humans 

has been documented; a mixture of anise and ylang ylang essential oils in coconut extract (Paranix ® ) 

was applied once to five children. Viable lice were not found after 7 days (Scanni and Bonifazi, 

2006). 

11.14 RECURRENT APHTHOUS STOMATITIS 

Recurrent aphthous stomatitis (RAS), also known as canker sores, are the most common oral 

mucosal lesions and although the process is sometimes self-limiting, the ulcer activity is mostly 

continuous and some forms may last for 20 years. Predisposing agents include bacteria and fungi, 

stress, mouth trauma, certain medications, and food allergies. Two essential oils both endemic to 

Iran have been investigated for treatment of this condition: Zataria multifl ora, a thyme-like plant 

containing thymol, carvacrol, and linalool as major components, and Satureja khuzistanica containing 

predominantly carvacrol. 

In a double-blind, randomized study, 60 patients with RAS received either 30 mL of an 

oral mouthwash composed of 60 mg of Zataria multifl ora essential oil in an aqueous-alcoholic solution 

or placebo thrice daily for 4 weeks. In the treatment group, 83% of patients responded well 

while 17% reported a deterioration of their condition. This was compared with 13% and 87% for the 

placebo group, respectively. A significant clinical improvement with regard to less pain and shorter 

duration of the condition was found in the essential oil group (Mansoori et al., 2002). 

Satureja khuzistanica essential oil 0.2% v/v was prepared in a hydroalcoholic solution and used 

in double-blind, randomized trial with 60 RAS patients. Its activity was compared with a 25% 

hydroalcoholic extract of the same plant and a hydroalcoholic placebo. A cotton pad was impregnated 

with 5 drops of preparation and placed on the ulcers for 1 min (fasting for 30 min afterwards) 

four times a day. The results of the extract and the essential oil groups were similar, with a significantly 

lower time for both pain elimination and complete healing of the ulcers in comparison with 

the placebo (Amanlou et al., 2007). The reported antibacterial, analgesic, antioxidant, and antiinflammatory 

activities of this essential oil (Abdollahi et al., 2003; Amanlou et al., 2004, 2005) 

were thought responsible for the result. 

11.15 RESPIRATORY TRACT 

Given the volatile nature of essential oils, it should come as no surprise that their ability to directly 

reach the site of intended activity via inhalation therapy has led to their use in the treatment of a 

range of respiratory conditions. Moreover, a number of components are effective when taken internally, 

since they are bioactive at the level of bronchial secretions during their excretion. With the 

exception of one report, all of the research has used the individual components of either 1,8-cineole 

or menthol, or has employed them in combination with several other isolated essential oil components 

within commercial preparations. 

11.15.1 MENTHOL 

Menthol-containing essential oils have been used in the therapy of respiratory conditions for many 

years and the individual component is present in a wide range of over-the-counter medications. Of 

the eight optical isomers of menthol, l-(-)-menthol is the most abundant in nature and imparts a 

cooling sensation to the skin and mucous membranes.


Menthol is known to react with a temperature-sensitive (8–28°C range) transient receptor potential 

channel, leading to an increase in intracellular calcium, depolarization and initiation of an 

action potential (Jordt et al., 2003). This channel, known as TRPM8, is expressed in distinct populations 

of afferent neurons; primarily thinly myelinated Ad cool fibers and to a lesser extent, unmyelinated 

C-fiber nociceptors (Thut et al., 2003). It is the interaction with the TRPM8 thermoreceptor 

that is responsible for the cooling effect of menthol when it is applied to the skin. This activity is not 

confined to the dermis, since the presence of TRPM8 has been demonstrated by animal 

experimentation in the squamous epithelium of the nasal vestibule (Clarke et al., 1992), the larynx 

(Sant’Ambrogio et al., 1991), and lung tissue (Wright et al., 1998). Thus the activation of cold receptors 

via inhaled menthol leads to a number of beneficial effects. 

11.15.1.1 Antitussive 

Despite being used as a component in cough remedies since the introduction of a “vaporub” in 1890, 

there are few human trials of menthol used alone as being effective. In a citric acid-induced cough 

model in healthy subjects, Packman and London (1980) found that menthol was effective, although 

1,8-cineole was more efficacious. The use of an aromatic unction rather than direct inhalation may 

have affected the results, since the inhalation of menthol has been shown in animal models to be 

significantly more effective at cough frequency reduction (28% and 56% at 10 and 30 g/l, respectively) 

compared to 1,8-cineole (Laude et al., 1994). 

A single-blind pseudorandomized crossover trial in 42 healthy children was used to compare the 

effect of an inhalation of either menthol or placebo on citric acid-induced cough. It was found that 

cough frequency was reduced in comparison with the baseline but not to that of the placebo (Kenia 

et al., 2008). However, the placebo chosen was eucalyptus oil, whose main component is 1,8-cineole 

and known to have similar antitussive properties to menthol. 

Along with other ion channel modulators, menthol is recognized as a potential “novel therapy” 

for the treatment of chronic cough (Morice et al., 2004, p. 489). It is not clear whether the antitussive 

activity of menthol is due solely to its stimulation of airway cold receptors; it may also involve pulmonary 

C-fibers (a percentage of which also express TRPM8) or there may be a specific interaction 

with the neuronal cough reflex. 

11.15.1.2 Nasal Decongestant 

Menthol is often thought of as a decongestant, but this effect is a sensory illusion. Burrow et al. 

(1983) and Eccles et al. (1988) showed that there was no change in nasal resistance to airflow during 

inhalation of menthol, although the sensation of nasal airflow was enhanced. In the former experiment, 

1,8-cineole and camphor were also shown to enhance the sensation of airflow, but to a lesser 

extent than menthol. 

In a double-blind, randomized trial subjects suffering from the common cold were given lozenges 

containing 11 mg of menthol. Posterior rhinomanometry could detect no change in nasal 

resistance to airflow after 10 min; however, there were significant changes in the nasal sensation of 

airflow (Eccles et al., 1990). 

A single-blind pseudorandomized crossover trial compared the effect of an inhalation of 

either menthol or placebo. The main outcome measures were nasal expiratory and inspiratory flows 

and volumes, as measured by a spirometer and the perception of nasal patency, assessed with a 

visual analogue scale. It was found that there was no effect of menthol on any of the spirometric 

measurements although there was a significant increase in the perception of nasal patency (Kenia 

et al., 2008). 

Thus it has been demonstrated that menthol is not a nasal decongestant. However, it is useful in 

therapy since stimulation of the cold receptors causes a subjective sensation of nasal decongestion 

and so relieves the feeling of a blocked nose. In commercial preparations that include menthol, a 

true decongestant such as oxymetazoline hydrochloride is often present.


11.15.1.3 Inhibition of Respiratory Drive and Respiratory Comfort 

When cold air was circulated through the nose in human breath-hold experiments, subjects were 

able to hold their breath longer (McBride and Whitelaw, 1981) and inhaling cold air was shown to 

inhibit normal breathing patterns (Burgess and Whitelaw, 1988). This indicated that cold receptors 

could be one source of monitoring inspiratory flow rate and volume. Several animal experiments 

demonstrated that the inhalation of cold air, warm air, plus menthol, or menthol alone (390 ng/mL) 

significantly enhanced ventilator inhibition (Orani et al., 1991; Sant’Ambrogio et al., 1992). 

Sloan et al. (1993) conducted breath-hold experiments with 20 healthy volunteers. The ingestion 

of a lozenge containing 11 mg of menthol significantly increased the hold time, indicating a 

depression of the ventilatory drive. It was later postulated by Eccles (2000) that in addition to 

chemoreceptors detecting oxygen and carbon dioxide in the blood, cold receptors in the respiratory 

tract may also modulate the drive to breathe. 

Eleven healthy subjects breathed through a device that had either an elastic load or a flowresistive 

load. Sensations of respiratory discomfort were compared using a visual analogue scale 

before, during, and after inhalation of menthol. It was found that the discomfort associated with 

loaded breathing was significantly reduced and was more effective during flow-resistive loading 

than elastic loading. Inhalation of another fragrance had no effect and so the result was attributed to 

a direct stimulation of cold receptors by menthol, a reduction in respiratory drive being perhaps 

responsible (Nishino et al., 1997). 

During an investigation of dyspnea, the effect of menthol inhalation on respiratory discomfort 

during loaded breathing was found to be inconsistent. Further tests found that the effect of menthol 

was most important during the first few minutes of inhalation and in the presence of high loads 

(Peiffer et al., 2001). The therapeutic application of menthol in the alleviation of dyspnea has yet to 

be described. 

11.15.1.4 Bronchodilation and Airway Hyperresponsiveness 

The spasmolytic activity of menthol on airway smooth muscle has been demonstrated in vitro 

(Taddei et al., 1988). To examine the bronchodilatory effects of menthol, a small trial was conducted 

on six patients with mild to moderate asthma. A poultice-containing menthol was applied daily for 

4 weeks and it was found that bronchoconstriction was decreased and airway hyperresponsiveness 

improved (Chiyotani et al., 1994b). 

A randomized, placebo-controlled trial examined the effects of menthol (10 mg nebulized twice 

daily for 4 weeks) on airway hyperresponsiveness in 23 patients with mild to moderate asthma. The 

diurnal variation in the peak expiratory flow rate (a value reflecting airway hyperexcitability) was 

decreased but the forced expiratory volume was not significantly altered. This indicated an improvement 

of airway hyperresponsiveness without affecting airflow limitation (Tamaoki et al., 1995). 

Later in vivo research examined the effect of menthol on airway resistance caused by capsaicin- and 

neurokinin-induced bronchoconstriction; there was a significant decrease in both cases by inhalation 

of menthol at 7.5 μg/L air concentration. The in vitro effect of menthol on bronchial rings was 

also studied. It was concluded that menthol attenuated bronchoconstriction by a direct action on 

bronchial smooth muscle (Wright et al., 1997). 

In cases of asthma, the beneficial effects of menthol seem to be mainly due to its bronchodilatory 

activity on smooth muscle; interaction with cold receptors and the respiratory drive may also play 

an important role. 

Recent in vitro studies have shown that a subpopulation of airway vagal afferent nerves expresses 

TRPM8 receptors and that activation of these receptors by cold and menthol excite these airway 

autonomic nerves. Thus, activation of TRPM8 receptors may provoke an autonomic nerve reflex to 

increase airway resistance. It was postulated that this autonomic response could provoke mentholor 

cold-induced exacerbation of asthma and other pulmonary disorders (Xing et al., 2008). Direct


cold stimulation or inhalation of menthol can cause immediate airway constriction and asthma in 

some people; perhaps the TRPM8 receptor expression is upregulated in these subjects. The situation 

is far from clear. 

11.15.1.5 Summary 

The respiratory effects of menthol that have been demonstrated are as follows: 

1. Antitussive at low concentration. 

2. Increases the sensation of nasal airflow giving the impression of decongestion. 

3. No physical decongestant activity. 

4. Depresses ventilation and the respiratory drive at comparatively higher concentration. 

5. Reduces respiratory discomfort and sensations of dyspnea. 

A number of in vitro and animal experiments have demonstrated the bronchomucotropic activity 

of menthol (Boyd and Sheppard, 1969; Welsh et al., 1980; Chiyotani et al., 1994a), whereas there 

have been conflicting reports as to whether menthol is a mucociliary stimulant (Das et al., 1970) or 

is ciliotoxic (Su et al., 1993). Apart from the inclusion of relatively small quantities of menthol in 

commercial preparations that have known beneficial mucociliary effects, there are no documented 

human trials to support the presence of these activities. 

11.15.2 1,8-CINEOLE 

This oxide has a number of biological activities that make it particularly useful in the treatment of 

the respiratory tract. 1,8-Cineole has been registered as a licensed medication in Germany for over 

20 years and is available as enteric-coated capsules (Soledum ® ). It is therefore not surprising that 

the majority of the trials originate from this country and use oral dosing of 1,8-cineole instead of 

inhalation. Rather than discuss specific pathologies, the individual activities will be examined and 

their relevance (alone or in combination) in treatment regimes should become apparent. 

11.15.2.1 Antimicrobial 

The anti-infectious properties of essential oils high in 1,8-cineole content may warrant their inclusion 

into a treatment regime but other components are more effective in this regard. 1,8-Cineole is 

often considered to have marginal or no antibacterial activity (Kotan et al., 2007), although it is very 

effective at causing leakage of bacterial cell membranes (Carson et al., 2002). It may thus allow 

more active components to enter the bacteria by permeabilizing their membranes. 

1,8-Cineole does possess noted antiviral properties compared to the common essential oil components 

of borneol, citral, geraniol, limonene, linalool, menthol, and thymol; only that of eugenol 

was greater (Bourne et al., 1999). However, in comparison with the potent thujone, the antiviral 

potential of 1,8-cineole was considered relatively low (Sivropoulou et al., 1997). 

A placebo-controlled, double-blind, randomized parallel-group trial examined the long-term treatment 

of 246 chronic bronchitics during winter with myrtol standardized Gelomyrtol ® forte. This 

established German preparation consists mainly of 15% a-pinene, 35% limonene, and 47% 1,8-cineole 

and was administered thrice daily in 300 mg capsules. It was found to reduce the requirement for 

antibiotics during acute exacerbations; 51.6% compared to 61.2% under placebo. Of those patients 

needing antibiotics, 62.5% in the test group required them for £7 days whereas 76.7% of patients in the 

placebo group needed antibiotics for more than 7 days. Moreover, 72% of patients remained without 

acute exacerbations in the test group compared to 53% in the placebo group (Meister et al., 1999). 

Although emphasis was given to antibiotic reduction, a significant antimicrobial effect by the 

preparation is unlikely to have paid an important contribution. Indeed, Meister et al. refer to reduced 

health impairment due to sputum expectoration and cough, and note other beneficial properties of 

1,8-cineole that will be discussed in Sections 11.15.2.2 through 11.15.2.6.


11.15.2.2 Antitussive 

The antitussive effects of 1,8-cineole were first proven by Packman and London in 1980, who 

induced coughing in 32 healthy human subjects via the use of an aerosol spray containing citric 

acid. This single-blind crossover study examined the effect of a commercially available chest rub 

containing, among others, eucalyptus essential oil. The rub was applied to the chest in a 7.5 mg dose 

and massaged for 10–15 s after which the frequency of the induced coughing was noted. It was 

found that the chest rub produced a significant decrease in the induced cough counts and that 

eucalyptus oil was the most active component of the rub. 

1,8-Cineole interacts with TRPM8, the cool-sensitive thermoreceptor that is primarily affected by 

menthol. In comparison with menthol, the effect of 1,8-cineole on TRPM8 (as measured by Ca 2+ influx 

kinetics) is much slower and declines more rapidly (Behrendt et al., 2004). In a similar manner to menthol, 

the antitussive activity of 1,8-cineole may be due in part to its stimulation of airway cold receptors. 

11.15.2.3 Bronchodilation 

In vitro tests using guinea pig trachea determined that the essential oil of Eucalyptus tereticornis 

had a myorelaxant, dose-dependent effect (10–1000 mg/mL) on airway smooth muscle, reducing 

tracheal basal tone and K + -induced contractions, as well as attenuating acetylcholine-induced 

contractions at higher concentrations (Coelho-de-Souza et al., 2005). This activity was found to 

be mainly due to 1,8-cineole, although the overall effect was thought due to a synergistic relationship 

between the oxide and a- and b-pinene. Similar results were obtained using the essential oil 

of Croton nepetaefolius, whose major component was also 1,8-cineole (Magalhães et al., 2003). 

A double-blind, randomized clinical trial over 7 days compared oral pure 1,8-cineole (3 ¥ 200 mg/ 

day) to Ambroxol (3 ¥ 30 mg/day) in 29 patients with chronic obstructive pulmonary disease (COPD). 

Vital capacity, airway resistance, and specific airway conductance improved significantly for both 

drugs, whereas the intrathoracic gas volume was reduced by 1,8-cineole but not by Ambroxol. All 

parameters of lung function, peak flow, and symptoms of dyspnea were improved by 1,8-cineole 

therapy, but were not statistically significant in comparison with Ambroxol due to the small number 

of patients. In addition to other properties, it was noted that the oxide seemed to have bronchodilatory 

effects (Wittman et al., 1998). 

11.15.2.4 Mucolytic and Mucociliary Effects 

Mucolytics break down or dissolve mucus and thus facilitate the easier removal of these secretions 

from the respiratory tract by the ciliated epithelium, a process known as mucociliary clearance. 

Some mucolytics also have a direct action on the mucociliary apparatus itself. 

Administered via steam inhalation to rabbits, 1,8-cineole in concentrations that produced a barely 

detectable scent (1–9 mg/kg) augmented the volume output of respiratory tract fluid from 9.5% to 

45.3% (Boyd and Sheppard, 1971), an effect that they described as “mucotropic.” Interestingly, in the 

same experiment fenchone at 9 mg/kg increased the output by 186.2%, thus confirming the strong 

effects of some ketones in this regard. Also using rabbits, Zanker (1983) found that oxygenated monoterpenoids 

reduced mucus deposition and partially recovered the activity of ciliated epithelium. 

Because of these early animal experiments, the beneficial effects of 1,8-cineole on mucociliary 

clearance have been clearly demonstrated in a number of human trials. Dorow et al. (1987) examined 

the effects of a 7-day course of either Gelomyrtol forte (4 ¥ 300 mg/day) or Ambroxol 

(3 ¥ 30 mg/day) in 20 patients with chronic obstructive bronchitis. Improved mucociliary clearance 

was observed in both groups, although improvement in lung function was not detected. 

Twelve patients with chronic obstructive bronchitis were given a 4-day treatment with 1,8- cineole 

(4 ¥ 200 mg/day). By measuring the reduction in percentage radioactivity of an applied radioaerosol, 

significant improvements in mucociliary clearance were demonstrated at the 60 and 120 min after 

each administration (Dorow, 1989). 

In a small double-blind study, the expectorant effect of Gelomyrtol forte (1 ¥ 300 mg/day, 

14 days) was examined in 20 patients with chronic obstructive bronchitis. The ability to expectorate,


frequency of coughing attacks, and shortness of breath were all improved by the therapy, as was 

sputum volume and color. Both patients and physicians rated the effects of Gelomyrtol forte as 

better than the placebo, but due to the small group size statistically significant differences could not 

be demonstrated (Ulmer and Schött, 1991). 

A randomized, double-blind, placebo-controlled trial was used to investigate the use of mucolytics 

to alleviate acute bronchitis (Mattys et al., 2000). They compared Gelomyrtol forte (4 ¥ 300 mg, 

days 1–14), with Ambroxol (3 ¥ 30 mg, days 1–3; 2 ¥ 30 mg, days 4–14) and Cefuroxime (2 ¥ 250 mg, 

days 1–6) in 676 patients. By monitoring cough frequency data, regression of the frequency of 

abnormal auscultation, hoarseness, headache, joint pain, and fatigue, it was shown that Gelomyrtol 

forte was very efficacious and comparable to the other active treatments. Overall, it scored slightly 

more than Ambroxol and Cefuroxime and was therefore considered to be a well-evidenced alternative 

to antibiotics for acute bronchitis. 

Several studies have demonstrated a direct effect of 1,8-cineole on the ciliated epithelium itself. 

Kaspar et al. (1994) conducted a randomized, double-blind three-way crossover 4-day study of the 

effects of 1,8-cineole (3 ¥ 200 mg/day) or Ambroxol (3 ¥ 30 mg/day) on mucociliary clearance in 

30 patients with COPD. Treatment with the oxide resulted in a statistically significant increase in the 

ciliary beat frequency of nasal cilia, a phenomenon that did not occur with the use of Ambroxol (an 

increase of 8.2% and 1.1%, respectively). A decrease of “saccharine-time” was clinically relevant 

and significant after 1,8-cineole therapy (241 s) but not after Ambroxol (48 s). Lung function parameters 

were significantly improved equally by both drugs. 

After the ingestion of Gelomyrtol forte (3 ¥ 1 capsule/day for 4 days) by four healthy persons and 

one person after sinus surgery, there was a strong increase in mucociliary transport velocity, as 

detected by movement of a radiolabeled component (Behrbohm et al., 1995). 

In sinusitis, the ciliated beat frequency is reduced and 30% of ciliated cells convert to mucussecreting 

goblet cells. The impaired mucociliary transport, excessive secretion of mucus, and edema 

block drainage sites leading to congestion, pain, and pressure. 

To demonstrate the importance of drainage and ventilation of sinuses as a therapeutic concept, 

Federspil et al. (1997) conducted a double-blind, randomized, placebo-controlled trial using 

331 patients with acute sinusitis. The secretolytic effects of Gelomyrtol forte (300 mg) over a 6-day 

period proved to be significantly better than the placebo. 

Kehrl et al. (2004) used the known stimulatory effects of 1,8-cineole on ciliated epithelium and its 

mucolytic effect as a rationale for treating 152 acute rhinosinusitis patients in a randomized, doubleblind, 

placebo-controlled study. The treatment group received 3 ¥ 200 mg 1,8-cineole daily for 7 

days. There was a clinically relevant and significant improvement in frontal headache, headache on 

bending, pressure point sensitivity of the trigeminal nerve, nasal obstruction, and rhinological secretions 

in the test group, as compared to the control group. It was concluded that 1,8-cineole was a safe 

and effective treatment for acute nonpurulent rhinosinusitis before antibiotics are indicated. 

11.15.2.5 Anti-Inflammatory Activity 

The effects of 1,8-cineole on stimulated human monocyte mediator production were studied in vitro 

and compared with that of budesonide, a corticosteroid agent with anti-inflammatory and immunosuppresive 

effects (Juergens et al., 1998a). At therapeutic levels, both substances demonstrated a 

similar inhibition of the inflammatory mediators leukotriene B 4 (LTB 4 ), PGE 2 , and interleukin-1b 

(IL-1b). This was the first evidence of a steroid-like inhibition of arachidonic acid metabolism and 

IL-1b production by 1,8-cineole. 

Later that year, the same team (Juergens et al., 1998b) reported a dose-dependent and highly 

significant inhibition of tumor necrosis factor-a (TNF-a), IL-1b, thromboxane B 2 , and LTB 4 production 

by 1,8-cineole from stimulated human monocytes in vitro. 

A third experiment combined ex vivo and in vivo testing; 10 patients with bronchial asthma were 

given 3 ¥ 200 mg of 1,8-cineole daily for 3 days. Lung function was measured before the first dose, at 

the end of the third dose and 4 days after discontinuation of the therapy. At the same time, blood


samples were taken from which monocytes were collected and stimulated ex vivo for LTB 4 and PGE 2 

production. Twelve healthy volunteers also underwent the treatment and their blood was taken for testing. 

It was found that by the end of the treatment and 4 days after, the production of LTB 4 and PGE 2 

from the monocytes of both asthmatics and healthy individuals was significantly inhibited. Lung 

function parameters of asthmatic patients were significantly improved (Juergens et al., 1998c). 

These three reports suggested a strong anti-inflammatory activity of 1,8-cineole via both the 

cyclooxygenase and 5-lipooxygenase pathways, and the possibility of a new, well-tolerated treatment 

of airway inflammation in obstructive airway disease. 

Juergens et al. (2003) conducted a double-blind, placebo-controlled clinical trial involving 

32 patients with steroid-dependent severe bronchial asthma. The subjects were randomly assigned 

to receive either a placebo or a 3 ¥ 200 mg 1,8-cineole daily for 12 weeks. Oral glucocorticosteroids 

were reduced by 2.5 mg increments every 3 weeks with the aim of establishing the glucocorticosteroid-sparing 

capacity of 1,8-cineole. The majority of asthma patients receiving oral 

1,8-cineole remained clinically stable despite a mean reduction of oral prednisolone dosage of 36%, 

equivalent to 3.8 mg/day. In the placebo group, where only four patients could tolerate a steroid 

decrease, the mean reduction was 7%, equivalent to 0.9 mg/day. Compared with the placebo group, 

1,8-cineole recipients maintained their lung function four times longer despite receiving lower 

doses of prednisolone. 

Increased mucus secretion often appears as an initial symptom in exacerbated COPD and asthma, 

where stimulated mediator cells migrate to the lungs to produce cytokines; of particular importance 

are TNF-a, IL-1b, IL-6, and IL-8 and those known to induce immunoglobulin E (IgE) antibody 

synthesis and maintain allergic eosinophilic inflammation (IL-4 and IL-5). Therefore, a study was 

conducted to investigate the role of 1,8-cineole in inhibiting cytokine production in stimulated 

human monocytes and lymphocytes in vitro (Juergens et al., 2004). It was shown that 1,8-cineole is 

a strong inhibitor of TNF-a and IL-1b in both cell types. At known therapeutic blood levels, it also 

had an inhibitory effect on the production of the chemotactic cytokines IL-8 and IL-5 and may 

possess additional antiallergic activity by blocking IL-4 production. 

A clinically relevant anti-inflammatory activity of 1,8-cineole has thus been proven for therapeutic 

use in airway diseases. 

11.15.2.6 Pulmonary Function 

An inhaler was used to apply 1,8-cineole (Soledum Balm) to 24 patients with asthma or chronic 

bronchitis in an 8-day-controlled trial. In all but one patient, an objective rise in expiratory peak 

flow values was demonstrated. The subjective experience of their illness was significantly improved 

for all subjects (Grimm, 1987). 

In an open trial of 100 chronic bronchitics using both inhaled (4 ¥ 200 mg) and oral (3 ¥ 200 mg) 

1,8-cineole over 7 days, the clinical parameters of forced vital capacity, forced expiratory volume, 

peak expiratory flow, and residual volume were all significantly improved when compared to initial 

values before treatment (Mahlo, 1990). 

In a randomized, double-blind, placebo-controlled study of 51 patients with COPD, 1,8-cineole 

(3 ¥ 200 mg/day) was given for 8 weeks. For the objective lung functions of “airway resistance” and 

“specific airway resistance,” there was a clinically significant reduction of 21% and 26%, respectively. 

The improvement was attributed to a positive influence on disturbed breathing patterns, 

mucociliary clearance, and anti-inflammatory effects (Habich and Repges, 1994). 

The majority of the in vivo trials involving 1,8-cineole report good, if not significant, changes in 

lung function parameters, whether the investigation concerns the common cold or COPD. This is 

not a convenient, accidental side effect of treatment but is a direct result of one or more of the factors 

already discussed that have direct effects on the pathophysiology of the airways. The ability to 

breathe more effectively and easily is an important consequence of the therapy that is sometimes 

minimized when dealing with the specific complexities of infection, inflammation, and so on. 

A compilation of human trials with 1,8-cineole is given in Table 11.3.


Table 11.3 

Summary of Human Trials Demonstrating the Beneficial Effects of 1,8-Cineole in Various 

Respiratory Conditions 

Patients Treatment Outcome Reference 

11 Cineole inhalation, 

8 days 

10 Cineole 3 ¥ 200 mg 

daily, 3 days 


daily, 12 weeks 

Asthma 

Objective rise in expiratory peak flow found. Subjective 

experiences of illness significantly improved 

LTB 4 and PGE 2 production by monocytes was 

significantly inhibited. Lung functions were 

significantly improved 

Twelve of 16 patients in cineole group remained 

stable despite a 36% reduction in oral steroid dosage 

Acute bronchitis 

60 Vaporub 3 min Decreased breathing frequency, suggesting “easier 

breathing” 

676 Gelomyrtol 

4 ¥ 300 mg daily, 

14 days 

9 Cineole inhalation, 

8 days 

100 Cineole 3 ¥ 200 mg 

daily, 7 days 

246 Gelomyrtol 

3 ¥ 300 mg daily, 

6 months 

20 Gelomyrtol 4 ¥ 0.3 g 

daily, 7 days 

20 Gelomyrtol 1 ¥ 0.3 g 

daily, 14 days 

12 Cineole 4 ¥ 200 mg, 

4 days 

51 including 

16 asthmatics 

Cineole 3 ¥ 200 mg 

daily, 8 weeks 


daily, 4 days 


daily, 7 days 

24 Eucalyptus oil (9% 

of a mixture) 

331 Gelomyrtol 300 mg, 

6 days 

152 Cineole 3 ¥ 200 mg 

daily, 7 days 

Coughing, sputum consistency, well-being, bronchial 

hyperreactivity, and associated symptoms all improved 

similarly by Gelomyrtol, Ambroxol, and Cefuroxime 

Chronic bronchitis 

Objective rise in expiratory peak flow found. Subjective 

experiences of illness significantly improved 

Grimm (1987) 

Juergens et al. 

(1998c) 

Juergens et al. 

(2003) 

Berger et al. 

(1978a) 

Mattys et al. 

(2000) 

Grimm (1987) 

All lung function parameters significantly improved Mahlo (1990) 

Reduced acute exacerbations, reduced requirement 

for antibiotics, reduced treatment times when 

antibiotics taken. Well-being significantly improved 

COPD 

Improved mucociliary clearance 

All parameters relating to coughing improved. 

Sputum volume increased 

Meister et al. 

(1999) 

Dorow et al. 

(1987) 

Ulmer and 

Schött (1991) 

Significant improvement of mucociliary clearance Dorow (1989) 

Significant improvement in airway resistance (21%), 

positive effects on sputum output and dyspnea 

Significant improvements in lung functions of 

FVC and FEV 1 (Ambroxol and cineole equieffective), 

significant increase in ciliary beat frequency 

All lung function parameters, peak flow and 

dyspnea improved from day 1 onward 

Common cold 

Reversed lung function abnormalities in small 

and large airways 

Sinusitis 

Effective treatment instead of antibiotics 

Effective reduction of symptoms without 

the need for antibiotics 

Habich and 

Repges 

(1994) 

Kaspar et al. 

(1994) 

Wittmann 

et al. (1998) 

Cohen and 

Dressler 

(1982) 

Federspil et al. 

(1997) 

Kehrl et al. 

(2004)


11.15.2.7 Summary 

Although discussed separately, the multifaceted activities of 1,8-cineole perform together in harmony 

to provide an effective intervention that can inherently adapt to the needs of the individual 

patient. As already described, 1,8-cineole is known to possess the following properties: 

1. Antimicrobial 

2. Antitussive 

3. Bronchodilatory 

4. Mucolytic 

5. Ciliary transport promotion 

6. Anti-inflammatory 

7. Lung function improvement. 

Therefore, it may be seen that a diverse range of respiratory conditions of varying complexities 

will benefit from the use of pure 1,8-cineole or from essential oils containing this oxide as a major 

component. 

11.15.3 TREATMENT WITH BLENDS CONTAINING BOTH MENTHOL AND 1,8-CINEOLE 

A study measured transthoracic impedance pneumographs of 60 young children (2–40 months) 

with acute bronchitis before and after a 3-min application of Vaporub ® to the back and chest. The 

data showed an early increase in amplitude up to 33%, which slowly descended during the 70-min 

post-treatment period to slightly above the control. Breathing frequency progressively decreased 

during the same period by 19.4%. Clinical observations combined with these results suggested a 

condition of “easier breathing” (Berger et al., 1978a). Currently, the active ingredients of Vaporub 

are camphor 4.8%, 1,8-cineole 1.2%, and menthol 2.6, but these components and percentages may 

have changed over the years. 

The same team employed a similar experiment but used the pneumographic data to examine the 

quiet periods, that is, parts of the pneumogram where changes in the baseline were at least half of the 

average amplitude in more than five consecutive breathing excursions. It was found that the application 

of Vaporub increased quiet periods by up to 213.8%, whereas the controls (petroleum jelly application 

or rubbing only) never exceeded 62.4%. Thus the breathing restlessness of children with 

bronchitis was diminished and this was confirmed by clinical observations (Berger et al., 1978b). 

By the measurement of lung and forced expiratory volumes, nasal, lower, and total airway resistances, 

closing volume data, the phase III slope of the alveolar plateau, and the maximum expiratory 

flow volume, peripheral airway dysfunction was confirmed in 24 adults with common colds. In a 

randomized, controlled trial, an aromatic mixture of menthol, eucalyptus oil, and camphor (56%, 

9%, and 35% w/w, respectively) were vaporized in a room where the subjects were seated. Respiratory 

function measurements were made at baseline, 20 and 60 min after exposure. After the last measurement, 

phenylephrine was sprayed into the nostrils and the measurements taken again 5–10 min later 

to determine potential airway responsiveness. The control consisted of tap water. The results showed 

significant changes in forced vital capacity, forced expiratory volume, closing capacity, and the phase 

III slope after aromatic therapy as compared to the control. It was concluded that the aromatic inhalation 

favorably modified the peripheral airway dysfunction (Cohen and Dressler, 1982). 

In a randomized, placebo-controlled trial of citric acid-induced cough in 20 healthy subjects, the 

inhalation of a combination of menthol and eucalyptus oil (75% and 25%, respectively) significantly 

decreased the cough frequency (Morice et al., 1994). 

The effect of an aromatic inunction (Vaporub) was studied by the inhalation of a radioaerosol in 

a randomized, single-blinded, placebo-controlled crossover trial with 12 chronic bronchitics. It was 

found that after the application of 7.5 g of the product to the chest, removal of the tracheobronchial


deposit was significantly enhanced at 30 and 60 min postinhalation, although further effects could 

not be demonstrated during the following 5 h, despite further application of the rub. During the first 

hour, mucociliary clearance was correlated with the concentration level of the aromatics (Hasani 

et al., 2003). 

Another commercial preparation Pinimenthol ® , a mixture of eucalyptus and pine needle oils plus 

menthol, reduced bronchospasm and demonstrated significant secretolytic effects when insufflated 

through the respiratory tract and when applied to the epilated skin of animals (Schäfer and Schäfer, 

1981). In addition to the known effects of menthol and 1,8-cineole, pine needle oil is considered to 

be weakly antiseptic and secretolytic (Approved Herbs, 1998). 

In a randomized, double-blind 14-day trial, 100 patients with chronic obstructive bronchitis 

received a combination of theophylline with b-adrenergica 2–3 times daily. The test group also 

received Pinimenthol. The parameters were investigated were objective (measurement of lung function 

and sputum) and subjective (cough, respiratory insufficiency, and pulmonary murmur). All 

differences in the subjective evaluations were statistically significant and of clinical importance; 

secretolysis was clearly shown. The addition of Pinimenthol showed a clear superiority to the basic 

combination therapy alone (Linsenmann and Swoboda, 1986). 

A postmarketing survey was conducted of 3060 patients prescribed Pinimenthol suffering from 

cold, acute or chronic bronchitis, bronchial catarrh, or hoarseness. The product was given by inunction 

(29.6%), inhalation (17.3%), or inunction and inhalation (53.1%). Only 22 patients reported 

adverse effects and the efficacy of the product was judged as excellent or good by 88.3% of physicians 

and 88.1% of patients (Kamin and Kieser, 2007). 

11.16 ALLERGIC RHINITIS 

In a proof of concept study, a nasal spray was made from the essential oil of Artemisia abrotanum 

L. (4 mg/mL) and flavonoid extracts (2.5 μg/mL) from the same plant. The essential oil consisted 

primarily of 1,8-cineole and davanone at approximately 40% and 50%, respectively. Apart 

from a spasmolytic activity (Perfumi et al., 1995), little is known about the biological activity of 

davanone. The flavonoids present were thought to inhibit histamine release and interfere with 

arachidonic acid metabolism. The nasal spray was self-administered by 12 patients with allergic 

rhinitis, allergic conjunctivitis, and/or bronchial obstructive disease. They were instructed to use 

1–2 puffs in each nostril at the first sign of symptoms, to a maximum of six treatments per day. All 

patients experienced rapid and significant relief of nasal symptoms and for those with allergic 

conjunctivitis, a significant relief of subjective eye symptoms was also experienced. Three of six 

patients with bronchial obstructive disease experienced rapid and clinically significant bronchial 

relief (Remberg et al., 2004). 

11.17 SNORING 

A blend of 15 essential oils was developed into a commercial product called “Helps stop snoring” 

and 140 adult snorers were recruited into a randomized trial using the product as a spray or gargle. 

Visual analogue scales were completed by the snorers’ partners relating to sleep disturbance each 

night. The treatment lasted for 14 days and results were compared to a pretrial period of the same 

length. The partners of 82% of the patients using the spray and 71% of patients using the gargle 

reported a reduction in snoring. This was compared to 44% of placebo users. The mode of action 

was postulated as being antispasmodic to the soft palate and pharynx (Pritchard, 2004). 

11.18 SWALLOWING DYSFUNCTION 

A delayed triggering of the swallowing reflex, mainly in elderly people, predisposes to aspiration 

pneumonia. To improve dysphagia, two different approaches using essential oils have been tried 

with success.


As black pepper is a strong appetite stimulant, it was postulated that nasal inhalation of the 

essential oil may stimulate cerebral blood flow in the insular cortex, the dysfunction of which has 

been reported to play a role in dysphagia. A randomized, controlled study of 105 elderly patients 

found that the inhalation of black pepper oil for 1 min significantly shortened the delayed swallowing 

time and increased the number of swallowing movements. Emission computed tomography 

demonstrated activation of the anterior cingulate cortex by the treatment. The inhalation of lavender 

essential oil or water had no effects (Ebihara et al., 2006a). 

A second study used the established stimulating effects of menthol on cold receptors, since 

cold stimulation was known to restore sensitivity to trigger the swallowing reflex in dysphagic patients. 

Menthol was introduced into the pharynx of patients with mild to moderate dysphagia via a nasal 

catheter. The latent time of swallowing reflex was reduced significantly by menthol in a concentrationdependent 

manner; 10 -2 menthol reduced the time to 9.4 s as compared to 13.8 s for distilled water. 

The use of a menthol lozenge before meals was thought appropriate (Ebihara et al., 2006b). 


It is apparent from the diverse range of conditions that have benefitted from the administration of 

essential oils that their therapeutic potential is vast and yet underdeveloped. Moreover, since they 

are not composed of a single “magic bullet” with one target, they often have multiple effects that 

have additive or synergistic properties within a treatment regime. 

A great many research papers investigating the bioactivity of essential oils conclude that the 

results are very encouraging and that clinical trials are the next step. For the majority, this step is 

never taken. The expense is one limiting factor and it is not surprising that clinical trials are mostly 

conducted once the essential oils have been formulated into a commercial product that has financial 

backing. It is evident that many of the claims made for essential oils in therapeutic applications have 

not been substantiated and an evidence base is clearly lacking. However, there is similarly a lack of 

research to demonstrate that essential oils are not effective interventions. 

With the continuing search for new medicaments from natural sources, especially in the realm 

of antimicrobial therapy, it is hoped that future research into the efficacy of essential oils will be 

both stimulated and funded. 

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12 

In Vitro Antimicrobial 

Activities of Essential Oils 

Monographed in the European 

Pharmacopoeia 6th Edition 

Alexander Pauli and Heinz Schilcher 

CONTENTS 

12.1 Introduction ..................................................................................................................... 353 

12.1.1 Agar Diffusion Test (ADT) ................................................................................ 354 

12.1.2 Dilution Test (DIL) ............................................................................................. 354 

12.1.3 Vapor Phase Test (VP) ....................................................................................... 355 

12.2 Results ............................................................................................................................. 534 

12.3 Discussion ....................................................................................................................... 537 

References .................................................................................................................................. 540 


One of the first systematic in vitro examinations of the antimicrobial activity of essential oils dates 

back to the late nineteenth century when Buchholtz studied the growth inhibitory properties of caraway 

oil, thyme oil, phenol, and thymol on bacteria having been cultivated in a tabac decoction. In 

this examination, thymol turned out to be 10-fold stronger than phenol (Buchholtz, 1875), which 

was in use as surgical antiseptic at that time (Ashhurst, 1927). The German pharmacopoeia 

“Deutsches Arzneibuch 6” (DAB 6) issued in 1926 and later supplements (1947, 1959) listed together 

26 essential oils, and by this it has become obvious that essential oils have a long history in pharmaceutical 

practice due to their pharmacological activities. The European Pharmacopoeia 6th edition 

issued in 2007 lists 28 essential oils. Among them are 20 oils already present in DAB 6 (anise, bitter 

fennel, caraway, cassia, cinnamon bark, citronella, clove, coriander, eucalyptus, juniper, lavender, 

lemon, matricaria, neroli, peppermint, pine needle, pumilio pine, rosemary, thyme, and turpentine), 

three oils have been previously listed in the British Pharmacopoeia in the year 1993 (dementholized 

mint, nutmeg, and sweet orange), one in the French Pharmacopoeia X (star anise), and five oils were 

added later (cinnamon leaf, clary sage, mandarin, star anise, and tea tree). 

Since essential oils are subject for pharmacological studies, tests on their antimicrobial activities 

have been done frequently. In consequence, a comprehensive data material exists, which, however, 

has never been compiled together for important essential oils, like such listed actually in the 

European Pharmacopoeia. Therefore, an attempt was done to collect and examine such information 

353


from scientific literature to obtain an insight into the variability of test parameters, data variation, 

and the significance of such factors in the interpretation of results. 

Among the testing methods used to characterize in vitro antimicrobial activity of essential oils, 

the three main methods turned out to be agar diffusion test, serial broth or agar dilution test, and the 

vapor phase test. Further tests comprise various kill-time studies, for example, the activity of a 

compound relative to phenol after 15 min (phenol coefficient) (Rideal et al., 1903), killing time 

determination after contact to a test compounds using contaminated silk threads (Koch, 1881), 

recording of growth curves and determination of the amount of a compound being effective to 

inhibit growth of 50% of test organisms (Friedman et al., 2004), poisoned food techniques in which 

the delay of microbial growth is determined in presence of growth inhibitors (Kurita et al., 1983; 

Reiss, 1982), spore germination, and short contact time studies in fungi (Smyth et al., 1932; Mikhlin 

et al., 1983). Other studies monitor presence or absence of growth by measuring metabolic CO 2 in 

yeast (Belletti et al., 2004) or visualize growth by indicators, such as sulfur salts from sulfursupplemented 

cow milk as growth medium (Geinitz, 1912), 2,3,5-triphenyltetrazolium chloride 

(Canillac et al., 1996), or p-iodonitrophenyltetrazolium violet (Al-Bayati, 2008; Weseler et al., 

2005). The bioautographic assay on thin-layer chromatography plates has been developed for identification 

of active compounds in plant extracts (Rahalison et al., 1994), but was later also taken for 

the examination of essential oils (Iscan et al., 2002). 

12.1.1 AGAR DIFFUSION TEST (ADT) 

In the agar diffusion test, the essential oil to be tested is placed on top of an agar surface. Two 

techniques exist: In the first one, the essential oil is placed onto a paper disk; in the second, a hole 

is made into the agar surface and the essential oil is put into the hole. In the following, the essential 

oil diffuses from its reservoir through the agar medium, which is seeded with microorganism. 

Antimicrobially active oils cause an inhibition zone around the reservoir after incubation, respectively, 

and normally the size of inhibition zone is regarded as measure for the antimicrobial 

potency of an essential oil. However, lipophilic compounds such as farnesol cause only small 

inhibition zones against Bacillus subtilis in the agar diffusion test (Weis, 1986), although the 

compound resulted in a strong inhibition in the serial dilution test (MIC = 12.5 μg/ml) (Kubo 

et al., 1983). Thus, strong inhibitors having low water solubility gave a poor or even negative 

result in the agar diffusion test. It is therefore wrong to conclude that an essential oil without 

resulting in an inhibition zone in the agar diffusion test is without any antimicrobially active 

constituents; or in other words, antimicrobially active compounds are easily overlooked by this 

method. Another problem is the interpretation of the size of inhibition zones, which depend on 

both, the diffusion coefficient plus antimicrobial activity of every compound present in an essential 

oil. Beside generally unknown diffusion coefficients of essential oil constituents, the size of 

inhibition zones is influenced by several other factors: volatilization of essential oil, disk or hole 

sizes, amount of compound given to disk or into hole, adsorption by the disk, agar type, agar–agar 

content, pH, volume of agar, microbial strains tested (Janssen et al., 1987). Taken together, this 

test method can be used as a pretest, but the results should not be over-rated. In the following data 

schedule the experimental conditions are briefly given (culture medium, incubation temperature, 

incubation time, disk or hole size, amount of essential oil on disk or hole). The amount of compound 

in microgram added to a reservoir (disk or hole) is recalculated from microliter with a 

density of 1 for all essential oils. 

12.1.2 DILUTION TEST (DIL) 

In the dilution test, the essential oil to be tested is incorporated in a semisolid agar medium or liquid 

broth in several defined amounts. Absence of growth in agar plates or test tubes is determined with

In Vitro Antimicrobial Activities of Essential Oils 355 

the naked eye after incubation. The minimum inhibitory concentration (MIC) is the concentration 

of essential oil present in the ungrown agar plate or test tube with the highest amount of test material. 

When essential oils are tested, the main difficulty is caused by their low water solubility. The addition 

of solvents (e.g., dimethylsulfoxide and ethanol) or detergents (e.g., Tween 20) to the growth 

medium are unavoidable, which however influences the MIC (Hili et al., 1997; Hammer et al., 1999; 

Remmal et al., 1993). Another problem is the volatilization of essential oils during incubation. 

Working in a chamber with saturated moistened atmosphere (Pauli, 2006) or high water activity 

levels (Guynot et al., 2005) improved the situation. The MIC is additionally influenced by the selection 

of growth media, for example, in RPMI 1640, the MIC toward yeast is about 15 times lower 

than in Sabouraud medium (Jirovetz et al., 2007; McCarthy et al., 1992). Further MIC-influencing 

test parameters are size of inoculum, pH of growth medium, and incubation time. Nevertheless, the 

serial dilution test in liquid broth was recommended for natural substances (Boesel, 1991; Hadacek 

et al., 2000; Pauli, 2007) and is standardized for the testing of antibacterial and antifungal drugs in 

liquid broth and agar plates (Clinical & Laboratory Standards Institute, 2008). Its use enables a link 

to data of pharmaceutical drugs and an easier interpretation of test results. In the data section, all 

concentrations are recalculated in μg/ml. To distinguish between the agar dilution test and the serial 

dilution test, the growth medium is abbreviated either as agar (A) or broth (B). Test parameters— 

exceptionally citations—are systematically given and comprise growth medium, incubation time, 

incubation temperature, and MIC in μg/ml or ppm. 

12.1.3 VAPOR PHASE TEST (VP) 

In the vapor phase, a standardized method does not exist among tests to study antimicrobial activity 

of essential oils. In most of the examinations, a reservoir (paper disk, cup, and glass) contains the 

sample of essential oil, and a seeded agar plate was inverted and covered the reservoir. After inoculation, 

an inhibition zone is formed, which is the measure of activity. Most of the data listed in the 

following tables have been worked out by such methods. Because these methods allow only the 

creation of relative values, a few examiners defined the MIC in atmosphere (MIC air ) by using airtight 

boxes (Inouye et al., 2001; Nakahara et al., 2003), which contained a seeded agar plate and the 

essential oil on the glass or paper. Otherwise, the results were estimated with +++ = normal growth, 

++ = reduced growth, + = visible growth, and NG = no growth. The test parameters given in the 

following tables are growth medium, incubation time, incubation temperature, and activity evaluation 

or MIC air in μg/ml or ppm. 

To give detailed information, the following abbreviations are used in Tables 12.1 through 12.80: 

(h), essential oil was given into a hole; BA, Bacto agar; BHA, brain–heart infusion agar; BlA, blood 

agar; CA, Czapek’s agar; CAB, Campylobacter agar base; CDA, Czapek Dox agar; DMSO, dimethylsulfoxide; 

EtOH, ethanol; EYA, Emerson’s Ybss agar; germ., germination; HIB, heart infusion 

broth; HS, horse serum; inh., inhibition; ISA, Iso-sensitest agar; KBA, King’s medium B agar; LA, 

Laury agar; LSA, Listeria selective agar; MA, malt agar; MAA, medium A agar; MBA, mycobiotic 

agar; MCA, MacConkey agar; MEB, malt extract broth; MHA, Mueller–Hinton agar; MHA, 

Mueller–Hinton broth; MIA, minimum inhibitory amount in μg per disk; MIC, minimum inhibitory 

concentration in μg/ml or ppm; MIC air , minimal inhibitory concentration in μg/ml or ppm in 

the vapor phase; MYA, malt extract–yeast extract–peptone–glucose agar; MYB, malt extract–yeast 

extract–glucose–peptone broth; NA, nutrient agar; NB, nutrient broth; OA, oat agar; PB, Pennassay 

broth; Ref., reference number; SA, Sabouraud agar; SB, Sabouraud broth; sd, saturated disk; SDA, 

Sabouraud dextrose agar; SGB, Sabouraud glucose broth; SMA, Sabouraud maltose agar; sol., solution; 

TYA, tryptone–glucose–yeast extract agar; THB, Todd–Hewitt broth; TSA, trypticase soy 

agar; TSB, trypticase soy broth; TYB, tryptone–glucose–yeast extract broth; VC, various conditions: 

not given in detail; WA, sucrose–peptone agar; WFA, wheat flour agar; YPB, and yeast 

extract–peptone–dextrose broth.


TABLE 12.1 

Inhibitory Data of Anise Oil Obtained in the Agar Diffusion Test 

Microorganism MO Class Test Parameters 

Disk Size (mm), 

Quantity (µg) 

Inhibition 

Zone (mm) Ref. 

Acinetobacter calcoaceticus Bac- ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987) 

Aerobacter aerogenes Bac- NA, 24 h, 37°C —, sd 0 Maruzzella and Lichtenstein (1956) 

Aeromonas hydrophila Bac- ISA, 48 h, 25°C 4 (h), 10,000 6 Deans and Ritchie (1987) 

Alcaligenes faecalis Bac- ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987) 

Beneckea natriegens Bac- ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987) 

Campylobacter jejuni Bac- TSA, 24 h, 42°C 4 (h), 25,000 4.5 Smith-Palmer et al. (1998) 

Citrobacter freundii Bac- ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987) 

Enterobacter aerogenes Bac- ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987) 

Enterococcus faecalis Bac- NA, 24 h, 37°C 5000 on agar 0 Di Pasqua et al. (2005) 

Erwinia carotovora Bac- ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987) 

Escherichia coli Bac- NA, 24 h, 37°C —, sd 0 Maruzzella and Lichtenstein (1956) 

Escherichia coli Bac- NA, 24 h, 37°C 10 (h), 100,000 0 Narasimha Rao and Nigam (1970) 

Escherichia coli Bac- ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987) 

Escherichia coli Bac- Cited 15, 2500 1 Pizsolitto et al. (1975) 

Escherichia coli Bac- Cited, 18 h, 37°C 6, 2500 7 Janssen et al. (1986) 

Escherichia coli Bac- TSA, 24 h, 35°C 4 (h), 25,000 4.5 Smith-Palmer et al. (1998) 

Escherichia coli Bac- NA, 18 h, 37°C 6 (h), pure 11 Yousef and Tawil (1980) 

Flavobacterium suaveolens Bac- ISA, 48 h, 25°C 4 (h), 10,000 6 Deans and Ritchie (1987) 

Klebsiella aerogenes Bac- NA, 24 h, 37°C 10 (h), 100,000 0 Narasimha Rao and Nigam (1970) 

Klebsiella pneumoniae Bac- ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987) 

Klebsiella sp. Bac- Cited 15, 2500 0 Pizsolitto et al. (1975) 

Moraxella sp. Bac- ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987) 

Neisseria perfl ava Bac- NA, 24 h, 37°C —, sd 1 Maruzzella and Lichtenstein (1956) 

Proteus sp. Bac- Cited 15, 2500 1 Pizsolitto et al. (1975) 

Proteus vulgaris Bac- ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987) 

Proteus vulgaris Bac- NA, 24 h, 37°C —, sd 1.5 Maruzzella and Lichtenstein (1956) 

Pseudomonas aeruginosa Bac- NA, 24 h, 37°C —, sd 0 Maruzzella and Lichtenstein (1956) 

Pseudomonas aeruginosa Bac- Cited 15, 2500 0 Pizsolitto et al. (1975)


Pseudomonas aeruginosa Bac- ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987) 

Pseudomonas aeruginosa Bac- NA, 18 h, 37°C 6 (h), pure 0 Yousef and Tawil (1980) 

Pseudomonas aeruginosa Bac- Cited, 18 h, 37°C 6, 2500 6.7 Janssen et al. (1986) 

Pseudomonas sp. Bac- NA, 24 h, 37°C 5000 on agar 0 Di Pasqua et al. (2005) 

Salmonella enteritidis Bac- TSA, 24 h, 35°C 4 (h), 25,000 4.5 Smith-Palmer et al. (1998) 

Salmonella pullorum Bac- ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987) 

Salmonella sp. Bac- Cited 15, 2500 0 Pizsolitto et al. (1975) 

Salmonella typhi Bac- NA, 24 h, 37°C 10 (h), 100,000 16 Narasimha Rao and Nigam (1970) 

Serratia marcescens Bac- ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987) 

Serratia marcescens Bac- NA, 24 h, 37°C —, sd 1 Maruzzella and Lichtenstein (1956) 

Serratia sp. Bac- Cited 15, 2500 1 Pizsolitto et al. (1975) 

Shigella sp. Bac- Cited 15, 2500 1 Pizsolitto et al. (1975) 

Vibrio cholerae Bac- NA, 24 h, 37°C 10 (h), 100,000 17 Narasimha Rao and Nigam (1970) 

Yersinia enterocolitica Bac- ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987) 

Bacillus mesentericus Bac+ NA, 24 h, 37°C —, sd 3 Maruzzella and Lichtenstein (1956) 

Bacillus sp. Bac+ Cited 15, 2500 1 Pizsolitto et al. (1975) 

Bacillus subtilis Bac+ ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987) 

Bacillus subtilis Bac+ NA, 24 h, 37°C —, sd 4 Maruzzella and Lichtenstein (1956) 

Bacillus subtilis Bac+ Cited, 18 h, 37°C 6, 2500 8 Janssen et al. (1986) 

Bacillus subtilis Bac+ NA, 18 h, 37°C 6 (h), pure 12.5 Yousef and Tawil (1980) 

Brevibacterium linens Bac+ ISA, 48 h, 25°C 4 (h), 10,000 9 Deans and Ritchie (1987) 

Brochotrix thermosphacta Bac+ NA, 24 h, 37°C 5000 on agar 0 Di Pasqua et al. (2005) 

Brochotrix thermosphacta Bac+ ISA, 48 h, 25°C 4 (h), 10,000 5.5 Deans and Ritchie (1987) 

Clostridium sporogenes Bac+ ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987) 

Corynebacterium diphtheriae Bac+ NA, 24 h, 37°C 10 (h), 100,000 0 Narasimha Rao and Nigam (1970) 

Lactobacillus delbrueckii Bac+ NA, 24 h, 37°C 5000 on agar 0 Di Pasqua et al. (2005) 

Lactobacillus plantarum Bac+ ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987) 

Lactobacillus plantarum Bac+ NA, 24 h, 37°C 5000 on agar 0 Di Pasqua et al. (2005) 

Lactococcus garvieae Bac+ NA, 24 h, 37°C 5000 on agar 0 Di Pasqua et al. (2005) 

Lactococcus lactis subsp. lactis Bac+ NA, 24 h, 37°C 5000 on agar 0 Di Pasqua et al. (2005) 

Leuconostoc cremoris Bac+ ISA, 48 h, 25°C 4 (h), 10,000 5 Deans and Ritchie (1987) 

continued






Inhibition 


Listeria monocytogenes Bac+ ISA, 48 h, 25°C 4 (h), 10,000 0 Lis-Balchin et al. (1998) 

Listeria monocytogenes Bac+ NA, 24 h, 37°C 5000 on agar 0 Di Pasqua et al. (2005) 

Listeria monocytogenes Bac+ TSA, 24 h, 35°C 4 (h), 25,000 4 Smith-Palmer et al. (1998) 

Micrococcus luteus Bac+ ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987) 

Mycobacterium phlei Bac+ NA, 18 h, 37°C 6 (h), pure 18 Yousef and Tawil (1980) 

Sarcina lutea Bac+ NA, 24 h, 37°C —, sd 0 Maruzzella and Lichtenstein (1956) 

Staphylococcus aureus Bac+ NA, 24 h, 37°C —, sd 0 Maruzzella and Lichtenstein (1956) 

Staphylococcus aureus Bac+ NA, 24 h, 37°C 10 (h), 100,000 0 Narasimha Rao and Nigam (1970) 

Staphylococcus aureus Bac+ ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987) 

Staphylococcus aureus Bac+ NA, 24 h, 37°C 5000 on agar 0 Di Pasqua et al. (2005) 

Staphylococcus aureus Bac+ Cited 15, 2500 1 Pizsolitto et al. (1975) 

Staphylococcus aureus Bac+ TSA, 24 h, 35°C 4 (h), 25,000 4 Smith-Palmer et al. (1998) 

Staphylococcus aureus Bac+ Cited, 18 h, 37°C 6, 2500 8.3 Janssen et al. (1986) 

Staphylococcus aureus Bac+ NA, 18 h, 37°C 6 (h), pure 14.5 Yousef and Tawil (1980) 

Staphylococcus epidermidis Bac+ Cited 15, 2500 4 Pizsolitto et al. (1975) 

Staphylococcus epidermidis Bac+ NA, 24 h, 37°C 10 (h), 100,000 12 Narasimha Rao and Nigam (1970) 

Streptococcus faecalis Bac+ ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987) 

Streptococcus sp. Bac+ NA, 24 h, 37°C 10 (h), 100,000 16 Narasimha Rao and Nigam (1970) 

Streptococcus viridans Bac+ Cited 15, 2500 0 Pizsolitto et al. (1975) 

Streptomyces venezuelae Bac+ SMA, 2–7 d, 20°C 6.35, sd 7 Maruzzella and Liguori (1958) 

Alternaria porri Fungi PDA, 72 h, 28°C 5, 5000 35 Pawar and Thaker (2007) 

Alternaria solani Fungi SMA, 2–7 d, 20°C 6.35, sd 9 Maruzzella and Liguori (1958) 

Aspergillus fl avus Fungi Cited —, pure 28 Gangrade et al. (1991) 

Aspergillus fumigatus Fungi SMA, 2–7 d, 20°C 6.35, sd 9 Maruzzella and Liguori (1958) 

Aspergillus niger Fungi PDA, 48 h, 28°C 5, 5000 0 Pawar and Thaker (2006) 

Aspergillus niger Fungi SMA, 2–7 d, 20°C 6.35, sd 13 Maruzzella and Liguori (1958) 

Aspergillus niger Fungi Cited —, pure 27 Gangrade et al. (1991)


Aspergillus niger Fungi SDA, 8 d, 30°C 6 (h), pure 60 Yousef and Tawil (1980) 

Fusarium oxysporum Fungi Cited —, pure 32 Gangrade et al. (1991) 

Fusarium oxysporum f.sp. cicer Fungi PDA, 72 h, 28°C 5, 5000 15 Pawar and Thaker (2007) 

Geotrichum sp. Fungi SDA, 2–7 d, 20°C 5 (h), 60,000 17 Kosalec et al. (2005) 

Helminthosporium sativum Fungi SMA, 2–7 d, 20°C 6.35, sd 10 Maruzzella and Liguori (1958) 

Microsporum canis Fungi SDA, 2–7 d, 20°C 5 (h), 60,000 27 Kosalec et al. (2005) 

Microsporum gypseum Fungi SDA, 2–7 d, 20°C 5 (h), 60,000 21 Kosalec et al. (2005) 

Mucor mucedo Fungi SMA, 2–7 d, 20°C 6.35, sd 6 Maruzzella and Liguori (1958) 

Mucor sp. Fungi SDA, 8 d, 30°C 6 (h), pure 12 Yousef and Tawil (1980) 

Nigrospora panici Fungi SMA, 2–7 d, 20°C 6.35, sd 12 Maruzzella and Liguori (1958) 

Penicillium chrysogenum Fungi SDA, 8 d, 30°C 6 (h), pure 60 Yousef and Tawil (1980) 

Penicillium digitatum Fungi SMA, 2–7 d, 20°C 6.35, sd 15 Maruzzella and Liguori (1958) 

Rhizopus nigricans Fungi SMA, 2–7 d, 20°C 6.35, sd 3 Maruzzella and Liguori (1958) 

Rhizopus sp. Fungi SDA, 8 d, 30°C 6 (h), pure 13 Yousef and Tawil (1980) 

Trichophyton mentagrophytes Fungi SDA, 2–7 d, 20°C 5 (h), 60,000 23 Kosalec et al. (2005) 

Trichophyton rubrum Fungi SDA, 2–7 d, 20°C 5 (h), 60,000 25 Kosalec et al. (2005) 

Candida albicans Yeast SMA, 2–7 d, 20°C 6.35, sd 2 Maruzzella and Liguori (1958) 

Candida albicans Yeast Cited, 18 h, 37°C 6, 2500 12 Janssen et al. (1986) 

Candida albicans Yeast SDA, 18 h, 30°C 6 (h), pure 15 Yousef and Tawil (1980) 

Candida albicans Yeast SDA, 2–7 d, 20°C 5 (h), 60,000 29 Kosalec et al. (2005) 

Candida glabrata Yeast SDA, 2–7 d, 20°C 5 (h), 60,000 21 Kosalec et al. (2005) 

Candida krusei Yeast SDA, 2–7 d, 20°C 5 (h), 60,000 

a 

Kosalec et al. (2005) 

Candida krusei Yeast SMA, 2–7 d, 20°C 6.35, sd 0 Maruzzella and Liguori (1958) 

Candida parapsilosis Yeast SDA, 2–7 d, 20°C 5 (h), 60,000 30 Kosalec et al. (2005) 

Candida pseudotropicalis Yeast SDA, 2–7 d, 20°C 5 (h), 60,000 

a 


Candida tropicalis Yeast SDA, 2–7 d, 20°C 5 (h), 60,000 

a 


Candida tropicalis Yeast SMA, 2–7 d, 20°C 6.35, sd 2 Maruzzella and Liguori (1958) 

Cryptococcus neoformans Yeast SMA, 2–7 d, 20°C 6.35, sd 9 Maruzzella and Liguori (1958) 

Cryptococcus rhodobenhani Yeast SMA, 2–7 d, 20°C 6.35, sd 0 Maruzzella and Liguori (1958) 

Saccharomyces cerevisiae Yeast SMA, 2–7 d, 20°C 6.35, sd 0 Maruzzella and Liguori (1958) 

a 

Fungicidal.


TABLE 12.2 

Inhibitory Data of Anise Oil Obtained in the Dilution Test 

Microorganism MO Class Test Parameters MIC (µg/mL) Ref. 

Campylobacter jejuni Bac- TSB, 24 h, 42°C >10,000 Smith-Palmer et al. (1998) 

Escherichia coli Bac- TSB, 24 h, 35°C >10,000 Smith-Palmer et al. (1998) 

Escherichia coli Bac- TSB, 24 h, 37°C >10,000 Di Pasqua et al. (2005) 

Escherichia coli Bac- MHB, DMSO, 24 h, 37°C >500 Al-Bayati (2008) 

Escherichia coli Bac- NB, Tween 20, 18 h, 37°C 50,000 Yousef and Tawil (1980) 

Helicobacter pylori Bac- Cited, 20 h, 37°C 294.7–589.4 Weseler et al. (2005) 

Klebsiella pneumoniae Bac- MHB, DMSO, 24 h, 37°C >500 Al-Bayati (2008) 

Proteus mirabilis Bac- MHB, DMSO, 24 h, 37°C 125 Al-Bayati (2008) 

Proteus vulgaris Bac- MHB, DMSO, 24 h, 37°C 62.5 Al-Bayati (2008) 

Pseudomonas aeruginosa Bac- MHB, DMSO, 24 h, 37°C >500 Al-Bayati (2008) 

Pseudomonas aeruginosa Bac- NB, Tween 20, 18 h, 37°C >50,000 Yousef and Tawil (1980) 

Salmonella enteritidis Bac- TSB, 24 h, 35°C >10,000 Smith-Palmer et al. (1998) 

Salmonella typhi Bac- MHB, DMSO, 24 h, 37°C 500 Al-Bayati (2008) 

Salmonella typhimurium Bac- TSB, 24 h, 37°C >10,000 Di Pasqua et al. (2005) 

Salmonella typhimurium Bac- MHB, DMSO, 24 h, 37°C 250 Al-Bayati (2008) 

Bacillus cereus Bac+ MHB, DMSO, 24 h, 37°C 62.5 Al-Bayati (2008) 

Bacillus subtilis Bac+ NB, Tween 20, 18 h, 37°C 1600 Yousef and Tawil (1980) 

Brochotrix thermosphacta Bac+ M17, 24 h, 20°C >10,000 Di Pasqua et al. (2005) 

Listeria monocytogenes Bac+ TSB, 24 h, 35°C >10,000 Smith-Palmer et al. (1998) 

Mycobacterium phlei Bac+ NB, Tween 20, 18 h, 37°C 200 Yousef and Tawil (1980) 

Staphylococcus aureus Bac+ TSB, 24 h, 35°C >10,000 Smith-Palmer et al. (1998) 

Staphylococcus aureus Bac+ MHB, DMSO, 24 h, 37°C 125 Al-Bayati (2008) 

Staphylococcus aureus Bac+ NB, Tween 20, 18 h, 37°C 25,000 Yousef and Tawil (1980) 

Alternaria alternata Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Alternaria tenuissima Fungi Cited 100% inh. 600 Shukla and Tripathi (1987)


Aspergillus awamorii Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Aspergillus fl avus Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon (1986) 

Aspergillus fl avus Fungi PDA, 7–14 d, 28°C 500 Soliman and Badeaa (2002) 

Aspergillus fl avus Fungi PDA, 6–8 h 20°C, spore germ. inh. 50–100 Thompson (1986) 

Aspergillus fumigatus Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Aspergillus niger Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Aspergillus niger Fungi NB, Tween 20, 8 d, 30°C 1600 Yousef and Tawil (1980) 

Aspergillus niger Fungi YES broth, 10 d 83% inh. 10,000 Lis-Balchin et al. (1998) 

Aspergillus ochraceus Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Aspergillus ochraceus Fungi PDA, 7–14 d, 28°C 500 Soliman and Badeaa (2002) 

Aspergillus ochraceus Fungi YES broth, 10 d 82% inh. 10,000 Lis-Balchin et al. (1998) 

Aspergillus oryzae Fungi Cited 250 Okazaki and Oshima (1953) 

Aspergillus parasiticus Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon (1986) 

Aspergillus parasiticus Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Aspergillus parasiticus Fungi PDA, 7–14 d, 28°C 500 Soliman and Badeaa (2002) 

Aspergillus parasiticus Fungi PDA, 6–8 h 20°C, spore germ. inh. 50–100 Thompson (1986) 

Aspergillus sydowi Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Aspergillus tamari Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Aspergillus terreus Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Botryodiplodia theobromae Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Cephalosporium sacchari Fungi OA, EtOH, 3 d, 20°C 20,000 Narasimba Rao et al. (1971) 

Ceratocystis paradoxa Fungi OA, EtOH, 3 d, 20°C 1000 Narasimba Rao et al. (1971) 

Cladosporium herbarum Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Colletotrichum capsici Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Curvularia lunata Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Curvularia lunata Fungi OA, EtOH, 3 d, 20°C 4000 Narasimba Rao et al. (1971) 

Curvularia pallescens Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Epicoccum nigrum Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Epidermophyton fl occosum Fungi SA, Tween 80, 21 d, 20°C




Fusarium accuminatum Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Fusarium culmorum Fungi YES broth, 10 d 69% inh. 10,000 Lis-Balchin et al. (1998) 

Fusarium equisiti Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Fusarium moniliforme Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Fusarium moniliforme Fungi PDA, 7–14 d, 28°C 500 Soliman and Badeaa (2002) 

Fusarium moniliforme Fungi PDA, 7 d, 23.5°C 52% inh. 10,000 Mueller-Ribeau et al. (1995) 

Fusarium moniliforme var. subglutinans Fungi OA, EtOH, 3 d, 20°C 2000 Narasimba Rao et al. (1971) 

Fusarium oxysporum Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Fusarium semitectum Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Fusarium udum Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Geotrichum sp. Fungi SGB, 3–7 d, 25°C 15,600 Kosalec et al. (2005) 

Helminthosporium sacchari Fungi OA, EtOH, 3 d, 20°C 4000 Narasimba Rao et al. (1971) 

Macrophomina phaesoli Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Microsporum canis Fungi SGB, 3–7 d, 25°C 1000 Kosalec et al. (2005) 

Microsporum gypseum Fungi SGB, 3–7 d, 25°C 2000 Kosalec et al. (2005) 

Mucor hiemalis Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon (1986) 

Mucor mucedo Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon (1986) 

Mucor racemosus Fungi Cited 250 Okazaki and Oshima (1953) 

Mucor racemosus f. racemosus Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon (1986) 

Mucor sp. Fungi NB, Tween 20, 8 d, 30°C 400 Yousef and Tawil (1980) 

Nigrospora oryzae Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Penicillium chrysogenum Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Penicillium chrysogenum Fungi NB, Tween 20, 8 d, 30°C 1600 Yousef and Tawil (1980) 

Penicillium chrysogenum Fungi Cited 250 Okazaki and Oshima (1953) 

Penicillium citrinum Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Physalospora tucumanensis Fungi OA, EtOH, 3 d, 20°C 2000 Narasimba Rao et al. (1971) 

Phytophthora capsici Fungi PDA, 7 d, 23.5°C 36% inh. 10,000 Mueller-Ribeau et al. (1995)


Rhizoctonia solani Fungi PDA, 7 d, 23.5°C 100% inh. 10,000 Mueller-Ribeau et al. (1995) 

Rhizopus 66-81-2 Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon (1986) 

Rhizopus arrhizus Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon (1986) 

Rhizopus chinensis Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon (1986) 

Rhizopus circinans Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon (1986) 

Rhizopus japonicus Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon (1986) 

Rhizopus kazanensis Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon (1986) 

Rhizopus nigricans Fungi Cited 100% inh. 600 Shukla and Tripathi (1987) 

Rhizopus oryzae Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon (1986) 

Rhizopus pymacus Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon (1986) 

Rhizopus sp. Fungi NB, Tween 20, 8 d, 30°C 3200 Yousef and Tawil (1980) 

Rhizopus stolonifer Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon (1986) 

Rhizopus tritici Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon (1986) 

Sclerotina sclerotiorum Fungi PDA, 7 d, 23.5°C 100% inh. 10,000 Mueller-Ribeau et al. (1995) 

Sclerotium rolfsii Fungi OA, EtOH, 6 d, 20°C 2000 Narasimba Rao et al. (1971) 

Trichophyton mentagrophytes Fungi SA, Tween 80, 21 d, 20°C 300–625 Janssen et al. (1988) 

Trichophyton mentagrophytes Fungi SGB, 3–7 d, 25°C 7800 Kosalec et al. (2005) 

Trichophyton rubrum Fungi SA, Tween 80, 21 d, 20°C


TABLE 12.3 

Inhibitory Data of Anise Oil Obtained in the Vapor Phase Test 

Microorganism MO Class Test Parameters Quantity (µg) Activity Ref. 

Escherichia coli Bac- NA, 24 h, 37°C ~20,000 +++ Kellner and Kober (1954) 

Neisseria sp. Bac- NA, 24 h, 37°C ~20,000 + Kellner and Kober (1954) 

Salmonella typhi Bac- NA, 24 h, 37°C ~20,000 ++ Kellner and Kober (1954) 

Salmonella typhi Bac- NA, 24 h, 37°C 6.35, sd +++ Maruzzella and Sicurella (1960) 

Bacillus megaterium Bac+ NA, 24 h, 37°C ~20,000 ++ Kellner and Kober (1954) 

Bacillus subtilis var. aterrimus Bac+ NA, 24 h, 37°C 6.35, sd +++ Maruzzella and Sicurella (1960) 

Corynebacterium diphtheriae Bac+ NA, 24 h, 37°C ~20,000 + Kellner and Kober (1954) 

Mycobacterium avium Bac+ NA, 24 h, 37°C 6.35, sd +++ Maruzzella and Sicurella (1960) 

Staphylococcus aureus Bac+ NA, 24 h, 37°C ~20,000 +++ Kellner and Kober (1954) 

Staphylococcus aureus Bac+ NA, 24 h, 37°C 6.35, sd +++ Maruzzella and Sicurella (1960) 

Streptococcus faecalis Bac+ NA, 24 h, 37°C ~20,000 ++ Kellner and Kober (1954) 

Streptococcus faecalis Bac+ NA, 24 h, 37°C 6.35, sd +++ Maruzzella and Sicurella (1960) 

Streptococcus pyogenes Bac+ NA, 24 h, 37°C ~20,000 + Kellner and Kober (1954) 

Aspergillus fl avus Fungi WFA, 42 d, 25°C Disk, 50,000 +++ Guynot et al. (2003) 

Aspergillus niger Fungi WFA, 42 d, 25°C Disk, 50,000 +++ Guynot et al. (2003) 

Eurotium amstelodami Fungi WFA, 42 d, 25°C Disk, 50,000 +++ Guynot et al. (2003) 

Eurotium herbarum Fungi WFA, 42 d, 25°C Disk, 50,000 +++ Guynot et al. (2003) 

Eurotium repens Fungi WFA, 42 d, 25°C Disk, 50,000 +++ Guynot et al. (2003) 

Eurotium rubrum Fungi WFA, 42 d, 25°C Disk, 50,000 +++ Guynot et al. (2003) 

Penicillium corylophilum Fungi WFA, 42 d, 25°C Disk, 50,000 + Guynot et al. (2003) 

Candida albicans Yeast NA, 24 h, 37°C ~20,000 + Kellner and Kober (1954)


TABLE 12.4 

Inhibitory Data of Bitter Fennel Obtained in the Agar Diffusion Test 




Inhibition 



Aeromonas hydrophila Bac- ISA, 48 h, 25°C 4 (h), 10,000 9.5 Deans and Ritchie (1987) 

Agrobacterium tumefaciens Bac- WA, 48 h, 25°C 6, 8000 MIA 2880 Cantore et al. (2004) 



Burkholderia gladioli pv. agaricicola Bac- WA, 48 h, 25°C 6, 8000 MIA 7680 Cantore et al. (2004) 



Enterobacter aerogenes Bac- MHA, 48 h, 27°C 6, 15,000 10 Ertürk et al. (2006) 


Erwinia carotovora subsp. atroseptica Bac- WA, 48 h, 25°C 6, 8000 MIA 7680 Cantore et al. (2004) 

Erwinia carotovora subsp. carotovora Bac- WA, 48 h, 25°C 6, 8000 MIA 7680 Cantore et al. (2004) 



Escherichia coli Bac- NA, 18 h, 37°C 5 (h), -30,000 14 Schelz et al. (2006) 

Escherichia coli Bac- MHA, 48 h, 27°C 6, 15,000 25 Ertürk et al. (2006) 

Escherichia coli Bac- WA, 48 h, 25°C 6, 8000 MIA >7680 Cantore et al. (2004) 



Moraxella sp. Bac- ISA, 48 h, 25°C 4 (h), 10,000 6.5 Deans and Ritchie (1987) 




Pseudomonas aeruginosa Bac- MHA, 48 h, 27°C 6, 15,000 18 Ertürk et al. (2006) 

Pseudomonas agarici Bac- KBA, 48 h, 25°C 6, 8000 MIA >7680 Cantore et al. (2004) 

Pseudomonas chichorii Bac- KBA, 48 h, 25°C 6, 8000 MIA >7680 Cantore et al. (2004) 

continued






Inhibition 


Pseudomonas corrugate Bac- KBA, 48 h, 25°C 6, 8000 MIA >7680 Cantore et al. (2004) 

Pseudomonas reactans Bac- KBA, 48 h, 25°C 6, 8000 MIA >7680 Cantore et al. (2004) 

Pseudomonas syringae pv. apata Bac- KBA, 48 h, 25°C 6, 8000 MIA >7680 Cantore et al. (2004) 

Pseudomonas syringae pv. apii Bac- KBA, 48 h, 25°C 6, 8000 MIA >7680 Cantore et al. (2004) 

Pseudomonas syringae pv. atrofaciens Bac- KBA, 48 h, 25°C 6, 8000 MIA 3840 Cantore et al. (2004) 

Pseudomonas syringae pv. Glycinea Bac- KBA, 48 h, 25°C 6, 8000 MIA 960 Cantore et al. (2004) 

Pseudomonas syringae pv. lachrymans Bac- KBA, 48 h, 25°C 6, 8000 MIA >7680 Cantore et al. (2004) 

Pseudomonas syringae pv. maculicola Bac- KBA, 48 h, 25°C 6, 8000 MIA >7680 Cantore et al. (2004) 

Pseudomonas syringae pv. phaseolicola Bac- KBA, 48 h, 25°C 6, 8000 MIA >7680 Cantore et al. (2004) 

Pseudomonas syringae pv. pisi Bac- KBA, 48 h, 25°C 6, 8000 MIA >7680 Cantore et al. (2004) 

Pseudomonas syringae pv. syringae Bac- KBA, 48 h, 25°C 6, 8000 MIA >7680 Cantore et al. (2004) 

Pseudomonas syringae pv. tomato Bac- KBA, 48 h, 25°C 6, 8000 MIA >7680 Cantore et al. (2004) 

Pseudomonas tolaasii Bac- KBA, 48 h, 25°C 6, 8000 MIA 7680 Cantore et al. (2004) 

Pseudomonas viridifl ava Bac- KBA, 48 h, 25°C 6, 8000 MIA >7680 Cantore et al. (2004) 


Salmonella typhimurium Bac- MHA, 48 h, 27°C 6, 15,000 8 Ertürk et al. (2006) 


Xanthomonas campestris pv. campestris Bac- WA, 48 h, 25°C 6, 8000 MIA 5760 Cantore et al. (2004) 

Xanthomonas campestris pv. phaesoli var. fuscans Bac- WA, 48 h, 25°C 6, 8000 MIA 720 Cantore et al. (2004) 

Xanthomonas campestris pv. phaesoli var. phaesoli Bac- WA, 48 h, 25°C 6, 8000 MIA 480 Cantore et al. (2004) 

Xanthomonas campestris pv. Vesicatoria Bac- WA, 48 h, 25°C 6, 8000 MIA 1440 Cantore et al. (2004) 


Bacillus megaterium Bac+ WA, 48 h, 25°C 6, 8000 MIA >7680 Cantore et al. (2004) 


Bacillus subtilis Bac+ NA, 18 h, 37°C 6 (h), pure 18 Yousef and Tawil (1980) 

Brevibacterium linens Bac+ ISA, 48 h, 25°C 4 (h), 10,000 7.5 Deans and Ritchie (1987) 

Brochotrix thermosphacta Bac+ ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987) 

Clavibacter michiganensis subsp. Michiganensis Bac+ WA, 48 h, 25°C 6, 8000 MIA 7680 Cantore et al. (2004)


Clavibacter michiganensis subsp. sepedonicus Bac+ WA, 48 h, 25°C 6, 8000 MIA 960 Cantore et al. (2004) 


Curtobacterium fl accunfaciens pv. betae Bac+ WA, 48 h, 25°C 6, 8000 MIA >7680 Cantore et al. (2004) 

Curtobacterium fl accunfaciens pv. fl accunfaciens Bac+ WA, 48 h, 25°C 6, 8000 MIA >7680 Cantore et al. (2004) 


Leuconostoc cremoris Bac+ ISA, 48 h, 25°C 4 (h), 10,000 9.5 Deans and Ritchie (1987) 



Rhodococcus fascians Bac+ WA, 48 h, 25°C 6, 8000 MIA 1920 Cantore et al. (2004) 

Staphylococcus aureus Bac+ ISA, 48 h, 25°C 4 (h), 10,000 7.5 Deans and Ritchie (1987) 

Staphylococcus aureus Bac+ MHA, 48 h, 27°C 6, 15,000 16 Ertürk et al. (2006) 

Staphylococcus aureus Bac+ NA, 18 h, 37°C 6 (h), pure 17 Yousef and Tawil (1980) 

Staphylococcus epidermidis Bac+ MHA, 48 h, 27°C 6, 15,000 12 Ertürk et al. (2006) 

Staphylococcus epidermidis Bac+ NA, 18 h, 37°C 5 (h), -30,000 15 Schelz et al. (2006) 



Absidia cormybifera Fungi EYA, 48 h, 45°C 5, sd 0 Nigam and Rao (1979) 


Alternaria sp. Fungi PDA, 18 h, 37°C 6, sd 12 Sharma and Singh (1979) 

Aspergillus candidus Fungi PDA, 18 h, 37°C 6, sd 0 Sharma and Singh (1979) 

Aspergillus fl avus Fungi PDA, 18 h, 37°C 6, sd 7 Sharma and Singh (1979) 

Aspergillus fumigatus Fungi PDA, 18 h, 37°C 6, sd 0 Sharma and Singh (1979) 


Aspergillus nidulans Fungi PDA, 18 h, 37°C 6, sd 9 Sharma and Singh (1979) 


Aspergillus niger Fungi PDA, 18 h, 37°C 6, sd 12 Sharma and Singh (1979) 

Aspergillus niger Fungi MHA, 48 h, 27°C 6, 15,000 12 Ertürk et al. (2006) 


Cladosporium herbarum Fungi PDA, 18 h, 37°C 6, sd 12.5 Sharma and Singh (1979) 

Cunninghamella echinulata Fungi PDA, 18 h, 37°C 6, sd 21 Sharma and Singh (1979) 

Fusarium oxysporum Fungi PDA, 18 h, 37°C 6, sd 0 Sharma and Singh (1979) 

Helminthosporium sacchari Fungi PDA, 18 h, 37°C 6, sd 16 Sharma and Singh (1979) 

continued






Inhibition 



Humicola grisea var. thermoidea Fungi EYA, 48 h, 45°C 5, sd 0 Nigam and Rao (1979) 

Microsporum gypseum Fungi PDA, 18 h, 37°C 6, sd 14.5 Sharma and Singh (1979) 


Mucor mucedo Fungi PDA, 18 h, 37°C 6, sd 12 Sharma and Singh (1979) 



Penicillium aculeatum Fungi CA, 48 h, 27°C 5, sd 0 Nigam and Rao (1979) 

Penicillium chrysogenum Fungi CA, 48 h, 27°C 5, sd 15 Nigam and Rao (1979) 


Penicillium digitatum Fungi PDA, 18 h, 37°C 6, sd 7 Sharma and Singh (1979) 


Penicillium javanicum Fungi CA, 48 h, 27°C 5, sd 25 Nigam and Rao (1979) 

Penicillium jensenii Fungi CA, 48 h, 27°C 5, sd 10 Nigam and Rao (1979) 

Penicillium lividum Fungi CA, 48 h, 27°C 5, sd 0 Nigam and Rao (1979) 

Penicillium notatum Fungi CA, 48 h, 27°C 5, sd 0 Nigam and Rao (1979) 

Penicillium obscurum Fungi CA, 48 h, 27°C 5, sd 15 Nigam and Rao (1979) 

Penicillium sp. I Fungi CA, 48 h, 27°C 5, sd 15 Nigam and Rao (1979) 

Penicillium sp. II Fungi CA, 48 h, 27°C 5, sd 10 Nigam and Rao (1979) 

Penicillium sp. III Fungi CA, 48 h, 27°C 5, sd 0 Nigam and Rao (1979) 


Rhizopus nigricans Fungi PDA, 18 h, 37°C 6, sd 7 Sharma and Singh (1979) 


Sporotrichum thermophile Fungi EYA, 48 h, 45°C 5, sd 25 Nigam and Rao (1979) 

Thermoascus aurantiacis Fungi EYA, 48 h, 45°C 5, sd 10 Nigam and Rao (1979)


Thermomyces lanuginosa Fungi EYA, 48 h, 45°C 5, sd 15 Nigam and Rao (1979) 

Thielava minor Fungi EYA, 48 h, 45°C 5, sd 0 Nigam and Rao (1979) 

Trichophyton rubrum Fungi PDA, 18 h, 37°C 6, sd 8 Sharma and Singh (1979) 

Trichothecium roseum Fungi PDA, 18 h, 37°C 6, sd 9 Sharma and Singh (1979) 

Brettanomyces anomalus Yeast MYA, 4 d, 30°C 5, 10% sol. sd 0 Conner and Beuchat (1984) 


Candida albicans Yeast MHA, 48 h, 27°C 6, 15,000 12 Ertürk et al. (2006) 



Candida lipolytica Yeast MYA, 4 d, 30°C 5, 10% sol. sd 0 Conner and Beuchat (1984) 




Debaryomyces hansenii Yeast MYA, 4 d, 30°C 5, 10% sol. sd 0 Conner and Beuchat (1984) 

Geotrichum candidum Yeast MYA, 4 d, 30°C 5, 10% sol. sd 0 Conner and Beuchat (1984) 

Hansenula anomala Yeast MYA, 4 d, 30°C 5, 10% sol. sd 0 Conner and Beuchat (1984) 

Kloeckera apiculata Yeast MYA, 4 d, 30°C 5, 10% sol. sd 10 Conner and Beuchat (1984) 

Kluyveromyces fragilis Yeast MYA, 4 d, 30°C 5, 10% sol. sd 0 Conner and Beuchat (1984) 

Lodderomyces elongisporus Yeast MYA, 4 d, 30°C 5, 10% sol. sd 0 Conner and Beuchat (1984) 

Metchnikowia pulcherrima Yeast MYA, 4 d, 30°C 5, 10% sol. sd 0 Conner and Beuchat (1984) 

Pichia membranaefaciens Yeast MYA, 4 d, 30°C 5, 10% sol. sd 0 Conner and Beuchat (1984) 

Rhodotorula rubra Yeast MYA, 4 d, 30°C 5, 10% sol. sd 7 Conner and Beuchat (1984) 

Saccharomyces cerevisiae Yeast MYA, 4 d, 30°C 5, 10% sol. sd 0 Conner and Beuchat (1984) 


Saccharomyces cerevisiae Yeast NA, 24 h, 20°C 5 (h), -30,000 11 Schelz et al. (2006) 

Torula glabrata Yeast MYA, 4 d, 30°C 5, 10% sol. sd 7 Conner and Beuchat (1984) 

Torula thermophila Yeast EYA, 48 h, 45°C 5, sd 20 Nigam and Rao (1979)


TABLE 12.5 

Inhibitory Data of Bitter Fennel Oil Obtained in the Dilution Test 


MIC 

(µg/mL) 

Ref. 

Enterobacter aerogenes Bac- MHB, 24 h, 37°C 4880 Ertürk et al. (2006) 

Escherichia coli Bac- MHB, 24 h, 37°C 1220 Ertürk et al. (2006) 

Escherichia coli Bac- TYB, 18 h, 37°C 3000 Schelz et al. (2006) 


Pseudomonas aeruginosa Bac- MHB, 24 h, 37°C 9760 Ertürk et al. (2006) 

Pseudomonas aeruginosa Bac- NB, Tween 20, 18 h, 37°C 25,000 Yousef and Tawil (1980) 

Salmonella typhimurium Bac- MHB, 24 h, 37°C 19,560 Ertürk et al. (2006) 



Staphylococcus aureus Bac+ MHB, 24 h, 37°C 4880 Ertürk et al. (2006) 


Staphylococcus epidermidis Bac+ TYB, 18 h, 37°C 3000 Schelz et al. (2006) 

Staphylococcus epidermidis Bac+ MHB, 24 h, 37°C 9760 Ertürk et al. (2006) 

Alternaria alternata Fungi MEB, 72 h, 28°C 1500 Mimica-Dukic et al. (2003) 

Aspergillus fl avus Fungi MEB, 72 h, 28°C 1800–2700 Mimica-Dukic et al. (2003) 

Aspergillus niger Fungi MEB, 72 h, 28°C 1700–2200 Mimica-Dukic et al. (2003) 


Aspergillus niger Fungi MHB, 24 h, 37°C 9760 Ertürk et al. (2006) 

Aspergillus ochraceus Fungi MEB, 72 h, 28°C 1800–2000 Mimica-Dukic et al. (2003) 


Aspergillus terreus Fungi MEB, 72 h, 28°C 1800–2200 Mimica-Dukic et al. (2003) 

Aspergillus versicolor Fungi MEB, 72 h, 28°C 2000–2200 Mimica-Dukic et al. (2003) 

Cladosporium cladosporoides Fungi MEB, 72 h, 28°C 1200–1500 Mimica-Dukic et al. (2003) 

Epidermophyton fl occosum Fungi MEB, 72 h, 28°C 1200–1300 Mimica-Dukic et al. (2003) 

Fusarium tricinctum Fungi MEB, 72 h, 28°C 800–1500 Mimica-Dukic et al. (2003) 

Microsporum canis Fungi MEB, 72 h, 28°C 1000–1200 Mimica-Dukic et al. (2003) 





Penicillium funiculosum Fungi MEB, 72 h, 28°C 2800–3000 Mimica-Dukic et al. (2003) 

Penicillium ochroyloron Fungi MEB, 72 h, 28°C 2500–2800 Mimica-Dukic et al. (2003) 

Phomopsis helianthi Fungi MEB, 72 h, 28°C 800–1300 Mimica-Dukic et al. (2003) 

Rhizopus sp. Fungi NB, Tween 20, 8 d, 30°C 12,500 Yousef and Tawil (1980) 

Trichoderma viride Fungi MEB, 72 h, 28°C 2700–3200 Mimica-Dukic et al. (2003) 

Trichophyton mentagrophytes Fungi MEB, 72 h, 28°C 1000–1200 Mimica-Dukic et al. (2003) 

Candida albicans Yeast NB, Tween 20, 18 h, 37°C 800 Yousef and Tawil (1980) 

Candida albicans Yeast MHB, 24 h, 37°C 4880 Ertürk et al. (2006) 

Saccharomyces cerevisiae Yeast YPB, 24 h, 20°C 800 Schelz et al. (2006)


TABLE 12.6 

Inhibitory Data of Bitter Fennel Oil Obtained in the Vapor Phase Test 


Escherichia coli Bac- NA, 24 h, 37°C ~20,000 ++ Kellner and Kober (1954) 

Neisseria sp. Bac- NA, 24 h, 37°C ~20,000 NG Kellner and Kober (1954) 

Salmonella typhi Bac- NA, 24 h, 37°C ~20,000 + Kellner and Kober (1954) 


Bacillus megaterium Bac+ NA, 24 h, 37°C ~20,000 + Kellner and Kober (1954) 




Staphylococcus aureus Bac+ NA, 24 h, 37°C ~20,000 + Kellner and Kober (1954) 

Streptococcus faecalis Bac+ NA, 24 h, 37°C ~20,000 + Kellner and Kober (1954) 


Streptococcus pyogenes Bac+ NA, 24 h, 37°C ~20,000 NG Kellner and Kober (1954) 

Candida albicans Yeast NA, 24 h, 37°C ~20,000 NG Kellner and Kober (1954)


TABLE 12.7 

Inhibitory Data of Caraway Oil Obtained in the Agar Diffusion Test 



Quantity (µg) Inhibition Zone (mm) Ref. 






Citrobacter freundii Bac- ISA, 48 h, 25°C 4 (h), 10,000 10.5 Deans and Ritchie (1987) 





Escherichia coli Bac- TYA, 18–24 h, 37°C 9.5, 2000 0 Morris et al. (1979) 

Escherichia coli Bac- NA, 24 h, 37°C 4, — 4 El-Gengaihi and Zaki (1982) 

Escherichia coli Bac- Cited, 18 h, 37°C 6, 2500 10.3 Janssen et al. (1986) 

Escherichia coli Bac- ISA, 48 h, 25°C 4 (h), 10,000 9.5 Deans and Ritchie (1987) 



Klebsiella pneumoniae Bac- ISA, 48 h, 25°C 4 (h), 10,000 5.5 Deans and Ritchie (1987) 



Proteus vulgaris Bac- NA, 24 h, 37°C —, sd 0 Maruzzella and Lichtenstein (1956) 

Proteus vulgaris Bac- ISA, 48 h, 25°C 4 (h), 10,000 9.5 Deans and Ritchie (1987) 





Pseudomonas fl uorescens Bac- NA, 24 h, 37°C 4, — 4 El-Gengaihi and Zaki (1982) 

Salmonella pullorum Bac- ISA, 48 h, 25°C 4 (h), 10,000 6.5 Deans and Ritchie (1987) 

Salmonella sp. Bac- NA, 24 h, 37°C 4, — 10 El-Gengaihi and Zaki (1982)




Yersinia enterocolitica Bac- ISA, 48 h, 25°C 4 (h), 10,000 8.5 Deans and Ritchie (1987) 

Actinomyces sp. Bac+ NA, 24 h, 37°C 4, — 10 El-Gengaihi and Zaki (1982) 


Bacillus subtilis Bac+ NA, 24 h, 37°C 4, — 4 El-Gengaihi and Zaki (1982) 



Bacillus subtilis Bac+ Cited, 18 h, 37°C 6, 2500 8.3 Janssen et al. (1986) 




Clostridium butyricum Bac+ NA, 24 h, 37°C 4, — 0 El-Gengaihi and Zaki (1982) 


Corynebacterium sp. Bac+ TYA, 18–24 h, 37°C 9.5, 2000 0 Morris et al. (1979) 

Lactobacillus delbrueckii Bac+ NA, 24 h, 37°C 5000 on agar 0 Di Pasqua et al. (2005) 

Lactobacillus plantarum Bac+ NA, 24 h, 37°C 5000 on agar 0 Di Pasqua et al. (2005) 


Lactococcus garvieae Bac+ NA, 24 h, 37°C 5000 on agar 0 Di Pasqua et al. (2005) 

Lactococcus lactis subsp. lactis Bac+ NA, 24 h, 37°C 5000 on agar 0 Di Pasqua et al. (2005) 


Listeria monocytogenes Bac+ NA, 24 h, 37°C 5000 on agar 0 Di Pasqua et al. (2005) 

Micrococcus luteus Bac+ ISA, 48 h, 25°C 4 (h), 10,000 8.5 Deans and Ritchie (1987) 

Micrococcus sp. Bac+ NA, 48 h, 37°C 4, — 4 El-Gengaihi and Zaki (1982) 

Mycobacterium phlei Bac+ NA, 24 h, 37°C 4, — 10 El-Gengaihi and Zaki (1982) 



Sarcina lutea Bac+ NA, 48 h, 37°C 4, — 4 El-Gengaihi and Zaki (1982) 


Staphylococcus aureus Bac+ TYA, 18–24 h, 37°C 9.5, 2000 0 Morris et al. (1979) 

Staphylococcus aureus Bac+ NA, 24 h, 37°C 4, — 4 El-Gengaihi and Zaki (1982) 

continued











Absidia corMYBifera Fungi EYA, 48 h, 45°C 5, sd 0 Nigam and Rao (1979) 

Alternaria porri Fungi PDA, 72 h, 28°C 5, 5000 7.4 Pawar and Thaker (2007) 


Alternaria sp. Fungi PDA, 18 h, 37°C 6, sd 0 Sharma and Singh (1979) 

Aspergillus candidus Fungi PDA, 18 h, 37°C 6, sd 13.5 Sharma and Singh (1979) 









Cladosporium herbarum Fungi PDA, 18 h, 37°C 6, sd 0 Sharma and Singh (1979) 




Helminthosporium sacchari Fungi PDA, 18 h, 37°C 6, sd 6.5 Sharma and Singh (1979) 



Microsporum gypseum Fungi PDA, 18 h, 37°C 6, sd 11 Sharma and Singh (1979) 




Nigrospora panici Fungi SMA, 2–7 d, 20°C 6.35, sd 15 Maruzzella and Liguori (1958)


Penicillium aculeatum Fungi CA, 48 h, 27°C 5, sd 0 Nigam and Rao (1979) 

Penicillium chrysogenum Fungi CA, 48 h, 27°C 5, sd 0 Nigam and Rao (1979) 




Penicillium javanicum Fungi CA, 48 h, 27°C 5, sd 10 Nigam and Rao (1979) 

Penicillium jensenii Fungi CA, 48 h, 27°C 5, sd 15 Nigam and Rao (1979) 

Penicillium lividum Fungi CA, 48 h, 27°C 5, sd 0 Nigam and Rao (1979) 

Penicillium notatum Fungi CA, 48 h, 27°C 5, sd 15 Nigam and Rao (1979) 

Penicillium obscurum Fungi CA, 48 h, 27°C 5, sd 20 Nigam and Rao (1979) 

Penicillium sp. I Fungi CA, 48 h, 27°C 5, sd 10 Nigam and Rao (1979) 

Penicillium sp. II Fungi CA, 48 h, 27°C 5, sd 15 Nigam and Rao (1979) 

Penicillium sp. III Fungi CA, 48 h, 27°C 5, sd 0 Nigam and Rao (1979) 





Thermoascus aurantiacis Fungi EYA, 48 h, 45°C 5, sd 10 Nigam and Rao (1979) 







Candida albicans Yeast TYA, 18–24 h, 37°C 9.5, 2000 0 Morris et al. (1979) 

Candida albicans Yeast NA, 24 h, 37°C 4, — 0 El-Gengaihi and Zaki (1982) 

Candida albicans Yeast Cited, 18 h, 37°C 6, 2500 9.7 Janssen et al. (1986) 





Candida utilis Yeast NA, 24 h, 37°C 4, — 0 El-Gengaihi and Zaki (1982) 


continued

















Saccharomyces cerevisiae Yeast NA, 24 h, 37°C 4, — 0 El-Gengaihi and Zaki (1982) 





TABLE 12.8 

Inhibitory Data of Caraway Oil Obtained in the Dilution Test 

Microorganism MO Class Test Parameters MIC (µg/ml) Ref. 

Bacteria Bac 5% Glucose, 9 d, 37°C 2000 Buchholtz (1875) 

Escherichia coli Bac- TYB, 18–24 h, 37°C >1000 Morris et al. (1979) 


Escherichia coli Bac- NA, 1–3 d, 30°C 2000 Farag et al. (1989) 

Escherichia coli Bac- NB, Tween 20, 18 h, 37°C 6400 Yousef and Tawil (1980) 

Helicobacter pylori Bac- Cited, 20 h, 37°C 273.1 Weseler et al. (2005) 


Pseudomonas fl uorescens Bac- NA, 1–3 d, 30°C 2000 Farag et al. (1989) 

Pseudomonas sp. Bac- TSB, 24 h, 37°C 6000 Di Pasqua et al. (2005) 

Salmonella typhimurium Bac- TSB, 24 h, 37°C >10,000 Di Pasqua et al. (2005) 

Serratia marcescens Bac- NA, 1–3 d, 30°C 2500 Farag et al. (1989) 

Bacillus subtilis Bac+ NA, 1–3 d, 30°C 1000 Farag et al. (1989) 


Brochotrix thermosphacta Bac+ M17, 24 h, 20°C 10,000 Di Pasqua et al. (2005) 

Corynebacterium sp. Bac+ TYB, 18–24 h, 37°C 500 Morris et al. (1979) 

Micrococcus sp. Bac+ NA, 1–3 d, 30°C 1000 Farag et al. (1989) 

Mycobacterium phlei Bac+ NA, 1–3 d, 30°C 750 Farag et al. (1989) 


Sarcina sp. Bac+ NA, 1–3 d, 30°C 1250 Farag et al. (1989) 

Staphylococcus aureus Bac+ TSB, 24 h, 37°C 10,000 Di Pasqua et al. (2005) 

Staphylococcus aureus Bac+ NA, 1–3 d, 30°C 1250 Farag et al. (1989) 

Staphylococcus aureus Bac+ TYB, 18–24 h, 37°C 500 Morris et al. (1979) 

Staphylococcus aureus Bac+ NB, Tween 20, 18 h, 37°C 6400 Yousef and Tawil (1980) 

Alternaria citri Fungi PDA, 8 d, 22°C >1000 Arras and Usai (2001) 




Aspergillus fl avus Fungi CA, 7 d, 28°C 84–96% inh. 500 Kumar et al. (2007) 


continued



Microorganism MO Class Test Parameters MIC (µg/ml) Ref. 





Botrytis cinera Fungi PDA, 8 d, 22°C >1000 Arras and Usai (2001) 

Epidermophyton fl occosum Fungi SA, Tween 80, 21 d, 20°C 300–625 Janssen et al. (1988) 







Penicillium digitatum Fungi PDA, 8 d, 22°C >1000 Arras and Usai (2001) 

Penicillium italicum Fungi PDA, 8 d, 22°C >1000 Arras and Usai (2001) 












Trichophyton mentagrophytes Fungi SA, Tween 80, 21 d, 20°C


TABLE 12.9 

Inhibitory Data of Caraway Oil Obtained in the Vapor Phase Test 


Escherichia coli Bac- NA, 24 h, 37°C ~20,000 NG Kellner and Kober (1954) 


Salmonella typhi Bac- NA, 24 h, 37°C ~20,000 NG Kellner and Kober (1954) 


Bacillus megaterium Bac+ NA, 24 h, 37°C ~20,000 NG Kellner and Kober (1954) 


Corynebacterium diphtheriae Bac+ NA, 24 h, 37°C ~20,000 NG Kellner and Kober (1954) 


Staphylococcus aureus Bac+ NA, 24 h, 37°C ~20,000 NG Kellner and Kober (1954) 

Streptococcus faecalis Bac+ NA, 24 h, 37°C ~20,000 NG Kellner and Kober (1954) 





TABLE 12.10 

Inhibitory Data of Cassia Oil Obtained in the Agar Diffusion Test 




Inhibition Zone 

(mm) Ref. 

Enterobacter aerogenes Bac- MHA, 24 h, 30°C 6, 15,000 18 Rossi et al. (2007) 

Escherichia coli Bac- BA, 24–48 h, 37°C 12.7, sd 14 Goutham and Purohit (1974) 

Escherichia coli Bac- MHA, 24 h, 30°C 6, 15,000 21 Rossi et al. (2007) 

Proteus vulgaris Bac- BA, 24–48 h, 37°C 12.7, sd 17.5 Goutham and Purohit (1974) 

Pseudomonas aeruginosa Bac- BA, 24–48 h, 37°C 12.7, sd 18 Goutham and Purohit (1974) 

Pseudomonas aeruginosa Bac- MHA, 24 h, 30°C 6, 15,000 19 Rossi et al. (2007) 

Salmonella pullorum Bac- BA, 24–48 h, 37°C 12.7, sd 13.5 Goutham and Purohit (1974) 

Bacillus subtilis Bac+ BA, 24–48 h, 37°C 12.7, sd 13.5 Goutham and Purohit (1974) 

Corynebacterium pyogenes Bac+ BA, 24–48 h, 37°C 12.7, sd 13.5 Goutham and Purohit (1974) 


Pasteurella multocida Bac+ BlA, 24–48 h, 37°C 12.7, sd 19 Goutham and Purohit (1974) 

Staphylococcus aureus Bac+ BA, 24–48 h, 37°C 12.7, sd 0 Goutham and Purohit (1974) 

Staphylococcus aureus Bac+ MHA, 24 h, 37°C 6, 15,000 30 Rossi et al. (2007) 



Fusarium oxysporum f.sp. cicer Fungi PDA, 72 h, 28°C 5, 5000 37 Pawar and Thaker (2007)


TABLE 12.11 

Inhibitory Data of Cassia Oil Obtained in the Dilution Test 


Escherichia coli Bac- NA, cited 75–600 Ooi et al. (2006) 

Escherichia coli O157:H7 Bac- BHI, 48 h, 35°C 500 Oussalah et al. (2006) 

Pseudomonas aeruginosa Bac- NA, cited 75–600 Ooi et al. (2006) 

Salmonella typhimurium Bac- NA, cited 75–600 Ooi et al. (2006) 

Salmonella typhimurium Bac- BHI, 48 h, 35°C 250 Oussalah et al. (2006) 

Vibrio cholerae Bac- NA, cited 75–600 Ooi et al. (2006) 

Vibrio parahaemolyticus Bac- NA, cited 75–600 Ooi et al. (2006) 

Yersinia enterocolitica Bac- MHA, Tween 20, 24 h, 37°C 300 Rossi et al. (2007) 

Listeria monocytogenes Bac+ BHI, 48 h, 35°C 300 Oussalah et al. (2006) 

Staphylococcus aureus Bac+ NA, cited 75–600 Ooi et al. (2006) 

Staphylococcus aureus Bac+ BHI, 48 h, 35°C 250 Oussalah et al. (2006) 

Alternaria alternata Fungi PDA, 7 d, 28°C 100% inh. 300 Feng and Zheng (2007) 




Aspergillus sp. Fungi NA, cited 75–150 Ooi et al. (2006) 


Fusarium sp. Fungi NA, cited 75–150 Ooi et al. (2006) 

Microsporum gypseum. Fungi NA, cited 18.8–37.5 Ooi et al. (2006) 



Trichophyton mentagrophytes Fungi NA, cited 18.8–37.5 Ooi et al. (2006) 

Trichophyton rubrum Fungi NA, cited 18.8–37.5 Ooi et al. (2006) 

Candida albicans Yeast NA, cited 100–450 Ooi et al. (2006) 

Candida glabrata Yeast NA, cited 100–450 Ooi et al. (2006) 

Candida krusei Yeast NA, cited 100–450 Ooi et al. (2006) 

Candida tropicalis Yeast NA, cited 100–450 Ooi et al. (2006)


TABLE 12.12 

Inhibitory Data of Cassia Oil Obtained in the Vapor Phase Test 





Salmonella typhi Bac- NA, 24 h, 37°C 6.35, sd ++ Maruzzella and Liguori (1958) 

Salmonella typhi Bac- NA, 24 h, 37°C 6.35, sd ++ Maruzzella and Sicurella (1960) 


Bacillus subtilis var. aterrimus Bac+ NA, 24 h, 37°C 6.35, sd ++ Maruzzella and Liguori (1958) 

Bacillus subtilis var. aterrimus Bac+ NA, 24 h, 37°C 6.35, sd ++ Maruzzella and Sicurella (1960) 


Mycobacterium avium Bac+ NA, 24 h, 37°C 6.35, sd NG Maruzzella and Liguori (1958) 

Mycobacterium avium Bac+ NA, 24 h, 37°C 6.35, sd + Maruzzella and Sicurella (1960) 


Staphylococcus aureus Bac+ NA, 24 h, 37°C 6.35, sd ++ Maruzzella and Liguori (1958) 

Staphylococcus aureus Bac+ NA, 24 h, 37°C 6.35, sd ++ Maruzzella and Sicurella (1960) 


Streptococcus faecalis Bac+ NA, 24 h, 37°C 6.35, sd + Maruzzella and Liguori (1958) 

Streptococcus faecalis Bac+ NA, 24 h, 37°C 6.35, sd + Maruzzella and Sicurella (1960) 




TABLE 12.13 

Inhibitory Data of Ceylon Cinnamon Bark Oil Obtained in the Agar Diffusion Test 





(mm) Ref. 

Acinetobacter calcoaceticus Bac- ISA, 48 h, 25°C 4 (h), 10,000 16.5 Deans and Ritchie (1987) 













Escherichia coli Bac- NA, 18 h, 37°C 6 (h), pure 15.5 Yousef and Tawil (1980) 


Escherichia coli Bac- NA, 24 h, 37°C 10 (h), 2000 35 Singh et al. (2007) 

Escherichia coli Bac- NA, 24 h, 30°C Drop, 5000 45 Hili et al. (1997) 









Pseudomonas aeruginosa Bac- Cited 15, 2500 2 Pizsolitto et al. (1975) 


continued







(mm) Ref. 



Pseudomonas aeruginosa Bac- NA, 18 h, 37°C 6 (h), pure 17.5 Yousef and Tawil (1980) 

Pseudomonas aeruginosa Bac- NA, 24 h, 30°C Drop, 5000 25 Hili et al. (1997) 

Pseudomonas aeruginosa Bac- NA, 24 h, 37°C 10 (h), 2000 60 Singh et al. (2007) 


Salmonella pullorum Bac- ISA, 48 h, 25°C 4 (h), 10,000 21.2 Deans and Ritchie (1987) 


Salmonella typhi Bac- NA, 24 h, 37°C 10 (h), 2000 41 Singh et al. (2007) 






Bacillus cereus Bac+ NA, 24 h, 37°C 10 (h), 2000 27 Singh et al. (2007) 






Bacillus subtilis Bac+ ISA, 48 h, 25°C 4 (h), 10,000 23.5 Deans and Ritchie (1987) 

Bacillus subtilis Bac+ NA, 24 h, 37°C 10 (h), 2000 56 Singh et al. (2007) 






Listeria monocytogenes Bac+ TSA, 24 h, 35°C 4 (h), 25,000 6.8 Smith-Palmer et al. (1998) 

Micrococcus luteus Bac+ ISA, 48 h, 25°C 4 (h), 10,000 18 Deans and Ritchie (1987)





Staphylococcus aureus Bac+ Cited, 18 h, 37°C 6, 2500 10 Janssen et al. (1986) 


Staphylococcus aureus Bac+ TSA, 24 h, 35°C 4 (h), 25,000 7.5 Smith-Palmer et al. (1998) 



Staphylococcus aureus Bac+ NA, 24 h, 30°C Drop, 5000 45 Hili et al. (1997) 

Staphylococcus aureus Bac+ NA, 24 h, 37°C 10 (h), 2000 57 Singh et al. (2007) 







Aspergillus fumigatus Fungi SDA, 3 d, 28°C 6, sd 18 Saksena and Saksena (1984) 







Keratinomyces afelloi Fungi SDA, 3 d, 28°C 6, sd 18 Saksena and Saksena (1984) 

Keratinophyton terreum Fungi SDA, 3 d, 28°C 6, sd 12 Saksena and Saksena (1984) 

Microsporum gypseum Fungi SDA, 3 d, 28°C 6, sd 21 Saksena and Saksena (1984) 








continued







(mm) Ref. 

Trichophyton equinum Fungi SDA, 3 d, 28°C 6, sd 28 Saksena and Saksena (1984) 

Trichophyton rubrum Fungi SDA, 3 d, 28°C 6, sd 28 Saksena and Saksena (1984) 


Candida albicans Yeast SDA, 3 d, 28°C 6, sd 14 Saksena and Saksena (1984) 



Candida albicans Yeast NA, 24 h, 30°C Drop, 5000 39 Hili et al. (1997) 





Candida tropicalis Yeast SDA, 3 d, 28°C 6, sd 21 Saksena and Saksena (1984) 














Saccharomyces cerevisiae Yeast NA, 24 h, 30°C Drop, 5000 53 Hili et al. (1997) 

Schizosaccharomyces pombe Yeast NA, 24 h, 30°C Drop, 5000 43 Hili et al. (1997) 


Torula utilis Yeast NA, 24 h, 30°C Drop, 5000 42 Hili et al. (1997)


TABLE 12.14 

Inhibitory Data of Ceylon Cinnamon Bark Oil Obtained in the Dilution Test 


Campylobacter jejuni Bac- TSB, 24 h, 42°C 500 Smith-Palmer et al. (1998) 

Escherichia coli Bac- NB, 16 h, 37°C 200 Lens-Lisbonne et al. (1987) 


Escherichia coli Bac- TSB, 24 h, 35°C 500 Smith-Palmer et al. (1998) 

Escherichia coli Bac- MYB, DMSO, 40 h, 30°C 67% inh. 500 Hili et al. (1997) 


Pseudomonas aeruginosa Bac- NB, 16 h, 37°C 300 Lens-Lisbonne et al. (1987) 

Pseudomonas aeruginosa Bac- NB, Tween 20, 18 h, 37°C 400 Yousef and Tawil (1980) 

Pseudomonas aeruginosa Bac- MYB, DMSO, 40 h, 30°C 85% inh. 500 Hili et al. (1997) 

Salmonella enteritidis Bac- TSB, 24 h, 35°C 500 Smith-Palmer et al. (1998) 



Listeria monocytogenes Bac+ TSB, 24 h, 35°C 300 Smith-Palmer et al. (1998) 

Listeria monocytogenes Bac+ TSB, 10 d, 4°C 300 Smith-Palmer et al. (1998) 




Staphylococcus aureus Bac+ NB, 16 h, 37°C 350 Lens-Lisbonne et al. (1987) 

Staphylococcus aureus Bac+ MHB, Tween 80, 24 h, 37°C 390 Bastide et al. (1987) 


Staphylococcus aureus Bac+ TSB, 24 h, 35°C 400 Smith-Palmer et al. (1998) 

Staphylococcus aureus Bac+ MYB, DMSO, 40 h, 30°C 70% inh. 500 Hili et al. (1997) 

Streptococcus faecalis Bac+ NB, 16 h, 37°C 200 Lens-Lisbonne et al. (1987) 


Aspergillus fl avus Fungi PDA, 5 d, 27°C 1000 Thompson and Cannon (1986) 





Aspergillus parasiticus Fungi PDA, 5 d, 27°C 1000 Thompson and Cannon (1986) 

continued





Botrytis cinera Fungi PDA, Tween 20, 7 d, 24°C 25% inh. 1000 Bouchra et al. (2003) 

Colletotrichum musae Fungi SMKY, EtOH, 7 d, 28°C 300 Ranasinghe et al. (2002) 



Fusarium proliferatum Fungi SMKY, EtOH, 7 d, 28°C 500 Ranasinghe et al. (2002) 

Geotrichum citri-aurantii Fungi PDA, Tween 20, 7 d, 24°C 30% inh. 1000 Bouchra et al. (2003) 

Lasiodiplodia theobromae Fungi SMKY, EtOH, 7 d, 28°C 350 Ranasinghe et al. (2002) 

Mucor hiemalis Fungi PDA, 5 d, 27°C 500 Thompson and Cannon (1986) 

Mucor mucedo Fungi PDA, 5 d, 27°C 1000 Thompson and Cannon (1986) 

Mucor racemosus f. racemosus Fungi PDA, 5 d, 27°C 1000 Thompson and Cannon (1986) 



Penicillium digitatum Fungi PDA, Tween 20, 7 d, 24°C 32% inh. 1000 Bouchra et al. (2003) 

Phytophthora citrophthora Fungi PDA, Tween 20, 7 d, 24°C 38% inh. 1000 Bouchra et al. (2003) 

Rhizopus 66-81-2 Fungi PDA, 5 d, 27°C 1000 Thompson and Cannon (1986) 

Rhizopus arrhizus Fungi PDA, 5 d, 27°C 500 Thompson and Cannon (1986) 

Rhizopus chinensis Fungi PDA, 5 d, 27°C 500 Thompson and Cannon (1986) 

Rhizopus circinans Fungi PDA, 5 d, 27°C 500 Thompson and Cannon (1986) 

Rhizopus japonicus Fungi PDA, 5 d, 27°C 1000 Thompson and Cannon (1986) 

Rhizopus kazanensis Fungi PDA, 5 d, 27°C 500 Thompson and Cannon (1986) 

Rhizopus oryzae Fungi PDA, 5 d, 27°C 1000 Thompson and Cannon (1986) 

Rhizopus pymacus Fungi PDA, 5 d, 27°C 500 Thompson and Cannon (1986) 


Rhizopus stolonifer Fungi PDA, 5 d, 27°C 500 Thompson and Cannon (1986) 

Rhizopus tritici Fungi PDA, 5 d, 27°C 1000 Thompson and Cannon (1986) 



TABLE 12.15 

Inhibitory Data of Ceylon Cinnamon Bark Oil Obtained in the Vapor Phase Test 



Escherichia coli Bac- BlA, 18 h, 37°C MIC air 12.5 Inouye et al. (2001) 

Haemophilus infl uenzae Bac- MHA, 18 h, 37°C MIC air 3.13 Inouye et al. (2001) 



Salmonella typhi Bac- NA, 24 h, 37°C 6.35, sd ++ Maruzzella and Sicurella (1960) 


Bacillus subtilis var. aterrimus Bac+ NA, 24 h, 37°C 6.35, sd ++ Maruzzella and Sicurella (1960) 


Mycobacterium avium Bac+ NA, 24 h, 37°C 6.35, sd + Maruzzella and Sicurella (1960) 


Staphylococcus aureus Bac+ NA, 24 h, 37°C 6.35, sd ++ Maruzzella and Sicurella (1960) 

Staphylococcus aureus Bac+ MHA, 18 h, 37°C MIC air 6.25 Inouye et al. (2001) 


Streptococcus faecalis Bac+ NA, 24 h, 37°C 6.35, sd + Maruzzella and Sicurella (1960) 

Streptococcus pneumoniae Bac+ MHA, 18 h, 37°C MIC air 1.56–3.13 Inouye et al. (2001) 


Streptococcus pyogenes Bac+ MHA, 18 h, 37°C MIC air 6.25 Inouye et al. (2001) 

Aspergillus fl avus Fungi CDA, 6 d 12, 6000 +++ Singh et al. (2007) 

Aspergillus niger Fungi CDA, 6 d 12, 6000 + Singh et al. (2007) 

Aspergillus ochraceus Fungi CDA, 6 d 12, 6000 ++ Singh et al. (2007) 

Aspergillus terreus Fungi CDA, 6 d 12, 6000 NG Singh et al. (2007) 

Fusarium graminearum Fungi CDA, 6 d 12, 6000 NG Singh et al. (2007) 

Fusarium moniliforme Fungi CDA, 6 d 12, 6000 NG Singh et al. (2007) 

Penicillium citrinum Fungi CDA, 6 d 12, 6000 NG Singh et al. (2007) 

Penicillium viridicatum Fungi CDA, 6 d 12, 6000 NG Singh et al. (2007) 



TABLE 12.16 

Inhibitory Data of Ceylon Cinnamon Leaf Oil Obtained in the Agar Diffusion Test 





(mm) Ref. 

Escherichia coli Bac- TYA, 18–24 h, 37°C 9.5, 2000 17 Morris et al. (1979) 

Escherichia coli Bac- NA, 24 h, 37°C 10 (h), 2000 25 Singh et al. (2007) 

Pseudomonas aeruginosa Bac- NA, 24 h, 37°C 10 (h), 2000 90 Singh et al. (2007) 

Salmonella typhi Bac- NA, 24 h, 37°C 10 (h), 2000 17 Singh et al. (2007) 

Bacillus cereus Bac+ NA, 24 h, 37°C 10 (h), 2000 32 Singh et al. (2007) 

Bacillus subtilis Bac+ NA, 24 h, 37°C 10 (h), 2000 90 Singh et al. (2007) 

Corynebacterium sp. Bac+ TYA, 18–24 h, 37°C 9.5, 2000 12 Morris et al. (1979) 


Staphylococcus aureus Bac+ TYA, 18–24 h, 37°C 9.5, 2000 18 Morris et al. (1979) 





Candida albicans Yeast TYA, 18–24 h, 37°C 9.5, 2000 14 Morris et al. (1979)


TABLE 12.17 

Inhibitory Data of Ceylon Cinnamon Leaf Oil Obtained in the Dilution Test 


Escherichia coli Bac- TYB, 18–24 h, 37°C 1000 Morris et al. (1979) 



Corynebacterium sp. Bac+ TYB, 18–24 h, 37°C 500 Morris et al. (1979) 


Staphylococcus aureus Bac+ TYB, 18–24 h, 37°C 500 Morris et al. (1979) 










Fusarium proliferatum Fungi SMKY, EtOH, 7 d, 28°C 500 Ranasinghe et al. (2002) 

Lasiodiplodia theobromae Fungi SMKY, EtOH, 7 d, 28°C 600 Ranasinghe et al. (2002) 














Candida albicans Yeast TYB, 18–24 h, 37°C 500 Morris et al. (1979)


TABLE 12.18 

Inhibitory Data of Ceylon Cinnamon Leaf Oil Obtained in the Vapor Phase Test 










Aspergillus fl avus Fungi WFA, 42 d, 25°C Disk, 50,000 NG Guynot et al. (2003) 

Aspergillus fl avus Fungi Bread, 14 d, 25°C 30,000 +++ Suhr and Nielsen (2003) 

Aspergillus fl avus Fungi CDA, 6 d 12, 6000 NG Singh et al. (2007) 

Aspergillus niger Fungi WFA, 42 d, 25°C Disk, 50,000 NG Guynot et al. (2003) 

Aspergillus niger Fungi CDA, 6 d 12, 6000 NG Singh et al. (2007) 

Aspergillus ochraceus Fungi CDA, 6 d 12, 6000 + Singh et al. (2007) 

Aspergillus terreus Fungi CDA, 6 d 12, 6000 + Singh et al. (2007) 

Endomyces fi lbuliger Fungi Bread, 14 d, 25°C 30,000 +++ Suhr and Nielsen (2003) 

Eurotium amstelodami Fungi WFA, 42 d, 25°C Disk, 50,000 NG Guynot et al. (2003) 

Eurotium herbarum Fungi WFA, 42 d, 25°C Disk, 50,000 NG Guynot et al. (2003) 

Eurotium repens Fungi WFA, 42 d, 25°C Disk, 50,000 NG Guynot et al. (2003) 

Eurotium repens Fungi Bread, 14 d, 25°C 30,000 +++ Suhr and Nielsen (2003) 

Eurotium rubrum Fungi WFA, 42 d, 25°C Disk, 50,000 NG Guynot et al. (2003) 

Fusarium graminearum Fungi CDA, 6 d 12, 6000 NG Singh et al. (2007) 


Penicillium citrinum Fungi CDA, 6 d 12, 6000 NG Singh et al. (2007) 

Penicillium corylophilum Fungi WFA, 42 d, 25°C Disk, 50,000 NG Guynot et al. (2003) 

Penicillium corylophilum Fungi Bread, 14 d, 25°C 30,000 +++ Suhr and Nielsen (2003) 

Penicillium roqueforti Fungi Bread, 14 d, 25°C 30,000 +++ Suhr and Nielsen (2003) 

Penicillium viridicatum Fungi CDA, 6 d 12, 6000 NG Singh et al. (2007) 



TABLE 12.19 

Inhibitory Data of Citronella Oil Obtained in the Agar Diffusion Test 

Microorganism MO Class Conditions Inhibition Zone (mm) Ref. 

Aerobacter aerogenes Bac- NA, 24 h, 37°C —, sd 0 a Maruzzella and Lichtenstein (1956) 

Aerobacter aerogenes Bac- NA, 24 h, 37°C 5 × 20, 1000 0 a Möse et al. (1957) 

Brucella abortus Bac- NA, 24 h, 37°C 5 × 20, 1000 2–5 a Möse et al. (1957) 

Campylobacter jejuni Bac- Cited 6, 15,000 40 a Wannissorn et al. (2005) 

Escherichia coli Bac- NA, 24 h, 37°C —, sd 0 a Maruzzella and Lichtenstein (1956) 

Escherichia coli Bac- NA, 24 h, 37°C 5 × 20, 1000 0–1 a Möse et al. (1957) 

Escherichia coli Bac- Cited 6, 15,000 10.5 a Wannissorn et al. (2005) 

Escherichia coli Bac- Cited 20,000 0 Lemos et al. (1992) 


Escherichia coli Bac- TGA, 18–24 h, 37°C 9.5, 2000 0 Morris et al. (1979) 

Escherichia coli Bac- Agar, 24 h, 37°C 6, 6000 7 Jirovetz et al. (2006) 

Klebsiella pneumonia Bac- NA, 24 h, 37°C 5 × 20, 1000 2–5 a Möse et al. (1957) 

Klebsiella pneumonia subsp. oceanae Bac- NA, 24 h, 37°C 5 × 20, 1000 2–5 a Möse et al. (1957) 

Klebsiella pneumoniae Bac- Agar, 24 h, 37°C 6, 6000 0 Jirovetz et al. (2006) 

Neisseria perfl ava Bac- NA, 24 h, 37°C —, sd 0 a Maruzzella and Lichtenstein (1956) 

Proteus OX19 Bac- NA, 24 h, 37°C 5 × 20, 1000 2–5 a Möse et al. (1957) 

Proteus vulgaris Bac- NA, 24 h, 37°C —, sd 0 a Maruzzella and Lichtenstein (1956) 

Proteus vulgaris Bac- NA, 24 h, 37°C 5 × 20, 1000 0 a Möse et al. (1957) 

Proteus vulgaris Bac- Agar, 24 h, 37°C 6, 6000 10 Jirovetz et al. (2006) 

Pseudomonas aeruginosa Bac- NA, 24 h, 37°C —, sd 0 a Maruzzella and Lichtenstein (1956) 

Pseudomonas aeruginosa Bac- NA, 24 h, 37°C 5 × 20, 1000 0 a Möse et al. (1957) 


Pseudomonas aeruginosa Bac- Agar, 24 h, 37°C 6, 6000 0 Jirovetz et al. (2006) 

Pseudomonas fl uorescens Bac- NA, 24 h, 37°C 5 × 20, 1000 0–1 a Möse et al. (1957) 

Pseudomonas mangiferae indicae Bac- NA, 36–48 h, 37°C 6, sd 11 Garg and Garg (1980) 

Salmonella enteritidis Bac- Cited 6, 15,000 12.8 a Wannissorn et al. (2005) 

Salmonella enteritidis Bac- NA, 24 h, 37°C 5 × 20, 1000 6–10 a Möse et al. (1957) 

continued




Salmonella paratyphi Bac- NA, 36–48 h, 37°C 6, sd 14 Garg and Garg (1980) 

Salmonella paratyphi B Bac- NA, 24 h, 37°C 5 × 20, 1000 1 a Möse et al. (1957) 

Salmonella sp. Bac- Agar, 24 h, 37°C 6, 6000 0 Jirovetz et al. (2006) 

Salmonella typhi Bac- NA, 24 h, 37°C 5 × 20, 1000 2–5 a Möse et al. (1957) 

Salmonella typhi Bac- NA, 36–48 h, 37°C 6, sd 18 Garg and Garg (1980) 

Salmonella typhimurium Bac- Cited 6, 15,000 21 a Wannissorn et al. (2005) 

Serratia marcescens Bac- NA, 24 h, 37°C 5 × 20, 1000 0 a Möse et al. (1957) 


Vibrio albicans Bac- NA, 24 h, 37°C 5 × 20, 1000 6–10 a Möse et al. (1957) 

Vibrio cholera Bac- NA, 24 h, 37°C 5 × 20, 1000 6–10 a Möse et al. (1957) 

Vibrio cholera Bac- NA, 36–48 h, 37°C 6, sd 0 Garg and Garg (1980) 

Bacillus anthracis Bac+ NA, 24 h, 37°C 5 × 20, 1000 1 a Möse et al. (1957) 

Bacillus cereus Bac+ Cited 20,000 20 Lemos et al. (1992) 

Bacillus mesentericus Bac+ NA, 24 h, 37°C —, sd 3 a Maruzzella and Lichtenstein (1956) 

Bacillus mycoides Bac+ NA, 36–48 h, 37°C 6, sd 12 Garg and Garg (1980) 

Bacillus pumilus Bac+ NA, 36–48 h, 37°C 6, sd 12 Garg and Garg (1980) 

Bacillus subtilis Bac+ NA, 24 h, 37°C 5 × 20, 1000 1 a Möse et al. (1957) 

Bacillus subtilis Bac+ NA, 24 h, 37°C —, sd 8 a Maruzzella and Lichtenstein (1956) 

Bacillus subtilis Bac+ NA, 36–48 h, 37°C 6, sd 12 Garg and Garg (1980) 


Clostridium perfringens Bac+ Cited 6, 15,000 39.5 a Wannissorn et al. (2005) 

Corynebacterium diphtheria Bac+ NA, 24 h, 37°C 5 × 20, 1000 6–10 a Möse et al. (1957) 

Enterococcus faecalis Bac+ Agar, 24 h, 37°C 6, 6000 10 Jirovetz et al. (2006) 

Listeria monocytogenes Bac+ NA, 24 h, 37°C 5 × 20, 1000 1 a Möse et al. (1957) 


Sarcina alba Bac+ NA, 24 h, 37°C 5 × 20, 1000 6–10 a Möse et al. (1957) 

Sarcina beige Bac+ NA, 24 h, 37°C 5 × 20, 1000 16–23 a Möse et al. (1957) 

Sarcina citrea Bac+ NA, 24 h, 37°C 5 × 20, 1000 0 a Möse et al. (1957) 

Sarcina lutea Bac+ NA, 24 h, 37°C —, sd 0 a Maruzzella and Lichtenstein (1956)


Sarcina lutea Bac+ NA, 36–48 h, 37°C 6, sd 0 Garg and Garg (1980) 

Sarcina rosa Bac+ NA, 24 h, 37°C 5 × 20, 1000 11–15 a Möse et al. (1957) 

Sporococcus sarc. Bac+ NA, 24 h, 37°C 5 × 20, 1000 26–33 a Möse et al. (1957) 

Staphylococcus albus Bac+ NA, 24 h, 37°C 5 × 20, 1000 6–10 a Möse et al. (1957) 

Staphylococcus aureus Bac+ NA, 24 h, 37°C —, sd 0 a Maruzzella and Lichtenstein (1956) 

Staphylococcus aureus Bac+ Cited 20,000 0 a Lemos et al. (1992) 

Staphylococcus aureus Bac+ NA, 24 h, 37°C 5 × 20, 1000 2–5 a Möse et al. (1957) 

Staphylococcus aureus Bac+ Agar, 24 h, 37°C 6, 6000 10 Jirovetz et al. (2006) 

Staphylococcus aureus Bac+ TGA, 18–24 h, 37°C 9.5, 2000 11 Morris et al. (1979) 

Staphylococcus aureus Bac+ NA, 36–48 h, 37°C 6, sd 12 Garg and Garg (1980) 


Staphylococcus epidermidis Bac+ Cited 20,000 12 a Lemos et al. (1992) 

Streptococcus haemolyticus Bac+ NA, 24 h, 37°C 5 × 20, 1000 6–10 a Möse et al. (1957) 

Streptococcus viridians Bac+ NA, 24 h, 37°C 5 × 20, 1000 1 a Möse et al. (1957) 

Streptomyces venezuelae Bac+ SMA, 2–7 d, 20°C sd 4 Maruzzella and Liguori (1958) 

Alternaria solani Fungi SMA, 2–7 d, 20°C sd 6 Maruzzella and Liguori (1958) 

Aspergillus fumigatus Fungi SMA, 2–7 d, 20°C sd 6 Maruzzella and Liguori (1958) 


Aspergillus niger Fungi SMA, 2–7 d, 20°C sd 7 Maruzzella and Liguori (1958) 

Aspergillus niger Fungi SDA, 7–10 d, 28°C 5, 5000 8 Saikia et al. (2001) 

Helminthosporium sativum Fungi SMA, 2–7 d, 20°C sd 2 Maruzzella and Liguori (1958) 

Microsporon gypseum Fungi SDA, 7–10 d, 28°C 5, 5000 34 Saikia et al. (2001) 

Mucor mucedo Fungi SMA, 2–7 d, 20°C sd 6 Maruzzella and Liguori (1958) 


Nigrospora panici Fungi SMA, 2–7 d, 20°C sd 6 Maruzzella and Liguori (1958) 


Penicillium digitatum Fungi SMA, 2–7 d, 20°C sd 5 Maruzzella and Liguori (1958) 

Rhizopus nigricans Fungi SMA, 2–7 d, 20°C sd 1 Maruzzella and Liguori (1958) 


Sporothrix schenkii Fungi SDA, 7–10 d, 28°C 5, 5000 12 Saikia et al. (2001) 

Candida albicans Yeast TGA, 18–24 h, 37°C 9.5, 2000 1 Morris et al. (1979) 

Candida albicans Yeast Cited 20,000 17 Lemos et al. (1992) 

continued





Candida albicans Yeast Agar, 24 h, 37°C 6, 6000 28 Jirovetz et al. (2006) 

Candida albicans Yeast SMA, 2–7 d, 20°C sd 6 Maruzzella and Liguori (1958) 

Candida albicans Yeast SDA, 7–10 d, 28°C 5, 5000 7 Saikia et al. (2001) 

Candida krusei Yeast SMA, 2–7 d, 20°C sd 0 Maruzzella and Liguori (1958) 

Candida tropicalis Yeast Cited 20,000 0 Lemos et al. (1992) 

Candida tropicalis Yeast SMA, 2–7 d, 20°C sd 3 Maruzzella and Liguori (1958) 

Cryptococcus neoformans Yeast SMA, 2–7 d, 20°C sd 2 Maruzzella and Liguori (1958) 

Cryptococcus rhodobenhani Yeast SMA, 2–7 d, 20°C sd 7 Maruzzella and Liguori (1958) 

Saccharomyces cerevisiae Yeast SMA, 2–7 d, 20°C sd 2 Maruzzella and Liguori (1958) 

a 

Citronella oil obtained from Cymbopogon nardus (Ceylon-type).


TABLE 12.20 

Inhibitory Data of Citronella Oil Obtained in the Dilution Test 

Microorganism MO Class Conditions MIC Ref. 

Escherichia coli Bac- MHB, 24 h, 36°C 2000–8000 Duarte et al. (2006) 


Escherichia coli O157:H7 Bac- BHI, 48 h, 35°C >8000 Oussalah et al. (2006) 

Pseudomonas aeruginosa Bac- NA, Tween 80, 24 h, 37°C >500 a Koba et al. (2004) 


Pseudomonas cepacia Bac- NA, Tween 80, 24 h, 37°C >500 a Koba et al. (2004) 







Staphylococcus intermedius Bac+ NA, Tween 80, 24 h, 37°C >500 a Koba et al. (2004) 

Aspergillus candidus Fungi Cited >250 a Nakahara et al. (2003) 

Aspergillus fl avus Fungi Cited >250 a Nakahara et al. (2003) 

Aspergillus fl avus Fungi PDA, 28 d, 21°C 4000–10,000 Thanaboripat et al. (2004) 

Aspergillus fumigatus Fungi NA, Tween 80, 24 h, 37°C 200 a Koba et al. (2004) 

Aspergillus niger Fungi SDB, 7–10 d, 28°C 2500 Saikia et al. (2001) 



Aspergillus versicolor Fungi Cited >250 a Nakahara et al. (2003) 

Eurotium amstelodami Fungi Cited >250 a Nakahara et al. (2003) 

Eurotium chevalieri Fungi Cited >250 a Nakahara et al. (2003) 

continued




Microsporon canis Fungi NA, Tween 80, 24 h, 37°C 200 a Koba et al. (2004) 

Microsporon gypseum Fungi NA, Tween 80, 24 h, 37°C 500 a Koba et al. (2004) 

Microsporon gypseum Fungi SDB, 7–10 d, 28°C 625 Saikia et al. (2001) 



Penicillium adametzii Fungi Cited >250 a Nakahara et al. (2003) 

Penicillium chrysogenum Fungi NB, Tween 20, 8 d, 30°C 12,500 Yousef and Tawil (1980) 


Penicillium citrinum Fungi Cited >250 a Nakahara et al. (2003) 

Penicillium griseofulvum Fungi Cited >250 a Nakahara et al. (2003) 

Penicillium islansicum Fungi Cited >250 a Nakahara et al. (2003) 


Sporothrix schenkii Fungi SDB, 7–10 d, 28°C 1250 Saikia et al. (2001) 

Trichophyton mentagrophytes Fungi NA, Tween 80, 24 h, 37°C 150 a Koba et al. (2004) 

Candida albicans Yeast NA, Tween 80, 24 h, 37°C >500 a Koba et al. (2004) 

Candida albicans Yeast SDB, 7–10 d, 28°C 1250 Saikia et al. (2001) 


Cryptococcus neoformans Yeast NA, Tween 80, 24 h, 37°C 500 a Koba et al. (2004) 

Malassezia pachydermis Yeast NA, Tween 80, 24 h, 37°C 150 a Koba et al. (2004) 

a 



TABLE 12.21 

Inhibitory Data of Citronella Oil Obtained in the Vapor Phase Test 

Microorganism MO Class Conditions Activity Ref. 

Escherichia coli Bac- NA, 24 h, 37°C ~20,000 + Kellner and Kober (1954) 


Salmonella typhi Bac- NA, 24 h, 37°C sd +++ a Maruzzella and Sicurella (1960) 



Bacillus subtilis var. aterrimus Bac+ NA, 24 h, 37°C sd + a Maruzzella and Sicurella (1960) 


Mycobacterium avium Bac+ NA, 24 h, 37°C sd NG a Maruzzella and Sicurella (1960) 

Staphylococcus aureus Bac+ NA, 24 h, 37°C sd +++ a Maruzzella and Sicurella (1960) 


Streptococcus faecalis Bac+ NA, 24 h, 37°C sd +++ a Maruzzella and Sicurella (1960) 



Aspergillus candidus Fungi PDA, 3–5 d, 27°C 250 mg/L air NG b Nakahara et al. (2003) 

Aspergillus fl avus Fungi PDA, 3–5 d, 27°C 250 mg/L air NG b Nakahara et al. (2003) 

Aspergillus fl avus Fungi PDA, 10 d, 25°C 50 ++ Sarbhoy et al. (1978) 

Aspergillus fumigatus Fungi PDA, 10 d, 25°C 50 NG Sarbhoy et al. (1978) 

Aspergillus sulphureus Fungi PDA, 10 d, 25°C 50 NG Sarbhoy et al. (1978) 

Aspergillus versicolor Fungi PDA, 3–5 d, 27°C 250 mg/L air NG b Nakahara et al. (2003) 

Eurotium amstelodami Fungi PDA, 3–5 d, 27°C 250 mg/L air NG b Nakahara et al. (2003) 

Eurotium chevalieri Fungi PDA, 3–5 d, 27°C 250 mg/L air NG b Nakahara et al. (2003) 

Mucor fragilis Fungi PDA, 10 d, 25°C 50 NG Sarbhoy et al. (1978) 

Penicillium adametzii Fungi PDA, 3–5 d, 27°C 250 mg/L air NG b Nakahara et al. (2003) 

Penicillium citrinum Fungi PDA, 3–5 d, 27°C 250 mg/L air NG b Nakahara et al. (2003) 

Penicillium griseofulvum Fungi PDA, 3–5 d, 27°C 250 mg/L air NG b Nakahara et al. (2003) 

Penicillium islansicum Fungi PDA, 3–5 d, 27°C 250 mg/L air NG b Nakahara et al. (2003) 

Rhizopus stolonifer Fungi PDA, 10 d, 25°C 50 ++ Sarbhoy et al. (1978) 

Candida albicans Yeast NA, 24 h, 37°C ~20,000 NG Kellner and Kober (1954) 

a 

Formosan citronella oil. 

b 



TABLE 12.22 

Inhibitory Data of Clary Sage Oil Obtained in the Agar Diffusion Test 


Escherichia coli Bac- MHA, 24 h, 37°C 6, 10,000 14 Yousefzadi et al. (2007) 

Klebsiella pneumoniae Bac- MHA, 24 h, 37°C 6, 10,000 10 Yousefzadi et al. (2007) 

Pseudomonas aeruginosa Bac- MHA, 24 h, 37°C 6, 10,000 0 Yousefzadi et al. (2007) 

Bacillus pumilus Bac+ MHA, 24 h, 37°C 6, 10,000 16 Yousefzadi et al. (2007) 

Bacillus subtilis Bac+ MHA, 24 h, 37°C 6, 10,000 17 Yousefzadi et al. (2007) 

Enterococcus faecalis Bac+ MHA, 24 h, 37°C 6, 10,000 9 Yousefzadi et al. (2007) 

Listeria monocytogenes Bac+ ISA, 48 h, 25°C 4 (h), 10,000 9–15 Lis-Balchin et al. (1998) 

Staphylococcus aureus Bac+ MHA, 24 h, 37°C 6, 10,000 15 Yousefzadi et al. (2007) 

Staphylococcus epidermidis Bac+ MHA, 24 h, 37°C 6, 10,000 15 Yousefzadi et al. (2007) 


Aspergillus niger Fungi MHA, 48 h, 30°C 6, 10,000 0 Yousefzadi et al. (2007) 



Candida albicans Yeast MHA, 48 h, 30°C 6, 10,000 0 Yousefzadi et al. (2007) 

Saccharomyces cerevisiae Yeast MHA, 48 h, 30°C 6, 10,000 0 Yousefzadi et al. (2007)


TABLE 12.23 

Inhibitory Data of Clary Sage Oil Obtained in the Dilution Test 


Acinetobacter baumannii Bac- MHA, Tween 20, 48 h, 35°C >20,000 Hammer et al. (1999) 

Aeromonas sobria Bac- MHA, Tween 20, 48 h, 35°C >20,000 Hammer et al. (1999) 

Erwinia carotovora Bac- NA, 48 h, 22°C >2000 Maruzzella (1963) 

Escherichia coli Bac- NA, 48 h, 37°C >2000 Maruzzella (1963) 

Escherichia coli Bac- MHA, Tween 20, 48 h, 35°C >20,000 Hammer et al. (1999) 

Escherichia coli Bac- MHB, Tween 80, 24 h, 37°C 15,000 Yousefzadi et al. (2007) 

Escherichia coli Bac- Cited 1500–2000 Peana et al. (1999) 

Klebsiella pneumoniae Bac- MHB, Tween 80, 24 h, 37°C >15,000 Yousefzadi et al. (2007) 

Klebsiella pneumoniae Bac- MHA, Tween 20, 48 h, 35°C >20,000 Hammer et al. (1999) 

Proteus vulgaris Bac- NA, 48 h, 37°C >2000 Maruzzella (1963) 

Pseudomonas aeruginosa Bac- MHA, Tween 20, 48 h, 35°C >20,000 Hammer et al. (1999) 

Salmonella typhi Bac- NA, 48 h, 37°C >2000 Maruzzella (1963) 

Salmonella typhimurium Bac- MHA, Tween 20, 48 h, 35°C >20,000 Hammer et al. (1999) 

Serratia marcescens Bac- MHA, Tween 20, 48 h, 35°C >20,000 Hammer et al. (1999) 

Bacillus cereus Bac+ NA, 48 h, 37°C 250 Maruzzella (1963) 

Bacillus circulans Bac+ NA, 48 h, 37°C 100 Maruzzella (1963) 

Bacillus megaterium Bac+ NA, 48 h, 37°C 250 Maruzzella (1963) 

Bacillus pumilus Bac+ MHB, Tween 80, 24 h, 37°C 7500 Yousefzadi et al. (2007) 

Bacillus subtilis Bac+ MHB, Tween 80, 24 h, 37°C 7500 Yousefzadi et al. (2007) 

Bacillus subtilis var. aterrimus Bac+ NA, 48 h, 37°C 100 Maruzzella (1963) 

Enterococcus faecalis Bac+ MHB, Tween 80, 24 h, 37°C >15,000 Yousefzadi et al. (2007) 

Enterococcus faecalis Bac+ MHA, Tween 20, 48 h, 35°C >20,000 Hammer et al. (1999) 

Staphylococcus aureus Bac+ NA, 48 h, 37°C >2000 Maruzzella (1963) 

Staphylococcus aureus Bac+ MHA, Tween 20, 48 h, 35°C >20,000 Hammer et al. (1999) 

Staphylococcus aureus Bac+ Cited 1500–2000 Peana et al. (1999) 

continued




Staphylococcus aureus Bac+ MHB, Tween 80, 24 h, 37°C 7500 Yousefzadi et al. (2007) 

Staphylococcus epidermidis Bac+ Cited 1500–2000 Peana et al. (1999) 

Staphylococcus epidermidis Bac+ MHB, Tween 80, 24 h, 37°C 7500 Yousefzadi et al. (2007) 

Alternaria alternata Fungi SDA, 6–8 h, 20°C 500, 62% inh. Dikshit et al. (1986) 

Aspergillus fl avus Fungi SDA, 6–8 h, 20°C 500, 52% inh. Dikshit et al. (1986) 

Aspergillus niger Fungi YES broth, 10 d - 92% inh. 10,000 Lis-Balchin et al. (1998) 

Aspergillus ochraceus Fungi YES broth, 10 d - >10,000 Lis-Balchin et al. (1998) 

Fusarium culmorum Fungi YES broth, 10 d - 69% inh. 10,000 Lis-Balchin et al. (1998) 

Fusarium oxysporum f.sp. dianthi Fungi PDA, Tween 20, 4 d, 23°C 72% inh. 2000 Pitarokili et al. (2002) 

Geotrichum candidum Fungi NA, 5 d, 22°C >2000 Maruzzella (1963) 

Gibberella fugikuroi Fungi NA, 5 d, 22°C >2000 Maruzzella (1963) 

Helminthosporium truicicum Fungi NA, 5 d, 22°C >2000 Maruzzella (1963) 

Microsporum gypseum Fungi SDA, 7 d, 30°C 400, 56% inh. Dikshit and Husain (1984) 

Microsporum gypseum Fungi SDA, 6–8 h, 20°C 500, 56% inh. Dikshit et al. (1986) 

Penicillium italicum Fungi SDA, 6–8 h, 20°C 500, 59% inh. Dikshit et al. (1986) 

Phoma betae Fungi NA, 5 d, 22°C >2000 Maruzzella (1963) 

Pityrosporum ovale Fungi NA, 5 d, 22°C >2000 Maruzzella (1963) 

Sclerotina cepivorum Fungi PDA, Tween 20, 4 d, 23°C 94% inh. 1000 Pitarokili et al. (2002) 

Sclerotina sclerotiorum Fungi PDA, Tween 20, 4 d, 23°C 1000 Pitarokili et al. (2002) 

Trichophyton equinum Fungi SDA, 7 d, 30°C 400, 30% inh. Dikshit and Husain (1984) 

Trichophyton mentagrophytes Fungi SDA, 6–8 h, 20°C 500, 40% inh. Dikshit et al. (1986) 

Trichophyton rubrum Fungi SDA, 7 d, 30°C 400, 43% inh. Dikshit and Husain (1984) 

Trichophyton rubrum Fungi SDA, 6–8 h, 20°C 500, 43% inh. Dikshit et al. (1986) 

Candida albicans Yeast NA, 48 h, 37°C >2000 Maruzzella (1963) 

Candida albicans Yeast MHA, Tween 20, 48 h, 35°C >20,000 Hammer et al. (1999) 

Candida albicans Yeast Cited 1500–2000 Peana et al. (1999)


TABLE 12.24 

Inhibitory Data of Clary Sage Oil Obtained in the Vapor Phase Test 


Escherichia coli Bac- NA, 48 h, 37°C 500 μL in cover +++ Maruzzella (1963) 

Proteus vulgaris Bac- NA, 48 h, 37°C 500 μL in cover NG Maruzzella (1963) 

Salmonella typhi Bac- NA, 24 h, 37°C sd +++ Maruzzella and Sicurella (1960) 

Bacillus subtilis var. aterrimus Bac+ NA, 48 h, 37°C 500 μL in cover + Maruzzella (1963) 

Bacillus subtilis var. aterrimus Bac+ NA, 24 h, 37°C sd +++ Maruzzella and Sicurella (1960) 

Mycobacterium avium Bac+ NA, 24 h, 37°C sd NG Maruzzella and Sicurella (1960) 

Staphylococcus aureus Bac+ NA, 48 h, 37°C 500 μL in cover NG Maruzzella (1963) 

Staphylococcus aureus Bac+ NA, 24 h, 37°C sd +++ Maruzzella and Sicurella (1960) 

Streptococcus faecalis Bac+ NA, 24 h, 37°C sd +++ Maruzzella and Sicurella (1960) 

Botrytis cinera Fungi PDA, 3 d, 25°C 1000 +++ Lee et al. (2007) 

Colletotrichum gleosporoides Fungi PDA, 3 d, 25°C 1000 +++ Lee et al. (2007) 

Fusarium oxysporum Fungi PDA, 3 d, 25°C 1000 +++ Lee et al. (2007) 

Geotrichum candidum Fungi NA, 5 d, 22°C 500 μL in cover +++ Maruzzella (1963) 

Gibberella fugikuroi Fungi NA, 5 d, 22°C 500 μL in cover +++ Maruzzella (1963) 

Helminthosporium truicicum Fungi NA, 5 d, 22°C 500 μL in cover +++ Maruzzella (1963) 

Phoma betae Fungi NA, 5 d, 22°C 500 μL in cover +++ Maruzzella (1963) 

Pityrosporum ovale Fungi NA, 5 d, 22°C 500 μL in cover +++ Maruzzella (1963) 

Pythium ultimum Fungi PDA, 3 d, 25°C 1000 +++ Lee et al. (2007) 

Rhizoctonia solani Fungi PDA, 3 d, 25°C 1000 +++ Lee et al. (2007) 

Candida albicans Yeast NA, 48 h, 37°C 500 μL in cover +++ Maruzzella (1963) 

Annotation: Clary sage absolute tested (Maruzzella, 1963).


TABLE 12.25 

Inhibitory Data of Clove Oil Obtained in the Agar Diffusion Test 

Microorganism MO Class Conditions 


(mm) Ref. 






Campylobacter jejuni Bac- TSA, 24 h, 42°C 4 (h), 25,000 9 Smith-Palmer et al. (1998) 

Campylobacter jejuni Bac- Cited 6, 15,000 22.5 Wannissorn et al. (2005) 


Citrobacter sp. Bac- NA, 24 h, 37°C 11, sd 0 Prasad et al. (1986) 

Enterobacter aerogenes Bac- ISA, 48 h, 25°C 4 (h), 10,000 8.5 Deans and Ritchie (1987) 

Enterobacter sp. Bac- NA, 24 h, 37°C 11, sd 16 Prasad et al. (1986) 

Erwinia carotovora Bac- ISA, 48 h, 25°C 4 (h), 10,000 14.5 Deans and Ritchie (1987) 


Escherichia coli Bac- NA, 24 h, 37°C 11, sd 0 Prasad et al. (1986) 







Escherichia coli Bac- Cited 6, 15,000 16.5 Wannissorn et al. (2005) 







Proteus sp. Bac- Cited 15, 2500 4 Pizsolitto et al. (1975)







Pseudomonas aeruginosa Bac- Cited, 18 h, 37°C 6, 2500 9 Janssen et al. (1986) 



Pseudomonas sp. Bac- NA, 24 h, 37°C 11, sd 0 Prasad et al. (1986) 


Salmonella enteritidis Bac- Cited 6, 15,000 14.3 Wannissorn et al. (2005) 


Salmonella saintpaul Bac- NA, 24 h, 37°C 11, sd 22 Prasad et al. (1986) 


Salmonella sp. B Bac- NA, 24 h, 37°C 11, sd 26 Prasad et al. (1986) 

Salmonella typhimurium Bac- Cited 6, 15,000 19.5 Wannissorn et al. (2005) 

Salmonella weltevreden Bac- NA, 24 h, 37°C 11, sd 22 Prasad et al. (1986) 






Bacillus anthracis Bac+ NA, 24 h, 37°C 11, sd 25 Prasad et al. (1986) 


Bacillus saccharolyticus Bac+ NA, 24 h, 37°C 11, sd 20 Prasad et al. (1986) 


Bacillus stearothermophilus Bac+ NA, 24 h, 37°C 11, sd 20 Prasad et al. (1986) 


continued





(mm) Ref. 



Bacillus subtilis Bac+ NA, 24 h, 37°C 11, sd 22 Prasad et al. (1986) 


Bacillus thurengiensis Bac+ NA, 24 h, 37°C 11, sd 19 Prasad et al. (1986) 



Clostridium perfringens Bac+ Cited 6, 15,000 20.5 Wannissorn et al. (2005) 


Corynebacterium sp. Bac+ TGA, 18–24 h, 37°C 9.5, 2000 15 Morris et al. (1979) 

Lactobacillus casei Bac+ NA, 24 h, 37°C 11, sd 22 Prasad et al. (1986) 


Lactobacillus plantarum Bac+ NA, 24 h, 37°C 11, sd 50 Prasad et al. (1986) 




Micrococcus glutamicus Bac+ NA, 24 h, 37°C 11, sd 44 Prasad et al. (1986) 




Sarcina lutea Bac+ NA, 24 h, 37°C 11, sd 32 Prasad et al. (1986) 







Staphylococcus aureus Bac+ NA, 18 h, 37°C 6 (h), pure 14.3 Yousef and Tawil (1980)



Staphylococcus aureus Bac+ NA, 24 h, 37°C 11, sd 30 Prasad et al. (1986) 


Staphylococcus sp. Bac+ NA, 24 h, 37°C 11, sd 26 Prasad et al. (1986) 

Streptococcus faecalis Bac+ ISA, 48 h, 25°C 4 (h), 10,000 8.5 Deans and Ritchie (1987) 


















Brettanomyces anomalus Yeast MPA, 4 d, 30°C 5, 10% sol. sd 18 Conner and Beuchat (1984) 







Candida lipolytica Yeast MPA, 4 d, 30°C 5, 10% sol. sd 27 Conner and Beuchat (1984) 



continued





(mm) Ref. 


Debaryomyces hansenii Yeast MPA, 4 d, 30°C 5, 10% sol. sd 26 Conner and Beuchat (1984) 

Geotrichum candidum Yeast MPA, 4 d, 30°C 5, 10% sol. sd 19 Conner and Beuchat (1984) 

Hansenula anomala Yeast MPA, 4 d, 30°C 5, 10% sol. sd 19 Conner and Beuchat (1984) 

Kloeckera apiculata Yeast MPA, 4 d, 30°C 5, 10% sol. sd 21 Conner and Beuchat (1984) 

Kluyveromyces fragilis Yeast MPA, 4 d, 30°C 5, 10% sol. sd 14 Conner and Beuchat (1984) 

Lodderomyces elongisporus Yeast MPA, 4 d, 30°C 5, 10% sol. sd 15 Conner and Beuchat (1984) 

Metchnikowia pulcherrima Yeast MPA, 4 d, 30°C 5, 10% sol. sd 29 Conner and Beuchat (1984) 

Pichia membranaefaciens Yeast MPA, 4 d, 30°C 5, 10% sol. sd 13 Conner and Beuchat (1984) 

Rhodotorula rubra Yeast MPA, 4 d, 30°C 5, 10% sol. sd 18 Conner and Beuchat (1984) 


Saccharomyces cerevisiae Yeast MPA, 4 d, 30°C 5, 10% sol. sd 19 Conner and Beuchat (1984) 



Torula glabrata Yeast MPA, 4 d, 30°C 5, 10% sol. sd 15 Conner and Beuchat (1984) 



TABLE 12.26 

Inhibitory Data of Clove Oil Obtained in the Dilution Test 


Acinetobacter baumannii Bac- MHA, Tween 20, 48 h, 35°C 2500 Hammer et al. (1999) 

Brucella abortus Bac- Cited 250 Okazaki and Oshima (1952) 

Brucella melitensis Bac- Cited 500 Okazaki and Oshima (1952) 

Brucella suis Bac- Cited 15 Okazaki and Oshima (1952) 


Escherichia coli Bac- LA, 18 h, 37°C 500–1000 Remmal et al. (1993) 


Escherichia coli Bac- Cited 500 Okazaki and Oshima (1952) 

Escherichia coli Bac- MPB, DMSO, 40 h, 30°C 74% inh. 500 Hili et al. (1997) 

Escherichia coli Bac- TGB, 18–24 h, 37°C 1000 Morris et al. (1979) 


Escherichia coli Bac- MHA, Tween 20, 48 h, 35°C 2500 Hammer et al. (1999) 



Klebsiella pneumoniae Bac- Cited >500 Okazaki and Oshima (1952) 

Klebsiella pneumoniae Bac- MHA, Tween 20, 48 h, 35°C 2500 Hammer et al. (1999) 

Proteus vulgaris Bac- Cited 250 Okazaki and Oshima (1952) 

Pseudomonas aeruginosa Bac- Cited >500 Okazaki and Oshima (1952) 

Pseudomonas aeruginosa Bac- MPB, DMSO, 40 h, 30°C 75% inh. 500 Hili et al. (1997) 





Salmonella haddar Bac- LA, 18 h, 37°C 500 Remmal et al. (1993) 

Salmonella paratyphi A Bac- Cited 500 Okazaki and Oshima (1952) 

Salmonella paratyphi B Bac- Cited >500 Okazaki and Oshima (1952) 



Serratia marcescens Bac- NA, 1–3 d, 30°C 1500 Farag et al. (1989) 

continued




Serratia marcescens Bac- MHA, Tween 20, 48 h, 35°C 2500 Hammer et al. (1999) 

Shigella dysenteriae I Bac- Cited 500 Okazaki and Oshima (1952) 

Shigella dysenteriae II Bac- Cited 500 Okazaki and Oshima (1952) 

Bacillus megaterium Bac+ LA, 18 h, 37°C 500–1000 Remmal et al. (1993) 


Bacillus subtilis Bac+ NB, Tween 20, 18 h, 37°C 50,000 Yousef and Tawil (1980) 

Corynebacterium sp. Bac+ TGB, 18–24 h, 37°C 500 Morris et al. (1979) 

Enterococcus faecalis Bac+ MHA, Tween 20, 48 h, 35°C 5000 Hammer et al. (1999) 







Mycobacterium tuberculosis Bac+ Cited 125 Okazaki and Oshima (1952) 



Staphylococcus aureus Bac+ Cited 500 Okazaki and Oshima (1952) 

Staphylococcus aureus Bac+ TGB, 18–24 h, 37°C 500 Morris et al. (1979) 


Staphylococcus aureus Bac+ MPB, DMSO, 40 h, 30°C 83% inh. 500 Hili et al. (1997) 


Staphylococcus aureus Bac+ LA, 18 h, 37°C 1000 Remmal et al. (1993) 

Staphylococcus aureus Bac+ MHA, Tween 20, 48 h, 35°C 2500 Hammer et al. (1999) 


Staphylococcus citreus Bac+ Cited 500 Okazaki and Oshima (1952) 

Achorion gypseum Fungi Cited, 15 d 125 Okazaki and Oshima (1952) 

Alternaria alternata Fungi RPMI, 1.5% EtOH, 7 d, 30°C 156–312 Tullio et al. (2006) 

Alternaria citri Fungi PDA, 8 d, 22°C 500 Arras and Usai (2001) 

Aspergillus fl avus Fungi PDA, 8 h, 20°C, spore germ. inh. 50–100 Thompson (1986)



Aspergillus fl avus Fungi RPMI, 1.5% EtOH, 7 d, 30°C 2500 Tullio et al. (2006) 

Aspergillus fl avus var. columnaris Fungi RPMI, 1.5% EtOH, 7 d, 30°C 1250 Tullio et al. (2006) 

Aspergillus fumigatus Fungi RPMI, 1.5% EtOH, 7 d, 30°C 5000 Tullio et al. (2006) 

Aspergillus niger Fungi RPMI, 1.5% EtOH, 7 d, 30°C 2500 Tullio et al. (2006) 





Aspergillus parasiticus Fungi PDA, 8 h, 20°C, spore germ. inh. 50–100 Thompson (1986) 


Botrytis cinera Fungi PDA, 8 d, 22°C 500 Arras and Usai (2001) 

Cladosporium cladosporoides Fungi RPMI, 1.5% EtOH, 7 d, 30°C 78–156 Tullio et al. (2006) 


Epidermophyton fl occosum Fungi RPMI, 1.5% EtOH, 7 d, 30°C 125 Tullio et al. (2006) 

Epidermophyton fl occosum Fungi SA, Tween 80, 21 d, 20°C 10,000 Tullio et al. (2006) 



Penicillium digitatum Fungi PDA, 8 d, 22°C 500 Arras and Usai (2001) 

Penicillium frequentans Fungi RPMI, 1.5% EtOH, 7 d, 30°C 1250 Tullio et al. (2006) 

Penicillium italicum Fungi PDA, 8 d, 22°C 500 Arras and Usai (2001) 

continued




Penicillium lanosum Fungi RPMI, 1.5% EtOH, 7 d, 30°C 5000 Tullio et al. (2006) 









Rhizopus sp. Fungi RPMI, 1.5% EtOH, 7 d, 30°C >10,000 Tullio et al. (2006) 




Scopulariopsis brevicaulis Fungi RPMI, 1.5% EtOH, 7 d, 30°C 10,000 Tullio et al. (2006) 

Trichophyton asteroides Fungi Cited, 15 d 125 Okazaki and Oshima (1952) 

Trichophyton interdigitale Fungi Cited, 15 d 125 Okazaki and Oshima (1952) 

Trichophyton mentagrophytes Fungi RPMI, 1.5% EtOH, 7 d, 30°C 250–500 Tullio et al. (2006) 



TABLE 12.27 

Inhibitory Data of Clove Oil Obtained in the Vapor Phase Test 

Microorganism 

MO 

Class Conditions Activity Ref. 




Salmonella typhi Bac- NA, 24 h, 37°C sd ++ Maruzzella and Sicurella 

(1960) 


Bacillus subtilis var. 

aterrimus 

Corynebacterium 

diphtheriae 

Bac+ NA, 24 h, 37°C sd +++ Maruzzella and Sicurella 

(1960) 

Bac+ NA, 24 h, 37°C ~20,000 NG Kellner and Kober (1954) 

Mycobacterium avium Bac+ NA, 24 h, 37°C sd + Maruzzella and Sicurella 

(1960) 


Staphylococcus aureus Bac+ NA, 24 h, 37°C sd +++ Maruzzella and Sicurella 

(1960) 


Streptococcus faecalis Bac+ NA, 24 h, 37°C sd +++ Maruzzella and Sicurella 

(1960) 


Alternaria alternata Fungi RPMI, 7 d, 30°C MIC air 312–625 Tullio et al. (2006) 


Aspergillus fl avus Fungi RPMI, 7 d, 30°C MIC air 625–1250 Tullio et al. (2006) 

Aspergillus fumigatus Fungi RPMI, 7 d, 30°C MIC air 625–1250 Tullio et al. (2006) 


Aspergillus niger Fungi RPMI, 7 d, 30°C MIC air 625–1250 Tullio et al. (2006) 

Cladosporium 

Fungi RPMI, 7 d, 30°C MIC air 78–156 Tullio et al. (2006) 

cladosporoides 





Fusarium oxysporum Fungi RPMI, 7 d, 30°C MIC air 312 Tullio et al. (2006) 

Microsporum canis Fungi RPMI, 7 d, 30°C MIC air 312–1250 Tullio et al. (2006) 

Microsporum gypseum Fungi RPMI, 7 d, 30°C MIC air 156–312 Tullio et al. (2006) 

Mucor sp. Fungi RPMI, 7 d, 30°C MIC air 625 Tullio et al. (2006) 


Penicillium frequentans Fungi RPMI, 7 d, 30°C MIC air >10,000 Tullio et al. (2006) 

Penicillium lanosum Fungi RPMI, 7 d, 30°C MIC air >10,000 Tullio et al. (2006) 

Rhizopus sp. Fungi RPMI, 7 d, 30°C MIC air 125 Tullio et al. (2006) 

Scopulariopsis 


brevicaulis 

Trichophyton 


mentagrophytes 



TABLE 12.28 

Inhibitory Data of Coriander Oil Obtained in the Agar Diffusion Test 


Inhibition 





Agrobacterium tumefaciens Bac- WA, 48 h, 25°C 6, 8000 MIA 435 Cantore et al. (2004) 



Burkholderia gladioli pv. agaricicola Bac- WA, 48 h, 25°C 6, 8000 MIA 3480 Cantore et al. (2004) 



Enterobacter aerogenes Bac- MHA, 48 h, 27°C 6, 15,000 12 Ertürk et al. (2006) 


Erwinia carotovora subsp. atroseptica Bac- WA, 48 h, 25°C 6, 8000 MIA 435 Cantore et al. (2004) 

Erwinia carotovora subsp. carotovora Bac- WA, 48 h, 25°C 6, 8000 MIA 435 Cantore et al. (2004) 







Escherichia coli Bac- MHA, 48 h, 27°C 6, 15,000 20 Ertürk et al. (2006) 

Escherichia coli Bac- WA, 48 h, 25°C 6, 8000 MIA 870 Cantore et al. (2004) 







Pseudomonas aeruginosa Bac- NA, 18 h, 37°C 6 (h), pure 0 Yousef and Tawil (1980)






Pseudomonas aeruginosa Bac- MHA, 48 h, 27°C 6, 15,000 12 Ertürk et al. (2006) 

Pseudomonas agarici Bac- KBA, 48 h, 25°C 6, 8000 MIA 3480 Cantore et al. (2004) 

Pseudomonas chichorii Bac- KBA, 48 h, 25°C 6, 8000 MIA 6960 Cantore et al. (2004) 

Pseudomonas corrugate Bac- KBA, 48 h, 25°C 6, 8000 MIA 3480 Cantore et al. (2004) 

Pseudomonas reactans Bac- KBA, 48 h, 25°C 6, 8000 MIA >6960 Cantore et al. (2004) 

Pseudomonas syringae pv. apata Bac- KBA, 48 h, 25°C 6, 8000 MIA 3480 Cantore et al. (2004) 

Pseudomonas syringae pv. atrofaciens Bac- KBA, 48 h, 25°C 6, 8000 MIA 6960 Cantore et al. (2004) 

Pseudomonas syringae pv. glycinea Bac- KBA, 48 h, 25°C 6, 8000 MIA 870 Cantore et al. (2004) 

Pseudomonas syringae pv. lachrymans Bac- KBA, 48 h, 25°C 6, 8000 MIA >6960 Cantore et al. (2004) 

Pseudomonas syringae pv. maculicola Bac- KBA, 48 h, 25°C 6, 8000 MIA 870 Cantore et al. (2004) 

Pseudomonas syringae pv. phaseolicola Bac- KBA, 48 h, 25°C 6, 8000 MIA 2610 Cantore et al. (2004) 

Pseudomonas syringae pv. pisi Bac- KBA, 48 h, 25°C 6, 8000 MIA 2610 Cantore et al. (2004) 

Pseudomonas syringae pv. syringae Bac- KBA, 48 h, 25°C 6, 8000 MIA 3480 Cantore et al. (2004) 

Pseudomonas syringae pv. tomato Bac- KBA, 48 h, 25°C 6, 8000 MIA 3480 Cantore et al. (2004) 

Pseudomonas tolaasii Bac- KBA, 48 h, 25°C 6, 8000 MIA >6960 Cantore et al. (2004) 

Pseudomonas viridifl ava Bac- KBA, 48 h, 25°C 6, 8000 MIA >6960 Cantore et al. (2004) 


Salmonella typhimurium Bac- MHA, 48 h, 27°C 6, 15,000 7 Ertürk et al. (2006) 


Serratia marcescens Bac- ISA, 48 h, 25°C 4 (h), 10,000 7.5 Deans and Ritchie (1987) 

Xanthomonas campestris pv. campestris Bac- WA, 48 h, 25°C 6, 8000 MIA 217 Cantore et al. (2004) 

Xanthomonas campestris pv. phaesoli 

var. fuscans 

Bac- WA, 48 h, 25°C 6, 8000 MIA 217 Cantore et al. (2004) 

Xanthomonas campestris pv. phaesoli 

Bac- WA, 48 h, 25°C 6, 8000 MIA 217 Cantore et al. (2004) 

var. phaesoli 

Xanthomonas campestris pv. vesicatoria Bac- WA, 48 h, 25°C 6, 8000 MIA 217 Cantore et al. (2004) 


continued




Inhibition 


Bacillus megaterium Bac+ WA, 48 h, 25°C 6, 8000 MIA 435 Cantore et al. (2004) 








Clavibacter michiganensis subsp. 

michiganensis 

Bac+ WA, 48 h, 25°C 6, 8000 MIA 374 Cantore et al. (2004) 

Clavibacter michiganensis subsp. 


sepedonicus 



Curtobacterium fl accunfaciens pv. betae Bac+ WA, 48 h, 25°C 6, 8000 MIA 632 Cantore et al. (2004) 

Curtobacterium fl accunfaciens pv. 

fl accunfaciens 






Rhodococcus fascians Bac+ WA, 48 h, 25°C 6, 8000 MIA 435 Cantore et al. (2004) 







Staphylococcus aureus Bac+ Cited, 18 h, 37°C 6, 2500 11.3 Janssen et al. (1986)


Staphylococcus aureus Bac+ MHA, 48 h, 27°C 6, 15,000 18 Ertürk et al. (2006) 

Staphylococcus epidermidis Bac+ MHA, 48 h, 27°C 6, 15,000 10 Ertürk et al. (2006) 





Alternaria sp. Fungi PDA, 18 h, 37°C 6, sd 9.5 Sharma and Singh (1979) 

Aspergillus candidus Fungi PDA, 18 h, 37°C 6, sd 0 Sharma and Singh (1979) 






Aspergillus niger Fungi MHA, 48 h, 27°C 6, 15,000 0 Ertürk et al. (2006) 




Cladosporium herbarum Fungi PDA, 18 h, 37°C 6, sd 21.5 Sharma and Singh (1979) 



Fusarium oxysporum f.sp. cicer Fungi PDA, 72 h, 28°C 5, 5000 11.5 Pawar and Thaker (2007) 

Helminthosporium sacchari Fungi PDA, 18 h, 37°C 6, sd 13 Sharma and Singh (1979) 


Microsporum gypseum Fungi PDA, 18 h, 37°C 6, sd 8 Sharma and Singh (1979) 





Penicillium chrysogenum Fungi SDA, 8 d, 30°°C 6 (h), pure 60 Yousef and Tawil (1980) 





continued




Inhibition 








Candida albicans Yeast MHA, 48 h, 27°C 6, 15,000 10 Ertürk et al. (2006) 

























TABLE 12.29 

Inhibitory Data of Coriander Oil Obtained in the Dilution Test 

Microorganism 

MO 

Class Conditions MIC Ref. 

Enterobacter aerogenes Bac- MHB, 24 h, 37°C 4315 Ertürk et al. (2006) 

Escherichia coli Bac- MPB, DMSO, 40 h, 30°C >500 Hili et al. (1997) 

Escherichia coli Bac- TGB, 18–24 h, 37°C >1000 Morris et al. (1979) 

Escherichia coli Bac- MHB, 24 h, 37°C 2150 Ertürk et al. (2006) 




Pseudomonas aeruginosa Bac- MHB, 24 h, 37°C 4350 Ertürk et al. (2006) 



Salmonella typhimurium Bac- MHB, 24 h, 37°C 17,260 Ertürk et al. (2006) 



Listeria monocytogenes Bac+ BHI, 48 h, 35°C >8000 Oussalah et al. (2006) 




Staphylococcus aureus Bac+ MHB, 24 h, 37°C 1070 Ertürk et al. (2006) 




Staphylococcus 

Bac+ MHB, 24 h, 37°C 8630 Ertürk et al. (2006) 

epidermidis 

Aspergillus flavus Fungi PDA, 8 h, 20°C, spore germ. 50–100 Thompson (1986) 

inh. 


Aspergillus fl avus Fungi CDA, cited 3000 Dubey et al. (1990) 


Aspergillus niger Fungi MHB, 24 h, 37°C >20,000 Ertürk et al. (2006) 

Aspergillus parasiticus Fungi PDA, 8 h, 20°C, spore germ. 50–100 Thompson (1986) 

inh. 


Epidermophyton 

Fungi SA, Tween 80, 21 d, 20°C 1000 Thompson and Cannon (1986) 


Mucor racemosus f. Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon (1986) 

racemosus 







continued



Inhibitory Data of Coriander Oil Obtained in the Dilution Test 

Microorganism 

MO 

Class Conditions MIC Ref. 








Trichophyton 

Fungi SA, Tween 80, 21 d, 20°C 300,625 Janssen et al. (1988) 


Trichophyton rubrum Fungi SA, Tween 80, 21 d, 20°C 1000 Morris et al. (1979) 

Candida albicans Yeast MHB, Tween 80, 48 h, 35°C 2500 Hammer et al. (1998) 

Candida albicans Yeast MHB, 24 h, 37°C 4310 Ertürk et al. (2006) 

Saccharomyces cerevisiae Yeast MPB, DMSO, 40 h, 30°C 68% inh. 500 Hili et al. (1997) 

Schizosaccharomyces Yeast MPB, DMSO, 40 h, 30°C 18% inh. 500 Hili et al. (1997) 

pombe 

Torula utilis Yeast MPB, DMSO, 40 h, 30°C 87% inh. 500 Hili et al. (1997) 

TABLE 12.30 

Inhibitory Data of Coriander Oil Obtained in the Vapor Phase Test 



Escherichia coli Bac- BLA, 18 h, 37°C MIC air 250 Inouye et al. (2001) 




Salmonella typhi Bac- NA, 24 h, 37°C sd ++ Maruzzella and Sicurella (1960) 







Staphylococcus aureus Bac+ MHA, 18 h, 37°C MIC air 50 Inouye et al. (2001) 



Streptococcus pneumoniae Bac+ MHA, 18 h, 37°C MIC air 25 Inouye et al. (2001) 


Streptococcus pyogenes Bac+ MHA, 18 h, 37°C MIC air 25 Inouye et al. (2001) 



TABLE 12.31 

Inhibitory Data of Dwarf Pine Oil Obtained in the Agar Diffusion Test 

Microorganism 

MO 

Class 

Conditions 

Inhibition 

Zone 

(mm) 

Ref. 



















Helminthosporium 

Fungi SMA, 2–7 d, 20°C sd 0 Maruzzella and Liguori (1958) 

sativum 









Cryptococcus 

Yeast SMA, 2–7 d, 20°C sd 0 Maruzzella and Liguori (1958) 

neoformans 

Cryptococcus 


rhodobenhani 

Saccharomyces 

cerevisiae 


TABLE 12.32 

Inhibitory Data of Dwarf Pine Oil Obtained in the Dilution Test 


Epidermophyton fl occosum Fungi SA, Tween 80, 21 d, 20°C >1250 Janssen et al. (1988) 

Trichophyton mentagrophytes Fungi SA, Tween 80, 21 d, 20°C >1250 Janssen et al. (1988) 

Trichophyton rubrum Fungi SA, Tween 80, 21 d, 20°C >1250 Janssen et al. (1988)


TABLE 12.33 

Inhibitory Data of Dwarf Pine Oil Obtained in the Vapor Phase Test 




Salmonella typhi Bac- NA, 24 h, 37°C ~20,000 +++ Kellner and Kober (1954) 






Candida albicans Yeast NA, 24 h, 37°C ~20,000 ++ Kellner and Kober (1954)


TABLE 12.34 

Inhibitory Data of Eucalyptus Oil Obtained in the Agar Diffusion Test 


Inhibition 






























continued




Inhibition 































Sarcina lutea Bac+ NA, 24 h, 37°C —, sd 4 Maruzzella and Lichtenstein (1956)



































Saccharomyces cerevisiae Yeast NA, 24 h, 20°C 5 (h), -30,000 16–21 Schelz et al. (2006) 

Saccharomyces cerevisiae Yeast SMA, 2–7 d, 20°C sd 8 Maruzzella and Liguori (1958)


TABLE 12.35 

Inhibitory Data of Eucalyptus Oil Obtained in the Dilution Test 


Campylobacter jejuni Bac- TSB, 24 h, 42°C >10,000 Smith-Palmer et al. (1998) 

Citrobacter freundii Bac- ISB, Tween 80, 20–24 h, 37°C 10,000 Harkenthal et al. (1999) 

Enterobacter aerogenes Bac- ISB, Tween 80, 20–24 h, 37°C 20,000 Harkenthal et al. (1999) 



Escherichia coli Bac- ISB, Tween 80, 20–24 h, 37°C >40,000 Harkenthal et al. (1999) 

Escherichia coli Bac- TGB, 18 h, 37°C 2800 Schelz et al. (2006) 


Klebsiella pneumoniae Bac- ISB, Tween 80, 20–24 h, 37°C 5000 Harkenthal et al. (1999) 

Proteus mirabilis Bac- ISB, Tween 80, 20–24 h, 37°C 20,000 Harkenthal et al. (1999) 

Pseudomonas aeruginosa Bac- ISB, Tween 80, 20–24 h, 37°C >40,000 Harkenthal et al. (1999) 


Salmonella choleraesuis Bac- ISB, Tween 80, 20–24 h, 37°C 20,000 Harkenthal et al. (1999) 


Shigella fl exneri Bac- ISB, Tween 80, 20–24 h, 37°C 2500 Harkenthal et al. (1999) 

Bacillus subtilis Bac+ ISB, Tween 80, 20–24 h, 37°C 10,000 Harkenthal et al. (1999) 


Corynebacterium pseudodiphtheriae Bac+ ISB, Tween 80, 20–24 h, 37°C 20,000 Harkenthal et al. (1999) 

Corynebacterium sp. Bac+ TGB, 18–24 h, 37°C >1000 Morris et al. (1979) 

Enterococcus durans Bac+ ISB, Tween 80, 20–24 h, 37°C 20,000 Harkenthal et al. (1999) 

Enterococcus faecalis Bac+ ISB, Tween 80, 20–24 h, 37°C 20,000 Harkenthal et al. (1999) 

Enterococcus faecium Bac+ ISB, Tween 80, 20–24 h, 37°C 20,000 Harkenthal et al. (1999) 

Listeria monocytogenes Bac+ ISB, Tween 80, 20–24 h, 37°C 2500 Harkenthal et al. (1999) 



Staphylococcus aureus Bac+ TGB, 18–24 h, 37°C >1000 Morris et al. (1979)



Staphylococcus aureus Bac+ ISB, Tween 80, 20–24 h, 37°C 20,000 Harkenthal et al. (1999) 


Staphylococcus epidermidis Bac+ TGB, 18 h, 37°C 2800 Schelz et al. (2006) 

Staphylococcus epidermidis Bac+ ISB, Tween 80, 20–24 h, 37°C 5000 Harkenthal et al. (1999) 

Staphylococcus saprophyticus Bac+ ISB, Tween 80, 20–24 h, 37°C 5000 Harkenthal et al. (1999) 

Staphylococcus xylosus Bac+ ISB, Tween 80, 20–24 h, 37°C 5000 Harkenthal et al. (1999) 


Aspergillus niger Fungi NB, Tween 20, 8 d, 30°C 50,000 Yousef and Tawil (1980) 


Aspergillus ochraceus Fungi YES broth, 10 d - 61% inh. 10,000 Lis-Balchin et al. (1998) 




Fusarium culmorum Fungi YES broth, 10 d - 78% inh. 10,000 Lis-Balchin et al. (1998) 


Mucor racemosus Fungi Cited >500 Okazaki and Oshima (1953) 

Mucor sp. Fungi NB, Tween 20, 8 d, 30°C 12,500 Yousef and Tawil (1980) 







Trichophyton rubrum Fungi SA, Tween 80, 21 d, 20°C 625–1250 Janssen et al. (1988) 

Candida albicans Yeast TGB, 18–24 h, 37°C 1000 Morris et al. (1979) 




TABLE 12.36 

Inhibitory Data of Eucalyptus Oil Obtained in the Vapor Phase Test 













Streptococcus faecalis Bac+ NA, 24 h, 37°C sd ++ Maruzzella and Sicurella (1960) 









TABLE 12.37 

Inhibitory Data of Juniper Oil Obtained in the Agar Diffusion Test 



(mm) Ref. 
























Aspergillus fl avus Fungi SDA, 72 h, 26°C 8, 25,000 0 Shin (2003) 


Aspergillus niger Fungi SDA, 72 h, 26°C 8, 25,000 0 Shin (2003) 

continued





(mm) Ref. 



Botrytis cinera Fungi PDA, few days, 24°C 12.7, sd 0 Angioni et al. (2003) 

Cercospora beticola Fungi PDA, few days, 24°C 12.7, sd 0 Angioni et al. (2003) 

Fusarium graminearum Fungi PDA, few days, 24°C 12.7, sd 0 Angioni et al. (2003) 


Fusarium oxysporum lycopersici Fungi PDA, few days, 24°C 12.7, sd 0 Angioni et al. (2003) 

Helminthosporium oryzae Fungi PDA, few days, 24°C 12.7, sd 0 Angioni et al. (2003) 





Phytophthora capsici Fungi PDA, few days, 24°C 12.7, sd 0 Angioni et al. (2003) 

Pyricularia oryzae Fungi PDA, few days, 24°C 12.7, sd 0 Angioni et al. (2003) 

Pythium ultimum Fungi PDA, few days, 24°C 12.7, sd 0 Angioni et al. (2003) 

Rhizoctonia solani Fungi PDA, few days, 24°C 12.7, sd 0 Angioni et al. (2003) 


Sclerotium rolfsii Fungi PDA, few days, 24°C 12.7, sd 0 Angioni et al. (2003) 

Septoria tritici Fungi PDA, few days, 24°C 12.7, sd 0 Angioni et al. (2003) 










TABLE 12.38 

Inhibitory Data of Juniper Oil Obtained in the Dilution Test 


Escherichia coli Bac- NB, DMSO, 24 h, 37°C >900 Angioni et al. (2003) 

Escherichia coli Bac- NB, 24 h, 37°C >900 Cosentino et al. (2003) 


Escherichia coli O157:H7 Bac- NB, 24 h, 37°C >900 Cosentino et al. (2003) 

Pseudomonas aeruginosa Bac- NB, DMSO, 24 h, 37°C >900 Angioni et al. (2003) 

Pseudomonas aeruginosa Bac- NB, 24 h, 37°C >900 Cosentino et al. (2003) 

Salmonella typhimurium Bac- NB, 24 h, 37°C >900 Cosentino et al. (2003) 

Yersinia enterocolitica Bac- NB, 24 h, 37°C >900 Cosentino et al. (2003) 

Yersinia enterocolitica Bac- MHA, Tween 20, 24 h, 37°C 2500 Rossi et al. (2007) a 

Bacillus cereus Bac+ NB, 24 h, 37°C >900 Cosentino et al. (2003) 

Enterococcus faecalis Bac+ NB, 24 h, 37°C >900 Cosentino et al. (2003) 

Enterococcus faecium VRE Bac+ HIB, Tween 80, 18 h, 37°C >20,000 Nelson (1997) 

Listeria monocytogenes Bac+ NB, 24 h, 37°C >900 Cosentino et al. (2003) 

Staphylococcus aureus Bac+ NB, DMSO, 24 h, 37°C >900 Angioni et al. (2003) 

Staphylococcus aureus Bac+ NB, 24 h, 37°C >900 Cosentino et al. (2003) 

Staphylococcus aureus MRSA Bac+ HIB, Tween 80, 18 h, 37°C >20,000 Nelson (1997) 

Staphylococcus epidermidis Bac+ TGB, 18 h, 37°C >11,300 Schelz et al. (2006) 

Alternaria alternate Fungi SDA, 6–8 h, 20°C 500, 13% inh. Dikshit et al. (1986) 

Aspergillus fl avus Fungi MYB, 72 h, 26°C >25,000 Shin (2003) 

Aspergillus fl avus Fungi RPMI, 24 h, 37°C >900 Cosentino et al. (2003) 

Aspergillus fl avus Fungi RPMI, DMSO, 72 h, 37°C 20,000 Cavaleiro et al. (2006) a 

Aspergillus fl avus Fungi SDA, 6–8 h, 20°C 500, 11% inh. Dikshit et al. (1986) 

Aspergillus fumigatus Fungi RPMI, DMSO, 72 h, 37°C 10,000 Cavaleiro et al. (2006) a 

continued




Aspergillus niger Fungi MYB, 72 h, 26°C >25,000 Shin (2003) 

Aspergillus niger Fungi RPMI, DMSO, 72 h, 37°C 10,000–20,000 Cavaleiro et al. (2006) a 


Epidermophyton fl occosum Fungi RPMI, DMSO, 72 h, 37°C 1250 Cavaleiro et al. (2006) a 

Microsporum canis Fungi RPMI, DMSO, 72 h, 37°C 1250 Cavaleiro et al. (2006) a 

Microsporum gypseum Fungi RPMI, DMSO, 72 h, 37°C 2500 Cavaleiro et al. (2006) a 

Microsporum gypseum Fungi SDA, 6–8 h, 20°C 500, 48% inh. Dikshit et al. (1986) 

Penicillium italicum Fungi SDA, 6–8 h, 20°C 500, 20% inh. Dikshit et al. (1986) 


Trichophyton mentagrophytes Fungi RPMI, DMSO, 72 h, 37°C 1250 Cavaleiro et al. (2006) a 

Trichophyton mentagrophytes Fungi SDA, 6–8 h, 20°C 500, 48% inh. Dikshit et al. (1986) 

Trichophyton rubrum Fungi SA, Tween 80, 21 d, 20°C >1250 Janssen et al. (1988) 

Trichophyton rubrum Fungi RPMI, DMSO, 72 h, 37°C 1250 Cavaleiro et al. (2006) a 

Trichophyton rubrum Fungi SDA, 6–8 h, 20°C 500, 53% inh. Dikshit et al. (1986) 

Candida albicans Yeast NB, DMSO, 24 h, 30°C >900 Angioni et al. (2003) 

Candida albicans Yeast RPMI, DMSO, 24 h, 37°C 1250–10,000 Cavaleiro et al. (2006) a 

Candida glabrata Yeast RPMI, DMSO, 24 h, 37°C 5000 Cavaleiro et al. (2006) a 

Candida krusei Yeast RPMI, DMSO, 24 h, 37°C 5000 Cavaleiro et al. (2006) a 

Candida parapsilosis Yeast RPMI, DMSO, 24 h, 37°C 5000 Cavaleiro et al. (2006) a 

Candida tropicalis Yeast RPMI, DMSO, 24 h, 37°C 20,000 Cavaleiro et al. (2006) a 

Saccharomyces cerevisiae Yeast SB, 24 h, 37°C >900 Cosentino et al. (2003) 

Saccharomyces cerevisiae Yeast YPB, 24 h, 20°C 2700 Schelz et al. (2006) 

a 

Juniper communis ssp. alpine.


TABLE 12.39 

Inhibitory Data of Juniper Oil Obtained in the Vapor Phase Test 

















TABLE 12.40 

Inhibitory Data of Lavender Oil Obtained in the Agar Diffusion Test 


Inhibition 















Escherichia coli Bac- Cited, 24 h, 37°C —



Shigella fl exneri Bac- Cited, 24 h, 37°C —




Inhibition 





Staphylococcus epidermidis Bac+ MHA, cited 9, 20,000 14 Pellecuer et al. (1980) 

Streptococcus D Bac+ MHA, cited 9, 20,000 0–29 Pellecuer et al. (1980) 


Streptococcus micros Bac+ MHA, cited 9, 20,000 26 Pellecuer et al. (1980) 





















Candida albicans Yeast SDA, 18 h, 30°C 6 (h), pure 11.5 Yousef and Tawil (1980) 








TABLE 12.41 

Inhibitory Data of Lavender Oil Obtained in the Dilution Test 


Bordetella bronchiseptica Bac- Cited 2000 Pellecuer et al. (1976) 

Escherichia coli Bac- Cited 500 Pellecuer et al. (1976) 




Haemophilus infl uenza Bac- Cited 2000 Pellecuer et al. (1976) 

Klebsiella pneumoniae Bac- Cited 1000 Pellecuer et al. (1976) 

Moraxella glucidolytica Bac- Cited 2000 Pellecuer et al. (1976) 

Neisseria catarrhalis Bac- Cited 250 Pellecuer et al. (1976) 

Neisseria fl ava Bac- Cited 500 Pellecuer et al. (1976) 


Salmonella typhimurium Bac- TSB, 24 h, 37°C 10,000 Di Pasqua et al. (2005) 


Bacillus subtilis Bac+ Cited 1000 Pellecuer et al. (1976) 

Corynebacterium pseudodiphtheriae Bac+ Cited 1000 Pellecuer et al. (1976) 


Enterococcus faecium VRE Bac+ HIB, Tween 80, 18 h, 37°C 5000–10,000 Nelson (1997) 

Lactobacillus sp. Bac+ MRS, cited 5 Pellecuer et al. (1980) 

Micrococcus fl avus Bac+ Cited 1000 Pellecuer et al. (1976) 

Micrococcus luteus Bac+ MHB, cited 1.25–2.5 Pellecuer et al. (1980) 

Micrococcus ureae Bac+ MHB, cited 2.5 Pellecuer et al. (1980) 


Sarcina lutea Bac+ Cited 2000 Pellecuer et al. (1976) 

Sarcina ureae Bac+ MHB, cited 0.31–0.62 Pellecuer et al. (1980) 

Staphylococcus aureus Bac+ TGB, 18–24 h, 37°C >1000 Morris et al. (1979) 

Staphylococcus aureus Bac+ Cited 2000 Pellecuer et al. (1976) 

continued




Staphylococcus aureus Bac+ TSB, 3% EtOH, 24 h, 37°C 10,000 Rota et al. (2004) 


Staphylococcus aureus Bac+ MHB, Tween 80, 24 h, 37°C 12,500 Bastide et al. (1987) 

Staphylococcus aureus MRSA Bac+ HIB, Tween 80, 18 h, 37°C 5000 Nelson (1997) 

Staphylococcus epidermidis Bac+ MHB, cited 5 Pellecuer et al. (1980) 

Staphylococcus epidermidis Bac+ Cited 2000 Pellecuer et al. (1976) 

Streptococcus D Bac+ MHB, cited 0.62–5 Pellecuer et al. (1980) 

Streptococcus micros Bac+ MHB, cited >5 Pellecuer et al. (1980) 

Streptococcus pyogenes Bac+ Cited 2000 Pellecuer et al. (1976) 

Alternaria alternata Fungi RPMI, 1.5% EtOH, 7 d, 30°C 5000 Tullio et al. (2006) 

Aspergillus fl avus Fungi CA, 7 d, 28 5–8% inh. 500 Kumar et al. (2007) 

Aspergillus fl avus Fungi MYB, 72 h, 26°C 3120 Shin (2003) 

Aspergillus fl avus Fungi RPMI, 1.5% EtOH, 7 d, 30°C 10,000 Tullio et al. (2006) 

Aspergillus fl avus var. columnaris Fungi RPMI, 1.5% EtOH, 7 d, 30°C >10,000 Tullio et al. (2006) 

Aspergillus fumigatus Fungi RPMI, 1.5% EtOH, 7 d, 30°C 10,000 Tullio et al. (2006) 

Aspergillus niger Fungi Cited 1000 Pellecuer et al. (1976) 

Aspergillus niger Fungi MYB, 72 h, 26°C 3120 Shin (2003) 


Aspergillus niger Fungi RPMI, 1.5% EtOH, 7 d, 30°C 10,000 Tullio et al. (2006) 



Cladosporium cladosporoides Fungi RPMI, 1.5% EtOH, 7 d, 30°C 2500–10,000 Tullio et al. (2006) 



Fusarium oxysporum Fungi RPMI, 1.5% EtOH, 7 d, 30°C 156 Tullio et al. (2006) 

Microsporum canis Fungi RPMI, 1.5% EtOH, 7 d, 30°C 2500–5000 Tullio et al. (2006) 

Microsporum canis Fungi MBA, Tween 80, 10 d, 30°C 75– >300 Perrucci et al. (1994) 

Microsporum gypseum Fungi RPMI, 1.5% EtOH, 7 d, 30°C 5000–10,000 Tullio et al. (2006) 

Microsporum gypseum Fungi MBA, Tween 80, 10 d, 30°C 50– >300 Perrucci et al. (1994) 


Mucor sp. Fungi RPMI, 1.5% EtOH, 7 d, 30°C >10,000 Tullio et al. (2006) 




Penicillium lanosum Fungi RPMI, 1.5% EtOH, 7 d, 30°C 10,000 Tullio et al. (2006) 





Trichophyton interdigitale Fungi Cited 1000 Pellecuer et al. (1976) 

Trichophyton mentagrophytes Fungi RPMI, 1.5% EtOH, 7 d, 30°C 5000–10,000 Tullio et al. (2006) 


Trichophyton rubrum Fungi SA, Tween 80, 21 d, 20°C


TABLE 12.42 

Inhibitory Data of Lavender Oil Obtained in the Vapor Phase Test 



Escherichia coli Bac- BLA, 18 h, 37°C MIC air >1600 Inouye et al. (2001) 

Haemophilus infl uenzae Bac- MHA, 18 h, 37°C MIC air 25 Inouye et al. (2001) 







Lactobacillus sp. Bac+ MRS, cited Disk, 20,000? +++ Pellecuer et al. (1980) 

Micrococcus luteus Bac+ MHB, cited Disk, 20,000? +++ Pellecuer et al. (1980) 

Micrococcus ureae Bac+ MHB, cited Disk, 20,000? ++ Pellecuer et al. (1980) 


Sarcina ureae Bac+ MHB, cited Disk, 20,000? + Pellecuer et al. (1980) 




Staphylococcus epidermidis Bac+ MHB, cited Disk, 20,000? ++ Pellecuer et al. (1980) 

Streptococcus D Bac+ MHB, cited Disk, 20,000? +++ Pellecuer et al. (1980)




Streptococcus micros Bac+ MHB, cited Disk, 20,000? +++ Pellecuer et al. (1980) 




Alternaria alternata Fungi RPMI, 7 d, 30°C MIC air 625–2500 Tullio et al. (2006) 

Aspergillus fl avus Fungi RPMI, 7 d, 30°C MIC air 2500 Tullio et al. (2006) 


Aspergillus niger Fungi RPMI, 7 d, 30°C MIC air 1250 Tullio et al. (2006) 

Cladosporium cladosporoides Fungi RPMI, 7 d, 30°C MIC air 156–312 Tullio et al. (2006) 



Microsporum gypseum Fungi RPMI, 7 d, 30°C MIC air 312 Tullio et al. (2006) 


Penicillium frequentans Fungi RPMI, 7 d, 30°C MIC air 625 Tullio et al. (2006) 

Penicillium lanosum Fungi RPMI, 7 d, 30°C MIC air 625 Tullio et al. (2006) 


Scopulariopsis brevicaulis Fungi RPMI, 7 d, 30°C MIC air 125 Tullio et al. (2006) 

Trichophyton mentagrophytes Fungi RPMI, 7 d, 30°C MIC air 312–625 Tullio et al. (2006) 



TABLE 12.43 

Inhibitory Data of Lemon Oil Obtained in the Agar Diffusion Test 



(mm) Ref. 


Aerobacter aerogenes Bac- NA, 24 h, 37°C 5 × 20, 1000 1–10 Möse et al. (1957) 





Brucella abortus Bac- NA, 24 h, 37°C 5 × 20, 1000 2–5 Möse et al. (1957) 


Campylobacter jejuni Bac- CAB, 24 h, 42°C Disk, 10,000 41 Fisher and Phillips (2006) 




Escherichia coli Bac- NA, 24 h, 37°C 5 × 20, 1000 0–10 Möse et al. (1957) 


Escherichia coli Bac- NA, 24 h, 37°C 10 (h), 100,000 0 Narasimha Rao and Nigam (1970) 




Escherichia coli Bac- TSA, 24 h, 35°C 4 (h), 25,000 4 Smith-Palmer et al. (1998) 

Escherichia coli Bac- NA, 24 h, 37°C Disk, 10,000 21 Fisher and Phillips (2006) 


Flavobacterium suaveolens Bac- ISA, 48 h, 25°C 4 (h), 10,000 13.5 Deans and Ritchie (1987) 


Klebsiella pneumonia Bac- NA, 24 h, 37°C 5 × 20, 1000 6–10 Möse et al. (1957) 

Klebsiella pneumonia subsp. oceanae Bac- NA, 24 h, 37°C 5 × 20, 1000 2–5 Möse et al. (1957) 


Klebsiella sp. Bac- Cited 15, 2500 0 Pizsolitto et al. (1975)




Proteus OX19 Bac- NA, 24 h, 37°C 5 × 20, 1000 2–5 Möse et al. (1957) 


Proteus vulgaris Bac- NA, 24 h, 37°C 5 × 20, 1000 2–5 Möse et al. (1957) 







Pseudomonas aeruginosa Bac- NA, 24 h, 37°C 5 × 20, 1000 1 Möse et al. (1957) 

Pseudomonas fl uorescens Bac- NA, 24 h, 37°C 5 × 20, 1000 1 Möse et al. (1957) 


Salmonella enteritidis Bac- NA, 24 h, 37°C 5 × 20, 1000 6–10 Möse et al. (1957) 

Salmonella enteritidis Bac- TSA, 24 h, 35°C 4 (h), 25,000 4 Smith-Palmer et al. (1998) 


Salmonella paratyphi B Bac- NA, 24 h, 37°C 5 × 20, 1000 6–10 Möse et al. (1957) 



Salmonella typhi Bac- NA, 24 h, 37°C 5 × 20, 1000 2–5 Möse et al. (1957) 

Salmonella typhi Bac- NA, 24 h, 37°C 10 (h), 10,0000 0 Narasimha Rao and Nigam (1970) 




Serratia marcescens Bac- NA, 24 h, 37°C 5 × 20, 1000 1 Möse et al. (1957) 



Vibrio albicans Bac- NA, 24 h, 37°C 5 × 20, 1000 11–13 Möse et al. (1957) 


Vibrio cholerae Bac- NA, 24 h, 37°C 5 × 20, 1000 6–10 Möse et al. (1957) 

continued





(mm) Ref. 

Vibrio cholerae Bac- NA, 24 h, 37°C 10 (h), 10,0000 22 Narasimha Rao and Nigam (1970) 


Bacillus anthracis Bac+ NA, 24 h, 37°C 5 × 20, 1000 6–10 Möse et al. (1957) 

Bacillus cereus Bac+ BHA, 24 h, 30°C Disk, 10,000 19 Fisher and Phillips (2006) 





Bacillus subtilis Bac+ NA, 24 h, 37°C 5 × 20, 1000 1 Möse et al. (1957) 








Corynebacterium diphtheria Bac+ NA, 24 h, 37°C 5 × 20, 1000 1 Möse et al. (1957) 

Corynebacterium diphtheria Bac+ NA, 24 h, 37°C 10 (h), 10,0000 0 Narasimha Rao and Nigam (1970) 




Listeria monocytogenes Bac+ NA, 24 h, 37°C 5 × 20, 1000 2–5 Möse et al. (1957) 



Listeria monocytogenes Bac+ LSA, 24 h, 37°C Disk, 10,000 41 Fisher and Phillips (2006) 



Sarcina alba Bac+ NA, 24 h, 37°C 5 × 20, 1000 2–5 Möse et al. (1957) 

Sarcina beige Bac+ NA, 24 h, 37°C 5 × 20, 1000 6–10 Möse et al. (1957)


Sarcina citrea Bac+ NA, 24 h, 37°C 5 × 20, 1000 2–5 Möse et al. (1957) 



Sarcina rosa Bac+ NA, 24 h, 37°C 5 × 20, 1000 6–10 Möse et al. (1957) 

Sporococcus sarc. Bac+ NA, 24 h, 37°C 5 × 20, 1000 6–10 Möse et al. (1957) 

Staphylococcus albus Bac+ NA, 24 h, 37°C 5 × 20, 1000 6–10 Möse et al. (1957) 

Staphylococcus aureus Bac+ NA, 24 h, 37°C 5 × 20, 1000 11–15 Möse et al. (1957) 








Staphylococcus aureus Bac+ BHA, 24 h, 37°C Disk, 10,000 14 Fisher and Phillips (2006) 





Streptococcus haemolyticus Bac+ NA, 24 h, 37°C 5 × 20, 1000 6–10 Möse et al. (1957) 



Streptococcus viridians Bac+ NA, 24 h, 37°C 5 × 20, 1000 1 Möse et al. (1957) 










continued





(mm) Ref. 




























Torula glabrata Yeast MPA, 4 d, 30°C 5, 10% sol. sd 0 Conner and Beuchat (1984)


TABLE 12.44 

Inhibitory Data of Lemon Oil Obtained in the Dilution Test 


Aerobacter aerogenes Bac- NA, pH 7 >2000 Subba et al. (1967) 

Campylobacter jejuni Bac- CAB, 24 h, 42°C >40,000 Fisher and Phillips (2006) 


Escherichia coli Bac- NA, 24 h, 37°C 10,000 Fisher and Phillips (2006) 


Escherichia coli Bac- NB, 24–72 h, 37°C 98% inh. 10,000 Dabbah et al. (1970) 


Pseudomonas aeruginosa Bac- NB, 24–72 h, 37°C 90% inh. 10,000 Dabbah et al. (1970) 

Salmonella schottmuelleri Bac- NA, pH 7 >2000 Subba et al. (1967) 

Salmonella senftenberg Bac- NB, 24–72 h, 37°C 98% inh. 10,000 Dabbah et al. (1970) 

Serratia marcescens Bac- NA, pH 7 >2000 Subba et al. (1967) 

Bacillus cereus Bac+ BHA, 24 h, 30°C 10,000 Fisher and Phillips (2006) 

Bacillus subtilis Bac+ NA, pH 7 2000 Subba et al. (1967) 



Lactobacillus plantarum Bac+ NA, pH 7 1000 Subba et al. (1967) 

Listeria monocytogenes Bac+ LSA, 24 h, 37°C 2500 Fisher and Phillips (2006) 

Micrococcus sp. Bac+ NA, pH 7 2000 Subba et al. (1967) 


Staphylococcus aureus Bac+ BHA, 24 h, 37°C >40,000 Fisher and Phillips (2006) 

Staphylococcus aureus Bac+ NB, 24–72 h, 37°C 10,000 Dabbah et al. (1970) 



continued




Streptococcus faecalis Bac+ NA, pH 7 1000 Subba et al. (1967) 

Aspergillus awamorii Fungi PDA, pH 4.5 >2000 Subba et al. (1967) 


Aspergillus fl avus Fungi PDA, pH 4.5 >2000 Subba et al. (1967) 

Aspergillus fl avus Fungi PDA, 8 h, 20°C, spore germ. inh. 50–100 Thompson (1986) 

Aspergillus niger Fungi PDA, pH 4.5 >2000 Subba et al. (1967) 




Aspergillus oryzae Fungi Cited >500 Okazaki and Oshima (1953) 












Penicillium chrysogenum Fungi Cited 500 Okazaki and Oshima (1953)



Penicillium digitatum Fungi SDB, 5 d, 20°C, MIC = ED50 500–1000 Caccioni et al. (1998) 

Penicillium italicum Fungi SDB, 5 d, 20°C, MIC = ED50 1000–2500 Caccioni et al. (1998) 













Candida albicans Yeast MHB, Tween 80, 48 h, 35°C 20,000 Hammer et al. (1998) 



Saccharomyces cerevisiae Yeast PDA, pH 4.5 500 Subba et al. (1967) 

Torula utilis Yeast PDA, pH 4.5 1000 Subba et al. (1967) 

Zygosaccharomyces mellis Yeast PDA, pH 4.5 >2000 Subba et al. (1967)


TABLE 12.45 

Inhibitory Data of Lemon Oil Obtained in the Vapor Phase Test 


Campylobacter jejuni Bac- CAB, 24 h, 42°C Disk, 10,000 +++ Fisher and Phillips (2006) 


Escherichia coli Bac- NA, 24 h, 37°C Disk, 10,000 +++ Fisher and Phillips (2006) 





Bacillus cereus Bac+ BHA, 24 h, 30°C Disk, 10,000 +++ Fisher and Phillips (2006) 



Listeria monocytogenes Bac+ LSA, 24 h, 37°C Disk, 10,000 +++ Fisher and Phillips (2006) 


Staphylococcus aureus Bac+ BHA, 24 h, 37°C Disk, 10,000 +++ Fisher and Phillips (2006) 















Penicillium corylophilum Fungi WFA, 42 d, 25°C Disk, 50,000 ++ Guynot et al. (2003) 





TABLE 12.46 

Inhibitory Data of Mandarin Oil Obtained in the Agar Diffusion Test 



(mm) Ref. 







Campylobacter jejuni Bac- Cited 6, 15,000 11 Wannissorn et al. (2005) 








Escherichia coli Bac- Cited 6, 15,000 0 Wannissorn et al. (2005) 








continued





(mm) Ref. 





Salmonella enteritidis Bac- Cited 6, 15,000 0 Wannissorn et al. (2005) 



Salmonella typhimurium Bac- Cited 6, 15,000 9 Wannissorn et al. (2005) 









Clostridium perfringens Bac+ Cited 6, 15,000 35 Wannissorn et al. (2005) 




Leuconostoc cremoris Bac+ ISA, 48 h, 25°C 4 (h), 10,000 7 Deans and Ritchie (1987)




























TABLE 12.47 

Inhibitory Data of Mandarin Oil Obtained in the Dilution Test 





Salmonella senftenberg Bac- NB, 24–72 h, 37°C >10,000 Dabbah et al. (1970) 



Staphylococcus aureus Bac+ TGB, 18–24 h, 37°C >1000 Morris et al. (1979) 

Staphylococcus aureus Bac+ NB, 24–72 h, 37°C 10,000 Dabbah et al. (1970) 


Annotation: Terpeneless mandarin oil tested Maruzzella and Liguori (1958). 

TABLE 12.48 

Inhibitory Data of Mandarin Oil Obtained in the Vapor Phase Test 








Penicillium corylophilum Fungi WFA, 42 d, 25°C Disk, 50,000 + Guynot et al. (2003)


TABLE 12.49 

Inhibitory Data of Matricaria Oil Obtained in the Agar Diffusion Test 


Inhibition 



Aerobacter aerogenes Bac- NA, 24 h, 37°C 5 × 20, 1000 0 Möse et al. (1957) 




Brucella abortus Bac- NA, 24 h, 37°C 5 × 20, 1000 0 Möse et al. (1957) 






Escherichia coli Bac- NA, 24 h, 37°C 5 × 20, 1000 0 Möse et al. (1957) 



Klebsiella pneumonia Bac- NA, 24 h, 37°C 5 × 20, 1000 2–5 Möse et al. (1957) 

Klebsiella pneumonia subsp. Oceanae Bac- NA, 24 h, 37°C 5 × 20, 1000 2–5 Möse et al. (1957) 



Proteus OX19 Bac- NA, 24 h, 37°C 5 × 20, 1000 0 Möse et al. (1957) 

Proteus vulgaris Bac- NA, 24 h, 37°C 5 × 20, 1000 0 Möse et al. (1957) 



Pseudomonas aeruginosa Bac- NA, 24 h, 37°C 5 × 20, 1000 0 Möse et al. (1957) 


Pseudomonas fl uorescens Bac- NA, 24 h, 37°C 5 × 20, 1000 0 Möse et al. (1957) 

continued




Inhibition 


Salmonella enteritidis Bac- NA, 24 h, 37°C 5 × 20, 1000 2–5 Möse et al. (1957) 

Salmonella paratyphi B Bac- NA, 24 h, 37°C 5 × 20, 1000 0 Möse et al. (1957) 


Salmonella typhi Bac- NA, 24 h, 37°C 5 × 20, 1000 2–5 Möse et al. (1957) 

Serratia marcescens Bac- NA, 24 h, 37°C 5 × 20, 1000 0 Möse et al. (1957) 


Vibrio albicans Bac- NA, 24 h, 37°C 5 × 20, 1000 1 Möse et al. (1957) 

Vibrio cholerae Bac- NA, 24 h, 37°C 5 × 20, 1000 2–5 Möse et al. (1957) 


Bacillus anthracis Bac+ NA, 24 h, 37°C 5 × 20, 1000 2–5 Möse et al. (1957) 

Bacillus subtilis Bac+ NA, 24 h, 37°C 5 × 20, 1000 0 Möse et al. (1957) 






Corynebacterium diphtheria Bac+ NA, 24 h, 37°C 5 × 20, 1000 1 Möse et al. (1957) 





Listeria monocytogenes Bac+ NA, 24 h, 37°C 5 × 20, 1000 2–5 Möse et al. (1957) 

Micrococcus luteus Bac+ ISA, 48 h, 25°C 4 (h), 10,000 0 Deans and Ritchie (1987)


Mycobacterium phlei Bac+ NA, 18 h, 37°C 6 (h), pure 15.5 Yousef and Tawil (1980) 

Sarcina alba Bac+ NA, 24 h, 37°C 5 × 20, 1000 0 Möse et al. (1957) 

Sarcina beige Bac+ NA, 24 h, 37°C 5 × 20, 1000 6–10 Möse et al. (1957) 

Sarcina citrea Bac+ NA, 24 h, 37°C 5 × 20, 1000 0 Möse et al. (1957) 

Sarcina rosa Bac+ NA, 24 h, 37°C 5 × 20, 1000 6–10 Möse et al. (1957) 

Sporococcus sarc. Bac+ NA, 24 h, 37°C 5 × 20, 1000 6–10 Möse et al. (1957) 

Staphylococcus albus Bac+ NA, 24 h, 37°C 5 × 20, 1000 2–5 Möse et al. (1957) 



Staphylococcus aureus Bac+ NA, 24 h, 37°C 5 × 20, 1000 2–5 Möse et al. (1957) 



Streptococcus haemolyticus Bac+ NA, 24 h, 37°C 5 × 20, 1000 2–5 Möse et al. (1957) 

Streptococcus viridians Bac+ NA, 24 h, 37°C 5 × 20, 1000 2–5 Möse et al. (1957) 









Candida albicans Yeast SDA, 18 h, 30°C 6 (h), pure 9.5 Yousef and Tawil (1980)


TABLE 12.50 

Inhibitory Data of Matricaria Oil Obtained in the Dilution Test 



Escherichia coli Bac- NB, Tween 80, 24 h, 37°C >8000 Aggag and Yousef (1972) 

Escherichia coli Bac- Agar, cited 10,000 Kedzia et al. (1991) 

Escherichia coli Bac- NB, Tween 20, 18 h, 37°C >50,000 Yousef and Tawil (1980) 

Helicobacter pylori Bac- Cited, 20 h, 37°C 35.7–70.4 Weseler et al. (2005) 

Klebsiella pneumoniae Bac- Agar, cited 10,000 Kedzia et al. (1991) 

Pseudomonas aeruginosa Bac- Agar, cited 7500 Kedzia et al. (1991) 



Bacillus subtilis Bac+ NB, Tween 80, 24 h, 37°C 6000 Aggag and Yousef (1972) 



Pseudomonas aeruginosa Bac+ NB, Tween 80, 24 h, 37°C >8000 Aggag and Yousef (1972) 


Staphylococcus aureus Bac+ Agar, cited 2500 Kedzia et al. (1991) 

Staphylococcus aureus Bac+ NB, Tween 80, 24 h, 37°C 7000 Aggag and Yousef (1972) 


Streptococcus faecalis Bac+ Agar, cited 2500 Kedzia et al. (1991)


Aspergillus fl avus Fungi PDA, 7–14 d, 28°C >3000 Soliman and Badeaa (2002) 

Aspergillus niger Fungi YES broth, 10 d -63% inh. 10,000 Lis-Balchin et al. (1998) 


Aspergillus ochraceus Fungi PDA, 7–14 d, 28°C >3000 Soliman and Badeaa (2002) 

Aspergillus ochraceus Fungi YES broth, 10 d -56% inh. 10,000 Lis-Balchin et al. (1998) 

Aspergillus parasiticus Fungi PDA, 7–14 d, 28°C >3000 Soliman and Badeaa (2002) 


Fusarium culmorum Fungi YES broth, 10 d -75% inh. 10,000 Lis-Balchin et al. (1998) 

Fusarium moniliforme Fungi PDA, 7–14 d, 28°C >3000 Soliman and Badeaa (2002) 

Microsporum gypseum Fungi Agar, cited 1000 Kedzia et al. (1991) 





Trichophyton mentagrophytes Fungi SDA, 21 d, 20°C 1000 Szalontai et al. (1977) 

Trichophyton mentagrophytes Fungi SDA, 21 d, 20°C 1000 Szalontai et al. (1977) 


Candida albicans Yeast SDA, 7 d, 37°C 1000 Szalontai et al. (1977) 

Candida albicans Yeast Agar, cited 5000 Kedzia et al. (1991) 

Candida albicans Yeast NB, Tween 80, 24 h, 37°C 7000 Aggag and Yousef (1972) 

Candida albicans Yeast NB, Tween 20, 18 h, 37°C 50,000 Yousef and Tawil (1980)


TABLE 12.51 

Inhibitory Data of Matricaria Oil Obtained in the Vapor Phase Test 



Neisseria sp. Bac- NA, 24 h, 37°C ~20,000 +++ Kellner and Kober (1954) 


Bacillus megaterium Bac+ NA, 24 h, 37°C ~20,000 ++ Kellner and Kober (1954) 



Streptococcus faecalis Bac+ NA, 24 h, 37°C ~20,000 ++ Kellner and Kober (1954) 

Streptococcus pyogenes Bac+ NA, 24 h, 37°C ~20,000 + Kellner and Kober (1954) 






Candida albicans Yeast NA, 24 h, 37°C ~20,000 +++ Kellner and Kober (1954)


TABLE 12.52 

Inhibitory Data of Mint Oil Obtained in the Agar Diffusion Test 


Inhibition 





Beneckea natriegens Bac- ISA, 48 h, 25°C 4 (h), 10,000 9.5 Deans and Ritchie (1987) 

Campylobacter jejuni Bac- Cited 6, 15,000 90 Wannissorn et al. (2005) 






Escherichia coli Bac- Cited 6, 15,000 14.5 Wannissorn et al. (2005) 

Escherichia coli O157 Bac- Cited 6, 15,000 13.5 Wannissorn et al. (2005) 









Salmonella agona Bac- Cited 6, 15,000 12 Wannissorn et al. (2005) 

Salmonella braenderup Bac- Cited 6, 15,000 13 Wannissorn et al. (2005) 

Salmonella derby Bac- Cited 6, 15,000 10 Wannissorn et al. (2005) 

Salmonella enteritidis Bac- Cited 6, 15,000 13.5 Wannissorn et al. (2005) 

Salmonella gallinarum Bac- Cited 6, 15,000 52.5 Wannissorn et al. (2005) 

continued




Inhibition 


Salmonella hadar Bac- Cited 6, 15,000 13.5 Wannissorn et al. (2005) 

Salmonella mbandaka Bac- Cited 6, 15,000 11.5 Wannissorn et al. (2005) 

Salmonella montevideo Bac- Cited 6, 15,000 12 Wannissorn et al. (2005) 


Salmonella saintpaul Bac- Cited 6, 15,000 12 Wannissorn et al. (2005) 

Salmonella schwarzergrund Bac- Cited 6, 15,000 11.5 Wannissorn et al. (2005) 

Salmonella senftenberg Bac- Cited 6, 15,000 12 Wannissorn et al. (2005) 


Salmonella typhimurium Bac- Cited 6, 15,000 47.5 Wannissorn et al. (2005) 









Clostridium perfringens Bac+ Cited 6, 15,000 90 Wannissorn et al. (2005) 


Lactobacillus plantarum Bac+ ISA, 48 h, 25°C 4 (h), 10,000 9 Deans and Ritchie (1987)



Micrococcus luteus Bac+ ISA, 48 h, 25°C 4 (h), 10,000 8.5 Deans and Ritchie (1987) 






Absidia corMYBifera Fungi EYA, 48 h, 45°C 5, sd 10 Nigam and Rao (1979) 

Aspergillus fl avus Fungi PDA, 10 d, 25°C 50 NG Sarbhoy et al. (1978) 

Aspergillus fumigatus Fungi PDA, 10 d, 25°C 50 NG Sarbhoy et al. (1978) 

Aspergillus sulphureus Fungi PDA, 10 d, 25°C 50 NG Sarbhoy et al. (1978) 


Mucor fragilis Fungi PDA, 10 d, 25°C 50 NG Sarbhoy et al. (1978) 

Rhizopus stolonifer Fungi PDA, 10 d, 25°C 50 NG Sarbhoy et al. (1978) 







TABLE 12.53 

Inhibitory Data of Mint Oil Obtained in the Dilution Test 


Escherichia coli Bac- Cited, 24 h 400 Imai et al. (2001) 

Helicobacter pylori Bac- Cited, 48 h 100 Imai et al. (2001) 

Salmonella enteritidis Bac- Cited, 24 h 800 Imai et al. (2001) 


Staphylococcus aureus MRSA Bac+ Cited, 24 h 400 Imai et al. (2001) 


Staphylococcus aureus MSSA Bac+ Cited, 24 h 400 Imai et al. (2001) 

Alternaria alternate Fungi SDA, 6–8 h, 20°C 63% inh. 500 Dikshit et al. (1986) 

Aspergillus fl avus Fungi CA, 7 d, 28°C 500 Kumar et al. (2007) 

Aspergillus fl avus Fungi Cited 1000 Kumar et al. (2007) 

Aspergillus fl avus Fungi SDA, 6–8 h, 20°C 65% inh. 500 Dikshit et al. (1986) 

Aspergillus niger Fungi CA, 7 d, 28°C 81% inh. 500 Kumar et al. (2007) 

Botryodiplodia theobromae Fungi CA, 7 d, 28°C 87% inh. 500 Kumar et al. (2007) 

Cladosporium cladosporoides Fungi CA, 7 d, 28°C >500 Kumar et al. (2007) 

Fusarium oxysporum Fungi CA, 7 d, 28°C 83% inh. 500 Kumar et al. (2007) 

Helminthosporium oryzae Fungi CA, 7 d, 28°C 500 Kumar et al. (2007) 

Helminthosporium oryzae Fungi MA, cited a 2000 Dikshit et al. (1979) 

Helminthosporium oryzae Fungi MA, pH 5.0, 6 d, 28°C a,b 2000 Dikshit et al. (1982) 

Helminthosporium oryzae Fungi MA, pH 4.0, 6 d, 28°C a,b 500 Dikshit et al. (1982) 

Macrophomina phaesoli Fungi CA, 7 d, 28°C 94% inh. 500 Kumar et al. (2007) 

Microsporum gypseum Fungi SDA, 6–8 h, 20°C 64% inh. 500 Dikshit et al. (1986) 

Penicillium italicum Fungi SDA, 6–8 h, 20°C 65% inh. 500 Dikshit et al. (1986) 

Sclerotium rolfsii Fungi CA, 7 d, 28°C 500 Kumar et al. (2007) 

Trichophyton mentagrophytes Fungi SDA, 6–8 h, 20°C 69% inh. 500 Dikshit et al. (1986) 

Trichophyton rubrum Fungi SDA, 6–8 h, 20°C 36% inh. 500 Dikshit et al. (1986) 

Candida albicans Yeast Cited, 48 h, 36°C a 1100 Duarte et al. (2005) 

a 

Mentha arvensis var. piperascens. 

b 

Dementholized oil.


TABLE 12.54 

Inhibitory Data of Neroli Oil Obtained in the Agar Diffusion Test 



(mm) Ref. 

Aerobacter aerogenes Bac- NA, 24 h, 37°C —, sd 0 Imai et al. (2001) 

Escherichia coli Bac- Cited 15, 2500 0 Imai et al. (2001) 

Escherichia coli Bac- NA, 24 h, 37°C —, sd 2 Imai et al. (2001) 

Escherichia coli Bac- NA, 18 h, 37°C 6 (h), pure 14 Nelson (1997) 

Escherichia coli Bac- NA, 24 h, 37°C 10 (h), 100,000 22 Imai et al. (2001) 

Klebsiella aerogenes Bac- NA, 24 h, 37°C 10 (h), 100,000 0 Nelson (1997) 

Klebsiella sp. Bac- Cited 15, 2500 0 Imai et al. (2001) 

Neisseria perfl ava Bac- NA, 24 h, 37°C —, sd 2 Dikshit et al. (1986) 

Proteus sp. Bac- Cited 15, 2500 0 Kumar et al. (2007) 

Proteus vulgaris Bac- NA, 24 h, 37°C —, sd 2 Kumar et al. (2007) 

Pseudomonas aeruginosa Bac- Cited 15, 2500 0 Dikshit et al. (1986) 

Pseudomonas aeruginosa Bac- NA, 24 h, 37°C —, sd 0 Kumar et al. (2007) 

Pseudomonas aeruginosa Bac- NA, 18 h, 37°C 6 (h), pure 12 Kumar et al. (2007) 

Salmonella sp. Bac- Cited 15, 2500 0 Kumar et al. (2007) 

Salmonella typhi Bac- NA, 24 h, 37°C 10 (h), 100,000 19 Kumar et al. (2007) 

Serratia marcescens Bac- NA, 24 h, 37°C —, sd 0 Kumar et al. (2007) 

Serratia sp. Bac- Cited 15, 2500 0 Dikshit et al. (1979) 

Shigella sp. Bac- Cited 15, 2500 1 Dikshit et al. (1982) 

Vibrio cholerae Bac- NA, 24 h, 37°C 10 (h), 100,000 0 Dikshit et al. (1982) 

Bacillus mesentericus Bac+ NA, 24 h, 37°C —, sd 7 Kumar et al. (2007) 

Bacillus sp. Bac+ Cited 15, 2500 1 Dikshit et al. (1986) 

Bacillus subtilis Bac+ NA, 24 h, 37°C —, sd 19 Dikshit et al. (1986) 

Bacillus subtilis Bac+ NA, 18 h, 37°C 6 (h), pure 22 Kumar et al. (2007) 

Corynebacterium diphtheriae Bac+ NA, 24 h, 37°C 10 (h), 100,000 18 Dikshit et al. (1986) 

Listeria monocytogenes Bac+ ISA, 48 h, 25°C 4 (h), 10,000 11–19 Dikshit et al. (1986) 

continued





(mm) Ref. 

Mycobacterium phlei Bac+ NA, 18 h, 37°C 6 (h), pure 20 Duarte et al. (2005) 

Sarcina lutea Bac+ NA, 24 h, 37°C —, sd 0 Imai et al. (2001) 

Staphylococcus aureus Bac+ NA, 24 h, 37°C 10 (h), 100,000 0 Imai et al. (2001) 

Staphylococcus aureus Bac+ NA, 24 h, 37°C —, sd 0 Imai et al. (2001) 

Staphylococcus aureus Bac+ Cited 15, 2500 2 Nelson (1997) 

Staphylococcus aureus Bac+ NA, 18 h, 37°C 6 (h), pure 13.6 Imai et al. (2001) 

Staphylococcus epidermidis Bac+ Cited 15, 2500 1 Nelson (1997) 

Staphylococcus epidermidis Bac+ NA, 24 h, 37°C 10 (h), 100,000 22 Imai et al. (2001) 

Streptococcus sp. Bac+ NA, 24 h, 37°C 10 (h), 100,000 0 Dikshit et al. (1986) 

Streptococcus viridans Bac+ Cited 15, 2500 0 Kumar et al. (2007) 

Streptomyces venezuelae Bac+ SMA, 2–7 d, 20°C sd 9 Kumar et al. (2007) 

Alternaria solani Fungi SMA, 2–7 d, 20°C sd 9 Dikshit et al. (1986) 

Aspergillus fumigatus Fungi SMA, 2–7 d, 20°C sd 5 Kumar et al. (2007) 

Aspergillus niger Fungi SMA, 2–7 d, 20°C sd 9 Kumar et al. (2007) 

Aspergillus niger Fungi SDA, 8 d, 30°C 6 (h), pure 26 Kumar et al. (2007) 

Helminthosporium sativum Fungi SMA, 2–7 d, 20°C sd 9 Kumar et al. (2007) 

Mucor mucedo Fungi SMA, 2–7 d, 20°C sd 9 Kumar et al. (2007) 

Mucor sp. Fungi SDA, 8 d, 30°C 6 (h), pure 22 Dikshit et al. (1979) 

Nigrospora panici Fungi SMA, 2–7 d, 20°C sd 5 Dikshit et al. (1982) 

Penicillium chrysogenum Fungi SDA, 8 d, 30°C 6 (h), pure 56 Dikshit et al. (1982) 

Penicillium digitatum Fungi SMA, 2–7 d, 20°C sd 9 Kumar et al. (2007) 

Rhizopus nigricans Fungi SMA, 2–7 d, 20°C sd 6 Dikshit et al. (1986) 

Rhizopus sp. Fungi SDA, 8 d, 30°C 6 (h), pure 21 Dikshit et al. (1986) 

Candida albicans Yeast SMA, 2–7 d, 20°C sd 8 Kumar et al. (2007) 

Candida albicans Yeast SDA, 18 h, 30°C 6 (h), pure 30 Dikshit et al. (1986) 

Candida krusei Yeast SMA, 2–7 d, 20°C sd 0 Dikshit et al. (1986) 

Candida tropicalis Yeast SMA, 2–7 d, 20°C sd 0 Duarte et al. (2005) 

Cryptococcus neoformans Yeast SMA, 2–7 d, 20°C sd 6 Imai et al. (2001) 

Cryptococcus rhodobenhani Yeast SMA, 2–7 d, 20°C sd 6 Imai et al. (2001) 

Saccharomyces cerevisiae Yeast SMA, 2–7 d, 20°C sd 4 Imai et al. (2001)


TABLE 12.55 

Inhibitory Data of Neroli Oil Obtained in the Dilution Test 





Mycobacterium phlei Bac+ NB, Tween 20, 18 h, 37°C 50,000 Yousef and Tawil (1980) 







Ceratocystis paradoxa Fungi OA, EtOH, 3 d, 20°C 20,000 Narasimba Rao et al. (1971) 

Curvularia lunata Fungi OA, EtOH, 3 d, 20°C 4000 Narasimba Rao et al. (1971) 

Fusarium culmorum Fungi YES broth, 10 d -71% inh. 10,000 Lis-Balchin et al. (1998) 

Fusarium moniliforme var. subglutinans Fungi OA, EtOH, 3 d, 20°C 2000 Narasimba Rao et al. (1971) 

Helminthosporium sacchari Fungi OA, EtOH, 3 d, 20°C 2000 Narasimba Rao et al. (1971) 





Physalospora tucumanensis Fungi OA, EtOH, 3 d, 20°C 20,000 Narasimba Rao et al. (1971) 


Sclerotium rolfsii Fungi OA, EtOH, 6 d, 20°C 20,000 Narasimba Rao et al. (1971) 

Candida albicans Yeast NB, Tween 20, 18 h, 37°C 3200 Yousef and Tawil (1980)


TABLE 12.56 

Inhibitory Data of Neroli Oil Obtained in the Vapor Phase Test 

















TABLE 12.57 

Inhibitory Data of Nutmeg Oil Obtained in the Agar Diffusion Test 



(mm) Ref. 



























continued





(mm) Ref. 


























Staphylococcus epidermidis Bac+ Cited 15, 2500 1 Pizsolitto et al. (1975)




























TABLE 12.58 

Inhibitory Data of Nutmeg Oil Obtained in the Dilution Test 


Campylobacter jejuni Bac- TSB, 24 h, 42°C >10,000 Smith-Palmer et al. 

(1998) 

Escherichia coli Bac- NB, Tween 20, 18 h, 800 Yousef and Tawil (1980) 

37°C 


Escherichia coli Bac- TSB, 24 h, 35°C >10,000 Smith-Palmer et al. 

(1998) 

Pseudomonas aeruginosa Bac- NB, Tween 20, 18 h, >50,000 Yousef and Tawil (1980) 

37°C 

Salmonella enteritidis Bac- TSB, 24 h, 35°C >10,000 Smith-Palmer et al. 

(1998) 

Bacillus subtilis Bac+ NB, Tween 20, 18 h, 6400 Yousef and Tawil (1980) 

37°C 


Listeria monocytogenes Bac+ TSB, 24 h, 35°C 10,000 Smith-Palmer et al. 

(1998) 

Staphylococcus aureus Bac+ NB, Tween 20, 18 h, 25,000 Yousef and Tawil (1980) 

37°C 


Aspergillus fl avus Fungi PDA, 8 h, 20°C, spore 50–100 Thompson (1986) 

germ. inh. 

Aspergillus fl avus Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon 

(1986) 


Aspergillus niger Fungi YES broth, 10 d -88% inh. Lis-Balchin et al. (1998) 

10,000 

Aspergillus ochraceus Fungi YES broth, 10 d -86% inh. Lis-Balchin et al. (1998) 

10,000 

Aspergillus parasiticus Fungi PDA, 8 h, 20°C, spore 50–100 Thompson (1986) 

germ. inh. 

Aspergillus parasiticus Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon 

(1986) 

Epidermophyton 

fl occosum 

Fungi SA, Tween 80, 21 d, 

20°C 

300–625 Janssen et al. (1988) 

Fusarium culmorum Fungi YES broth, 10 d ->10,000 Lis-Balchin et al. (1998) 

Mucor hiemalis Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon 

(1986) 

Mucor mucedo Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon 

(1986) 

continued



Inhibitory Data of Nutmeg Oil Obtained in the Dilution Test 


Mucor racemosus f. 

racemosus 

Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon 

(1986) 



Rhizopus 66-81-2 Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon 

(1986) 

Rhizopus arrhizus Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon 

(1986) 

Rhizopus chinensis Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon 

(1986) 

Rhizopus circinans Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon 

(1986) 

Rhizopus japonicus Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon 

(1986) 

Rhizopus kazanensis Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon 

(1986) 

Rhizopus oryzae Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon 

(1986) 

Rhizopus pymacus Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon 

(1986) 


Rhizopus stolonifer Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon 

(1986) 

Rhizopus tritici Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon 

(1986) 

Trichophyton 


Fungi SA, Tween 80, 21 d, 

20°C 

625–1250 Janssen et al. (1988) 

Trichophyton rubrum Fungi SA, Tween 80, 21 d, 300–625 Janssen et al. (1988) 

20°C 


Candida albicans Yeast NB, Tween 20, 18 h, 

37°C 

3200 Yousef and Tawil (1980) 

TABLE 12.59 

Inhibitory Data of Nutmeg Oil Obtained in the Vapor Phase Test 




Mycobacterium avium Bac+ NA, 24 h, 37°C sd ++ Maruzzella and Sicurella (1960) 


Streptococcus faecalis Bac+ NA, 24 h, 37°C sd +++ Maruzzella and Sicurella (1960)


TABLE 12.60 

Inhibitory Data of Peppermint Oil Obtained in the Agar Diffusion Test 





Alcaligenes faecalis Bac- ISA, 48 h, 25°C 4 (h), 10,000 8.5 Deans and Ritchie (1987) 

Beneckea natriegens Bac- ISA, 48 h, 25°C 4 (h), 10,000 16.5 Deans and Ritchie (1987) 


























Salmonella enteritidis Bac- TSA, 24 h, 35°C 4 (h), 25,000 6.3 Smith-Palmer et al. (1998)


Salmonella paratyphi Bac- NA, 24 h, 37°C 6, sd 18 Dube and Rao (1984) 







Vibrio cholera Bac- NA, 24 h, 37°C 6, sd 16 Dube and Rao (1984) 


Bacillus anthracis Bac+ NA, 24 h, 37°C 6, sd 17 Dube and Rao (1984) 


Bacillus pumilus Bac+ NA, 24 h, 37°C 6, sd 13 Dube and Rao (1984) 






















continued




Staphylococcus aureus Bac+ NA, 24 h, 37°C 6, sd 13 Dube and Rao (1984) 








Absidia cormybifera Fungi EYA, 48 h, 45°C 5, sd 0 Nigam and Rao (1979) 



Aspergillus fl avus Fungi PDA, 10 d, 25°C 10 – Sarbhoy et al. (1978) 

Aspergillus fumigatus Fungi PDA, 10 d, 25°C 10 – Sarbhoy et al. (1978) 


Aspergillus fumigatus Fungi SDA, 3 d, 28°C 6, sd 10 Saksena and Saksena (1984) 




Aspergillus sulphureus Fungi PDA, 10 d, 25°C 10 – Sarbhoy et al. (1978) 




Keratinomyces afelloi Fungi SDA, 3 d, 28°C 6, sd 18 Saksena and Saksena (1984) 

Keratinophyton terreum Fungi SDA, 3 d, 28°C 6, sd 0 Saksena and Saksena (1984) 

Microsporum gypseum Fungi SDA, 3 d, 28°C 6, sd 10 Saksena and Saksena (1984) 

Mucor fragilis Fungi PDA, 10 d, 25°C 10 – Sarbhoy et al. (1978) 





Penicillium digitatum Fungi SMA, 2–7 d, 20°C sd 8 Maruzzella and Liguori (1958)




Rhizopus stolonifer Fungi PDA, 10 d, 25°C 10 – Sarbhoy et al. (1978) 





Trichophyton equinum Fungi SDA, 3 d, 28°C 6, sd 14 Saksena and Saksena (1984) 

Trichophyton rubrum Fungi SDA, 3 d, 28°C 6, sd 16 Saksena and Saksena (1984) 






Candida albicans Yeast SDA, 3 d, 28°C 6, sd 18 Saksena and Saksena (1984) 




Candida tropicalis Yeast SDA, 3 d, 28°C 6, sd 23 Saksena and Saksena (1984) 


















TABLE 12.61 

Inhibitory Data of Peppermint Oil Obtained in the Dilution Test 


Treponema denticola Bac HS, 72 h, 37°C 1000 Shapiro et al. (1994) 

Treponema vincentii Bac HS, 72 h, 37°C 2000 Shapiro et al. (1994) 

Actinobacillus 

Bac- HS, 72 h, 37°C 3000 Shapiro et al. (1994) 

actinomycetemcomitans 


Capnocytophaga sp. Bac- HS, 72 h, 37°C 3000 Shapiro et al. (1994) 

Eikenella corrodens Bac- HS, 72 h, 37°C 2000 Shapiro et al. (1994) 

Escherichia coli Bac- Cited, 24 h 800 Imai et al. (2001) 




Escherichia coli Bac- MHB, 24 h, 36°C >10,000 Duarte et al. (2006) 


Fusobacterium nucleatum Bac- HS, 72 h, 37°C 2000 Shapiro et al. (1994) 

Helicobacter pylori Bac- Cited, 48 h 100 Imai et al. (2001) 


Porphyromonas gingivalis Bac- HS, 72 h, 37°C 2000 Shapiro et al. (1994) 

Prevotella buccae Bac- HS, 72 h, 37°C 2000 Shapiro et al. (1994) 

Prevotella intermedia Bac- HS, 72 h, 37°C 3000 Shapiro et al. (1994) 

Prevotella nigrescens Bac- HS, 72 h, 37°C 2000 Shapiro et al. (1994) 


Salmonella enteritidis Bac- Cited, 24 h 400 Imai et al. (2001) 


Selenomonas artemidis Bac- HS, 72 h, 37°C 1000 Shapiro et al. (1994) 

Actinomyces viscosus Bac+ HS, 16–24, 37°C 5000 Shapiro et al. (1994) 




Mycobacterium phlei Bac+ NB, Tween 20, 18 h, 37°C 400 Yousef and Tawil (1980)


Peptostreptococcus anaerobius Bac+ HS, 72 h, 37°C 2000 Shapiro et al. (1994) 




Staphylococcus aureus MRSA Bac+ Cited, 24 h 200 Imai et al. (2001) 

Staphylococcus aureus MSSA Bac+ Cited, 24 h 200 Imai et al. (2001) 


Streptococcus sanguinis Bac+ HS, 16–24, 37°C 6000 Shapiro et al. (1994) 

Streptococcus sobrinus Bac+ HS, 16–24, 37°C 3000 Shapiro et al. (1994) 











Fusarium culmorum Fungi YES broth, 10 d - >10,000 Lis-Balchin et al. (1998) 













continued
















Trichophyton rubrum Fungi SDA, 7 d, 30°C 400, 61% inh. Dikshit and Husain (1984) 



Candida albicans Yeast TGB, 18–24 h, 37°C >1000 Morris et al. (1979) 

Candida albicans Yeast MHB, Tween 80, 48 h, 35°C 5000 Hammer et al. (1998) 



TABLE 12.62 

Inhibitory Data of Peppermint Oil Obtained in the Vapor Phase Test 









Bacillus subtilis var. aterrimus Bac+ NA, 24 h, 37°C sd ++ Maruzzella and Sicurella (1960) 




Staphylococcus aureus Bac+ NA, 24 h, 37°C sd ++ Maruzzella and Sicurella (1960) 











Eurotium amstelodami Fungi WFA, 42 d, 25°C Disk, 50,000 ++ Guynot et al. (2003) 


Eurotium repens Fungi WFA, 42 d, 25°C Disk, 50,000 ++ Guynot et al. (2003) 






Candida albicans Yeast NA, 24 h, 37°C ~20,000 + Kellner and Kober (1954)


TABLE 12.63 

Inhibitory Data of Pinus sylvestris Oil Obtained in the Agar Diffusion Test 



Bordetella bronchiseptica Bac- MHA, 18 h, 37°C 6, 17,500 0 Schales et al. (1993) 

Citrobacter freundii Bac- MHA, 18 h, 37°C 6, 17,500 0 Schales et al. (1993) 



Escherichia coli 1 Bac- MHA, 18 h, 37°C 6, 17,500 8 Schales et al. (1993) 

Escherichia coli 2 Bac- MHA, 18 h, 37°C 6, 17,500 0 Schales et al. (1993) 

Klebsiella pneumoniae Bac- MHA, 18 h, 37°C 6, 17,500 0 Schales et al. (1993) 


Proteus mirabilis Bac- MHA, 18 h, 37°C 6, 17,500 0 Schales et al. (1993) 



Pseudomonas aeruginosa Bac- MHA, 18 h, 37°C 6, 17,500 0 Schales et al. (1993) 




Bacillus sp. Bac+ MHA, 18 h, 37°C 6, 17,500 11 Schales et al. (1993) 



Clostridium perfringens Bac+ MHA, 18 h, 37°C 6, 17,500 23 Schales et al. (1993)


Enterococcus sp. Bac+ MHA, 18 h, 37°C 6, 17,500 15 Schales et al. (1993) 




Staphylococcus sp. Bac+ MHA, 18 h, 37°C 6, 17,500 6 Schales et al. (1993) 


















TABLE 12.64 

Inhibitory Data of Pinus sylvestris Oil Obtained in the Dilution Test 


Acinetobacter baumannii Bac- MHA, Tween 20, 48 h, 35°C 20,000 Hammer et al. (1999) 

Aeromonas sobria Bac- MHA, Tween 20, 48 h, 35°C 20,000 Hammer et al. (1999) 

Escherichia coli Bac- MHB, Tween 80, 24 h, 37°C >29,000 Chalchat et al. (1989) 

Escherichia coli Bac- MHA, Tween 20, 48 h, 35°C 20,000 Hammer et al. (1999) 

Escherichia coli Bac- MHB, Tween 80, 24 h, 37°C 64,300 Bastide et al. (1987) 

Klebsiella pneumonia Bac- MHB, Tween 80, 24 h, 37°C 3500 Chalchat et al. (1989) 

Klebsiella pneumoniae Bac- MHA, Tween 20, 48 h, 35°C >20,000 Hammer et al. (1999) 

Proteus mirabilis Bac- MHB, Tween 80, 24 h, 37°C >29,000 Chalchat et al. (1989) 


Pseudomonas aeruginosa Bac- MHB, Tween 80, 24 h, 37°C >29,000 Chalchat et al. (1989) 



Bacillus sp. Bac+ CA, 7 d, 25°C 5000 Motiejunaite and Peciulyte (2004) 


Rhodococcus sp. Bac+ CA, 7 d, 25°C 5000 Motiejunaite and Peciulyte (2004) 

Staphylococcus aureus Bac+ MHA, Tween 20, 48 h, 35°C >20,000 Hammer et al. (1999) 

Staphylococcus aureus Bac+ MHB, Tween 80, 24 h, 37°C 3500 Chalchat et al. (1989) 


Alternaria alternata Fungi RPMI, 1.5% EtOH, 7 d, 30°C 10,000 Tullio et al. (2006) 



Aspergillus fumigatus Fungi RPMI, 1.5% EtOH, 7 d, 30°C 5000–10,000 Tullio et al. (2006) 

Aspergillus niger Fungi RPMI, 1.5% EtOH, 7 d, 30°C 5000 Tullio et al. (2006) 

Aspergillus niger Fungi CA, 7 d, 25°C 7500–15,000 Motiejunaite and Peciulyte (2004) 

Aspergillus versicolor Fungi CA, 7 d, 25°C 7500–15,000 Motiejunaite and Peciulyte (2004)


Aureobasidum pullulans Fungi CA, 7 d, 25°C 5000–7500 Motiejunaite and Peciulyte (2004) 

Chaetomium globosum Fungi CA, 7 d, 25°C 5000 Motiejunaite and Peciulyte (2004) 


Cladosporium cladosporoides Fungi CA, 7 d, 25°C 5000 Motiejunaite and Peciulyte (2004) 

Epidermophyton fl occosum Fungi RPMI, 1.5% EtOH, 7 d, 30°C 1250 Tullio et al. (2006) 

Fusarium oxysporum Fungi RPMI, 1.5% EtOH, 7 d, 30°C 312 Tullio et al. (2006) 


Microsporum gypseum Fungi RPMI, 1.5% EtOH, 7 d, 30°C 2500–5000 Tullio et al. (2006) 

Mucor sp. Fungi RPMI, 1.5% EtOH, 7 d, 30°C Tullio et al. (2006) 

Paecilomyces variotii Fungi CA, 7 d, 25°C >10,000 Thanaboripat et al. (2004) 

Motiejunaite and Peciulyte (2004) 

Penicillium chrysogenum Fungi CA, 7 d, 25°C 10,000–25,000 Motiejunaite and Peciulyte (2004) 


Penicillium lanosum Fungi RPMI, 1.5% EtOH, 7 d, 30°C 10,000 Tullio et al. (2006) 

Phoma glomerata Fungi CA, 7 d, 25°C 10,000–25,000 Motiejunaite and Peciulyte (2004) 

Phoma sp. Fungi CA, 7 d, 25°C 7500 Motiejunaite and Peciulyte (2004) 


Rhizopus stolonifer Fungi CA, 7 d, 25°C 5000–7500 Motiejunaite and Peciulyte (2004) 


Stachybotrys chartarum Fungi CA, 7 d, 25°C 10,000–15,000 Motiejunaite and Peciulyte (2004) 

Trichoderma viride Fungi CA, 7 d, 25°C 5500 Motiejunaite and Peciulyte (2004) 

Trichophyton mentagrophytes Fungi RPMI, 1.5% EtOH, 7 d, 30°C 2500–5000 Tullio et al. (2006) 

Candida albicans Yeast MHB, Tween 80, 24 h, 37°C 14,000 Chalchat et al. (1989) 

Candida albicans Yeast MHA, Tween 20, 48 h, 35°C 20,000 Hammer et al. (1999) 

Candida lipolytica Yeast CA, 7 d, 25°C 5000 Motiejunaite and Peciulyte (2004) 

Geotrichum candida Yeast CA, 7 d, 25°C 3500–5000 Motiejunaite and Peciulyte (2004)


Table 12.65 

Inhibitory Data of Pinus sylvestris Oil Obtained in the Vapor Phase Test 






Rhizoctonia solani Fungi PDA, 3 d, 25°C 1000 +++ Lee et al. (2007)


TABLE 12.66 

Inhibitory Data of Rosemary Oil Obtained in the Agar Diffusion Test 




Aerobacter tumefaciens Bac- Cited (h) 20,000 — Hethenyi et al. (1989) 









Escherichia coli Bac- Cited (h) 20,000 - to +++ Hethenyi et al. (1989) 





Escherichia coli Bac- Cited, 24 h, 37°C —




Flavobacterium suaveolens Bac- ISA, 48 h, 25°C 4 (h), 10,000 8.5 Deans and Ritchie (1987) 

Haemophilus infl uenza Bac- Cited (h) 20,000 — Hethenyi et al. (1989) 






Peptobacterium carotovorum Bac- Cited (h) 20,000 +++ Hethenyi et al. (1989) 


Proteus vulgaris Bac- Cited w 20,000 +++ Hethenyi et al. (1989) 



Pseudomonas aeruginosa Bac- Cited (h) 20,000 +++ Hethenyi et al. (1989) 








Pseudomonas pisi Bac- Cited (h) 20,000 ++ Hethenyi et al. (1989) 

Pseudomonas tabaci Bac- Cited (h) 20,000 +++ Hethenyi et al. (1989) 

Salmonella enteritidis Bac- TSA, 24 h, 35°C 4 (h), 25,000 9.3 Smith-Palmer et al. (1998)


Salmonella enteritidis Bac- Cited, 24 h, 37°C —




Lactobacillus sp. Bac+ MRS, cited 9, 20,000 16.5 – >90 Pellecuer et al. (1980) 




Micrococcus luteus Bac+ MHA, cited 9, 20,000 18–26 Pellecuer et al. (1980) 


Micrococcus ureae Bac+ MHA, cited 9, 20,000 21 Pellecuer et al. (1980) 


Pneumococcus sp. Bac+ Cited (h) 20,000 ++ Hethenyi et al. (1989) 


Sarcina ureae Bac+ MHA, cited 9, 20,000 >90 Pellecuer et al. (1980) 

Staphylococcus aureus Bac+ Cited (h) 20,000 — Hethenyi et al. (1989) 














Streptococcus aegui Bac+ Cited (h) 20,000 + Hethenyi et al. (1989) 

Streptococcus D Bac+ MHA, cited 9, 20,000 0–30 Pellecuer et al. (1980)


Streptococcus faecalis Bac+ Cited (h) 20,000 ++ Hethenyi et al. (1989) 


Streptococcus haemolyticus Bac+ Cited (h) 20,000 + Hethenyi et al. (1989) 

Streptococcus micros Bac+ MHA, cited 9, 20,000 18 Pellecuer et al. (1980) 

Streptococcus sp. Bac+ Cited (h) 20,000 + Hethenyi et al. (1989) 












Fusarium moniliforme Fungi Cited (h) 20,000 — Hethenyi et al. (1989) 


Fusarium solani Fungi Cited (h) 20,000 — Hethenyi et al. (1989) 





Ophiobolus graminis Fungi Cited (h) 20,000 — Hethenyi et al. (1989) 






continued




Candida albicans Yeast Cited (h) 20,000 — Hethenyi et al. (1989) 



















Saccharomyces cerevisiae Yeast Cited (h) 20,000 — Hethenyi et al. (1989) 



Saccharomyces cerevisiae Yeast NA, 24 h, 20°C 5 (h), -30,000 10 Schelz et al. (2006) 






TABLE 12.67 

Inhibitory Data of Rosemary Oil Obtained in the Dilution Test 


Acinetobacter baumannii Bac- MHA, Tween 20, 48 h, 35°C 10,000 Hammer et al. (1999) 

Aeromonas sobria Bac- MHA, Tween 20, 48 h, 35°C 5000 Hammer et al. (1999) 



Escherichia coli Bac- TSB, Tween 80, 48 h, 35°C 40 Panizzi et al. (1993) 


Escherichia coli Bac- NB, DMSO, 24 h, 37°C >900 Angioni et al. (2004) 




Escherichia coli Bac- MHA, Tween 20, 48 h, 35°C 10,000 Hammer et al. (1999) 



Helicobacter pylori Bac- Cited, 20 h, 37°C 137 Weseler et al. (2005) 


Klebsiella pneumoniae Bac- MHA, Tween 20, 48 h, 35°C 20,000 Hammer et al. (1999) 




Pseudomonas aeruginosa Bac- TSB, Tween 80, 48 h, 35°C >40 Panizzi et al. (1993) 


Pseudomonas aeruginosa Bac- NB, DMSO, 24 h, 37°C >900 Angioni et al. (2004) 



Pseudomonas fl uorescens Bac- NA, 1–3 d, 30°C Thanaboripat et al. (2004) 

Farag et al. (1989) 


continued




Salmonella typhimurium Bac- TSB, 3% EtOH, 24 h, 37°C 10,000–13,000 Rota et al. (2004) 


Serratia marcescens Bac- NA, 1–3 d, 30°C 11,000 Farag et al. (1989) 


Shigella fl exneri Bac- TSB, 3% EtOH, 24 h, 37°C 20,000 Rota et al. (2004) 

Yersinia enterocolitica Bac- TSB, 3% EtOH, 24 h, 29°C 10,000–15,000 Rota et al. (2004) 


Actinomyces viscosus Bac+ HS, 16–24, 37°C >6000 Shapiro et al. (1994) 

Bacillus subtilis Bac+ TSB, Tween 80, 48 h, 35°C 10 Panizzi et al. (1993) 






Lactobacillus sp. Bac+ MRS, cited 5 Pellecuer et al. (1980) 

Listeria monocytogenes Bac+ TSB, 3% EtOH, 24 h, 37°C 7000–10,000 Rota et al. (2004) 



Micrococcus luteus Bac+ MHB, cited 2.5–5 Pellecuer et al. (1980) 


Micrococcus ureae Bac+ MHB, cited 5 Pellecuer et al. (1980) 





Sarcina ureae Bac+ MHB, cited 2.5 Pellecuer et al. (1980) 

Staphylococcus aureus Bac+ TSB, 3% EtOH, 24 h, 37°C 30,000–50,000 Rota et al. (2004) 

Staphylococcus aureus Bac+ TSB, Tween 80, 48 h, 35°C 20 Panizzi et al. (1993) 


Staphylococcus aureus Bac+ MPB, DMSO, 40 h, 30°C 1% inh. 500 Hili et al. (1997)


Staphylococcus aureus Bac+ NB, DMSO, 24 h, 37°C >900 Angioni et al. (2004) 


Staphylococcus aureus Bac+ Cited 1250 Pellecuer et al. (1976) 

Staphylococcus aureus Bac+ MHA, Tween 20, 48 h, 35°C 10,000 Hammer et al. (1999) 


Staphylococcus epidermidis Bac+ MHB, cited 5 Pellecuer et al. (1980) 

Staphylococcus epidermidis Bac+ NB, DMSO, 24 h, 37°C >900 Angioni et al. (2004) 

Staphylococcus epidermidis Bac+ Cited 1250 Pellecuer et al. (1976) 

Streptococcus D Bac+ MHB, cited 2.5–5 Pellecuer et al. (1980) 

Streptococcus micros Bac+ MHB, cited 5 Pellecuer et al. (1980) 

Streptococcus pyogenes Bac+ Cited 625 Pellecuer et al. (1976) 

Streptococcus sanguinis Bac+ HS, 16–24, 37°C >6000 Shapiro et al. (1994) 

Streptococcus sobrinus Bac+ HS, 16–24, 37°C >6000 Shapiro et al. (1994) 

Absidia glauca Fungi Cited 2000 Pellecuer et al. (1976) 

Aspergillus chevalieri Fungi Cited 2000 Pellecuer et al. (1976) 

Aspergillus clavatus Fungi Cited 2000 Pellecuer et al. (1976) 



Aspergillus fl avus Fungi Cited 2000 Pellecuer et al. (1976) 

Aspergillus fl avus Fungi MYB, 72 h, 26°C 12,500 Shin (2003) 

Aspergillus giganteus Fungi Cited 2000 Pellecuer et al. (1976) 



Aspergillus niger Fungi MYB, 72 h, 26°C 12,500 Shin (2003) 




Aspergillus oryzae Fungi Cited 2000 Pellecuer et al. (1976) 



Aspergillus repens Fungi Cited 2000 Pellecuer et al. (1976) 


continued




Ceratocystis paradoxa Fungi OA, EtOH, 3 d, 20°C 4000 Narasimba Rao et al. (1971) 

Cladosporium herbarum Fungi Cited 2000 Pellecuer et al. (1976) 

Curvularia lunata Fungi OA, EtOH, 3 d, 20°C 20,000 Narasimba Rao et al. (1971) 


Epidermophyton fl occosum Fungi Cited >4000 Pellecuer et al. (1976) 


Fusarium moniliforme var. subglutinans Fungi OA, EtOH, 3 d, 20°C 20,000 Narasimba Rao et al. (1971) 

Helminthosporium sacchari Fungi OA, EtOH, 3 d, 20°C 20,000 Narasimba Rao et al. (1971) 

Microsporum canis Fungi MBA, Tween 80, 10 d, 30°C 300– >300 Perrucci et al. (1994) 

Microsporum gypseum Fungi MBA, Tween 80, 10 d, 30°C >300 Perrucci et al. (1994) 


Mucor mucedo Fungi Cited 1000 Pellecuer et al. (1976) 






Penicillium chrysogenum Fungi Cited 2000 Pellecuer et al. (1976) 


Penicillium liliacinum Fungi Cited 2000 Pellecuer et al. (1976) 

Penicillium rubrum Fungi Cited 2000 Pellecuer et al. (1976) 

Physalospora tucumanensis Fungi OA, EtOH, 3 d, 20°C 20,000 Narasimba Rao et al. (1971) 







Rhizopus nigricans Fungi Cited 2000 Pellecuer et al. (1976) 

Rhizopus oryzae Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon (1986)






Sclerotium rolfsii Fungi OA, EtOH, 6 d, 20°C 4000 Narasimba Rao et al. (1971) 

Scopulariopsis brevicaulis Fungi Cited 2000 Pellecuer et al. (1976) 

Syncephalastrum racemosum Fungi Cited 2000 Pellecuer et al. (1976) 

Trichophyton interdigitale Fungi Cited >4000 Pellecuer et al. (1976) 


Trichophyton rubrum Fungi SA, Tween 80, 21 d, 20°C 900 Angioni et al. (2004) 

Candida albicans Yeast Cited 1000 Pellecuer et al. (1976) 


Candida albicans Yeast MHA, Tween 20, 48 h, 35°C 10,000 Hammer et al. (1999) 

Candida mycoderma Yeast Cited 2000 Pellecuer et al. (1976) 

Candida paraspilosis Yeast Cited 1000 Pellecuer et al. (1976) 

Candida pelliculosa Yeast Cited 2000 Pellecuer et al. (1976) 

Candida tropicalis Yeast Cited 2000 Pellecuer et al. (1976) 

Geotrichum asteroides Yeast Cited 4000 Pellecuer et al. (1976) 

Geotrichum candidum Yeast Cited 2000 Pellecuer et al. (1976) 

Hansenula sp. Yeast Cited 2000 Pellecuer et al. (1976) 

Saccharomyces carlsbergensis Yeast Cited 4000 Pellecuer et al. (1976) 

Saccharomyces cerevisiae Yeast SDB, Tween 80, 48 h, 35°C 5 Panizzi et al. (1993) 


Saccharomyces cerevisiae Yeast NA, 1–3 d, 30°C 2000 Farag et al. (1989) 

Schizosaccharomyces pombe Yeast MPB, DMSO, 40 h, 30°C 86% inh. 500 Hili et al. (1997) 


Escherichia coli Bac- TGB, 18 h, 37°C 11,300 Schelz et al. (2006) 

Staphylococcus epidermidis Bac+ TGB, 18 h, 37°C 11,300 Schelz et al. (2006) 



TABLE 12.68 

Inhibitory Data of Rosemary Oil Obtained in the Vapor Phase Test 


Aerobacter tumefaciens Bac- Cited Disk, 20,000 NG Hethenyi et al. (1989) 


Escherichia coli Bac- Cited Disk, 20,000 NG to +++ Hethenyi et al. (1989) 

Haemophilus infl uenza Bac- Cited Disk, 20,000 NG Hethenyi et al. (1989) 


Peptobacterium carotovorum Bac- Cited Disk, 20,000 +++ Hethenyi et al. (1989) 

Proteus vulgaris Bac- Cited Disk, 20,000 +++ Hethenyi et al. (1989) 

Pseudomonas aeruginosa Bac- Cited Disk, 20,000 +++ Hethenyi et al. (1989) 

Pseudomonas pisi Bac- Cited Disk, 20,000 +++ Hethenyi et al. (1989) 

Pseudomonas tabaci Bac- Cited Disk, 20,000 +++ Hethenyi et al. (1989) 



Shigella sonnei Bac- Cited Disk, 20,000 +++ Hethenyi et al. (1989) 

Xanthomonas versicolor Bac- Cited Disk, 20,000 +++ Hethenyi et al. (1989) 


Bacillus subtilis Bac+ Cited Disk, 20,000 NG Hethenyi et al. (1989) 



Corynebacterium fascians Bac+ Cited Disk, 20,000 +++ Hethenyi et al. (1989) 

Lactobacillus sp. Bac+ MRS, cited Disk, 20,000? ++ Pellecuer et al. (1980) 

Micrococcus luteus Bac+ MHB, cited Disk, 20,000? ++ Pellecuer et al. (1980) 

Micrococcus ureae Bac+ MHB, cited Disk, 20,000? +++ Pellecuer et al. (1980) 

Mycobacterium avium Bac+ NA, 24 h, 37°C sd ++ Maruzzella and Sicurella (1960) 

Pneumococcus sp. Bac+ Cited Disk, 20,000 NG Hethenyi et al. (1989) 

Sarcina ureae Bac+ MHB, cited Disk, 20,000? ++ Pellecuer et al. (1980) 


Staphylococcus aureus Bac+ Cited Disk, 20,000 NG Hethenyi et al. (1989) 

Staphylococcus epidermidis Bac+ MHB, cited Disk, 20,000? +++ Pellecuer et al. (1980)


Streptococcus aegui Bac+ Cited Disk, 20,000 NG Hethenyi et al. (1989) 

Streptococcus D Bac+ MHB, cited Disk, 20,000? ++ Pellecuer et al. (1980) 


Streptococcus faecalis Bac+ Cited Disk, 20,000 NG Hethenyi et al. (1989) 


Streptococcus haemolyticus Bac+ Cited Disk, 20,000 NG Hethenyi et al. (1989) 


Streptococcus sp. Bac+ Cited Disk, 20,000 NG Hethenyi et al. (1989) 










Eurotium repens Fungi Bread, 14 d, 25°C 30,000 +++ Suhr and Nielsen (2003) 


Fusarium moniliforme Fungi Cited Disk, 20,000 NG Hethenyi et al. (1989) 


Fusarium solani Fungi Cited Disk, 20,000 NG Hethenyi et al. (1989) 

Ophiobolus graminis Fungi Cited Disk, 20,000 NG Hethenyi et al. (1989) 

Penicillium corylophilum Fungi WFA, 42 d, 25°C Disk, 50,000 ++ Guynot et al. (2003) 





Candida albicans Yeast NA, 24 h, 37°C ~20,000 NG Kellner and Kober (1954) 

Candida albicans Yeast Cited Disk, 20,000 NG Hethenyi et al. (1989) 

Saccharomyces cerevisiae Yeast Cited Disk, 20,000 NG Hethenyi et al. (1989)


TABLE 12.69 

Inhibitory Data of Star Anise Oil Obtained in the Agar Diffusion Test 











Escherichia coli Bac- NA, 24 h, 37°C 10 (h), 2000 18.5 Singh et al. (2006) 







Pseudomonas aeruginosa Bac- NA, 24 h, 37°C 10 (h), 2000 20.3 Singh et al. (2006) 


Salmonella typhi Bac- NA, 24 h, 37°C 10 (h), 2000 30.1 Singh et al. (2006) 



Bacillus cereus Bac+ NA, 24 h, 37°C 10 (h), 2000 0 Singh et al. (2006)




Bacillus subtilis Bac+ NA, 24 h, 37°C 10 (h), 2000 26.2 Singh et al. (2006) 


























TABLE 12.70 

Inhibitory Data of Star Anise Oil Obtained in the Dilution Test 





TABLE 12.71 

Inhibitory Data of Star Anise Oil Obtained in the Vapor Phase Test 










Aspergillus fl avus Fungi CDA, 6 d 12, 6000 + Singh et al. (2006) 

Aspergillus niger Fungi CDA, 6 d 12, 6000 ++ Singh et al. (2006) 

Aspergillus ochraceus Fungi CDA, 6 d 12, 6000 +++ Singh et al. (2006) 

Aspergillus terreus Fungi CDA, 6 d 12, 6000 +++ Singh et al. (2006) 

Fusarium graminearum Fungi CDA, 6 d 12, 6000 +++ Singh et al. (2006) 


Penicillium citrinum Fungi CDA, 6 d 12, 6000 + Singh et al. (2006) 

Penicillium viridicatum Fungi CDA, 6 d 12, 6000 ++ Singh et al. (2006) 



TABLE 12.72 

Inhibitory Data of Sweet Orange Oil Obtained in the Agar Diffusion Test 

Microorganism MO Class Conditions Inhibition Zone in mm Ref. 






Campylobacter jejuni Bac- CAB, 24 h, 42°C Disk, 10,000 0 Fisher and Phillips (2006) 













Escherichia coli Bac- NA, 24 h, 37°C Disk, 10,000 18 Fisher and Phillips (2006) 









continued













Pseudomonas mangiferae indicae Bac- NA, 24 h, 28°C 6, sd 9 Kindra and Satyanarayana (1978) 



Salmonella paratyphi Bac- NA, 24 h, 28°C 6, sd 19 Kindra and Satyanarayana (1978) 









Vibrio cholera Bac- NA, 24 h, 28°C 6, sd 13 Kindra and Satyanarayana (1978) 

Xanthomonas campestris Bac- NA, 24 h, 28°C 6, sd 20.5 Kindra and Satyanarayana (1978) 


Bacillus anthracis Bac+ NA, 24 h, 28°C 6, sd 23 Kindra and Satyanarayana (1978) 

Bacillus cereus Bac+ BHA, 24 h, 30°C Disk, 10,000 36 Fisher and Phillips (2006) 



Bacillus mycoides Bac+ NA, 24 h, 28°C 6, sd 17 Kindra and Satyanarayana (1978)



Bacillus pumilus Bac+ NA, 24 h, 28°C 6, sd 16 Kindra and Satyanarayana (1978) 







Bacillus subtilis Bac+ NA, 24 h, 28°C 6, sd 21.5 Kindra and Satyanarayana (1978) 









Listeria monocytogenes Bac+ LSA, 24 h, 37°C Disk, 10,000 >90 Fisher and Phillips (2006) 


Mycobacterium phlei Bac+ NA, 18 h, 37°C 6 (h), pure 23.5 Yousef and Tawil (1980) 












continued




Staphylococcus aureus Bac+ BHA, 24 h, 37°C Disk, 10,000 46 Fisher and Phillips (2006) 

Staphylococcus epidermidis Bac+ NA, 18 h, 37°C 5 (h), –30,000 0 Schelz et al. (2006) 



















Rhizopus sp. Fungi SDA, 8 d, 30°C 6 (h), pure 0 Yousef and Tawil (1980)


























TABLE 12.73 

Inhibitory Data of Sweet Orange Oil Obtained in the Dilution Test 


Aerobacter aerogenes Bac- NA, pH 7 2000 Subba et al. (1967) 



Escherichia coli Bac- NA, 24 h, 37°C 10,000 Fisher and Phillips (2006) 


Escherichia coli Bac- TGB, 18 h, 37°C >11,300 Schelz et al. (2006) 




Salmonella heidelberg Bac- NB, 1–2 d, 35–37°C 90% inh., 1000 Dabbah et al. (1970) 

Salmonella montevideo Bac- NB, 1–2 d, 35–37°C 90% inh., 1000 Dabbah et al. (1970) 

Salmonella oranienburg Bac- NB, 1–2 d, 35–37°C 90% inh., 1000 Dabbah et al. (1970) 

Salmonella schottmuelleri Bac- NA, pH 7 1000 Subba et al. (1967) 

Salmonella senftenberg Bac- NB, 24–72 h, 37°C 93% inh. 10,000 Dabbah et al. (1970) 

Salmonella typhimurium Bac- NB, 1–2 d, 35–37°C 90% inh., 1000 Dabbah et al. (1970) 

Serratia marcescens Bac- NA, pH 7 >2000 Subba et al. (1967) 


Bacillus cereus Bac+ BHA, 24 h, 30°C >40,000 Fisher and Phillips (2006) 

Bacillus subtilis Bac+ NA, pH 7 2000 Subba et al. (1967) 



Lactobacillus plantarum Bac+ NA, pH 7 1000 Subba et al. (1967) 

Listeria monocytogenes Bac+ LSA, 24 h, 37°C 2500 Fisher and Phillips (2006) 

Micrococcus sp. Bac+ NA, pH 7 1000 Subba et al. (1967) 




Staphylococcus aureus Bac+ NB, 24–72 h, 37°C 10,000 Dabbah et al. (1970)


Staphylococcus epidermidis Bac+ TGB, 18 h, 37°C >11,300 Schelz et al. (2006) 

Streptococcus faecalis Bac+ NA, pH 7 1000 Subba et al. (1967) 

Aspergillus awamorii Fungi PDA, pH 4.5 2000 Subba et al. (1967) 

Aspergillus fl avus Fungi PDA, pH 4.5 2000 Subba et al. (1967) 



Aspergillus niger Fungi PDA, pH 4.5 2000 Subba et al. (1967) 








Cephalosporium sacchari Fungi OA, EtOH, 3 d, 20°C >20,000 Narasimba Rao et al. (1971) 

Ceratocystis paradoxa Fungi OA, EtOH, 3 d, 20°C 20,000 Narasimba Rao et al. (1971) 

Curvularia lunata Fungi OA, EtOH, 3 d, 20°C 20,000 Narasimba Rao et al. (1971) 



Fusarium moniliforme var. subglutinans Fungi OA, EtOH, 3 d, 20°C >20,000 Narasimba Rao et al. (1971) 


Helminthosporium sacchari Fungi OA, EtOH, 3 d, 20°C 20,000 Narasimba Rao et al. (1971) 








Penicillium digitatum Fungi SDB, 5 d, 20°C, MIC = ED50 1000–2400 Caccioni et al. (1998) 


Penicillium italicum Fungi SDB, 5 d, 20°C, MIC = ED50 3000–5500 Caccioni et al. (1998) 

continued




Physalospora tucumanensis Fungi OA, EtOH, 3 d, 20°C 4000 Narasimba Rao et al. (1971) 













Sclerotium rolfsii Fungi OA, EtOH, 6 d, 20°C 20,000 Narasimba Rao et al. (1971) 


Trichophyton rubrum Fungi SA, Tween 80, 21 d, 20°C >1250 Janssen et al. (1988) 



Saccharomyces cerevisiae Yeast PDA, pH 4.5 1000 Subba et al. (1967) 


Torula utilis Yeast PDA, pH 4.5 1000 Subba et al. (1967) 

Zygosaccharomyces mellis Yeast PDA, pH 4.5 1000 Subba et al. (1967)


TABLE 12.74 

Inhibitory Data of Sweet Orange Oil Obtained in the Vapor Phase Test 


Campylobacter jejuni Bac- CAB, 24 h, 42°C Disk, 10,000 +++ Fisher and Phillips (2006) 


Escherichia coli Bac- NA, 24 h, 37°C Disk, 10,000 +++ Fisher and Phillips (2006) 



Bacillus cereus Bac+ BHA, 24 h, 30°C Disk, 10,000 +++ Fisher and Phillips (2006) 



Listeria monocytogenes Bac+ LSA, 24 h, 37°C Disk, 10,000 +++ Fisher and Phillips (2006) 


Staphylococcus aureus Bac+ BHA, 24 h, 37°C Disk, 10,000 +++ Fisher and Phillips (2006) 



Aspergillus fl avus Fungi Bread, 14 d, 25°C 30,000 NG Suhr and Nielsen (2003) 








Eurotium repens Fungi Bread, 14 d, 25°C 30,000 + Suhr and Nielsen (2003) 











TABLE 12.75 

Inhibitory Data of Tea Tree Oil Obtained in the Agar Diffusion Test 







Staphylococcus aureus MRSA Bac+ MHA, 24 h, 37°C 12.7, 30,000 21–33 Carson et al. (1995) 













TABLE 12.76 

Inhibitory Data of Tea Tree Oil Obtained in the Dilution Test 


Mycoplasma fermentans Bac Cited 100–600 Furneri et al. (2006) 

Mycoplasma hominis Bac Cited 600–1200 Furneri et al. (2006) 

Mycoplasma pneumoniae Bac Cited 100 Furneri et al. (2006) 

Acinetobacter baumannii Bac- HIB, Tween 80, 24 h, 35°C 600–10,000 Hammer et al. (1996) 

Actinobacillus actinomycetemcomitans Bac- HS, 72 h, 37°C 1100 Shapiro et al. (1994) 


Bacteroides sp. Bac- VC, Tween 80 300–5000 Hammer et al. (1999) 

Citrobacter freundii Bac- ISB, Tween 80, 20–24 h, 37°C 5000 Harkenthal et al. (1999) 

Coliform bacilli Bac- MHB, Tween 80, 24 h, 37°C 10,000–20,000 Banes-Marshall et al. (2001) 

Coliform bacilli Bac- BA, 24 h, 37°C 5000 Banes-Marshall et al. (2001) 

Enterobacter aerogenes Bac- ISB, Tween 80, 20–24 h, 37°C 2500 Harkenthal et al. (1999) 

Enterococcus faecalis Bac- ISB, Tween 20, 24 h, 37°C 5000–7500 Griffin et al. (2000) 

Escherichia coli Bac- ISB, Tween 20, 24 h, 37°C 2000 Griffin et al. (2000) 


Escherichia coli Bac- ISB, Tween 80, 16–20 h, 37°C 2500 Gustafson et al. (1998) 

Escherichia coli Bac- ISB, Tween 80, 20–24 h, 37°C 2500 Harkenthal et al. (1999) 



Fusobacterium nucleatum Bac- HS, 72 h, 37°C >6000 Shapiro et al. (1994) 

Fusobacterium sp. Bac- VC, Tween 80 600–2500 Hammer et al. (1999) 

Gardnerella vaginalis Bac- VC, Tween 80 600 Hammer et al. (1999) 

Klebsiella pneumoniae Bac- HIB, Tween 80, 24 h, 35°C 1200–50,000 Hammer et al. (1996) 

Klebsiella pneumoniae Bac- ISB, Tween 80, 20–24 h, 37°C 2500 Harkenthal et al. (1999) 

Klebsiella pneumoniae Bac- ISB, Tween 20, 24 h, 37°C 3000 Griffin et al. (2000) 


Porphyromonas gingivalis Bac- HS, 72 h, 37°C 1100 Shapiro et al. (1994) 

Prevotella sp. Bac- VC, Tween 80 300–2500 Hammer et al. (1999) 

continued




Proteus mirabilis Bac- ISB, Tween 80, 20–24 h, 37°C 2500 Harkenthal et al. (1999) 

Proteus vulgaris Bac- ISB, Tween 20, 24 h, 37°C 3000 Griffin et al. (2000) 

Pseudomonas aeruginosa Bac- MCA, 24 h, 37 >20,000 Banes-Marshall et al. (2001) 

Pseudomonas aeruginosa Bac- ISB, Tween 80, 20–24 h, 37°C >40,000 Harkenthal et al. (1999) 

Pseudomonas aeruginosa Bac- ISB, Tween 20, 24 h, 37°C 10,000– >20,000 Griffin et al. (2000) 

Pseudomonas aeruginosa Bac- MHB, Tween 80, 24 h, 37°C 10,000–80,000 Banes-Marshall et al. (2001) 

Pseudomonas aeruginosa Bac- HIB, Tween 80, 24 h, 35°C 20,000–50,000 Hammer et al. (1996) 

Pseudomonas aeruginosa Bac- MHB, 18–24 h, 37°C 40,000 Papadopoulos et al. (2006) 

Pseudomonas aeruginosa Bac- MHA, Tween 20, 48 h, 35°C 5000 Hammer et al. (1999) 


Pseudomonas fl uorescens Bac- MHB, 18–24 h, 37°C 40,000 Papadopoulos et al. (2006) 

Pseudomonas putida Bac- ISB, Tween 20, 24 h, 37°C >20,000 Griffin et al. (2000) 

Pseudomonas putida Bac- MHB, 18–24 h, 37°C 10,000 Papadopoulos et al. (2006) 

Salmonella choleraesuis Bac- ISB, Tween 80, 20–24 h, 37°C 2500 Harkenthal et al. (1999) 

Salmonella typhimurium Bac- MHA, Tween 20, 48 h, 35°C 5000 Hammer et al. (1999) 

Serratia marcescens Bac- ISB, Tween 20, 24 h, 37°C 1000–3000 Griffin et al. (2000) 

Serratia marcescens Bac- HIB, Tween 80, 24 h, 35°C 2500–50,000 Hammer et al. (1996) 


Shigella fl exneri Bac- ISB, Tween 80, 20–24 h, 37°C 2500 Harkenthal et al. (1999) 

Actinomyces viscosus Bac+ HS, 16–24, 37°C 6000 Shapiro et al. (1994) 

Anaerobic cocci Bac+ VC, Tween 80 600–2500 Hammer et al. (1999) 

Bacillus cereus Bac+ ISB, Tween 20, 24 h, 37°C 3000 Griffin et al. (2000) 

Bacillus subtilis Bac+ ISB, Tween 80, 20–24 h, 37°C 2500 Harkenthal et al. (1999) 

Bacillus subtilis Bac+ ISB, Tween 20, 24 h, 37°C 3000 Griffin et al. (2000) 

Corynebacterium pseudodiphtheriae Bac+ ISB, Tween 80, 20–24 h, 37°C 5000 Harkenthal et al. (1999) 

Corynebacterium sp. Bac+ ISB, Tween 20, 24 h, 37°C 2000–3000 Griffin et al. (2000) 

Corynebacterium sp. Bac+ HIB, Tween 80, 24 h, 35°C 600–20,000 Hammer et al. (1996) 

Enterococcus durans Bac+ ISB, Tween 80, 20–24 h, 37°C 1000 Harkenthal et al. (1999) 

Enterococcus faecalis Bac+ ISB, Tween 80, 20–24 h, 37°C 1000 Harkenthal et al. (1999)


Enterococcus faecalis Bac+ MHA, Tween 20, 48 h, 35°C 10,000 Hammer et al. (1999) 

Enterococcus faecalis Bac+ MHB, Tween 80, 24 h, 37°C 80,000 Banes-Marshall et al. (2001) 

Enterococcus faecium Bac+ ISB, Tween 80, 20–24 h, 37°C 1000 Harkenthal et al. (1999) 


Listeria monocytogenes Bac+ ISB, Tween 80, 20–24 h, 37°C 2500 Harkenthal et al. (1999) 

Micrococcus luteus Bac+ ISB, Tween 20, 24 h, 37°C 2000–3000 Griffin et al. (2000) 

Micrococcus luteus Bac+ HIB, Tween 80, 24 h, 35°C 600–5000 Hammer et al. (1996) 

Micrococcus sp. Bac+ HIB, Tween 80, 24 h, 35°C 600–5000 Hammer et al. (1996) 

Micrococcus varians Bac+ HIB, Tween 80, 24 h, 35°C 5000–10,000 Hammer et al. (1996) 

Mobiluncus sp. Bac+ VC, Tween 80 300–600 Hammer et al. (1999) 

Peptostreptococcus anaerobius Bac+ HS, 72 h, 37°C 2000 Shapiro et al. (1994) 

Peptostreptococcus anaerobius Bac+ VC, Tween 80 600–2500 Hammer et al. (1999) 

Propionibacterium acnes Bac+ Agar, 24 h, 37°C 3100–6300 Raman et al. (1995) 

Propionibacterium acnes Bac+ ISB, Tween 20, 24 h, 37°C 5000 Griffin et al. (2000) 

Staphylococcus aureus Bac+ BA, 24 h, 37°C 10,000 Banes-Marshall et al. (2001) 

Staphylococcus aureus Bac+ HIB, Tween 80, 24 h, 35°C 1200–5000 Hammer et al. (1996) 

Staphylococcus aureus Bac+ ISB, Tween 20, 24 h, 37°C 2000 Griffin et al. (2000) 

Staphylococcus aureus Bac+ MHB, Tween 80, 24 h, 37°C 20,000 Banes-Marshall et al. (2001) 

Staphylococcus aureus Bac+ ISB, Tween 80, 20–24 h, 37°C 2500 Harkenthal et al. (1999) 


Staphylococcus aureus Bac+ MHA, Tween 20, 48 h, 35°C 5000 Hammer et al. (1999) 

Staphylococcus aureus Bac+ MHB, Tween 80, 24 h, 37°C 5000 Banes-Marshall et al. (2001) 

Staphylococcus aureus Bac+ Agar, 24 h, 37°C 6300–12,500 Raman et al. (1995) 

Staphylococcus aureus MRSA Bac+ BA, 24 h, 37°C 10,000 Banes-Marshall et al. (2001) 

Staphylococcus aureus MRSA Bac+ MHB, Tween 80, 24 h, 37°C 20,000–40,000 Banes-Marshall et al. (2001) 

Staphylococcus aureus MRSA Bac+ ISB, Tween 20, 24 h, 37°C 2000–3000 Griffin et al. (2000) 


Staphylococcus aureus MRSA Bac+ Cited 2500 Carson and Messager (2005) 

Staphylococcus aureus MRSA Bac+ HIB, Tween 80, 24 h, 37°c 2500–5000 Carson et al. (1995) 

Staphylococcus capitis Bac+ HIB, Tween 80, 24 h, 35°C 10,000–10,0000 Hammer et al. (1996) 

Staphylococcus capitis Bac+ ISB, Tween 80, 20–24 h, 37°C 1200–2500 Harkenthal et al. (1999) 

continued




Staphylococcus epidermidis Bac+ HIB, Tween 80, 24 h, 35°C 1200–40,000 Hammer et al. (1996) 

Staphylococcus epidermidis Bac+ ISB, Tween 80, 20–24 h, 37°C 2500 Harkenthal et al. (1999) 

Staphylococcus epidermidis Bac+ ISB, Tween 20, 24 h, 37°C 5000 Griffin et al. (2000) 


Staphylococcus epidermidis Bac+ Agar, 24 h, 37°C 6300–12,500 Raman et al. (1995) 

Staphylococcus haemolyticus Bac+ HIB, Tween 80, 24 h, 35°C 10,000–40,000 Hammer et al. (1996) 

Staphylococcus haemolyticus Bac+ ISB, Tween 80, 20–24 h, 37°C 2500–5000 Harkenthal et al. (1999) 

Staphylococcus hominis Bac+ HIB, Tween 80, 24 h, 35°C 10,000–40,000 Hammer et al. (1996) 

Staphylococcus hominis Bac+ ISB, Tween 80, 20–24 h, 37°C 1200 Harkenthal et al. (1999) 

Staphylococcus saprophyticus Bac+ HIB, Tween 80, 24 h, 35°C 20,000–30,000 Hammer et al. (1996) 

Staphylococcus saprophyticus Bac+ ISB, Tween 80, 20–24 h, 37°C 2500–5000 Harkenthal et al. (1999) 

Staphylococcus warneri Bac+ HIB, Tween 80, 24 h, 35°C 20,000–80,000 Hammer et al. (1996) 

Staphylococcus xylosus Bac+ HIB, Tween 80, 24 h, 35°C 10,000–30,000 Hammer et al. (1996) 

Staphylococcus xylosus Bac+ ISB, Tween 80, 20–24 h, 37°C 2500 Harkenthal et al. (1999) 

Streptococci beta-haemolytic Bac+ BA, 24 h, 37°C 1200–5000 Banes-Marshall et al. (2001) 

Streptococci beta-haemolytic Bac+ MHB, Tween 80, 24 h, 37°C 80,000 Banes-Marshall et al. (2001) 

Streptococci beta-haemolytic Gp.D Bac+ MHB, Tween 80, 24 h, 37°C 5000–20,000 Banes-Marshall et al. (2001) 

Streptococci, faecal Bac+ MHB, Tween 80, 24 h, 37°C >80,000 Banes-Marshall et al. (2001) 

Streptococci, faecal Bac+ BA, 24 h, 37°C 10,000 Banes-Marshall et al. (2001) 

Streptococcus equi Bac+ THB, Tween 80, 24 h, 35°C 1200 Carson et al. (1996) 

Streptococcus equisimilis Bac+ THB, Tween 80, 24 h, 35°C 1200 Carson et al. (1996) 

Streptococcus pyogenes Bac+ THB, Tween 80, 24 h, 35°C 1200 Carson et al. (1996) 

Streptococcus pyogenes Bac+ MHB, Tween 80, 24 h, 37°C 20,000 Banes-Marshall et al. (2001) 

Streptococcus sobrinus Bac+ HS, 16–24, 37°C 6000 Shapiro et al. (1994) 

Streptococcus sp. group G Bac+ THB, Tween 80, 24 h, 35°C 1200 Carson et al. (1996) 

Streptococcus zooepidemicus Bac+ THB, Tween 80, 24 h, 35°C 600 Carson et al. (1996) 


Alternaria sp. Fungi Cited data 160–1200 Carson et al. (2006) 

Alternaria sp. Fungi RPMI, Tween 80, 48 h, 30°C 160–1200 Carson and Riley (2002) 

Aspergillus fl avus Fungi Cited data 3100–7000 Carson et al. (2006)


Aspergillus fl avus Fungi MYB, 72 h, 26°C 3120 Shin (2003) 

Aspergillus fl avus Fungi MA, Tween 20, 24 h, 30°C 4000–5000 Griffin et al. (2000) 

Aspergillus fl avus Fungi MA, Tween 20, 24 h, 30°C 5000–7000 Griffin et al. (2000) 

Aspergillus fl avus Fungi RPMI, Tween 80, 48 h, 35°C 600–1200 Carson and Riley (2002) 

Aspergillus fumigatus Fungi RPMI, Tween 80, 48 h, 35°C 600–1200 Carson and Riley (2002) 

Aspergillus niger Fungi Cited data 160–4000 Carson et al. (2006) 

Aspergillus niger Fungi MA, Tween 20, 24 h, 30°C 3000–4000 Griffin et al. (2000) 

Aspergillus niger Fungi MYB, 72 h, 26°C 3120 Shin (2003) 

Aspergillus niger Fungi RPMI, Tween 80, 48 h, 35°C 600–1200 Carson and Riley (2002) 

Aspergillus niger Fungi YES broth, 10 d >10,000 Lis-Balchin et al. (1998) 


Blastoschizomyces capitatus Fungi Cited data 2500 Carson et al. (2006) 

Cladosporium sp. Fungi RPMI, Tween 80, 72 h, 30°C 160–1200 Carson and Riley (2002) 

Cladosporium sp. Fungi Cited data 80–1200 Carson et al. (2006) 

Epidermophyton fl occosum Fungi RPMI, Tween 80, 96 h, 30°C 80–300 Carson and Riley (2002) 

Epidermophyton fl occosum Fungi Cited data 80–7000 Carson et al. (2006) 


Fusarium sp. Fungi Cited data 80–2500 Carson et al. (2006) 

Fusarium sp. Fungi RPMI, Tween 80, 48 h, 35°C 80–2500 Carson and Riley (2002) 

Malassezia sympodialis Fungi Cited data 160–1200 Carson et al. (2006) 

Microsporum canis Fungi Cited data 300–5000 Carson et al. (2006) 

Microsporum canis Fungi RPMI, Tween 80, 96 h, 30°C 40–300 Carson and Riley (2002) 

Microsporum gypseum Fungi RPMI, Tween 80, 96 h, 30°C 160–300 Carson and Riley (2002) 

Penicillium sp. Fungi Cited data 300–600 Carson et al. (2006) 

Penicillium sp. Fungi RPMI, Tween 80, 48 h, 35°C 300–600 Carson and Riley (2002) 

Pleurotus ferulae Fungi SDA, 7 d, 25°C 72–82% inh. 1000 Angelini et al. (2008) 

Pleurotus nebrodensis Fungi SDA, 7 d, 25°C 64–88% inh. 1000 Angelini et al. (2008) 

Pleurotus nebrodensis Fungi SDA, 7 d, 25°C 83–88% inh. 1000 Angelini et al. (2008) 

Trichophyton interdigitale Fungi RPMI, Tween 80, 96 h, 30°C 80–300 Carson and Riley (2002) 

Trichophyton mentagrophytes Fungi Cited data 1100–4400 Carson et al. (2006) 

Trichophyton mentagrophytes Fungi MA, Tween 20, 24 h, 30°C 3000–4000 Griffin et al. (2000) 

Trichophyton mentagrophytes Fungi RPMI, Tween 80, 96 h, 30°C 80–600 Carson and Riley (2002) 

Trichophyton rubrum Fungi MA, Tween 20, 24 h, 30°C 10,000 Banes-Marshall et al. (2001) 

continued




Trichophyton rubrum Fungi Cited data 300–6000 Carson et al. (2006) 

Trichophyton rubrum Fungi RPMI, Tween 80, 96 h, 30°C 80–300 Carson and Riley (2002) 

Trichophyton tonsurans Fungi Cited data 40–160 Carson et al. (2006) 

Trichophyton tonsurans Fungi RPMI, Tween 80, 96 h, 30°C 40–160 Carson and Riley (2002) 

Candida albicans Yeast RPMI, Tween 80, 48 h, 30°C 1250 Oliva et al. (2003) 

Candida albicans Yeast MEB, Tween 20, 24 h, 37 2000 Griffin et al. (2000) 

Candida albicans Yeast RPMI, Tween 80, 48 h, 35°C 2500 Mondello et al. (2006) 

Candida albicans Yeast MHB, Tween 80, 48 h, 35°C 2500–5000 Hammer et al. (1998) 

Candida albicans Yeast MPB, DMSO, 40 h, 30°C 37% inh. 500 Hili et al. (1997) 

Candida albicans Yeast MHA, Tween 20, 48 h, 35°C 5000 Hammer et al. (1999) 

Candida albicans Yeast SDA, 24 h, 37°C 5000–10,000 Banes-Marshall et al. (2001) 

Candida capitatus Yeast RPMI, Tween 80, 48 h, 30°C 1250–2500 Oliva et al. (2003) 

Candida famata Yeast SDA, 24 h, 37°C 2500 Banes-Marshall et al. (2001) 

Candida glabrata Yeast MHB, Tween 80, 48 h, 35°C 1200–5000 Hammer et al. (1998) 

Candida glabrata Yeast SDA, 24 h, 37°C 2500–5000 Banes-Marshall et al. (2001) 

Candida glabrata Yeast RPMI, Tween 80, 48 h, 30°C 300–1250 Oliva et al. (2003) 

Candida glabrata Yeast Cited data 300–8000 Carson et al. (2006) 

Candida glabrata Yeast RPMI, Tween 80, 48 h, 35°C 600 Mondello et al. (2006) 

Candida guilliermondii Yeast RPMI, Tween 80, 48 h, 30°C 125ß Oliva et al. (2003) 

Candida incospigua Yeast RPMI, Tween 80, 48 h, 30°C 300 Oliva et al. (2003) 

Candida krusei Yeast RPMI, Tween 80, 48 h, 30°C 1250 Oliva et al. (2003) 

Candida krusei Yeast RPMI, Tween 80, 48 h, 35°C 2500 Mondello et al. (2006) 

Candida krusei Yeast SDA, 24 h, 37°C 5000 Banes-Marshall et al. (2001) 

Candida lipolytica Yeast RPMI, Tween 80, 48 h, 30°C 600–1250 Oliva et al. (2003)


Candida lusitaniae Yeast RPMI, Tween 80, 48 h, 30°C 1250 Oliva et al. (2003) 

Candida parapsilosis Yeast RPMI, Tween 80, 48 h, 35°C 1250 Mondello et al. (2006) 

Candida parapsilosis Yeast MHB, Tween 80, 48 h, 35°C 2500–5000 Hammer et al. (1998) 

Candida parapsilosis Yeast Cited data 300–5000 Carson et al. (2006) 

Candida parapsilosis Yeast RPMI, Tween 80, 48 h, 30°C 600–1250 Oliva et al. (2003) 

Candida sp. Yeast MHB, Tween 80, 48 h, 35°C 1200–5000 Hammer et al. (1998) 

Candida sp. Yeast MHB, Tween 80, 24 h, 37°C 5000 Banes-Marshall et al. (2001) 

Candida tropicalis Yeast Cited data 1200–20,000 Carson et al. (2006) 

Candida tropicalis Yeast RPMI, Tween 80, 48 h, 35°C 600 Mondello et al. (2006) 

Cryptococcus neoformans Yeast Cited data 150–600 Carson et al. (2006) 

Cryptococcus neoformans Yeast RPMI, Tween 80, 48 h, 35°C 300 Mondello et al. (2006) 

Malassezia furfur Yeast MMA, Tween 20, 7 d 1200–2500 Hammer et al. (2000) 

Malassezia furfur Yeast Cited data 300–1200 Carson et al. (2006) 

Malassezia globosa Yeast MMA, Tween 20, 7 d 300–1200 Hammer et al. (2000) 

Malassezia obtusa Yeast MMA, Tween 20, 7 d 1200 Hammer et al. (2000) 

Malassezia slooffi ae Yeast MMA, Tween 20, 7 d 1200–2500 Hammer et al. (2000) 

Malassezia sympodialis Yeast MMA, Tween 20, 7 d 160–2500 Hammer et al. (2000) 

Rhodotorula rubra Yeast Cited data 600 Carson et al. (2006) 

Saccharomyces cerevisiae Yeast MEB, Tween 20, 24 h, 37°C 2000 Griffin et al. (2000) 

Saccharomyces cerevisiae Yeast Cited data 2500 Carson et al. (2006) 



Schizosaccharomyces pombe Yeast MPB, DMSO, 40 h, 30°C 74% inh. 500 Hili et al. (1997) 


Trichophyton tonsurans Yeast Cited data 1200–2200 Carson et al. (2006)


TABLE 12.77 

Inhibitory Data of Tea Tree Oil Obtained in the Vapor Phase Test 


Escherichia coli Bac- BLA, 18 h, 37°C MIC air 50 Inouye et al. (2001) 














Rhizoctonia solani Fungi PDA, 3 d, 25°C 1000 +++ Lee et al. (2007)


TABLE 12.78 

Inhibitory Data of Thyme Oil Obtained in the Agar Diffusion Test 













Escherichia coli Bac- NA, 24 h, 37°C 8 (h), pure 16 Fawzi (1991) 
















continued





Pseudomonas aeruginosa Bac- NA, 24 h, 37°C 8 (h), pure 8 Fawzi (1991) 



Pseudomonas aeruginosa Bac- ISA, 48 h, 25°C 4 (h), 10,000 22.5 Deans and Ritchie (1987) 









Bacillus cereus Bac+ NA, 24 h, 37°C 8 (h), pure 21 Fawzi (1991) 











Lactobacillus sp. Bac+ MRS, cited 9, 20,000 25– >90 Pellecuer et al. (1980) 





Micrococcus luteus Bac+ MHA, cited 9, 20,000 40 Pellecuer et al. (1980)


Micrococcus ureae Bac+ MHA, cited 9, 20,000 40 Pellecuer et al. (1980) 


Sarcina ureae Bac+ MHA, cited 9, 20,000 >90 Pellecuer et al. (1980) 




Staphylococcus aureus Bac+ NA, 24 h, 37°C 8 (h), pure 19 Fawzi (1991) 

Staphylococcus aureus Bac+ NA, 18 h, 37°C 5 (h), -30,000 24 Schelz et al. (2006) 







Streptococcus D Bac+ MHA, cited 9, 20,000 17– >90 Pellecuer et al. (1980) 

Streptococcus faecalis Bac+ ISA, 48 h, 25°C 4 (h), 10,000 19.5 Deans and Ritchie (1987) 

Streptococcus micros Bac+ MHA, cited 9, 20,000 >90 Pellecuer et al. (1980) 








Aspergillus niger Fungi SDA, 3 d, 30 8 (h), pure 35 Fawzi (1991) 







continued







Candida albicans Yeast SDA, 24 h, 37°C 8 (h), pure 37 Fawzi (1991) 

























TABLE 12.79 

Inhibitory Data of Thyme Oil Obtained in the Dilution Test 


Bacteria Bac 5% Glucose, 9 d, 37 500–1000 Buchholtz (1875) 

Acinetobacter baumannii Bac- MHA, Tween 20, 48 h, 35°C 1200 Hammer et al. (1999) 




Enterococcus faecalis Bac- M17, 24 h, 20°C >10,000 Di Pasqua et al. (2005) 



Escherichia coli Bac- TSB, Tween 80, 48 h, 35°C 2 Panizzi et al. (1993) 

Escherichia coli Bac- NB, 16 h, 37°C 200 Lens-Lisbonne et al. (1987) 

Escherichia coli Bac- TSB, 3% EtOH, 24 h, 37°C 3000–8000 Rota et al. (2004) 

Escherichia coli Bac- LA, 18 h, 37°C 375–500 Remmal et al. (1993) 

Escherichia coli Bac- NB, 24 h, Tween 20, 37 400 Fawzi (1991) 

Escherichia coli Bac- TGB, 18–24 h, 37°C 500 Morris et al. (1979) 



Escherichia coli Bac- MHB, DMSO, 24 h, 37°C 62.5 Al-Bayati (2008) 

Escherichia coli Bac- TSB, 24 h, 37°C 700 Di Pasqua et al. (2005) 



Escherichia coli Bac- MHB, 24 h, 36°C >10,000 Oussalah et al. (2006) 

Escherichia coli O157:H7 Bac- BHI, 48 h, 35°C >8000 Oussalah et al. (2006) 






continued





Klebsiella pneumoniae Bac- MHB, DMSO, 24 h, 37°C 500 Al-Bayati (2008) 





Proteus mirabilis Bac- MHB, DMSO, 24 h, 37°C 62.5 Al-Bayati (2008) 

Proteus vulgaris Bac- MHB, DMSO, 24 h, 37°C 31.2 Al-Bayati (2008) 


Pseudomonas aeruginosa Bac- TSB, Tween 80, 48 h, 35°C >40 Panizzi et al. (1993) 

Pseudomonas aeruginosa Bac- MHB, DMSO, 24 h, 37°C >500 Al-Bayati (2008) 

Pseudomonas aeruginosa Bac- NB, 24 h, Tween 20, 37°C >50,000 Fawzi (1991) 


Pseudomonas aeruginosa Bac- NB, 16 h, 37°C 800 Lens-Lisbonne et al. (1987) 


Pseudomonas sp. Bac- TSB, 24 h, 37°C 1500 Di Pasqua et al. (2005) 


Salmonella enteritidis Bac- TSB, 3% EtOH, 24 h, 37°C 4000–8000 Rota et al. (2004) 

Salmonella haddar Bac- LA, 18 h, 37°C 500 Remmal et al. (1993) 

Salmonella typhi Bac- MHB, DMSO, 24 h, 37°C 250 Al-Bayati (2008) 



Salmonella typhimurium Bac- MHB, DMSO, 24 h, 37°C 125 Al-Bayati (2008) 


Salmonella typhimurium Bac- TSB, 24 h, 37°C 300 Di Pasqua et al. (2005) 



Salmonella typhimurium Bac- TSB, 3% EtOH, 24 h, 37°C 5000 Rota et al. (2004) 

Serratia marcescens Bac- NA, 1–3 d, 30°C 1250 Farag et al. (1989)



Shigella fl exneri Bac- TSB, 3% EtOH, 24 h, 37°C 3000–8000 Rota et al. (2004) 

Yersinia enterocolitica Bac- TSB, 3% EtOH, 24 h, 29°C 3000–5000 Rota et al. (2004) 

Bacillus cereus Bac+ NB, 24 h, Tween 20, 37°C >50,000 Fawzi (1991) 

Bacillus cereus Bac+ MHB, DMSO, 24 h, 37°C 15.6 Al-Bayati (2008) 

Bacillus megaterium Bac+ LA, 18 h, 37°C 375–500 Remmal et al. (1993) 

Bacillus subtilis Bac+ TSB, Tween 80, 48 h, 35°C 2 Panizzi et al. (1993) 



Brochotrix thermosphacta Bac+ M17, 24 h, 20°C 2500 Di Pasqua et al. (2005) 



Enterococcus faecalis Bac+ MHA, Tween 20, 48 h, 35°C 5000 Hammer et al. (1999) 


Lactobacillus delbrueckii Bac+ MRS, 24 h, 37°C >10,000 Di Pasqua et al. (2005) 

Lactobacillus plantarum Bac+ MRS, 24 h, 30°C >10,000 Di Pasqua et al. (2005) 

Lactobacillus sp. Bac+ MRS, cited 310–620 Pellecuer et al. (1980) 

Lactococcus garvieae Bac+ M17, 24 h, 20°C >10,000 Di Pasqua et al. (2005) 

Lactococcus lactis subsp. lactis Bac+ M17, 24 h, 20°C >10,000 Di Pasqua et al. (2005) 

Listeria monocytogenes Bac+ TSB, 3% EtOH, 24 h, 37°C 8000 Oussalah et al. (2006) 

Listeria monocytogenes Bac+ BHI, 48 h, 35°C >8000 Oussalah et al. (2006) 

Listeria monocytogenes Bac+ TSB, 24 h, 37°C 1000 Di Pasqua et al. (2005) 






Micrococcus luteus Bac+ MHB, cited 150 Pellecuer et al. (1980) 


Micrococcus ureae Bac+ MHB, cited 150 Pellecuer et al. (1980) 

continued







Sarcina ureae Bac+ MHB, cited 78 Pellecuer et al. (1980) 

Staphylococcus aureus Bac+ TSB, 3% EtOH, 24 h, 37°C



Alternaria citri Fungi PDA, 8 d, 22°C 500 Arras and Usai (2001) 

Aspergillus chevalieri Fungi Cited 250 Pellecuer et al. (1976) 

Aspergillus clavatus Fungi Cited 1000 Pellecuer et al. (1976) 

Aspergillus fl avus Fungi Cited 1000 Pellecuer et al. (1976) 





Aspergillus fl avus Fungi CA, 7 d, 28°C 74–76% inh. 500 Kumar et al. (2007) 


Aspergillus fumigatus Fungi RPMI, 1.5% EtOH, 7 d, 30°C 2500 Tullio et al. (2006) 

Aspergillus giganteus Fungi Cited 1000 Pellecuer et al. (1976) 


Aspergillus niger Fungi SB, 72 h, Tween 20, 37°C 200 Fawzi (1991) 

Aspergillus niger Fungi RPMI, 1.5% EtOH, 7 d, 30°C 2500–5000 Tullio et al. (2006) 




Aspergillus oryzae Fungi Cited 2000 Pellecuer et al. (1976) 




Aspergillus repens Fungi Cited 250 Pellecuer et al. (1976) 

Botrytis cinera Fungi PDA, 8 d, 22°C 500 Arras and Usai (2001) 


Cladosporium herbarum Fungi Cited 1000 Pellecuer et al. (1976) 





Microsporum canis Fungi MBA, Tween 80, 10 d, 30°C 12.5– >300 Perrucci et al. (1994) 


Microsporum gypseum Fungi RPMI, 1.5% EtOH, 7 d, 30°C 1250–2500 Tullio et al. (2006) 

Microsporum gypseum Fungi MBA, Tween 80, 10 d, 30°C 25– >300 Perrucci et al. (1994) 



Mucor mucedo Fungi Cited 1000 Pellecuer et al. (1976) 


Mucor sp. Fungi RPMI, 1.5% EtOH, 7 d, 30°C >10,000 Tullio et al. (2006) 

Penicillium chrysogenum Fungi Cited 1000 Pellecuer et al. (1976) 

Penicillium digitatum Fungi PDA, 8 d, 22°C 1000 Arras and Usai (2001) 


Penicillium italicum Fungi PDA, 8 d, 22°C 1000 Arras and Usai (2001) 

Penicillium lanosum Fungi RPMI, 1.5% EtOH, 7 d, 30°C 2500 Tullio et al. (2006) 

Penicillium liliacinum Fungi Cited 500 Pellecuer et al. (1976) 

Penicillium rubrum Fungi Cited 1000 Pellecuer et al. (1976) 







Rhizopus nigricans Fungi Cited 500 Pellecuer et al. (1976) 






Scopulariopsis brevicaulis Fungi Cited 1000 Pellecuer et al. (1976) 


Syncephalastrum racemosum Fungi Cited 500 Pellecuer et al. (1976)


Trichophyton interdigitale Fungi Cited 1000 Pellecuer et al. (1976) 



TABLE 12.80 

Inhibitory Data of Thyme Oil Obtained in the Vapor Phase Test 



Escherichia coli Bac- BLA, 18 h, 37°C MIC air 12.5 Inouye et al. (2001) 




Salmonella typhi Bac- NA, 24 h, 37°C sd ++ Maruzzella and Sicurella (1960) 


Bacillus subtilis var. aterrimus Bac+ NA, 24 h, 37°C sd ++ Maruzzella and Sicurella (1960) 


Lactobacillus sp. Bac+ MRS, cited Disk, 20,000 ? ++ Pellecuer et al. (1980) 

Micrococcus luteus Bac+ MHB, cited Disk, 20,000 ? ++ Pellecuer et al. (1980) 

Micrococcus ureae Bac+ MHB, cited Disk, 20,000 ? ++ Pellecuer et al. (1980) 

Mycobacterium avium Bac+ NA, 24 h, 37°C sd + Maruzzella and Sicurella (1960) 

Sarcina ureae Bac+ MHB, cited Disk, 20,000 ? + Pellecuer et al. (1980) 


Staphylococcus aureus Bac+ NA, 24 h, 37°C sd ++ Maruzzella and Sicurella (1960) 

Staphylococcus aureus Bac+ MHA, 18 h, 37°C MIC air 6.25–12.5 Inouye et al. (2001) 

Staphylococcus epidermidis Bac+ MHB, cited Disk, 20,000 ? ++ Pellecuer et al. (1980) 

Streptococcus D Bac+ MHB, cited Disk, 20,000 ? ++ Pellecuer et al. (1980) 


Streptococcus faecalis Bac+ NA, 24 h, 37°C sd ++ Maruzzella and Sicurella (1960) 

Streptococcus pneumoniae Bac+ MHA, 18 h, 37°C MIC air 3.13–6.25 Inouye et al. (2001) 


Streptococcus pyogenes Bac+ MHA, 18 h, 37°C MIC air 3.13–6.25 Inouye et al. (2001) 

Alternaria alternata Fungi RPMI, 7 d, 30°C MIC air 78 Tullio et al. (2006) 



Aspergillus fl avus Fungi RPMI, 7 d, 30°C MIC air 156–312 Tullio et al. (2006)




Aspergillus niger Fungi RPMI, 7 d, 30°C MIC air 78–156 Tullio et al. (2006) 


Cladosporium cladosporoides Fungi RPMI, 7 d, 30°C MIC air 78 Tullio et al. (2006) 

Colletotrichum gleosporoides Fungi PDA, 3 d, 25°C 1000 + Lee et al. (2007) 





Eurotium repens Fungi Bread, 14 d, 25°C 30,000 NG Suhr and Nielsen (2003) 


Fusarium oxysporum Fungi PDA, 3 d, 25°C 1000 + Lee et al. (2007) 



Microsporum gypseum Fungi RPMI, 7 d, 30°C MIC air 78 Tullio et al. (2006) 




Penicillium frequentans Fungi RPMI, 7 d, 30°C MIC air 156 Tullio et al. (2006) 

Penicillium lanosum Fungi RPMI, 7 d, 30°C MIC air 312 Tullio et al. (2006) 



Rhizoctonia solani Fungi PDA, 3 d, 25°C 1000 + Lee et al. (2007) 


Scopulariopsis brevicaulis Fungi RPMI, 7 d, 30°C MIC air 78 Tullio et al. (2006) 

Trichophyton mentagrophytes Fungi RPMI, 7 d, 30°C MIC air 39–78 Tullio et al. (2006) 



12.2 RESULTS 

The results of antimicrobial testing of the 28 essential oils listed in the European Pharmacopoeia 

6th edition are shown in Tables 12.1 through 12.80. Although literature is not fully covered, the 

quantity of available information was in part unexpected. 

ANISE OIL, ANISI AETHEROLEUM 

Definition: Essential oil obtained by steam distillation of the dry ripe fruits of Pimpinella 

anisum L. 

BITTER-FENNEL FRUIT OIL, FOENICULI AMARI FRUCTUS AETHEROLEUM 

Definition: Essential oil obtained by steam distillation of the ripe fruits of Foeniculum vulgare 

Miller, ssp. vulgare var. vulgare. 

Content: fenchone: 12–25%, trans-anethole: 55–75%. 

CARAWAY OIL, CARVI AETHEROLEUM 

Definition: Essential oil obtained by steam distillation of the dry fruits of Carum carvi L. 

CASSIA OIL, CINNAMOMI CASSIA AETHEROLEUM 

Definition: Cassia oil is obtained by steam distillation of the leaves and young branches of 

Cinnamomum cassia Blume (Cinnamomum aromaticum Nees). 

CEYLON CINNAMON BARK OIL, CINNAMOMI ZEYLANICII CORTICIS AETHEROLEUM 

Definition: Ceylon cinnamon bark oil is obtained by steam distillation of the bark of the shoots of 

Cinnamomum zeylanicum Nees (Cinnamomum verum J.S. Presl.). 

CEYLON CINNAMON LEAF OIL, CINNAMOMI ZEYLANICI FOLII AETHEROLEUM 

Definition: Essential oil obtained by steam distillation of the leaves of Cinnamomum verum J.S. 

Presl. 

CITRONELLA OIL, CITRONELLAE AETHEROLEUM 

Definition: Essential oil obtained by steam distillation of the fresh or partially dried aerial parts of 

Cymbopogon winterianus Jowitt. 

CLARY SAGE OIL, SALVIAE SCLAERAE AETHEROLEUM 

Definition: Essential oil obtained by steam distillation of the fresh or dried flowering stems of 

Salvia sclarea L. 

CLOVE OIL, CARYOPHYLLI FLORIS AETHEROLEUM 

Definition: Clove oil is obtained by steam distillation of the dried flower buds of Syzygium aromaticum 

(L.) Merill et L. M. Perry (Eugenia caryophyllus C. Spreng. Bull. et Harr.).


CORIANDER OIL, CORIANDRI AETHEROLEUM 

Definition: Essential oil obtained by steam distillation of the fruits of Coriandrum sativum L. 

DWARF-PINE OIL, PINI PUMILIONIS AETHEROLEUM 

Definition: Essential oil obtained by steam distillation of the fresh needles, twigs, and branches of 

Pinus mugo ssp. mugo ZENARI and/or Pinus mugo ssp. pumilio (HAENKE) FRANCO. 

Pinus mugo Turra [Pinus mugo var. pumilio (Haenke) Zenari], plant part: leaves and twigs. 

EUCALYPTUS OIL, EUCALYPTI AETHEROLEUM 

Definition: Eucalyptus oil is obtained by steam distillation and rectification of the fresh leaves or the 

fresh terminal branchlets of various species of Eucalyptus rich in 1,8-cineole. The species mainly 

used are Eucalyptus globulus Labill., Eucalyptus polybractea R.T. Baker, and Eucalyptus smithii 

R.T. Baker. 

JUNIPER OIL, JUNIPERI AETHEROLEUM 

Definition: Essential oil obtained by steam distillation of the ripe, nonfermented berry cones of 

Juniperus communis L. 

LAVENDER OIL, LAVANDULAE AETHEROLEUM 

Definition: Essential oil obtained by steam distillation of the flowering tops of Lavandula angustifolia 

Miller (Lavandula offi cinalis Chaix). 

LEMON OIL, LIMONIS AETHEROLEUM 

Definition: Essential oil obtained by suitable mechanical means, without the aid of heat, from the 

fresh peel of Citrus limon (L.) Burman fil. 

MANDARIN OIL, CITRI RETICULATAE AETHEROLEUM 

Definition: Essential oil obtained without heating, by suitable mechanical treatment of the fresh 

peel of the fruit of Citrus reticulata Blanco var. mandarin. 

MATRICARIA OIL, MATRICARIAE AETHEROLEUM 

Definition: Blue essential oil obtained by steam distillation of the fresh or dried flower heads or 

flowering tops of Matricaria recutita L. (Chamomilla recutita L. Rauschert). There are two 

types of matricaria oils, which are characterized as rich in bisabolol oxides or rich in 

(-)-a-bisabolol. 

MINT OIL, MENTHAE ARVENSIS AETHEROLEUM PARTIM MENTHOL PRIVUM 

Definition: Essential oil obtained by steam distillation of the fresh, flowering aerial parts, 

recently gathered from Mentha canadensis L. [syn. Mentha arvensis L. var. glabrata (Benth) Fern., 

Mentha arvensis var. piperascens Malinv. ex Holmes], followed by partial separation of menthol by 

crystallization.


NEROLI OIL, NEROLI AETHEROLEUM 

Definition: Neroli oil is obtained by steam distillation of the fresh flowers of Citrus aurantium L. 

subsp. aurantium L. (Citrus aurantium L. subsp. amara Engl.). 

NUTMEG OIL, MYRISTICAE FRAGRANTIS AETHEROLEUM 

Definition: Nutmeg oil is obtained by steam distillation of the dried and crushed kernels of Myristica 

fragrans Houtt. 

PEPPERMINT OIL, MENTHAE PIPERITAE AETHEROLEUM 

Definition: Essential oil obtained by steam distillation of the fresh aerial parts of the flowering plant 

of Mentha piperita L. 

PINUS SYLVESTRIS OIL, PINI SYLVESTRIS AETHEROLEUM 

Definition: Essential oil obtained by steam distillation of the fresh needles, twigs, and branches of 

Pinus sylvestris L. 

TURPENTINE OIL, PINUS PINASTER TYPE, TEREBINTHI AETHEROLEUM AB PINUM PINASTRUM 

Definition: Essential oil obtained by steam distillation, followed by rectification at a temperature 

below 180°C, from the oleoresin obtained by tapping Pinus pinaster Aiton. 

ROSEMARY OIL, ROSMARINUM AETHEROLEUM 

Definition: Essential oil obtained by steam distillation of the flowering aerial parts of Rosmarinus 

offi cinalis L. 

STAR ANISE OIL, ANISI STELLATI AETHEROLEUM 

Definition: Essential oil obtained by steam distillation of the dry ripe fruits of Illicium verum 

Hook. fil. 

SWEET ORANGE OIL, AURANTII DULCIS AETHEROLEUM 

Definition: Essential oil obtained without heating, by suitable mechanical treatment of the fresh 

peel of the fruit of Citrus sinensis (L.) Osbeck (Citrus aurantium L. var. dulcis L.). 

TEA TREE OIL, MELALEUCAE AETHEROLEUM 

Definition: Essential oil obtained by steam distillation of the foliage and terminal branchlets of 

Melaleuca alternifolia (Maiden and Betch) Cheel, Melaleuca linariifolia Smith, Melaleuca dissitifl 

ora F. Mueller, and/or other species of Melaleuca. 

THYME OIL, THYMI AETHEROLEUM 

Definition: Essential oil obtained by steam distillation of the fresh flowering aerial parts of Thymus 

vulgaris L., T. zygis Loefl. ex L. or a mixture of both species.


12.3 DISCUSSION 

The in vitro most antimicrobially active essential oils regularly (or normally) contain substances as 

main components, which are themselves known to exhibit pronounced antimicrobial properties. 

These are cinnamic aldehyde (cinnamon bark and cassia oil) and the phenolic compounds eugenol 

(clove and cinnamon leaf oil) and thymol (thyme oil) (Pauli, 2001). All these essential oils reveal a 

broadband spectrum of activity in various in vitro test systems (agar diffusion, dilution, and vapor 

phase) due to their considerable water solubility and volatility. The evaluated antimicrobial inhibitory 

data of the essential oils obtained in agar dilution tests (ADT), serial dilution tests (DIL), and 

vapor phase (VP) tests are summarized in Table 12.81. 

A few essential oils exhibit limited activities and act against a class of microorganism, for example, 

anise and bitter fennel oil specifically inhibit the growth of filamentous fungi (Table 12.81), or 

act well against microbial species belonging to an identical genus, for example, caraway oil inhibits 

Trichophyton species. Some essential oils are active only in a specific test system against a defined 

group of microorganism, for example, dwarf-pine oil and juniper oil preferably inhibit gram-positive 

bacteria in the vapor phase, but both were of low activity in the agar diffusion or dilution tests. 

Similarly, citronella oil was not inhibitory toward nine different fungal species in the agar dilution 

test, but inhibited all of them in the vapor phase test (Nakahara et al., 2003). Yeasts turned out to be 

susceptible toward essential oil vapors, which is in agreement to observations made with volatile 

esters and monoterpenes. Possibly, in yeast, the biosynthesis of chitin is inhibited by voilatile compounds 

emitted from plants (Pauli, 2006). 

The inhibitory data itself differ considerably from each other when results of different examiners 

are compared as it can be seen, for example, by the activities of lemon oil toward Escherichia coli 

(5 tests inactive and 4 tests active) in the agar diffusion test (Table 12.43) or by the MIC values 

of rosemary oil against Staphylococcus aureus (Table 12.67), which cover a range from 20 to 

50,000 μg/mL in nine examinations. Even if the unit of the low value of 20 was confused—this 

might had happened in references Panizzi et al. (1993) and Pellecuer et al. (1980)—the activity 

range of rosemary oil is 400–50,000 μg/mL. Taken together, exceptionally the above-mentioned 

relatively strong-acting essential oils, the results are not coordinated and cover frequently the complete 

activity spectrum from inactive to strong active (evaluation from 0 to 3 in Table 12.81). In part, 

the results are contradictory and no general rules can be raised from the data shown in Tables 12.1 

through 12.80. 

Several reasons can be made responsible for this undesirable, but at least normal situation: 

• Natural variability in the composition of essential oils. 

• Natural variability in the susceptibility of microorganism. 

• Different parameters in microbiological testing methods. 

• Unknown history of the tested essential oils: production, storage, and age. 

• Unsufficient knowledge about exact phytochemical composition. 

The composition of essential oils depends on several factors: plant part used, place of growth, climate, 

natural variation (varieties, subspecies, and chemotypes), harvesting time, production, storage 

conditions, and analysis parameters in compound identification. Because some of these influencing 

factors differ from year to year, no constant composition of an essential can be expected, even when 

it is grown and produced at the same place. Chemotypes possess in part a completely different composition, 

for example, the MICs of four thyme oil chemotypes (linalool-, thuyanol-, carvacrol, and 

thymol-type) toward S. aureus (Table 12.79) differ from 250 to 4000 μg/mL and low MIC values 

depended on the presence of thymol (Oussalah et al., 2006). In some literature works, the botanical 

description of investigated plant material is not defined exactly. The characterization of “fennel oil” 

is not sufficient to decide between sweet or bitter fennel. The same is true for orange oil, where 

sweet orange (Citrus sinensis) and bitter orange (Citrus aurantium) refer to two different botanical


TABLE 12.81 

Summary of Results of Antimicrobial Activities of Essential Oils Listed in the European 

Pharmacopoeia 6th Edition 

Essential Oil Bac- Bac+ Fungi Yeast 

Test Method ADT DIL VP ADT DIL VP ADT DIL VP ADT DIL VP 

Anise 0–1 0–3 0–2 0–1 0–3 0–2 2–3 0–3 0–2 0–1 1–2 0 

Bitter fennel 0–2 1–2 0–3 0–2 1–3 0–2 0–2 2–3 ND 0–2 2 3 

Caraway 0–1 0–3 0–3 0–1 1–3 0–3 1–3 0–3 ND 0–1 2 3 

Cassia 2 2–3 1–3 2 3 1–3 3 1–3 ND ND 3 3 

Cinnamon bark 1–2 3 1–3 1–3 3 1–3 2–3 2–3 0–3 2–3 2–3 3 

Cinnamon leaf 2–3 2 3 2–3 2 3 3 1–3 0–3 2 2 3 

Citronella 0–1 1–2 0–3 0–1 2–3 0–3 1–3 1–3 3 1–2 1–3 3 

Clary sage 0–1 0–1 0–3 1–2 0–3 0–3 0–1 0–2 0 0 0–2 0 

Clove 0–2 0–3 1–3 1–3 0–3 0–3 1–3 0–3 3 1–3 0–2 3 

Coriander 0–2 0–2 3 0–2 1–3 0–3 0–2 0–3 ND 0–2 2 3 

Dwarf pine 0–1 ND 0–3 0–1 ND 2–3 0 ND ND 0–1 0 1 

Eucalyptus 0–1 0–1 0–3 0–1 1 0–3 0–1 0–2 0 1 2 3 

Juniper 0–1 0–2 1–3 0–1 0 2–3 0–1 0–2 0 0–1 2 3 

Lavender 0–1 0–3 0–3 0–3 1–3 0–3 0–2 0–3 3 0–1 2 3 

Lemon 0–1 0–1 0–3 0–1 0–2 0–3 0–1 0–3 0–1 0–1 2 3 

Mandarin 0–1 0–2 ND 0–1 0–1 ND 1 ND 0–2 0–1 2 ND 

Matricaria 0–1 0–3 0 0–1 0–3 1–2 0–1 0–2 0 0–1 0–2 0 

Mint 0–3 2–3 ND 0–1 2–3 ND 0–3 2 ND 1 2 ND 

Neroli 0–1 0 0–3 0–1 1–2 0–3 1–2 1–3 ND 0–1 2 3 

Nutmeg 0–1 0–2 0 0–1 0–3 0–1 0–3 0–3 ND 0–1 2 ND 

Peppermint 0–1 0–3 0–3 0–1 1–3 2–3 0–3 0–3 1–3 0–1 2–3 2 

Pinus sylvestris 0–1 0–1 ND 1 0–2 ND 1 0–2 0 1 1–2 ND 

Pinus pinaster 0–1 0–2 1–3 0–1 2 1–3 0–1 2 ND 0–1 0–3 2 

Rosemary 0–1 0–3 0–3 0–2 0–3 0–3 0–2 0–2 0–3 0–1 0–2 3 

Star anise 0–1 ND 2–3 0–1 ND 2–3 ND ND 0–3 0–1 2–3 3 

Sweet orange 0–2 0–3 0–3 0–2 0–2 0–3 0–2 0–3 0–3 0–1 2 3 

Tea tree 1 0–3 0–3 1–2 0–3 0–3 1 1–3 0 1–3 1–3 ND 

Thyme 1–2 1–3 2–3 0–3 0–3 1–3 1–3 1–3 0–3 1–3 2–3 3 

Evaluation: Agar diffusion test (ADT): 0 = inactive, 1 = weak (most of the inhibition zones up to ~15 mm), 2 = moderate 

(most of the inhibition zones between 16 and 30 mm), 3 = strong inhibitory (most of the inhibition 

zones >30 mm); dilution test (DIL): 0 = MIC > 20,000 μg/mL, 1 = >5000–20,000 μg/mL, 2 = 500–5000 μg/ 

mL, 3 =


Questions concerning disinfectant activity of essential oils, for example, the minimum time 

needed to kill a given microbial species or the determination of microbial survivors after short time 

contact, are not answered by agar diffusion or dilution tests. In older literature, the killing concentration 

relative to phenol was determined after 15 or 30 min exposure of the respective microbials 

species to the compound to be tested. The so-called carboxylic acid coefficient or phenol coefficient 

was introduced in 1903 (Rideal et al., 1903) and was also taken for the characterization of the 

killing activity of essential oils toward microorganism (Martindale, 1910). 

Differences in the susceptibility exist between organisms of the identical species as it was shown 

in experiments with three Escherichia coli strains. One of them was inhibited by eugenol methyl 

ether at a low concentration (MIC = 550 μg/mL), while two other strains tolerated still 8000 μg/mL 

without any visible growth reduction. Remarkably the MIC of eugenol toward all three strains was 

almost equal (550–600 μg/mL) (Pauli, 1994). Because these Escherichia coli strains never had had 

the opportunity to develop a specific resistance against eugenol methyl ether before, it is evident that 

a natural variation of susceptibility toward natural antimicrobials exists. 

To improve the data situation of in vitro antimicrobial data of essential oils, all aforementioned 

biological and experimental parameters should be controlled as best as possible. An appropriate 

microbiological test system should be taken, which allows comparison of inhibitory data with drugs 

used in the therapy of human infectious diseases. Such worked out and standardized serial dilution 

tests (Clinical & Laboratory Standards Institute 2008) are already utilized in the examination of 

natural substances, for example Hostettmann et al. (1999) and Jirovetz et al. (2007). To avoid 

complications by strains with unknown susceptibilities toward antimicrobials and to make the 

results from different laboratories comparable to each other, available standard strains from collections 

(e.g., American Type Culture Collection, ATCC; Deutsche Sammlung für Mikroorganismen 

und Zellkulturen, DSMZ; and Institute for Fermentation Osaka, IFO) should be taken in the routine 

analysis of antimicrobial activities of natural compounds and essential oils. Antimicrobials tests 

should include a greater number of different organisms belonging to the groups: gram-positive, 

gram-negative bacteria, filamentous fungi, and yeasts. 

Two principal reasons for performing the in vitro tests are as follows: 

1. Identification of antimicrobially active compounds. 

2. Control of microbial susceptibilities toward approved antibiotics and antimycotics. 

The procedure from identification of antimicrobially active compounds to their use in humans to 

treat infectious diseases is a multistep pathway, which includes pharmacological (concentration of 

the active compound at the site of action, half-life time, serum levels, dose–response relationship, 

etc.) and toxicological (e.g., toxicity, allergic responses, and interactions) aspects. 

Essential oils consist frequently of over 100 individual compounds, which themselves plus their 

metabolic transformation products cannot be followed up in the living body. This fact may explain 

why pharmacological studies with entire essential oils never have been in the focus of pharmaceutical 

research. 

In animal experiments, it was demonstrated by Imura over 80 years ago that the in vivo protection 

against tuberculosis was not parallel with results in vitro (Table 12.82). 

The superior protection of tuberculosis-infected guinea pigs by anethole and lemon oil is 

contradictory to their weak in vitro antitubercular activity. By means of these results obtained 

parallelly in vivo and in vitro it is obvious that in vitro inhibitory data alone cannot be used as 

an information basis for the treatment of infectious diseases in humans (Table 12.81). Therefore, 

the recording of so-called aromatograms with patient isolates and a greater number of essential 

oils—as it is common in “aromatherapy”—is critical (Anonymus, 2008; DHZ-Spektrum, 2007). 

The selection of the most active essential oil by using in vitro testing methods for the therapy of 

infectious diseases may have success, but at least there is no rational relationship between in vitro 

testing and in vivo success (Table 12.81). Besides that, many factors influence the results obtained


TABLE 12.82 

Comparison of In Vitro Growth Inhibition of Human Type of 

Mycobacterium tuberculosis with the In Vivo Protection of 

Tuberculosis-Infected Guinea Pigs by Essential Oils and 

Components Thereof 

Test Materials 

Lymphnodes 

In Vivo a 

Viscera 

In Vitro b 

Anethole 33 50 2500 

Lemon oil 49 55 1250 

Terpineol 59 57 625 

Nutmeg oil, expressed 61 73 312 

Geraniol 96 71 625 

Eugenol 89 84 1250 

Source: Imura, K., 1935. J. Shanghai Sci. Institute/Section, 4, 1: 235–270. 

a 

Grade of tuberculous change in percent was observed after 10 weeks in lymphnodes and 

viscera in groups of five infected guinea pigs fed with ~250 mg/kg (recalculated from 

experimental section) test material per day for 1 week, respectively. 

b 

Growth reduction (dry weight tubercle bacilli 5 mg) by a given concentration in μg/mL 

determined in glycerin-bouillon after 3 weeks incubation at 37°C. 

in the agar diffusion test and may lead to wrong interpretation of the results concerning the 

antimicrobial strength of an essential oil. 

It seems to be suitable to discuss a special status of essential oils in concern of their pharmacological 

activities, since essential oils cannot be followed up by classical pharmacological methods 

due to their complex nature. Recently, successes of wound treatment with essential oils have 

been demonstrated under clinical conditions, which cannot be realized with pharmaceuticals 

(Warnke et al., 2005). In another case, a deep infection of a hip arthoplasty was successfully 

treated with a pure compound occurring in matricaria oil: (-)-a-bisabolol (Pauli et al., 2007, 

2009). Due to its lipophilic nature (-)-a-bisabolol is thought to be taken up by the skin and it 

enters the blood circulatory system, which should be measureable with pharmacological methods. 

Interestingly, the toxicity of essential oils toward mammalians decreases significantly with 

increase of average lipophilicity of their components (Pauli, 2008), while simultaneously the toxicity 

toward bacteria and fungi increases significantly with increasing lipophilicity (Pauli, 2007), 

which points to the extraordinary role of essential oils among natural compounds, especially of 

their highly lipophilic constituents. 

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13 

Aromatherapy with 



CONTENTS 

13.1 Introduction ..................................................................................................................... 550 

13.1.1 Aromatherapy Practice in the United Kingdom and the United States ............. 550 

13.2 Definitions of Aromatherapy .......................................................................................... 550 

13.3 Introduction to Aromatherapy Concepts ......................................................................... 551 

13.3.1 Aromatherapy, Aromatology, and Aromachology ............................................. 551 

13.3.2 Scientifically Accepted Benefits of Essential Oils versus the 

Lack of Evidence for Aromatherapy .................................................................. 551 

13.4 Historical Background to Aromatherapy ........................................................................ 553 

13.4.1 Scented Plants used as Incense in Ancient Egypt ............................................. 554 

13.5 Perfume and Cosmetics: Precursors of Cosmetological Aromatherapy ......................... 554 

13.5.1 Three Methods of Producing Perfumed Oils by the Egyptians ........................ 555 

13.6 Medicinal Uses: Precursors of Aromatology or “Clinical” Aromatherapy .................... 555 

13.6.1 Middle Ages: Use of Aromatics and Quacks ..................................................... 556 

13.7 Modern Perfumery .......................................................................................................... 557 

13.8 Aromatherapy Practice ................................................................................................... 557 

13.8.1 Methods of Application of Aromatherapy Treatment ........................................ 558 

13.9 Massage using Essential Oils .......................................................................................... 559 

13.9.1 Massage Techniques .......................................................................................... 559 

13.10 Aromatherapy: Blending of Essential Oils ..................................................................... 561 

13.10.1 Fixed Oils ........................................................................................................... 561 

13.11 Internal Usage of Essential Oils by Aromatherapists ..................................................... 561 

13.12 Use of Pure or Synthetic Components ............................................................................ 562 

13.13 Therapeutic Claims for the Application of Essential Oils .............................................. 562 

13.13.1 False Claims Challenged in Court ..................................................................... 563 

13.14 Physiological and Psychological Responses to Essential Oils and Psychophysiology .... 563 

13.15 Placebo Effect of Aromatherapy ..................................................................................... 564 

13.16 Safety Issue in Aromatherapy ......................................................................................... 565 

13.17 Toxicity in Humans ......................................................................................................... 566 

13.17.1 Increase in Allergic Contact Dermatitis in Recent Years .................................. 566 

13.17.2 Photosensitizers .................................................................................................. 567 

13.17.3 Commonest Allergenic Essential Oils and Components ................................... 567 

13.17.4 Toxicity in Young Children: A Special Case ..................................................... 568 

13.17.5 Selected Toxicities of Common Essential Oils and Their Components ............ 568 

13.18 Clinical Studies of Aromatherapy .................................................................................. 569 

13.19 Recent Clinical Studies ................................................................................................... 570 

13.19.1 Aromatherapy in Dementia ................................................................................ 570 

549


13.20 Past Clinical Studies ....................................................................................................... 571 

13.20.1 Critique of Selected Clinical Trials ................................................................... 572 

13.21 Use of Essential Oils Mainly as Chemical Agents and Not for Their Odor ................... 575 

13.21.1 Single-Case Studies .......................................................................................... 576 

13.22 Conclusion ....................................................................................................................... 576 

References .................................................................................................................................. 577 


13.1.1 AROMATHERAPY PRACTICE IN THE UNITED KINGDOM AND THE UNITED STATES 

Aromatherapy has become more of an art than a science. This is mostly due to the health and beauty 

industries, which have taken over the original concept as a money-spinner in the United Kingdom, 

United States, and almost all other parts of the world. There are virtually thousands of “aromatherapy” 

products in pharmacies, high street shops, supermarkets, hair salons, and beauty salons. 

The products are supposedly made with “essential oils” (which are usually perfumes) and include 

skin creams, hair shampoos, shower gels, moisturizers, bath salts, lotions, candles, as well as essential 

oils themselves. 

Many aromatherapy products, such as perfumes, are also linked with sexual attractiveness. There 

are numerous “health and beauty” salons or clinics that offer aromatherapy as part of their “treatments” 

together with waxing, electrolysis, massage (of various types, including “no-hands massage”), 

facial treatments including botox, manicures and pedicures, eyes and eyebrow shaping, ear-piercing, 

tanning, and makeup application. Often hundreds of these “therapies” are offered in one small shop, 

with aromatherapy thrown in. Most people, especially men, consider aromatherapy to be a sensual 

massage with some perfumes given all over the body by a young lady. This is often the case, although 

aromatherapy massage is often provided just on the back or even just on the face and hands for busy 

people. The use of pure essential oils both in such beauty massage and all the aromatherapy products 

on sale everywhere is very doubtful (because of the cost) but the purchaser believes the advertisements 

assuring pure oil usage. Beauty consultants/therapists use massage skills and a nice odor 

simply for relaxation; they sometimes include beautifying treatments using specific essential oils as 

initiated by Marguerite Maury (1989). Aromatherapy has thus become an art. 

However, aromatherapists (who have studied the “science” for 3 h, a week, a year, or even did a 

3-year degree) are keen to bring science into this alternative “treatment.” The multitude of books 

written on the subject, aromatherapy journals, and the web sites all consider that there has been 

enough proof of the scientific merit of aromatherapy. They quote studies that have shown no positive 

or statistically significant effects as proof that aromatherapy works. The actual validity of these 

claims will be discussed later and several publications criticized this on scientific grounds. 

Aromatherapy is often combined with “counseling” by a “qualified” therapist, with no counseling 

qualifications. Massaging is carried out using very diluted plant essential oils (2–5 drops per 10 mL 

of carrier oil, such as almond oil) on the skin—that is, in almost homeopathic dilutions! But they 

believe that the essential oils are absorbed and go straight to the target organ where they exert the 

healing effect. Many aromatherapists combine their practice with cosmology, crystals, colors, music, 

and so on. These may also be associated with a commercial sideline in selling “own trademark” 

essential oils and associated items, including diffusers, scented candles, and scented jewelry. 

13.2 DEFINITIONS OF AROMATHERAPY 

Aromatherapy is defined as “the use of aromatic plant extracts and essential oils in massage and other 

treatment” (Concise Oxford Dictionary, 1995). However, there is no mention of massage or the 

absorption of essential oils through the skin and their effect on the target organ (which is the mainframe

Aromatherapy with Essential Oils 551 

of aromatherapy in the United Kingdom and the United States) in Aromatherapie (Gattefossé, 1937/1993). 

This was where the term “aromatherapy” was coined after all, by the “father of aromatherapy”—but 

was actually based on the odor of essential oils and perfumes and their antimicrobial, physiological, and 

cosmetological properties (Gattefossé, 1928, 1952, 1937/1993). “Pure” essential oils were of no concern 

to Gattefossé. Recently, definitions have begun to encompass the effects of aromatherapy on the mind 

as well as on the body (Lawless, 1994; Worwood, 1996, 1998; Hirsch, 1998). 

13.3 INTRODUCTION TO AROMATHERAPY CONCEPTS 

The original concept of modern aromatherapy was based on the assumption that the volatile, fatsoluble 

essential oil was equivalent in bioactivity to that of the whole plant when inhaled or massaged 

onto the skin. Information about the medicinal and other properties of the plants was taken 

from old English herbals (e.g., Culpeper, 1653), combined with some more esoteric nuances involving 

the planets and astrology (Tisserand, 1977). 

This notion is clearly flawed. As an example, a whole orange differs from just the essential oil 

(extracted from the rind alone) as the water-soluble vitamins (thiamine, riboflavin, nicotinic acid, and 

vitamins C and A) are excluded, as are calcium, iron, proteins, carbohydrates, and water. Substantial 

differences in bioactivity are found in different fractions of plants, for example, the essential oils of 

Pelargonium species produced a consistent relaxation of the smooth muscle of the guinea pig in vitro, 

whereas the water-soluble extracts did not (Lis-Balchin, 2002b). Botanical misinterpretations are 

also common in many aromatherapy books, for example, “geranium oil” bioactivity is based on Herb 

Robert, a hardy Geranium species found widely in European hedgerows, whereas geranium oil is 

distilled from species of the South African genus Pelargonium (Lis-Balchin, 2002a). 

13.3.1 AROMATHERAPY, AROMATOLOGY, AND AROMACHOLOGY 

Aromatherapy can now be divided into three “sciences”: aromatherapy, aromatology, and 

aromachology. 

Aromachology [coined by the Sense of Smell Institute (SSI), USA, 1982] is based on the interrelationship 

of psychology and odor, that is, its effect on specific feelings (e.g., relaxation, exhilaration, 

sensuality, happiness, and achievement) by its direct effect on the brain. 

Aromatherapy is defined by the SSI as “the therapeutic effects of aromas on physical conditions 

(such as menstrual disorders, digestive problems, etc.) as well as psychological conditions (such as 

chronic depression).” The odor being composed of a mixture of fat-soluble chemicals may thus have 

an effect on the brain via inhalation, skin absorption, or even directly via the nose. 

Aromatology is concerned with the internal use of oils (SSI). This is similar to the use of aromatherapy 

in most of Europe, excluding the United Kingdom; it includes the effect of the chemicals 

in the essential oils via oral intake, or via the anus, vagina, or any other possible opening by medically 

qualified doctors or at least herbalists, using essential oils as internal medicines. 

There is a vast difference between aromatherapy in the United Kingdom and that in continental 

Europe (aromatology): the former is “alternative” while the latter is “conventional.” The “alternative” 

aromatherapy is largely based on “healing,” which is largely based on belief (Millenson, 1995; 

Benson and Stark, 1996; Lis-Balchin, 1997). This is credited with a substantial placebo influence. 

However, the placebo effect can be responsible for results in both procedures. 

13.3.2 SCIENTIFICALLY ACCEPTED BENEFITS OF ESSENTIAL OILS VERSUS THE LACK OF EVIDENCE 

FOR AROMATHERAPY 

There is virtually no scientific evidence, as yet, regarding the direct action of essential oils, applied 

through massage on the skin, on specific internal organs—rather than through the odor pathway 

leading into the mid-brain’s “limbic system” and then through the normal sympathetic and


parasympathetic pathways. This is despite some evidence that certain components of essential oils 

can be absorbed either through the skin or lungs (Buchbauer et al., 1992; Jager et al., 1992; Fuchs 

et al., 1997). 

Many fragrances have been shown to have an effect on mood and, in general, pleasant odors 

generate happy memories, more positive feelings, and a general sense of well-being (Knasko et al., 

1990; Knasko, 1992; Warren and Warrenburg, 1993) just like perfumes. Many essential oil vapors 

have been shown to depress contingent negative variation (CNV) brain waves in human volunteers 

and these are considered to be sedative (Torii et al., 1988). Others increase CNV and are considered 

stimulants (Kubota et al., 1992). An individual with anosmia showed changes in cerebral blood flow 

on inhaling certain essential oils, just as in people able to smell (Buchbauer et al., 1993c), showing 

that the oil had a positive brain effect despite the patient’s inability to smell it. There is some evidence 

that certain essential oils (e.g., nutmeg) can lower high blood pressure (Warren and Warrenburg, 

1993). Externally applied essential oils (e.g., tea tree) can reduce/eliminate acne (Bassett et al., 

1990) and athlete’s foot (Tong et al., 1992). This happens, however, using conventional chemical 

effects of essential oils rather than aromatherapy. 

Most clients seeking out aromatherapy are suffering from some stress-related conditions, and 

improvement is largely achieved through relaxation. An alleviation of suffering and possibly pain, 

due to gentle massage and the presence of someone who cares and listens to the patient, could be 

beneficial in such cases as in cases of terminal cancer; the longer the time spent by the therapist with 

the patient, the stronger the belief imparted by the therapist and the greater the willingness of the 

patient to believe in the therapy, the greater the effect achieved (Benson and Stark, 1996). There is 

a need for this kind of healing contact, and aromatherapy with its added power of odor fits this 

niche, as the main action of essential oils is probably on the primitive, unconscious, limbic system 

of the brain (Lis-Balchin, 1997), which is not under the control of the cerebrum or higher centers 

and has a considerable subconscious effect on the person. However, as mood and behavior can be 

influenced by odors, and memories of past odor associations could also be dominant, aromatherapy 

should not be used by aromatherapists, unqualified in psychology, and so on in the treatment of 

Alzheimer’s or other diseases of aging (Lis-Balchin, 2006). 

Proven uses of essential oils and their components are found in industry, for example, foods, 

cosmetic products, household products, and so on. They impart the required odor or flavor to food, 

cosmetics and perfumery, tobacco, and textiles. Essential oils are also used in the paint industry, 

which capitalizes on the exceptional “cleaning” properties of certain oils. This, together with their 

embalming properties, suggests that essential oils are very potent and dangerous chemicals—not 

the sort of natural products to massage into the skin! 

Why, therefore, should essential oils be of great medicinal value? They are, after all, just 

chemicals. However, essential oils have many functions in everyday life ranging from their use 

in dentistry (e.g., cinnamon and clove oils), as decongestants (e.g., Eucalyptus globulus, camphor, 

peppermint, and cajuput) to their use as mouthwashes (e.g., thyme), also external usage as 

hyperemics (e.g., rosemary, turpentine, and camphor) and anti-inflammatories (e.g., German 

chamomile and yarrow). Some essential oils are used internally as stimulants of digestion (e.g., 

anise, peppermint, and cinnamon) and as diuretics (e.g., buchu and juniper oils) (Lis-Balchin, 

2006). 

Many plant essential oils are extremely potent antimicrobials in vitro (Deans and Ritchie, 1987; 

Bassett et al., 1990; Lis-Balchin, 1995; Lis-Balchin et al., 1996; Deans, 2002). Many are also strong 

antioxidant agents and have recently been shown to stop some of the symptoms of aging in animals 

(Dorman et al., 1995a, 1995b). The use of camphor, turpentine oils, and their components as rubefacients, 

causing increased blood flow to a site of pain or swelling when applied to the skin, is well 

known and is the basis of many well-known medicaments such as Vicks VapoRub and Tiger Balm. 

Some essential oils are already used as orthodox medicines: peppermint oil is used for treating 

irritable bowel syndrome and some components of essential oils, such as pinene, limonene, camphene, 

and borneol, given orally have been found to be effective against certain internal ailments,


such as gallstones (Somerville et al., 1985) and ureteric stones (Engelstein et al., 1992). Many essential 

oils have been shown to be active on many different animal tissues in vitro (Lis-Balchin et al., 

1997b). There are many examples of the benefits of using essential oils by topical application for 

acne, Alopecia areata, and Athlete’s foot (discussed later in Section 13.21), but this is a treatment 

using chemicals rather than aromatherapy treatment. 

Future scientific studies, such as those on Alzheimer’s syndrome (Perry et al., 1998, 1999), may 

reveal the individual benefits of different essential oils for different ailments, but in practice this 

may not be of utmost importance as aromatherapy massage for relief from stress. Aromatherapy has 

had very little scientific evaluation to date. As with so many alternative therapies, the placebo effect 

may provide the largest percentage benefit to the patient (Benson and Stark, 1996). Many aromatherapists 

have not been greatly interested in scientific research and some have even been antagonistic 

to any such research (Vickers, 1996; Lis-Balchin, 1997). Animal experiments, whether maze 

studies using mice or pharmacology using isolated tissues, are considered unacceptable and only 

essential oils that are “untested on animals” are acceptable, despite all essential oils having been 

already tested on animals (denied by assurances of essential oil suppliers) because this is required 

by law before they can be used in foods. 

The actual mode of action of essential oils in vivo is still far from clear, and clinical studies to 

date have been scarce and mostly rather negative (Stevenson, 1994; Dunn et al., 1995; Brooker et al., 

1997; Anderson et al., 2000). The advent of scientific input into the clinical studies, rather than 

aromatherapist-led studies, has recently yielded some more positive and scientifically acceptable 

data (Smallwood et al., 2001; Ballard et al., 2002; Burns et al., 2000; Holmes et al., 2002; Kennedy 

et al., 2002). The main difficulty in clinical studies is that it is virtually impossible to do randomized 

double-blind studies involving different odors as it is almost impossible to provide an adequate 

control as this would have to be either odorless or else of a different odor, neither of which is satisfactory. 

In aromatherapy, as practiced, there is a variation in the treatment for each client, based on 

“holistic” principles, and each person can be treated by an aromatherapist with one to five or more 

different essential oil mixtures on subsequent visits, involving one to four or more different essential 

oils in each mixture. This makes scientific evaluation almost useless, as seen by studies during 

childbirth (Burns and Blaney, 1994; see also Section 13.19). There is also the belief among alternative 

medicine practitioners that if the procedure “works” in one patient, there is no need to study 

it using scientific double-blind procedures. There is therefore a great bias when clinical studies in 

aromatherapy are conducted largely by aromatherapists. 

Recent European regulations (the seventh Amendment to the European Cosmetic Directive 

76/768/EEC, 2002; see Appendices 27 and 28) have listed 26 sensitizers found in most of the 

common essential oils used: this could be a problem for aromatherapists as well as clients, both in 

possibly causing sensitization and also resulting in legal action regarding such an eventuality in the 

case of the client. Care must be taken regarding the sensitization potential of the essential oils, especially 

when massaging patients with cancer or otherwise sensitive skin. It should also be borne in 

mind when considering the use of essential oils during childbirth and in other clinical studies (Burns 

and Blaney, 1994; Burns et al., 2000) that studies in animals have indicated that some oils cause a 

decrease in uterine contractions (Lis-Balchin and Hart, 1997). 

13.4 HISTORICAL BACKGROUND TO AROMATHERAPY 

The advent of “aromatherapy” has been attributed to both the Ancient Egyptians and Chinese over 

4500 years ago, as scented plants and their products were used in religious practices, as medicines, 

perfumes, and embalming agents (Manniche, 1989, 1999), and to bring out greater sexuality (Schumann 

Antelme and Rossini, 2001). But essential oils as such were unlikely to have been used. In Ancient 

Egypt, crude plant extracts of frankincense, myrrh, or galbanum, and so on were used in an oily 

vegetable or animal fat that was massaged onto the bodies of workers building the pyramids or the 

rich proletariat after their baths (Manniche, 1999). These contained essential oils, water-soluble


extractives, and pigments. Incense smoke from resinous plant material provided a more sacrosanct 

atmosphere for making sacrifices, both animal and human, to the gods. The incense was often 

mixed with narcotics like cannabis to anesthetize the sacrificial animals, especially with humans 

(Devereux, 1997). The frankincense extract in oils (citrusy odor) was entirely different to that burnt 

(church-like) in chemical composition (Arctander, 1960), and therefore would have entirely different 

functions. 

13.4.1 SCENTED PLANTS USED AS INCENSE IN ANCIENT EGYPT 

Frankincense (Boswellia carterii; Boswellia thurifera) (Burseraceae), Myrrh (Commiphora myrrha; 

Balsamodendron myrrha; Balsamodendron opobalsamum) (Burseraceae), Labdanum (Cistus 

ladaniferus), Galbanum (Ferula galbaniflua), Styrax (Styrax officinalis), or Liquidambar orientalis, 

Balm of Gilhead (Commiphora opobalsamum), Sandalwood (Santalum album), and Opoponax 

(Opoponax chironium). 

Uses included various concoctions of kyphi, burnt three times a day to the sun god Ra: morning, 

noon, and sunset, in order for him to come back. The ingredients included raisins, juniper, cinnamon, 

honey, wine, frankincense, myrrh, burnt resins, cyperus, sweet rust, sweet flag, and aspalanthus 

in a certain secret proportion (Loret, 1887; Manniche, 1989; Forbes, 1955), as shown on the 

walls of the laboratory in the temples of Horus at Edfu and Philae. Embalming involved odorous 

plants such as juniper, cassia, cinnamon, cedarwood, and myrrh, together with natron to preserve 

the body and ensure safe passage to the afterlife. The bandages in which the mummy was wrapped 

were drenched in stacte (oil of myrrh) and sprinkled with other spices (for further descriptions and 

uses, see Lis-Balchin, 2006). 

The Chinese also used an incense, hsiang, meaning “aromatic,” made from a variety of plants, 

with sandalwood being particularly favored by Buddhists. In India, fragrant flowers including jasmine 

and the root of spikenard giving a sweet scent were used. The Hindus obtained cassia from 

China and were the first to organize trading routes to Arabia where frankincense was exclusively 

found. The Hebrews traditionally used incense for purification ceremonies. The use of incense probably 

spread to Greece from Egypt around the eighth century bc. The Indians of Mesoamerica used 

copal, a hard, lustrous resin, obtained from pine trees and various other tropical trees by slicing the 

bark (Olibanum americanum). Copal pellets bound to corn-husk tubes would be burnt in hollows on 

the summits of holy hills and mountains, and these places, blackened by centuries of such usage, are 

still resorted to by today’s Maya in Guatemala (Janson, 1997) and used medicinally to treat diseases 

of the respiratory system and the skin. 

Anointing also involves incense (Unterman, 1991). Queen Elizabeth II underwent the ritual in 

1953 at her coronation, with a composition of oils originated by Charles I: essential oils of roses, 

orange blossom, jasmine petals, sesame seeds, and cinnamon combined with gum benzoin, musk, 

civet, and ambergris were used (Ellis, 1960). Similarly, musk, sandalwood, and other fragrances 

were used by the Hindus to wash the effigies of their gods, and this custom was continued by the 

early Christians. This probably accounts for the divine odor frequently reported when the tombs of 

early Christians were opened (Atchley and Cuthbert, 1909). The Christian Church was slow to 

adopt the use of incense until medieval times, when it was used for funerals (Genders, 1972). The 

reformation reversed the process as it was considered to be of pagan origin but it still survives in the 

Roman Catholic Church. Aromatic substances were also widely used in magic (Pinch, 1994). 

13.5 PERFUME AND COSMETICS: PRECURSORS OF COSMETOLOGICAL 

AROMATHERAPY 

The word “perfume” is derived from the Latin per fumare: “by smoke.” The preparation of perfumes 

in Ancient Egypt was done by the priests, who passed on their knowledge to new priests 

(Manniche, 1989, 1999). Both high-class people like Nefertiti and Cleopatra used huge amounts of


fragranced materials as unguents, powders, and perfumes and the workers building the great pyramids, 

who even went on strike when they were denied their allocation of “aromatherapy massage 

oil” (Manniche, 1999). 

13.5.1 THREE METHODS OF PRODUCING PERFUMED OILS BY THE EGYPTIANS 

Enfleurage involved steeping the flowers or aromatics in oils or animal fats (usually goat) until the 

scent from the materials was imparted to the fat. The impregnated fat was often molded into cosmetic 

cones and used for perfuming hair wigs, worn on festive occasions, which could last for 3 

days; the fat would soften and start melting, spreading the scented grease not only over the wig, but 

also over the clothes and body—more pleasing than the stench of stale wine, food, and excrement 

(Manniche, 1999). 

Maceration was used principally for skin creams and perfumes: flowers, herbs, spices, or resins 

were chopped up and immersed in hot oils. The oil was strained and poured into alabaster (calcite) 

containers and sealed with wax. These scented fatty extracts were also massaged onto the skin 

(Manniche, 1999). 

Expression involved putting flowers or herbs into bags or presses, which extracted the aromatic 

oils. Expression is now only used for citrus fruit oils (Lis-Balchin, 1995). Wine was often included 

in the process and the resulting potent liquid was stored in jars. These methods are still used 

today. 

Megaleion, an Ancient Greek perfume described by Theophrastus who believed it to be good for 

wounds, was made of burnt resins and balanos oil, and boiled for 10 days before adding cassia, cinnamon, 

and myrrh (Groom, 1992). Rose, marjoram, sage, lotus flower, and galbanum perfumes 

were also made. Apart from these, aromatic oils from basil, celery, chamomile, cumin, dill, fenugreek, 

fir, henna, iris, juniper, lily, lotus, mandrake, marjoram, myrtle, pine, rose, rue, and sage 

were sometimes used in perfumes or as medicines taken internally and externally. 

Dioscorides, in his De Materia Medica, discussed the components of perfumes and their medicinal 

properties, providing detailed perfume formulae. Alexandrian chemists were divided into three 

schools, one of which was the school of Maria the Jewess, which produced pieces of apparatus for 

distillation and sublimation, such as the bain Marie, useful for extracting the aromatic oils from 

plant material. Perfumes became more commonly known in medieval Europe as knights returning 

from the Crusades brought back musk, floral waters, and a variety of spices. 

13.6 MEDICINAL USES: PRECURSORS OF AROMATOLOGY OR “CLINICAL” 

AROMATHERAPY 

The ancient use of plants, not essential oils, can be found in fragments of Egyptian herbals. The 

names of various plants, their habitats, characteristics, and the purposes for which they were used 

are included in the following: Veterinary papyrus (ca. 2000 b.c.), Gynaecological papyrus (ca. 

2000 b.c.), Papyrus Edwin Smith (an army surgeon’s manual, ca. 1600 b.c.), Papyrus Ebers 

(includes remedies for health, beauty, and the home, ca. 1600 b.c.), Papyrus Hearst (with prescriptions 

and spells, ca. 1400 b.c.), and Demotic medical papyri (second century b.c. to first 

century a.d.). 

Magic was often used as part of the treatment and gave the patient the expectation of a cure and 

thus provided a placebo effect (Pinch, 1994). The term “placing the hand” appears frequently in a 

large number of medical papyri; this probably alludes to the manual examination in order to reach 

a diagnosis but could also imply cure by the “laying on of hands,” or even both (Nunn, 1997). This 

could be the basis of modern massage (with or without aromatherapy). It is certainly the basis of 

many alternative medicine practices at present (Lis-Balchin, 1997). 

Plants were used in numerous ways. Onions were made into a paste with wine and inserted into 

the vagina to stop a woman menstruating. Garlic ointment was used to keep away serpents and


snakes, heal dog-bites, and bruises; raw garlic was given to asthmatics; fresh garlic and coriander in 

wine was a purgative and an aphrodisiac! Juniper mixed with honey and beer was used orally to 

encourage defecation; and origanum was boiled with hyssop for a sick ear (Manniche, 1989). 

Egyptians also practiced inhalation by using a double-pot arrangement whereby a heated 

stone was placed in one of the pots and a liquid herbal remedy poured over it. The second pot, 

with a hole in the bottom through which a straw was inserted, was placed on top of the first pot, 

allowing the patient to breathe in the steaming remedy (Manniche, 1989), that is, aromatherapy 

by inhalation. 

13.6.1 MIDDLE AGES: USE OF AROMATICS AND QUACKS 

In the twelfth century, the Benedictine Abbess Hildegard of Bingen (1098–1179) was authorized by 

the Church to publish her visions on medicine (Causae et Curae), dealing with the causes and remedies 

for illness (Brunn and Epiney-Burgard, 1989). The foul smell of refuse in European towns in 

the seventeenth century was thought to be the major cause of disease, including the plague (Classen 

et al., 1994), and aromatics were used for both preventing and in some cases curing diseases; herbs 

such as rosemary were in great demand and sold for exorbitant prices as a prophylactic against the 

plague (Wilson, 1925). People forced to live near victims of the plague would carry a pomander, 

which contained a mixture of aromatic plant extracts. Medical practitioners carried a small cassolette 

or “perfume box” on the top of their walking sticks, when visiting contagious patients, which 

was filled with aromatics (Rimmel, 1865). Some physicians wore a device filled with herbs and 

spices over their nose when they examined plague patients (Wilson, 1925). These became known as 

“beaks” and it is from this that the term “quack” developed. 

Apothecaries were originally wholesale merchants and spice importers, and in 1617 the 

Worshipful Society of Apothecaries was formed, under the control of the London Royal College of 

Physicians, which produced an “official” pharmacopoeia specifying the drugs the apothecaries 

were allowed to dispense. The term “perfumer” occurs in some places instead of “apothecary” 

(Rimmel, 1865). 

John Gerard (1545–1612) and Nicholas Culpeper (1616–1654) were two of the better-known 

apothecaries of their time. Nicholas Culpeper combined healing herbs with astrology as he believed 

that each plant, like each part of the body, and each disease, was governed or under the influence of 

one of the planets: rosemary was believed to be ruled by the Sun, lavender by Mercury, and spearmint 

by Venus. Culpeper also adhered to the Doctrine of Signatures, introduced by Paracelsus in the 

sixteenth century, and mythology played a role in many of the descriptive virtues in Culpeper’s 

herbal. This astrological tradition is carried through by many aromatherapists today, together with 

other innovations such as ying and yang, crystals, and colors. 

Culpeper’s simple or distilled waters and oils (equivalent to the present hydrosols) were prepared 

by the distillation of herbs in water in a pewter still, and then fractionating them to separate out the 

essential or “chymical” oil from the scented plants. The plant waters were the weakest of the herbal 

preparations and were not regarded as being beneficial. Individual plants such as rose or elderflower 

were used to make the corresponding waters, or else mixtures of herbs were used to make compound 

waters (Culpeper, 1826/1981; Tobyn, 1997). Essential oils of single herbs were regarded by 

Culpeper as too strong to be taken alone, due to their vehement heat and burning, but had to be 

mixed with other medicinal preparations. Two or three drops were used in this way at a time. 

Culpeper mentioned the oils of wormwood, hyssop, marjoram, the mints, oregano, pennyroyal, 

rosemary, rue, sage, thyme, chamomile, lavender, orange, and lemon. Spike lavender, not Lavandula 

angustifolia, is used in aromatherapy nowadays. Herbs such as dried wormwood and rosemary were 

also steeped in wine and set in the sun for 30–40 days to make a “physical wine.” The “herbal 

extracts” mentioned in the herbals were mostly water soluble and at best, alcoholic extracts, none of 

which are equivalent to essential oils, which contain many potent chemical components are not 

found in essential oils.


13.7 MODERN PERFUMERY 

In the fourteenth century, alcohol was used for the extraction and preservation of plants, and oleum 

mirable, an alcoholic extract of rosemary and resins, was later popularized as “Hungary water,” 

without the resins (Müller et al., 1984). 

In the sixteenth century, perfumes were made using animal extracts, which were the base notes 

or fixatives, and made the scent last longer (Piesse, 1855). Among these ingredients were ambergris, 

musk, and civet. 

Perfumes came into general use in England during the reign of Queen Elizabeth (1558–1603). 

Many perfumes, such as rose water, benzoin, and storax, were used for sweetening the heavy ornate 

robes of the time, which were impossible to wash. Urinals were treated with orris powder, damask 

rose powder, and rose water. Bags of herbs, musk, and civet were used to perfume bath water. 

Elizabeth I carried a pomander filled with ambergris, benzoin, civet, damask rose, and other 

perfumes (Rimmel, 1865) and used a multitude of perfumed products in later life. Pomanders, from 

the French pomme d’amber (“ball of ambergris”), were originally hung in silver perforated balls 

from the ceiling to perfume the room. The ingredients such as benzoin, amber, labdanum, storax, 

musk, civet, and rose buds could be boiled with gum tragacanth and kneaded into balls; the small 

ones were made into necklaces. 

Various recipes were used for preparing aromatic waters, oils, and perfumes. Some of these were 

for perfumes and some undoubtedly for alcoholic beverages, as one of the major ingredients for many 

concoctions was a bottle or two of wine, which when distilled produced a very alcoholic brew. 

Ambergris, musk, and civet went out of fashion, as the excremental odors could not be reconciled 

with modesty (Corbin, 1986). The delicate floral perfumes became part of the ritual of bodily 

hygiene, gave greater variety, and allowed Louis XV a different perfume every day. Today the sentiment 

“odours are carried in bottles, for fear of annoying those who do not like them” (Dejeans, 

1764) is reemerging as more and more people are becoming sensitive to odors, giving them headaches, 

asthma, and migraines. 

The Victorians liked simple perfumes made of individual plant extracts. Particular favorites 

were rose, lavender, and violet. These would be steam distilled or extracted with solvents. The 

simple essential oils produced would often be blended together to produce perfumes like eau de 

Cologne (1834). 

The first commercial scent production was produced in the United Kingdom, in Mitcham, Surrey, 

in the seventeenth century, using lavender (Festing, 1989). In 1865, cinnamaldehyde, the first 

synthetic, was made. Adulteration and substitution by the essential oil or component of another 

plant species became rampant. Aroma chemicals synthesized from coal, petroleum by-products, 

and terpenes are much cheaper than the equivalent plant products, so perfumes became cheap. 

The way was now open for the use of scent in the modern era. It seems therefore a retrograde step 

to use pure essential oils in “aromatherapy,” especially as the “father of aromatherapy,” René- 

Maurice Gattefossé, used scents or deterpenated essential oils. 

13.8 AROMATHERAPY PRACTICE 

Aromatherapists usually treat their clients (patients) after an initial full consultation, which usually 

involves taking down a full medical case history. The aromatherapist then decides what treatment 

to give, which usually involves massage with three essential oils, often one each chosen from those 

with top, middle, and base perfumery notes, which balances the mixture. Sometimes only “specific” 

essential oils for the “disease” are used. Most aromatherapists arrange to see the client 3–5 times 

and the mixture will often be changed on the next visit, if not on each visit, in order to treat all 

the possible symptoms presented by the client (holistically), or simply as a substitute when no 

improvement was initially obtained. Treatment may involve other alternative medicine procedures, 

including chakras.


Many aromatherapists offer to treat any illness, as they are convinced that essential oils have 

great powers. They embark on the treatment of endometriosis, infertility, asthma, diabetes, and 

arthritis, even cancer, as they are convinced of the therapeutic nature of essential oils, but are often 

without the necessary scientific and medical knowledge. “Psychoneuroimmunology” treatment is 

the current buzzword. 

Although aromatherapists consider themselves professionals, there is no Hippocratic oath 

involved. The aromatherapist, being nonmedically qualified, may not even be acquainted with most 

of the illnesses or symptoms, so there could be a very serious mistake made as potentially serious 

illnesses could be adversely affected by being “treated” by a layperson. Some, but not all, aromatherapists 

ask the patients to tell their doctor of the aromatherapy treatment. Counseling is greatly 

recommended by aromatherapy schools. Aromatherapists are not necessarily, however, trained in 

counseling, and with few exceptions could do more damage than good, especially when dealing 

with psychiatric illness, cases of physical or drug abuse, people with learning difficulties, and so on, 

where their “treatment” should only be complementary and under a doctor’s control (Lis-Balchin, 

2006). 

13.8.1 METHODS OF APPLICATION OF AROMATHERAPY TREATMENT 

Various methods are used to apply the treatment in aromatherapy. The most usual methods are the 

following: 

• A diffuser, usually powered by electricity, giving out a fine mist of the essential oil. 

• A burner, with water added to the fragrance to prevent burning of the essential oil. About 

1–4 drops of essential oil are added to about 10 mL water. The burner can be warmed by 

candles or electricity. The latter would be safer in a hospital or a children’s room or even a 

bedroom. 

• Ceramic or metal rings, placed on an electric light bulb with a drop or two of essential oil. 

This results in a rapid burnout of the oil and lasts for a very short time due to the rapid volatilization 

of the essential oil in the heat. 

• A warm bath with drops of essential oil added. This results in the slow volatilization of the 

essential oil, and the odor is inhaled via the mouth and nose. Any effect is not likely to be 

through the absorption of the essential oil through the skin as stated in aromatherapy 

books, as the essential oil does not mix with water. Droplets either form on the surface of 

the water, often coalescing, or else the essential oil sticks to the side of the bath. Pouring in 

an essential oil mixed with milk serves no useful purpose as the essential oil still does not 

mix with water, and the premixing of the essential oil in a carrier oil, as for massage, just 

results in a nasty oily scum around the bath. 

• A bowl of hot water with drops of essential oil, often used for soaking feet or used as a 

bidet. Again the essential oil does not mix with the water. This is, however, a useful method 

for inhaling essential oils in respiratory conditions and colds; the essential oil can be 

breathed in when the head is placed over the container and a towel placed over the head and 

container. This is an established method of treatment and has been used successfully with 

Vicks VapoRub, obas oil, and Eucalyptus oils for many years, so it is not surprising that it 

works with aromatherapy essential oils! 

• Compresses using essential oil drops on a wet cloth, either hot or cold, to relieve 

inflammation, treat wounds, and so on. Again, the essential oil is not able to mix with the 

water and can be concentrated in one or two areas, making it a possible health hazard. 

• Massage of hands, feet, back, or all over the body using 2–4 drops of essential oil (single 

essential oil or mixture) diluted in 10 mL carrier oil (fixed, oily), for example, almond oil 

or jojoba oil, grapeseed, wheat-germ oils, and so on. The massage applied is usually by 

gentle effleurage with some petrissage (kneading), with and without some shiatsu, lymph


drainage in some cases, and is more or less vigorous, according to the aromatherapist’s 

skills and beliefs. 

• Oral intake is more like conventional than “alternative” usage of essential oils. Although it 

is practiced by a number of aromatherapists, this is not to be condoned unless the aromatherapist 

is medically qualified. Essential oil drops are “mixed” in a tumbler of hot water 

or presented on a sugar cube or “mixed” with a teaspoonful of honey and taken internally. 

The inability of the essential oil to mix with aqueous solutions presents a health hazard, as 

do the other methods, as such strong concentrations of essential oils are involved. 

13.9 MASSAGE USING ESSENTIAL OILS 

The most popular method of using aromatherapy is through massage. The first written records referring 

to massage date back to its practice in China more than 4000 years and in Egypt. Hippocrates, 

the father of modern medicine, wrote, “the physician must be experienced in many things, but most 

assuredly in rubbing.” 

Massage has been used for centuries in Ayurvedic medicine in India as well as in China and 

shiatsu, acupressure, reflexology, and many other contemporary techniques have their roots in these 

sources. Massage was used for conventional therapeutic purposes in hospitals before World War II 

and is still used by physiotherapists for various conditions including sports injuries. 

René-Maurice Gattefossé, credited as being the founding father of modern aromatherapy, never 

made a connection between essential oils and massage. It was Marguerite Maury who advocated the 

external use of essential oils combined with carrier oils (Maury, 1989). She used carefully selected 

essential oils for cleansing the skin, including that in acne, using a unique blend of oils for each client 

created specifically for the person’s temperament and health situation. Maury’s main focus was 

on rejuvenation; she was convinced that aromas could be used to slow down the aging process if the 

correct oils were chosen. In recent experiments on animals, it has been shown that the oral intake of 

some antioxidant essential oils can appear to defer aging, as indicated by the composition of membranes 

in various tissues (Youdim and Deans, 2000). 

Massage per se can be a relaxing experience and can help to alleviate the stresses and strains of 

daily life. In a review of the literature on massage, Vickers (1996) found that in most studies massage 

had no psychological effect, in a few studies there was arousal, and in an even smaller number 

of studies there was sedation; some massage has both local and systemic effects on blood flow and 

possibly on lymph flow and reduction of muscle tension. 

It may be that these variable responses are directly related to the variability of massage techniques, 

of which there are over 200. Massage can be given over the whole body or limited to the 

face, neck, or just hands, feet, legs—depending on the patient and his or her condition or illness, for 

example, patients with learning disabilities and many psychiatric patients are often only able to have 

limited body contact for a short time. 

13.9.1 MASSAGE TECHNIQUES 

Massage is customarily defined as the manual manipulation of the soft tissues of the body for therapeutic 

purposes, using strokes that include gliding, kneading, pressing, tapping, and/or vibrating 

(Tisserand, 1977; Price and Price, 1999). Massage therapists may also cause movement within the 

joints, apply heat or cold, use holding techniques, and/or advise clients on exercises to improve 

muscle tone and range of motion. Some common massage techniques include Swedish massage, 

acupressure, craniosacral therapy, deep tissue massage, infant massage, lymph system massage, 

polarity therapy, reflexology, reiki, rolfing, shiatsu, and therapeutic touch. 

Massage usually involves the use of a lubricating oil to help the practitioner’s hands glide more 

evenly over the body. The addition of perfumed essential oils further adds to its potential to relax.


In most English-speaking countries, massage is nowadays seen as an alternative or complementary 

treatment. However, before World War II, it was regarded as a conventional treatment (Goldstone, 

1999, 2000), as it is now in continental Europe. In Austria, for example, most patients with back pain 

receive (and are usually reimbursed for) massage treatment (Ernst, 2003a). 

Not all massage treatments are free of risk. Too much force can cause fractures of osteoporotic 

bones, and even rupture of the liver and damage to nerves have been associated with massage 

(Ernst, 2003b). These events are rarities, however, and massage is relatively safe, provided that welltrained 

therapists observe the contraindications: phlebitis, deep vein thrombosis, burns, skin infections, 

eczema, open wounds, bone fractures, and advanced osteoporosis (Ernst et al., 2001). 

It is not known exactly how massage works, although many theories abound (Vickers, 1996; 

Ernst et al., 2001). The mechanical action of the hands on cutaneous and subcutaneous structures 

enhances circulation of blood and lymph, resulting in increased supply of oxygen and removal of 

waste products or mediators of pain (Goats, 1994). Certain massage techniques have been shown to 

increase the threshold for pain (Dhondt et al., 1999). Also, most importantly from the standpoint of 

aromatherapy, a massage can relax the mind and reduce anxiety, which could positively affect the 

perception of pain (Vickers, 1996; Ernst, 2003a). Many studies have been carried out, most of which 

are unsatisfactory. It appears that placebo-controlled, double-blind trials may not be possible, yet 

few randomized clinical trials have been forthcoming. 

Different client groups require proper recognition before aromatherapy trials are started or aromatherapy 

massage is given. For example, for cancer patients, guidelines must be observed 

(Wilkinson et al., 1999): special care must be taken for certain conditions such as autoimmune disease 

(where there are tiny bruises present); low blood cell count, which makes the patient lethargic 

and needing nothing more than very gentle treatment; and lymphoedema, which should not be 

treated unless the therapist has special knowledge and where enfleurage toward the lymph nodes 

should not be used. 

Recent individual studies to investigate the benefit of massage for certain complaints have 

given variable results. Many are positive, although the standard of the studies has, in general, 

been poor (Vickers, 1996). The most successful applications of massage or aromatherapy massage 

have been in cancer care, and about a third of patients with cancer use complementary/ 

alternative medicine during their illness (Ernst and Cassileth, 1998). Massage is commonly provided 

within UK cancer services (Kohn, 1999), and although only anecdotal and qualitative evidence 

is available, it is considered by patients to be beneficial. Only a few small-scale studies 

among patients with cancer have identified short-term benefits from a course of massage, mainly 

in terms of reduced anxiety (Corner et al., 1995; Kite et al., 1998; Wilkinson et al., 1999). These 

studies have been criticized by scientists; however, as they were either nonrandomized, had inadequate 

control groups or were observational in design (Cooke and Ernst, 2000). Complementary 

therapy practitioners have criticized medical research for not being sufficiently holistic in 

approach, focusing on efficacy of treatments in terms of tumor response and survival, rather than 

quality of life (Wilkinson, 2003). 

A general study of the clinical effectiveness of massage by Ernst (1994) used numerous trials, 

with and without control groups. A variety of control interventions were used in the controlled studies 

including placebo, analgesics, transcutaneous electrical nerve stimulation (TENS), and so on. 

There were some positive effects of vibrational or manual massage, assessed as improvements in 

mobility, Doppler flow, expiratory volume, and reduced lymphoedema in controlled studies. 

Improvements in musculoskeletal and phantom limb pain, but not cancer pain, were recorded in 

controlled studies. Uncontrolled studies were invariably positive. Adverse effects included thrombophlebitis 

and local inflammation or ulceration of the skin. 

Different megastudies included massage for delayed-onset muscle soreness—seven trials were 

included with 132 patients in total (Ernst, 1998); effleurage backrub for relaxation—nine trials were 

included with a total of 250 patients (Labyak and Metzger, 1997), and massage for low back pain 

(Ernst, 1999a, 1999b). All gave positive and negative outcomes.


13.10 AROMATHERAPY: BLENDING OF ESSENTIAL OILS 

There are numerous suggestions for the use of particular essential oils for treating specific illnesses 

in books on aromatherapy. However, when collated, each essential oil can treat each illness (Vickers, 

1996; compare also individual essential oil monographs in Lis-Balchin, 2006). 

A few drops of the essential oil or oils chosen are always mixed with a carrier oil before being 

applied to the skin for an aromatherapy massage. The exact dilution of the essential oils in the 

carrier oil is often controversial and can be anything from 0.5% to 20% and more. Either 5, 10, or 

20 mL of carrier oil is first poured into a (usually brown) bottle with a stoppered dropper. The essential 

oil is then added dropwise into the carrier oil, either as a single essential oil or as a mixture of 

2–3 different essential oils, and then stoppered. 

Volumes of essential oils used for dilutions vary widely in different aromatherapies and the fact 

that even the size of a “dropper” varies raised the question of possible safety problems (Lis-Balchin, 

2006), and a recent article in a nursing journal makes a request for standardization of the measurement 

of the dropper size (Ollevant et al., 1999). 

13.10.1 FIXED OILS 

Many fixed oils are used for dilution and all provide a lubricant; many have a high vitamin E and A 

content. By moistening the skin, they can assist in a variety of mild skin conditions especially where 

the skin is rough, cracked, or dry (Healey and Aslam, 1996). 

Almond (Prunus amygdalus var. dulcis)—sweet, cheapest, and most commonly used. Others 

include apricot kernel (Prunus armeniaca), borage seed (Borago officinalis), calendula (Calendula 

officinalis), coconut oil (Cocos nucifera), evening primrose (Oenothera biennis), grapeseed (Vitis 

vinifera), macadamia nut (Macadamia integrifolia), olive (Olea europaea), rose hip seed (Rosa 

mosqueta, etc.), soya bean (Glycine soya), sunflower (Helianthus annuus), wheatgerm (Triticum 

vulgare), and jojoba (Simmondsia californica). The latest oil in vogue is emu oil (Dromiceius 

novaehol-landiae), which comes from a thick pad of fat on the bird’s back. For centuries, the aborigines 

of Australia have been applying emu oil to their wounds with excellent results. It is now found 

in muscle pain relievers, skin care products, and natural soaps. 

The exact method of mixing is controversial, but most aromatherapists are taught not to shake 

the bottle containing the essential oil(s) and the diluent fixed oils, but to gently mix the contents by 

turning the bottle in the hand. Differences in the actual odor and thereby presumable benefits of 

the diluted oils made by different aromatherapists can just be due to the different droppers (Lis- 

Balchin, 2006). 

13.11 INTERNAL USAGE OF ESSENTIAL OILS BY AROMATHERAPISTS 

Oral intake of essential oils is not true “aromatherapy” as the odor has virtually no effect past the 

mouth and the effect of the chemical components takes over as odors cannot influence the internal 

organs (Lis-Balchin, 1998a). Therapy with essential oils is dealt with in another chapter. Most aromatherapists 

consider that essential oils should only be prescribed by primary care practitioners 

such as medical doctors or medical herbalists who have intimate knowledge of essential oil toxicology 

(Tisserand and Balacs, 1995). In the United Kingdom, such “clinical aromatherapy” is rare, 

unlike on the continent. Maladies treated include arthritis, bronchitis, rheumatism, chilblains, 

eczema, high blood pressure, and venereal diseases. In clinical aromatherapy, there is a real risk of 

overdosage due to variable droppers on bottles, which can differ by as much as 200% (Lis-Balchin, 

2006); this may be the cause of asphyxiation of a baby, as already shown by peppermint oil (Bunyan, 

1998). It is possible that aromatherapists would not be covered by their insurance if there were 

adverse effects. However, most of us ingest small amounts of essential oils and their components 

daily in almost all processed foods and drinks, but it does not make us all healthy.


Conventional drugs involving essential oils and their components have been used internally for a 

long time, for example, decongestants containing menthol, camphor and pine, and various throat drops 

containing components from essential oils such as lemon, thyme, peppermint, sage, and hyssop. 

Essential oils in processed foods are used in very minute amounts of 10 ppm, but can be 1000 ppm 

in mint confectionery or chewing gum (Fenaroli, 1997). This contrasts greatly with the use of drops 

of undiluted essential oils on sugar lumps for oral application, or on suppositories in anal or vaginal 

application. Damage to mucous membranes could result due to the high concentration of the essential 

oils in certain areas of the applicator. 

Essential oils and their components are incorporated into enterically coated capsules to prevent 

damage and used for treating irritable bowel syndrome (peppermint in Colpermin), a mixture of 

monoterpenes for treating gallstones (Rowatol) and ureteric stones (Rowatinex); these are under 

product licenses as medicines (Somerville et al., 1984, 1985; Engelstein et al., 1992). 

Some aromatherapists support the use of essential oils in various venereal conditions. However, 

aromatherapists are either qualified to treat venereal disease conditions, nor can make an accurate 

diagnosis in the first place, unless they are also medically qualified. Tea tree oil (2–3 drops undiluted) 

was used on a tampon for candidiasis with apparently very encouraging results (Zarno, 1994). 

Candida treatments also include chamomile, lavender, bergamot, and thyme (Schnaubelt, 1999). 

Essential oils used in this way, sometimes for months, often produced extremely painful reactions 

and putrid discharges due to damage to delicate mucosal membranes. 

13.12 USE OF PURE OR SYNTHETIC COMPONENTS 

Does it really matter whether the essential oil is pure or a synthetic mixture as long as the odor is 

the same? The perfumers certainly do not see any difference, and even prefer the synthetics as they 

remain constant. Many of the so-called pure essential oils used today are, however, adulterated 

(Which Report, 2001; Lis-Balchin et al., 1996, 1998). There is often a difference in the proportion 

of different enantiomers of individual components that often have different odors and different biological 

properties (Lis-Balchin, 2002a, 2002b). This was not, however, appreciated by Gattefosse 

(1937/1993), who worked with perfumes and not with the “pure plant essential oils” (Formulaires 

de Parfumerie Gattefossé, 1906). He studied the antimicrobial and wound-healing properties of 

essential oils on soldiers during World War I (Arnould-Taylor, 1981). He later worked in hospitals on 

the use of perfumes and essential oils as antiseptics and other (unstated) applications, and also in 

dermatology, which led to advances in the development of beauty products and treatments and the 

publication of Physiological Aesthetics and Beauty Products in 1936 (Gattefosse, 1992). 

Gattefossé promoted the deterpenization of essential oils because, being a perfumer, he was 

aware that his products must be stable, have a long shelf-life, and not go cloudy when diluted in 

alcohol. Terpenes also oxidize rapidly, often giving rise to toxic oxidation products (e.g., limonene 

of citrus essential oils). But this goes against the use of pure essential oils, as their wholeness or 

natural synergy is apparently destroyed (Price, 1993). Bergamot and other citrus essential oils 

obtained by expression are therefore recommended, despite their phototoxicity (Price and Price, 

1999). There is no reason why a toxic essential oil should be preferentially used if the nontoxic 

furanocoumarin-free (FCF) alternative is available. If adverse effects resulted, it is possible that 

there could be legal implications for the therapist. 

13.13 THERAPEUTIC CLAIMS FOR THE APPLICATION OF ESSENTIAL OILS 

There are a wide range of properties ascribed to each essential oil in aromatherapy books, without 

any scientific proof of effectiveness (Vickers, 1996; Lis-Balchin, 2006). The following are a few 

examples. 

Diabetes can be treated by eucalyptus, geranium, and juniper (Tisserand, 1977); clary sage, 

eucalyptus, geranium, juniper, lemon, pine, red thyme, sweet thyme, vetiver, and ylang ylang (Price,


1993); eucalyptus, geranium, juniper, and onion (Valnet, 1982); and eucalyptus, geranium, cypress, 

lavender, hyssop, and ginger (Worwood, 1991). 

Allergies can be treated by immortelle, chamomile, balm, and rose (Fischer-Rizzi, 1990); lemon 

balm, chamomile (German and Roman), helichrysum, true lavender, and spikenard (Lawless, 1992); 

and chamomile, jasmine, neroli, and rose (Price, 1983). 

No botanical names are, however, given in the lists, even when there are several possible species. 

No indication is provided as to why these particular essential oils are used and how they are supposed 

to affect the condition. Taking the case of diabetes, where there is a lack of the hormone 

insulin, it is impossible to say how massage with any given essential oil could cure the condition, 

without giving the hormone itself in juvenile-type diabetes or some blood glucose-decreasing 

drugs in late-onset diabetes. Unfortunately, constant repetition of a given statement often lends it 

credence—at least to the layperson, who does not require scientific evidence of its validity. 

13.13.1 FALSE CLAIMS CHALLENGED IN COURT 

The false promotion of products for treating not only medical conditions but also well-being generally 

is now being challenged in the law courts. For example, in 1997, Los Angeles attorney Morsé 

Mehrban charged that Lafabre and Aroma Vera had violated the California Business and Professions 

Code by advertising that their products could promote health and well-being, relax the body, relax 

the mind, enhance mood, purify the air, are antidotes to air pollution, relieve fatigue, tone the body, 

nourish the skin, promote circulation, alleviate feminine cramps, and do about 50 other things 

(Barrett, 2000). In September 2000, the case was settled out of court with a $5700 payment to 

Mehrban and a court-approved stipulation prohibiting the defendants from making 57 of the disputed 

claims in advertising within California (Horowitz, 2000). 

13.14 PHYSIOLOGICAL AND PSYCHOLOGICAL RESPONSES TO ESSENTIAL 

OILS AND PSYCHOPHYSIOLOGY 

Many examples of essential oil effects abound in animal studies, for example, the sedative action of 

lavender on the overall activity of mice decreased when exposed to lavender vapor (Lavandula 

angustifolia P. Miller); its components linalool and linalyl acetate showed a similar effect (Buchbauer 

et al., 1992). A possible explanation for the observed sedative effects was shown by Linalool, which 

produced a dose-dependent inhibition of the binding of glutamate (an excitatory neurotransmitter in 

the brain) to its receptors on membranes of the rat cerebral cortex (Elisabetsky et al., 1995). More 

recently, this action was related to an anticonvulsant activity of linalool in rats (Elisabetsky et al., 

1999). Other oils with sedative activity were found to be neroli and sandalwood; active components 

included citronellal, phenylethyl acetate, linalool, linalyl acetate, benzaldehyde, -terpineol, and 

isoeugenol (in order of decreasing activity). 

Stimulant oils included jasmine, patchouli, ylang ylang, basil, and rosemary; active components 

included fenchone, 1,8-cineole, isoborneol, and orange terpenes (Lis-Balchin, 2006). There was 

considerable similarity in the sedative and stimulant effects of some essential oils studied physiologically 

(e.g., their effect on smooth muscle of the guinea pig in vitro) and in various psychological 

assessments, mostly on humans (Lis-Balchin, 2006). 

1,8-Cineole when inhaled, showed a decreased blood flow through the brain (measured using 

computerized tomography) although no changes were found with lavender oil or linalyl acetate 

(Buchbauer et al., 1993c). Changing electrical activity, picked up by scalp electrodes, in response to 

lavender odors was considered a measure of brain activity (EEG) (Van Toller et al., 1993). The most 

consistent responses to odors were in the theta band (Klemm et al., 1992). Many essential oil vapors 

have been shown to depress CNV brain waves (an upward shift in EEG waves that occurs when 

people are expecting something to happen) in human volunteers and these are considered to be 

sedatives; others increase CNV and are considered stimulants: lavender was found to have a sedative


effect on humans (Torii et al., 1988; Kubota et al., 1992; Manley, 1993) and had a “positive” effect 

on mood, EEG patterns, and maths computations (Diego et al., 1998). It also caused reduced motility 

in mice (Kovar et al., 1987; Ammon, 1989; Buchbauer et al., 1992, 1993a, 1993b, 1993c; Jaeger 

et al., 1992). However, Karamat et al. (1992) found that lavender had a stimulant effect on decision 

times in human experiments. 

A large workplace in Japan with odorized air via the whole building showed that citrus smells 

refreshed the workers first thing in the morning and after the lunch break, and floral smells improved 

their concentration in between. In the lunch break and during late afternoon, woodland scents were 

circulated to relax the workers and this increased productivity (Van Toller and Dodd, 1991). It is 

also possible that the use of a general regime of odorants could have very negative effects on some 

members of the workforce or on patients in hospital wards, where the use of pleasant odors could 

mask the usual unpleasant odors providing the smell of fear. Ambient odors have an effect on creativity, 

mood, and perceived health (Knasco, 1992, 1993) and so does feigned odor (Knasco et al., 

1990). 

It is very difficult to make simple generalizations concerning the effects of any fragrance on 

psychological responses, which are based on the immediate perceptual effects, rather than the longer 

term pharmacological effects because the pharmacological effect is likely to affect people similarly, 

but the additional psychological mechanisms will create complex effects at the individual level. 

Odors are perceptible even during sleep, as shown in another experiment; college students were 

tested with fragrances during the night and the day (Badia, 1991). 

Various nonscientific studies have been published in perfumery journals on the treatment of 

psychiatric patients by psychoaromatherapy in the 1920s (Gatti and Cajola, 1923a, 1923b, 1929; 

Tisserand, 1997) but there was virtually no information on their exact illnesses. Sedative essential 

oils or essences were identified as chamomile, melissa, neroli, petitgrain, opoponax, asafoetida, and 

valerian. Stimulants were angelica, cardamom, lemon, fennel, cinnamon, clove, and ylang ylang. 

Many aromatherapists have also written books on the effect of essential oils on the mind, giving 

directives for the use of specific plant oils for treating various conditions, without any scientific 

proof (Lawless, 1994; Worwood, 1996, 1998; Hirsch, 1998). 

13.15 PLACEBO EFFECT OF AROMATHERAPY 

The placebo effect is an example of a real manifestation of mind over matter. It does not confine 

itself to alternative therapies, but there is a greater likelihood of the placebo effect accounting for 

over 90% of the effect in the latter (Millenson, 1995). Reasons for the potency of the placebo effect 

are either the patient’s belief in the method; the practitioner’s belief in the method; or the patient and 

practitioner’s belief in each other, that is, the strength of their relationship (Weil, 1983). 

Placebo effects have been shown to relieve postoperative pain, induce sleep or mental awareness, 

bring about drastic remission in both symptoms and objective signs of chronic diseases, initiate the 

rejection of warts, and other abnormal growths, and so on (Weil, 1983). Placebo affects headaches, 

seasickness, and coughs, as well as have beneficial effects on pathological conditions such as rheumatoid 

and degenerative arthritis, blood cell count, respiratory rates, vasomotor function, peptic 

ulcers, hay fever, and hypertension (Cousins, 1979). There can also be undesirable side effects, such 

as nausea, headaches, skin rashes, allergic reactions, and even addiction, that is, a nocebo effect. 

This is almost akin to voodoo death threats or when patients are mistakenly told that their illness is 

hopeless—both are said to cause death soon after. 

Rats were found to have increased levels of opioids in their brains after inhaling certain essential 

oils. Opioids are a factor in pain relief (Lis-Balchin, 1998b) and can be increased in the body by 

autosuggestion, relaxation, belief, and so on. 

The use of aromatherapy for pain relief is best achieved through massage, personal concern and 

touch of the patient, and also listening to their problems. The extra benefit of real “healers” found 

among aromatherapists is an added advantage.


13.16 SAFETY ISSUE IN AROMATHERAPY 

Many aromatherapists and laymen consider natural essential oils to be completely safe. This is 

based on the misconception that all herbs are safe—because they are “natural,” which is a fallacy. 

The toxicity of essential oils can also be entirely different to that of the herb, not only because of 

their high concentration, but also because of their ability to pass across membranes very efficiently 

due to their lipophilicity. 

Some aromatherapists erroneously believe that aromatherapy is self-correcting, unlike conventional 

therapy with medicines, and if errors are made in aromatherapy, they may be resolved through 

discontinuation of the wrongful application of the oil (e.g., Schnaubelt, 1999). 

Many essential oils are inherently toxic at very low concentrations due to very toxic components; 

these are not normally used in aromatherapy. Many essential oils that are considered to be nontoxic 

can have a toxic effect on some people; this can be influenced by previous sensitization to a given 

essential oil, a group of essential oils containing similar components, or some adulterant in the essential 

oil. It can also be influenced by the age of the person; babies and young children are especially 

vulnerable and so are very old people. The influence of other medicaments, both conventional and 

herbal, is still in the preliminary stages of being studied. It is possible that these medicaments, and 

also probably household products, including perfumes and cosmetics, can influence the adverse reactions 

to essential oils. 

Aromatherapists themselves have also been affected by sensitization (Crawford et al., 2004); in 

a 12-month period under study, prevalence of hand dermatitis in a sample of massage therapists was 

15% by self-reported criteria and 23% by a symptom-based method and included the use of aromatherapy 

products in massage oils, lotions, or creams. In contrast, the suggestion that aromatherapists 

have any adverse effects to long-term usage of essential oils was apparently disproved by a 

nonscientific survey (Price and Price, 1999). 

As most essential oils were tested over 30 years ago, the toxicity data may now be meaningless, 

as different essential oils are now used, some of which contain different quantities of many different 

synthetic components (Lis-Balchin, 2006). 

The major drawbacks of trying to extrapolate toxicity studies in animals to humans concern 

feelings—from headaches to splitting migraines; feeling sick, vertigo, profound nausea; tinnitus; 

sadness, melancholia, suicidal thoughts; feelings of hate—which are clearly impossible to measure 

in animals (Lis-Balchin, 2006). The toxicity of an individual essential oil/component is also 

tested in isolation in animals and disregards the possibility of modification by other substances, 

including food components and food additive chemicals, the surrounding atmosphere with gaseous 

and other components, fragrances used in perfumes, domestic products, in the car, in public transport 

(including the people), workplace, and so on. These could cause modification of the essential 

oil/component, its bioavailability, and possibly the enhancement or loss of its function. The 

detoxification processes in the body are all directed to the production of a more polar product(s), 

which can be excreted mainly by the kidneys regardless of whether this/these are more toxic or less 

toxic than the initial substance and differ in different animals. 

Most essential oils have GRAS (generally recognized as safe) status granted by the Flavor and 

Extract Manufacturers Association (FEMA) and approved by the US Food and Drug Administration 

(FDA) for food use, and many appear in the food chemical codex. This was reviewed in 1996 after 

evaluation by the expert panel of the FEMA. The assessment was based on data of exposure, and as 

most flavor ingredients are used at less than 100 ppm, predictions regarding their safety can be assessed 

from data on their structurally related group(s) (Munro et al., 1996). The no-observed-adverse-effect 

levels (NOELs) are more than 100,000 times their exposure levels from use as flavor ingredients 

(Adams et al., 1996). Critical to GRAS assessment are data of metabolic fate and chronic studies rather 

than acute toxicity. Most essential oils and components have an LD50 of 1–20 g/kg body weight or 

roughly 1–20 mL/kg, with a few exceptions as follows: Boldo leaf oil 0.1/0.9 (oral/dermal); Calamus 

0.8–9/5; Chenopodium 0.2/0.4; Pennyroyal 0.4/4; and Thuja 0.8/4.


Research Institute for Fragrance Materials (RIFM) testing is generally limited to acute oral and 

dermal toxicity, irritation and dermal sensitization, and phototoxicity of individual materials, and 

there is little effort to address synergistic and modifying effects of materials in combination 

(Johansen et al., 1998). 

Many materials that were widely used for decades in the past had severe neurotoxic properties 

and accumulated in body tissues (Spencer et al., 1979; Furuhashi et al., 1994) but most fragrance 

materials have never been tested for neurological effects, despite the fact that olfactory pathways 

provide a direct route to the brain (Hastings et al., 1991). 

13.17 TOXICITY IN HUMANS 

The most recent clinical review of the adverse reactions to fragrances (de Groot and Frosch, 1997) 

showed many examples of cutaneous reactions to essential oils reported elsewhere (Guin, 1982, 

1995). In the United States, about 6 million people have a skin allergy to fragrance and this has a 

major impact on their quality of life. Symptoms include headaches, dizziness, nausea, fatigue, shortness 

of breath, and difficulty in concentrating. Fragrance materials are readily absorbed into the 

body via the respiratory system and once absorbed they cause systemic effects. 

Migraine headaches are frequently triggered by fragrances that can act on the same receptors in 

the brain as alcohol and tobacco, altering mood and function [Institute of Medicine USA, sponsored 

by the Environmental Protection Agency (EPA)]. Perfumes and fragrances are recognized as triggers 

for asthma by the American Lung Association. The vast majority of materials used in fragrances 

are respiratory irritants and there are a few that are known to be respiratory sensitizers. 

Most have not been evaluated for their effects on the lungs and the respiratory system. 

Respiratory irritants are known to make the airways more susceptible to injury and allergens, as 

well as to trigger and exacerbate conditions such as asthma, allergies, sinus problems, and other 

respiratory disorders. In addition, there is a subset of asthmatics that is specifically triggered by 

fragrances (Shim and Williams, 1986; Bell et al., 1993; Baldwin et al., 1999), which suggests that 

fragrances not only trigger asthma, they may also cause it in some cases (Millqvist and Lowhagen, 

1996). Placebo-controlled studies using perfumes to challenge people with asthma-like symptoms 

showed that asthma could be elicited with perfumes without the presence of bronchial obstruction 

and these were not transmitted by the olfactory nerve as the patients were unaware of the smell 

(Millqvist and Lowhagen, 1996). 

Adverse reactions to fragrances are difficult or even impossible to link to a particular chemical— 

often due to secrecy rules of the cosmetic/perfumery companies and the enormous range of synthetic 

components, constituting about 90% of flavor and fragrance ingredients (Larsen, 1998). The 

same chemicals are used in foods and cosmetics—there is, therefore, a greater impact due to the 

three different modes of entry: oral, inhalation, and skin. 

13.17.1 INCREASE IN ALLERGIC CONTACT DERMATITIS IN RECENT YEARS 

A study of 1600 adults in 1987 showed that 12% reacted adversely to cosmetics and toiletries, 

4.3% of which were used for their odor (i.e., they contained high levels of fragrances). Respiratory 

problems worsened with prolonged fragrance exposure (e.g., at cosmetic/perfumery counters) and 

even in churches. In another study, 32% of the women tested had adverse reactions and 80% of 

these had positive skin tests for fragrances (deGroot and Frosch, 1987). Problems with essential 

oils have also been increasing. For example, contact dermatitis and allergic contact dermatitis 

(ACD) caused by tea tree oil has been reported, which was previously considered to be safe (Carson 

and Riley, 1995). It is unclear whether eucalyptol was responsible for the allergenic response 

(Southwell, 1997); out of seven patients sensitized to tea tree oil, six reacted to limonene, five to 

a-terpinene and aromadendrene, two to terpinen-4-ol, and one to p-cymene and a-phellandrene 

(Knight and Hausen, 1994).


Many studies on ACD have been done in different parts of the world (deGroot and Frosch, 1987) 

and recently more studies have appeared: 

• Japan (Sugiura et al., 2000): The patch test with lavender oil was found to be positive in 

increased numbers and above that of other essential oils in 10 years. 

• Denmark (Johansen et al., 2000): There was an 11% increase in the patch test in the last 

year and of 1537 patients, 29% were allergic to scents. 

• Hungary (Katona and Egyud, 2001): Increased sensitivity to balsams and fragrances was 

noted. 

• Switzerland (Kohl et al., 2002): ACD incidence has increased over the years and recently 

36% of 819 patch tests were positive to cosmetics. 

• Belgium (Kohl et al., 2002): Increased incidence of ACD has been noted. 

Occupational increases have also been observed. Two aromatherapists developed ACD: one to 

citrus, neroli, lavender, frankincense, and rosewood and the other to geraniol, ylang ylang, and 

angelica (Keane et al., 2000). Allergic airborne contact dermatitis from the essential oils used in 

aromatherapy was also reported (Schaller and Korting, 1995). ACD occurred in an aromatherapist 

due to French marigold essential oil, Tagetes (Bilsland and Strong, 1990). A physiotherapist developed 

ACD to eugenol, cloves, and cinnamon (Sanchez-Perez and Garcia Diez, 1999). 

There is also the growing problem that patients with eczema are frequently treated by aromatherapists 

using massage with essential oils. A possible allergic response to a variety of essential oils 

was found in children with atopic eczema, who were massaged with or without the oils. At first, both 

massages proved beneficial, though not significantly different; but on reapplying the essential oil 

massage after a month’s break, there was a notable adverse effect on the eczema, which could suggest 

sensitization (Anderson et al., 2000). 

13.17.2 PHOTOSENSITIZERS 

Berlocque dermatitis is frequently caused by bergamot or other citrus oil applications on the skin 

(often due to their inclusion in eau de Cologne) followed by exposure to UV light. This effect is caused 

by psolarens or furanocoumarins (Klarmann, 1958). Citrus essential oils labeled FCF have no phototoxic 

effect, but are suspected carcinogens (Young et al., 1990). Other phototoxic essential oils include 

yarrow and angelica, neroli, petitgrain, cedarwood, rosemary, cassia, calamus, cade, eucalyptus (species 

not stated), orange, anise, bay, bitter almond, ylang ylang, carrot seed, and linaloe (the latter probably 

due to linalool, which, like citronellol, has a sensitizing methylene group exposed) (Guin, 1995). 

Photosensitizer oils include cumin, rue, dill, sandalwood, lemon (oil and expressed), lime (oil and 

expressed), opoponax, and verbena (the latter being frequently adulterated) (Klarmann, 1958). Even 

celery soup eaten before UV irradiation has been known to cause severe sunburn (Boffa et al., 1996). 

Many of these photosensitizers are now banned or restricted. New International Fragrance 

Research Association (IFRA) proposals for some phototoxic essential oils include rue oil to be 

0.15% maximum in consumer products, marigold oil and absolute to be 0.01%, and petitgrain 

mandarin oil to be 0.165%. 

13.17.3 COMMONEST ALLERGENIC ESSENTIAL OILS AND COMPONENTS 

The most common fragrance components causing allergy are cinnamic alcohol, hydroxycitronellal, 

musk ambrette, isoeugenol, and geraniol (Scheinman, 1996). These are included in the eight 

commonest markers used to check for ACD, usually as a 2% mix. Other components considered 

allergenic are benzyl salicylate, sandalwood oil, anisyl alcohol, benzyl alcohol, and coumarin. 

IFRA and RIFM have forbidden the use of several essential oils and components, including 

costus root oil, dihydrocoumarin, musk ambrette, and balsam of Peru (Ford, 1991); a concentration


limit is imposed on the use of isoeugenol, cold-pressed lemon oil, bergamot oil, angelica root oil, 

cassia oil, cinnamic alcohol, hydroxycitronellal, and oakmoss absolute. Cinnamic aldehyde, citral, 

and carvone oxide can only be used with a quenching agent. 

Photosensitivity and phototoxicity occurs with some allergens such as musk ambrette and 

6-methyl coumarin that are now removed from skin care products. Children were often found to be 

sensitive to Peru balsam, probably due to the use of baby-care products containing this (e.g., talcum 

powder used on nappy rash). 

Fragrance materials have been found to interact with food flavorings, for example, a “balsam of 

Peru-free diet” has been devised in cases where cross reactions are known to occur (Veien et al., 

1985). “Newer” sensitizers include ylang ylang (Romaguera and Vilplana, 2000), sandalwood oil 

(Sharma et al., 1994) but much of this essential oil is adulterated or completely synthetic, lyral 

(Frosch et al., 1999; Hendriks et al., 1999), and eucalyptol (Vilaplana and Romaguera, 2000). 

Some sensitizers have been shown to interact with other molecules. For example, cinnamaldehyde 

interacts with proteins (Weibel et al., 1989), indicating how the immunogenicity occurs. 

There have been very few published reports on neurotoxic aromachemicals such as musk ambrette 

(Spencer et al., 1984), although many synthetic musks took over as perfume ingredients when public 

opinion turned against the exploitation of animal products. Musk ambrette was found to have neurotoxic 

properties in orally fed mice in 1967 and was readily absorbed through the skin. A similar story 

occurred with acetylethyltetramethyltetralin (AETT), another synthetic musk, also known as versalide, 

patented in the early 1950s. During routine tests for irritancy in 1975, it was noted that with 

repeated applications, the skin of the mice turned bluish and they exhibited signs of neurotoxicity. The 

myelin sheath was damaged irreversibly in a manner similar to that which occurs with multiple sclerosis. 

Musk xylene, one of the commonest fragrance materials, is found in blood samples from the 

general population (Kafferlein et al., 1998) and bound to human hemoglobin (Riedel et al., 1999). 

These musk products have been found to have an effect on the life stages of experimental animals such 

as the frog, Xenopus laevis, the zebra fish, Danio rerio (Chou and Dietrich, 1999), and the rat (Christian 

et al., 1999). The hepatotoxic effect of musks is under constant study (Steinberg et al., 1999). 

13.17.4 TOXICITY IN YOUNG CHILDREN: A SPECIAL CASE 

Many aromatherapy books give dangerous advice on the treatment of babies and children, for example, 

5–10 drops of “chamomile oil” three times a day in a little warmed milk given to their babies to 

treat colic with no indication as to which of the three commercially available chamomile oils is to 

be used and because, depending on the dropper size, the dose could easily approach the oral LD50 

for the English and German chamomile oils, this could result in a fatality. Peppermint, often mentioned, 

could possibly be given by mothers in the form of oil, and has been known to kill a 1-weekold 

baby (Evening Standard, 1998). Dosages given in terms of drops can vary widely according to 

the size of the dropper in an essential oil. 

Many “cosmetics” designed for use by children contain fragrance allergens (Rastogi et al., 1999). 

In Denmark, samples of children’s cosmetics were found to contain geraniol, hydroxycitronellol, 

isoeugenol, and cinnamic alcohol (Rastogi et al., 1999). Children are more susceptible than adults 

to any chemical, so the increase in childhood asthma reported in recent years could be caused by 

fragrance components also found in fast foods. Aromatherapy therefore could be dangerous. 

13.17.5 SELECTED TOXICITIES OF COMMON ESSENTIAL OILS AND THEIR COMPONENTS 

Limonene and Linalool are found in a multitude of the commonest aromatherapy oils. 

Limonene is a common industrial cleaner and is also the main citrus oil component, which 

causes ACD, particularly when aged (Chang et al., 1997; Karlberg and Dooms-Goossens, 1997). 

The major volatile component of lactating mothers’ milk in the USA was found to contain d-limonene 

and the component is used as a potential skin penetration promoter for drugs such as indometacin,


especially when mixed with ethanol (Falk-Filipsson et al., 1993). Lastly, cats and dogs are very 

susceptible to insecticides and baths containing d-limonene, giving rise to neurological symptoms 

including ataxia, stiffness, apparent severe CNS depression, tremors, and coma (von Burg, 1995; see 

also Beasley, 1999). 

Linalool, when oxidized for just 10 weeks, the linalool content fell to 80% and the remaining 

20% consisted of a range of breakdown chemicals including linalool hydroperoxide, which was 

confirmed as a sensitizing agent. The fresh linalool was not a sensitizer; therefore, the EC regulations 

that are warnings about sensitization potential are looking for potential harm even on storage 

(Skoeld et al., 2002a, 2002b). 

Most cosmetics and perfumes are tested on human “guinea pigs” using similar tests to those 

described for animals. These are demanded by the RIFM as a final test before marketing a product. 

Further data are accumulated from notifications from disgruntled consumers who report dermatitis, 

itching, or skin discoloration after use. These notifications can result in legal claims, although most 

cases are probably settled out of court and not reported to the general public. 

The internet has made it possible for a trusting, although often ill-informed, public to purchase a 

wide range of dubious plant extracts and essential oils. Even illegal essential oils can now be 

obtained. Furthermore, unqualified people can offer potentially dangerous advice, such as internal 

usage or the use of undiluted essential oils on the skin for “mummification,” or in order to rid the 

body of toxic waste. The latter can result in excruciating pain from the burns produced and the 

subsequent loss of layers of skin. 

There is a recipe for suntan oil, including bergamot, carrot seed, and lemon essential oils (all 

phototoxic) in an aromatherapy book (Fischer-Rizzi, 1990). The author then advises that bergamot 

oil is added to suntan lotion to get the bonus of the substance called “furocumarin,” which lessens 

the skin’s sensitivity to the sun while it helps one to tan quickly. This could cause severe burns. 

Elsewhere, sassafras (Ocotea pretiosa) was said to be only toxic for rats, due to its metabolism and 

not dangerous to humans (Pénoel, 1991a, 1991b) and a 10% solution in oil was suggested for treating 

muscular and joint pain and sports injuries. Safrole (and sassafras oil) is, however, controlled under 

the Controlled Drugs Regulations (1993) and listed as a Category 1 substance, as it is a precursor to 

the illicit manufacture of hallucinogenic, narcotic, and psychotropic drugs like ecstasy. 

French practitioners and other therapists have apparently become “familiar” with untested oils 

(Guba, 2000). The use of toxicologically untested Nepalese essential oils and the like includes 

lichen resinoids, sugandha kokila oil, jatamansi oil, and Nepalese lemongrass (Cymbopogon 

flexuosa), also Tagetes oil (Basnyet, 1999). Melaleuca rosalina (Melaleuca ericifolia), 1,8-cineole 

18–26%, is apparently especially useful for the respiratory system (Pénoel, 1998), but it is untested 

and could be a sensitizer. 

The Medicines and Healthcare Products Regulatory Agency in the United Kingdom may bring 

about changes in aromatherapy practice similar to their threat on herbal remedies. Aromatherapists 

are now using some potentially harmful products in their therapy. This immediately places them at 

serious risk if there is any untoward reaction to their specific treatment. It virtually means that 

bottles and containers of essential oils now rank with domestic bleach for labeling purposes and 

companies are now obliged to self-classify their essential oils on their labels and place them in suitable 

containers; this applies both to large distributing companies as well as individual aromatherapists 

reselling essential oils under their own name. Finally, new legislation has gone to the Council 

of Ministers and may imply that only qualified people will be able to use essential oils, and retail 

outlets for oils will be pharmacies. Their definition of “qualified” is limited to academic 

qualifications—doctors or pharmacists. 

13.18 CLINICAL STUDIES OF AROMATHERAPY 

Very few scientific clinical studies on the effectiveness of aromatherapy have been published to 

date. Perhaps the main reason is that until recently scientists were not involved and people engaging


in aromatherapy clinical studies had accepted the aromatherapy doctrine in its entirety, precluding 

any possibility of a nonbiased study. This has been evident in the design and execution of the studies; 

the main criterion has usually been the use of massage with essential oils and not the effect of 

the odorant itself. The latter is considered by most aromatherapists as irrelevant to clinical aromatherapy, 

which implies that it is simply the systemic action of essential oils absorbed through the 

skin that exerts an effect on specific organs or tissues. Odorant action is considered to be just “aromachology,” 

despite its enormous psychological and physiological impact (Lis-Balchin, 2006). In 

some studies, attempts are even made to bypass the odorant effect entirely by making the subjects 

wear oxygen masks throughout (Dunn et al., 1995). 

The use of particular essential oils for certain medical conditions is also adhered to, despite the 

wide assortment of supposed functions for each essential oil claimed by different aromatherapy 

source materials. In many studies, it is even unclear exactly which essential oil was used; as often 

the correct nomenclature, chemical composition, and exact purity are not given. 

Many aromatherapists feel that they know that aromatherapy works as they have enormous numbers 

of case studies to prove it. But the production of lists of “positive” results on diverse clients, 

with diverse ailments, using diverse essential oils in the treatments, and diverse methods of application 

(which also frequently change from visit to visit for the same client) does not satisfy scientific 

criteria. 

Negative results must surely be among the positive ones, due to the change in essential oils during 

the course of the treatment, which suggests that they did not work, but these are never stated. 

There are also no controls in case studies and no attempt to control the bias of the individual aromatherapist 

and clients. 

Double-blind studies are not possible in individual case studies. Physiological or psychological 

changes due to the treatment are not properly defined and loose phrases such as “the client felt 

better” or “happier” are inappropriate for a scientific study. 

These faults in the design and interpretation of results of aromatherapy research have been 

pointed out many times, for example, in Vickers (1996) Kirk-Smith (1996a), Nelson (1997), and 

Lis-Balchin (2002b). However, the lack of statistically significant results does not prevent many 

aromatherapists from accepting vaguely positive clinical research results and numerous poor-grade 

clinical studies are now quoted as factual confirmations that aromatherapy works. 

It is almost impossible to do a double-blind study using odorants, as the patient and treatment 

provider would experience the odor differences and would inevitably react knowingly or unknowingly 

to that factor alone. The psychological effect(s) could be very diverse, as recall of odors can 

bring about very acute reactions in different people, depending on the individual’s past experiences 

and on the like (Lis-Balchin, 2006). Lastly, there is potential bias as patients receiving aromatherapy 

treatment could be grateful for the attention given to them and, not wanting to upset the givers 

of such attention, would state that they were better and happier than before. 

13.19 RECENT CLINICAL STUDIES 

13.19.1 AROMATHERAPY IN DEMENTIA 

A meticulously conducted double-blind study involved 72 dementia patients with clinically 

significant agitation treated with melissa oil (Ballard et al., 2002). Agitation included anxiety and 

irritability, motor restlessness, and abnormal vocalization—symptoms that often lead to disturbed 

behaviors such as pacing, wandering, aggression, shouting, and night-time disturbance, all 

characterized by appropriate inventories. 

Ten percent (by weight) melissa oil (active) or sunflower oil (placebo), combined with a base 

lotion (Prunus dulcis oil, glycerine, stearic acid, cetearyl alcohol, and tocopheryl acetate), was dispensed 

in metered doses and applied to the face and both arms twice daily for 4 weeks by a care


assistant, the process taking 1–2 min. The patients also received neuroleptic treatment and other 

conventional treatments when necessary; this was therefore a study of complementary aromatherapy 

treatment—not an alternative treatment. 

The “melissa group” showed a higher significant improvement in reducing aggression than the 

control group by week 4; the total Cohen–Mansfield Agitation Inventory (CMAI) scores had 

decreased significantly in both groups, from a mean of 68 to 45 (35%; P < .0001) in the treatment 

group and from 61 to 53 (11%; P < .005) in the placebo group. Clinically significant reduction in 

agitation occurred in 60% of the melissa group compared with 14% of placebo responders (P < .0001). 

Neuropsychiatric Inventory (NPI) scores also declined with melissa treatment, and quality of life 

was improved, with less social isolation and more involvement in activities. The latter was in contrast 

to the usual neuroleptic treatment effects. 

The authors concluded that the melissa treatment was successful, but pointed out that there was 

also a significant, but lower, improvement in the control group and suggested that a stronger odor 

should have been used. 

The effect of the melissa oil was probably on cholinergic receptors as shown by previous in vitro 

studies (Perry et al., 1999; Wake et al., 2000). The authors also concluded that as most people with 

severe dementia have lost any meaningful sense of smell, a direct placebo effect due to a pleasantsmelling 

fragrance, although possible, is an unlikely explanation for the positive effects of melissa 

in this study but others may disagree with this conclusion as it has been shown that subliminal odors 

can have an effect. The fragrance may have had some impact upon the care staff, and influenced 

ratings to some degree on the informant schedules. 

A further recent study found no support for the use of a purely olfactory form of aromatherapy to 

decrease agitation in severely demented patients using lavender and thyme oil (Snow et al., 2004). 

Other research (Burns et al., 2002) suggested that aromatherapy and light therapy were more 

effective and gentler alternatives to the use of neuroleptics in patients with dementia. Three studies 

were analyzed in each category; in the aromatherapy section, it included the study above, plus the 

use of 2% lavender oil via inhalation in a double-blind study for 10 days (Holmes et al., 2002) and a 

2-week single-blind study using either aromatherapy plus massage, aromatherapy plus conversation 

or massage alone (Smallwood et al., 2001). All of the interventions in the aromatherapy groups 

proved significantly beneficial. However, so did the light treatment, where patients sat in front of a 

light box that beamed out 10,000 lux of artificial light, which adjusts the body’s melatonin levels, 

affects the body clock, and is used in the treatment of SAD (seasonal affective disorder). 

13.20 PAST CLINICAL STUDIES 

In contrast to more recent studies, past clinical trials were often very defective in design and also 

outcomes. In a recent review, Cooke and Ernst (2000) included only those aromatherapy trials that 

were randomized and included human patients; they excluded those with no control group or if only 

local effects (e.g., antiseptic effects of tea tree oil) or preclinical studies on healthy volunteers 

occurred. The six trials included massage with or without aromatherapy (Buckle, 1993; Stevenson, 

1994; Corner et al., 1995; Dunn et al., 1995; Wilkinson, 1995; Wilkinson et al., 1999) and were based 

on their relaxation outcomes. The authors concluded that the effects of aromatherapy were probably 

not strong enough for it to be considered for the treatment of anxiety or for any other indication. 

A further study included trials with no replicates, and contained six studies. It showed that in five 

out of six cases the main outcomes were positive; however, these were limited to very specific criteria, 

such as small airways resistance for common colds (Cohen and Dressler, 1982), prophylaxis 

of bronchi for bronchitis (Ferley et al., 1989), lessening smoking withdrawal symptoms (Rose and 

Behm, 1993, 1994), relief of anxiety (Morris et al., 1995), and treatment of alopecia areata (Hay 

et al., 1998). The alleviation of perineal discomfort (Dale and Cornwell, 1994) was not significant. 

Psychological effects, which include inhalation of essential oils and behavioral changes, were 

already discussed.


13.20.1 CRITIQUE OF SELECTED CLINICAL TRIALS 

The following clinical studies attempted to show that aromatherapy was more efficient than massage 

alone but they showed mainly negative results; however, in some cases, the authors clearly 

emphasized some very small positive results and this was then accepted and the report was welcomed 

in aromatherapy journals as a positive trial that supported aromatherapy. 

Massage, aromatherapy massage, or a period of rest in 122 patients in an intensive care unit 

(ICU) (Dunn et al., 1995) showed no difference between massage with or without lavender oil and 

no treatment in the physiological parameters and all psychological parameters showed no effects 

throughout, bar a significantly greater improvement in mood and in anxiety levels between the rest 

group and essential oil massage group after the first session. The trial had a large number of changeable 

parameters: it involved patients in the ICU for about 5 days (age range 2–92 years), who received 

1–3 therapy sessions in 24 h given by six different nurses. Massage was performed on the back or 

outside of limbs or scalp for 15–30 min with lavender (Lavandula vera at 1% in grapeseed oil, which 

was the only constant parameter). The patients wore oxygen masks, for some of the time. It seems 

unlikely that confused patients in ICU could remember the massage or its effects and a child of 

2 years could not be expected to answer any pertinent questions. 

Massage with and without Roman chamomile in 51 palliative care patients (Wilkinson, 1995) 

showed that both groups experienced the same decrease in symptoms and severity after three full 

body massages in 3 weeks. There was, however, a statistically significant difference between the 

two groups after the first aromatherapy massage and also an improvement in the “quality of life” 

from pre- to postmassage. German chamomile was likely to have been used, not Roman chamomile 

as stated, according to the chemical composition and potential bioactivity given. 

Aromatherapy with and without massage, and massage alone on disturbed behavior in four 

patients with severe dementia (Brooker et al., 1997), was an unusual single-case study evaluating 

the use of “true” aromatherapy (using inhaled lavender oil) for 10 treatments of each, randomly 

given to each patient over a 3-month period and assessed against 10 no-treatment periods. Two 

patients became more agitated following their treatment sessions and only one patient seemed to 

have benefited. According to the staff providing the treatment, however, the use of all the treatments 

seemed to have been beneficial to the patients, suggesting pronounced bias. 

An investigation of the psychophysiological effects of aromatherapy massage following cardiac 

surgery (Stevenson, 1994) showed experimenter bias due to the statement that “neroli is also especially 

valuable in the relief of anxiety, it calms palpitations, has an antispasmodic effect and an 

anti-inflammatory effect … it is useful in the treatment of hysteria, as an antidepressant and a gentle 

sedative.” None of this has been scientifically proven, but as this was not a double-blind study and 

presumably the author did the massaging, communicating, and collating information alone, bias is 

probable. Statistical significances were not shown, nor the age ranges of the 100 patients, and no 

differences between the aromatherapy-only and massage-only groups were shown, except for an 

immediate increase in respiratory rate when the two control groups (20 min chat or rest) were compared 

with the aromatherapy massage and massage-only groups. 

Atopic eczema in 32 children treated by massage with and without essential oils (Anderson et al., 

2000) in a single-case experimental design across subjects showed that this complementary therapy 

provided no statistically significant differences between the two groups after 8 weeks of treatment. 

This indicated that massage and thereby regular parental contact and attention showed positive 

results, which was expected in these children. However, a continuation of the study, following a 

3-month period of rest, using only the essential oil massage group showed a possible sensitization 

effect, as the symptoms worsened. 

Massage using two different types of lavender oil on postcardiotomy patients (Buckle, 1993) was 

proclaimed to be a “double-blind” study but had no controls and the results by the author did not 

appear to be assessed correctly (Vickers, 1996). The author attempted to show that the “real” lavender 

showed significant benefits in the state of the patients compared with the other oil. However,


outcome measures were not described and the chemical composition and botanical names of the 

“real” and “not real” lavender remains a mystery, as three lavenders were stated in the text. Although 

the results were insignificant, this paper is quoted widely as proof that only “real” essential oils 

work through aromatherapy massage. 

Aromatherapy trails in childbirth have been of dubious design and low scientific merit and, not 

surprisingly, have yielded confusing results (Burns and Blaney, 1994), mainly due to the numerous 

parameters incorporated. In the study by Burns and Blaney (1994), many different essential 

oils were used in various uncontrolled ways during childbirth and assessed using possibly biased 

criteria as to their possible benefits to the mother and midwife. The first pilot study used 585 

women in a delivery suite over a 6-month period using lavender, clary sage, peppermint, eucalyptus, 

chamomile, frankincense, jasmine, lemon, and mandarin. These oils were either used singly 

or as part of a mixture where they could be used as the first, second, third, or fourth essential oil. 

The essential oils were applied in many different ways and at different times during parturition, 

for example, sprayed in a “solution” in water onto a face flannel, pillow, or bean bag; in a bath; 

foot bath; an absorbent card for inhalation; or in almond oil for massage. Peppermint oil was 

applied as an undiluted drop on the forehead and frankincense onto the palm. 

Midwives and mothers filled in a form as to the effects of the essential oils including their relaxant 

value, effect on nausea and vomiting, analgesic action, mood enhancer action, accelerator, or not 

of labor. The results were inconclusive and there was a bias toward the use of a few oils, for example, 

lavender was stated to be “oestrogenic and used to calm down uterine tightenings if a woman was 

exhausted and needed sleep” and clary sage was given to “encourage the establishment of labor.” 

This shows complete bias and a belief in unproven clinical attributes by the authors and presumably 

those carrying out the study. Which of the lavender, peppermint, eucalyptus, chamomile, or frankincense 

species were used remains a mystery. 

The continuation of this study (Burns et al., 2000) on 8058 mothers during childbirth was 

intended to show that aromatherapy would “relieve anxiety, pain, nausea and/or vomiting, or 

strengthen contractions.” Data from the unit audit were compared with those of 15,799 mothers not 

given aromatherapy treatment. The results showed that 50% of the aromatherapy group mothers 

found the intervention “helpful” and only 14% “unhelpful.” The use of pethidine over the year 

declined from 6% to 0.2% by women in the aromatherapy group. The study also (apparently) showed 

that aromatherapy may have the potential to augment labor contractions for women in dysfunctional 

labor, in contrast to scientific data showing that the uterine contractions decrease due to administration 

of any common essential oils (Lis-Balchin and Hart, 1997). 

It is doubtful whether a woman would in her first labor, or in subsequent ones, be able to judge 

whether the contractions were strengthened or the labor shortened due to aromatherapy. It seems 

likely that there was some placebo effect (itself a very powerful effector) due to the bias of the 

experimenters and the “suggestions” made to the aromatherapy group regarding efficacy of essential 

oils, which were obviously absent in the case of the control group. 

Lavender oil (volatilized from a burner during the night in their hospital room) has been successful 

in replacing medication to induce sleep in three out of four geriatrics (Hardy et al., 1995). There 

was a general deterioration in the sleep patterns when the medication was withdrawn, but lavender 

oil seemed to be as good as the original medication. However, the deterioration in the sleep patterns 

(due to “rebound insomnia”?) may simply have been due to recovery of normal sleep patterns when 

lavender was given (Vickers, 1996). 

The efficacy of peppermint oil was studied on postoperative nausea in 18 women after gynecological 

operations (Tate, 1997) using peppermint oil or a control, peppermint essence (obviously of 

similar odor). A statistically significant difference was found between the controls and the test group. 

The test group required less antiemetics and received less opioid analgesia. However, the use of a 

peppermint essence as a control seems rather like having two test groups as inhalation was used. 

A group of 313 patients undergoing radiotherapy were randomly assigned to receive either 

carrier oil with fractionated oils, carrier oil only, or pure essential oils of lavender, bergamot, and


cedarwood administered by inhalation concurrently with radiation treatment. There were no 

significant differences in Hospital Anxiety and Depression Score (HADS) and other scores between 

the randomly assigned groups. Aromatherapy, as administered in this study, was not found to be 

beneficial (Graham et al., 2003). 

Heliotropin, a sweet, vanilla-like scent, reduced anxiety during magnetic resonance imaging 

(Redd and Manne, 1991), which causes distress to many patients as they are enclosed in a “coffin”-like 

apparatus. Patients experienced approximately 63% less overall anxiety than a control group of 

patients. 

A double-blind randomized trial was conducted on 66 women undergoing abortions (Wiebe, 

2000). Ten minutes were spent sniffing a numbered container with either a mixture of the essential 

oils (vetivert, bergamot, and geranium) or a hair conditioner (placebo). Aromatherapy involving 

essential oils was no more effective than having patients sniff other pleasant odors in reducing preoperative 

anxiety. 

An audit into the effects of aromatherapy in palliative care (Evans, 1995) showed that the most 

frequently used oils were lavender, marjoram, and chamomile. These were applied over a period of 

6 months by a therapist available for 4 h on a weekly basis in the ward. Relaxing music was played 

throughout, each session to allay fears of the hands-on massage. The results revealed that 81% of the 

patients stated that they either felt “better” or “very relaxed” after the treatment; most appreciated 

the music greatly. The researchers themselves confessed that it is uncertain whether the benefits 

were the result of the patient being given individual attention, talking with the therapist, the effects 

of touch and massage, the effects of the aromatherapy essential oils, or the effects of the relaxation 

music. 

Aromatherapy massage studied in eight cancer patients did not show any psychological benefit. 

However, there was a statistically significant reduction in all of the four physical parameters, which 

suggests that aromatherapy massage affects the autonomic nervous system, inducing relaxation. 

This finding was supported by the patients themselves, all of whom stated during interview that they 

felt “relaxed” after aromatherapy massage (Hadfield, 2001). 

Forty-two cancer patients were randomly allocated to receive weekly massages with lavender 

essential oil in carrier oil (aromatherapy group), carrier oil only (massage group), or no intervention 

for 4 weeks (Soden et al., 2004). Outcome measures included a visual analogue scale (VAS) of pain 

intensity, the Verran and Snyder–Halpern Sleep Scale (VSH), the Hospital Anxiety and Depression 

Scale (HADS), and the Rotterdam Symptom Checklist (RSCL). No significant long-term benefits of 

aromatherapy or massage in terms of improving pain control, anxiety, or quality of life were shown. 

However, sleep scores improved significantly in both the massage and the combined massage (aromatherapy 

and massage) groups. There were also statistically significant reductions in depression 

scores in the massage group. In this study of patients with advanced cancer, the addition of lavender 

essential oil did not appear to increase the beneficial effects of massage. 

A randomized controlled pilot study was carried out to examine the effects of adjunctive aromatherapy 

massage on mood, quality of life, and physical symptoms in patients with cancer attending 

a specialist unit (Wilcock et al., 2004). Patients were randomized to conventional day care 

alone, or day care plus weekly aromatherapy massage using a standardized blend of oils for 4 weeks. 

At baseline and at weekly intervals, patients rated their mood, quality of life, and the intensity and 

bother of two symptoms most important to them. However, although 46 patients were recruited to 

the study, only 11 of 23 (48%) patients in the aromatherapy group and 18 of 23 (78%) in the control 

group completed all 4 weeks. Mood, physical symptoms, and quality of life improved in both groups 

but there was no statistically significant difference between groups, but all patients were satisfied 

with the aromatherapy and wished to continue it. 

Aromatherapy sessions in deaf and deaf–blind people became an accepted, enjoyable, and therapeutic 

part of the residents’ lifestyle in an uncontrolled series of case studies. It appeared that this 

gentle, noninvasive therapy could benefit deaf and deaf–blind people, especially as their intact 

senses can be heightened (Armstrong and Heidingsfeld, 2000).


A scientifically unacceptable study of the effect of aromatherapy on endometriosis, reported only 

at an aromatherapy conference (Worwood, 1996), involved 22 aromatherapists who treated a total 

of 17 women in two groups over 24 weeks. One group was initially given massage with essential oils 

and then not “touched” for the second period, while the second group had the two treatments 

reversed. Among the many parameters measured were constipation, vaginal discharge, fluid retention, 

abdominal and pelvic pain, degree of feeling well, renewed vigor, depression, and tiredness. 

The data were presented as means (or averages, possibly, as this was not stated) but without standard 

errors of mean (SEM) and lacked any statistical analyses. Unfortunately, the study has been accepted 

by many aromatherapists as being a conclusive proof of the value in treating endometriosis using 

aromatherapy. 

In all the trials above, there was a more positive outcome for aromatherapy if there were no stringent 

scientific double-blind and randomized control measures, suggesting that in the latter case, bias 

is removed. 

13.21 USE OF ESSENTIAL OILS MAINLY AS CHEMICAL AGENTS 

AND NOT FOR THEIR ODOR 

The efficacy and safety of capsules containing peppermint oil (90 mg) and caraway oil (50 mg), 

when studied in a double-blind, placebo-controlled, multicenter trial in patients with nonulcer dyspepsia 

was shown by May et al. (1996). Intensity of pain was significantly improved for the experimental 

group compared with the placebo group after 4 weeks. 

Six drops of pure lavender oil included in the bath water for 10 days following childbirth was 

assessed against “synthetic” lavender oil and a placebo (distilled water containing an unknown GRAS 

additive) for perineal discomfort (Cornwell and Dale, 1995). No significant differences between 

groups were found for discomfort, but lower scores in discomfort means for days 3 and 5 for the 

lavender group were seen. This was very unsatisfactory as a scientific study, mainly because essential 

oils do not mix with water and there was no proof whether the lavender oil itself was pure. 

Alopecia areata was treated in a randomized trial using “aromatherapy” carried out over 7 

months. The test group massaged a mixture of 2 drops of Thymus vulgaris, 3 drops Lavandula 

angustifolia, 3 drops of Rosmarinus officinalis, and 2 drops of Cedrus atlantica in 3 mL of jojoba 

and 20 mL grapeseed oil into the scalp for 2 min minimum every night. The control group massaged 

the carrier oils alone (Hay et al., 1998). There was a significant improvement in the test group 

(44%) compared with the control group (15%). The smell of the essential oils (psychological/physiological) 

and/or their chemical nature on the scalp may have achieved these long-term results. On 

the other hand, the scalp may have healed naturally anyway after 7 months. 

Ureterolithiasis was treated with Rowatinex, a mixture of terpenes smelling like Vicks VapoRub 

in 43 patients against a control group treated with a placebo. The overall expulsion rate of the ureteric 

stones was greater in the Rowatinex group (Engelstein et al., 1992). Similar mixes have shown 

both positive and negative results on gallstones over the years. 

In a double-blind, placebo-controlled, randomized crossover study involving 332 healthy subjects, 

four different preparations were used to treat headaches (Gobel et al., 1994). Peppermint oil, 

eucalyptus oil (species not stated), and ethanol were applied to large areas of the forehead and 

temples. A combination of the three increased cognitive performance, muscle relaxation, and mental 

relaxation, but had no influence on pain. Peppermint oil and ethanol decreased the headache. The 

reason for the success could have been the intense coldness caused by the application of the latter 

mixture, which was followed by a warming up as the peppermint oil caused counterirritation on the 

skin; the essential oils were also inhaled. 

A clinical trial on 124 patients with acne, randomly distributed to a group treated with 5% tea 

tree oil gel or a 5% benzoyl peroxide lotion group (Bassett et al., 1990), showed improvement in both 

groups and fewer side effects in the tea tree oil group. The use of tea tree oil has, however, had 

detrimental effects in some people (Lis-Balchin, 2006, Chapter 7).


A 10% tea tree oil was used on 104 patients with athlete’s foot Tinea pedis) in a randomized 

double-blind study against 1% tolnaflate and placebo creams. The tolnaflate group showed a better 

effect; tea tree oil was as effective in improving the condition, but was no better than the placebo at 

curing it (Tong et al., 1992). Surprisingly, tea tree oil is sold as a cure for athlete’s foot. 

13.21.1 SINGLE-CASE STUDIES 

In the past few years, the theme of the case studies (reported mainly in aromatherapy journals) has 

started to change and most of the aromatherapists are no longer announcing that they are “curing” 

cancer and other serious diseases. Emphasis has swung toward real complementary treatment, often 

in the area of palliative care. However, the so-called clinical aromatherapists persist in attempting 

to cure various medical conditions using high doses of oils mainly by mouth, vagina, anus, or on the 

skin. Many believe that healing wounds using essential oils is also classed as aromatherapy (Guba, 

2000) despite the evidence that odor does not kill germs and any effect is due to the chemical activity 

alone. 

Because of the lack of scientific evidence in many studies, we could assume that aromatherapy is 

mainly based on faith; it works because the aromatherapist believes in the treatment and because the 

patient believes in the supposed action of essential oils, that is, the placebo effect. 

Decreased smoking withdrawal symptoms in 48 cigarette smokers were achieved by black pepper 

oil puffed out of a special instrument for 3 h after an overnight cigarette deprivation against 

mint/menthol or nothing (Rose and Behm, 1994). 

Chronic respiratory infection was successfully treated when the patient was massaged with tea 

tree, rosemary, and bergamot oils while on her second course of antibiotics and taking a proprietary 

cough medicine. She also used lavender and rosemary oils in her bath, a drop of eucalyptus oil and 

lavender oil on her tissue near the pillow at night, 3 drops of eucalyptus and ginger for inhalations 

daily, and reduced her dairy products and starches. In a week, her cough was better and by 3 weeks, 

it had gone (Laffan, 1992). It is unclear which treatment actually helped the patient, and as it took a 

long time, the infection may well have gone away by then, or sooner, without any medicinal aid. 

After just one treatment of aromatherapy massage using rose oil, bergamot, and lavender at 2.5% 

in almond oil, a 36-year-old woman managed to get pregnant after being told she was possibly infertile 

following the removal of her right fallopian tube (Rippon, 1993)! 

Aromatherapy can apparently help patients with multiple sclerosis, especially for relaxation, in 

association with many other changes in the diet and also use of conventional medicines (Barker, 

1994). French basil, black pepper, and true lavender in evening primrose oil with borage oil was 

used to counteract stiffness and also to stimulate; this mixture was later changed to include relaxing 

and sedative oils such as Roman chamomile, ylang ylang, and melissa. 

Specific improvements in clients given aromatherapy treatment in dementia include increased 

alertness, self-hygiene, contentment, initiation of toileting, sleeping at night, and reduced levels of 

agitation, withdrawal, and wandering. Family carers reported less distress, improved sleeping patterns, 

and calmness (Kilstoff and Chenoweth, 1998). Other patients with dementia were monitored 

over a period of 2 months, and then for a further 2 months during which they received aromatherapy 

treatments in a clinical trial; they showed a significant improvement in motivational behavior during 

the period of aromatherapy treatment (MacMahon and Kermode, 1998). 


Aromatherapy, using essential oils as an odorant by inhalation or massage onto the skin, has not 

been shown to work better than massage alone or a control. No failures have, however, been reported, 

although treatment is invariably changed on each visit. Many patients feel better, even if their disease 

is getting worse, due to their belief in an alternative therapist and this is a good example of 

“mind over matter,” that is, the placebo effect. This effect has been recommended by some members


of the House of Lords Select Committee on Science and Technology, Sixth Report (2000), as a good 

basis for retaining complementary and alternative medicine, but other members argued that scientific 

proof of effects is necessary. 

It is hoped that aromatherapists do not try to convince their patients of a cure, especially in the 

case of serious ailments such as cancer, which often recede naturally for a time on their own. 

Conventional treatment should always be advised in the first instance and retained during aromatherapy 

treatment with the consent of the patient’s primary healthcare physician or consultant. 

Aromatherapy can provide a useful complementary medical service both in healthcare settings and 

in private practice, and should not be allowed to become listed as a bogus cure in alternative 

medicine. 

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Worwood, V., 1998. The Fragrant Heavens. The Spiritual Dimension of Fragrance and Aromatherapy. Novato, 

CA: New World Library. 

Youdim, K.A. and S.G. Deans, 2000. Effect of thyme oil and thymol dietary supplementation on the antioxidant 

status and fatty acid composition of the ageing rat brain. Br. J. Nutr., 83: 87–93. 

Young, A.R., et al., 1990. Phototumorigenesis studies of 5-methoxypsoralen in bergamot oil: Evaluation and 

modification of risk of human use in an albino mouse skin model. J. Phytochem. Photobiol., 7: 

231–250. 

Zarno, V., 1994. Candidiasis: A holistic view. Int. J. Aromather., 6(2): 20–23.

14 

Biotransformation of 

Monoterpenoids by 

Microorganisms, Insects, 

and Mammals 

Yoshiaki Noma and Yoshinori Asakawa 

CONTENTS 

14.1 Introduction ..................................................................................................................... 586 

14.2 Metabolic Pathways of Acyclic Monoterpenoids ............................................................ 587 

14.2.1 Acyclic Monoterpene Hydrocarbons ................................................................. 587 

14.2.1.1 Myrcene ............................................................................................ 587 

14.2.1.2 Citronellene ....................................................................................... 588 

14.2.2 Acyclic Monoterpene Alcohols and Aldehydes ................................................. 588 

14.2.2.1 Geraniol, Nerol, (+)- and (-)-Citronellol, Citral, and 

(+)- and (-)-Citronellal ..................................................................... 588 

14.2.2.2 Linalool and Linalyl Acetate ............................................................ 596 

14.2.2.3 Dihydromycenol ................................................................................ 602 

14.3 Metabolic Pathways of Cyclic Monoterpenoids ............................................................. 603 

14.3.1 Monocyclic Monoterpene Hydrocarbon ............................................................ 603 

14.3.1.1 Limonene .......................................................................................... 603 

14.3.1.2 Isolimonene ...................................................................................... 614 

14.3.1.3 p-Menthane ....................................................................................... 614 

14.3.1.4 1-p-Menthene .................................................................................... 614 

14.3.1.5 3-p-Menthene .................................................................................... 615 

14.3.1.6 α-Terpinene ....................................................................................... 616 

14.3.1.7 γ-Terpinene ........................................................................................ 616 

14.3.1.8 Terpinolene ....................................................................................... 616 

14.3.1.9 α-Phellandrene ................................................................................ 616 

14.3.1.10 p-Cymene ......................................................................................... 616 

14.3.2 Monocyclic Monoterpene Aldehyde .................................................................. 619 

14.3.2.1 Perillaldehyde ................................................................................... 619 

14.3.2.2 Phellandral and 1,2-Dihydrophellandral .......................................... 620 

14.3.2.3 Cuminaldehyde ................................................................................ 621 

14.3.3 Monocyclic Monoterpene Alcohol .................................................................... 621 

14.3.3.1 Menthol ............................................................................................. 621 

14.3.3.2 Neomenthol ...................................................................................... 627 

14.3.3.3 (+)-Isomenthol .................................................................................. 627 

14.3.3.4 Isopulegol ......................................................................................... 627 

585


14.3.3.5 α-Terpineol ....................................................................................... 629 

14.3.3.6 (-)-Terpinen-4-ol .............................................................................. 631 

14.3.3.7 Thymol and Thymol Methyl Ether ................................................... 631 

14.3.3.8 Carvacrol and Carvacrol Methyl Ether ............................................ 633 

14.3.3.9 Carveol ............................................................................................. 634 

14.3.3.10 Dihydrocarveol ................................................................................ 639 

14.3.3.11 Piperitenol ........................................................................................ 643 

14.3.3.12 Isopiperitenol ................................................................................... 643 

14.3.3.13 Perillyl Alcohol ................................................................................. 645 

14.3.3.14 Carvomenthol ................................................................................... 646 

14.3.4 Monocyclic Monoterpene Ketone ...................................................................... 648 

14.3.4.1 a, b-Unsaturated Ketone .................................................................. 648 

14.3.4.2 Saturated Ketone ............................................................................... 667 

14.3.4.3 Cyclic Monoterpene Epoxide ........................................................... 670 

14.4 Metabolic Pathways of Bicyclic Monoterpenoids ........................................................... 677 

14.4.1 Bicyclic Monoterpene ........................................................................................ 677 

14.4.1.1 a-Pinene ........................................................................................... 677 

14.4.1.2 b-Pinene ............................................................................................ 680 

14.4.1.3 (±)-Camphene .................................................................................. 686 

14.4.1.4 3-Carene and Carane ........................................................................ 688 

14.4.2 Bicyclic Monoterpene Aldehyde ........................................................................ 689 

14.4.2.1 Myrtenal and Myrtanal ..................................................................... 689 

14.4.3 Bicyclic Monoterpene Alcohol .......................................................................... 690 

14.4.3.1 Myrtenol ........................................................................................... 690 

14.4.3.2 Myrtanol ........................................................................................... 691 

14.4.3.3 Pinocarveol ....................................................................................... 691 

14.4.3.4 Pinane-2,3-diol ................................................................................. 692 

14.4.3.5 Isopinocampheol (3-Pinanol) ............................................................ 693 

14.4.3.6 Borneol and Isoborneol ................................................................... 696 

14.4.3.7 Fenchol and Fenchyl Acetate ............................................................ 697 

14.4.3.8 Verbenol ............................................................................................ 699 

14.4.3.9 Nopol and Nopol Benzyl Ether ......................................................... 699 

14.4.4 Bicyclic Monoterpene Ketones .......................................................................... 700 

14.4.4.1 a-, b-Unsaturated Ketone ................................................................ 700 

14.4.4.2 Saturated Ketone ............................................................................... 701 

14.5 Summary ......................................................................................................................... 709 

14.5.1 Metabolic Pathways of Monoterpenoids by Microorganisms ........................... 709 

14.5.2 Microbial Transformation of Terpenoids as Unit Reaction ............................... 720 

References .................................................................................................................................. 726 


A large number of monoterpenoids have been detected in or isolated from essential oils and solvent 

extracts of fungi, algae, liverworts, and higher plants, but the presence of monoterpenoids in fern is 

negligible. Vegetables, fruits, and spices contain monoterpenoids; however, their fate in human and 

other animal bodies has not yet been fully investigated systematically. The recent development of 

analytical instruments makes it easy to analyze the chemical structures of very minor components, 

and the essential oil chemistry field has dramatically developed.

Biotransformation of Monoterpenoids by Microorganisms, Insects, and Mammals 587 

Since monoterpenoids, in general, show characteristic odor and taste, they have been used as 

cosmetic materials; food additives; and often for insecticides, insect repellents, and attractant drugs. 

In order to obtain much more functionalized substances from monoterpenoids, various chemical 

reactions and microbial transformations of commercially available and cheap synthetic monoterpenoids 

have been carried out. On the other hand, insect larva and mammals have been used for direct 

biotransformations of monoterpenoids to study their fate and safety or toxicity in their bodies. 

The biotransformation of a-pinene (4) by using the black fungus Aspergillus niger was reported 

by Bhattacharyya et al. (1960) half a century ago. During that period, many scientists studied the 

biotransformation of a number of monoterpenoids by using various kinds of bacteria, fungi, insects, 

mammals, and cultured cells of higher plants. In this chapter, the microbial transformation of 

monoterpenoids using bacteria and fungi is discussed. Furthermore, the biotransformation by using 

insect larva, mammals, microalgae, as well as suspended culture cells of higher plants is also summarized. 

In addition, several biological activities of biotransformed products are also represented. 

At the end of this chapter, the metabolite pathways of representative monoterpenoids for further 

development on biological transformation of monoterpenoids are demonstrated. 

14.2 METABOLIC PATHWAYS OF ACYCLIC MONOTERPENOIDS 

14.2.1 ACYCLIC MONOTERPENE HYDROCARBONS 

14.2.1.1 Myrcene 

The microbial biotransformation of myrcene (302) was described with Diplodia gossypina ATCC 

10936 (Abraham et al., 1985). The main reactions were hydroxylation, as shown in Figure 14.1. On 

oxidation, myrcene (302) gave the diol (303) (yield up to 60%) and also a side-product (304) that 

possesses one carbon atom less than the parent compound, in yields of 1–2%. 

One of the publications dealing with the bioconversion of myrcene (Busmann and Berger, 

1994) described its transformation to a variety of oxygenated metabolites, with Ganoderma 

applanatum, Pleurotus fl abellatus, and Pleurotus sajor-caju possessing the highest transformation 

activities. One of the main metabolites was myrcenol (305) (2-methyl-6-methylene-7-octen- 

2-ol), which gives a fresh, flowery impression and dominates the sensory impact of the mixture 

(see Figure 14.1). 

D. gosssypina 

OH 

OH 

G. applanatum 

Pleurotus sp. 

OH 

OH 

303 304 

302 

OH 

305 

FIGURE 14.1 Biotransformation of myrcene (302) by Diplodia gossypina (Abraham et al., 1985), Ganoderma 

applanatum, and Pleurotus sp. (Modified from Busmann, D. and R.G. Berger, 1994. J. Biotechol., 37: 39–43.)


OH 

O 

OH 

302 307 308 

FIGURE 14.2 Biotransformation of myrcene (302) by Spodoptera litura. (Modified from Miyazawa, M. 

et al., 1998. Proc. 42nd TEAC, pp. 123–125.) 

b-Myrcene (302) was converted by common cutworm larvae, Spodoptera litura, to give 

myrcene-3,(10)-glycol (308) via myrcene-3,(10)-epoxide (307) (Figure 14.2) (Miyazawa et al., 

1998). 

14.2.1.2 Citronellene 

(-)-Citronellene (309) and (+)-citronellene (309¢) were biotransformed by the cutworm Spodoptera 

litura to give (3R)-3,7-dimethyl-6-octene-1,2-diol (310) and (3S)-3,7-dimethyl-6-octene-1,2-diol 

(310¢), respectively (Takeuchi and Miyazawa, 2005) (Figure 14.3). 

14.2.2 ACYCLIC MONOTERPENE ALCOHOLS AND ALDEHYDES 

14.2.2.1 Geraniol, Nerol, (+)- and (-)-Citronellol, Citral, and (+)- and (-)-Citronellal 

CH 2 OH 

CH 2 OH 

CHO 

CHO 

COOH 

COOH 

258 (R)- (+) 258' (S)- (–) 261 (R)- (+) 261' (S)- (–) 262 (R)- (+) 262' (S)- (–) 

Citronellol 

Citronellal 

Citronellic acid 

CH 2 OH 

CHO 

COOH 

CH 2 OH 

CHO 

COOH 

271 

Geraniol 

272 

Nerol 

276 

Geranial 

275 

Neral 

278 

Geranic acid 

277 

Neric acid 

275 & 276 

Citral 

The microbial degradation of the acyclic monoterpene alcohols citronellol (258), nerol (272), geraniol 

(271), citronellal (261), and citral (equal mixture of 275 and 276) was reported in the early part 

of 1960 (Seubert and Remberger, 1963; Seubert et al., 1963; Seubert and Fass, 1964a, 1964b). 

Pseudomonas citronellolis metabolized citronellol (258), citronellal (261), geraniol (271), and 

geranic acid (278). The metabolism of these acyclic monoterpenes is initiated by the oxidation of the


S. litura 

OH 

OH 

309 

310 

S. litura 

OH 

OH 

309' 

310' 

FIGURE 14.3 Biotransformation of (-)-citronellene (309) and (+)-citronellene (309¢) by Spodoptera litura. 

(Modified from Takeuchi, H. and M. Miyazawa, 2005. Proc. 49th TEAC, pp. 426–427.) 

primary alcohols group to the carboxyl group, followed by the carboxylation of the C-10 methyl 

group (b-methyl) by a biotin-dependent carboxylase (Seubert and Remberger, 1963). The carboxymethyl 

group is eliminated at a later stage as acetic acid. Further degradation follows the b-oxidation 

pattern. The details of the pathway are shown in Figure 14.4 (Seubert and Fass, 1964a). 

CH 2 

OH 

CHO 

COOH 

258 

261 

262 

CH 2 

COOH 

CH 2 

COOH 

OH 

CH 2 

OH 

CHO 

COOH 

CO 2 

COOH 

H 2 

O 

COOH 

272 

275 

277 281 

282 

CH 2 

OH 

CHO 

COOH 

CH 3 

COOH 

O 

β-Oxidation 

COOH 

271 

276 

278 

FIGURE 14.4 Biotransformation of citronellol (258), nerol (272), and geraniol (271) by Pseudomonas 

citronellolis. [Modified from Madyastha, K.M. 1984. Proc. Indian Acad. Sci. (Chem. Sci.), 93: 677–686.] 

283


The microbial transformation of citronellal (261) and citral (275 and 276) was reported by way 

of Pseudomonas aeruginosa (Joglekar and Dhavlikar, 1969). This bacterium, capable of utilizing 

citronellal (261) or citral (275 and 276) as the sole carbon and energy source, has been isolated 

from soil by the enrichment culture technique. It metabolized citronellal (261) to citronellic acid 

(262) (65%), citronellol (258) (0.6%), dihydrocitronellol (259) (0.6%), 3,7-dimethyl-1,7-octanediol 

(260) (1.7%), and menthol (137) (0.75%) (Figure 14.5). The metabolites of citral (275 and 276) were 

geranic acid (278) (62%), 1-hydroxy-3,7-dimethyl-6-octen-2-one (279) (0.75%), 6-methyl-5- 

heptenoic acid (280) (0.5%), and 3-methyl-2-butenoic acid (286) (1%) (Figure 14.5). In a similar 

way, Pseudomonas convexa converted citral (275 and 276) to geranic acid (278) (Hayashi et al., 

1967). The biotransformation of citronellol (258) and geraniol (271) by Pseudomonas aeruginosa, 

Pseudomonas citronellolis, and Pseudomonas mendocina was also reported by another group 

(Cantwell et al., 1978). 

A research group in Czechoslovakia patented the cyclization of citronellal (261) with subsequent 

hydrogenation to menthol by Penicillium digitatum in 1952. Unfortunately the optical purities of the 

intermediates pulegol and isopulegol were not determined and presumably the resulting menthol 

was a mixture of enantiomers. Therefore, it cannot be excluded that this extremely interesting cyclization 

is the result of a reaction primarily catalyzed by the acidic fermentation conditions and only 

partially dependent on enzymatic reactions (Babcka et al., 1956) (Figure 14.6). 

Based on previous data (Madyastha et al., 1977; Rama and Bhattacharyya, 1977a), two pathways 

for the degradation of geraniol (271) were proposed by Madyastha (1984) (Figure 14.7). Pathway A 

involves an oxidative attack on the 2,3-double bond, resulting in the formation of an epoxide. 

Opening of the epoxide yields the 2,3-dihydroxygeraniol (292), which upon oxidation forms 2-oxo, 

3-hydroxygeraniol (293). The ketodiol (293) is then decomposed to 6-methyl-5-hepten-2-one (294) 

by an oxidative process. Pathway B is initiated by the oxidation of the primary alcoholic group 

to geranic acid (278) and further metabolism follows the mechanism as proposed earlier for 

Pseudomonas citronellolis (Seubert and Remberger, 1963; Seubert et al., 1963). In the case of nerol 

(272), the Z-isomer of geraniol (271), degradative pathways analogous to pathways A and B as in 

geraniol (271) are observed. It was also noticed that Pseudomonas incognita metabolizes acetates 

of geraniol (271), nerol (272), and citronellol (258) much faster than their respective alcohols 

(Madyastha and Renganathan, 1983). 

CHO 

P. aeruginosa 

COOH CH 2 

OH CH 2 

OH CH 2 

OH 

OH 

OH 

261 262 

258 

259 

260 

137 

COOH 

CHO 

CHO 

P. aeruginosa 

COOH 

O 

CH 2 

OH 

HOOC 

275 276 

278 

279 

280 

286 

FIGURE 14.5 Biotransformation of citronellal (261) and citral (275 and 276) by Pseudomonas aeruginosa. 

(Modified from Joglekar, S.S. and R.S. Dhavlikar, 1969. Appl. Microbiol., 18: 1084–1087.)


OH 

CHO 

P. digitatum 263 

OH 

261 

OH 

137 

267 

FIGURE 14.6 Biotransformation of citronellal to menthol by Penicillium digitatum. (Modified from 

Babcka, J. et al., 1956. Patent 56-9686b.) 

OH 

OH 

CH 2 

OH CH 2 

OH CH 2 

OH 

A 

OH 

O 

271 292 

293 

B 

294 

CH 2 

COOH 

O 

CO 2 

H 2 

O 

CH 2 

COOH 

OH 

CHO 

COOH 

COOH 

CO 2 

β-Oxidation 

COOH 

H 2 

O 

COOH 

276 278 277 

281 

282 

CH 3 

COOH 

O 

COOH 

FIGURE 14.7 Metabolism of geraniol (271) by Pseudomonas incognita. [Modified from Madyastha, K.M. 

1984. Proc. Indian Acad. Sci. (Chem. Sci.), 93: 677–686.] 

Euglena gracilis Z converted citral (275 and 276, 56:44, peak area in GC) to geraniol (271) and 

nerol (272), respectively, of which geraniol (271) was further transformed to (+)- and (-)-citronellol 

(258 and 258¢). On the other hand, when either geraniol (271) or nerol (272) was added, both compounds 

were isomerized to each other and, then, geraniol (271) was transformed to citronellol. 

These results showed that Euglena could distinguish between the stereoisomers geraniol (271) and 

nerol (272) and hydrogenated geraniol (271) selectively. (+)-, (-)-, and (±)-Citronellal (261, 261¢, and 

283


COOH 

COOH 

278 

262 

142a 

OH 

OH 

CHO 

CH 2 OH 

CH 2 OH 

CHO 

OH 

OH 

276 

271 

258 

261 

142b 

CHO 

CH 2 OH 

CH 2 OH 

CHO 

OH 

OH 

275 

272 

258' 

261' 

142b' 

277 

COOH 

262' 

COOH 

FIGURE 14.8 Metabolic pathways of citral (275 and 276) and its metabolites by Euglena gracilis Z. 

(Modified from Noma, Y. et al., 1991a. Phytochem., 30: 1147–1151.) 

142a' 

OH 

OH 

261 and 261¢) were also transformed to the corresponding (+)-, (-)-, and (±)-citronellol (258, 258¢, 

and 258 and 258¢) as the major products and (+)-, (-)-, and (±)-citronellic acids (262, 262¢, and 262 

and 262¢) as the minor products, respectively (Noma et al., 1991a) (Figure 14.8). 

Dunaliella tertiolecta also reduced citral (geranial (276) and neral (275) = 56:44), (+)-, (-)-, 

and (±)-citronellal (261, 261¢, and 261 and 261¢) to the corresponding alcohols, namely, geraniol 

(271), nerol (272), (+)-, (-)-, and (±)-citronellol (258, 258¢, and 258 and 258¢) (Noma et al., 1991b, 

1992a). 

Citral (a mixture of geranial (276) and neral (275), 56:44 peak area in GC) is easily transformed 

to geraniol (271) and nerol (272), respectively, of which geraniol (32) is further hydrogenated to 

(+)-citronellol (258) and (-)-citronellol (258¢). Geranic acid (278) and neric acid (277) as the minor 

products are also formed from 276 and 275, respectively. On the other hand, when either 271 or 

272 is used as a substrate, both compounds are isomerized to each other, and then 271 is transformed 

to citronellol (258 or 258¢). These results showed the Euglena could distinguish between


the stereoisomers, 271 and 272 and hydrogenated selectively 271 to citronellol (258 or 258¢). (+)-, 

(-)-, and (±)-Citronellal (261, 261¢, and equal mixture of 261 and 261¢) are also transformed to the 

corresponding citronellol and p-menthane-trans- and cis-3,8-diols (142a, b, a¢ and b¢) as the major 

products, which are well known as mosquito repellents and plant growth regulators (Nishimura 

et al., 1982; Nishimura and Noma, 1996), and (+)-, (-)-, and (±)-citronellic acids (262, 262¢, and 

equal mixture of 262 and 262¢) as the minor products, respectively. 

Streptomyces ikutamanensis, Ya-2–1, also reduced citral (geranial (276) and neral (275) = 56:44), 

(+)-, (-)-, and (±)-citronellal (261, 261¢, and 261 and 261¢) to the corresponding alcohols, namely, 

geraniol (271), nerol (272), (+)-, (-)-, and (±)-citronellol (258, 258¢, 258 and 258¢). Compounds 271 

and 272 were isomerized to each other. Furthermore, terpene alcohols (258¢, 272, and 271) were 

epoxidized to give 6,7-epoxygeraniol (274), 6,7-epoxynerol (273), and 2,3-epoxycitronellol (268). 

On the other hand, (+)- and (±)-citronellol (258 and 258 and 258¢) were not converted at all (Noma 

et al., 1986) (Figure 14.9). 

CHO 

CH 2 OH 

CH 2 OH 

276 

271 

274 

O 

CHO 

CH 2 OH 

CH 2 OH 

O 

275 

272 

273 

CHO 

CH 2 OH 

CH 2 OH 

261' 

258' 

268 

O 

CHO 

CH 2 OH 

261 

258 

CHO 

CH 2 OH 

261 & 261' 

258 & 258' 

FIGURE 14.9 Reduction of terpene aldehydes and epoxidation of terpene alcohols by Streptomyces 

ikutamanensis, Ya-2-1. (Modified from Noma, Y. et al., 1986. Proc. 30th TEAC, pp. 204–206.)


A strain of Aspergillus niger, isolated from garden soil, was able to transform geraniol (271), 

citronellol (258 and 258¢), and linalool (206) to their respective 8-hydroxy derivatives. This reaction 

was called “w-hydroxylation” (Madyastha and Krishna Murthy, 1988a, 1988b). 

Fermentation of citronellyl acetate with Aspergillus niger resulted in the formation of a major 

metabolite, 8-hydroxycitronellol, accounting for approximately 60% of the total transformation products, 

accompanied by 38% citronellol. Fermentation of geranyl acetate with Aspergillus niger gave 

geraniol and 8-hydroxygeraniol (50% and 40%, respectively, of the total transformation products). 

One of the most important examples of fungal bioconversion of monoterpene alcohols is the 

biotransformation of citral by Botrytis cinerea. Botrytis cinerea is a fungus of high interest in winemaking 

(Rapp and Mandery, 1988). In an unripe state of maturation the infection of grapes by 

Botrytis cinerea is very much feared, as the grapes become mouldy (“gray rot”). With fully ripe 

grapes, however, the growth of Botrytis cinerea is desirable; the fungus is then called “noble rot” 

and the infected grapes deliver famous sweet wines, such as, for example, Sauternes of France or 

Tokay Aszu of Hungary (Brunerie et al., 1988). 

One of the first reports in this area dealt with the biotransformation of citronellol (258) by Botrytis 

cinerea (Brunerie et al., 1987a, 1988). The substrate was mainly metabolized by w-hydroxylation. 

The same group also investigated the bioconversion of citral (275 and 276) (Brunerie et al., 1987b). A 

comparison was made between grape must and a synthetic medium. When using grape must, no volatile 

bioconversion products were found. With a synthetic medium, biotransformation of citral (275 and 276) 

was observed yielding predominantly nerol (272) and geraniol (271) as reduction products and some 

w-hydroxylation products as minor compounds. Finally, the bioconversion of geraniol (271) and nerol 

(272) was described by the same group (Bock et al., 1988). When using grape must, a complete bioconversion 

of geraniol (271) was observed mainly yielding w-hydroxylation products. 

The most important metabolites from geraniol (271), nerol (272), and citronellol (258) are summarized 

in Figure 14.9. In the same year the biotransformation of these monoterpenes by Botrytis 

cinerea in model solutions was described by another group (Rapp and Mandery, 1988). Although 

the major metabolites found were w-hydroxylation compounds, it is important to note that some 

new compounds that were not described by the previous group were detected (Figure 14.9). Geraniol 

(271) was mainly transformed to (2E,5E)-3,7-dimethyl-2,5-octadiene-1,7-diol (318), (E)-3,7- 

dimethyl-2,7-octadiene-1,6-diol (319), and (2E,6E)-2,6-dimethyl-2,6-octadiene-1,8-diol (300); nerol 

(272) to (2Z,5E)-3,7-dimethyl-2,5-octadiene-1,7-diol (314), (Z)-3,7-dimethyl-2,7-octadiene-1,6-diol 

(315), and (2E,6Z)-2,6-dimethyl-2,6-octadiene-1,8-diol (316). Furthermore, a cyclization product 

(318) that was not previously described was formed. Finally, citronellol (258) was converted to 

trans- (312) and cis-rose oxide (313) (a cyclization product not identified by the other group), (E)-3,7- 

dimethyl-5-octene-1,7-diol (311), 3,7-dimethyl-7-octene-1,6-diol (260), and (E)-2,6-dimethyl-2- 

octene-1,8-diol (265) (Miyazawa et al., 1996a) (Figure 14.10). 

One of the latest reports in this area described the biotransformation of citronellol by the plant 

pathogenic fungus Glomerella cingulata to 3,7-dimethyl-1,6,7-octanetriol (Miyazawa et al., 1996a). 

The ability of fungal spores of Penicillium digitatum to biotransform monoterpene alcohols, 

such as geraniol (271) and nerol (272) and a mixture of the aldehydes, that is, citral (276 and 275), 

has only been discovered very recently by Demyttenaera and coworkers (Demyttenaera et al., 1996, 

2000; Demyttenaera and De Pooter, 1996, 1998). Spores of Penicillium digitatum were inoculated 

on solid media. After a short incubation period, the spores germinated and a mycelial mat was 

formed. After 2 weeks, the culture had completely sporulated and bioconversion reactions were 

started. Geraniol (271), nerol (272), or citral (276 and 275) were sprayed onto the sporulated surface 

culture. After 1 or 2 days, the period during which transformation took place, the cultures were 

extracted. Geraniol and nerol were transformed into 6-methyl-5-hepten-2-one by sporulated surface 

cultures of Pencillium digitatum. The spores retained their activity for at least 2 months. An overall 

yield of up to 99% could be achieved. 

The bioconversion of geraniol (271) and nerol (272) was also performed with sporulated surface 

cultures of Aspergillus niger. Geraniol (271) was converted to linalool (206), a-terpineol (34), and


H 

H 

258 

CH 2 OH 

311 

5 6 7 

CH 2 OH CH 2 OH 4 CH 2 OH 

3 

OH 

260 

OH 

2 

265 

1 

OH 

O 

312 

O 

313 

272 

CH 2 OH 

314 

CH 2 OH CH 2 OH CH 2 OH 

OH 

OH 

315 

316 

8 

1 

OH 

O 

317 

271 

CH 2 OH CH 2 OH CH 2 OH 3 

CH 2 OH 

4 2 

OH 

5 

6 

318 

OH 

319 

300 

7 

8 

OH 

FIGURE 14.10 Biotransformation of geraniol (271), nerol (272), and citronellol (258) by Botrytis cinerea. 

(Modified from Miyazawa, M. et al., 1996a. Nat. Prod. Lett., 8: 303–305.) 

limonene (68), and nerol (272) was converted mainly to linalool (206) and a-terpineol (34) 

(Demyttenaera et al., 2000). 

The biotransformation of geraniol (271) and nerol (272) by Catharanthus roseus suspension 

cells was carried out. It was found that the allylic positions of geraniol (271) and nerol (272) were 

hydroxylated and reduced to double bond and ketones (Figure 14.11). Geraniol (271) and nerol (272) 

were isomerized to each other. Geraniol (271) and nerol (272) were hydroxylated at C10 to 

CH 2 OH CH 2 OH CH 2 OH CH 2 OH 

258 271 

300 CH 2 OH 

265 

CH 2 OH 

CH 2 OH 

CH 2 OH 

272 

320 CH 2 OH 

FIGURE 14.11 The biotransformation of geraniol (271) and nerol (272) by Catharanthus roseus. (Modified 

from Hamada, H. and H. Yasumune, 1995. Proc. 39th TEAC, pp. 375–377.)


8-hydroxygeraniol (300) and 8-hydroxynerol (320), respectively. 8-Hydroxygeraniol (300) was 

hydrogenated to 10-hydroxycitronellol (265). Geraniol (271) was hydrogenated to citronellol (258) 

(Hamada and Yasumune, 1995). 

Cyanobacterium converted geraniol (271) to geranic acid (278) via geranial (276), followed by 

hydrogenation to give citronellic acid (262) via citronellal (261). Furthermore, the substrate 271 was 

isomerized to nerol (272), followed by oxidation, reduction, and further oxidation to afford neral 

(275), citronellal (261), citronellic acid (262), and nerolic acid (277) (Kaji et al., 2002; Hamada 

et al., 2004) (Figure 14.12). 

Plant suspension cells of Catharanthus roseus converted geraniol (271) to 8-hydroxygeraniol 

(300). The same cells converted citronellol (258) to 8- (265) and 10-hydroxycitronellol (264) 

(Hamada et al., 2004) (Figure 14.13). 

Nerol (272) was converted by the insect lavae Spodoptera litura to give 8-hydroxynerol (320), 

10-hydroxynerol (321), 1-hydroxy-3,7-dimethyl-(2E,6E)-octadienal (322), and 1-hydroxy-3,7- 

dimethyl-(2E,6E)-octadienoic acid (323) (Takeuchi and Miyazawa, 2004) (Figure 14.14). 

14.2.2.2 Linalool and Linalyl Acetate 

(+)-Linalool (206) [(S)-3,7-dimethyl-1,6-octadiene-3-ol] and its enantiomer (206¢) ((R)-3,7-dimethyl- 

1,6-octadiene-3-ol) occur in many essential oils, where they are often the main component. (S)-(+)- 

Linalool (206) makes up 60–70% of coriander oil. (R)-(-)-linalool (206¢), for example, occurs at a 

concentration of 80–85% in Ho oils from Cinnamomum camphora; rosewood oil contains ca. 80% 

(Bauer et al., 1990). 

CH 2 OH 

CHO 

COOH 

271 276 278 

CH 2 OH 

CHO 

COOH 

258 

261 262 

CH 2 OH 

CHO 

COOH 

272 275 277 

OH 

206 

FIGURE 14.12 Biotransformation of geraniol (271) and citronellol (258) by Cyanobacterium.


CH 2 OH 

CH 2 OH 

Catharanthus 

roseus 

271 300 

CH 2 OH 

CH 2 OH 

Catharanthus 

roseus 

CH 2 OH 

+ 

CH 2 OH 

258 265 

CH 2 OH 

HOH 2 C 

264 

FIGURE 14.13 Biotransformation of geraniol (271), citronellol (258), and linalool (206) by plant suspension 

cells of Catharanthus roseus. (Modified from Hamada, H. et al., 2004. Proc. 48th TEAC, pp. 393–395.) 


CH 2 OH 

CHO 

320 322 323 

COOH 

CH 2 OH 

272 

CH 2 OH 

HOH 2 C 

321 

FIGURE 14.14 Biotransformation of nerol (272) by Spodoptera litura. (Modified from Takeuchi, H. and 

M. Miyazawa, 2004. Proc. 48th TEAC, pp. 399–400.) 

Catharanthus roseus converted (+)-linalool (206) to 8-hydroxylinalool (219) (Hamada et al., 

2004) (Figure 14.15). 

The biodegradation of (+)-linalool (206) by Pseudomonas pseudomallei (strain A), which grows 

on linalool as the sole carbon source, was described in 1973 (Murakami et al., 1973) (Figure 14.16). 

Madyastha et al. (1977) isolated a soil Pseudomonad, Pseudomonas incognita, by the enrichment 

culture technique with linalool as the sole carbon source. This microorganism, the “linalool strain” 

as it was called, was also capable of utilizing limonene (68), citronellol (258), and geraniol (271) but 

failed to grow on citral (275 and 276), citronellal (261), and 1,8-cineole (122). Fermentation was 

carried out with shake cultures containing 1% linalool (206) as the sole carbon source. It was suggested 

by the authors that linalool (206) was metabolized by at least three different pathways of 

biodegradation (Figure 14.19). One of the pathways appeared to be initiated by the specific oxygenation 

of C-8 methyl group of linalool (206), leading to 8-hydroxylinalool (219), which was further 

oxidized to linalool-8-carboxylic acid (220). The presence of furanoid linalool oxide (215) and 

2-methyl-2-vinyltetrahydrofuran-5-one (216) as the unsaturated lactone in the fermentation medium


OH 

OH 

206 (S)- 

(+)-linalool 

206' (R)- 

(–)-linalool 

OH 

OH 

Catharanthus 

roseus 

206 

219 

CH 2 

OH 

FIGURE 14.15 Biotransformation of linalool (206) by plant suspension cells of Catharanthus roseus. 

(Modified from Hamada, H. et al., 2004. Proc. 48th TEAC, pp. 393–395.) 

OH 

OH 

OH 

COOHC OOH COOH 

CH 2 OH 

COOH 

COOH 

206 219 

220 221 222 223 224 

FIGURE 14.16 Degradative metabolic pathway of (+)-linalool (206) by Pseudomonas pseudomallei. 

(Modified from Murakami, T. et al., 1973. Nippon Nogei Kagaku Kaishi, 47: 699–703.) 

HO 

H 

3 6 

O 

215a 

trans 

3R, 6R 

HO 

H 

O 

215b 

cis 

3S, 6R 

H 

H 

HO 

O 

215a' 

trans 

3S, 6S 

FIGURE 14.17 Four stereoisomers of furanoid linalool oxides. (Modified from Noma, Y. et al., Proc. 30th 

TEAC, pp. 204–206.) 

suggested another mode of utilization of linalool (206). The formation of these compounds was 

believed to proceed through the epoxidation of the 6,7-double bond giving rise to 6,7-epoxylinalool 

(214), which upon further oxidation yielded furanoid linalool oxide (215) and 2-methyl-2-vinyltetrahydrofuran-5-one 

(216) (Figure 14.19). 

The presence of oleuropeic acid (204) in the fermentation broth suggested a third pathway. Two 

possibilities were proposed: (3a) water elimination giving rise to a monocyclic cation (33), yielding 

HO 

O 

215b' 

cis 

3R, 6S


HO 

HO 

3 

6 

O 

217a 

trans 

3 S, 6 R 

HO 

HO 

O 

217b' 

cis 

3 R, 6 R 

O 

O 

217b 

cis 

3 S, 6 S 

217a' 

trans 

3 R, 6 S 

FIGURE 14.18 Four stereoisomers of pyranoid linalool oxides. 

a-terpineol (34), which upon oxidation gave oleuropeic acid (204); (3b) oxidation of the C-10 methyl 

group of linalool (206) before cyclization, giving rise to oleuropeic acid (204). This last pathway 

was also called the “prototropic cyclization” (Madyastha, 1984). 

Racemic linalool (206 and 206¢) is cyclized into cis- and trans-linalool oxides by various microorganisms 

such as Streptomyces albus NRRL B1865, Streptomyces hygroscopicus NRRL B3444, 

Streptomyces cinnamonnensis ATCC 15413, Streptomyces griseus ATCC 10137, and Beauveria 

sulfurescens ATACC 7159 (David and Veschambre, 1984) (Figure 14.19). 

Aspergillus niger isolated from garden soil biotransformed linalool and its acetates to give linalool 

(206), 2,6-dimethyl-2,7-octadiene-1,6-diol (8-hydroxylinalool (219a), a-terpineol (34), geraniol (271), 

and some unidentified products in trace amounts (Madyastha and Krishna Murthy, 1988a, 1988b). 

OH 

OH 

OH 

see 

Fig.15 

O 

HO 

O 

215 

O 

O 

216 

COOH 

CH 2 OH 

220 219 214 

2 

1 

COOH 

OH 

OH 

COOH 

COOH 

3b 

+ 

3a 

206 

211 

+ 

213 

+ 

COOH 

+ 

+ 

OH 

207 208 33 34 

204 

OH 

FIGURE 14.19 Biotransformation of linalool (206) by Pseudomonas incognita (Madyastha et al., 1977) and 

Streptomyces albus NRRL B1865. (Modified from David, L. and H. Veschambre, 1984. Tetrahadron Lett., 

25: 543–546.)


OH 

OH 

219a 

OH 

HO 

219b 

324 

O 

H 

H 

HO 

O 

215a 

HO 

O 

215b 

O 

O 

216 

HO 

HO 

O 

O 

O 

O 

O 

O 

O 

O 

217a 217b 217a-Ac 217b-Ac 

FIGURE 14.20 Biotransformation products of linalool (206) by Botrytis cinerea. (Modified from Bock, G. 

et al., 1986. J. Food Sci., 51: 659–662.) 

The biotransformation of linalool (206) by Botrytis cinerea was carried out and identified transformation 

products such as (E)-(219a) and (Z)-2,6-dimethyl-2,7-octadiene-1,6-diol (219b), trans- 

(215a) and cis-furanoid linalool oxide (215b), trans- (217a) and cis-pyranoid linalool oxide (217b) 

(Figure 14.18) and their acetates (217a-Ac, 217b-Ac), 3,9-epoxy-p-menth-1-ene (324) and 2-methyl- 

2-vinyl-tetrahydrofuran-5-one (216) (unsaturated lactone) (Bock et al., 1986) (Figure 14.20). 

Quantitative analysis, however, showed that linalool (206) was predominantly (90%) metabolized to 

(E)-2,6-dimethyl-2,7-octadiene-1,6-diol (219a) by Botrytis cinerea. The other compounds were 

only found as by-products in minor concentrations. 

The bioconversion of (S)-(+)-linalool (206) and (R)-(-)-linalool (206¢) was investigated with 

Diplodia gossypina ATCC 10936 (Abraham et al., 1990). The biotransformation of (±)-linalool (206 

and 206¢) by Aspergillus niger ATCC 9142 with submerged shaking culture yielded a mixture of 

cis- (215b) and trans-furanoid linalool oxide (215a) (yield 15–24%) and cis- (217b) and transpyranoid 

linalool oxide (217a) (yield 5–9%) (Demyttenaere and Willemen, 1998). The biotransformation 

of (R)-(-)-linalool (206a) with Aspergillus niger ATCC 9142 yielded almost pure 

trans-furanoid linalool oxide (215a) and trans-pyranoid linalool oxide (217a) (ee > 95) (Figure 

14.21). These conversions were purely biocatalytic, since in acidified water (pH < 3.5) almost 50% 

linalool (206) was recovered unchanged, the rest was evaporated. The biotransformation was also 

carried out with growing surface cultures. 

OH 

OH 

HO 

A. niger ATCC 9142 

O 

H 

+ 

HO O O 

215a 

217a 

206' 214' 

FIGURE 14.21 Biotransformation of (R)-(-)-linalool (206¢) by Aspergillus niger ATCC 9142. (Modified 

from Demyttenaere, J.C.R. and H.M. Willemen, 1998. Phytochemistry, 47: 1029–1036.)


OH 

OH 

S. ikutamanensis, 

Ya-2-1 

O 

HO 

H 

3 6 

O 

215a 

HO 

H 

O 

215b 

206a 

214a 

OH 

OH 

H 

H 


Ya-2-1 

O 

HO 

O 

215a' 

HO 

O 

215b' 

206b 

214b 

H 

H 

OH 

OH 

HO 

O 

215a 

HO 

O 

215b 


Ya-2-1 

O 

HO 

O 

HO 

O 

206ab 

214ab 

215a' 

215b' 

FIGURE 14.22 Metabolic pathway of (+)- (206), (-)- (206¢), and racemic linalool (206 and 206¢) by 

Streptomyces ikutamanensis, Ya-2-1. (Modified from Noma, Y. et al., 1986. Proc. 30th TEAC, pp. 204–206.) 

H 

H 

Streptomyces ikutamanensis, Ya-2-1 also converted (+)- (206), (-)- (206¢), and racemic linalool 

(206 and 206¢) via corresponding 2,3-epoxides (214 and 214¢) to trans- and cis-furanoid linalool 

oxides (215a, b, a¢ and b¢) (Noma et al., 1986) (Figure 14.22). The absolute configuration at C-3 and 

C-6 of trans- and cis-linalool oxides are shown in Figure 14.17. 

Biotransformation of racemic trans-pyranoid linalool oxide (217a and a¢) and racemic cislinalool-pyranoid 

(217b and b¢) has been carried out using fungus Glomerella cingulata 

(Miyazawa et al., 1994a). trans and cis-Pyranoid linalool oxide (217a and 217b) were transformed 

to trans- (217a¢-1) and cis-linalool oxide-3-malonate (217b¢-1), respectively. In the 

biotransformation of racemic cis-linalool oxide-pyranoid, (+)-(3R,6R)-cis-pyranoid linalool oxide 

(217a and a¢) was converted to (3R,6R)-pyranoid-cis-linalool oxide-3-malonate (217a¢-1). (-)-(3S, 

6S)-cis-Pyranoid linalool oxide-pyranoid (217a¢) was not metabolized. On the other hand, in the 

biotransformation of racemic trans-pyranoid linalool oxide (217b and b¢), (-)-(3R,6S)-trans-linalool 

oxide (217b¢) was transformed to (3R,6S)-trans-linalool oxide-3-malonate (217b¢-1) 

(Figure 14.23). (+)-(3S,6S)-trans-Pyranoid-linalool oxide (217b) was not metabolized. These 

facts showed that Glomerella cingulata recognized absolute configuration of the secondary 

hydroxyl group at C-3. On the basis of this result, it has become apparent the optical resolution of 

racemic pyranoid linalool oxide proceeded in the biotransformation with Glomerella cingulata 

(Miyazawa et al., 1994a). 

Linalool (206) and tetrahydrolinalool (325) were converted by suspension cells of Catharanthus 

roseus to give 1-hydroxylinalool (219) from linalool (206) and 3,7-dimethyloctane-3,5-diol (326), 

3,7-dimethyloctane-3,7-diol (327), and 3,7-dimethyloctane-3,8-diol (328) from tetrahydrolinalool 

(325) (Hamada and Furuya, 2000; Hamada et al., 2004) (Figure 14.24).


HO 

3 

8 

O 

217a&a' 

217a: (3S, 6R) 

217a': (3R, 6S) 

G. cingulata 

RO 

O 

O 

O 

+ 

HO 

O 

O 

217a' 

217a 

(3R, 6S) 

217a: (3S, 6R) 

217a'-1: R-H 

217a'-2: R=CH 3 

HO 

3 

8 

O 

217b&b' 

217b: (3S, 6S) 

217b': (3R, 6R) 

G. cingulata 

RO 

O 

O 

O 

O 

217b' 

(3S, 6R) 

217b'-1: R-H 

217b'-2: R=CH 3 

+ 

HO 

O 

217b 

217a': (3S, 6S) 

FIGURE 14.23 Biotransformation of racemic trans-linalool oxide-pyranoid (217a and a¢) and racemic 

cis-linalool-pyranoid (217b and b¢) by Glomerella cingulata. (Modified from Miyazawa, M. et al., 1994a. 

Proc. 38th TEAC, pp. 101–102.) 

OH 

OH 

C. roseus 

206 

219 

CH 2 OH 

OH 

OH 

OH 

OH 

C. roseus 

+ + 

HO 

OH 

325 

326 327 328 

CH 2 OH 

FIGURE 14.24 Biotransformation of linalool (206) and tetrahydrolinalool (325) by Catharanthus roseus. 

(Modified from Hamada, H. and T. Furuya, 2000. Proc. 44th TEAC, pp. 167–168; Hamada, H. et al., 2004. 

Proc. 48th TEAC, pp. 393–395.) 

(±)-Linalyl acetate (206-Ac) was hydrolyzed to (+)-(S)-linalool (206) and (±)-linallyl acetate 

(206-Ac) by Bacillus subtilis, Trichoderma S, Absidia glauca, and Gibberella fujikuroi as shown in 

Figure 14.25. But, (±)-dihydrolinallyl acetate (469-Ac) was not hydrolyzed by the above microorganisms 

(Oritani and Yamashita, 1973a). 

14.2.2.3 Dihydromycenol 

Dihydromyrcenol (329) was fed by Spodptera litura to give 1,2-epoxydihydro-myrcenol (330) as a 

main products and 3b-hydroxydidyromyrcerol (331) as a minor product. Dihydromyrcenyl acetate 

(332) was converted to 1,2-dihydroxydihydromyrcenyl acetate (333) (Murata and Miyazawa, 1999) 

(Figure 14.26).


OAc 

OH 

OAc 

B. subtilis 

+ 

206-Ac 

OAc 

206 

(+)-(S) 

206-Ac 

(+ –) 

469-Ac 

FIGURE 14.25 Hydrolysis of (±)-linalyl acetate (206-Ac) by microorganisms. (Modified from Oritani, T. 

and K. Yamashita, 1973a. Agric. Biol. Chem., 37: 1923–1928.) 

OH 

329 

OH 

330 

OH 

O 

+ 

331 

OH 

OH 

OAc 

OH 

OAc 

FIGURE 14.26 Biotransformation of dihydromyrcenol (329) and dihydromyrcenyl acetate (332) by 

Spodptera litura. (Modified from Murata, T. and M. Miyazawa, 1999. Proc. 43rd TEAC, pp. 393–394.) 

14.3 METABOLIC PATHWAYS OF CYCLIC MONOTERPENOIDS 

14.3.1 MONOCYCLIC MONOTERPENE HYDROCARBON 

14.3.1.1 Limonene 

332 333 

68 

(R)-limonene 

68' 

(S)-limonene 

Limonene is the most widely distributed terpene in nature after a-pinene (4) (Krasnobajew, 1984). 

(4R)-(+)-Limonene (68) is present in Citrus peel oils at a concentration of over 90%; a low concentration 

of the (4S)-(-)-limonene (68¢) is found in oils from the Mentha species and conifers


(Bauer et al., 1990). The first microbial biotransformation on limonene was carried out by using a soil 

Pseudomonad. The microorganism was isolated by the enrichment culture technique on limonene as 

the sole source of carbon (Dhavalikar and Bhattacharyya, 1966). The microorganism was also capable 

of growing on a-pinene (4), b-pinene (1), 1-p-menthene (62), and p-cymene (178). The optimal level 

of limonene for growth was 0.3–0.6% (v/v) although no toxicity was observed at 2% levels. 

Fermentation of limonene (68) by this bacterium in a mineral-salts medium resulted in the formation 

of a large number of neutral and acidic products such as dihydrocarvone (64), carvone (61), carveol 

(60), 8-p-menthene-1,2-cis-diol (65b), 8-p-menthen-1-ol-2-one (66), 8-p-menthene-1,2-trans-diol 

(65a), and 1-p-menthene-6,9-diol (62). Perillic acid (69), b-isopropenyl pimeric acid (72), 2-hydroxy- 

8-p-menthen-7-oic acid (70), and 4,9-dihydroxy-1-p-menthen-7-oic acid (73) were isolated and 

identified as acidic compounds. Based on these data three distinct pathways for the catabolism of 

limonene (68) by the soil Pseudomonad were proposed by Dhavalikar et al. (1966), involving allylic 

oxygenation (pathway1), oxygenation of the 1,2-double bond (pathway 2), and progressive oxidation 

of the 7-methyl group to perillic acid (82) (pathway 3) (Figure 14.27) (Krasnobajew, 1984). Pathway 

O 

OH 

93 

OH 

Pathway 1 

108 

90 

OH 

O 

O 

Pathway 2 

68 

69 101 

HO 

Pathway 3 

(main pathway) 

OH 

O 

OH 

COOH 

HOOC 

COOH 

HO 

71 

OH 

72 83 

86 

CH 2 OH 

CHO 

COOH COOH COOH 

HO 

O 

74 78 82 84 85 

FIGURE 14.27 Pathways for the degradation of limonene (68) by a soil Pseudomonad sp. strain (L). (Modified 

from Krasnobajew, V., 1984. In: Biotechnology, K. Kieslich, ed., Vol. 6a, pp. 97–125. Weinheim: Verlag Chemie.)


2 yields (+)-dihydrocarvone (101) via intermediate limonene epoxide (69) and 8-p-menthen-1-ol-2- 

one (72) as oxidation product of limonene-1,2-diol (71). The third and main pathway leads to perillyl 

alcohol (74), perillaldehyde (78), perillic acid (82), constituents of various essential oils and used in 

the flavour and fragrance industry (Fenaroli, 1975), 2-oxo-8-p-menthen-7-oic acid (85), b-isopropenyl 

pimeric acid (86), and 4,9-dihydroxy-1-p-menthene-7-oic acid (83). 

(+)-Limonene (68) was biotransformed via limonene-1,2-epoxide (69) to 8-p-menthene 1,2- 

trans-diol (71b). On the other hand, (+)-carvone (93) was biotransformed via (-)-isodihydrocarvone 

(101b) and 1a-hydroxydihydrocarvone (72) to (+)-8-p-menthene-1,2-trans-diol (71a) (Noma et al., 

1985a, 1985b) (Figure 14.28). A soil Pseudomonad formed 1-hydroxydihydrocarvone (72), 

8-p- menthene-1,2-trans-diol (71b) from (+)-limonene (68). Dhavalikar and Bhattacharyya (1966) 

considered that the formation of 1-hydroxy-dihydrocarvone (66) is from dihydrocarvone (64). 

Pseudomonas gladioli was isolated by an enrichment culture technique from pine bark and sap 

using a mineral salts broth with limonene as the sole carbon source (Cadwallander et al., 1989; 

Cadwallander and Braddock, 1992). Fermentation was performed during 4–10 days in shake flasks 

at 25∞C using a pH 6.5 mineral salts medium and 1.0% (+)-limonene (68). Major products were 

identified as (+)-a-terpineol (34) and (+)-perillic acid (82). This was the first report of the microbial 

conversion of limonene to (+)-a-terpineol (34). 

The first data on fungal bioconversion of limonene (68) date back to the late 1960s (Kraidman 

et al., 1969; Noma, 2007). Three soil microorganisms were isolated on and grew rapidly in mineral 

salts media containing appropriate terpene substrates as sole carbon sources. The microorganisms 

belonged to the class Fungi Imperfecti, and they had been tentatively identified as Cladosporium 

O 

O 

OH 

O 

96 

O 

97 

O 

103 

O 

HO 

OH 

HO 

OH 

98 

O 

O 

102 

105 

O 

OH 

94 

OH 

93 

OH 

101 

OH 

O 

OH 

OH 

92 

O 

OH 

81 

O 

62 

OH 

HO 

OH 

HO 

72 

OH 

71 

OH 

68 

69 

71 

334 

FIGURE 14.28 Formation of (+)-8-p-menthene-1,2-trans-diol (71b) in the biotransformation of (+)-limonene 

(68) and (+)-carvone (93) by Aspergillus niger TBUYN-2. (Modified from Noma, Y. et al., 1985a. Annual Meeting 

of Agricultural and Biological Chemistry, Sapporo, p. 68; Noma, Y. et al., Proc. 29th TEAC, pp. 235–237.)


species. One of these strains, designated as Cladosporium sp. T 7 was isolated on (+)-limonene 

(68a). The growth medium of this strain contained 1.5 g/L of trans-limonene-1,2-diol (71a). Minor 

quantities of the corresponding cis-1,2-diol (71b) were also isolated. The same group isolated a 

fourth microorganism from a terpene-soaked soil on mineral salts media containing (+)-limonene 

as the sole carbon source (Kraidman et al., 1969). The strain, Cladosporium, designated T 12 , was 

capable of converting (+)-limonene (68a) into an optically active isomer of a-terpineol (34) in yields 

of approximately 1.0 g/L. 

a-Terpineol (34) was obtained from (+)-limonene (68) by fungi such as Penicillium digitatum, 

Pencillium italicum, and Cladosporium and several bacteria (Figure 14.29). (+)-cis-Carveol (81b), 

(+)-carvone (93) [an important constituent of caraway seed and dill-seed oils (Fenaroli, 1975; 

Bouwmester et al., 1995), and 1-p-menthene-6,9-diol (90) were also obtained by Pencillium digitatum 

and Pencillium italicum. (+)-(S)-Carvone (93) is a natural potato sprout inhibiting, fungistatic, 

and bacteriostatic compound (Oosterhaven et al., 1995a, 1995b). It is important to note that 

(-)-carvone (93¢, the “spearmint flavour”] was not yet described in microbial transformation 

(Krasnobajew, 1984). However, the biotransformation of limonene to (-)-carvone (93¢) was patented 

by a Japanese group (Takagi et al., 1972). Corynebacterium species grown on limonene was able to 

produce about 10 mg/L of 99% pure (-)-carvone (93¢) in 24–48 h. 

Mattison et al. (1971) isolated Penicillium sp. cultures from rotting orange rind that utilized limonene 

(68) and converted it rapidly to a-terpineol (34). Bowen (1975) isolated two common Citrus moulds, 

Penicillium italicum and Penicillium digitatum, responsible for the postharvest diseases of Citrus fruits. 

Fermentation of Penicillium italicum on limonene (68) yielded cis- (81b) and trans-carveol (81a) (26%) 

as the main products, together with cis- and trans-p-mentha-2,8-dien-1-ol (73) (18%), (+)-carvone (93¢) 

(6%), p-mentha-1,8-dien-4-ol (80) (4%), perillyl alcohol (74) (3%), and 8-p-menthene-1,2-diol (71) (3%). 

Conversion of 68 by Penicillium digitatum yielded the same products in lower yields (Figure 14.29). 

The biotransformation of limonene (68) by Aspergillus niger is a very important example of fungal 

bioconversion. Screening for fungi capable of metabolizing the bicyclic hydrocarbon terpene a-pinene 

(4) yielded a strain of Aspergillus niger NCIM 612 that was also able to transform limonene (68) (Rama 

Devi and Bhattacharyya, 1978). This fungus was able to carry out three types of oxygenative rearrangements 

a-terpineol (34), carveol (81), and p-mentha-2,8-dien-1-ol (73) (Rama Devi and Bhattacharyya, 

1978) (Figure 14.30). In 1985, Abraham et al. (1985) investigated the biotransformation of (R)-(+)- 

limonene (68a) by the fungus Penicillium digitatum. A complete transformation for the substrate to 

a-terpineol (34) by Penicillium digitatum DSM 62840 was obtained with 46% yield of pure product. 

OH 

OH 

O 

OH 

HO 

OH 

+ + + + + 

OH 

68 73 

80 

81 

93 

74 

71 

FIGURE 14.29 Biotransformation products of limonene (68) by Penicillium digitatum and Penicillium 

italicum. (Modified from Bowen, E.R., 1975. Proc. Fla. State Hortic. Soc., 88: 304–308.) 

A. niger 

OH 

+ + 

OH 

OH 

68 34 

81 

73 

FIGURE 14.30 Biotransformation of limonene (68) by Aspergillus niger NCIM 612. (Modified from Rama 

Devi, J. and P.K. Bhattacharyya, 1978. J. Indian Chem. Soc., 55: 1131–1137.)


The production of glycols from limonene (68) and other terpenes with a 1-menthene skeleton was 

reported by Corynespora cassiicola DSM 62475 and Diplodia gossypina ATCC 10936 (Abraham 

et al., 1984). Accumulation of glycols during fermentation was observed. An extensive overview on 

the microbial transformations of terpenoids with a 1-p-menthene skeleton was published by Abraham 

et al. (1986). 

The biotransformation of (+)-limonene (68) was carried out by using Aspergillus cellulosae M-77 

(Noma et al., 1992b) (Figure 14.32). It is important to note that (+)-limonene (68a) was mainly 

OH 

OH 

O 

CH 2 OH 

HO 

HO 

O 

HO 

HO 

68 

81a 

81b 

O 

93 

O 

74 

HO 

HO 

111 

71a 

1S,2S,4R 

OH 

OH 

OH 

OH 

334 

OH 

34 

OH 

101a 

OH 

101b 

OH 

71b 

1S,2R,4R 

71 c II-P 

1S,2S,4S 

102a 

OH 

102b 

OH 

102c 

OH 

102d 

OH 

HO 

HO 

HO 

HO 

102a' 

102b' 

102c' 

102d' 

71b ' 

1R,2S,4S 

71c ' 

1R,2R,4R 

OH 

OH 

O 

CH 2 OH 

HO 

HO 

O 

HO 

68' 

81a' 

81b' 

93' 

74' 

111' 

71a ' 

1R,2R,4S 

HO 

O 

O 

OH OH 

34' 

334' 

101b' 

101a' 

FIGURE 14.31 (+)- and (-)-limonenes (68 and 68¢) and related compounds.


A. cellulosae 

O 

+ 

CH 2 OH 

+ 

OH 

+ 

HO 

HO 

68 

111 74 

FIGURE 14.32 Biotransformation of (+)-limonene (68) by Aspergillus cellulosae IFO4040. (Modified from 

Noma, Y. et al., 1992b. Phytochemistry, 31: 2725–2727.) 

converted to (+)-isopiperitenone (111) (19%) as new metabolite, (1S,2S,4R)-(+)-limonene-1,2-transdiol 

(71a) (21%), (+)-cis-carveol (81b) (5%), and (+)-perillyl alcohol (74) (12%) (Figure 14.32). 

(+)-Limonene (68) was biotransformed by a kind of Citrus pathogenic fungi, Penicillium digitatum 

(Pers.; Fr.) Sacc. KCPYN. to isopiperitenone (111, 7% GC ratio), 2a-hydroxy-1,8-cineole (125b, 7%), 

(+)-limonene-1,2-trans-diol (71a, 6%), and (+)-p-menthane-1b,2a,8-triol (334, 45%) as main products 

and (+)-trans-sobrerol (95a, 2%), (+)-trans-carveol (81a), (+)-carvone (93), (-)-isodihydrocarvone 

(101b), and (+)-trans-isopiperitenol (110a) as minor products (Noma and Asakawa, 2006a, 2007a) 

(Figure 14.33). The metabolic pathways of (+)-limonene by Penicillium digitatum is shown in 

Figure 14.34. 

On the other hand, (-)-limonene (68¢) was also biotransformed by a kind of Citrus pathogenic fungi, 

Penicillium digitatum (Pers.; Fr.) Sacc. KCPYN. to give isopiperitenone (111¢), 2a-hydroxy-1,8-cineole 

(125b¢), (-)-limonene-1,2-trans-diol (71¢), and p-menthane-1,2,8-triol (334¢) as main products together 

with (+)-trans-sobrerol (80¢), (+)-trans-carveol (81a¢), (-)-carvone (93¢), (-)-dihydrocarvone 

(101a¢), and (+)-isopiperitenol (110a¢) as minor products (Noma and Asakawa, 2007b) (Figure 14.35.) 

Newly isolated unidentified red yeast, Rhodotorula sp., converted (+)-limonene (68) mainly to 

(+)-limonene-1,2-trans-diol (71a), (+)-trans-carveol (81a), (+)-cis-carveol (81b), and (+)-carvone 

(93¢) together with (+)-limonene-1,2-cis-diol (71b) as minor product (Noma and Asakawa, 2007b) 

(Figure 14.36). 

Cladosporium sp. T 7 was cultivated with (+)-limonene (68) as the sole carbon source; it converted 

68 to trans-p-menthane-1,2-diol (71a) (Figure 14.36) (Mukherjee et al., 1973). 

On the other hand, the same red yeast converted (-)-limonene (68¢) mainly to (-)-limonene-1,2- 

trans-diol (71a¢), (-)-trans-carveol (81a¢), (-)-cis-carveol (81b¢), and (-)-carvone (93¢) together with 

(-)-limonene-1,2-cis-diol (71b¢) as minor product (Noma and Asakawa, 2007b) (Figure 14.37). 

The biotransformation of (+)- and (-)-limonene (68 and 68¢), (+)- and (-)-a-terpineol (34 and 

34¢), (+)- and (-)-limonene-1,2-epoxide (69 and 69¢), and caraway oil was carried out by Citrus 

81 

71a 

HO 

HO 

HO 

HO 

HO 

O 

O 

OH 

68 

111 

125b 

71a 

334 

OH 

OH 

O 

O 

OH 

HO 

95a 81a 93 101a 110a 

FIGURE 14.33 Metabolites of (+)-limonene (68) by a kind of Citrus pathogenic fungi, Penicillium digitatum 

(Pers.; Fr.) Sacc. KCPYN. (Modified from Noma, Y. and Y. Asakawa, 2006a. Proc. 50th TEAC, pp. 431–433; 

Noma, Y. and Y. Asakawa, 2007a. Book of Abstracts of the 38th ISEO, p. 7.)


HO 

HO 

OH 

O 

O 

O 

HO 

71a 

HO 

69 

81a 93 101b 

OH 

HO 

334 

34 

OH 

HO 

O 

68 110a 111 

O 

125b 

FIGURE 14.34 Biotransformation of (+)-limonene (68) by Citrus pathogenic fungi, Penicillium digitatum 

(Pers.; Fr.) Sacc. KCPYN. (Modified from Noma, Y. and Y. Asakawa, 2006a. Proc. 50th TEAC, pp. 431–433; 

Noma, Y. and Y. Asakawa, 2007a. Book of Abstracts of the 38th ISEO, p. 7.) 

HO 

HO 

O 

OH 

O 

O 

HO HO 

71a' 

69' 

81a' 93' 101a' 

OH 

334' 

OH 

34' 

OH 

68' 

OH 

HO 

110a' 

O 

111' 

O 

OH 

125b' 

95a' 

FIGURE 14.35 Biotransformation of (-)-limonene (68¢) by Citrus pathogenic fungi, Penicillium digitatum 

(Pers.; Fr.) Sacc. KCPYN. (Modified from Noma, Y. and Y. Asakawa, 2007b. Proc. 51st TEAC, pp. 299–301.) 

pathogenic fungi Penicillium (Pers.; Fr.) Sacc. KCPYN and newly isolated red yeast, a kind of 

Rhodotorula sp. Penicillium digitatum KCPYN converted limonenes (68 and 68¢) to the corresponding 

isopiperitone (111 and 111¢), 1a-hydroxy-1,8-cineole (125b and 125b¢), limonene-1,2- 

trans-diol (71a and 71a¢), p-menthane-1,2,8-triol (334 and 334¢), and trans-sobrerol as main 

products. (+)- and (-)-a-Terpineol (34 and 34¢) were the precursors of 2a-hydroxy-1,8-cineole 

(125b and b¢) and p-menthane-1,2,8-triol (334). (+)- and (-)-Limonene-1,2-epoxide (69 and 69¢) 

were also the precursor of limonene-1,2-trans-diol (71a). Rhodotorula sp. also biotransformed (+)- 

and (-)-limonene (68 and 68¢) to the corresponding trans- and cis-carveols (81a and b) as main 

products. This microbe also converted caraway oil, equal mixture of (+)-limonene (68) and (+)-carvone 

(93). (+)-Limonene (68) disappeared and (+)-carvone (93) was produced and accumulated in 

the cultured broth (Noma and Asakawa, 2007b).


HO 

OH 

OH 

71a 

81a 

O 

HO 

OH 

68 

OH 

93 

71b 

FIGURE 14.36 Biotransformation of (+)-limonene (68) by red yeast, Rhodotorula sp. and Cladosporium sp. 

T 7 . (Modified from Mukherjee, B.B. et al., 1973. Appl. Microbiol., 25: 447–453; Noma, Y. and Y. Asakawa, 

2007b. Proc. 51st TEAC, pp. 299–301.) 

81b 

HO 

OH 

OH 

71a' 

81a' 

O 

HO 

OH 

68' 

OH 

93' 

71b' 

FIGURE 14.37 Biotransformation of (-)-limonene (68¢) by a kind of Rhodotorula sp. (Modified from 

Noma, Y. and Y. Asakawa, 2007b. Proc. 51st TEAC, pp. 299–301.) 

(4S)-(-)- (68¢) and (4R)-(+)-Limonene (68) and their epoxides (69 and 69¢) were incubated by 

Cyanobacterium. It was found that the transformation was enantio- and regioselective. Cyanobacterium 

biotransformed only (4S)-limonene (68¢) to (-)-cis- (81b¢, 11.1%) and (-)-trans-carveol (81a¢, 5%) in 

low yield. On the other hand, (4R)-limonene oxide (69) was converted to limonene-1,2-trans-diol 

(71a¢) and 1-hydroxy-(+)-dihydrocarvone (72a¢). However, (4R)-(+)-limonene (68) and (4S)-limonene 

oxide (69¢) were not converted at all (Figure 14.38) (Hamada et al., 2003). 

(+)-Limonene (68) was fed by Spodptera litura to give (+)-limonene-7-oic acid (82), (+)-limonene- 

9-oic acid (70), and (+)-limonene-8,9-diol (79); (-)-limonene (68¢) was converted to (-)-linonene-7- 

oic acid (82¢), (-)-limonene-9-oic acid (70¢), and (-)-limonene-8,9-diol (79¢) (Figure 14.39) 

(Miyazawa et al., 1995a). 

Kieslich et al. (1985) found a nearly complete microbial resolution of a racemate in the biotransformation 

of (±)-limonene by Penicillium digitatum (DSM 62840). The (R)-(+)-limonene (68) is converted 

to the optically active (+)-a-terpineol, [a] D = +99∞, while the (S)-(-)-limonene (68¢) is presumably 

adsorbed onto the mycelium or degraded via unknown pathways (Kieslich et al., 1985) (Figure 14.40). 

(4S)- and (4R)-Limonene epoxides (69a¢ and a) were biotransformed by Cyanobacterium to give 

8-p-menthene-1a,2b-ol (71a, 68.4%) and 1a-hydroxy-8-p-menthen-2-one (72, 31.6%) (Hamada 

et al., 2003) (Figure 14.41). 

81b'


O 

OH 

OH 

OH 

O 

68 

69' 

71a' 

72a' 

OH 

+ 

OH 

O 

68' 81a' 81b' 

69 

FIGURE 14.38 Biotransformation of (+)- and (-)-limonene (68 and 68¢) and limonene epoxide (69 and 69¢) 

by Cyanobacterium. (Modified from Hamada, H. et al., 2003. Proc. 47th TEAC, pp. 162–163.) 

COOH 

S. litura 

+ + 

68 

82 

70 

COOH 

OH 

OH 

79 

COOH 

S. litura 

+ + 

68' 

82' 

COOH 

OH 

OH 

79' 

FIGURE 14.39 Biotransformation of (+)-limonene (68) and (-)-limonene (68¢) by Spodptera litura. 

(Modified from Miyazawa, M. et al., 1995a. Proc. 39th TEAC, pp. 362–363.) 

70' 

P. digitatum 

OH 

68 

34 

68' 

FIGURE 14.40 Microbial resolution of racemic limonene (68 and 68¢) and the formation of optically active 

a-terpineol by Penicillium digitatum. (Modified from Kieslich, K. et al., 1985. In: Topics in fl avor research, 

R.G. Berger, S. Nitz, and P. Schreier, eds, pp. 405–427. Marzling Hangenham: Eichborn.)


O 

Cyanobacterium 

OH 

OH 

+ 

OH 

O 

69a 

71a 72 

O 

69a' 

FIGURE 14.41 Enantioselective biotransformation of (4S)- (69a¢) and (4R)-limonene epoxides (69a) by 

Cyanobacterium. (Modified from Hamada, H. et al., 2003. Proc. 47th TEAC, pp. 162–163.) 

O 

2 R 

1 

S 

O 

2 S 

1 

R 

O 

2 S 

1 

R 

O 

2 R 

1 

S 

R 

4 

R 

4 

S 

S 

4 4 

(1S,2R,4R)-(+)-trans 

(1R,2S,4R)-(+)-cis 

(1R,2S,4S)-(–)-trans 

(1S,2R,4S)-(–)-cis 

The mixture of (+)-trans- (69a) and cis- (69b), and the mixture of (-)-trans- (69a¢) and cislimonene-1,2-epoxide 

(69b¢) were biotransformed by Citrus pathogenic fungi, Penicillium digitatum 

(Pers.; Fr.) Sacc. KCPYN to give (1R,2R,4R)- (-)-trans-(71a) and (1S,2S,4S)-(+)-8-p-menthene-1,2- 

trans-diol (71a¢) and (-)-p-menthane-1,2,8-triols (334a and 334a¢) (Noma and Asakawa, 2007b) 

(Figure 14.42). 

Biotransformation of 1,8-cineole (122) by Aspergillus niger gave racemic 2a-hydroxy-1,8-cineole 

(125b and b¢) (Nishimura et al., 1982). When racemic 2a-hydroxy-1,8-cineole (125b and b¢) 

was biotransformed by Glomerella cingulata, only (-)-2a-hydroxy-1,8-cineole (125b¢) was selectively 

esterifized with malonic acid to give its malonate (125b¢-Mal). The malonate was hydrolyzed 

to give optical pure 125b¢ (Miyazawa et al., 1995b). On the other hand, Citrus pathogenic fungi, 

Penicillium digitatum, biotransformed limonene (68) to give optical pure 125b (Noma and Asakawa, 

2007b) (Figure 14.43). 

HO 

HO 

R 

R 

HO 

HO 

R 

R 

HO 

HO 

S 

S 

HO 

HO 

S 

S 

R 

R 

S 

S 

HO 

HO 

71a 

(1R,2R,4R)-(–)-trans 

334a 

(1R,2R,4R)-(–)-trans 

71a' 

(1S,2S,4S)-(+)-trans 

334a' 

(1S,2S,4S)-(+)-trans 

FIGURE 14.42 Biotransformation of (+)-trans- (69a) and cis- (69b), and (-)-trans- (69a¢) and cis-limonene- 

1,2-epoxide (69b¢) by Citrus pathogenic fungi, Penicillium digitatum (Pers.; Fr.) Sacc. KCPYN and their 

metabolites. (Modified from Noma, Y. and Y. Asakawa, 2007b. Proc. 51st TEAC, pp. 299–301.)


O 

122 

A. niger 

68 

P. digitatum 

O 

O 

HO 

O 

125b 

O 

125b' 

OH 

G. cingulata 

HO 

FIGURE 14.43 Formation of optical pure (+)- and (-)-2a-hydroxy-1,8-cineole (125b and b¢) from the 

biotransformation of 1,8-cineole (122) and (+)-limonene (68) by Citrus pathogenic fungi, Penicillium 

digitatum (Pers.; Fr.) Sacc. KCPYN and Aspergillus niger TBUYN-2. (Modified from Nishimura, H. et al., 

1982. Agric. Biol. Chem., 46: 2601–2604; Miyazawa, M. et al., 1995b. Proc. 39th TEAC, pp. 352–353; Noma, 

Y. and Y. Asakawa, 2007b. Proc. 51st TEAC, pp. 299–301.) 

When monoterpenes, such as limonene (68), a-pinene (4), and 3-carene (336), were administered 

to the cultured cells of Nicotiana tabacum, they were converted to the corresponding epoxides 

enantio- and stereoselectively. The enzyme (p38) concerning with the epoxidation reaction was purified 

from the cultured cells by cation exchanged chromatography. The enzyme had not only epoxidation 

activity but also peroxidase activity. Amino acid sequence of p38 showed 89% homology in their 

9 amino acid overlap with horseradish peroxidase (Yawata et al., 1998) (Figure 14.44). It was found 

that limonene and carene were converted to the corresponding epoxides in the presence of hydrogen 

O 

(1S, 2S, 4R)-125b 

[α] D +29.6 

+ 

O 

O 

125b'-Mal 

Hydrolysis 

125b' 

OH 

H 2 O 2 

H2 O 

OH 

Peroxidase 

OH O 2 

O 

O 

. . 

O 

O 

68 

Polymerization 

O 

O 

O 

. 

OH 

OH 

O 

O 

69 

FIGURE 14.44 Proposed mechanism for the epoxidation of (+)-limonene (68) with p38 from the cultured 

cells of Nicotiana tabacum. (Modified from Yawata, T. et al., 1998. Proc. 42nd TEAC, pp. 142–144.) 

. 

O


p38 

O O 

O 

p38 

+ + 

O 

68 

69a' 

10.7% 

69b' 

14.2% 

68' 

69b 

9.4% 

69a 

8.0% 

p38 

O 

p38 

O 

p38 

O 

4 

33 

trace 

4' 

335' 

trace 

337 

12.8% 

FIGURE 14.45 Epoxidation of limonene (68), a-pinene (4) and 3-carene (336) with p38 from the cultured 

cells of Nicotiana tabacum. (Modified from Yawata, T. et al., 1998. Proc. 42nd TEAC, pp. 142–144.) 

336 

peroxide and p-cresol by a radical mechanism with the peroxidase. (R)-limonene (68), (S)-limonene 

(68¢), (1S,5R)-a-pinene (4), (1R,5R)-a-pinene (4), and (1R,6R)-3-carene (336) were oxidized by 

cultured cells of Nicotiana tabacum to give corresponding epoxides enantio- and stereoselectively 

(Yawata et al., 1998) (Figure 14.45). 

14.3.1.2 Isolimonene 

Spodptera litura converted (1R)-trans-isolimonene (338) to (1R,4R)-p-menth-2-ene-8,9-diol (339) 

(Miyazawa et al., 1996b) (Figure 14.46). 

14.3.1.3 p-Menthane 

Hydroxylation of trans- and cis-p-menthane (252a and b) by microorganisms is also very interesting 

from the viewpoint of the formation of the important perfumes such as (-)-menthol (137b¢), 

(-)-carvomenthol (49b¢), etc., plant growth regulators, and mosquito repellents such as p-menthanetrans-3,8-diol 

(142a), p-menthane-cis-3,8-diol (142b) (Nishimura and Noma, 1996), and p- 

menthane-2,8-diol (93) (Noma, 2007). Pseudomonas mendocina strain SF biotransformed 

252b stereoselectively to p-cis-menthan-1-ol (253) (Tsukamoto et al., 1975) (Figure 14.47). 

On the other hand, the biotransformation of the mixture of p-trans- (252a) and cis-menthane (252b) 

(45:55, peak area in GC) by Aspergillus niger gave p-cis-menthane-1,9-diol (254) via p-cis-menthan- 

1-ol (253). No metabolite was obtained from 252a at all (Noma et al., 1990) (Figure 14.47). 

14.3.1.4 1-p-Menthene 

Concentrated cell suspension of Pseudomonas sp. strain (PL) was inoculated to the medium containing 

1-p-menthene (62) as the sole carbon source. It was degraded to give b-isopropyl pimelic 

acid (248) and methylisopropyl ketone (251) (Hungund et al., 1970) (Figure 14.48). 

S. litura 

OH 

OH 

338 

339 

FIGURE 14.46 Biotransformation of (1R)-trans-isolimonene (338) by Spodoptera litura. (Modified from 

Miyazawa, M. et al., 1996b. Proc. 40th TEAC, pp. 80–81.)


252a 

P. mendocina 

A. niger 

OH 

A. niger 

OH 

OH 

25b2 

253 

254 

FIGURE 14.47 Biotransformation of the mixture of trans- (252a) and cis-p-menthane (252b) by 

Pseudomonas mendocina SF and Aspergillus niger TBUYN-2. (Modified from Tsukamoto, Y. et al., 1974. 

Proc. 18th TEAC, pp. 24–26; Tsukamoto, Y. et al., 1975. Agric. Biol. Chem., 39: 617–620; Noma, Y., 2007. 

Aromatic Plants from Asia their Chemistry and Application in Food and Therapy, L. Jiarovetz, N.X. Dung, 

and V.K. Varshney, pp. 169–186. Dehradun: Har Krishan Bhalla & Sons.) 

COOH 

Pseudomonas 

sp. strain (PL) 

COOH 

+ 

O 

62 248 

251 

FIGURE 14.48 Biodegradation of (4R)-1-p-menthene (62) by Pseudomonas sp. strain (PL). (Modified from 

Hungund, B.L. et al., 1970. Indian J. Biochem., 7: 80–81.) 

S. litura 

COOH 

Cladosporiuum 

sp. T 1 

HO 

HO 

62 65 

62' 54 

FIGURE 14.49 Biotransformation of (4R)-p-menth-1-ene (62) by Spodoptera litura and Cladosporium sp. 

T 1 . (Modified from Miyazawa, M. et al., 1996b. Proc. 40th TEAC, pp. 80–81; Mukherjee, B.B. et al., 1973. 

Appl. Microbiol., 25: 447–453.) 

As shown in Figure 14.49, Spodoptera litura converted (4R)-p-menth-1-ene (62) at C-7 position 

to (4R)-phellandric acid (65) (Miyazawa et al., 1996b). On the other hand, when Cladosporium sp. 

T 1 was cultivated with (+)-limonene (68) as the sole carbon source, it converted 62¢ to transp-menthane-1,2-diol 

(54) (Mukherjee et al., 1973). 

14.3.1.5 3-p-Menthene 

When Cladosporium sp. T 8 was cultivated with 3-p-menthene (147) as the sole carbon source, it was 

converted to trans-p-menthane-3,4-diol (141) as shown in Figure 14.50 (Mukherjee et al., 1973).


Cladosporiuum 

sp. T 8 

OH 

OH 

147 141 

FIGURE 14.50 Biotransformation of p-Menth-3-ene (147) by Cladosporium sp. T 8 . (Modified from 

Mukherjee, B.B. et al., 1973. Appl. Microbiol., 25: 447–453.) 

14.3.1.6 α-Terpinene 

a-Terpinene (340) was converted by Spodoptera litura to give a-terpinene-7-oic acid (341) and 

p-cymene-7-oic acid (194, cuminic acid) (Miyazawa et al., 1995a) (Figure 14.51). 

A soil Pseudomonad has been found to grow with p-mentha-1,3-dien-7-al (463) as the sole carbon 

source and to produce a-terpinene-7-oic acid (341) in a mineral salt medium (Kayahara et al., 1973) 

(Figure 14.51). 

14.3.1.7 γ-Terpinene 

g-Terpinene (344) was converted by Spodoptera litura to give g-terpinene-7-oic acid (345) and 

p-cymene-7-oic acid (194, cuminic acid) (Miyazawa et al., 1995a) (Figure 14.52). 

14.3.1.8 Terpinolene 

Terpinolene (346) was converted by Aspergillus niger to give (1R)-8-hydroxy-3-p-menthen-2-one 

(347), (1R)-1,8-dihydroxy-3-p-menthen-2-one (348), and 5b-hydroxyfenchol (350b¢). In case of 

Corynespora cassiicola it was converted to terpinolene-1,2-trans-diol (351) and terpinolene-4,8-diol 

(352). Furthermore, in case of rabbit terpinolene-9-ol (353) and terpinolene-10-ol (354) were formed 

from 346 (Asakawa et al., 1983). Spodoptera litura also converted 346 to give 1-p-menthene-4,8-diol 

(352), cuminic acid (194, 29% main product), and terpinolene-7-oic acid (357) (Figure 14.53). 

14.3.1.9 α-Phellandrene 

a-Phellandrene (355) was converted by Spodoptera litura to give a-phellandrene-7-oic acid (356) 

and p-cymene-7-oic acid (194, cuminic acid) (Miyazawa et al., 1995a) (Figure 14.54). 

14.3.1.10 p-Cymene 

Pseudomonas sp. strain (PL) was cultivated with p-cymene (178) as the sole carbon source to give 

cumyl alcohol (192), cumic acid (194), 3-hydroxycumic acid (196), 2,3-dihydroxycumic acid (197), 

COOH 

COOH 

S. litura 

+ 

340 341 194 

CHO 

Pseudomonad 

463 

FIGURE 14.51 Biotransformation of a-terpinene (340) by Spodoptera litura and p-mentha-1,3-dien-7-al 

(463) by a soil Pseudomonad. (Modified from Kayahara, H. et al., 1973. J. Ferment. Technol., 51: 254–259; 

Miyazawa, M. et al., 1995a. Proc. 39th TEAC, pp. 362–363.)


COOH 

COOH 

S. litura 

+ 

344 345 194 

FIGURE 14.52 Biotransformation of g-terpinene (344) by Spodoptera litura. (Modified from Miyazawa, 

M. et al., 1995a. Proc. 39th TEAC, pp. 362–363.) 

OH 

OH 

OH 

OH 

352 

HO 

O 

A.n. 

O 

A.n. 

C. cassiicola 

S. litura 

351 

C. cassiicola 

348 

OH 

347 

OH 

Rabbit 

+ 

OH 

A.n. 

346 

S. litura 

HO 

353 354 

OH 

HO 

COOH 

COOH 

350b' 

357 

194 

FIGURE 14.53 Biotransformation of terpinolene (346) by Aspergillus niger (Asakawa et al., 1991), 

Corynespora cassiicola (Abraham et al., 1985), rabbit (Asakawa et al., 1983), and Spodoptera litura. (Modified 

from Miyazawa, M. et al., 1995a. Proc. 39th TEAC, pp. 362–363.) 

COOH 

COOH 

S. litura 

+ 

355 

356 194 

FIGURE 14.54 Biotransformation of a-phellandrene (355) by Spodoptera litura. (Modified from 

Miyazawa, M. et al., 1995a. Proc. 39th TEAC, pp. 362–363.)


2-oxo-4-methylpentanoic acid (201), 9-hydroxy-p-cymene (189), and p-cymene-9-oic acid (190) as 

shown in Figure 14.55 (Madyastha and Bhattacharyya, 1968). On the other hand, p-cymene (178) 

was converted regiospecifically to cumic acid (194) by Pseudomonas sp., Pseudomonas desmolytica, 

and Nocardia salmonicolor (Madyastha and Bhattacharyya, 1968) (Figure 14.56). 

p-Cymene (178) is converted to thymoquinone (358) and analogues, 179 and 180, by various 

kinds of microorganisms (Demirci et al., 2007) (Figure 14.57). 

CH 2 

OH CHO 


OH 

OH 

OH 

178 192 193 

194 

196 

197 

COOH 

O 

CH 2 

OH 

189 190 COOH 

FIGURE 14.55 Biotransformation of p-cymene (178) by Pseudomonas sp. strain (PL). (Modified from 

Madyastha, K.M. and P.K. Bhattacharyya, 1968. Indian J. Biochem., 5: 161–167.) 

201 

COOH 

178 194 

FIGURE 14.56 Biotransformation of p-cymene (178) to cumic acid (194) by Pseudomonas sp., Pseudomonas 

desmolytica and Nocardia salmonicolor. (Modified from Yamada, K. et al., 1965. Agric. Biol. Chem., 29: 

943–948; Madyastha, K.M. and P.K. Bhattacharyya, 1968. Indian J. Biochem., 5: 161–167; Noma, Y., 2000. 

unpublished data.) 

HO 

O 

OH 

OH 

O 

178 179 180 358 

FIGURE 14.57 Biotransformation of p-cymene (178) to thymoquinone (358) and analogues by microorganisms. 

(Modified from Demirci, F. et al., 2007. Book of Abstracts of the 38th ISEO, SL-1, p. 6.)


14.3.2 MONOCYCLIC MONOTERPENE ALDEHYDE 

COOH 

CHO 


CHO 

COOH 

HO 

360a 

1,2-dihydro 

perillic acid 

359a 

1,2-dihydroperillaldehde 

361a 

8-OH-s 

hisool 

75a 

1RS,4RS 

shisool 

74' 

(+)-4R 

perillyl 

alcohol 

78' (+)-4R 

perillaldehyde 

82' 

(+)-4R 

perillic 

acid 

COOH 

CHO 

CH 2 OH 

CH 2 OH 

CH 2 OH 

CHO 

COOH 

HO 

360b 

CH 2 OH 

359b 

361b 

75b 

74 (–)-4S 

perillyl 

alcohol 

78 (–)-4S 

perillalde 

hyde 

82 (–)-4S 

perillic 

acid 

O 

77 

(–)-8,9-epoxy 

perillyl 

alcohol 

14.3.2.1 Perillaldehyde 

Biotransformation of (-)-perillaldehyde (78), (+)-perillaldehyde (78¢), (-)-perillyl alcohol (74), 

trans-1,2-dihydroperillaldehyde (359a) and cis-1,2-dihydroperillaldehyde (359b), and trans-shisoic 

acid (360a) and cis-shisoic acid (360b) was carried out by Euglena gracilis Z. (Noma et al., 1991a), 

Dunaliella tertiolecta (Noma et al., 1991b, 1992a), Chlorella ellipsoidea IAMC-27 (Noma et al., 

1997), Streptomyces ikutamanensis Ya-2-1 (Noma et al., 1984, 1986), and other microorganisms 

(Kayahara et al., 1973) (Figure 14.58). 

(-)-Perillaldehyde (78) is easily transformed to give (-)-perillyl alcohol (74) and trans-shisool 

(75a), which is well known as a fragrance, as the major product, and (-)-perillic acid (82) as the 

minor product. (-)-Perillyl alcohol (74) is also transformed to trans-shisool (75a) as the major product 

with cis-shisool (75b) and 8-hydroxy-cis-shisool (361b). Furthermore, trans-shisool (75a) and 

cis-shisool (75b) are hydroxylated to 8-hydroxy-trans-shisool (361a) and 8-hydroxy-cis-shisool 

(361b), respectively. trans-1,2-Dihydroperillaldehyde (359a) and cis-1,2-dihydroperillaldehyde 

(359b) are also transformed to 75a and 75b as the major products and trans-shisoic acid (360a) and 

cis-shisoic acid (360b) as the minor products, respectively. Compound 360a was also formed from 

75a. In the biotransformation of (±)-perillaldehyde (74 and 74¢), the same results were obtained as 

described in the case of 74. In the case of Streptomyces ikutamanensis Ya-2-1, (-)-perillaldehyde 

(78) was converted to (-)-perillic acid (82), (-)-perillyl alcohol (74), and (-)-perillyl alcohol-8,9- 

epoxide (77) which was the major product.


COOH 

CHO 

360a 

CH 2 OH 

E.g. 

D.t. 

C.e. 

359a 

E.g. D.t. C.e. 

CH 2 OH 

CH 2 OH 

CHO 

COOH 

HO 

E.g. 

D.t. 

C.e. 

E.g. 

D.t. 

C.e. 

E.g. 

D.t. 

C.e. 

361a 

75a 

74' 78' 82' 


CHO 

COOH 

HO 

E.g. 

D.t. 

C.e. 

E.g. 

D.t. 

C.e. 

E.g. 

D.t. 

C.e. 

361b 

COOH 

75b 

E.g. 

D.t. C.e. 

CHO 

74 78 82 

S.i. Ya21 

CH 2 OH 

O 

360b 

359b 

77 

FIGURE 14.58 Metabolic pathways of perillaldehyde (78 and 78¢) by Euglena gracilis Z (Noma et al., 

1991a), Dunaliella tertiolecta (Noma et al., 1991b; 1992a), Chlorella ellipsoidea IAMC-27 (Noma et al., 1997), 

Streptomyces ikutamanensis Ya-2-1 (Noma et al., 1984, 1986), a soil Pseudomonad (Kayahara et al., 1973), and 

rabbit (Ishida et al., 1981a). 

A soil Pseudomonad has been found to grow with (-)-perillaldehyde (78) as the sole carbon 

source and to produce (-)-perillic acid (82) in a mineral salt medium (Kayahara et al., 1973). 

On the other hand, rabbit metabolized (-)-perillaldehyde (78) to (-)-perillic acid (82) along with 

minor shisool (75a) (Ishida et al., 1981a). 

14.3.2.2 Phellandral and 1,2-Dihydrophellandral 

Biotransformation of (-)-phellandral (64), trans-tetrahydroperillaldehyde (362a), and cis-tetrahydroperillaldehyde 

(362b) was carried out by microorganisms (Noma et al., 1986, 1991a, 1991b, 

1997). (-)-Phellandral (64) was metabolized mainly via (-)-phellandrol (63) to trans-tetrahydroperillyl 

alcohol (66a). trans-Tetrahydroperillaldehyde (362a) and cis-tetrahydroperillaldehyde (362b) were 

also transformed to trans-tetrahydroperillyl alcohol (66a) and cis-tetrahydroperillyl alcohol (66b)


COOH 

CHO 

CH 2 

OH 

363a 

COOH 

362a 

CHO 

E.g. 

D.t. 

C.e. 

66a 

CH 2 

OH 

E.g. 

D.t. 

C.e. 

CH 2 

OH 

E.g. 

D.t. 

C.e. 

CHO 

63 64 65 

COOH 

E.g. 

D.t. 

C.e. 

363b 

362b 

66b 

FIGURE 14.59 Metabolic pathways of (-)-phellandral (64) by microorganisms. (Modified from Noma, Y. 

et al., 1986. Proc. 30th TEAC, pp. 204–206; Noma, Y. et al., 1991a. Phytochem., 30: 1147–1151; Noma, Y. 

et al., 1991b. Proc. 35th TEAC, pp. 112–114; Noma, Y. et al., 1997. Proc. 41st TEAC, pp. 227–229.) 

as the major products and trans-tetrahydroperillic acid (363a) and cis-tetrahydro perillic acid (363b) 

as the minor products, respectively (Figure 14.59). 

14.3.2.3 Cuminaldehyde 

Cumin aldehyde (193) is transformed by Euglena (Noma et al., 1991a), Dunaliella (Noma et al., 

1991b), and Streptomyces ikutamanensis (Noma et al., 1986) to give cumin alcohol (192) as the 

major product and cuminic acid (194) as the minor product (Figure 14.60). 

14.3.3 MONOCYCLIC MONOTERPENE ALCOHOL 

14.3.3.1 Menthol 

OH 

OH 

OH 

OH 

137a 

(1R,3S,4S) 

(+)-Neomenthol 

137b 

(1R,3R,4S) 

(–)-Menthol 

137c 

(1S,3R,4S) 

(–)-Isomenthol 

137d 

(1S,3S,4S) 

(–)-Neoisomenthol 

OH 

OH 

OH 

OH 

137a' 

(1S,3R,4R) 

(–)-Neomenthol 

137b' 

(1S,3S,4R) 

(+)-Menthol 

137c' 

(1R,3S,4R) 

(+)-Isomenthol 

137d' 

(1R,3R,4R) 

(+)-Neoisomenthol


CH 2 OH CHO COOH 

192 193 194 

FIGURE 14.60 Metabolic pathway of cumin aldehyde (193) by microorganism. (Modified from Noma, Y. 

et al., 1986. Proc. 30th TEAC, pp. 204–206; Noma, Y. et al., 1991a. Phytochem., 30: 1147–1151; Noma, Y. 

et al., 1991b. Proc. 35th TEAC, pp. 112–114.) 

Menthol (137) is one of the rare naturally occurring monocyclic monoterpene alcohols that have not 

only various physiological properties, such as sedative, anesthetic, antiseptic, gastric, and antipruritic, 

but also characteristic fragrance (Bauer et al., 1990). There are in fact eight isomers with a 

menthol (p-menthan-3-ol) skeleton; (-)-menthol (137b) is the most important one, because of its 

cooling and refreshing effect. It is the main component of peppermint and cornmint oils obtained 

from the Mentha piperita and Mentha arvensis species. Many attempts have been made to produce 

(-)-menthol (137b) from inexpensive terpenoid sources, but these sources also unavoidably yielded 

the (±)-isomers (137b and 137b¢): isomenthol (137c), neomenthol (137a), and neoisomenthol (137d) 

(Krasnobajew, 1984). Japanese researchers have been active in this field, maybe because of the large 

demand for (-)-menthol (137b) in Japan itself, namely 500 t/year (Janssens et al., 1992). Indeed, 

most literature deals with the enantiomeric hydrolysis of (±)-menthol (137b and 137b¢) esters to 

optically pure l-menthol (137b). The asymmetric hydrolysis of (±)-menthyl chloroacetate by an 

esterase of Arginomonas non-fermentans FERM-P-1924 has been patented by the Japanese Nippon 

Terpene Chemical Co. (Watanabe and Inagaki, 1977a, 1977b). Investigators from the Takasago 

Perfumery Co. Ltd. claim that certain selected species of Absidia, Penicillium, Rhizopus, 

Trichoderma, Bacillus, Pseudomonas, and others asymmetrically hydrolyze esters of (±)-menthol 

isomers such as formates, acetates, propanoates, caproates, and esters of higher fatty acids (Moroe 

et al., 1971; Yamaguchi et al., 1977) (Figure 14.61). 

Numerous investigations into the resolution of the enantiomers by selective hydrolysis with microorganisms 

or enzymes were carried out. Good results were described by Yamaguchi et al. (1977) with 

the asymmetric hydrolysis of (±)-methyl acetate by a mutant of Rhodotorula mucilaginosa, yielding 

44 g of (-)-menthol (137b) form a 30% (±)-menthyl acetate mixture per liter of cultured medium for 

24 h. The latest development is the use of immobilized cells of Rhodotorula minuta in aqueous saturated 

organic solvents (Omata et al., 1981) (Figure 14.62). 

Besides the hydrolysis of menthyl esters, the biotransformation of menthol and its enantiomers has 

also been published (Shukla et al., 1987; Asakawa et al., 1991). The fungal biotransformation of (-)- 

(137b) and (+)-menthols (137b¢) by Aspergillus niger and Aspergillus cellulosae was described 

microorgansims 

O-Ac 

O-Ac 

OH 

O-Ac 

137b-Ac 

137b'-Ac 

137b 

137b'-Ac 

FIGURE 14.61 Asymmetric hydrolysis of racemic menthyl acetate (137b-Ac and 137b¢-Ac) to obtain pure 

(-)-menthol (137b). (Modified from Watanabe, Y. and T. Inagaki, 1977a. Japanese Patent 77.12.989. No. 187696x; 

Watanabe, Y. and T. Inagaki, 1977b. Japanese Patent 77.122.690. No. 87656g; Moroe, T. et al., 1971. Japanese 

Patent, 2.036. 875. no. 98195t; Oritani, T. and Yamashita, K. 1973b. Agric. Biol. Chem., 37: 1695–1700.)


137b-succinate 

O-COCH 2 CH 2 COOH 

Rodotrula 

minuta 

137b 

OH 

O-COCH 2 CH 2 COOH 

137b'-succinate 

FIGURE 14.62 Asymmetric hydrolysis of racemic menthyl succinate (137b- and 137b¢-succinates) to obtain 

pure (-)-menthol (137b). (Modified from Yamaguchi, Y. et al., 1977. J. Agric. Chem. Soc. Jpn., 51: 411–416,) 

(Asakawa et al., 1991). Aspergillus niger converted (-)-menthol (137b) to 1- (138b), 2- (140b), 6- (139b), 

7- (143b), 9-hydroxymenthols (144b), and the mosquito repellent-active 8-hydroxymenthol (142b), 

whereas (+)-menthol (137b¢) was smoothly biotransformed by the same microorganism to 7-hydroxymenthol 

(143b). The bioconversion of (+)- (137a¢) and (-)-neomenthol (137a) and (+)-isomenthol 

(137c¢) by Aspergillus niger was studied later by Takahashi et al. (1994), mainly giving hydroxylated 

products. Noma and Asakawa (1995) reviewed the schematic menthol hydroxylation in detail. 

Incubation of (-)-menthol (137b) with Cephalosporium aphidicola for 12 days yielded 10- 

acetoxymenthol (144bb-Ac), 1a-hydroxymenthol (138b), 6a-hydroxymenthol (139bb), 7-hydroxymenthol 

(143b), 9-hydroxymenthol (144ba), and 10-hydroxymenthol (144bb) (Atta-ur-Rahman 

et al., 1998) (Figure 14.63). 

Aspergillus niger TBUYN-2 converted (-)-menthol (137b) to 1a- (138b), 2a- (140b), 4b- (141b), 

6a- (139bb), 7- (143b)-, 9-hydroxymenthols (144ba), and the mosquito repellent-active 8-hydroxymenthol 

(142b) (Figure 14.64). Aspergillus cellulosae M-77 biotransformed (-)-menthol (137b) 

to 4b-hydroxymenthol (141b) predominantly. The formation of 141b is also observed in 

Aspergillus cellulosae IFO 4040 and Aspergillus terreus IFO 6123, but its yield is much less than 

that obtained from 137b by Aspergillus cellulosae M-77 (Asakawa et al., 1991) (Table 14.1). 

OH 

OH 

+ + 

OH 

HO 

OH 

OH 

C. aphidicola 

O-COCH 3 

144bb-Ac 143b 139bb 

OH 

137b 

OH 

+ + 

OH 

OH 

144bb 

OH 

138b 

HO 

144ba 

FIGURE 14.63 Biotransformation of (-)-menthol (137b) by Cephalosporium aphidicola. (Modified from 

Atta-ur-Rahman, M. et al., 1998. J. Nat. Prod., 61: 1340–1342.)


OH 

OH 

OH 

OH 

OH 

138b 

OH 

143b 

OH 

OH 

142b 

7 

6 

1 2 

5 4 

3 

OH 

8 

9 

137b 

10 

140bb 

HO 

139bb 

OH 

OH 

OH 

142b 

OH 

144ba 

OH 

FIGURE 14.64 Metabolic pathways of (-)-menthol (137b) by Aspergillus niger. (Modified from Asakawa, Y. 

et al., 1991. Phytochemistry, 30: 3981–3987.) 

On the other hand, (+)-menthol (137b¢) was smoothly biotransformed by Aspergillus niger to 

give 1b-hydroxymenthol (138b¢), 6b-hydroxymenthol (139ba¢), 2b-hydroxymenthol (140ba¢), 

4a-hydroxymenthol (141b¢), 7-hydroxymenthol (143b¢), 8-hydroxymenthol (142b¢), and 9-hydroxymenthol 

(144ba¢) (Figure 14.65) (Table 14.2). 

Spodoptera litura converted (-)- and (+)-menthols (137b and 137b¢) gave the corresponding 

10-hydroxy products (143b and 143b¢) (Miyazawa et al., 1997a) (Figure 14.66). 

TABLE 14.1 

Metabolites of (−)-Menthol (137b) by Various Aspergillus spp. (Static Culture) 

Microorganisms 138b 142b 139bb 143b 139bb 144ba 141b 

A. awamori IFO 4033 + a ++ − + ++ +++ − 

A. fumigatus IFO 4400 − + − + + + − 

A. sojae IFO 4389 ++ + + − − ++++ − 

A. usami IFO 4338 − − − + − +++ − 

A. cellulosae M-77 + − − + − ++ ++++ 

A. cellulosae IFO 4040 − + − − − ++ ++ 

A. terreus IFO 6123 + + + − + + − 

A. niger IFO 4049 − + − + − +++ − 

A. niger IFO 4040 − + − +++ − +++ − 

A. niger TBUYN-2 + ++ + + ++ ++ − 

a 

Symbols +, ++, +++, etc. are relative concentrations estimated by GC-MS.


OH 

OH 

OH 

OH 

OH 

138b' 

OH 

143b' 

7 

140ba' 

6 

1 

2 

HO 

141b' 

OH 

OH 

5 4 

3 

OH 

8 

9 

137b' 

10 

139ba' 

OH 

OH 

OH 

OH 

OH 

142b' 

144ba' 

FIGURE 14.65 Metabolic pathways of (+)-menthol (137b¢) by Aspergillus niger. (Modified from Noma, Y. 

et al., 1989. Proc. 33rd TEAC, pp. 124–126; Asakawa, Y. et al., 1991. Phytochemistry, 30: 3981–3987.) 

(-)-Menthol (137b) was glycosylated by Eucalyptus perriniana suspension cells to (-)-menthol 

diglucoside (364, 26.6%) and another menthol glycoside. On the other hand, (+)-menthol (137b¢) 

was glycosylated by Eucalyptus perriniana suspension cells to (+)-menthol di- (364¢, 44.0%) and 

triglucosides (365, 6.8%) (Hamada et al., 2002) (Figure 14.67). 

TABLE 14.2 

Metabolites of (+)-Menthol (137b¢) by Various Aspergillus spp. (Static Culture) 

Microorganisms 138b¢ 142b¢ 140ba¢ 143b¢ 139ba¢ 144ba¢ 141b¢ 

A. awamori IFO 4033 + a ++ – +++ – +++ – 

A. fumigatus IFO 4400 + ++ – + – ++ – 

A. sojae IFO 4389 + ++ – – – +++ – 

A. usami IFO 4338 + – – + – +++ – 

A. cellulosae M-77 – + – – – ++ ++++ 

A. cellulosae IFO 4040 + + – – ++ + + 

A. terreus IFO 6123 + +++ + + + ++ – 

A. niger IFO 4049 + – – – + +++ – 

A. niger IFO 4040 + ++ – + – ++ – 

A. niger TBUYN-2 ++ + – +++++ + + – 

a 

Symbols +, ++, +++, etc. are relative concentrations estimated by GC-MS.


OH 

OH 

S. litura 

S. litura 

OH 

OH 

OH 

OH 

137b 

143b 

FIGURE 14.66 Biotransformation of (-)- (137b) and (+)-menthol (137b¢) by Spodoptera litura. (Modified 

from Miyazawa, M. et al., 1997a. Proc. 41st TEAC, pp. 391–392.) 

137b' 

143b' 

OH 

E. perriniana 

+ 

O- 1' Glc 6' - 1'' Glc 

Another menthol glycoside 

137b 364 

OH 

E. perriniana 

+ 

O- 1' Glc 6' - 1'' Glc 

O- 1' 2' Glc6' - 1'' Glc 

1''' Glc 

137b' 

364' 365 

FIGURE 14.67 Biotransformation of (-)- (137b) and (+)-menthol (137b¢) by Eucalyptus perriniana suspension 

cells. (Modified from Hamada, H. et al., 2002. Proc. 46th TEAC, pp. 321–322.) 

Human CYP2A6 

Human CYP2A6 

OH 

OH 

OH 

OH 

137b 

142b 

OH 

FIGURE 14.68 Biotransformation of (-)-menthol (137b) and its enantiomer (137b¢) by human CYP 2A6. 

(Modified from Nakanishi, K. and M. Miyazawa, 2005. Proc. 49th TEAC, pp. 423–425.) 

137b' 

142b' 

OH 

(-)-Menthol (137b) and its enantiomer (137b¢) were converted to their corresponding 8-hydroxy 

derivatives (142b and 142b¢) by human CYP 2A6 (Nakanishi and Miyazawa, 2005) (Figure 14.68). 

By various assays, cytochrome P450 molecular species responsible for the metabolism of (-)- (137b) 

and (+)-menthol (137b¢) was determined to be CYP 2A6 and CYP2B1 in human and rat, respectively. 

Also, kinetic analysis showed that K, and V max values for the oxidation of (-)- (137b) and (+)-menthol 

(137b¢) recombinant CYP2A6 and CYP2B1 were determined to be 28 mM and 10.33 nmol/min/nmol 

P450 and 27 mM, 5.29 nmol/min/nmol P450, 28 mM and 3.58 nmol/min/nmol P450, and 33 mM and 

5.3 nmol/min/nmol P450, respectively (Nakanishi and Miyazawa, 2005) (Figure 14.68).


14.3.3.2 Neomenthol 

(+)-Neomenthol (137a) is biotransformed by Aspergillus niger TBUYN-2 to give five kinds of diols 

(138a, 143a, 144aa, 144ab, and 142a) and two kinds of triols (145a and 146a) as shown in 

Figure 14.69 (Takahashi et al., 1994). 

(-)-Neomenthol (137a¢) is biotransformed by Aspergillus niger to give six kinds of diols (140a¢, 

139a¢, 143a¢, 144aa¢, 144ab¢, and 142a¢) and a triol (146a¢) as shown in Figure 14.70 (Takahashi 

et al., 1994). 

14.3.3.3 (+)-Isomenthol 

(+)-Isomenthol (137c) is biotransformed to give two kinds of diols such as 1b-hydroxy- (138c) and 

6b-hydroxyisomenthol (139c) by Aspergillus niger (Takahashi et al., 1994) (Figure 14.71). 

(±)-Isomenthyl acetate (137c-Ac and 137c¢-Ac) was asymmetrically hydrolyzed to (-)-iso menthol 

(137c) with (+)-isomenthol acetate (137c¢-Ac) by many microorganisms and esterases (Oritani and 

Yamashita, 1973b) (Figure 14.72). 

14.3.3.4 Isopulegol 

(-)-Isopulegol (366) was biotransformed by Spodoptera litura larvae to give 7-hydroxy-(-)-isopulegol 

(367), 9-hydroxy-(-)-menthol (144ba) and 10-hydroxy-(-)-isopulegol (368). On the other 

hand, (+)-isopulegol (366¢) was biotransformed by the same larvae in the same manner to give 

7-hydroxy-(+)-isopulegol (367¢), 9-hydroxy-(+)-menthol (144ba¢), and 10-hydroxy-(+)-isopulegol 

(368¢) (Ohsawa and Miyazawa, 2001) (Figure 14.73). 

Microbial resolution of (±)-isopulegyl acetate (366-Ac and 366¢-Ac) was studied by microorganisms. 

(±)-Isopulegyl acetate (366-Ac and 366¢-Ac) was hydrolyzed asymmetrically to give a mixture 

of (-)-isopulegol (366) and (+)-isopulegyl acetate (366¢-Ac) (Oritani and Yamashita, 1973c) 

(Figure 14.74). 

OH 

OH 

OH 

OH 

146a 

OH 

138a 

OH 

OH 

OH 

145a 

142a 

OH 

OH 

OH 

OH 

7 

6 1 

2 

5 

3 

4 

OH 

8 

9 

137a 

10 

HO 

144aa 

OH 

OH 

OH 

143a 

144ab 

FIGURE 14.69 Metabolic pathways of (+)-neomenthol (137a) by Aspergillus niger. (Modified from 

Takahashi, H. et al., 1994. Phytochemistry, 35: 1465–1467.)


OH 

OH 

OH 

OH 

OH 

OH 

OH 

146a' 

138a' 

OH 

OH 

OH 

143a' 

6 

7 

1 2 

5 4 

3 

OH 

8 

9 

137a' 

10 

140a' 

HO 

OH 

139a' 

OH 

142a' 

HO 

144ab' 

144aa' 

FIGURE 14.70 Metabolic pathways of (-)-neomenthol (137a¢) by Aspergillus niger. (Modified from 

Takahashi, H. et al., 1994. Phytochemistry, 35: 1465–1467.) 

OH 

OH 

OH 

HO 

OH 

OH 

OH 

OH 

139c 

137c 

138c 

FIGURE 14.71 Metabolic pathways of (+)-isomenthol (137c) by Aspergillus niger. (Modified from 

Takahashi, H. et al., 1994. Phytochemistry, 35: 1465–1467.) 

Microorgansims 

O-Ac 

O-Ac 

OH 

O-Ac 

137c-Ac 

137c'-Ac 

137c 

137c'-Ac 

FIGURE 14.72 Microbial resolution of (±)-isomenthyl acetate (137c-Ac and 137c¢-Ac) by microbial esterase. 

(Modified from Oritani, T. and Yamashita, K. 1973b. Agric. Biol. Chem., 37: 1695–1700.)


OH 

OH 

S. litura 

OH 

+ + 

OH 

OH 

366 

367 

OH 

144ba 

OH 

HO 

368 

OH 

S. litura 

OH 

+ + 

OH 

OH 

366' 

367' 

144ba' 

FIGURE 14.73 Biotransformation of (-)- (366) and (+)-isopulegol (366¢) by Spodoptera litura. (Modified 

from Ohsawa, M. and Miyazawa, M. 2001. Proc. 45th TEAC, pp. 375–376.) 

OH 

HO 

368' 

OAc 

OAc 

OH 

OAc 

366-Ac 

366'-Ac 

FIGURE 14.74 Microbial resolution of (±)-isopulegyl acetate (366-Ac and 366¢-Ac) by microorganisms. 

(Modified from Oritani, T. and K. Yamashita, 1973c. Agric. Biol. Chem., 37: 1687–1689.) 

366 

366'-Ac 

14.3.3.5 α-Terpineol 

Pseudomonas pseudomonalli strain T was cultivated with a-terpineol (34) as the sole carbon source 

to give 8,9-epoxy-p-menthan-1-ol (58) via epoxide (369) and diepoxide (57) as intermediates 

(Hayashi et al., 1972) (Figure 14.75). 

(+)-a-Terpineol (34) was formed from (+)-limonene (34) by Citrus pathogenic Pencillium digitatum 

(Pers.; Fr.) Sacc. KCPYN, which was further biotransformed to p-menthane-1b,2a,8-triol 

(334), 2a-hydroxy-1,8-cineole (125b), and (+)-trans-sobrerol (95a) (Noma and Asakawa 2006a, 

2007a) (Figure 14.76). Penicillium sp. YuzuYN also biotransformed 34 to 334. Furthermore, 

Aspergillus niger Tiegh, CBAYN and Catharanthus roseus biotransformed 34 to give 95a and 

(+)-oleuropeyl alcohol (204), respectively (Hamada et al., 2001; Noma and Asakawa 2006a, 2007a) 

(Figure 14.76). 

Gibberella cyanea DSM 62719 biotransformed (-)-a-terpineol (34¢) to give p-menthane-1- 

b,2a,8-triol (334¢), 2a-hydroxy-1,8-cineole (125b¢), 1,2-epoxy-a-terpineol (369¢), (-)-oleuropeyl 

alcohol (204¢), (-)-trans-sobrerol (95a¢), and cis-sobrerol (95b¢) (Abraham et al., 1986) (Figure 

14.76). In cases of Pencillium digitatum (Pers. Fr.) Sacc. KCPYN, Penicillium sp. YuzuYN, 

Aspergillus niger Tiegh, CBAYN 34¢ was biotransformed to give 369¢, 95a¢, and 334¢, respectively 

(Noma and Asakawa 2006a, 2007a) (Figure 14.77). Catharanthus roseus biotransformed 34¢ to give 

95a¢ and 204¢ (Hamada et al., 2001) (Figure 14.77).


OH 

34, 

4R-(+) 

34', 

4S-(–) 

OH 

O 

O 

HO 

P. pseudomonalli 

OH 

OH 

O 

O 

34 

369 

57 58 

FIGURE 14.75 Biotransformation of (+)-a-terpineol (34) to 8,9-epoxy-p-menthan-1-ol (58) by Pseudomonas 

pseudomonalli strain T. (Modified from Hayashi, T. et al., 1972. Biol. Chem., 36: 690–691.) 

P. digitatum 

P. digitatum HO 

Yuzu Penicillium 

HO 

OH 

OH 

68 

OH 

A. niger Tiegh 

C. roses 

34 


P. digitatum 

C. roses 

P. digitatum 

HO 

334 

OH 

O 

OH 

204 

95a 

OH 

125b 

FIGURE 14.76 Biotransformation of (+)-a-terpineol (34) by Citrus pathogenic fungi, Pencillium digitatum 

(Pers.; Fr.) Sacc. KCPYN, Penicillium sp. YuzuYN, Aspergillus niger Tiegh, CBAYN. (Modified from 

Noma, Y. and Y. Asakawa, 2006a. Proc. 50th TEAC, pp. 431–433; Noma, Y. and Y. Asakawa, 2007a. Book of 

Abstracts of the 38th ISEO, p. 7.)


OH 

O 

O 

OH 

G. cyanea 

P. digitatum 

G. cyanea 

125b' 

OH 

OH 

369' 

OH 

Yuzu Penicillium 

G. cyanea 

OH 

G. cyanea 

C. roses 

OH 

34' 


G. cyanea G. cyanea 

C. roses 

334' 

OH 

204' 

HO 

HO 

95b' 

OH 

95a' 

OH 

FIGURE 14.77 Biotransformation of (-)-a-terpineol (34¢) by Gibberella cyanea DSM 62719, Pencillium 

digitatum (Pers. Fr.) Sacc. KCPYN, Penicillium sp. Yuzu YN, Aspergillus niger Tiegh, CBAYN. (Modified 

from Abraham, W.-R. et al., 1986. Appl. Microbiol. Biotechnol., 24: 24–30; Noma, Y. and Y. Asakawa, 2006a. 

Proc. 50th TEAC, pp. 431–433; Noma, Y. and Y. Asakawa, 2007a. Book of Abstracts of the 38th ISEO, p. 7.) 

14.3.3.6 (-)-Terpinen-4-ol 

342, 

4S-(+) 

OH 

342', 

4R-(–) 

OH 

Gibberella cyanea DSM 62719 biotransformed (S)-(-)-terpinen-4-ol (342) (1-p-menthen-4-ol) to 

give 2a-hydroxy-1,4-cineole (132b), 1-p-menthene-4a,6-diol (372), and p-menthane-1b,2a,4atriol 

(371) (Abraham et al., 1986). On the other hand, Aspergillus niger TBUYN-2 also biotransformed 

(-)-terpinen-4-ol (342) to give 2a-hydroxy-1,4-cineole (132b) and (+)-p-menthane-1b,2a,4a-triol 

(371) (Noma and Asakawa 2007b) (Figure 14.78). On the other hand, Spodoptera litura biotransformed 

(R)-terpinen-4-ol (342¢) to (4R)-p-menth-1-en-4,7-diol (373¢) (Kumagae and Miyazawa, 1999) 

(Figure 14.78). 

14.3.3.7 Thymol and Thymol Methyl Ether 

Thymol (179) was converted at the concentration of 14% by Streptomyces humidus, Tu-1 to give 

(1R,2S)- (181a) and (1R,2R)-2-hydroxy-3-p-menthen-5-one (181b) as the major products (Noma 

et al., 1988a) (Figure 14.79). On the other hand, in a Pseudomonas, thymol (179) was biotransformed 

to 6-hydroxy- (180), 7-hydroxy- (479), 9-hydroxy- (480), 7,9-dihydroxythymol (482), thymol-7-oic 

acid (481), and thymol-9-oic acid (483) (Chamberlain and Dagley, 1968) (Figure 14.79).


OH 

OH 

HO 

OH 

G. cyanea 

A. niger 

+ + 

O 

OH 

OH 

OH 

342 371 372 

132b 

OH 

S. litura 

OH 

OH 

342' 

373' 

FIGURE 14.78 Biotransformation of (-)-terpinen-4-ol (342) by Gibberella cyanea DSM 62719, Aspergillus 

niger TBUYN-2, and Spodoptera litura. (Modified from Abraham, W.-R. et al., 1986. Appl. Microbiol. 

Biotechnol., 24: 24–30; Kumagae, S. and M. Miyazawa, 1999. Proc. 43rd TEAC, pp. 389–390; Noma, Y. and 

Y. Asakawa, 2007b. Proc. 51st TEAC, pp. 299–301.) 

HO 

HO 

HO 

OH 

OH 

S. humidus, 

Tu-1 

O 

+ 

O 

180 

179 181a 181b 

COOH 

OH 

OH 

OH 

OH 

OH 

OH 

OH 

OH 

OH 

481 479 480 483 

482 

COOH 

OH 

OMe 

fungus 

+ 

OMe 

OMe 

459 

460 

461 

OH 

FIGURE 14.79 Biotransformation of thymol (179) and thymol methyl ether (459) by actinomycetes Streptomyces 

humidus, Tu-1 and fungi Aspergillus niger, Mucor ramannianus, Rhizopus arrhizus, and Trichothecium 

roseum. (Modified from Chamberlain, E.M. and S. Dagley, 1968. Biochem. J., 110: 755–763; Noma, Y. et al., 

1988a. Proc. 28th TEAC, pp. 177–179; Demirci, F. et al., 2001. XII Biotechnology Congr., Book of abstracts, p. 47.)


Thymol methyl ether (459) was converted by fungi, Aspergillus niger, Mucor ramannianus, 

Rhizopus arrhizus, and Trichothecium roseum to give 7-hydroxy- (460) and 9-hydroxythymol 

methyl ether (461) (Demirci et al., 2001) (Figure 14.79). 

14.3.3.8 Carvacrol and Carvacrol Methyl Ether 

When cultivated in a liquid medium with carvacrol (191), as a sole carbon source, the bacterial 

isolated from savory and pine consumed the carvacrol in the range of 19–22% within 5 days of 

cultivation. The fungal isolates grew much slower and after 13 days of cultivation consumed 

7.1–11.4% carvacrol (191). Pure strains belonging to the bacterial genera of Bacterium, Bacillus 

and Pseudomonas as well as fungal strain from Aspergillus, Botrytis, and Geotrichum genera, 

were also tested for their ability to grow in medium containing carvacrol (191). Among them, 

only in Bacterium sp. and Pseudomonas sp. Carvacrol (191) uptake was monitored. Both 

Pseudomonas sp. 104 and 107 consumed the substrate in the amount of 19%. These two strains 

also exhibited the highest cell mass yield and the highest productivity (1.1 and 1.2 g/L per day) 

(Schwammle et al., 2001). 

Carvacrol (191) was biotransformed to 3-hydroxy- (470), 9-hydroxy (471), 7-hydroxy- (475), and 

8-hydroxycarvacrol (474), 8,9-dehydrocarvacrol (473), carvacrol-9-oic acid (472), carvacrol-7-oic 

acid (476), and 8,9-dihydroxycarvacrol (477) by rats (Ausgulen et al., 1987) and microorganisms 

(Demirci, 2000) including Trichothecium roseum and Cladosporium sp. (Figure 14.80). Furthermore, 

carvacrol methyl ether (191-Me) was converted by the same fungi to give 7-hydroxy- (475-Ac) and 

OH 

OH 

Cladosporium sp. 

OH 

OH 

OH 

OH 

470 191 471 472 

T. roseum 

COOH 

OH 

OH 

COOH 

OH 

OH 

OH 

OH 

OH 

OH 

473 

474 

476 475 

477 

OH 

OH 

OH 

OMe 

OMe 

OMe 

OMe 

+ + 

191-Me 

OH 

475-Me 471-Me 

478 

OH 

FIGURE 14.80 Biotransformation of carvacrol (191) and carvacrol methyl ether (191-Me) by rats (Modified 

from Ausgulen, L.T. et al., 1987. Pharmacol. Toxicol., 61: 98–102) and microorganisms (Modified from 

Demirci, F., 2000. Microbial transformation of bioactive monoterpenes. Ph.D. thesis, pp. 1–137. Anadolu 

University, Eskisehir, Turkey).


9-hydroxycarvacrol methyl ether (471-Me) and 7,9-dihydroxycarvacrol methyl ether (478) (Demirci, 

2000) (Figure 14.80). 

14.3.3.9 Carveol 

OH 

OH 

OH 

OH 

81a 

81b 

81a' 

81b' 

At first, soil Pseudomonad biotransformed (+)-limonene (68) to (+)-carvone (93) and (+)-1-pmenthene-6,9-diol 

(90) via (+)-cis-carveol (81b) as shown in Figure 14.81 (Dhavalikar and 

Bhattacharyya, 1966; Dhavalikar et al., 1966). 

Secondary, Pseudomonas ovalis, strain 6-1 (Noma, 1977) biotransformed the mixture of (-)-ciscarveol 

(81b¢) and (-)-trans-carveol (81a¢) (94:6, GC ratio) to (-)-carvone (93’) (Noma, 1977), 

which was further metabolized reductively to give (+)-dihydrocarvone (101a¢), (+)-isodihydrocarvone 

(101b¢), (+)-neodihydrocarveol (102a), and (-)-dihydrocarveol (102b) (Noma et al., 1984). 

Hydrogenation at C1, 2-position did not occur, but the dehydrogenation at C6-position occured to 

give (-)-carvone (93) (Figure 14.82). 

On the other hand, in Streptomyces, A-5-1 and Nocardia, 1-3-11, which were isolated from 

soil, (-)-carvone (93¢) was reduced to give mainly (-)-trans-carveol (81a¢) and (-)-cis-carveol 

(81b¢), respectively. On the other hand, (-)-trans-carveol (81a¢) and (-)-cis-carveol (81b¢) were 

dehydrogenated to give 93¢ by strain 1-3-11 and other microorganisms (Noma et al., 1986). The 

reaction between trans- and cis-carveols (81a¢ and 81b¢) and (-)-carvone (93¢) is reversible 

(Noma, 1980) (Figure 14.82). 

Thirdly, the investigation for the biotransformation of the mixture of (-)-trans- (81a¢) and (-)-ciscarveol 

(81b¢) (60:40 in GC ratio) was carried out by using 81 strains of soil actinomycetes. All 

actinomycetes produced (-)-carvone (93¢) from the mixture of (-)-trans- (81a¢) and (-)-cis-carveol 

(81b¢) (60:40 in GC ratio). However, 41 strains of actinomycetes converted (-)-cis-carveol (81b¢) to 

give (4R,6R)-(+)-6,8-oxidomenth-1-en-9-ol (92a¢), which is named as bottrospicatol after the name 

of the microorganism, Streptomyces bottropensis ÈBottro˚, and (-)-cis-carveol (81b¢) containing 

Mentha spicata Èspicat˚ and alcohol Èol˚ (Nishimura et al., 1983a) (Figure 14.83). 

(+)-Bottrospicatol (92a¢) was prepared by epoxidation of (-)-carvone (93¢) with mCPBA to 

(-)-carvone-8,9-epoxide (96¢), followed by stereoselective reduction with NaBH 4 to alcohol, which 

was immediately cyclized with 0.1 N H 2 SO 4 to give diastereo mixture of bottrospicatol (92a¢ and b¢) 

(Nishimura et al., 1983a) (Figure 14.84). 

O 

OH 

93 

68 

81b 

OH 

OH 

90 

FIGURE 14.81 Proposed metabolic pathway of (+)-limonene (68) and (+)-cis-carveol (81b) by soil 

Pseudomonad. (Modified from Dhavalikar, R.S. and P.K. Bhattacharyya, 1966. Indian J. Biochem., 3: 144–157; 

Dhavalikar, R.S. et al., 1966. Indian J. Biochem., 3: 158–164.)


OH 

O 

P. ovalis P. ovalis 

OH 

81a' 

93' 81b' 

OH 

O 

OH 

Streptomyces 

Nocardia 

81a' 

93' 81b' 

FIGURE 14.82 Biotransformation of (-)-trans- (81a¢) and (-)-cis-carveol (81b¢) (6:94, GC ratio) by 

Pseudomonas ovalis, strain 6-1, Streptomyces , A-5-1, and Nocardia, 1-3-11. (Modified from Noma, Y., 1977. 

Nippon Nogeikagaku Kaishi, 51: 463–470; Noma, Y., 1980. Agric. Biol. Chem., 44: 807–812.) 

OH 

O 

81a' & b' 

93' 

OH 

81a' & b' 

92a' 

O 

OH 

FIGURE 14.83 The Metabolic pathways of cis-carveol (81b¢) by Pseudomonas ovalis, strain 6-1 (Modified 

from Noma, Y., 1977. Nippon Nogeikagaku Kaishi, 51: 463–470) and Streptomyces bottropensis SY-2-1 and 

other microorganisms (Modified from Noma, Y. et al., 1982. Agric. Biol. Chem., 46: 2871–2872; Nishimura, H. 

et al., 1983a. Proc. 27th TEAC, pp. 107–109). 

O 

mCPBA 

O 

NaBH 4 

OH 

H + 

92a' 

O 

OH 

O 

O 

93' 

96' 

91b' 

O 

HO 

92b' 

FIGURE 14.84 Preparation of (+)-bottrospicatol (92a¢) and (+)-isobottrospicatol (92b¢) from (-)-carvone (93¢) 

with mCPBA. (Modified from Nishimura, H. and Y. Noma, 1996. Biotechnology for Improved Foods and Flavors, 

G.R. Takeoka, et al., ACS Symp. Ser. 637, pp. 173–187. American Chemical Society, Washington, DC.)


OH 

O 

O 

OH 

HO 

HO 

O 

100aa' 

98a' 

93' 

81a' 

HO 

92b' 

O 

OH 

OH 

O 

96' 

HO 

94ba' 

FIGURE 14.85 Biotransformation of (-)-trans- (81a¢) and (-)-cis-carveol (81b¢) by Streptomyces bottropensis 

SY-2-1 and Streptomyces ikutamanensis Ya-2-1. (Modified from Noma, Y. et al., 1982. Agric. Biol. 

Chem., 46: 2871–2872; Noma, Y. and H. Nishimura, 1984. Proc. 28th TEAC, pp. 171–173; Noma, Y. and H. 

Nishimura, 1987. Agric. Biol. Chem., 51: 1845–1849.) 

Further investigation showed Streptomyces bottropensis SY-2-1 (Noma and Iwami, 1994) has 

different metabolic pathways for (-)-trans-carveol (81a¢) and (-)-cis-carveol (81b¢). Namely, 

Streptomyces bottropensis SY-2-1 converted (-)-trans-carveol (81a¢) to (-)-carvone (93¢), (-)-carvone-8,9-epoxide 

(96¢), (-)-5b-hydroxycarvone (98a¢), and (+)-5b-hydroxyneodihydrocarveol 

(100aa¢) (Figure 14.85). On the other hand, Streptomyces bottropensis SY-2-1 converted (-)-ciscarveol 

(81b¢) to give (+)-bottrospicatol (92a¢) and (-)-5b-hydroxy-cis-carveol (94ba¢) as main 

products together with (+)-isobottrospicatol (92b¢) as the minor product as shown in Figure 14.85. 

In the metabolism of cis-carveol by microorganisms there are four pathways (pathways 1–4) 

as shown in Figure 14.86. At first, cis-carveol (81) is metabolized to carvone (93) by C2 dehydrogenation 

(Noma, 1977, 1980) (pathway 1). Secondly, cis-carveol (81b) is metabolized via epoxide 

as intermediate to bottrospicatol (92) by rearrangement at C2 and C8 (Noma et al., 1982; Nishimura 

et al., 1983a, 1983b; Noma and Nishimura, 1987) (pathway 2). Thirdly, cis-carveol (81b) is hydroxylated 

at C5 position to give 5-hydroxy-cis-carveol (94) (Noma and Nishimura, 1984) (pathway 3). 

81b' 

92a' 

O 

OH 

OH 

O 

HO 

OH 

94 

93 

90 

OH 

OH 

81b 

FIGURE 14.86 General metabolic pathways of carveol (81) by microorganisms. (Modified from Noma, Y. 

et al., 1982. Agric. Biol. Chem., 46: 2871–2872; Noma, Y. and H. Nishimura, 1984. Proc. 28th TEAC, pp. 

171–173; Noma, Y. and H. Nishimura, 1987. Agric. Biol. Chem., 51: 1845–1849; Nishimura, H. and Y. Noma, 

1996. Biotechnology for Improved Foods and Flavors, G.R. Takeoka, et al., ACS Symp. Ser. 637, pp.173–187. 

American Chemical Society, Washington, DC.) 

O 

91 

OH 

92 

O 

OH


TABLE 14.3 

Effects of (−)-cis- (81b¢) and (−)-trans-Carveol (81a¢) Conversion Products 

by Streptomyces bottropensis SY-2-1 on the Germination of Lettuce Seeds 

Germination Rate (%) 

Compounds 24 h 48 h 

(-)-Carvone (93¢) 47 89 

(+)-Bottrospicatol (92¢) 3 48 

(-)-Carvone-8,9-epoxide (96¢) 2 77 

5b-Hydroxyneodihydrocarveol (102aa¢) 86 96 

5b-Hydroxycarvone (98a¢) 91 96 

Control 95 96 

Note: Concentration of each compound was adjusted at 200 ppm. 

Finally, cis-carveol (81b) is metabolized to 1-p-menthene-2,9-diol (90) by hydroxylation at C9 

position (Dhavalikar and Bhattacharyya, 1966; Dhavalikar et al., 1966) (pathway 4). 

Effects of (-)-cis- (81b¢) and (-)-trans-carveol (81a¢) conversion products by Streptomyces 

bottropensis SY-2-1 on the germination of lettuce seeds was examined and the result is shown in 

Table 14.3. (+)-Bottrospicatol (92¢) and (-)-carvone-8,9-epoxide (96¢) showed strong inhibitory 

activity for the germination of lettuce seeds. 

Streptomyces bottropensis SY-2-1 has also different metabolic pathways for (+)-trans-carveol 

(81a) and (+)-cis-carveol (81b) (Noma and Iwami, 1994). Namely, Streptomyces bottropensis SY-2-1 

converted (+)-trans-carveol (81a) to (+)-carvone (93), (+)-carvone-8,9-epoxide (96), and (+)-5ahydroxycarvone 

(98a) (Noma and Nishimura, 1982, 1984) (Figure 14.87). On the other hand, 

Streptomyces bottropensis SY-2-1 converted (+)-cis-carveol (81b) to give (-)-isobottrospicatol (92b) 

and (+)-5-hydroxy-cis-carveol (94b) as the main products and (-)-bottrospicatol (92a) as the minor 

product as shown in Figure 14.88 (Noma et al., 1980, Noma and Nishimura, 1987; Nishimura and 

Noma, 1996). 

Biological activities of (+)-bottrospicatol (92a¢) and related compounds for plant’s seed germination 

and root elongation were examined towards barnyard grass, wheat, garden cress, radish, green 

foxtail, and lettuce (Nishimura and Noma, 1996). 

O 

O 

OH 

HO 

O 

98a 

93 

81a 

HO 

92b 

O 

OH 

OH 

O 

96 

HO 

94ba 

81b 

92a 

O 

OH 

FIGURE 14.87 Metabolic pathways of (+)-trans- (81a) and (+)-cis-carveol (81b) by Streptomyces bottropensis 

SY-2-1. (Modified from Noma, Y. and H. Nishimura, 1987. Agric. Biol. Chem., 51: 1845–1849; Nishimura, 

H. and Y. Noma, 1996. Biotechnology for Improved Foods and Flavors, G.R. Takeoka, et al., ACS Symp. Ser. 

637, pp.173–187. American Chemical Society, Washington, DC.)


OH 

OH 

OH 

OH 

91b 

O 

O 

HO 

92b 

O 

HO 

OH 

94ba 

81b 

O 

OH 

O 

92a 

91a 

FIGURE 14.88 Metabolic pathways of (+)-cis-carveol (81b) by Streptomyces bottropensis SY-2-1 and 

Streptomyces ikutamanensis, Ya-2-1. (Modified from Noma, Y. and H. Nishimura, 1987. Agric. Biol. Chem., 

51: 1845–1849; Nishimura, H. and Y. Noma, 1996. Biotechnology for Improved Foods and Flavors, G.R. 

Takeoka, et al., ACS Symp. Ser. 637, pp.173–187. American Chemical Society, Washington, DC.) 

Isomers and derivatives of bottrospicatol were prepared by the procedure shown in Figure 14.89. 

The chemical structure of each compound was confirmed by the interpretation of spectral data. The 

effects of all isomers and derivatives on the germination of lettuce seeds were compared. The germination 

inhibitory activity of (+)-bottrospicatol (92a¢) was the highest of isomers. Interestingly, (-)-isobottrospicatol 

(92b) was not effective even in a concentration of 500 ppm. (+)-Bottrospicatol methyl 

ether (92a¢-methyl ether) and esters [92a¢-methyl (ethyl and n-propyl) ester] exhibited weak inhibitory 

activities. The inhibitory activity of (-)-isodihydrobottrospicatol (105c¢) was as high as that of 

(+)-bottrospicatol (92a¢). Furthermore, an oxidized compound, (+)-bottrospicatal (374a¢), exhibited 

higher activity than (+)-bottrospicatol (92a¢). So, the germination inhibitory activity of (+)-bottrospicatal 

(374a¢) against several plant seeds, lettuce, green foxtail, radish, garden cress, wheat, and 

92a' 

methyl 

ether 

O 

OCH 3 

MeI,NaH 

O 

1stmH 2 

.PtO 2 

AcOH 

105c' 

O 

OH 

O 

OCOR 

(RCO) 2 

Oor 

RCOCl/Py 

92a' 

92a' methyl (ethyl, n-propyl) ester 

OH 

CrO 3 

/Py 

374a' 

O 

CHO 

R=CH 3 ,C 2 H 5 ,n-C 3 H 7 

FIGURE 14.89 Preparation of (+)-bottrospicatol (92a¢) derivatives. (Modified from Nishimura, H. and Y. 

Noma, 1996. Biotechnology for Improved Foods and Flavors, G.R. Takeoka, et al., ACS Symp. Ser. 637, 

pp. 173–187. American Chemical Society, Washington, DC.)


barnyard grass was examined. The result indicates that (+)-bottrospicatal (374a¢) is a selective germination 

inhibitor as follows: lettuce > green foxtail > radish > garden cress > wheat > barnyard grass. 

Enantio- and diastereoselective biotransformation of trans- (81a and 81a¢) and cis-carveols by 

Euglena gracilis Z. (Noma and Asakawa 1992) and Chlorella pyrenoidosa IAM C-28 was studied 

(Noma et al., 1997). 

In the biotransformation of racemic trans-carveol (81a and 81a¢), Chlorella pyrenoidosa IAM 

C-28 showed high enantioselectivity for (-)-trans-carveol (81a¢) to give (-)-carvone (93¢), while 

(+)-trans-carveol (81a) was not converted at all. The same Chlorella pyrenoidosa IAM C-28 

showed high enantioselectivity for (+)-cis-carveol (81b) to give (+)-carvone (93) in the biotransformation 

of racemic cis-carveol (81b and 81b¢). (-)-cis-Carveol (81b¢) was not converted at all. The 

same phenomenon was observed in the biotransformation of mixture of (-)-trans- and (-)-ciscarveol 

(81a¢ and 81b¢) and the mixture of (+)-trans- and (+)-cis-carveol (81a and 81b) as shown 

in Figure 14.90. The high enantioselectivity and the high diastereoselectivity for the dehydrogenation 

of (-)-trans- and (+)-cis-carveols (81a and 81b¢) were shown in Euglena gracilis Z. (Noma 

and Asakawa, 1992), Chlorella pyrenoidosa IAM C-28 (Noma et al., 1997), Nicotiana tabacum, 

and other Chlorella spp. 

On the other hand, the high enantioselecivity for 81a¢ was observed in the biotransformation of 

racemic (+)-trans-carveol (81a) and (-)-trans-carveol (81a¢) by Chlorella sorokiniana SAG to give 

(-)-carvone (93¢). 

It was considered that the formation of (-)-carvone (93¢) from (-)-trans-carveol (81a¢) by 

diastereo- and enantioselective dehydrogenation is a very interesting phenomenon in order to 

produce mosquito repellent (+)-p-menthane-2,8-diol (50a¢) (Noma, 2007). 

(4R)-trans-Carveol (81a¢ ) was converted by Spodptera litura to give 1-p-menthene-6,8,9-triol 

(375) (Miyazawa et al., 1996b) (Figure 14.91). 

14.3.3.10 Dihydrocarveol 

(+)-Neodihydrocarveol (102a¢) was converted to p-menthane-2,8-diol (50a¢), 8-p-menthene-2, 

8-diol (107a¢), and p-menthane-2,8,9-triols (104a¢ and b¢) by Aspergillus niger TBUYN-2 (Noma 

et al., 1985a, 1985b; Noma and Asakawa, 1995) (Figures 14.92 and 14.93). In case of Euglena 

gracilis Z. mosquito repellent (+)-p-menthane-2,8-diol (50a¢) was formed stereospecifically from 

(-)-carvone (93¢) via (+)-dihydrocarvone (101a¢) and (+)-neodihydrocarveol (102a¢) (Noma et al., 

1993; Noma, 2007). (-)-Neodihydrocarveol (102a) was also easily and stereospecifically converted 

by Euglena gracilis Z. to give (-)-p-menthane-2,8-diol (50a) (Noma et al., 1993). 

On the other hand, Absidia glauca converted (-)-carvone (93¢) stereospecifically to give (+)-8-pmenthene-2,8-diol 

(107a¢) via (+)-dihydrocarvone (101a¢) and (+)-neodihydrocarveol (102a¢) 

(Demirci et al., 2004) (Figure 14.93). 

OH 

OH 

81b 

O 

81a' 

O 

OH 

OH 

93 

OH 

93' 

OH 

50' 

81a 

81b' 

FIGURE 14.90 Enantio- and diastereoselective biotransformation of trans- (81a and a¢) and cis-carveols 

(81b and b¢) by Euglena gracilis Z and Chlorella pyrenoidosa IAM C-28. (Modified from Noma, Y., and 

Y. Asakawa, 1992. Phytochem., 31: 2009–2011; Noma, Y. et al., 1997. Proc. 41st TEAC, pp. 227–229.)


OH 

S. litura 

OH 

OH 

OH 

81a' 375' 

FIGURE 14.91 Biotransformation of (4R)-trans-carveol (81a¢) by Spodptera litura. (Modified from 

Miyazawa, M. et al., 1996b. Proc. 40th TEAC, pp. 80–81.) 

(+)- (102b) and (-)-Dihydrocarveol (102b¢) were converted by 10 kinds of Aspergillus spp. to 

give mainly (+)- (107b¢) and (-)-10-hydroxydihydrocarveol (107b, 8-p-menthene-2,10-diol) and 

(+)- (50b¢) and (-)-8-hydroxydihydrocarveol (50b, p-menthane-2,8-diol), respectively (Figure 14.94). 

The metabolic pattern of dihydrocarveols is shown in Table 14.4. 

In case of the biotransformation of Streptomyces bottropensis, SY-2-1 (+)-dihydrocarveol 

(102b) was converted to (+)-dihydrobottrospicatol (105aa) and (+)-dihydroisobottrospicatol 

(105ab), whereas (-)-dihydrocarveol (102b¢) was metabolized to (-)-dihydrobottrospicatol 

(105aa¢) and (-)-dihydroisobottrospicatol (105ab¢). (+)-Dihydroisobottrospicatol (105ab) and 

(-)-dihydrobottrospicatol (105aa¢) are the major products (Noma, 1984) (Figure 14.95). 

Euglena gracilis Z. converted (-)-iso- (102c) and (+)-isodihydrocarveol (102c¢) to give the 

corresponding 8-hydroxyisodihydrocarveols (50c and 50c¢), respectively (Noma et al., 1993) 

(Figure 14.96). 

In case of the biotransformation of Streptomyces bottropensis, SY-2-1 (-)-neoisodihydrocarveol 

(102d) was converted to (+)-isodihydrobottrospicatol (105ba) and (+)-isodihydroisobottrospicatol 

(105bb), whereas (+)-neoisodihydro-carveol (102d¢) was metabolized to (-)-isodihydrobottrospicatol 

(105ba¢) and (-)-isodihydroisobottrospicatol (105bb¢). (+)-Isodihydroisobottrospicatol (105bb) 

and (-)-isodihydrobottrospicatol (105ba¢) are the major products (Noma, 1984) (Figure 14.97). 

OH 

OH 

OH 

OH 

102a 

(1S,2R,4R) 

(–)-Neo 

102b 

(1S,2S,4R) 

(+)-Dihydrocarveol 

102c 

(1R,2S,4R) 

(+)-Iso 

102d 

(1R,2R,4R) 

(+)-Neoiso 

OH 

OH 

OH 

OH 

102a' 

(1R,2S,4S) 

(+)-Neo 

102b' 

(1R,2R,4S) 

(–)-Dihydrocarveol 

102c' 

(1S,2R,4S) 

(–)-Iso 

102d' 

(1S,2S,4S) 

(–)-Neoiso 

FIGURE 14.92 Chemical structure of eight kinds of dihydrocarveols.


OH 

OH 

OH 

102a 

50a 

OH 

OH 

OH 

OH 

OH 

+ + 

+ 

102a' 

50a' 

OH 

107a' 

FIGURE 14.93 Biotransformation of (-)- and (+)-neodihydrocarveol (102a and a¢) by Euglena gracilis Z, 

Aspergillus niger TBUYN-2, and Absidia glauca. (Modified from Noma, Y. et al., 1985a. Annual Meeting 

of Agricultural and Biological Chemistry, Sapporo, p. 68; Noma, Y. et al., 1985b. Proc. 29th TEAC, pp. 

235–237; Noma, Y. et al., 1993. Proc. 37th TEAC, pp. 23–25; Noma, Y., 2007. Aromatic Plants from Asia their 

Chemistry and Application in Food and Therapy, L. Jiarovetz, N.X. Dung, and V.K. Varshney, pp. 169–186. 

Dehradun: Har Krishan Bhalla & Sons; Noma, Y. and Y. Asakawa, 1995. Biotechnology in Agriculture and 

Forestry, Vol. 33. Medicinal and Aromatic Plants VIII, Y.P.S. Bajaj, ed., pp. 62–96. Berlin: Springer; Demirci, 

F. et al., 2004. Naturforsch., 59c: 389–392.) 

OH 

104a' 

OH 

OH HO 

104b' 

OH 

Euglena gracilis Z. converted (-)- (102d) and (+)-neoisodihydrocarveol (102d¢) to give the 

corresponding 8-hydroxyneoisodihydrocarveols (50d and 50d¢), respectively (Noma et al., 1993) 

(Figure 14.98). 

Eight kinds of 8-hydroxydihydrocarveols (50a–d and 50a¢–d¢; 8-p-menthane-2,8-diols) were 

obtained from carvone (93 and 93¢), dihydrocarvones (101a–b and 101a¢–b¢), and dihydrocarveols 

(102a–d, 102a¢–d¢) by Euglena gracilis Z as shown in Figure 14.99 (Noma et al., 1993). 

OH 

Aspergillus sp. 

E.g 

OH 


OH 

OH 

50b 102b 107b 

OH 

OH 

OH 


E.g 


OH 

50b' 102b' 107b' 

OH 

OH 

FIGURE 14.94 Biotransformation of (+)- (102b) and (-)-dihydrocarveol (102b¢) by 10 kinds of Aspergillus 

spp. (Modified from Noma, Y., 1988. The Meeting of Kansai Division of The Agricultural and Chemical 

Society of Japan, Kagawa, p. 28) and Euglena gracilis Z (Modified from Noma, Y. et al., 1993. Proc. 37th 

TEAC, pp. 23–25).


TABLE 14.4 

Metabolic Pattern of Dihydrocarveols (102b and 102b¢) by 10 Kinds of 

Aspergillus spp. 

Compounds 

Microorganisms 107b¢ 50b¢ C.r. (%) 107b 50b C.r. (%) 

A. awamori, IFO 4033 0 98 99 3 81 94 

A. fumigatus, IFO 4400 0 14 34 + 6 14 

A. sojae, IFO 4389 0 47 59 1 50 85 

A. usami, IFO 4338 0 32 52 + 5 7 

A. cellulosae, M-77 0 27 52 + 7 14 

A. cellulosae, IFO 4040 0 30 55 1 5 8 

A. terreus, IFO 6123 0 79 92 + 18 46 

A. niger, IFO 4034 0 29 49 + 8 12 

A. niger, IFO 4049 4 50 67 9 34 59 

A. niger, TBUYN-2 29 68 100 30 53 100 

C.r.—conversion ratio. 

OH 

OH 

OH 

105aa 

O 

OH 

O 

102b 

O 

HO 

105ab 

O 

OH 

OH 

OH 

105aa' 

O 

OH 

O 

102b' 

O 

HO 

105ab' 

FIGURE 14.95 Biotransformation of (+)- (102b) and (-)-dihydrocarveol (102b¢) by Streptomyces bottropensis, 

SY-2-1. (Modified from Noma, Y., 1984. Kagaku to Seibutsu, 22: 742–746.) 

O 

OH 

OH 

OH 

OH 

E. graclis 

E. graclis 

OH 

OH 

102c 

50c 

102c' 

50c' 

FIGURE 14.96 Biotransformation of (+)-iso- (102c) and (-)-dihydrocarveol (102c¢) by Euglena gracilis Z. 

(Modified from Noma, Y. et al., 1993. Proc. 37th TEAC, pp. 23–25.)


OH 

OH 

OH 

105ba 

O 

OH 

O 

102d 

O 

HO 

105bb 

O 

OH 

OH 

OH 

105ba' 

O 

OH 

O 

102d' 

O 

HO 

105bb' 

O 

FIGURE 14.97 Formation of dihydroisobottrospicatols (105) from neoisodihydrocarveol (102d and d¢) by 

Streptomyces bottropensis, SY-2-1. (Modified from Noma, Y., 1984. Kagaku to Seibutsu, 22: 742–746.) 

OH 

OH 

OH 

OH 

E. graclis 

E. graclis 

OH 

OH 

102d 

50d 

102d' 

50d' 

FIGURE 14.98 Biotransformation of (+)- (102c) and (-)-neoisodihydrocarveol (102c¢) by Euglena gracilis 

Z. (Modified from Noma, Y. et al., 1993. Proc. 37th TEAC, pp. 23–25.) 

14.3.3.11 Piperitenol 

HO 

Incubation of piperitenol (458) with Aspergillus niger gave a complex metabolites whose structures 

have not yet been determined (Noma, 2000). 

14.3.3.12 Isopiperitenol 

458 

HO 

110


OH 

OH 

O 

102a 

50a 

OH 

OH 

OH 

101a 

O 

OH 

102b 

50b 

93 

OH 

OH 

O 

102c 

50c 

OH 

101b' 

OH 

OH 

OH 

102d 

50d 

OH 

OH 

O 

OH 

102a' 

50a' 

OH 

OH 

101a' 

O 

102b' 

50b' 

OH 

93' 

OH 

OH 

O 

102c' 

50c' 

OH 

101b 

OH 

OH 

OH 

102d' 

50d' 

FIGURE 14.99 Formation of eight kinds of 8-hydroxydihydrocarveols (50a–50d, 50a¢–50d¢), dihydrocarvones 

(101a–101b and 101a¢–101b¢), and dihydrocarveols (102a–102d and 102a¢–102d¢) from (+)- (93) and 

(-)-carvone (93¢) by Euglena gracilis Z. (Modified from Noma, Y. et al., 1993. Proc. 37th TEAC, pp. 23–25.)


Piperitenol (458) was metabolized by Aspergillus niger to give a complex alcohol mixtures whose 

structures have not yet been determined (Noma, 2000). 

14.3.3.13 Perillyl Alcohol 

OH 

OH 

74 

R-(+)- 

74' 

S-(–)- 

(-)-Perillyl alcohol (74¢) was epoxidized by Streptomyces ikutamanensis Ya-2-1 to give 8,9-epoxy- 

(-)-perillyl alcohol (77¢) (Noma et al., 1986) (Figure 14.100). 

(-)-Perillyl alcohol (74¢) was glycosylated by Eucalyptus perriniana suspension cells to 

(-)-perillyl alcohol monoglucoside (376¢) and diglucoside (377¢) (Hamada et al., 2002; Yonemoto 

et al., 2005) (Figure 14.101). 

Furthermore, 1-perillly-b-glucopyranoside (376) was converted into the corresponding oligosaccharides 

(377–381) using a cyclodextrin glucanotransferase (Yonemoto et al., 2005) 

(Figure 14.102). 

OH 

OH 

S. ikutamanensis 

O 

74' 77' 

FIGURE 14.100 Biotransformation of (-)-perillyl alcohol (74¢) by Streptomyces ikutamanensis, Ya-2-1. 

(Modified from Noma, Y. et al., 1986. Proc. 30th TEAC, pp. 204–206.) 

OH O-Glc O-Glc-Glc 

E. perriniana 

+ 

74' 376' 377' 

FIGURE 14.101 Biotransformation of (-)-perillyl alcohol (74¢) by Eucalyptus perriniana suspension cell. 

(Modified from Hamada, H. et al., 2002. Proc. 46th TEAC, pp. 321–322; Yonemoto, N. et al., 2005. Proc. 49th 

TEAC, pp. 108–110.)


O-Glc 

O-Glc-(Glc) n 

CGTase 

n=1–5 

55°C, pH 5.5 

376 

377–381 

FIGURE 14.102 Biotransformation of (-)-perillyl alcohol monoglucoside (376) by CGTase. (Modified from 

Yonemoto, N. et al., 2005. Proc. 49th TEAC, pp. 108–110.) 

14.3.3.14 Carvomenthol 

OH 

OH 

OH 

OH 

49a 

(1S,2R,4R) 

(–)-Neo 

49b 

(1S,2S,4R) 

(+)-Carvomenthol 

49c 

(1R,2S,4R) 

(–)-Iso 

49d 

(1R,2R,4R) 

(–)-Neoiso 

OH 

OH 

OH 

OH 

49a' 

(1R,2S,4S) 

(+)-Neo 

49b' 

(1R,2R,4S) 

(–)-Carvomenthol 

49c' 

(1S,2R,4S) 

(+)-Iso 

49d' 

(1S,2S,4S) 

(+)-Neoiso 

(+)-Iso- (49c) and (+)-neoisocarvomenthol (49d) were formed from (+)-carvotanacetone (47) via 

(-)-isocarvomenthone (48b) by Pseudomonas ovalis, strain 6-1, whereas (+)-neocarvomenthol 

(49a¢) and (-)-carvomenthol (49b¢) were formed from (-)-carvotanacetone (47¢) via (+)-carvomenthone 

(48a¢) by the same bacteria; of which 48b, 48a¢, and 49d were the major products (Noma et al., 

1974a) (Figure 14.103). 

Microbial resolution of carvomenthols was carried out by selected microorganisms such as 

Trichoderma S and Bacillus subtilis var. niger (Oritani and Yamashita, 1973d). Racemic carvomenthyl 

acetate, racemic isocarvomenthyl acetate, and racemic neoisocarvomenthyl acetate 

were asymmetrically hydrolyzed to (-)-carvomenthol (49b¢) with (+)-carvomenthyl acetate, 

(-)-isocarvomenthol (49c) with (+)-isocarvomenthyl acetate, and (+)-neoisocarvomenthol (49d¢) 

with (-)-neoisocarvomenthyl acetate, respectively; racemic neocarvomenthyl acetate was not 

hydrolyzed (Oritani and Yamashita, 1973d) (Figure 14.104).


O 

P. ovalis 6-1 

OH 

+ 

OH 

47 

49c 

49d 

O 

P. ovalis 6-1 

OH 

+ 

OH 

47' 

49a' 

49b' 

FIGURE 14.103 Formation of (-)-iso- (49c), (-)-neoiso- (49d), (+)-neo- (49a¢), and (-)-carvomenthol (49b¢) 

from (+)- (47) and (-)-carvotanacetone (47¢) by Pseudomonas ovalis, strain 6-1. (Modified from Noma, Y. 

et al., 1974a. Agric. Biol. Chem., 38: 1637–1642.) 

OAc 

OAc 

Trichoderma S 

B. subtilis 

Not hydroliyzed 

49a' 

49a' 

OAc 

OAc 

OAc 

OH 

Trichoderma S 

B. subtilis 

49b' 

49b' 

49b 

49b' 

OAc 

OAc 

OH 

OAc 

Trichoderma S 

B. subtilis 

49c 

OAc 

49c 

OAc 

49c 

OAc 

49c 

' 

OH 

Trichoderma S 

B. subtilis 

49d 

49d 

Racemic acetates 

FIGURE 14.104 Microbial resolution of carvomenthols by Trichoderma S and Bacillus. subtilis var. niger. 

(Modified from Oritani, T. and K. Yamashita, 1973d. Agric. Biol. Chem., 37: 1691–1694.) 

49d 

49d'


14.3.4 MONOCYCLIC MONOTERPENE KETONE 

14.3.4.1 α, β-Unsaturated Ketone 

14.3.4.1.1 Carvone 

O 

O 

S 

R 

4 

93 

(4S)-(+)-form 

93' 

(4R)-(–)-form 

Carvone occurs as (+)-carvone (93), (-)-carvone (93¢), or racemic carvone. (S)-(+)-Carvone (93) is 

the main component of caraway oil (ca. 60%) and dill oil and has a herbaceous odour reminiscent 

of caraway and dill seeds. (R)-(-)-Carvone (93¢) occurs in spearmint oil at a concentration of 

70–80% and has a herbaceous odour similar to spearmint (Bauer et al., 1990). 

The distribution of carvone convertible microorganisms is summarized in Table 14.5. When 

ethanol was used as a carbon source, 40% of bacteria converted (+)- (93) and (-)-carvone (93’). On 

the other hand, when glucose was used, 65% of bacteria converted carvone. In case of yeasts, 75% 

converted (+)- (93) and (-)-carvone (93¢). Of fungi, 90% and 85% of fungi converted 93 and 93¢, 

respectively. In actinomycetes, 56% and 90% converted 93 and 93¢, respectively. 

Many microorganisms except for some strains of actinomycetes were capable of hydrogenating the 

C=C double bond at C-1, 2 position of (+)- (93) and (-)-carvone (93¢) to give mainly (-)-isodihydrocarvone 

(101b) and (+)-dihydrocarvone (101a¢), respectively (Noma and Tatsumi, 1973; Noma et al., 

1974b; Noma and Nonomura, 1974; Noma, 1976, 1977) (Figure 14.105) (Tables 14.6 and 14.7). 

Furthermore, it was found that (-)-carvone (93¢) was converted via (+)-isodihydrocarvone (101b¢) 

to (+)-isodihydrocarveol (102c¢) and (+)-neoisodihydrocarveol (102d¢) by some strains of actinomycetes 

(Noma, 1979a, 1979b). (-)-Isodihydro- carvone (101b) was epimerized to (-)-dihydrocarvone 

(101a) after the formation of (-)-isodihydrocarvone (101b) from (+)-carvone (93) by the growing cells, 

the resting cells, and the cell-free extracts of Pseudomonas fragi, IFO 3458 (Noma et al., 1975). 

TABLE 14.5 

The Distribution of (+)- (93) and (-)-Carvone (93¢) Convertible 

Microorganisms 


Number of 


Used 

Numbers of Carvone 

Convertible 

Microorganisms Ratio (%) 

Bacteria 40 16 (Ethanol, 93) 40 

16 (Ethanol, 93¢) 40 

26 (Glucose, 93) 65 

26 (Glucose, 93¢) 65 

Yeasts 68 51 (93) 75 

51 (93¢) 75 

Fungi 40 34 (93) 85 

36 (93¢) 90 

Actinomycetes 48 27 (93) 56 

43 (93¢) 90 

Source: Noma, Y. et al., 1993. Part VIII. Proc. 37th TEAC, pp. 23–25.


OH 

OH 

O 

O 

102a' 

102a 

OH 

OH 

101a' 

101a 

O 

O 

102b' 

102b 

93' 

OH 

93 

OH 

O 

102c' 

O 

102c 

101b' 

OH 

101b 

OH 

102d' 

102d 

FIGURE 14.105 Biotransformation of (+)- (93) and (-)-carvone (93¢) by various kinds of microorganisms. 

(Modified from Noma, Y. and C. Tatsumi, 1973. Nippon Nogeikagaku Kaishi, 47: 705–711; Noma, Y. et al., 

1974b. Agric. Biol. Chem., 38: 735–740; Noma, Y. et al., 1974c. Proc. 18th TEAC, pp. 20–23; Noma, Y. and S. 

Nonomura, 1974. Agric. Biol. Chem., 38: 741–744; Noma, Y., 1976. Ann. Res. Stud. Osaka Joshigakuen Junior 

College, 20: 33–47; Noma, Y., 1977. Nippon Nogeikagaku Kaishi, 51: 463–470.) 

Consequently, the metabolic pathways of carvone by microorganisms were summarized as the 

following eight groups (Figure 14.105). 

Group 1. (-)-Carvone (93¢)- (+)-dihydrocarvone (101a¢)-(+)-neodihydrocarveol (102a¢) 

Group 2. 93¢–101a¢-(-)-Dihydrocarveol (102b¢) 

TABLE 14.6 

Ratio of Microorganisms that Carried Out the Hydrogenation 

of C=C Double Bond of Carvone by Si Plane Attack toward 

Microorganisms that Converted Carvone 

Microorganisms Ratio (%) 

Bacteria 

100 a 

96 b 

Yeasts 74 

Fungi 80 

Actinomycetes 39 

a 

When ethanol was used. 

b 

When glucose was used.


Group 3. 93¢–101a¢-102a¢ and 102b¢ 

Group 4. 93¢-(+)-Isodihydrocarvone (101b¢)–102c¢ and 102d’ 

Group 5. (+)-Carvone (93)-(-)-isodihydrocarvone (101b)-(-)-neoisodihydrocarveol (102d) 

Group 6. 93–101b–102c 

Group 7. 93–101b–102c and 102d 

Group 8. 93–101b–101a 

The result of the mode action of both the hydrogenation of carvone and the reduction for dihydrocarvone 

by microorganism is as follows. In bacteria, only two strains were able to convert 

(-)-carvone (93¢) via (+)-dihydrocarvone (101a¢) to (-)-dihydrocarveol (102b¢) as the major product 

(Group 3, when ethanol was used as a carbon source, 12.5% of (-)-carvone (93¢) convertible 

microorganisms belonged to this group and when glucose was used, 8% belonged to this group) 

(Noma and Tatsumi 1973; Noma et al., 1975), whereas when (+)-carvone (93) was converted, one 

strain converted it to a mixture of (-)-isodihydrocarveol (102c) and (-)-neoisodihydrocarveol 

(102d) (Group 7, 6% and 4% of 93 convertible bacteria belonged to this group, when ethanol and 

glucose were used, respectively.) and four strains converted it via (-)-isodihydrocarvone (101b) to 

(-)-dihydrocarvone (101a) (Group 8, 6% and 15% of (+)-carvone (93¢) convertible bacteria 

belonged to this group, when ethanol and glucose were used, respectively.) (Noma et al., 1975). In 

yeasts, 43% of carvone convertible yeasts belong to group 1, 14% to group 2, and 33% to group 3 

(of this group, three strains are close to group 1) and 12% to group 5, 4% to group 6, and 27% to 

group 7 (of this group, three strains are close to group 5 and one strain is close to group 6). In 

fungi, 51% of fungi metabolizing (-)-carvone (93¢) by way of group 1 and 3% via group 3, but 

there was no strain capable of metabolizing (-)-carvone (93¢) via group 2, whereas 20% of fungi 

metabolized (+)-carvone (93) via group 5 and 29% via group 7, but there was no strain capable of 

metabolizeing (+)-carvone (93) via group 6. In actinomycetes, (-)-carvone (93¢) was converted to 

dihydrocarveols via group 1 (49%), group 2 (0%), group 3 (9%), and group 4 (28%), whereas 

(+)-carvone (93) was converted to dihydrocarveols via group 5 (7%), group 6 (0%), group 7 (19%), 

and group 8 (0%). 

Furthermore, (+)-neodihydrocarveol (102a¢) stereospecifically formed from (-)-carvone (93¢) by 

Aspergillus niger TBUYN-2 was further biotransformed to mosquito repellent (1R,2S,4R)-(+)-pmenthane-2,8-diol 

(50a¢), (1R,2S,4R)-(+)-8-p-menthene-2,10-diol (107a¢), and the mixture of 

(1R,2S,4R,8S/R)-(+)-p-menthane-2,8,9-triols (104aa¢ and 104ab¢), while Absidia glauca ATCC 

22752 gave 107a¢ stereoselectively from 102a¢ (Demirci et al., 2001) (Figure 14.106). 

On the other hand, (-)-carvone (93¢) was biotransformed stereoselectively to (+)-neodihydrocarveol 

(102a¢) via (+)-dihydrocarvone (101a¢) by a strain of Aspergillus niger (Noma and Nonomura 

1974), Euglena gracilis Z. (Noma et al., 1993), and Chlorella miniata (Gondai et al., 1999). 

Furthermore, in Euglena gracilis Z., mosquito repellent (1R,2S,4R)-(+)-p-menthane-2,8-diol (50a¢) 

was obtained stereospecifically from (-)-carvone (93¢) via 101a¢ and 102a¢ (Figure 14.107). 

As the microbial method for the formation of mosquito repellentl 50a¢ was established, the production 

of (+)-dihydrocarvone (101a¢) and (+)-neodihydrocarveol (102a¢) as the precursor of mosquito 

repellent 50a¢ was investigated by using 40 strains of bacteria belonging to Escherichia, 

Aerobacter, Serratia, Proteus, Alcaligenes, Bacillus, Agrobacterium, Micrococcus, Staphylococcus, 

Corynebacterium, Sarcina, Arthrobacter, Brevibacterium, Pseudomonas, and Xanthomonas spp., 

68 strains of yeasts belonged to Schzosaccharomyces, Endomycopsis, Saccharomyces, 

Schwanniomyces, Debaryomyces, Pichia, Hansenula, Lipomyces, Torulopsis, Saccharomycodes, 

Cryptococcus, Kloeckera, Trigonopsis, Rhodotorula, Candida, and Trichosporon spp., 40 strains of 

fungi belonging to Mucor, Absidia, Penicillium, Rhizopus, Aspergillus, Monascus, Fusarium, 

Pullularia, Keratinomyces, Oospora, Neurospora, Ustilago, Sporotrium, Trichoderma, Gliocladium, 

and Phytophythora spp., and 48 strains of actinomycetes belonging to Streptomyces, Actinoplanes, 

Nocardia, Micromonospora, Microbispora, Micropolyspora, Amorphosporangium, Thermopolyspora, 

Planomonospora, and Streptosporangium spp.


O 

O 

OH 

OH 

OH 

+ 

93' 

101a' 

102a' 

S OH 

OH 

104aa' 

HO 

R 

104ab' 

OH 

OH 

OH 

OH 

OH 

107a' 

50a' 

FIGURE 14.106 Metabolic pathways of (-)-carvone (93¢) by Aspergillus niger TBUYN-2 and Absidia glauca 

ATCC 22752. (Modified from Demirci, F. et al., 2001. XII Biotechnology Congr., Book of abstracts, p. 47.) 

O 

A.n. 

E.g. 

C.p. 

O 

A.n. 

E.g. 

C.p. 

OH 

E.g. 

OH 

OH 

93' 101a' 102a' 

50a' 

FIGURE 14.107 Metabolic pathway of (-)-carvone (93¢) by Aspergillus niger, Euglena gracilis Z, and 

Chlorella miniata. (Modified from Noma, Y. and S. Nonomura, 1974. Agric. Biol. Chem., 38: 741–744; 

Noma, Y. et al., 1993. Proc. 37th TEAC, pp. 23–25; Gondai, T. et al., 1999. Proc. 43rd TEAC, pp. 217–219.) 

As a result, 65% of bacteria, 75% of yeasts, 90% of fungi, and 90% of actinomycetes converted 

(-)-carvone (93¢) to (+)-dihydrocarvone (101a¢) or (+)-neodihydrocarveol (102a¢) (Figure 14.105). 

Many microorganisms are capable of converting (-)-carvone (93¢) to (+)-neodihydrocarveol (102a¢) 

stereospecifically. Some of the useful microorganisms are listed in Tables 14.7 and 14.8. There is no 

good chemical method to obtain (+)-neodihydrocarveol (102a¢) in large quantity. It was considered 

that the method utilizing microorganisms is a very useful means and better than the chemical 

synthesis for the production of mosquito repellent precursor (+)-neodihydrocarveol (102a¢). 

(-)-Carvone (93) was biotransformed by Aspergillus niger TBUYN-2 to give mainly (+)-8-hydroxyneodihydrocarveol 

(50a¢), (+)-8,9-epoxyneodihydrocarveol (103a¢), and (+)-10-hydroxyneodihydrocarveol 

(107a¢) via (+)-dihydrocarvone (101a¢) and (+)-neodihydrocarveol (102a¢). Aspergillus 

niger TBUYN-2 dehydrogenated (+)-cis-carveol (81b) to give (+)-carvone (93), which was further 

converted to (-)-isodihydrocarvone (101b). Compound 101b was further metabolized by four pathways 

to give 10-hydroxy- (-)-isodihydrocarvone (106b), (1S,2S,4S)-p-menthane-1,2-diol (71d) via 

1a-hydroxy- (-)-isodihydrocarvone (72b) as intermediate, (-)-isodihydrocarveol (102c), and 

(-)-neoisodihydrocarveol (102d). Compound 102d was further converted to isodihydroisobottrospicatol 

(105bb) via 8,9-epoxy-(-)-neoisodihydrocarveol (103d); Compound 105¢ was a major 

product (Noma et al., 1985a) (Figure 14.109). 

In case of the plant pathogenic fungus Absidia glauca (-)-carvone (93¢) was metabolized to give 

the diol, 10-hydroxy-(+)-neodihydrocarveol (107a¢) (Nishimura et al., 1983b). 

(+)-Carvone (93) was converted by five bacteria and one fungus (Verstegen-Haaksma et al., 1995) to 

give (-)-dihydrocarvone (101a), (-)-isodihydrocarvone (101b), and (-)-neoisodihydrocarveol (102d).


OH 

O 

102d 

OH 

101b 

O 

102c 

93 

OH 

O 

102b 

OH 

101a 

102a 

FIGURE 14.108 Metabolic pathways of (+)-carvone (93) by Pseudomonas ovalis, strain 6-1 and other many 

microorganisms. (Modified from Noma, Y. et al., 1974b. Agric. Biol. Chem., 38: 735–740.) 

TABLE 14.7 

Summary of Microbial and Chemical 

Hydrogenation of (−)-Carvone (93¢) for the 

Formation of (+)-Dihdyrocarvone (101a¢) and 

(+)-Isodihydrocarvone (101b¢) 

Compounds 

Microorganisms 101a¢ 101b¢ 

Amorphosporangium auranticolor 100 0 

Microbiospora rosea IFO 3559 86 0 

Bacillus subtilis var. niger 85 13 

Bacillus subtilis IFO 3007 67 11 

Pseudomonas polycolor IFO 3918 75 15 

Pseudomonas graveolens IFO 3460 74 17 

Arthrobacter pascens IFO 121139 73 12 

Picha membranaefaciens IFO 0128 70 16 

Saccharomyces ludwigii IFO 1043 69 18 

Alcalygenes faecalis IAM B-141-1 70 13 

Zn-25% KOH–EtOH 73 27 

Raney-10% NaOH 71 19 

Source: Noma, Y., 1976. Ann. Res. Stud. Osaka Joshigakuen 

Junior College, 20: 33–47.


TABLE 14.8 

Summary of Microbial and Chemical Reduction of (-)-Carvone (93¢) for the 

Formation of (+)-Neodihydrocarveol (102a¢) 

Compounds 

Microorganisms 101a¢ 101b¢ 102a¢ 102b¢ 102c¢ 102d¢ 

Torulopsis xylinus IFO 454 0 0 100 0 0 0 

Monascus anka var. rubellus IFO 5965 0 0 100 0 0 0 

Fusarium anguioides Sherbakoff IFO 4467 0 0 100 0 0 0 

Phytophthora infestans IFO4872 0 0 100 0 0 0 

Kloeckera magna IFO 0868 0 0 98 2 0 0 

Kloeckera antillarum IFO 0669 19 4 72 0 0 0 

Streptomyces rimosus + 0 98 0 0 0 

Penicillium notatum Westling IFO 464 6 2 92 0 0 0 

Candida pseudotropicalis IFO 0882 17 4 79 0 0 0 

Candida parapsilosis IFO 0585 16 4 80 0 0 0 

LiAlH 4 0 0 17 67 2 13 

Meerwein–Ponndorf–Verley reduction 0 0 29 55 9 5 

Source: Noma, Y., 1976. Ann. Res. Stud. Osaka Joshigakuen Junior College, 20: 33–47. 

Sensitivity of the microorganism to (+)-carvone (93) and some of the products prevented yields exceeding 

0.35 g/L in batch cultures. The fungus Trychoderma pseudokoningii gave the highest yield of 

(-)-neoisodihydrocarveol (102d) (Figure 14.110). (+)-Carvone (93) is known to inhibit fungal growth of 

Fusarium sulphureum when it was administered via the gas phase (Oosterhaven et al., 1995a, 1995b). 

Under the same conditions, the related fungus, Fusarium solani var. coeruleum was not inhibited. 

In liquid medium, both fungi were found to convert (+)-carvone (93), with the same rate, mainly to 

(-)-isodihydrocarvone (101b), (-)-isodihydrocarveol (102c), and (-)-neoisodihydrocarveol (102d). 

14.3.4.1.1.1 Biotransformation of Carvone to Carveols by Actinomycetes The distribution of 

actinomycetes capable of reducing carbonyl group of carvone containing a, b-unsaturated ketone to 

(-)-trans- (81a¢) and (-)-cis-carveol (81b¢) was investigated. Of 93 strains of actinomycetes, 63 

strains were capable of converting (-)-carvone (93¢) to carveols. The percentage of microorganisms 

that produced carveols from (-)-carvone (93¢) to total microorganisms was about 71%. Microorganisms 

that produced carveols were classified into three groups according to the formation of (-)-transcarveol 

(81a¢) and (-)-cis-carveol (81b¢): group 1, (-)-carvone-81b¢ only; group 2, (-)-carvone-81a¢ 

only; and group 3, (-)-carvone-mixture of 81a¢ and 81b¢. Three strains belonged to group 1 (4.5%), 

34 strains belonged to group 2 (51.1%), and 29 strains belonged to group 3 (44%; of this group two 

strains were close to group 1 and 14 strains were close to group 2). 

Streptomyces, A-5-1 isolated from soil converted (-)-carvone (93¢) to 101a¢–102d¢ and (-)-transcarveol 

(81a¢), whereas Nocardia, 1-3-11 converted (-)-carvone (93¢) to (-)-cis-carveol (81b¢) together 

with 101a¢–81a¢ (Noma, 1980). In case of Nocardia, the reaction between 93¢ and 81a¢ was reversible 

and the direction from 81a¢ to 93¢ is predominantly (Noma, 1979a, 1979b; 1980) (Figure 14.111). 

(-)-Carvone (93¢) was metabolized by actinomycetes to give (-)-trans- (81a¢) and (-)-cis-carveol 

(81b¢) and (+)-dihydrocarvone (101a¢) as reduced metabolites. Compound 81b¢ was further 

metabolized to (+)-bottrospicatol (92a¢). Furthermore, 93¢ was hydroxylated at C-5 position and 

C-8, 9 position to give 5b-hydroxy-(-)-carvone (98a¢) and (-)-carvone-8,9-epoxide (96¢), respectively. 

Compound 98a¢ was further metabolized to 5b-hydroxyneodihydrocarveol (100aa¢) via 

5b-hydroxy-dihydrocarvone (99a¢) (Noma, 1979a, 1979b; 1980) (Figure 14.111). 

Metabolic pattern of (+)-carvone (93) is similar to that of (-)-carvone (93¢) in Streptomyces bottropensis. 

(+)-Carvone (93) was converted by Streptomyces bottropensis to give (+)-carvone-8,9-epoxide


O 

O 

OH 

OH 

OH 

93' 

101a' 

102a' 

O 

103a' 

OH 

50a' 

OH 

OH 

107a' 

OH 

HO 

OH 

104a' 

O 

OH 

O 

OH 

OH 

OH 

71d 

106b 

72b 

OH 

OH 

O 

O 

102c 

81b 

93 

101b 

OH 

OH 

102d 

O 

103d 

HO 

O 

105bb 

FIGURE 14.109 Possible main metabolic pathways of (-)-carvone (93¢) and (+)-carvone (93) by Aspergillus 

niger TBUYN-2. (Modified from Noma, Y. et al., 1985a. Annual Meeting of Agricultural and Biological 

Chemistry, Sapporo, p. 68.) 

(96) and (+)-5a-hydroxycarvone (98a) (Figure 14.112). (+)-Carvone-8,9-epoxide (96) has light 

sweet aroma and has strong inhibitory activity for the germination of lettuce seeds (Noma and 

Nishimura, 1982). 

The investigation of (-)-carvone (93¢) and (+)-carvone (93) conversion pattern was carried out by 

using rare actinomycetes. The conversion pattern was classified as follows (Figure 14.113): 

Group 1. Carvone (93)–dihydrocarvones (101)–dihydrocarveol (102)–dihydrocarveol-8,9- 

epoxide (103)–dihydrobottrospicatols (105)–5-hydroxydihydrocarveols (100) 

Group 2. Carvone (93)–carveols (89)–bottrospicatols (92)–5-hydroxy-cis-carveols (12) 

O 

O 

OH 

93 

101b 

102d 

FIGURE 14.110 Biotransformation of (+)-carvone (93) by Trychoderma pseudokoningii. (Modified from 

Verstegen-Haaksma, A.A. et al., 1995. Ind. Crops Prod., 4: 15–21.)


OH 

OH 

81a' 

O 

O 

92a' 

OH 

81b' 

O 

101a' 

OH 

O 

O 

93' 

O 

HO 

HO 

HO 

O 

100aa' 

99a' 

FIGURE 14.111 Metabolic pathways of (-)-carvone (93¢) by Streptomyces bottropensis SY-2-1, Streptomyces 

ikutamanensis Ya-2-1, Streptomyces, A-5-1, and Nocardia, 1-3-11. (Modified from Noma, Y., 1979a. Nippon 

Nogeikagaku Kaishi, 53: 35–39; Noma, Y., 1979b. Ann. Res. Stud. Osaka Joshigakuen Junior College, 23: 

27–31; Noma, Y., 1980. Agric. Biol. Chem., 44: 807–812; Noma, Y. and H. Nishimura, 1983a. Annual Meeting 

of Agricultural and Biological Chemical Society, Book of abstracts, p. 390; Noma, Y. and H. Nishimura, 

1983b. Proc. 27th TEAC, pp. 302–305.) 

98a' 

96' 

OH 

O 

O 

HO 

92b 

81b 

O 

101b 

O 

93 

O 

HO 

O 

98a 

FIGURE 14.112 Metabolic pathways of (+)-carvone (93¢) by Streptomyces bottropensis SY-2-1 and 

Streptomyces ikutamanensis Ya-2-1. (Modified from Noma, Y. and H. Nishimura, 1982. Proc. 26th TEAC, 

pp. 156–159l; Noma, Y. and H. Nishimura, 1983a. Annual Meeting of Agricultural and Biological Chemical 

Society, Book of abstracts, p. 390; Noma, Y. and H. Nishimura, 1983b. Proc. 27th TEAC, pp. 302–305; 

Noma, Y., 1984. Kagaku to Seibutsu, 22: 742–746.) 

96


OH 

OH 

O 

102a 

O 

103a 

OH 

101a 

O 

102b 

O 

OH 

105aa 

HO 

O 

105ab 

93 

OH 

OH 

O 

O 

102c 

103c 

101b 

102d 

OH 

O 

OH 

105ba 

HO 

105bb 

O 

OH 

OH 

O 

O 

102a' 

103a' 

OH 

O 

101a' 

102b' 

105aa' 

O 

OH 

HO 

O 

105ab' 

93' 

OH 

OH 

O 

102c' 

O 

103c' 

101b' 

102d' 

OH 

O 

OH 

105ba' 

HO 

105bb' 

FIGURE 14.113 Metabolic pathways of (+)- (93) and (-)-carvone (93¢) and dihydrocarveols (102a-d and 

102a¢-d¢) by Streptomyces bottropensis, SY-2-1 and Streptomyces ikutamanesis, Ya-2-1. (Modified from 

Noma, Y., 1984. Kagaku to Seibutsu, 22: 742–746.) 

O


Group 3. Carvone (93)–5-hydroxycarvone (98)–5-hydroxyneodihydrocarveols (15) 

Group 4. Carvone (93)–carvone-8,9-epoxides (96). 

Of 50 rare actinomycets, 22 strains (44%) were capable of converting (-)-carvone (93¢) to give 

(-)-carvone-8,9-epoxide (96¢) via pathway 4 and (+)-5b-hydroxycarvone (98a¢), (+)-5a-hydroxycarvone 

(98b¢), and (+)-5b-hydroxyneodihydrocarveol (100aa¢) via pathway 3 (Noma and Sakai, 1984). 

On the other hand, in case of (+)-carvone (93) conversion, 44% of rare actinomycetes were 

capable of converting (+)-carvone (93) to give (+)-carvone-8,9-epoxide (96) via pathway 4 and 

(-)-5a-hydroxycarvone (98a), (-)-5b-hydroxycarvone (98b), and (-)-5a-hydroxyneodihydrocarveol 

(100aa) via pathway 3 (Noma and Sakai, 1984). 

14.3.4.1.1.2 Biotransformation of Carvone by Citrus Pathogenic Fungi, Aspergillus niger Tiegh 

TBUYN Citrus pathogenic Aspergillus niger Tiegh (CBAYN) and Aspergillus niger TBUYN-2 

hydrogenated C=C double bond at C-1, 2 position of (+)-carvone (93) to give (-)-isodihydrocarvone 

(101b) as the major product together with a small amount of (-)-dihydrocarvone (101a), of which 

101b was further metabolized through two kinds of pathways as follows; namely one is the pathway 

to give (+)-1a-hydroxyneoisodihydrocarveol (71) via (+)-1a-hydroxyisodihydrocarvone (72) and 

the other one is the pathway to give (+)-4a-hydroxy-isodihydrocarvone (378) (Noma and Asakawa, 

2008) (Figure 14.114). 

The biotransformation of enones such as (-)-carvone (93¢) by the cultured cells of Chlorella 

miniata was examined. It was found that the cells reduced stereoselectively the enones from si-face 

at a-position of the carbonyl group and then the carbonyl group from re-face (Figure 14.115). 

Stereospecific hydrogenation occurs independent of the configuration and the kinds of the substituent 

at C-4 position, so that the methyl group at C-1 position is fixed mainly at R configuration. 

[2- 2 H]- (-)-Carvone ([2- 2 H]-93¢) was synthesized in order to clear up the hydrogenation mechanism 

at C-2 by microorganisms. (Compound [2- 2 H]-93 was also easily biotransformed to [2- 2 H]-8- 

hydroxy-(+)-neodihydro-carveol (50a¢) via [2- 2 H]-(+)-neodihydrocarveol (102a¢). On the basis of 

1 

H-NMR spectral data of compounds 102a¢ and 50a¢, the hydrogen addition of the carbon–carbon 

double bond at the C 1 and C 2 position by Aspergillus niger TBUYN-2, Euglena gracilis Z., and 

Dunaliella tertiolecta occurs from the si face and re face, respectively, namely, anti addition (Noma 

et al., 1995) (Figure 14.115) (Table 14.9). 

OH 

OH 

O O O 

OH 

93 

101b 

72 

71 

O 

O 

OH 

101a 

378 

FIGURE 14.114 Metabolic pathways of (+)-carvone (93) by Citrus pathogenic fungi, Aspergillus niger 

Tiegh CBAYN and Aspergillus niger TBUYN-2. (Modified from Noma, Y. and Y. Asakawa, 2008. Proc. 

52nd TEAC, pp. 206–208.)


H from si face 

2 H 

re re si 

O 

2 H 

H 

H 

O 

2 H 

H 

H 

OH 

2 H 

H 

H 

OH 

H 

from re face 

OH 

93' 101a' 102a' 50a' 

FIGURE 14.115 The stereospecific hydrogenation of the C=C double bond of a, b-unsaturated ketones, the 

reduction of saturated ketone, and the hydroxylation by Euglena gracilis Z. (Modified from Noma, Y. et al., 1995. 

Proc. 39th TEAC, pp. 367–368; Noma, Y. and Y. Asakawa, 1998. Biotechnology in Agriculture and Forestry, 

Vol. 41. Medicinal and Aromatic Plants X, Y.P.S. Bajaj, ed., pp. 194–237. Berlin Heidelberg: Springer.) 

14.3.4.1.1.3 Hydrogenation Mechanisms of C=C Double Bond and Carbonyl Group In 

order to understand the mechanism of the hydrogenation of a-, b-unsaturated ketone of (-)-carvone 

(93¢) and the reduction of carbonyl group of dihydrocarvone (101a¢) (-)-carvone (93¢), 

(+)-dihydrocarvone (101a¢) and the analogues of (-)-carvone (93¢) were chosen and the conversion 

of the analogues was carried out by using Pseudomonas ovalis, strain 6-1. As the analogues of 

carvone (93 and 93¢), (-)- (47¢) and (+)-carvotanacetone (47), 2-methyl-2-cyclohexenone (379), the 

mixture of (-)-cis- (81b¢) and (-)-trans-carveol (81a¢), 2-cyclohexenone, racemic menthenone 

(148), (-)-piperitone (156), (+)-pulegone (119), and 3-methyl-2-cyclohexenone (381) were chosen. 

Of these analogues, (-)- (47¢) and (+)-carvotanacetone (47) were reduced to give (+)-carvomenthone 

(48a¢) and (-)-isocarvomenthone (48b¢), respectively. 2-Methyl-2-cyclohexenone (379) was 

mainly reduced to (-)-2-methylcyclohexanone. But other compounds were not reduced. 

The efficient formation of (+)-dihydrocarvone (101a), (-)-isodihydrocarvone (101b¢), (+)-carvomenthone 

(48a), (-)-isocarvomenthone (48b¢), and (-)-2-methylcyclohexanone from (-)-carvone 

(93), (+)-carvone (93¢), (-)-carvotanacetone (47), (+)-carvotanacetone (47¢), and 2-methyl-2-cyclohexenone 

(379) suggested at least that C=C double bond conjugated with carbonyl group may be 

hydrogenated from behind (si plane) (Noma, 1977; Noma et al., 1974b) (Figure 14.116). 

14.3.4.1.1.4 What is Hydrogen Honor in the Hydrogenation of Carvone to Dihydrocarvone? 

What is Hydrogen Donor in Carvone Reductase? Carvone reductase prepared from 

Euglena gracilis Z, which catalyzes the NADH-dependent reduction of the C=C bond adjacent to the 

carbonyl group, was characterized with regard to the stereochemistry of the hydrogen transfer into 

the substrate. The reductase was isolated from Euglena gracilis Z and was found to reduce stereospecifically 

the C=C double bond of carvone by anti-addition of hydrogen from the si face at a-position 

to the carbonyl group and the re face at b-position (Table 14.9). The hydrogen atoms participating in 

the enzymatic reduction at a- and b-position to the carbonyl group originate from the medium and 

the pro-4R hydrogen of NADH, respectively (Shimoda et al., 1998) (Figure 14.117). 

TABLE 14.9 

The Summary for the Stereospecificity of the Reduction of the C=C Double 

Bond of [2- 2 H]-(-)-Carvone ([2- 2 H]-93) by Various Kinds of Microorganisms 

Stereochemistry at C-2H of Compounds 

Microorganisms 102a 50a 

Aspergillus niger TBUYN-2 

b 

Euglena gracilis Z b b 

Dunaliella tertiolecta 

b 

The cultured cells of Nicotiana tabacum (Suga et al., 1986) b


O 

O 

O 

O 

O 

O 

O 

O 

93' 

93 

47' 

47 

O 

96' 

O 

96 

44' 

OH 

44 

OH 

HO 

O 

O 

O 

O 

O 

O 

O 

98a' 

379 

380 

148 

148 

381 

119 

FIGURE 14.116 Substrates used for the hydrogenation of C=C double bond with Pseudomonas ovalis, strain 

6-1, Streptomyces bottropensis SY-2-1, Streptomyces ikutamanensis Ya-2-1, and Euglena gracilis Z. 

TABLE 14.10 

Purification of the Reductase from Euglena gracilis Z. 

Total 

Protein (mg) 

Total Activity 

Unit ¥ 10 4 

Sp. Act Units per 

Gram Protein 

Crude extract 125 2.2 1.7 1 

DEAE Toyopearl 7 1.5 21 12 

AF-Blue Toyopearl 0.1 0.03 30 18 

Fold 

In the case of biotransformation by using Cyanobacterium (+)- (93) and (-)-carvone (93¢) were 

converted with a different type of pattern to give (+)-isodihydrocarvone (101b¢, 76.6%) and 

(-)- dihydrocarvone (101a, 62.2%), respectively (Kaji et al., 2002) (Figure 14.118). On the other hand, 

Catarantus rosea cultured cell biotransformed (-)-carvone (93¢) to give 5b-hydroxy- (+)-neodihydrocarveol 

(100aa¢, 57.5%), 5a-hydroxy-(+)-neodihydrocarveol (100ab¢, 18.4%), 5a-hydroxy-(-)- 

carvone (98b¢), 4b-hydroxy-(-)-carvone (384¢, 6.3%), 10-hydroxycarvone (390¢), 5b-hydroxycarvone 

(98¢), 5a-hydroxyneodihydrocarveol (100ab¢), 5b-hydroxyneodihydrocarveol (100aa¢), and 5ahydroxydihydrocarvone 

(99b¢) as the metabolites as shown in Figure 14.119, whereas (+)-carvone 

(93) gave 5a-hydroxy-(+)-carvone (98a, 65.4%) and 4a-hydroxy-(+)-carvone (384, 34.6%) (Hamada 

and Yasumune, 1995; Hamada et al., 1996; Kaji et al., 2002) (Figure 14.119) (Table 14.11). 

(-)-Carvone (93¢) was incubated with Cyanobacterium, enone reductase (43 kDa) isolated from the 

bacterium and microsomal enzyme to afford (+)-isodihydrocarvone (101b¢) and (+)-dihydro carvone 

Hs 

NADH 

H R 

CONH 2 

N-R 

O 

H 

H 

O 

si 

re 

93' 

anti-addition 

FIGURE 14.117 Stereochemistry in the reduction of (-)-carvone (93¢) by the reductase from Euglena 

gracilis Z. (Modified from Shimoda, K. et al., 1998. Phytochem., 49: 49–53.) 

O 

101a'


O 

O 

O 

O 



93 

101a 

93' 101b' 

FIGURE 14.118 Biotransformation of (-)- and (+)-carvone (93 and 93¢) by Cyanobacterium. (Modified 

from Kaji, M. et al., 2002. Proc. 46th TEAC, pp. 323–325.) 

O 

Catharanthus 

roseus 

O 

OH 

+ 

HO 

O 

+ + 

HO 

OH 

HO 

OH 

93' 384' 

98b' 

100aa' 

100ab' 

O 

+ 

HO 

O 

+ 

HO 

O 

390' 

OH 

98a' 

99ab' 

O O O 

Catharanthus 

roseus 

OH 

+ 

HO 

93 384 

98a 

FIGURE 14.119 Biotransformation of (+)- and (-)-carvone (93 and 93¢) by Cataranthus roseus. (Modified 

from Hamada, H. and H. Yasumune, 1995. Proc. 39th TEAC, pp. 375–377; Hamada, H. et al., 1996. Proc. 40th 

TEAC, pp. 111–112; Kaji, M. et al., 2002. Proc. 46th TEAC, pp. 323–325.) 

(101a¢). Cyclohexenone derivatives (379 are 385) were treated in the same enone reductase with 

microsomal enzyme to give the dihydro derivative (382a, 386a) with R-configuration in excellent ee 

(over 99%) and the metabolites (382b, 386b) with S-configuration in relatively high ee (85% and 80%) 

(Shimoda et al., 2003) (Figure 14.120). 

TABLE 14.11 

Enantioselectivity in the Reduction of Enones (379 and 385) by 

Enone Reductase 

Microsomal Enzyme Substrate Product ee Configuration a 

– 379 382a >99 R 

– 385 386a >99 R 

+ 379 382b 85 S 

+ 385 386b 80 S 

a 

Preferred configuration at a-position to the carbonyl group of the products.


O 

Enone 

reductase 

O 

Enone 

reductase 

O 

Microsomal 

enzyme 

101b' 

93' 

101a' 

O 

O 

O 

382b 

379 

382a 

O 

O 

O 

386b 

385 386a 

FIGURE 14.120 Biotransformation of 2-methyl-2-cyclohexenone (379) and 2-ethyl-2-cyclohexenone (385) 

by enone reductase. 

In contrast, almost all the yeasts tested showed reduction of carvone, although the enzyme 

activity varied. The reduction of (-)-carvone (93¢) was often much faster than the reduction of 

(+)-carvone (93). Some yeasts only reduced the carbon–carbon double bond to yield the dihydrocarvone 

isomers (101a¢ and b¢ and 101a and b) with the stereochemistry at C-1 with R configuration, 

while others also reduced the ketone to give the dihydrocarveols with the stereochemistry at C-2 

always with S for (-)-carvone (93¢), but sometimes S and sometimes R for (+)-carvone (93). In the 

case of (-)-carvone (93¢) yields increased up to 90% within 2 h (van Dyk et al., 1998). 

14.3.4.1.2 Carvotanacetone 

O 

O 

47 

S-(+)- 

47' 

R-(–) 

In the conversion of (+)- (47) and (-)-carvotanacetone (47¢) by Pseudomonas ovalis, strain 6-1, 

(-)-carvotanacetone (47¢) is converted stereospecifically to (+)-carvomenthone (48a¢) an the latter 

compound is further converted to (+)-neocarvomenthol (49a¢) and (-)-carvomenthol (49b¢) in small 

amounts, whereas (+)-carvotanacetone (47) is converted mainly to (-)-isocarvomenthone (48b) and 

(-)-neoisocarvomenthol (49d), forming (-)-carvomenthone (48a) and (-)-isocarvomenthol (49c) in 

small amounts as shown in Figure 14.121 (Noma et al., 1974a). 

Biotransformation of (-)-carvotanacetone (47) and (+)-carvotanacetone (47¢) by Streptomyces 

bottropensis, SY-2-1 was carried out (Noma et al., 1985c). 

As shown in Figure 14.122, (+)-carvotanacetone (47) was converted by Streptomyces bottr opensis, 

SY-2-1 to give 5b-hydroxy-(+)-neoisocarvomenthol (139db), 5a-hydroxy-(+)-carvotanacetone 

(51a), 5b-hydroxy- (-)-carvomenthone (52ab), 8-hydroxy-(+)-carvotanacetone (44), and 8-hydroxy- 

(-)-carvomenthone (45a), whereas (-)-carvotanacetone (47¢) was converted to give 5b-hydroxy- 

(-)-carvotanacetone (51a¢) and 8-hydroxy-(-)-carvotanacetone (44¢). 

Aspergillus niger TBUYN-2 converted (-)-carvotanacetone (47¢) to (+)-carvomenthone (48a¢), 

(+)-carvomenthone (49a¢), diastereoisomeric p-menthane-2,9-diols [55aa¢ (8R) and 55ab¢ (8S) in the


O 

O 

OH 

O 

O 

OH 

47' 49a' 

47 

48a' 

48b 

49d 

OH 

O 

OH 

49b' 

48a 

49c 

FIGURE 14.121 Metabolic pathways of (-)-carvotanacetone (47¢) and (+)-carvotanacetone (47) by 

Pseudomonas ovalis, strain 6-1. (Modified from Noma, Y. et al., 1974a. Agric. Biol. Chem., 38: 1637–1642.) 

O 

O 

OH 

HO 

51b 

HO 

52bb 

HO 

139db 

HO 

O 

O 

O 

O 

O 

51a' 

47 

HO 

51a 

HO 

52ab 

47' 

O 

OH 

O 

O 

44' 

44 

OH 

45a 

OH 

FIGURE 14.122 Proposed the metabolic pathways of (+)-carvotanacetone (47) and (-)-carvotanacetone 

(47¢) by Streptomyces bottropensis, SY-2-1. (Modified from Noma, Y. et al., 1985c. Proc. 29th TEAC, 

pp. 238–240.) 

ratio of 3:1], and 8-hydroxy-(+)-neocarvomenthol (102a¢). On the other hand, the same fungus converted 

(+)-carvotanacetone (47) to (-)-isocarvomenthone (48b), 1a-hydroxy-(+)- neoisocarvo menthol 

(54) via 1a-hydroxy-(+)-isocarvomenthone (53) and 8-hydroxy-(-)-isocarvomenthone (45b) as shown 

in Figure 14.123 (Noma et al., 1988b). 

14.3.4.1.3 Piperitone 

O 

O 

156 

R-(–) 

156' 

S-(+)


O 

O 

OH 

3 

OH 

47' 

48a' 

49a' 

1 

H R 

55aa' 

OH 

OH 

OH 

O 

O 

OH 

102a' 

OH 

O 

S H 

OH 

55ab' 

OH 

OH 

47 

48b 

53 

54 

O 

FIGURE 14.123 Proposed metabolic pathways of (-)-carvotanacetone (47) and (+)-carvotanacetone (47¢) 

by Aspergillus niger TBUYN-2. (Modified from Noma, Y. et al., 1988b. Proc. 32nd TEAC, pp. 146–148.) 

45b 

OH 

A large number of yeasts were screened for the biotransformation of (-)-piperitone (156). A relatively 

small number of yeasts gave hydroxylation products of (-)-piperitone (156). Products obtained 

from (-)-piperitone (156) were 7-hydroxypiperitone (161), cis-6-hydroxypiperitone (158b), trans-6- 

hydroxypiperitone (158a), and 2-isopropyl-5-methylhydroquinone (180). Yields for the hydroxylation 

reactions varied between 8% and 60%, corresponding to the product concentrations of 

0.04–0.3 g/L. Not one of the yeasts tested reduced (-)-piperitone (156) (van Dyk et al., 1998). 

During the initial screen with (-)-piperitone (156) only hydroxylation products were obtained. The 

hydroxylation products (161, 158a, and 158b) obtained with nonconventional yeasts from the genera 

OH 

O 

HO 

HO 

+ 

O O OH 

156 

161 158a 180 

HO 

O 

158b 

FIGURE 14.124 Hydroxylation products of (R)-(-)-piperitone (156) by yeast. (Modified from van Dyk, M.S. 

et al., 1998. J. Mol. Catal. B: Enzym., 5: 149–154.)


Arxula, Candida, Yarrowia, and Trichosporon have recently been described (van Dyk et al., 1998) 

(Figure 14.124). 

14.3.4.1.4 Pulegone 

(R)-(+)-Pulegone (119), with a mint-like odour monoterpene ketone, is the main component (up to 

80–90%) of Mentha pulegium essential oil (Pennyroyal oil), which is sometimes used in beverages 

and food additive for human consumption and occasionally in herbal medicine as an abortifacient 

drug. The biotransformation of (+)-pulegone (119) by fungi was investigated (Ismaili-Alaoui et al., 

1992). Most fungal strains grown in a usual liquid culture medium were able to metabolize (+)-pulegone 

(119) to some extent in a concentration range of 0.1–0.5 g/L; higher concentrations were generally 

toxic, except for a strain of Aspergillus sp. isolated from mint leaves infusion, which was able 

to survive to concentrations of up to 1.5 g/L. The predominant product was generally l-hydroxy-(+)- 

pulegone (384) (20–30% yield). Other metabolites were present in lower amounts (5% or less) (see 

Figure 14.125). The formation of 1-hydroxy-(+)-pulegone (387) was explained by hydroxylation at a 

tertiary position. Its dehydration to piperitenone (112), even under the incubation conditions, during 

isolation or derivative reactions precluded any tentative determination of its optical purity and absolute 

configuration. 

Botrytis allii converted (+)-pulegone (119) to (-)-(1R)-8-hydroxy-4-p-menthen-3-one (121) and 

piperitenone (112) (Miyazawa et al., 1991a, 1991b). Hormonema isolate (UOFS Y-0067) quantitatively 

reduced (+)-pulegone (119) and (-)-menthone (149a) to (+)-neomenthol (137a) (van Dyk et al., 

1998) (Figure 14.125). 

Biotrasformation by the recombinant reductase and the transformed Escherichia coli cells were 

examined with pulegone, carvone, and verbenone as substrates (Figure 14.126). The recombinant 

reductase catalyzed the hydrogenation of the exocylic C=C double bond of pulegone (119) to give 

menthone derivatives (Watanabe et al., 2007) (Tables 14.12 and 14.13). 

O 

OH 

149a 

OH 

H + 

137a 

O 

O 

O 

119 

387 112 

HO 

O 

O 

O 

OH – 

OH 

OH 

388 121 

389 

FIGURE 14.125 Biotransformation of (+)-pulegone (119) by Aspergillus sp., Botrytis allii, and H. isolate 

(UOFS Y-0067). (Modified from Miyazawa, M. et al., 1991a. Chem. Express, 6: 479–482; Miyazawa, M. 

et al., 1991b. Chem. Express, 6: 873; Ismaili-Alaoui, M. et al., 1992. Tetrahedron Lett., 33: 2349–2352; van 

Dyk, M.S. et al., 1998. J. Mol. Catal. B: Enzym., 5: 149–154.)


O 

O 

O 

O 

O 

O 

24 24' 

119 119' 93' 93 

O 

O 

O 

O 

149a 

149b 

149a' 

149b' 

FIGURE 14.126 Chemical structures of substrate reduced by the recombinant pulegone reductase and the 

transformed Esherichia coli cells. 

14.3.4.1.5 Piperitenone and Isopiperitenone 

Piperitenone (112) is metabolized to 5-hydroxypiperitenone (117), 7-hydroxypiperitenone (118), and 7,8- 

dihydroxypiperitone (157). Isopiperitenol (110) is reduced to give isopiperitenone (111), which is further 

metabolized to piperitenone (112), 7-hydroxy- (113), 10-hydroxy- (115), 4-hydroxy- (114), and 5-hydroxyisopiperitenone 

(116). Compounds 111 and 112 are isomerized to each other. Pulegone (119) was metabolized 

to 112, 8,9-dehydromenthenone (120) and 8-hydroxymenthenone (121) as shown in the 

biotransformation of the same substrate using Botrytis allii (Miyazawa et al., 1991b) (Figure 14.127). 

H. isolate (UOFS Y-0067) reduced (4S)-isopiperitenone (111) to (3R,4S)-isopiperitenol (110), a 

precursor of (-)-menthol (137b) (van Dyk et al., 1998) (Figure 14.128). 

TABLE 14.12 

Substrate Specificity in the Reduction of Eenones with the Recombinant 

Pulegone Reductase 

Entry No. (Reaction Time) Substrates Products Conversions (%) 

1 (3 h) (R)-(+)-Pulegone (119) (1R, 4R)-Isomenthone (149b) 4.4 

2 (12 h) (R)-Pulegone (119) (1S, 4R)-Menthone (149a) 6.8 

3 (3 h) (S)-(-)-Pulegone (119¢) (1R, 4R)-Isomenthone (149b) 14.3 

4 (12 h) (S)-Pulegone (119¢) (1S, 4R)-Menthone (149a) 15.7 

5 (12 h) (R)-(-)-Carvone (93¢) (1S, 4S)-Isomenthone (149b¢) 0.3 

6 (12 h) (S)-(+)-Carvone (93) (1R, 4S)-Menthone (149a¢) 0.5 

7 (12 h) (1S, 5S)-Verbenone (24) (1S, 4S)-Isomenthone (149b¢) 1.6 

8 (12 h) (1R, 5R)-Verbenone (24¢) (1R, 4S)-Menthone (149a¢) 2.1 

N.d.—denotes not detected. 

N.d. 

— N.d. 

— N.d. 

— N.d. 

—


TABLE 14.13 

Biotransformation of Pulegone (119 and 119¢) with the 

Transformed Escherichia coli cells a 

Substrates Products Conversion (%) 

(R)-(+)-Pulegone (119) (1R, 4R)-Isomenthone (149b) 26.8 

(S)-(-)-Pulegone (119¢) (1S, 4R)-Menthone (149a) 30.0 

(1S, 4S)-Isomenthone (149b¢) 32.3 

(1R, 4S)-Menthone (149a¢) 7.1 

a 

Reaction times of the transformation reaction are 12 h. 

OH 

OH 

O 

O 

HO 

O 

113 

157 

OH 

117 

OH 

OH 

O 

O 

O 

O 

110 

111 

112 

118 

120 

HO 

O 

HO 

O 

O 

O 

O 

116 

114 

115 

OH 

119 

121 

OH 

FIGURE 14.127 Biotransformation of isopiperitenone (111) and piperitenone (112) by Aspergillus niger 

TBUYN-2. (Modified from Noma, Y. et al., 1992c. Proc. 37th TEAC, pp. 26–28.) 

O 

OH 

OH 

111 

110a 

137b 

FIGURE 14.128 Biotransformation of isopiperitenone (111) by H. isolate (UOFS Y-0067). (Modified from 

van Dyk, M.S. et al., 1998. J. Mol. Catal. B: Enzym., 5: 149–154.)


14.3.4.2 Saturated Ketone 

14.3.4.2.1 Dihydrocarvone 

O O O 

O 

101a' 

(1R,4S) 

(+) 

101b' 

(1S,4S) 

(+)-Iso 

101a 

(1S,4R) 

(–) 

101b 

(1R,4R) 

(–)-Iso 

In the reduction of saturated carbonyl group of dihydrocarvone by microorganism, (+)- dihydrocarvone 

(101a¢) is converted stereospecifically to either (+)-neodihydrocarveol (102a¢) or (-)-dihydrocarveol 

(102b¢) or nonstereospecifically to the mixture of 102a¢ and 102b¢, whereas (-)-isodihydrocarvone 

(101b) is converted stereospecifically to either (-)-neoisodihydrocarveol (102d) or (-)- isodihydro carveol 

(102c) or nonstereospecifically to the mixture of 102c and 102d by various microorganisms (Noma 

and Tatsumi, 1973; Noma et al., 1974c; Noma and Nonomura 1974; Noma, 1976, 1977). 

(+)-Dihydrocarvone (101a¢) and (+)-isodihydrocarvone (101b¢) are easily isomerized chemically 

to each other. In the microbial transformation of (-)-carvone (93¢), the formation of (+)- dihydrocarvone 

(101a¢) is predominant. (+)-Dihydrocarvone (101a¢) was reduced to both/either (+)- neodihydrocarveol 

(102a¢) and/or (-)-dihydrocarveol (102b), whereas in the biotransformation of (+)-carvone (93), 

(+)-isodihydrocarvone (101b) was formed predominantly. (+)-Isodihydrocarvone (101b) was reduced 

to both (+)-isodihydrocarveol (102c) and (+)-neoisodihydrocarveol (102d) (Figure 14.129). 

OH 

O 

102d 

OH 

O 

101b 

epimerization 

102c 

93 

OH 

O 

102b 

OH 

101a 

102a 

FIGURE 14.129 Proposed metabolic pathways of (+)-carvone (93) and (-)-isodihydrocarvone (101b) by 

Pseudomonas fragi, IFO 3458. (Modified from Noma, Y. et al., 1975. Agric. Biol. Chem., 39: 437–441.)


However, Pseudomonas fragi, IFO 3458, Pseudomonas fl uorescens, IFO 3081, and Aerobacter 

aerogemes, IFO 3319 and IFO 12059, formed (-)-dihydrocarvone (101a) predominantly from 

(+)-carvone (93). In the time course study of the biotransformation of (+)-carvone (93), it appeared 

that predominant formation of (-)-dihydrocarvone is due to the epimerization of (-)- isodihydrocarvone 

(101b¢) by epimerase of Pseudomonas fragi IFO 3458 (Noma et al., 1975). 

14.3.4.2.2 Isodihydrocarvone Epimerase 

14.3.4.2.2.1 Preparation of Isodihydrocarvone Epimerase The cells of Pseudomonas fragi 

IFO 3458 were harvested by centrifugation and washed five times with 1/100 M KH 2 PO 4 –Na 2 HPO 4 

buffer (pH 7.2). Bacterial extracts were prepared from the washed cells (20 g from 3-L medium) by 

sonic lysis (Kaijo Denki Co., Ltd., 20Kc., 15 min, at 5–7∞C) in 100 mL of the same buffer. Sonic 

extracts were centrifuged at 25, 500 g for 30 min at -2∞C. The opalescent yellow supernatant fluid 

had the ability to convert (-)-isodihydrocarvone (101b) to (-)-dihydrocarvone (101a). On the other 

hand, the broken cell preparation was incapable of converting (-)-isodihydrocarvone (101b) to 

(-)-dihydrocarvone (101a). The enzyme was partially purified from this supernatant fluid about 

56-fold with heat treatment (95–97∞C for 10 min), ammonium sulfate precipitation (0.4–0.7 saturation), 

and DEAE-Sephadex A-50 column chromatography. 

The reaction mixture consisted of a mixture of (-)-isodihydrocarvone (101b) and (-)-dihydrocarvone 

(101a) (60:40 or 90:10), 1/30 M KH 2 PO 4 –Na 2 HPO 4 buffer (pH 7.2), and the crude or partially 

purified enzyme solution. The reaction was started by the addition of the enzyme solution and stopped 

by the addition of ether. The ether extract was applied to analytical GLC (Shimadzu Gas Chromatograph 

GC-4A 10% PEG-20M, 3 m ¥ 3 mm, temperature 140–170∞C at the rate of 1∞C a min, N 2 35 mL/ 

min), and epimerization was assayed by measuring the peak areas of (-)-isodihydro carvone (101b) 

and (-)-dihydrocarvone (101a) in gas liquid chromatography (GLC) before and after the reaction. 

The crude extract and the partially purified preparation were found to be very stable to heat treatment; 

66% and 36% of the epimerase activity remained after treatment at 97∞C for 60 and 120 min, 

respectively (Noma et al., 1975). 

A strain of Aspergillus niger TBUYN-2 hydroxylated at C-1 position of (-)-isodihydrocarvone 

(101b) to give 1a-hydroxyisodihydrocarvone (72b), which was easily and smoothly reduced to 

(1S, 2S, 4S)-(-)-8-p-menthene-1,2-trans-diol (71d), which was also obtained from the biotransformation 

of (-)-cis-limonene-1,2-epoxide (69) by microorganisms and decomposition by 20% HCl 

(Figure 14.127) (Noma et al., 1985a, 1985b). Furthermore, Aspergillus niger TBUYN-2 and 

Aspergillus niger Tiegh (CBAYN) biotransformed (-)-isodihydrocarvone (101b) to give (-)-4ahydroxyisodihydrocarvone 

(378b) and (-)-8-p-menthene-1,2-trans-diol (71d) as the major products 

together with a small amount of 1a-hydroxyisodihydrocarvone (72b) (Noma and Asakawa, 2008) 

(Figure 14.130). 

14.3.4.2.3 Menthone and Isomenthone 

O 

O 

O 

O 

149a 

(1R,4S) 

(–)-Menthone 

149b 

(1S,4S) 

(–)-Isomenthone 

149a' 

(1S,4R) 

(+)-Menthone 

149b' 

(1R,4R) 

(+)-Isomenthone 

The growing cells of Pseudomonas fragi IFO 3458 epimerized 17% of racemic isomenthone 

(149b and b¢) to menthone (149a and a¢) (Noma et al., 1975). (-)-Menthone (149a) was converted


O 

O 

OH 

O 

OH 

OH 

O 

OH 

378b 

101b 

72b 

71d 

69b 

FIGURE 14.130 Biotransformation of (+)-carvone (93), (-)-isodihydrocarvone (101b), and (-)-cis-limonene- 

1,2-epoxide (69b) by Aspergillus niger TBUYN-2 and Aspergillus niger Tiegh (CBAYN). (Modified from 

Noma, Y. et al., 1985a. Annual Meeting of Agricultural and Biological Chemistry, Sapporo, p. 68; Noma, Y. 

and Y. Asakawa, 2008. Proc. 52nd TEAC, pp. 206–208.) 

by Pseudomonas fl uorescens M-2 to (-)-3-oxo-4-isopropyl-1-cyclohexanecarboxylic acid (164a), 

(+)-3-oxo-4-isopropyl-1-cyclohexanecarboxylic acid (164b), and (+)-3-hydroxy-4-isopropyl-1-cyclohexanecarboxylic 

acid (165ab). On the other hand, (+)-menthone (149a¢) was converted to give 

(+)-3-oxo-4-isopropyl-1-cyclohexane carboxylic acid (164a¢) and (-)-3-oxo-4-isopropyl-1- 

cyclohexane carboxylic acid (164b¢). Racemic isomenthone (149b and b¢) was converted to give 

racemic 1-hydroxy-1-methyl-4-isopropylcyclohexane-3-one (150), racemic piperitone (156), racemic 

3-oxo-4-isopropyl-1-cyclohexene-1-carboxylic acid (162), 3-oxo-4-isopropyl-1-cyclohexane 

carboxylic acid (164b), 3-oxo-4-isopropyl-1-cyclohexane carbxylic acid (164a), and (+)-3-hydroxy- 

4-isopropyl-1-cyclohexane carboxylic acid (165ab) (Figure 14.131). 

Soil plant pathogenic fungi, Rhizoctonia solani 189 converted (-)-menthone (149a) to 4b-hydroxy- 

(-)-menthone (392, 29%) and 1 a, 4 b-dihydroxy-(-)-menthone (393, 71%) (Nonoyama et al., 1999) 

(Figure 14.131). (-)-Menthone (149a) was transformed by Spodoptera litura to give 7- hydroxymenthone 

(151a), 7-hydroxyneomenthol (165c), and 7-hydroxy-9-carboxymenthone (394a) (Hagiwara et al., 

2006) (Figure 14.132). (-)-Menthone (149a) gave 7-hydroxymenthone (151a) and (+)-neomenthol 

(137c) by human liver microsome (CYP2B6). Of 11 recombinant human P450 enzymes (express in 

Trichoplusia ni cells) tested, CYP2B6 catalyzed oxidation of (-)-menthone (149a) to 7-hydroxymenthone 

(151a) (Nakanishi and Miyazawa, 2004) (Figure 14.132). 

14.3.4.2.4 Thujone 

O 

O 

4 5 

O 

4 5 

1 

1 

28a' 

28b 

28b' 

O 

4 5 

O 

4 5 

O 

O 

1 

1 

28a 

1S,4S,5R 

(+)-3- 

28a' 

1R,4R,5S 

(–)-3- 

28b 

1S,4R,5R 

(–)-3-iso 

28b' 

1R,4S,5S 

(+)-3-iso 

b-Pinene (1) is metabolized to 3-thujone (28) via a-pinene (4) (Gibbon and Pirt, 1971). a-Pinene (4) 

is metabolized to give thujone (28). Thujone (28) was biotransformed to thujoyl alcohol (29) by 

Aspergillus niger TBUYN-2 (Noma, 2000). Furthermore, (-)-3-isothujone (28b) prepared from 

Armois oil was biotransformed by plant pathogenic fungus, Botrytis allii IFO 9430 to give 

4- hydroxythujone (30) and 4,6-dihydroxythujone (31) (Miyazawa et al., 1992a) (Figure 14.133).


COOH 

COOH 

OH 

O 

O 

165ab 

COOH 

149a 

164a 

O 

CH 2 OH 

164b 

COOH 

COOH 

O 

O 

O 

O 

149a' 

151 164a' 

164b' 

HO 

COOH 

O 

O 

O 

O 

150 

156 

162 

149a & a' 

COOH 

COOH 

COOH 

O 

O 

OH 

164b 

164a 

165 

FIGURE 14.131 Biotransformation of (-)- (149a) and (+)-menthone (149a¢) and racemic isomenthone (149b 

and 149b¢) by Pseudomonas fl uorescens M-2. (Modified from Sawamura, Y. et al., 1974. Proc. 18th TEAC, 

pp. 27–29.) 

14.3.4.3 Cyclic Monoterpene Epoxide 

14.3.4.3.1 1,8-Cineole 

1,8-Cineole (122) is a main component of the essential oil of Eucalyptus adiata var. australiana leaves, 

comprising ca. 75% in the oil, which corresponds to 31 mg/g fr.wt. leaves (Nishimura et al., 1980). 

The most effective utilization of 122 is very important in terms of renewable biomass production. 

It would be of interest, for example, to produce more valuable substances, such as plant growth regulators, 

by the microbial transformation of 122. The first reported utilization of 122 was presented by 

MacRae et al. (1979), who showed that it was a carbon source for Pseudomonas fl ava growing on 

Eucalyptus leaves. Growth of the bacterium in a mineral salt medium containing 122 resulted in the 

oxidation at the C-2 position of 122 to give the metabolites (1S,4R,6S)-(+)-2a-hydroxy-1,8-cineole 

(225a), (1S,4R,6R)-(-)-2b-hydroxy-1,8-cineole (125a), (1S,4R)-(+)-2-oxo-1,8-cineole (126), and 

(-)-(R)-5,5-dimethyl-4-(3¢-oxobutyl)-4,5-dihydrofuran-2(3H)-one (128) (Figure 14.134).


OH 

R. solani 

+ 

O 

OH 

O 

OH 

392 393 

OH 

OH 

O 

S. litura 

O 

+ + 

OH 

O 

149a 

151a 165c 

394a COOH 

OH 

CYP2B6 

O 

+ 

OH 

151a 

FIGURE 14.132 Metabolic pathway of (-)-menthone (149a) by Rhizoctonia solani 189, Spodptera litura and 

human liver microsome (CYP2B6). (Modified from Nonoyama, H. et al., 1999. Proc. 43rd TEAC, pp. 387– 

388; Nakanishi, K. and M. Miyazawa, 2004. Proc. 48th TEAC, pp. 401–402; Hagiwara, Y. et al., 2006. Proc. 

50th TEAC, pp. 279–280.) 

137c 

O 

A.n. 

OH 

28 

O 

B. allii 

29 

OH 

O 

+ 

HO 

OH 

O 

28b 

30b 

FIGURE 14.133 Biotransformation of (-)-3-isothujone (28b) by Aspergillus niger TBUYN-2 and plant 

pathogenic fungus, Botrytis allii IFO 9430. (Modified from Gibbon, G.H. and S.J. Pirt, 1971. FEBS Lett., 18: 

103–105; Miyazawa, M. et al., 1992a. Proc. 36th TEAC, pp. 197–198.) 

31b 

P. flava 

HO 

HO 

+ 

+ 

O 

+ 

O 

O 

O 

O 

122 125a 125b 

126 

O 

O 

O 

128 

H 

FIGURE 14.134 Biotransformation of 1,8-cineole (84) by Pseudomonas fl ava. (Modified from MacRae, I.C. 

et al., 1979. Aust. J. Chem., 32: 917–922.)


HO 

S. bottropensis 

O 

+ 

HO 

O 

125b Major 

123b Minor 

122 

O 

S. ikutamanensis 

HO 

O 

+ 

HO 

O 

123b 

46% 

123a 

29% 

FIGURE 14.135 Biotransformation of 1,8-cineole (122) by Streptomyces bottropensis SY-2-1 and 

Streptomyces ikutamanensis Ya-2-1. (Modified from Noma, Y. and H. Nishimura, 1980. Annual Meeting of 

Agricultural and Biological Chemical Society, Book of abstracts, p. 28; Noma, Y. and H. Nishimura, 1981. 

Annual Meeting of Agricultural and Biological Chemical Society, Book of abstracts, p. 196.) 

Streptomyces bottropensis, SY-2-1 biotransformed 1,8-cineole (122) stereochemically to 

(+)-2a-hydroxy-1,8-cineole (125b) as the major product and (+)-3a-hydroxy-1,8-cineole (123b) as 

the minor product. Recovery ratio of 1,8-cineole metabolites as ether extract was ca. 30% in 

Streptomyces bottropensis, SY-2-1 (Noma and Nishimura, 1980, 1981) (Figure 14.135). 

In case of Streptomyces ikutamanensis, Ya-2-1 1,8-cineole (122) was biotransformed regioselectively 

to give (+)-3a-hydroxy-1,8-cineole (123b, 46%) and (+)-3b-hydroxy-1,8-cineole (123b, 29%) 

as the major product. Recovery ratio as ether extract was ca. 8.5% in Streptomyces ikutamanensis, 

Ya-2-1 (Noma and Nishimura, 1980, 1981) (Figure 14.135). 

When (+)-3a-hydroxy-1,8-cineole (123b) was used as substrate in the cultured medium of 

Streptomyces ikutamanensis, Ya-2-1, (+)-3b-hydroxy-1,8-cineole (123a, 32%) was formed as the major 

product together with a small amount of (+)-3-oxo-1,8-cineole (126a, 1.6%). When (+)-3b-hydroxy-1,8- 

cineole (123a) was used, (+)-3-oxo-1,8-cineole (126a, 9.6%) and (+)-3a-hydroxy-1,8-cineole (123b, 

2%) were formed. When (+)-3-oxo-1,8-cineole (126a) was used, (+)-3a-hydroxy- (123b, 19%) and 

(+)-3b-hydroxy-1,8-cineole (123a, 16%) were formed. 

Based on the above results, it is obvious that (+)-3b-hydroxy-1,8-cineole (123b) is formed mainly 

in the biotransformation of 1,8-cineole (122), (+)-3a-hydroxy-1,8-cineole (123b), and (+)-3-oxo-1,8- 

cineole (126a) by Streptomyces ikutamanensis, Ya-2-1. The production of (+)-3b-hydroxy-1,8-cineole 

(123b) is interesting, because it is a precursor of mosquito repellent, p-menthane-3,8-diol 

(142aa¢) (Noma and Nishimura, 1981) (Figure 14.136). 

When Aspergillus niger TBUYN-2 was cultured in the presence of 1,8-cineole (122) for 7 days, 

it was transformed to three alcohols [racemic 2a-hydroxy-1,8-cineoles (125b and b¢), racemic 

3a-hydroxy- (123b and b¢), and racemic 3b-hydroxy-1,8-cineoles (123a and 123a¢)] and two ketones 

[racemic 2-oxo- (126 and 126¢) and racemic 3-oxo-1,8-cineoles (124 and 124¢)] (Figure 14.135). The 

formation of 3a-hydroxy- (123b and b¢) and 3b-hydroxy-1,8-cineoles (123a and 123a¢) is of great 

interest not only due to the possibility of the formation of p-menthane-3,8-diol (142 and 142¢), the 

mosquito repellents and plant growth regulators that are synthesized chemically from 3a-hydroxy- 

(123b and b¢) and 3b-hydroxy-1,8-cineoles (123a and 123a¢), respectively, but also from the viewpoint 

of the utilization of Eucalyptus adiata var. australiana leaves oil as biomass. An Et 2 O extract 

of the culture broth (products and 122 as substrate) was recovered in 57% of substrate (w/w) 

(Nishimura et al., 1982; Noma et al., 1996) (Figure 14.137).


HO 

O 

122 

O 

46% 

29% 

123b 

O 

126a 

O 

HO 

O 

123a 

HO 

HO 

142aa' 

FIGURE 14.136 Biotransformation of 1,8-cineole (122), (+)-3a-hydroxy-1,8-cineole (123b), (+)-3b-hydroxy- 

1,8-cineole(123a), and (+)-3-oxo-1,8-cineole (126a) by Streptomyces ikutamanensis, Ya-2-1. (Modified from 

Noma, Y. and H. Nishimura, 1981. Annual Meeting of Agricultural and Biological Chemical Society, Book 

of abstracts, p. 196.) 

HO 

OH 

O 

O 

O 

O 

126 

O 

HO 

125a 

125a' 

OH 

126' 

O 

O 

125b 

122 

O 

125b' 

O 

HO 

O 

OH 

O 

123a 

123a' 

O 

O 

124 

HO 

O 

OH 

124' 

123b 

123b' 

FIGURE 14.137 Biotransformation of 1,8-cineole (122) by Aspergillus niger TBUYN-2. (Modified from 

Nishimura, H. et al., 1982. Agric. Biol. Chem., 46: 2601–2604; Noma, Y. et al., 1996. Proc. 40th TEAC, 

pp. 89–91.)


HO 

B. dothidea 

O 

+ 

O 

OH 

+ 

HO 

O 

125b 

123b' 

123b 

OH 

122 

O 

S. litura 

HO 

S. typhimurium 

HO 

125b 

123a 

O 

O 

+ 

+ 

HO 

HO 

125a 

O 

O 

+ + + 

HO 

O O 

O 

HO 

123b 395 127b 

FIGURE 14.138 Biotransformation of 1,8-cineole (122) by Botryosphaeria dothidea, Spodptera litura, and 

Salmonella typhimurium. (Modified from Noma, Y. et al., 1996. Proc. 40th TEAC, pp. 89–91; Saito, H. and 

M. Miyazawa, 2006. Proc. 50th TEAC, pp. 275–276; Hagiwara, Y. and M. Miyazawa, 2007. Proc. 51st TEAC, 

pp. 304–305.) 

125a 

Plant pathogenic fungus Botryosphaeria dothidea converted 1,8-cineole (122) to optical pure 

(+)-2a-hydroxy-1,8-cineole (125b) and racemic 3a-hydroxy-1,8-cineole (123b and b¢), which were 

oxidized to optically active 2-oxo- (126) (100% ee) and racemic 3-oxo-1,8-cineole (124 and 124¢), 

respectively (Table 14.14). Cytochrome P-450 inhibitor, 1-aminobenzotriazole, inhibited the hydroxylation 

of the substrate (Noma et al., 1996) (Figure 14.138). Spodptera litura also converted 1,8- 

cineole (122) to give three secondary alcohols (123b, 125a, and b) and two primary alcohols (395 

and 127) (Hagihara and Miyazawa, 2007). Salmonella typhimurium OY1001/3A4 and NADPH- 

P450 reductase hydroxylated 1,8-cineole (122) to 2b-hydroxy-1,8-cineole (125a, [a] D + 9.3, 65.3% 

ee) and 3b-hydroxy-1, 8-cineole (123a, [a] D -27.8, 24.7% ee) (Saito and Miyazawa, 2006). 

Extraction of the urinary metabolites from brushtail possums (Trichosurus vulpecula) maintained 

on a diet of fruit impregnated with 1,8-cineole (122) yielded p-cresol (129) and the novel C-9 

oxidated products 9-hydroxy-1,8-cineole (127a) and 1,8-cineole-9-oic acid (462a) (Flynn and 

Southwell, 1979; Southwell and Flynn, 1980) (Figure 14.139). 

1,8-Cineole (122) gave 2b-hydroxy-1,8-cineole (125a) by CYP-450 human and rat liver 

microsome. Cytochrome P450 molecular species responsible for metabolism of 1,8-cineole (122) 

was determined to be CYP3A4 and CYP3A1/2 in human and rat, respectively. Kinetic analysis 

showed that K m and V max values for the oxidation of 1,8-cineole (122) by human and rat treated with 

T. vulpecula 

+ + 

O 

122 127a 

O 

OH 

O 

COOH 

462a 

OH 

129 

FIGURE 14.139 Metabolism of 1,8-cineole in Trichosurus vulpecula. (Modified from Southwell, I.A. and 

T.M. Flynn, 1980. Xenobiotica, 10: 17–23.)


O 

OH 

122 

68 

68 

A. niger 

A. niger 

B. dothidea 

P. flava 

G. cyanea 

P. digitatum 

O 

O 

HO 

OH 

G. cingulata 

HO 

+ 

O 

OH 

O 

O 

O 

O 

hydrolysis 

125b 

125b' 

125b 

130b' 

125b' 

(1S, 2S, 4R) 

[α] D + 29.6 

FIGURE 14.140 Formation of 2a-hydroxy-1,8-cineoles (125b and b¢) from 1,8-cineole (122) and optical 

resolution by Glomerella cingulata and Aspergillus niger TBUYN-2 and 125b¢ from (+)-limonene (68) 

by Pencillium digitatum. (Modified from Nishimura, H. et al., 1982. Agric. Biol. Chem., 46: 2601–2604; 

Abraham, W.-R. et al., 1986. Appl. Microbiol. Biotechnol., 24: 24–30; Miyazawa, M. et al., 1995b. Proc. 39th 

TEAC, pp. 352–353; Noma, Y. et al., 1986. Proc. 30th TEAC, pp. 204–206; Noma, Y. and Y. Asakawa, 2007a. 

Book of Abstracts of the 38th ISEO, p. 7.) 

pregnenolone-16a-carbonitrile (PCN), recombinant CYP3A4 were determined to be 50 mM and 

90.9 nmol/min/nmol P450, 20 mM and 11.5 nmol/min/nmol P450, and 90 mM and 47.6 nmol/min/ 

nmol P450, respectively (Shindo et al., 2000). 

Microbial resolution of racemic 2a-hydroxy-1,8-cineoles (125b and b¢) was carried out by using 

Glomerella cingulata. The mixture of 125b and b¢ was added to a culture of Glomerella cingulata 

and esterified to give after 24 h (1R,2R,4S)-2a-hydroxy-1,8-cineole-2-yl-malonate (130b¢) in 45% 

yield (ee 100%). The recovered alcohol showed 100% ee of the (1S,2S,4R)-enantiomer (125b) 

(Miyazawa et al., 1995b). On the other hand, optically active (+)-2a-hydroxy-1,8-cineole (125b) was 

also formed from (+)-limonene (68) by a strain of Citrus pathogenic fungus Penicillium digitatum 

(Saito and Miyazawa 2006, Noma and Asakawa 2007a) (Figure 14.140). 

Esters of racemic 2a-hydroxy-1,8-cineole (125b and b¢) were prepared by a convenient method 

(Figure 14.141). Their odours were characteristic. Then products were tested against antimicrobial 

activity and their microbial resolution was studied (Hashimoto and Miyazawa, 2001) (Table 14.15). 

1,8-Cineole (122) was glucosylated by Eucalyptus perriniana suspension cells to 2a-hydroxy-1,8- 

cineole monoglucoside (404, 16.0% and 404¢, 16.0%) and diglucosides (405, 1.4%) (Hamada et al., 

2002) (Figure 14.142). 

14.3.4.3.2 1,4-Cineole 

Regarding the biotransformation of 1,4-cineole (131), Streptomyces griseus transformed it to 

8-hydroxy-1,4-cineole (134), whereas Bacillus cereus transformed 1,4-cineole (131) to 2a-hydroxy-1,4- 

cineole (132b, 3.8%) and 2b-hydroxy-1,4-cineoles (132a, 21.3%) (Liu et al., 1988) (Figure 14.144). On 

the other hand, a strain of Aspergillus niger biotransformed 1,4-cineole (131) regiospecifically to 

2a-hydroxy-1,4-cineole (132b) (Miyazawa et al., 1991c) and (+)-3a-hydroxy-1,4-cineole (133b) 

(Miyazawa et al., 1992b) along with the formation of 8-hydroxy-1,4-cineole (134) and 9-hydroxy-1,4- 

cineole (135) (Miyazawa et al., 1992c) (Figure 14.144).


HO 

OH 

MCPBA in dichloromethane 

O 

O 

Acid chloride, Pyridine 

HO 

OH 

34a 

34a' 

125b' 

125b 

O 

O 

R O 

O R R=CH 3 (396) 

O 

125b's Ester 

O 

125b Ester 

C 2 H 5 (397) 

C 3 H 7 (398) 

C 4 H 9 (399) 

CH(CH 3 )2(400) 

C(CH 3 ) 3 (401) 

CH 2 CH(CH 3 ) 2 (402) 

CH 2 C(CH 3 ) 3 (403) 

FIGURE 14.141 Chemical synthesis of esters of racemic 2a-hydroxy-1,8-cineole (125b and b¢). (Modified 

from Hashimoto Y. and M. Miyazawa, 2001. Proc. 45th TEAC, pp. 363–365.) 

Microbial optical resolution of racemic 2a-hydroxy-1,4-cineoles (132b and b¢) was carried out 

by using Glomerella cingulata (Liu et al., 1988). The mixture of 2a-hydroxy-1,4-cineoles (132b and 

b¢) was added to a culture of Glomerella cingulata and esterified to give after 24 h (1R,2R,4S)-2ahydroxy-1,4-cineole-2-yl 

malonate (136¢) in 45% yield (ee 100%). The recovered alcohol showed 

an ee of 100% of the (1S,2S,4R)-enantiomer (132b). On the other hand, optically active (+)-2ahydroxy-1,4-cineole 

(132b) was also formed from (-)-terpinen-4-ol (342) by Gibberella cyanea 

DSM (Abraham et al., 1986) and Aspergillus niger TBUYN-2 (Noma and Asakawa, 2007b) 

(Figure 14.145). 

TABLE 14.14 

Stereoselectivity in the Biotransformation of 1,8-Cineole (122) by Aspergillus 

niger, Botryosphaeria dothidea, and Pseudomonas flava 

Products 

Microorganisms 125a and a′, 125b and b′, 123b and b′, 123a and a′ 

Aspergillus niger TBUYN-2 2:43:49:6 

50:50 41:59 

Botryosphaeria dothidea 4:59:34:3 

100:0 53:47 

Pseudomonas fl ava 29:71:0:0 

100:0 

Source: Noma, Y. et al., 1996. Proc. 40th TEAC, pp. 89–91.


TABLE 14.15 

Yield and Enantiomer Excess of Esters of Racemic 2α-Hydroxy- 

1,8-Cineole (125b and b¢) on the Microbial Resolution by 

Glomerella cingulata 

0 h 24 h 48 h 

Compounds %ee %ee Yield (%) %ee Yield (%) 

396 (−)36.3 (+)85.0 24.0 (+)100 14.1 

397 (−)36.9 (+)73.8 18.6 (+)100 8.6 

398 (−)35.6 (+)33.2 13.7 (+)75.4 3.5 

399 (−)36.8 (+)45.4 14.4 (+)100 2.3 

400 (−)35.4 (−)21.4 25.2 (+)20.6 8.0 

401 (−)36.7 (−)37.8 31.5 (−)40.6 15.2 

402 (−)36.1 (−)29.8 46.8 (−)15.0 24.0 

403 (−)36.3 (−)37.6 72.2 (−)39.0 36.9 

Source: Hashimoto Y. and M. Miyazawa, 2001. Proc. 45th TEAC, pp. 363–365. 

14.4 METABOLIC PATHWAYS OF BICYCLIC MONOTERPENOIDS 

14.4.1 BICYCLIC MONOTERPENE 

14.4.1.1 α-Pinene 

4 4' 

(+)-α-Pinene (–)-α-Pinene 

a-Pinene (4 and 4¢) is the most abundant terpene in nature and obtained industrially by fractional 

distillation of turpentine (Krasnobajew, 1984). (+)-a-Pinene (4) occurs in oil of Pinus palustris 

Mill. at concentrations of up to 65%, and in oil of Pinus caribaea at concentrations of 70% (Bauer 

et al., 1990). On the other hand, Pinus caribaea contains (-)-a-pinene (4¢) at the concentration of 

70–80% (Bauer et al., 1990). 

The biotransformation of (+)-a-pinene (4) was investigated by Aspergillus niger NCIM 612 

(Bhattacharyya et al., 1960, Prema and Bhattacharyya, 1962). A 24 h shake culture of this strain 

metabolized 0.5% (+)-a-pinene (4) in 4–8 h. After the fermentation of the culture broth contained 

(+)-verbenone (24) (2–3%), (+)-cis-verbenol (23b) (20–25%), (+)-trans-sobrerol (43a) (2–3%), and 

Glu-Glu-O 

Glu-O 

O-Glc 

+ 

E. perriniana E. perriniana 

O 

O O O 

405 404 

122 

404' 

FIGURE 14.142 Biotransformation of 1,8-cineole (122) by Eucalyptus perriniana suspension cell. (Modified 

from Hamada, H. et al., 2002. Proc. 46th TEAC, pp. 321–322.)


HO 

O 

O 

OH 

HO 

132a 

(1S, 2R, 4R) 

O 

132a' 

(1R, 2S, 4S) 

OH 

O 

132b 

[α] D +19.6 (1S, 2S, 4R) 

O 

132b' 

(1R, 2R, 4S) [α] D –19.6 

131 

HO 

O 

O 

OH 

133a 

(1S, 3S, 4R) 

133a' 

(1R, 3R, 4S) 

HO 

O 

133b 

(1S, 3R, 4R) 

O 

OH 

133b' 

(1R, 3S, 4S) 

FIGURE 14.143 Metabolic pathways of 1,4-cineole (131) by microorganisms 

HO 

OH 

OH 

B. cereus 

O 

+ 

O 

OH 

O 

406 

A.n. 

131 

O 

S. griseus 

A.n. 

A.n. 

B. cereus 

HO 

132a 

O 

132b 

+ 

132a' 

O 

132b' 

OH 

OH 

134 

O 

A.n. 

O 

O 

135 

OH 

HO 

133b 

+ 

133b' 

OH 

FIGURE 14.144 Metabolic pathways of 1,4-cineole (131) by Aspergillus niger TBUYN-2, Bacillus cereus, 

and Streptomyces griseus. (Modified from Liu, W. et al., 1988. J. Org. Chem., 53: 5700–5704; Miyazawa, M. 

et al., 1991c. Chem. Express, 6: 771–774; Miyazawa, M. et al., 1992b. Chem. Express, 7: 305–308; Miyazawa, 

M. et al., 1992c. Chem. Express, 7: 125–128; Miyazawa, M. et al., 1995b. Proc. 39th TEAC, pp. 352–353.)


O 

OH 

131 

342 

HO 

A. niger 

O 

O 

A. niger 

OH 

G. cingulata 

HO 

A. niger 

G. cyanea 

O 

+ 

O 

O 

O 

O 

OH 

132b 132b' 

rac-132b & b' 

[α] D +0.13 

132b' 

132b 

136' 

(1R, 2R, 4S) 

(1S, 2S, 4R) 

[α] D –19.6 

[α] D +19.6 

FIGURE 14.145 Formation of optically active 2a-hydroxycineole from 1,4-cineole (131) and terpinene- 

4-ol (342) by Aspergillus niger TBUYN-2, Gibberella cyanea, and Glomerella cingulata. (Modified from 

Abraham, W.-R. et al., 1986. Appl. Microbiol. Biotechnol., 24: 24–30; Miyazawa, M. et al., 1991c. Chem. 

Express, 6: 771–774; Miyazawa, M. et al., 1995b. Proc. 39th TEAC, pp. 352–353; Noma, Y. and Y. Asakawa, 

2007b. Proc. 51st TEAC, pp. 299–301.) 

(+)-8-hydroxycarvotanacetone (44) (Bhattacharyya et al., 1960; Prema and Bhattacharyya 1962 

(Figure 14.146). 

The degradation of (+)-a-pinene (4) by a soil Pseudomonas sp. (PL strain) was investigated by 

Hungund et al. (1970). A terminal oxidation pattern was proposed, leading to the formation of 

organic acids through ring cleavage. (+)-a-Pinene (4) was fermented in shake cultures by a soil 

Pseudomonas sp. (PL strain) that is able to grow on (+)-a-pinene (4) as the sole carbon source, and 

borneol (36), myrtenol (5), myrtenic acid (84), and a-phellandric acid (65) (Shukla and Bhattacharyya, 

1968) (Figure 14.147) were obtained. 

The degradation of (+)-a-pinene (4) by Pseudomonas fl uorescens NCIMB11671 was studied and 

a pathway for the microbial breakdown of (+)-a-pinene (4) was proposed as shown in Figure 14.148 

(Best et al., 1987; Best and Davis, 1988). The attack of oxygen is initiated by enzymatic oxygenation 

of the 1,2-double bond to form a-pinene epoxide (38), which then undergoes rapid rearrangement 

to produce a unsaturated aldehyde, occurring as two isomeric forms. The primary product of the 

reaction (Z)-2-methyl-5-isopropylhexa-2,5-dien-1-al (39, isonovalal) can undergo chemical isomerization 

to the E-form (novalal, 40). Isonovalal (39), the native form of the aldehyde, possesses 

citrus, woody, spicy notes, whereas novallal (40) has woody, aldehydic, and cyclone notes. The same 

biotransformation was also carried out by Nocardia sp. strain P18.3 (Griffiths et al., 1987a, b). 

Pseudomonas PL strain and PIN 18 degradated a-pinene (4) by the pathway proposed in 

Figure 14.149 to give two hydrocarbon, limonene (68) and terpinolene (346), and neutral metabolite, 

+ + + 

OH 

O 

4 23b 24 

OH 

FIGURE 14.146 Biotransformation of (+)-a-pinene (4) by Aspergillus niger NCIM 612. (Modified from 

Bhattacharyya, P.K. et al., 1960. Nature, 187: 689–690; Prema, B.R. and P.K. Bhattachayya, 1962. Appl. 

Microbiol., 10: 524–528.) 

OH 

43a 44 

OH 

O


OH 

OH COOH COOH 

+ + + 

4 36 5 7 

FIGURE 14.147 Biotransformation of (+)-a-pinene (4) by Pseudomonas sp. (PL strain). (Modified from 

Shukla, O.P., and P.K. Bhattacharyya, 1968. Indian J. Biochem., 5: 92–101.) 

65 

CHO 

O 

CHO 

FIGURE 14.148 Biotransformation of (+)-a-pinene (4) by Pseudomonas fl uorescens NCIMB 11671. 

(Modified from Best, D.J. et al., 1987. Biotransform., 1: 147–159.) 

borneol (36). A probable pathway has been proposed for the terminal oxidation of b-isopropylpimelic 

acid (248) in the PL strain and PIN 18 (Shukala and Bhattacharyya, 1968). 

Pseudomonas PX 1 biotransformed (+)-a-pinene (4) to give (+)-cis-thujone (29) and (+)-transcarveol 

(81a) as major compounds. Compounds 81a, 171, 173, and 178 have been identified as 

fermentation products (Gibbon and Pirt, 1971; Gibbon et al., 1972) (Figure 14.150). 

Aspergillus niger TBUYN-2 biotransformed (-)-a-pinene (4¢) to give (-)-a-terpineol (34¢) and 

(-)-trans-sobrerol (43a¢) (Noma et al., 2001). The mosquitocidal (+)-(1R,2S,4R)-1-p-menthane-2,8- 

diol (50a¢) was also obtained as a crystal in the biotransformation of (-)-a-pinene (4¢) by Aspergillus 

niger TBUYN-2 (Noma et al., 2001; Noma, 2007) (Figure 14.151). 

(1R)-(+)-a-Pinene (4) and its enantiomer (4¢) were fed to Spodptera litura to give the corresponding 

(+)- and (-)-verbenones (24 and 24¢) and (+)- and (-)-myrtenols (5 and 5¢) (Miyazawa 

et al., 1996c) (Figure 14.152). 

(-)-a-Pinene (4¢) was treated in human liver microsomes CYP 2B6 to afford (-)-trans-verbenol 

(23¢) and (-)-myrtenol (5¢) (Sugie and Miyazawa, 2003) (Figure 14.153). 

In rabbit, (+)-a-pinene (4) was metabolized to (-)-trans-verbenols (23) as the main metabolites 

together with myrtenol (5) and myrtenic acid (7). The purities of (-)-verbenol (23) from (-)- (4¢), 

(+)- (4), and (+/-)-a-pinene (4 and 4¢) was 99%, 67%, and 68%, respectively. This means that the 

biotransformation of (-)-4¢ in rabbit is remarkably efficient in the preparation of (-)-trans-verbenol 

(23a) (Ishida et al., 1981b) (Figure 14.154). 

(-)-a-Pinene (4¢) was biotransformed by the plant pathogenic fungus Botrytis cinerea to afford 

3a-hydroxy-(-)-b-pinene (2a¢, 10%), 8-hydroxy-(-)-a-pinene (434¢, 12%), 4b-hydroxy-(-)-pinene- 

6-one (468¢, 16%), and (-)-verbenone (24¢) (Farooq et al., 2002) (Figure 14.155). 

14.4.1.2 β-Pinene 

4 38 39 40 

1 

(+)-β-Pinene 

1' 

(–)-β-Pinene


OH 

COOH 

4 

5 

7 

+ 

+ 

HO 

COOH 

1 32 

COOH 

35 

36 

(–)-from 1 

(+)-from 4 

COOH 

(–)-from 1 

(+)-from 4 

86 

68 

+ 

33 

61 

OH 

COOH 

COOH 

COOH 

OH 

OH 

? 

OH 

+ 

H – 

346 

83 

COOH 

OH 

COOH 

464 

463 

59 

COOH 

62 65 248 

(–)-from 1 and 4 

FIGURE 14.149 Metabolic pathways of degradation of a- and b-pinene by a soil Pseudomonad (PL strain) 

and Pseudomonas PIN 18. (Modified from Shukla, O.P., and P.K. Bhattacharyya, 1968. Indian J. Biochem., 

5: 92–101.) 

(+)-b-Pinene (1) is found in many essential oils. Optically active and racemic b-pinene are present 

in turpentine oils, although in smaller quantities than (+)-a-pinene (4) (Bauer et al., 1990). 

Shukla et al. (1968) obtained a similarly complex mixture of transformation products from 

(-)-b-pinene (1¢) through degradation by a Pseudomonas sp/(PL strain). On the other hand, 

Bhattacharyya and Ganapathy (1965) indicated that Aspergillus niger NCIM 612 acts differently 

and more specifically on the pinenes by preferably oxidizing (-)-b-pinene (1¢) in the allylic position 

to form the interesting products pinocarveol (2¢) and pinocarvone (3¢), besides myrtenol (5¢) 

(see Figure 14.156). Furthermore, the conversion of (-)-b-pinene (1¢) by Pseudomonas putidaarvilla 

(PL strain) gave borneol (36¢) (Rama Devi and Bhattacharyya, 1978) (Figure 14.156). 

Pseudomonas pseudomallai isolated from local sewage sludge by the enrichment culture technique 

utilized caryophyllene as the sole carbon source (Dhavlikar et al., 1974). Fermentation of 

(-)-b-pinene (1¢) by Pseudomonas pseudomallai in a mineral salt medium (Seubert’s medium) at


O 

4 

29 

OH 

OH 

OH 

O 

81a 

+ 

42 46a 

47 

COOH 

COOH 

COOH 

O 

O 

173 

172 

171 

56 

COOH 

HO 

COOH 

O 

COOH 

COOH 

174 175 176 

178 

FIGURE 14.150 Proposed metabolic pathways for (+)-a-pinene (4) degradation by Pseudomonas PX 1. 

(Modified from Gibbon, G.H. and S.J. Pirt, 1971. FEBS Lett., 18: 103–105; Gibbon, G.H. et al., 1972. Proc. IV 

IFS, Ferment. Technol. Today, pp. 609–612.) 

HO 

HO 

A. niger A. niger A. niger 

OH 

OH 

OH 

4' 

34' 

43a' 

50a' 

FIGURE 14.151 Biotransformation of (-)-a-pinene (4) by Aspergillus niger TBUYN-2. (Modified from 

Noma, Y. et al., 2001. Proc. 45th TEAC, pp. 88–90.) 

30 ∞ C with agitation and aeration for 4 days yielded camphor (37¢), borneol (36a¢), isoborneol (36b¢), 

a-terpineol (34¢), and b-isopropyl pimelic acid (248¢) (see Figure 14.154). Using modified Czapek- 

Dox medium and keeping the other conditions the same, the pattern of the metabolic products was 

dramatically changed. The metabolites were trans-pinocarveol (2¢), myrtenol (5¢), a-fenchol (11¢), 

a-terpineol (34¢), myrtenic acid (7¢), and two unidentified products (see Figure 14.157). 

(-)-b-Pinene (1¢) was converted by plant pathogenic fungi, Botrytis cinerea, to give four new compounds 

such as (-)-pinane-2a,3a-diol (408¢), (-)-6b-hydroxypinene (409¢), (-)-4a,5-dihydroxypinene 

(410¢), and (-)-4a-hydroxypinen-6-one (411¢) (Figure 14.158). 

This study progressed further biotransformation of (-)-pinane-2a,3a-diol (408¢) and related 

compounds by microorganisms as shown in Figure 14.158.


OH 

S. litura 

O 

4 24 5 

+ 

OH 

4' 

S. litura 

O 

FIGURE 14.152 Biotransformation of (+)- (4) and (-)-a-pinene (4¢) by Spodptera litura. (Modified from 

Miyazawa, M. et al., 1996c. Proc. 40th TEAC, pp. 84–85.) 

24' 

+ 

5' 

OH 

4' 

P450 2B6 

HO 

FIGURE 14.153 Biotransformation of (-)-a-pinene (4¢) by human liver microsomes CYP 2B6. (Modified 

from Sugie, A. and M. Miyazawa, 2003. Proc. 47th TEAC, pp. 159–161.) 

23' 

+ 

5' 

OH 

COOH 

Rabbit 

OH 

4 23a 5 

+ 

+ 

7 

Rabbit 

4' 

HO 

23a' 

FIGURE 14.154 Biotransformation of a-pinene by rabbit. (Modified from Ishida, T. et al., 1981b. J. Pharm. 

Sci., 70: 406–415.) 

B. cinerea 

HO 

+ 

+ 

O 

4' 

24' 

2a' 

OH 

434' 

HO 

O 

468' 

FIGURE 14.155 Microbial transformation of (-)-a-pinene (4¢) by Botrytis cinerea. (Modified from Farooq, 

A. et al., 2002. Z. Naturforsch., 57c: 686–690.) 

+


OH 

HO 

A. niger 

NCIM 

612 

O 

+ + 

2' 3' 5' 

1' 

P. pudidaarvilla 

PL strain 

OH 

36' 

FIGURE 14.156 Biotransformation of (-)-b-pinene (1¢) by Aspergillus niger NCIM 612 and Pseudomonas 

putida-arvilla (PL strain). (Modified from Bhattacharyya, P.K. and K. Ganapathy, 1965. Indian J. Biochem., 

2: 137–145; Rama Devi, J. and P.K. Bhattacharyya, 1978. J. Indian Chem. Soc., 55: 1131–1137.) 

COOH 

P.p. 

Seubert 

OH 

OH 

+ + 

O 

+ 

OH 

+ 

COOH 

36b' 36a' 37' 

34' 

248' 

1' 

P.p. 

HO 

Czapek-dox 

2' 

+ 

5' 

OH 

COOH 

+ + 

FIGURE 14.157 Biotransformation of (-)-b-pinene (1¢) by Pseudomonas pseudomallai. (Modified from 

Dhavalikar, R.S. et al., 1974. Dragoco Rep., 3: 47–49.) 

7' 

HO 

11b' 

+ 

34' 

OH 

HO 

OH 

1' 

B.c. 

+ + + 

OH HO 

HO 

408' 409' 410' OH 

411' 

O 

FIGURE 14.158 Biotransformation of (-)-b-pinene (1¢) by Botrytis cinerea. (Modified from Farooq, A. 

et al., 2002. Z. Naturforsch., 57c: 686–690.) 

As shown in Figure 14.159, (+)- (1) and (-)-b-pinenes (1¢) were biotransformed by Aspergillus 

niger TBUYN-2 to give (+)-a-terpineol (34) and (+)-oleuropeyl alcohol (204) and their antipodes 

(34¢ and 204¢), respectively. The hydroxylation process of a-terpineol (34) to oleuropeyl alcohol 

(204) was completely inhibited by 1-aminotriazole as cyt.P-450 inhibitor. 

(-)-b-Pinene (1¢) was at first biotransformed by Aspergillus niger TBUYN-2 to give (+)-transpinocarveol 

(2a¢) (274). (+)-trans-Pinocarveol (2a¢) was further transformed by three pathways: 

firstly, (+)-trans-pinocarveol (2a¢) was metabolized to (+)-pinocarvone (3¢), (-)-3-isopinanone 

(413¢), (+)-2a-hydroxy-3-pinanone (414¢), and (+)-2a,5-dihydroxy-3-pinanone (415¢). Secondly, 

(+)-trans-pinocarveol (2a¢) was metabolized to (+)-6b-hydroxyfenchol (349ba¢) and thirdly 

(+)-trans-pinocarveol (2a¢) was metabolized to (-)-6b,7-dihydroxyfenchol (412ba¢) via epoxide 

and diol as intermediates (Noma and Asakawa, 2005a) (Figure 14.160).


OH 

A.n. 

A.n. 

1 

OH 

34 204 

OH 

OH 

A.n. 

A.n. 

1' 

OH 

OH 

34' 

204' 

FIGURE 14.159 Biotransformation of (+)- (1) and (-)-b-pinene (1¢) by Aspergillus niger TBUYN-2. 

(Modified from Noma, Y. et al., 2001. Proc. 45th TEAC, pp. 88–90.) 

HO 

O 

O 

O 

OH 

O 

OH 

1' 

HO 

2a' 

OH 

H+ 

3' 

O 

OH 

413' 

OH 

OH 

414' 

HO 

OH 

415' 

OH 

OH 

349ba' 

412ba' 

FIGURE 14.160 The metabolism of (-)-b-pinene (1¢) and (+)-trans-pinocarveol (2a¢) by Aspergillus niger 

TBUYN-2. (Modified from Noma, Y. and Y. Asakawa, 2005a. Book of Abstracts of the 36th ISEO, p. 32.) 

(-)-b-Pinene (1¢) was metabolized by Aspergillus niger TBUYN-2 with three pathways as shown 

in Figure 14.154 to give (-)-a-pinene (4¢), (-)-a-terpineol (34’), and (+)-trans-pinocarveol (2a¢). 

(-)-a-Pinene (4¢) is further metabolized by three pathways. At first (-)-a-pinene (4¢) was metabolized 

via (-)-a-pinene epoxide (38¢), trans-sobrerol (43a¢), (-)-8-hydroxycarvotanacetone (44¢), 

(+)-8-hydroxycarvomenthone (45a) to (+)-p-menthane-2,8-diol (50a¢), which was also metabolized 

in (-)-carvone (93¢) metabolism. Secondly, (-)-a-pinene (4¢) is metabolized to myretenol (83¢), 

which is metabolized by rearrangement reaction to give (-)-oleuropeyl alcohol (204¢). (-)-a-Terpineol 

(34¢), which is formed from b-pinene (1¢), was also metabolized to (-)-oleuropeyl alcohol 

(204¢) and (+)-trans-pinocarveol (2a¢), formed from (-)-b-pinene (1¢), was metabolized to pinocarvone 

(3¢), 3-pinanone (413¢), 2a-hydroxy-3-pinanone (414¢), 2a,5-dihydroxy-3-pinanone (415¢), and 

2a,9-dihydroxy-3-pinanone (416¢). Furthermore, (+)-trans-pinocarveol (2a¢) was metabolized by 

rearrangemet reaction to give 6b-hydroxyfenchol (349ba¢) and 6b,7-dihydroxyfenchol (412ba¢) 

(Noma and Asakawa, 2005a) (Figure 14.161). 

(-)-b-Pinene (1¢) was metabolized by Aspergillus niger TBUYN-2 to give (+)-trans-pinocarveol 

(2a¢), which was further metabolized to 6b-hydroxyfenchol (349ba¢) and 6b, 7-dihydroxyfenchol


OH 

o 

HO 

O 

O 

HO 

HO 

83' 

OH 

38' 

HO 

OH 

43a' 

OH 

OH 

44' 

HO 

OH 

OH 

OH 

45a' 50a' 102a' 

OH 

O 

O 

HO 

204' 

OH 

4' 

412ba' 

417' 

418' 

93' 101a' 

HO 

O 

O 

O 

OH 

HO 

OH 

34' 

OH 

1' 

2b' 

3' 

413' 

414' 

OH 

419' 

HO 

HO 

OH 

O 

OH 

OH 

O 

OH 

11b' 

349ba' 

416' 

OH 

415' 

FIGURE 14.161 Biotransformation of (-)-b-pinene (1¢), (-)-a-pinene (4¢), and related compounds by 

Aspergillus niger TBUYN-2. (Modified from Noma, Y. and Y. Asakawa, 2005a. Book of Abstracts of the 36th 

ISEO, p. 32.) 

(412ba¢) by rearrangement reaction (Noma and Asakawa, 2005a) (Figure 14.162). 6b-Hydroxyfenchol 

(349ba¢) was also obtained from (-)-fenchol (11b¢). (-)-Fenchone was hydroxylated by the 

same fungus to give 6b- (13a¢) and 6a-hydroxy-(-)-fenchone (13b¢). There is a close relationship 

between the metabolism of (-)-b-pinene (1¢) and those of (-)-fenchol (11¢) and (-)-fenchone (12¢). 

(-)-b-Pinene (1¢) and (-)-a-pinene (4¢) were isomerized to each other. Both are metabovlized 

via (-)-a-terpineol (34’) to (-)-oleuropeyl alcohol (204¢) and (-)-oleuropeic acid (61¢). (-)-Myrtenol 

(5¢) formed from (-)-a-pinene (1¢) was further metabolized via cation to (-)-oleuropeyl alcohol 

(204¢) and (-)-oleuropeic acid (61¢). (-)-a-Pinene (4¢) is further metabolized by Aspergillus niger 

TBUYN-2 via (-)-a-pinene epoxide (38¢) to trans-sobrerol (43a¢), (-)-8-hydroxycarvotanacetone 

(44¢), (+)-8-hydroxycarvomenthone (45a), and mosquitocidal (+)-p-menthane-2,8-diol (50a¢) 

(Battacharyya et al., 1960; Noma et al., 2001, 2002, 2003) (Figure 14.163). 

The major metabolites of (-)-b-pinene (1¢) were trans-10-pinanol (myrtanol) (8ba¢) (39%) and 

(-)-1-p-menthene-7,8-diol (oleuropeyl alcohol) (204¢) (30%). In addition, (+)-trans-pinocarveol 

(2a¢) (11%) and (-)-a-terpineol (34¢) (5%), verbenol (23a and 23b) and pinocarveol (2a¢) were 

oxidation products of a- (4) and b-pinene (1), respectively, in bark beetle, Dendroctonus frontalis. 

(-)-Cis- (23b¢) and (+)-trans-verbenols (23a¢) have pheromonal activity in Ips paraconfussus and 

Dendroctonus brevicomis, respectively (Ishida et al., 1981b) (Figure 14.164). 

14.4.1.3 (±)-Camphene 

Racemate camphene (437 and 437¢) is a bicyclic monoterpene hydrocarbon found in Liquidamar 

species, Chrysanthemum, Zingiber offi cinale, Rosmarinus offi cinalis, and among other plants. It


HO 

HO 

OH 

OH 

1' 2b' 

412ba' 

HO 

HO 

OH 

O 

OH 

11b' 

349ba' 

13b' 

O 

OH 

O 

13a' 

12' 

FIGURE 14.162 Relationship of the metabolism of (-)-b-pinene (1¢), (+)-fenchol (11¢) and (-)-fenchone 

(12¢) by Aspergillus niger TBUYN-2. (Modified from Noma, Y. and Y. Asakawa, 2005a. Book of Abstracts 

of the 36th ISEO, p. 32.) 

HO 

HO 

+ 

OH 

OH – 

OH 

+ 

+ 

OH 

HO 

HO 

HOH 

OH + 

OH 

H + 

OH 

CHO 

50a' 

OH 

43a' 

OH 

4' 

5' 

OH 

204' 205' 

OH 

O 

O 

OH 

COOH 

1' 34' 

45a' 

OH 

OH 

44' 

61' 

OH 

FIGURE 14.163 Metabolic pathways of (-)-b-pinene (1¢) and related compounds by Aspergillus niger 

TBUYN-2 . (Modified from Bhattacharyya, P.K. et al., 1960. Nature, 187: 689–690; Noma, Y. et al., 2001. 

Proc. 45th TEAC, pp. 88–90; Noma, Y. et al., 2002. Book of Abstracts of the 33rd ISEO, p. 142; Noma, Y. 

et al., 2003. Proc. 47th TEAC, pp. 91–93.) 

was administered into rabbits. Six metabolites, camphene-2,10-glycols (438a, 438b), which were 

the major metabolites, together with 10-hydroxytricyclene (438c), 7-hydroxycamphene (438d), 

6-exo-hydroxycamphene (438e), and 3-hydroxytricyclene (438f) were obtained (Ishida et al., 1979). 

On the basis of the production of the glycols (438a and 438b) in good yield, these alcohols might be 

formed through their epoxides as shown in Figure 14.165. The homoallyl camphene oxidation 

products (438c–f) apparently were formed through the non-classical cation as the intermediate.


HO 

1' 

8ba' 

OH 

HO 

23b' 

HO 

4' 

2a' 

34' 

OH 

204' 

OH 

HO 

23a' 

FIGURE 14.164 Metabolism of b-pinene (1) by bark beetle, Dendroctonus frontalis. (Modified from Ishida, 

T. et al., 1981b. J. Pharm. Sci., 70: 406–415.) 

437 & 437' 

O 

O 

HO 

OH 

438c 

HO 

HO 

OH 

438a 

OH 

438b 

OH 

OH 

438d 

438e 

438f 

FIGURE 14.165 Biotransformation of (±)-camphene (437 and 437¢) by rabbits. (Modified from Ishida, T. 

et al., 1979. J. Pharm. Sci., 68: 928–930.) 

14.4.1.4 3-Carene and Carane 

1 

6 

6 

1 

439 1S,6R 

(+)-3-carene 

439' 1R,6S 

(–)-3-carene 

1 

6 

1 

6 

6 

1 

6 

1 

439b 1S,3S,6R 

(–)-cis-carane 

439a 1S,3S,6R 

(+)-trans-carane 

439a' 1R,3R,6S 

(–)-trans-carane 

439b' 1R,3R,6S 

(+)-cis-carane


6 

4 

1 

OH 

440 OH 441 

1 

6 

439 

1 

1 

1 

1 

6 

6 

6 

6 

HO 

HOOC 

OH 

9 

COOH 

9 

COOH 

COOH 

9 

442 

9 

443 444 445 

FIGURE 14.166 Metabolic pathways of (+)-3-carene (439) by rabbit (Modified from Ishida, T. et al., 1981b. 

J. Pharm. Sci., 70: 406–415). 3-(+)-Carene (439) was converted by Aspergillus niger NC 1M612 to give either 

hydroxylated compounds of 3-carene-2-one or 3-carene-5-one, which was not fully identified. (Modified from 

Noma, Y. et al., 2002. Book of Abstracts of the 33rd ISEO, p. 142) (Figure 14.167). 

(+)-3-Carene (439) was biotransformed by rabbits to give m-mentha-4,6-dien-8-ol (440) (71.6%) as 

the main metabolite together with its aromatized m-cymen-8-ol (441). The position of C-5 in the 

substrate is thought to be more easily hydroxylated than C-2 by enzymatic systems in the rabbit 

liver. In addition to ring opening compound, 3-carene-9-ol (442), 3-carene-9-carboxylic acid (443), 

3-carene-9,10-dicarboxilic acid (445), chamic acid, and 3-caren-10-ol-9-carboxylic acid (444) were 

formed. The formation of such compounds is explained by stereoselective hydroxylation and 

carboxylation of gem-dimethyl group (Ishida et al., 1981b) (Figure 14.166). In case of (-)-cis-carane 

(446), two C-9 and C-10 methyl groups were oxidized to give dicarboxylic acid (447) (Ishida et al., 

1981b) (Figure 14.166). 

3-(+)-Carene (439) was converted by Aspergillus niger NC 1M612 to give either hydroxylated 

compounds of 3-carene-2-one or 3-carene-5-one, which was not fully identified (Noma et al., 2002) 

(Figure 14.167). 

14.4.2 BICYCLIC MONOTERPENE ALDEHYDE 

14.4.2.1 Myrtenal and Myrtanal 

CHO CHO CHO CHO 

COH 

CHO 

6 

(+)- 

Myrtenal 

6 ' 

(–)- 

435b 

(+)-cis- 

Binihiol 

435a 

(–)-trans- 

Myrtanal 

435b' 

(–)-cis- 

435a' 

(+)-trans- 

Euglena gracilis Z biotransformed (-)-myrtenal (6¢) to give (-)-myrtenol (5¢) as the major product 

and (-)-myetenoic acid (7¢) as the minor product. However, further hydrogenation of (-)-myrtenol 

(5¢) to trans- and cis-myrtanol (8a and 8b) did not occur even at a concentration less than ca. 50 mg/L. 

(S)-Trans and (R)-cis-myrtanal (435a¢ and 435b¢) were also transformed to trans- and cis-myrtanol 

(8a¢ and 8b¢) as the major products and (S)-trans- and (R)-cis-myrtanoic acid (436a¢ and 436b¢) as 

the minor products, respectively (Noma et al., 1991a) (Figure 14.168).


1 

6 

O 

1 

6 

OH 

or 

HO 

1 

6 

O 

439 

FIGURE 14.167 Metabolic pathways of (+)-3-Carene (439) by Aspergillus niger NC 1M612. (Modified from 

Noma, Y. et al., 2002. Book of Abstracts of the 33rd ISEO, p. 142.) 

OH 

CHO 

COOH 

COOH CHO OH 

7' 

6' 

5' 

8b' 

OH 

435b' 

CHO 

436b' 

COOH 

OH 

OH 

8a' 

435a' 

436a' 

OH 

OH 

O 

25' 

34' 

204' 

FIGURE 14.168 Biotransformation of (-)-myrtenal (6¢) and (+)-trans- (435a¢) and (-)-cis-myrtanal (435b¢) 

by microorganisms. (Modified from Noma, Y. et al., 1991a. Phytochem., 30: 1147–1151; Noma, Y. and 

Y. Asakawa, 2005b. Proc. 49th TEAC, pp. 78–80; Noma, Y. and Y. Asakawa, 2006b. Book of Abstracts of the 

37th ISEO, p. 144.) 

In case of Aspergillus niger TBUYN-2, Aspergillus sojae, and Aspergillus usami, (-)-myrtenol 

(5¢) was further metabolized to 7-hydroxyverbenone (25¢) as a minor product together with (-)-oleuropeyl 

alcohol (204¢) as a major product (279, 280). (-)-Oleuropeyl alcohol (204¢) is also formed 

from (-)-a-terpineol (34) by Aspergillus niger TBUYN-2 (Noma et al., 2001) (Figure 14.168). 

Rabbits metabolized myrtenal (6¢) to myrtenic acid (7¢) as the major metabolite and myrtanol 

(8a¢ or 8b¢) as the minor metabolite (Ishida et al., 1981b) (Figure 14.168). 

14.4.3 BICYCLIC MONOTERPENE ALCOHOL 

14.4.3.1 Myrtenol 

OH 

OH 

5 

5'


OH 

OH 

OH 

O 

25' 

5' 

OH 

OH 

204' 

34' 

FIGURE 14.169 Biotransformation of (-)-myrtenol (5¢) and (-)-a-terpineol (34¢) by Aspergillus niger 

TBUYN-2. (Modified from Noma, Y. and Y. Asakawa, 2005b. Proc. 49th TEAC, pp. 78–80.) 

(-)-Myrtenol (5¢) was biotransformed mainly to (-)-oleuropeyl alcohol (204¢), which was formed 

from (-)-a-terpineol (34¢) as a major product by Aspergillus niger, TBUYN-2. In case of Aspergillus 

sojae IFO 4389 and Aspergillus usami IFO 4338, (-)-myrtenol (5¢) was metabolized to 7-hydroxyverbenone 

(25¢) as a minor product together with (-)-oleuropeyl alcohol (204¢) as a major product 

(Noma and Asakawa, 2005b) (Figure 14.169). 

14.4.3.2 Myrtanol 

Spodoptera litura converted (-)-trans-myrtanol (8a) and its enantiomer (8a¢) to give the corresponding 

myrtanic acid (436 and 436¢) (Miyazawa et al., 1997b) (Figure 14.170). 

14.4.3.3 Pinocarveol 

OH 

OH 

HO 

HO 

2a 

1S,3R,5S 

(–)-trans 

2b 

1S,3S,5S 

(+)-cis 

2a' 

1R,3S,5R 

(+)-trans 

2b' 

1R,3R,5R 

(–)-cis 

Pinocarveol 

(+)-trans-Pinocarveol (2a¢) was biotransformed by Aspergillus niger TBUYN-2 to the following 

two pathways. Namely, (+)-trans-pinocarveol (2a¢) was metabolized via (+)-pinocarvone (3¢), 

OH 

COOH 

S. litura 

8a 

OH 

436 

COOH 

S. litura 

8a' 

FIGURE 14.170 Biotransformation of (-)-trans-myrtanol (8a) and its enantiomer (8a¢) by Spodptera litura. 

(Modified from Miyazawa, M. et al., 1997b. Proc. 41st TEAC, pp. 389–390.) 

436'


(-)-3-isopinanone (413¢), and (+)-2a-hydroxy-3-pinanone (414¢) to (+)-2a,5-dihydroxy-3-pinanone 

(415¢) (pathway 1). Furthermore, (+)-trans-pinocarveol (2a¢) was metabolized to epoxide followed 

by rearrangement reaction to give 6b-hydroxyfenchol (349ba¢) and 6b,7-dihydroxyfenchol (412ba¢) 

(Noma and Asakawa, 2005a) (Figure 14.171). Spodoptera litura converted (+)-trans-pinocarveol 

(2a¢) to (+)-pinocarvone (3¢) as a major product (Miyazawa et al., 1995c) (Figure 14.171). 

14.4.3.4 Pinane-2,3-diol 

OH 

OH 

OH 

OH 

OH 

OH 

HO 

HO 

418aa 

418ab 

418ab' 

418aa' 

()-Pinane-2,3-diol 

1S,2S,3S,5S 

(+)-Pinane-2,3-diol 

1S,2S,3R,5S 

(–)-Pinane-2,3-diol 

1R,2R,3S,5R 

()-Pinane-2,3-diol 

1R,2R,3R,5R 

This results led us to study the biotransformation of (-)-pinane-2,3-diol (418ab¢) and (+)-pinane-2,3- 

diol (418ab) by Aspergillus niger TBUYN-2. (-)-Pinane-2,3-diol (418ab¢) was easily biotransformed 

to give (-)-pinane-2,3,5-triol (419ab¢) and (+)-2,5-dihydroxy-3-pinanone (415a¢) as the 

major products and (+)-2-hydroxy-3-pinanone (414a¢) as the minor product. 

On the other hand, (+)-pinane-2,3-diol (418ab) was also biotransformed easily to give (+)-pinane- 

2,3,5-triol (419ab) and (-)-2,5-dihydroxy-3-pinanone (415a) as the major products and 

(-)-2-hydroxy-3-pinanone (414a) as the minor product (Noma et al., 2003) (Figure 14.172). Glomerella 

cingulata transformed (-)-pinane-2,3-diol (418ab¢) to a small amount of (+)-2a-hydroxy-3-pinanone 

(414ab¢, 5%) (Kamino and Miyazawa, 2005), whereas (+)-pinane-2,3-diol (418ab) was transformed 

to a small amount of (-)-2a-hydroxy-3-pinanone (414ab, 10%) and (-)-3-acetoxy-2a-pinanol 

(433ab-Ac, 30%) (Kamino et al., 2004) (Figure 14.172). 

OH 

OH 

HO 

O 

O 

O 

O 

A.n. 

S. litula. 

A.n. A.n. A. n. 

2a' 

3' 

412' 

413' 

OH 

414' 

H+ 

OH 

O 

OH 

HO 

OH 

HO 

OH 

A.n. 

A.n. 

349ba' 

412ba' 

FIGURE 14.171 Biotransformation of (+)-trans-pinocarveol (2a¢) by Aspergillus niger TBUYN-2 and 

Spodptera litura. (Modified from Miyazawa, M. et al., 1995c. Proc. 39th TEAC, pp. 360–361; Noma, Y. and 

Y. Asakawa, 2005a. Book of Abstracts of the 36th ISEO, p. 32.)


OH 

OH 

OH 

OAc 

OH 

OH 

A.n. 

OH 

G.c. 

A.n. 

419ab 

OH 

OH 

418ab'-Ac 

418ab 

G.c. 

O 

A.n. 

O 

414a 

OH 

415a 

OH 

OH 

OH 

OH 

HO 

HO 

O 

O 

A.n. A.n. A.n. 

G.c. 

OH 

419ab' 

418ab' 

414a' 

OH 

415a' 

FIGURE 14.172 Biotransformation of (+)-pinane-2,3-diol (418ab¢) and (-)-pinane-2,3-diol (418ab¢) by 

Aspergillus niger TBUYN-2(276)] and Glomerella cingulata. (Modified from Noma, Y. et al., 2003. Proc. 

47th TEAC, pp. 91–93; Kamino, F. et al., 2004. Proc. 48th TEAC, pp. 383–384; Kamino, F. and M. Miyazawa, 

2005. Proc. 49th TEAC, pp. 395–396.) 

14.4.3.5 Isopinocampheol (3-Pinanol) 

OH 

OH 

OH 

OH 

420 ba 

(–)-isopino 

1R,2R,3R,5S 

420bb 

(+)-neoiso 

1R,2R,3S,5S 

420aa 

(–)-neo 

1R,2S,3R,5S 

420ab 

(+)-pinocampherol 

1R,2S,3S,5S 

HO HO HO HO 

420ba' (+)- 

1S,2S,3S,5R 

420bb' (–)- 

1S,2S,3R,5R 

420aa' (+)-neo 

1S,2R,3S,5R 

420ab' 

1S,2R,3R,5R 

14.4.3.5.1 Chemical Structure of (-)-Isopinocampheol (420ba) and (+)-Isopinocampheol 

(420ba¢) 

Biotransformation of isopinocampheol (3-pinanol) with 100 bacterial and fungal strains yielded 

1-, 2-, 4-, 5-, 7-, 8-, and 9-hydroxyisopinocampheol besides three rearranged monoterpenes, one


of them bearing the novel isocarene skeleton. A pronounced enantioselectivity between (-)- 

(420ba) and (+)-isopinocampheol (420ba¢) was observed. The phylogenetic position of the individual 

strains could be seen in their ability to form the products from (+)-isopinocampheol 

(420ba¢). The formation of 1,3-dihydroxypinane (421ba¢) is a domain of bacteria, while 3,5- 

(415ba¢) or 3,6-dihydroxypinane (428baa¢) was mainly formed by fungi, especially those of the 

phylum Zygomycotina. The activity of Basidiomycotina towards oxidation of isopinocampheol 

was rather low. Such informations can be used in a more effective selection of strains for screening 

(Wolf-Rainer, 1994) (Figure 14.173). 

(+)-Isopinocampheol (420ba¢) was metabolized to 4b-hydroxy-(+)-isopino-campheol (424¢), 

2b-hydroxy-(+)-isopinocampherol acetate (425ba¢-Ac), and 2a-methyl,3-(2-methyl-2-hydroxypropyl)-cyclopenta-1b-ol 

(432¢) (Wolf-Rainer, 1994) (Figure 14.174). 

RO 

HO 

HO 

OH 

OH 

HO 

427ba',R:H 

427ba'-Ac,R:Ac 

426ba' 

418ab' 

HO 

OH 

HO 

OH 

HO 

429' 

421ba' 

420ba' 

HO 

OH 

HO 

RO 

O 

430' 

OH 

428baa' 

OH 

415ba',R:H 

415ba'-Ac,R:Ac 

OH 

431b' 

FIGURE 14.173 Metabolic pathways of (+)-isopinocampheol (420ba¢) by microorganisms. (Modified from 

Wolf-Rainer, A., 1994. Naturforsch., 49c: 553–560.) 

OH 

HO 

AcO 

OH 

OH 

432' 

420ba' 

425ba'-Ac 

HO 

HO 

424' 

FIGURE 14.174 Metabolic pathways of (+)-isopinocampheol (420ba¢) by microorganisms. (Modified from 

Wolf-Rainer, A., 1994. Naturforsch., 49c: 553–560.)


HO 

OH 

S. litura 

OH 

HO 

HO 

S. litura 

OH 

HO 

423ba 

OH 

S. litura 

420ba 

420ba' 

423ba' 

418ab 

FIGURE 14.175 Biotransformation of (-)- (420ba) and (+)-isopinocampheol (420ba¢) by Spodptera litura. 

(Modified from Miyazawa, M. et al., 1997c. Phytochemistry, 45: 945–950.) 

(-)-Isopinocampheol (420ba) was converted by Spodoptera litura to give (1R,2S,3R,5S)-pinane- 

2,3-diol (418ba) and (-)-pinane-3,9-diol (423ba), whereas (+)-isopinocampheol (420ba¢) was converted 

to (+)-pinane-3,9-diol (423ba¢) (Miyazawa et al., 1997c) (Figure 14.175). 

(-)-Isopinocampheol (420ba) was biotransformed by Aspergillus niger TBUYN-2 to give 

(+)-(1S,2S,3S,5R)-pinane-3,5-diol (422ba, 6.6%), (-)-(1R,2R,3R,5S)-pinane-1,3-diol (421ba, 11.8%), 

and pinane-2,3-diol (418ba, 6.6%), whereas (+)-isopinocampheol (420ba¢) was biotransformed by 

Aspergillus niger TBUYN-2 to give (+)-(1S,2S,3S,5R)-pinane-3,5-diol (422ba¢, 6.3%) and 

(-)-(1R,2R,3R,5S)-pinane-1,3,-diol (421ba¢, 8.6%) (Noma et al., 2009) (Figure 14.176). On the other 

hand, Glomerella cingulata converted (-)- (420ba) and (+)-isopinocampheol (420ba¢) mainly to 

(1R,2R,3S,4S,5R)-3,4-pinanediol (484ba) and (1S,2S,3S,5R,6R)-3,6-pinanediol (485ba¢), respectively, 

together with (418ba), (422ba), (422ba¢), and (486ba¢) as minor products (Miyazawa et al., 1997c) 

(Figure 14.176). Some similarities exist between the main metabolites with Glomerella cingulata and 

Rhizoctonia solani (Miyazawa et al., 1997c) (Figure 14.176). 

OH 

OH 

OH 

HO 

HO 

OH 

422ba 

G.c. 

A.n. 

418ba 

A.n. 

G.c. 

485ba' 

G.c. 

OH 

A.n. 

G.c. 

OH 

422ba' 

HO 

OH 

A.n. 

OH 

HO HO OH 

A.n. 

421ba 

G.c. 

420ba 

420ba' 

G.c. 

421ba' 

OH 

HO 

484ba 

OH 

486ba' 

FIGURE 14.176 Biotransformation of (-)- (420ba) and (+)-isopinocampheol (420ba¢) by Aspergillus niger 

TBUYN-2 and Glomerella cingulata. (Modified from Miyazawa, M. et al., 1997c. Phytochemistry, 45: 945– 

950; Noma, Y. et al., 2009. unpublished data.) 

OH

E. gracilis 


14.4.3.6 Borneol and Isoborneol 

HO 

HO 

OH 

OH 

36a 

(1R,2S)-(+)- 

borneol 

(36a) 

36b' 

(1R,2R)-(–)- 

isoborneol 

(36b') 

36b' 

(1S,2R)-(–)- 

borneol 

(36a') 

36b 

(1S,2S)-(+)- 

isoborneol 

(36b) 

(-)-Borneol (36a¢) was biotransformed by Pseudomonas pseudomonallei strain H to give (-)-camphor 

(37¢), 6-hydroxycamphor (228¢), and 2,6-diketocamphor (229¢) (Hayashi et al., 1969) (Figure 14.177). 

Euglena gracilis Z. showed enantio- and diastereoselectivity in the biotransformation of (+)- 

(36a), (-)- (36a¢), and (±)-racemic borneols (equal mixture of 36a and 36a¢) and (+)- (36b), 

(-)- (36b¢), and (±)-isoborneols (equal mixture of 36b and 36b¢). The enantio- and diastereoselective 

dehydrogenation for (-)-borneol (36a¢) was carried out to give (-)-camphor (37¢) at ca. 50% 

yield (Noma et al., 1992d; Noma and Asakawa, 1998). The conversion ratio was always ca. 50% 

even at different kinds of concentration of (-)-borneol (36a¢). When (-)-camphor (37¢) was used as 

a substrate, it was also converted to (-)-borneol (36a¢) in 22% yield for 14 days. Furthermore, 

(+)-camphor (37) was also reduced to (+)-borneol (36a) in 4% and 18% yield for 7 and 14 days, 

respectively (Noma et al., 1992d, Noma and Asakawa, 1998) (Figure 14.178). 

O 

HO 

O 

OH 

37' 

P. pseudomonari 

O 

O 

36b' 

HO 

OH 

O 

OH 

229' 

228' 

FIGURE 14.177 Biotransformation of (-)-borneol (36a¢) by Pseudomonas pseudomonallei strain. (Modified 

from Hayashi, T. et al., 1969. J. Agric. Chem. Soc. Jpn., 43: 583–587.) 

HO 

OH 

36b' 

O 

O 

E. gracilis 

36b 

HO 

E. gracilis 

37 (+) 

37' 

(–) 

E. gracilis 

OH 

36a 

36a' 

FIGURE 14.178 Enantio- and diastereoselectivity in the biotransformation of (+)- (36a) and (-)- borneols 

(36a¢) by Euglena gracilis Z. (Modified from Noma, Y. et al., 1992d. Proc. 36th TEAC, pp. 199–201; Noma, Y. 

and Y. Asakawa, 1998. Biotechnology in Agriculture and Forestry, Vol. 41. Medicinal and Aromatic Plants X, 

Y.P.S. Bajaj, ed., pp. 194–237. Berlin Heidelberg: Springer.)


HO 

HO 

OH 

S. litura S. litura 

OH 

HO 

OH 

36a 

370 

36a' 

370' 

FIGURE 14.179 Biotransformation of (+)- (36a) and (-)-borneols (36a¢) by Spodptera litura. (Modified 

from Miyamoto, Y. and M. Miyazawa, 2001. Proc. 45th TEAC, pp. 377–378.) 

(+)- (36a) and (-)-Borneols (36a¢) were biotransformed by Spodoptera litura to (+)- (370a) and 

(-)-bornane-2,8-diols (370a¢), respectively (Miyamoto and Miyazawa, 2001) (Figure 14.179). 

14.4.3.7 Fenchol and Fenchyl Acetate 

10 

HO 1 

2 

6 

8 3 

7 

5 

9 4 

(1R,2R,4S) 

(+)-endo 

(+)-α-fenchol 

11a 

HO 

(1R,2S,4S) 

(+)-exo 

(+)-β-fenchol 

11b 

OH 

(1S,2S,4R) 

(–)-endo 

(–)-α-fenchol 

11a' 

OH 

(1S,2R,4R) 

(–)-exo 

(–)-β-fenchol 

11b' 

(1R,2R,4S)-(+)-Fenchol (11a) was converted by Aspergillus niger TBUYN-2 and Aspergillus 

cellulosae IFO 4040 to give (-)-fenchone (12), (+)-6b-hydroxyfenchol (349ab), (+)-5b-hydroxyfenchol 

(350ab) and 5a-hydroxyfenchol (350aa) (Noma and Asakawa, 2005a) (Figure 14.180). 

HO 

OH 

HO 

OH 

O 

467a 

HO 

HO 

465a 

HO 

12 

S. litura 

349ab 

A.n. 10 

A.n. 

HO 

1 

S. litura 

HO 

S. litura 

6 

2 

A.n. 

8 

7 

3 

5 

9 4 

11a 

A.n. 

350ab 

S. litura 

HO 

OH 

HO 

466a 

350aa 

OH 

FIGURE 14.180 Biotransformation of (+)-fenchol (11a) by Aspergillus niger TBUYN-2, Aspergillus 

cellulosae IFO 4040, and the larvae of common cutworm, Spodptera litura. (Modified from Miyazawa, M. 

and Y. Miyamoto, 2004. Tetrahadron, 60: 3091–3096; Noma, Y. and Y. Asakawa, 2005a. Book of Abstracts 

of the 36th ISEO, p. 32.)


The larvae of common cutworm, Spodoptera litura, converted (+)-fenchol (11a) to (+)-10-hydroxyfenchol 

(467a), (+)-8-hydroxyfenchol (465a), (+)-6b-hydroxyfenchol (349ab), and (-)-9-hydroxyfenchol 

(466a) (Miyazawa and Miyamoto, 2004) (Figure 14.180). 

(+)-trans-Pinocarveol (2), which was formed from (-)-b-pinene (1), was metabolized by Aspergillus 

niger TBUYN-2 to 6b-hydroxy- (+)-fenchol (349ab) and 6b,7-dihydroxy-(+)-fenchol (412ba¢). 

(-)-Fenchone (12) was also metabolized to 6a-hydroxy- (13b) and 6b-hydroxy- (-)-fenchone (13a). 

(+)-Fenchol (11) was metabolized to 6b-hydroxy-(+)-fenchol (349ab) by Aspergillus niger TBUYN-2. 

Relationship of the metabolisms of (+)-trans-pinocarveol (2), (-)-fenchone (12), and (+)-fenchol (11) 

by Aspergillus niger TBUYN-2 is shown in Figure 14.181 (Noma and Asakawa 2005a). 

(+)-a-Fencyl acetate (11a-Ac) was metabolized by Glomerella cingulata to give (+)-5-b-hydroxya-fencyl 

acetate (350a-Ac, 50%) as the major metabolite and (+)-fenchol (11a, 20%) as the minor 

metabolite (Miyazato and Miyazawa 1999). On the other hand, (-)-a-fencyl acetate (11a¢-Ac) was 

metabolized to (-)-5-b-hydroxy-a-fencyl acetate (350a¢-Ac, 70%) and (-)-fenchol (11a¢, 10%) as 

the minor metabolite by Glomerella cingulata (Miyazato and Miyazawa, 1999) (Figure 14.182). 

HO 

1 2a 

OH 

HO 

HO 

OH 

HO 

OH 

11a 

349ab 

412ba' 

O 

OH 

O 

O 

OH 

13a 

12 

13b 

FIGURE 14.181 Metabolism of (+)-trans-pinocarveol (2), (-)-fenchone (12), and (+)-fenchol (11) by Aspergillus 

niger TBUYN-2. (Modified from Noma, Y. and Y. Asakawa, 2005a. Book of Abstracts of the 36th ISEO, p. 32.) 

HO 

11a 

20% 

AcO 

8 

9 

10 

1 

2 

6 

3 

7 

5 

4 

11a-Ac 

AcO 

OH 

350a-Ac 

50% 

11a' 

10% 

OH 

11a'-Ac 

FIGURE 14.182 Biotransformation of (+)- (11a-Ac) and (-)-a-fencyl acetate (11a¢-Ac) by Glomerella 

cingulata. (Modified from Miyazato, Y. and M. Miyazawa, 1999. Proc. 43rd TEAC, pp. 213–214.) 

OAc 

HO 

5 

2 

350a'-Ac 

70% 

OAc


14.4.3.8 Verbenol 

HO 

HO 

OH 

OH 

23a' 

(–)-transverbenol 

23b' 

(–+)-cisverbenol 

23a 

(+)-transverbenol 

23b 

(–)-cisverbenol 

(-)-trans-Verbenol (23a¢) was biotransformed by Spodoptera litura to give 10-hydroxyverbenol 

(451a¢). Furthermore, (-)-verbenone (24¢) was also biotransformed in the same manner to give 

10-hydroxyverbenone (25¢) (Yamanaka and Miyazawa, 1999) (Figure 14.183). 

14.4.3.9 Nopol and Nopol Benzyl Ether 

Biotransformation of (-)-nopol (452¢) was carried out at 30∞C for 7 days at the concentration of 

100 mg/200 mL medium by Aspergillusniger TBUYN-2, Aspergillus sojae IFO 4389, and 

Aspergillus usami IFO 4338. (-)-Nopol (452¢) was incubated with Aspergillus niger TBUYN-2 to 

give 7-hydroxymethyl-1-p-menthen-8-ol (453¢). In cases of Aspergillus sojae IFO 4389 and 

Aspergillus usami IFO 4338, (-)-nopol (452¢) was metabolized to 3-oxonopol (454¢) as a minor 

product together with 7-hydroxymethyl-1-p-menthen-8-ol (453¢) as a major product (Noma and 

Asakawa, 2005b; 2006c) (Figure 14.184). 

Biotransformation of (-)-nopol benzyl ether (455¢) was carried out at 30∞C for 8–13 days at the 

concentration of 277 mg/200 mL medium by Aspergillus niger TBUYN-2, Aspergillus sojae IFO 

4389, and Aspergillus usami IFO 4338. (-)-Nopol benzyl ether (455¢) was biotransformed by 

Aspergillus niger TBUYN-2 to give 4-oxonopl-2¢, 4¢-dihydroxy benzyl ether (456¢), and (-)-oxonopol 

(454¢). 7-Hydroxymethyl-1-p-menthen-8-ol benzyl ether (457¢) was not formed at all (Figure 14.185). 

OH 

OH 

S. litura 

S. litura 

HO 

23a' 

(–)-transverbenol 

HO 

451a' 

24' 

(–)-verbenone 

FIGURE 14.183 Metabolism of (-)-trans-verbenol (23a¢) and (-)-verbenone (24¢) by Spodptera litura. 

(Modified from Yamanaka, T. and M. Miyazawa, 1999. Proc. 43rd TEAC, pp. 391–392.) 

O 

O 

25' 

OH 

OH 

OH 

O 

454' 

452' 

OH 

FIGURE 14.184 Biotransformation of (-)-nopol (452¢) by Aspergillus niger, TBUYN-2, Aspergillus sojae 

IFO 4389 and Aspergillus usami IFO 4338. (Modified from Noma, Y. and Y. Asakawa, 2005b. Proc. 49th 

TEAC, pp. 78–80; Noma, Y. and Y. Asakawa, 2006c. Proc. 50th TEAC, pp. 434–436.) 

453'


OH 

O 

O 

OH 

OH 

O 

+ 

O 

455' 

456' 

454' 

FIGURE 14.185 Biotransformation of (-)-Nopol benzyl ether (455¢) by Aspergillus niger TBUYN-2. 

(Modified from Noma, Y. and Y. Asakawa, 2006b. Book of Abstracts of the 37th ISEO, p. 144; Noma, Y. and 

Y. Asakawa, 2006c. Proc. 50th TEAC, pp. 434–436.) 

2' 

O 

O 

4' 

4 

455' 

OH 

457 

OH 

OH 

OH 

O 

O 

O 

OH 

OH 

HO 

HO 

O 

456' 

O 

454' 

FIGURE 14.186 Proposed metabolic pathways of (-)-nopol benzyl ether (455¢) by microorganisms. 

(Modified from Noma, Y. and Y. Asakawa, 2006b. Book of Abstracts of the 37th ISEO, p. 144; Noma, Y. and 

Y. Asakawa, 2006c. Proc. 50th TEAC, pp. 434–436.) 

4-Oxonopol-2’,4’-dihydroxybenzyl ether (456¢) shows strong antioxidative activity (IC 50 30.23 mM). 

Antioxidative activity of 4-oxonopol-2’,4’-dihydroxybenzyl ether (456¢) is the same as that of butyl 

hydroxyl anisol (BHA) (Noma and Asakawa, 2006b,c). 

Citrus pathogenic fungi, Aspergillus niger Tiegh (CBAYN) also transformed (-)-nopol (452¢) to 

(-)-oxonopol (454¢) and 4-oxonopol-2’,4’-dihydroxybenzyl ether (456¢) (Noma and Asakawa, 

2006b,c) (Figure 14.186). 

14.4.4 BICYCLIC MONOTERPENE KETONES 

14.4.4.1 α-, β-Unsaturated Ketone 

14.4.4.1.1 Verbenone 

O 

24 

(+)-verbenone 

O 

24' 

(–)-verbenone


O 

N. tabacum 

verbenone 

reductase O 

24' 

458b' 

FIGURE 14.187 Hydrogenation of (-)-verbenone (24¢) to (-)-isoverbanone (458b¢) by verbenone reductase of 

Nicotiana tabacum. (Modified from Suga, T. and T. Hirata, 1990. Phytochemistry, 29: 2393–2406; Shimoda, K. 

et al., 1996. J. Chem. Soc., Perkin Trans. 1, 355–358; Shimoda, K. et al., 1998. Phytochem., 49: 49–53; Shimoda, K. 

et al., 2002. Bull. Chem. Soc. Jpn., 75: 813–816; Hirata, T. et al., 2000. Chem. Lett., 29: 850–851.) 

OH 

O 

O 

O 

OH 

O 

3' 413b' 414b' 

O 

OH 

415b' 

OH 

OH 

FIGURE 14.188 Biotransformation of (+)-pinocarvone (3¢) by Aspergillusniger TBUYN-2. (Modified from 

Noma, Y. and Y. Asakawa, 2005a. Book of Abstracts of the 36th ISEO, p. 32.) 

(-)-Verbenone (24¢) was hydrogenated by reductase of Nicotiana tabacum to give (-)-isoverbanone 

(458b¢) (Suga and Hirata, 1990; Shimoda et al., 1996, 1998, 2002; Hirata et al., 2000) (Figure 14.187). 

14.4.4.1.2 Pinocarvone 

416b' 

O 

O 

3 

(–)-Pinocarvone 

3' 

(+)-Pinocarvone 

Aspergillus niger TBUYN-2 transformed (+)-pinocarvone (3¢) to give (-)-isopinocamphone (413b¢), 

2a-hydroxy-3-pinanone (414b¢), 2a, 5-dihydroxy-3-pinanone (415b¢) together with small amounts 

of 2a, 10-dihydroxy-3-pinanone (416b¢) (Noma and Asakawa, 2005a) (Figure 14.188). 

14.4.4.2 Saturated Ketone 

14.4.4.2.1 Camphor 

O 

O 

37 

(1R)-(+)-camphor 

(37) 

37' 

(1S)-(–)-camphor 

(37')


(+)- (37) and (-)-Camphor (37¢) are found widely in nature, of which (+)-camphor (37) is more 

abundant. It is the main component of oils obtained from the camphor tree Cinnamomum camphora 

(Bauer et al., 1990). The hydroxylation of (+)-camphor (37) by Pseudomonas putida C 1 was described 

(Abraham et al., 1988). The substrate was hydroxylated exclusively in its 5-exo- (235b) and 

6-exo- (228b) positions. 

Although only limited success was achieved in understanding the catabolic pathways of (+)-camphor 

(37), key roles for methylene group hydroxylation and biological Baeyer–Villiger monooxygenases 

in ring cleavage strategies were established (Trudgill, 1990). A degradation pathway of 

(+)-camphor (37) by Pseudomonas putida ATCC 17453 and Mycobacaterium rhodochorus T 1 was 

proposed (Trudgill, 1990). 

The metabolic pathway of (+)-camphor (37) by microorganisms is shown in Figure 14.189. 

(+)-Camphor (37) is metabolized to 3-hydroxy- (243), 5-hydroxy- (235), 6-hydroxy- (228), and 

9- hydroxycamphor (225) and 1,2- campholide (237). 6-Hydroxycamphor (228) is degradatively 

metabolized to 6-oxocamphor (229) and 4-carboxymethyl-2,3,3-trimethylcyclopentanone (230), 

4-carboxymethyl-3,5,5-trimethyltetrahydro-2-pyrone (231), isohydroxy-camphoric acid (232), 

isoketocamphoric acid (233), and 3,4,4-trimethyl-5-oxo-trans-2-hexenoic acid (234), whereas 1,2- 

campholide (237) is also degradatively metabolized to 6-hydroxy-1,2-campholide (238), 6-oxo-1, 

2-campholide (239), and 5-carboxymethyl-3,4,4-trimethyl-2-cyclopentenone (240), 6-carboxymethyl- 

4,5,5-trimethyl-5,6-dihydro-2-pyrone (241) and 5-carboxymethyl-3,4,4-trimethyl-2-heptene-1,7-dioic 

acid (242). 5-Hydroxycamphor (235) is metabolized to 6-hydroxy-1,2-campholide (238), 5-oxocamphor 

(236), and 6-oxo-1,2-campholide (239). 3-Hydroxycamphor (243) is also metabolized to 

camphorquinone (244) and 2-hydroxyepicamphor (245) (Bradshaw et al., 1959; Conrad et al., 1961, 

1965a, 1965b; Gunsalus et al., 1965; Chapman et al., 1966; Hartline and Gunsalus, 1971; Oritani and 

Yamashita, 1974) (Figure 14.189). 

Human CYP 2A6 converted (+)-camphor (37) and (-)-camphor (37¢) to 6-endo-hydroxycamphor 

(228a) and 5-exo-hydroxycamphor (235b), while rat CYP 2B1 did 5-endo- (235a), 5-exo- (235b) 

and 6-endo-hydroxycamphor (228a) and 8-hydroxycamphor (225) (Gyoubu and Miyazawa 2006) 

(Figure 14.190). 

(+)-Camphor (37) was glycosylated by Eucalyptus perriniana suspension cells to (+)-camphor 

monoglycoside (3 new, 11.7%) (Hamada et al., 2002) (Figure 14.191). 

14.4.4.2.2 Fenchone 

O 

O 

12 (+)- 

12' (–)- 

(+)-Fenchone (12) was incubated with Corynebacterium sp. (Chapman et al., 1965) and Absidia 

orchidis (Pfrunder and Tamm, 1969a) give 6b-hydroxy- (13a) and 5b-hydroxyfenchones (14a) 

(Figure 14.191). On the other hand, Aspergillus niger biotransformed (+)-fenchone (12) to (+)-6a- 

(13b) and (+)-5a-hydroxyfenchones (14b) (Miyazawa et al., 1990a, 1990b) and 5-oxofenchone (15), 

9-formylfenchone (17b), and 9-carboxyfenchone (18b) (Miyazawa et al., 1990a, 1990b) 

(Figure 14.192). 

Furthermore, Aspergillus niger biotransformed (-)-fenchone (12¢) to 5a-hydroxy- (14b¢) and 

6a-hydroxyfenchones (13b¢) (Yamamoto et al., 1984) (Figure 14.193). 

(+)- and (-)-Fenchone (12 and 12¢) were converted to 6b-hydroxy- (13a, 13a¢), 6a-hydroxyfenchone 

(13b, 13b¢), and 10 hydroxyfenchone (4, 4¢) by P-450. Of the 11 recombinant human P450 

enzymes tested, CYP2A6, CYP2B6 catalyzed oxidation of (+)- (12) and (-)-fenchone (12¢) (Gyoubu 

and Miyazawa, 2005) (Figure 14.194).


O 

HO 

HO 

AcO 

225a 

36a&b 

226a&b 

O 

OH 

O 

O 

O 

HO 

225b 

37 

HO 

O 

O 

243 244 245 

O 

O 

o 

O 

OH 

COOH 

235 

OH 

237 

228 

391 

COOH 

O 

O 

o 

O 

O 

236 

O 

238 

OH 

229 

O 

o 

O 

O 

O 

239 

O 

230 

COOH 

231 

COOH 

HO 

COOH 

O 

240 

COOH 

O 

241 

O 

COOH 

O 

COOH 

232 

OH 

COOH 

242 

COOH 

COOH 

O 

COOH 

233 

234 

H 

COOH 

FIGURE 14.189 Metabolic pathways of (+)-camphor (37) by Pseudomonas putida and Corynebacterium diphtheroides. 

(Modified from Bradshaw, W.H. et al., 1959. J. Am. Chem. Soc., 81: 5507; Conrad, H.E. et al., 1961. 

Biochem. Biophys. Res. Commun., 6: 293–297; Conrad, H.E. et al., 1965a. J. Biol. Chem., 240: 495–503; Conrad, 

H.E. et al., 1965b. J. Biol. Chem., 240: 4029–4037; Gunsalus, I.C. et al., 1965. Biochem. Biophys. Res. Commun., 

18: 924–931; Chapman, P.J. et al., 1966. J. Am. Chem. Soc., 88: 618–619; Hartline, R.A. and I.C. Gunsalus, 1971. 

J. Bacteriol., 106: 468–478; Oritani, T. and K. Yamashita, 1974. Agric. Biol. Chem., 38: 1961–1964.)


O O O O OH O 

rat CYP2B1 

+ + + 

OH 

OH 

37 235a 235b 228b 225 

OH 

O 

O 

human CYP2A6 

OH 

37 228b 37' 

O 

human CYP2A6 

FIGURE 14.190 Biotransformation of (+)-camphor (37) by rat P450 enzyme (above) and (+)- (37) and 

(-)-camphor (37¢) by human P450 enzymes. 

HO 

235a' 

O 

O 

O 

E. perriniana 

OH 

E. perriniana 

O 

O-Glc 

+ 

Another camphor glycoside 

37 

228 3new 

FIGURE 14.191 Biotransformation of (+)-camphor (37) by Eucalyptus perriniana suspension cell. 

HO 

6 

13b 

O 

A.n 

HO 

A.n 

5 

14b 

O 

A.n 

O 

5 

15 

O 

HO 

6 

13a 

O 

A. orchidis 

Corynebacterium sp. 

12 

O 

Corynebacterium sp. 

A. orchidis 

HO 

5 

14a 

O 

A.n 

O 

O 

CHO 

A.n 

COOH 

17b 

FIGURE 14.192 Metabolic pathways of (+)-fenchone (12) by Corynebacterium sp., A. orchidis and 

Aspergillus niger TBUYN-2. (Modified from Chapman, P.J. et al., 1965. Biochem. Biophys. Res. Commun., 

20: 104–108; Pfrunder, B. and Ch. Tamm, 1969a. Helv. Chim. Acta., 52: 1643–1654; Miyazawa, M. et al., 

1990a. Chem. Express, 5: 237–240; Miyazawa, M. et al., 1990b. Chem. Express, 5: 407–410.) 

18b


O 

14b' 

OH 

O 

8 

9 

2 

3 

10 

1 

4 

12' 

FIGURE 14.193 Metabolic pathways of (-)-fenchone (12¢) by Aspergillus niger TBUYN-2. (Modified from 

Yamamoto, K. et al., 1984. Proc. 28th TEAC, pp. 168–170.) 

7 

6 

5 

O 

13b' 

OH 

OH 

O 

P450 

HO 

6 

O 

HO 

6 

O 

+ + 

O 

12 

13a 13b 4 

OH 

O 

P450 

O 

OH O 

OH 

+ + 

12' 

13a' 13b' 4' 

FIGURE 14.194 Biotransformation of (+)-fenchone (12) and (-)-fenchone (12¢) by P-450 enzymes. (Modified 

from Gyoubu, K. and M. Miyazawa, 2005. Proc. 49th TEAC, pp. 420–422.) 

14.4.4.2.3 3-Pinanone (Pinocamphone and Isopinocamphone) 

O 

O 

O 

O 

413a 1R2S5S 

(+)-pinocamphone 

413b 1R,2R,5S 

(+)-isopinocamphone 

413a' 1S2R5R 

(–)-pinocamphone 

413b' 1S2S5R 

(–)-isopinocamphone 

(+)- (413) and (-)-Isopinocamphone (413¢) were biotransformed by Aspergillus niger to give (-)- 

(414) and (+)-2-hydroxy-3-pinanone (414¢) as the main products, respectively, which inhibit strongly 

germination of lettuce seeds, and (-)- (415) and (+)-2,5-dihydroxy-3-pinanone (415¢) as the minor 

components, respectively (Noma et al., 2003, 2004) (Figure 14.195). 

14.4.4.2.4 2-Hydroxy-3-Pinanone 

OH 

OH 

OH 

OH 

O 

O 

O 

O 

(1S,2R,5S) 

(–)-2-OH-3- 

pinanone 

414a 

(1S,2R,5S) 

(+)-2-OH-3- 

pinanone 

414b 

(1R,2R,5R) 

(+)-2-OH-3- 

pinanone 

414a' 

(1R,2S,5R) 

(+)-2-OH-3 

-pinanone 

414b'


O 

OH 

O 

OH 

O 

413 414 

OH 

415 

O 

O 

OH 

O 

OH 

413' 

414' 

FIGURE 14.195 Biotransformation of (+)-isopinocamphone (413b) and (-)-isopinocamphone (413b¢) by 

Aspergillus niger TBUYN-2. (Modified from Noma, Y. et al., 2003. Proc. 47th TEAC, pp. 91–93; Noma, Y. 

et al., 2004. Proc. 48th TEAC, pp. 390–392.) 

(-)-2a-Hydroxy-3-pinanone (414) was incubated with Aspergillus niger TBUYN-2 to give (-)-2a, 

5-dihydroxy-3-pinanone (415) predominantly, whereas the fungus converted (+)-2a-hydroxy-3- 

pinanone (414¢) mainly to 2a, 5-dihydroxy-3-pinanone (415¢), 2a,9-dihydroxy-3-pinanone (416¢), 

and (-)-pinane-2a,3a,5-triol (419ba¢) (Noma et al., 2003, 2004) (Figure 14.196). 

Aspergillus niger TBUYN-2 metabolized b-pinene (1), isopinocamphone (414b), 2a-hydroxy-3- 

pinanone (414a), and pinane-2,3-diol (419ab) as shown in Figure 14.197. On the other hand, 

Aspergillus niger TBUYN-2 and Botrytis cinerea metabolized b-pinene (1¢), isopinocamphone 

(414b¢), 2a-hydroxy-3-pinanone (414a¢), and pinane-2,3-diol (419ab¢) as shown in Figure 14.198. 

Relationship of the metabolism of b-pinene (1, 1¢), isopinocamphone (414b, 414b¢), 2a-hydroxy-3- 

pinanone (414a, 414a¢), and pinane-2,3-diol (419ab, 419ab¢) in Aspergillus niger TBUYN-2 and 

Botrytis cinerea is shown in Figures 14.197 and 14.198. 

OH 

415' 

OH 

O 

A. niger 

OH 

O 

414 

OH 

415 

O 

OH 

HO 

A. niger 

OH 

O 

OH 

A. niger 

A. niger 

OH 

415' 

OH 

OH 

419' 

414' 

O 

OH 

416' 

FIGURE 14.196 Biotransformation of (-)- (414) and (+)-2-hydroxy-3-pinanone (414¢) by Aspergillus niger 

TBUYN-2. (Modified from Noma, Y. et al., 2003. Proc. 47th TEAC, pp. 91–93; Noma, Y. et al., 2004. Proc. 

48th TEAC, pp. 390–392.)


OH 

OH 

A.n. 

OH 

OH 

OH 

O 

1 

OH 

A.n. 

418ab 

O 

A.n. 

A.n. 

OH 

415ab 

A.n. 

OH 

O 

A.n. 

OH 

415 

OH 

O 

420ba 

414b 

414a 

HO 

416b 

FIGURE 14.197 Relationship of the metabolism of b-pinene (1), isopinocamphone (414b), 2a-hydroxy-3- 

pinanone (414a), and pinane-2,3-diol (419ab) in Aspergillus niger TBUYN-2. (Modified from Noma, Y. et al., 

2003. Proc. 47th TEAC, pp. 91–93; Noma, Y. et al., 2004. Proc. 48th TEAC, pp. 390–392.) 

HO 

OH 

HO 

OH 

A.n. 

O 

OH 

B.c. 

A.n. 

1' 

A.n. 

418ab' 

A.n. 

OH 

419ab' 

A.n. 

OH 

415a' 

HO 

O 

O 

A.n. 

OH 

A.n. 

O 

OH 

OH 

414a' 

416b' 

420ba' 

414b' 

FIGURE 14.198 Relationship of the metabolism of b-pinene (1¢), isopinocamphone (414b¢), 2a-hydroxy- 

3-pinanone (414a¢), and pinane-2,3-diol (419ab¢) in Aspergillus niger TBUYN-2 and Botrytis cinerea. 

(Modified from Noma, Y. et al., 2003. Proc. 47th TEAC, pp. 91–93; Noma, Y. et al., 2004. Proc. 48th TEAC, 

pp. 390–392.) 

14.4.4.2.4.1 Mosquitocidal and Knock-Down Activity Knock-down and mortality activity 

toward mosquito, Culex quinequefasciatus, was carried out for the metabolites of (+)- (418ab) and 

(-)-pinane-2,3-diols (418ab¢) and (+)- and (-)-2-hydroxy-3-pinanones (414 and 414¢) by Dr. Radhika 

Samarasekera, Industrial Technology Institute, Sri Lanka. (-)-2-Hydroxy-3-pinanone (414¢) showed 

the mosquito knock-down activity and the mosqutocidal activity at the concentration of 1% and 2% 

(Table 14.16). 

14.4.4.2.4.2 Antimicrobial Activity The microorganisms were refreshed in Mueller Hilton 

Broth (Merck) at 35–37∞C, and inoculated on Mueller Hinton Agar (Mast Diagnostics, Merseyside, 

UK) media for preparation of inoculum. Escherichia coli (NRRL B-3008), Pseudomonas aeruginosa 

(ATCC 27853), Enterobacter aerogenes (NRRL 3567), Salmonella typhimurium (NRRL 

B-4420), Staphylococcus epidermidis (ATCC 12228), Methicillin-resistant Staphylococcus aureus 

(MRSA, Clinical isolate, Osmangazi University, Faculty of Medicine, Eskisehir, Turkey), and 

Candida albicans (Clinical Isolate, Osmangazi University, Faculty of Medicine, Eskisehir, Turkey)


TABLE 14.16 

Knock-down and Mortality Activity Toward Mosquito a 

Compounds Knock-Down (%) Mortality (%) 

(+)-2,5-Dihydroxy-3-pinanone (415, 2%) 27 20 

(-)-2,5-Dihydroxy-3-pinanone (415¢, 2%) NT 7 

(+)-2-Hydroxy-3-pinanone (414, 2%) 40 33 

(-)-2-Hydroxy-3-pinanone (414¢, 2%) 100 40 

(-)-2-Hydroxy-3-pinanone (414¢, 1%) 53 7 

(+)-Pinane-2,3,5-triol (419, 2%) NT NT 

(-)-Pinane-2,3,5-triol (419, 2%) 13 NT 

(+)-Pinane-2,3-diol (418, 2%) NT NT 

(-)-Pinane-2,3-diol (418¢, 2%) NT NT 

a 

The results are against Culex quinequefasciatus. 

were used as pathogen test microorganisms. Microdilution broth susceptibility assay (R1, R2) was 

used for the antimicrobial evaluation of the samples. Stock solutions were prepared in DMSO 

(Carlo-Erba). Dilution series were prepared from 2 mg/mL in sterile distilled water in micro-test 

tubes from where they were transferred to 96-well micro-titer plates. Overnight grown bacterial and 

candial suspensions in double strength Mueller–Hilton broth (Merck) was standardized to approximately 

10 8 CFU/mL using McFarland No:0.5 (10 6 CFU/mL for Candida albicans). A volume of 

100 mL of each bacterial suspension was then added to each well. The last row containing only the 

serial dilutions of samples without microorganism was used as negative control. Sterile distilled 

water, medium, and microorganisms served as a positive growth control. After incubation at 37∞C 

for a 24 h the first well without turbidity was determined as the minimal inhibition concentration 

(MIC), chloramphenicol (Sigma), ampicillin (sigma), and ketoconazole (Sigma) were used as standard 

antimicrobial agents (Koneman et al., 1997; Amsterdam, 1997) (Table 14.17). 

TABLE 14.17 

Biological Activity of Pinane-2,3,5-Triol (419 and 419¢), 2,5-Dihydroxy-3-Pinanone 

(415 and 415¢), and 7-Hydroxymethyl-1-p-menthene-8-ol (453¢) Toward MRSA 

MIC (mg/mL) 

Compounds 

Control 

Microorganisms 419 415¢ 415 419¢ 453¢ ST1 ST2 ST3 

Escherichia coli 0.5 0.5 0.25 0.5 0.25 0.007 0.0039 Nt 

Pseudomonas aeruginosa 0.5 0.125 0.125 0.25 0.25 0.002 0.0078 Nt 

Enterobacter aerogenes 0.5 0.5 0.25 0.5 1.00 0.007 0.0019 Nt 

Salmonella typhimurium 0.25 0.125 0.125 0.25 0.25 0.01 0.0019 Nt 

Candida albicans 0.5 0.125 0.125 0.25 1.00 Nt Nt 0.0625 

Staphylococcus epidermidis 0.5 0.5 0.25 0.5 1.00 0.002 0.0009 Nt 

MRSA 0.25 0.125 0.125 0.25 0.125 0.5 0.031 Nt 

Source: Iscan (2005, unpublished data). 

MRSA, methicillin-resistant Staphylococcus aureus; Nt, not tested; ST1, ampicillin-Na (Sigma); ST2, chloramphenicol 

(Sigma); ST3, ketoconazole (sigma).


14.5 SUMMARY 

14.5.1 METABOLIC PATHWAYS OF MONOTERPENOIDS BY MICROORGANISMS 

About 50 years are over since the hydroxylation of a-pinene (4) was reported by Aspergillus niger 

in 1960 (Bhattacharyya et al., 1960). During these years many investigators have studied the biotransformation 

of a number of monoterpenoids by using various kinds of microorganisms. Now we 

summarize the microbiological transformation of monoterpenoids according to the literatures listed 

in the references including the metabolic pathways (Figures 14.199 through 14.206) for the further 

development of the investigation on microbiological transformation of terpenoids. 

Metabolic pathways of b-pinene (1), a-pinene (4), fenchol (11), fenchone (12), thujone (28), 

carvotanacetone (47), and sobrerol (43) are summarized in Figure 14.199. In general, b-pinene (1) 

is metabolized by six pathways. At first, b-pinene (1) is metabolized via a-pinene (4) to many 

metabolites such as myrtenol (5) (Shukla et al., 1968; Shukla and Bhattacharyya, 1968), verbenol 

(23) (Bhattacharyya et al., 1960; Prema and Bhattacharyya, 1962), and thujone (28) (Gibbon and 

Pirt, 1971). Myrtenol (5) is further metabolized to myrtenal (6) and myrtenic acid (7). Verbenol 

(23) is further metabolized to verbenone (24), 7-hydroxyverbenone (25), 7-hydroxyverbanone 

(26), and 7-formyl verbanone (27). Thujone (28) is further metabolized to thujoyl alcohol (29), 

1-hydroxythujone (30), and 1,3-dihydroxythujone (31). Secondly, b-pinene (1) is metabolized to 

pinocarveol (2) and pinocarvone (3) (Ganapathy and Bhattacharyya, unpublished data). 

Pinocarvone (3) is further metabolized to isopinocamphone (413), which is further hydroxylated 

to give 2-hydroxy-3-pinanone (414). Compound 414 is further metabolized to give pinane-2,3- 

diol (419), 2,5- dihydroxy- (415), and 2,9-dihydroxy-3-pinanone (416). Thirdly, b-pinene (1) is 

metabolized to a-fenchol (11) and fenchone (12) (Dhavlikar et al., 1974), which are further 

metabolized to 6-hydroxy- (13) and 5-hydroxyfenchone (14), 5-oxofenchone (15), fenchone-9-al 

(17), fenchone-9-oic acid (18) via 9-hydroxyfenchone (16), 2,3-fencholide (21), and 1,2-fencholide 

(22) (Pfrunder and Tamm, 1969a, 1969b; Yamamoto, et al., 1984; Christensen and Tuthill, 1985; 

Miyazawa et al., 1990a, 1969b). Fenchol (12) is also metabolized to 9-hydroxyfenchol (466) and 

7-hydroxyfenchol (467), 6-hydroxyfenchol (349), and 6,7-dihydroxyfenchol (412). Fourthly, 

b-pinene (1) is metabolized via fenchoquinone (19) to 2-hydroxyfenchone (20) (Pfrunder and 

Tamm 1969b; Gibbon et al., 1972). Fifthly, b-pinene (1) is metabolized to a-terpineol (34) via 

pinyl cation (32) and 1-p-menthene-8-cation (33) (Hosler, 1969; Hayashi et al., 1972; Saeki and 

Hashimoto, 1968, 1971). a-Terpineol (34) is metabolized to 8,9-epoxy-1-p-menthanol (58) via 

diepoxide (57), terpine hydrate (60), and oleuropeic acid (204) (Shukla et al., 1968; Shukla and 

Bhattacharyya, 1968; Hosler 1969; Hungund et al., 1970; Hayashi et al., 1972; Saeki and 

Hashimoto, 1968, 1971). As shown in Figure 14.202, oleuropeic acid (204) is formed from linalool 

(206) and a-terpineol (34) via 204, 205, and 213 as intermediates (Shukla et al., 1968; Shukla 

and Bhattacharyya, 1968; Hungund et al., 1970) and degradatively metabolized to perillic acid 

(82), 2-hydroxy-8-p-menthen-7-oic acid (84), 2-oxo-8-p-menthen-7-oic acid (84), 2-oxo-8-p-menthen-1-oic 

acid (85), and b-isopropyl pimelic acid (86) (Shukla et al., 1968; Shukla and 

Bhattacharyya, 1968; Hungund et al., 1970). Oleuropeic acid (204) is also formed from b-pinene 

(1) via a-terpineol (34) as the intermediate (Noma et al., 2001). Oleuropeic acid (204) is also 

formed from myrtenol (5) by rearrangement reaction by Aspergillus niger TBUYN-2 (Noma and 

Asakawa 2005b). Finally, b-pinene (1) is metabolized to borneol (36) and camphor (37) via two 

cations (32 and 35) and to 1-p-menthene (62) via two cations (33 and 59) (Shukla and Bhattacharyya, 

1968). 1-p-Menthene (62) is metabolized to phellandric acid (65) via phellandrol (63) and 

phellandral (64), which is further degradatively metabolized through 246–251 and 89 to water 

and carbon dioxide as shown in Figure 14.204 (Shukla et al., 1968). Phellandral (64) is easily 

reduced to give phellandrol (63) by Euglena sp. and Dunaliella sp. (Noma et al., 1984, 1986, 

1991a, 1991b; 1992d). Furthermore, 1-p-menthene (62) is metabolized to 1-p-menthen-2-ol (46) 

and p-menthane-1,2-diol (54) as shown in Figure 14.204. Perillic acid (82) is easily formed from 

perillandehyde (78) and perillyl alcohol (74) (Figure 14.19) (Swamy et al., 1965; Dhavalikar and


OH 

OH 

OH 

OH 

OH 

O 

O 

COOH 

OH 

OH 

CHO 

OH 

419 

O 

418 

OH 

O 

OH 

416 

OH 

414 

OH 

415 

OH 

O 

+ 

413 

3 

O 

OH 

7 

CHO 

6 

OH 

OH 

204 

O 

24 

O 

26 27 

25 

OH 

O 

OH 

O 

O 

HO 

OH 

O 

OH 

37 

36 

35 

+ 

2 

5 

23 

OH 

O 

30 

OH 

31 

453 

O 

HO 

HO 

OH 

HO 

O 

O 

OH 

34 

13 

412 

OH 

466 

349 

14 

15 

OH 

OH 

OH 

OH 

OH 

O 

+ 

33 

HO 

HO 

O 

O 

O 

O 

OH 

11 

12 

17 

CHO 

18 

COOH 

32 

OH 

476 

16 

O 

O 

o 

o 

1 

11 

12 

19 

20 

22 

21 

OH 

O 

O 

O 

OH 

O 

4 

67 COOH 

+ 

32 

+ 

33 

OH 

O 

OH 

45 

OH 

OH 

O 

OH 

OH 

OH 

O 

28 

O 

Fig. 201 

COOH 

177 

38 

46 

81 

HO 

COOH 

OH 

O 

OH 

CHO 

COOH 

CHO 

39 40 

176 

175 

174 

FIGURE 14.199 Metabolic pathways of b-pinene (1), a-pinene (4), fenchone (9), thujone (28), and carvotanacetone 

(44) by microorganisms. 

34 

Fig. 201 

& 203 

50 

OH 

42 + 

43 

44 

OH 

93 

29 

47 

68 

O 

OH 

OH 

OH 

334 

Fig. 205 

& 202 

171 

56 

172 

173 

COOH 

O 

O 

O 

COOH 

COOH 

COOH


O 

O 

OH 

121 

120 

OH 

OH OH CHO COOH 

O 

O 

119 

OH 

O 

O 

114 

O 

OH 

O 

116 

OH O 

HO 

54 

115 

OH 

+ 

33 

62 

O 

63 64 65 

OH 

HO 

O 

OH 

66 

378 

68 

OH 

OH 

OH 

O 

O 

118 

112 

OH 

111 

OH 

110 

69 

71 72 

OH 

101 

OH 

O 

OH O 

117 

OH O 

O 

OH 

157 

113 

68 

HO 

HO 

68 

OH 

O 

91 

OH 

92 

HO 

O 

OH 

O 

100 

O 

O 

O 

O 

58 57 

COOH OH 

61 

Fig.205 

OH 

204 

122 

OH 

O 

OH 

OH 

334 

OH 

COOH 

HO 

HO 

COOH 

O 

34 

125 

OH 

O 

76 

O 

OH 

HO 

HO 

77 

OH 

COOH 

90 

75 

OH 

74 

CHO 

OH 

O 

O 

81 

81 

96 

97 

HO 

OH 

OH 

O 

O 

94 

93 

105 

HO 

O 

O 

OH 

OH 

HO 

98 99 

O 

O 

OH 

378 

101 

O 

OH 

106 

OH 

OH 

102 

OH 

81 

OH OH 

Fig.205 

O 

OH 

44 

O 

70 COOH OAc 

COOH 

COOH 

85 

86 87 

84 

COOH 

COOH 

COOH 

82 

88 89 

COOH 

78 

COOH 

83 

OH 

OH 

O 

109 

O OH OH 

HO 

OH 

103 104 50 

45 

FIGURE 14.200 Metabolic pathways of limonene (68), perillyl alcohol (74), carvone (93), isopiperitenone 

(111), and piperitenone (112) by microorganisms.


HO 

COOH 

162 

O 

COOH 

89 

199 

COOH 

O 

COOH 

198 

COOH 

197 

OH 

COOH 

O 

201 

OH 

O 

O 

O 

OH 

OH 

158 159 157 155 

OH 

OH 

OH 

COOH 

200 

OH 

O 

HO 

COOH 

196 

COOH 

O 

161 

169 

OH 

CHO 

O 

170 

160 

OH 

O 

O 

COOH 

185 

COOH 

195 

O 

168 

OH 

OH 

OH 

O 

COOH 

194 

477 

OH 

OH 

156 

O 

167 

O 

OH 

COOH 

O 

O 

CHO 

166 

OH 

OH 

O 

151 

164 163 

OH 

OH 

O 

193 

475 

358 

OH 

OH 

O 

150 

O 

OH 

OH 

O 

CHO 

HO 

O 

OH 

192 

O 

HO 

OH 

191 

O 

HOOC 

154 

OH 

149 

148 

181 

180 

179 

178 

OH 

OH 

O 

O 

O 

O 

HO 

OH 

OH 

471 

146 

O 

144 

143 

OH 

OH 

OH 

OH 

OH 

145 

OH 

OH 

OH 

OH 

137 

OH 

OH 

188 

OH 

OH 

OH 

O 

OH 

138 

142 

HO 

OH 

HO 

OH 

OH 

OH 

O 

O 

OH 

182 184 

OH 

OH 

Fig. 200 

183 185 

189 

OH 

47 

139 

140 

141 

137-Ac 

OH 

COOH 

O 

O 

COOH 

O 

OH OH 

OH 

O 

O 

OH 

OH 

OH 

OAc 

OMe 

51 

52 

147 

COOH 

186 

O 

COOH 

89 

CH 3 COOH 

187 

346 351 

352 

OH 

OH 

OH 

OH 

OH 

OMe 

470 

OH 

OH 

474 

OH 

477 

OH 

472 

COOH 

190 

COOH 

475-Me 

OH 

202 

OMe 

OMe 

473 

478 

OH 

OH 

471=Me 

FIGURE 14.201 Metabolic pathways of menthol (137), menthone (149), p-cymene (178), thymol (179), 

carvacrol methyl ether (201), and carvotanacetone (47) by microorganisms and rabbit.


COOH 

COOH 

COOH 

O 

COOH 

OH 

COOH 

COOH 

CHO 

86 

85 

OH 

84 

CHO 

82 

HOOC 

OH 

OH 

61 

COOH 

OH 

205 

OH 

O 

O 

OH 

O 

+ 

216 

215 

74 

78 

212 213 

HO 

O 

HO 

O O O 

O 

O 

218 

217 

214 209 210 211 

HO HO HO 

+ 

HOOC 

HOOC 

HOOC HOOC 

+ 

224 223 

222 

221 

COOH OH 

220 219 206 207 208 

204 

34 

OH 

OH 

125 

43 

Fig.200 

&2003 

O 

O 

HO 

O 

O 

O 

O 

O HO 

O 

OAc 

234 COOH HOOC COOH HOOC COOH 

233 

232 

COOH 

231 

COOH 

O 

O 

O 

OH 

O O 

O 

HO 

239 

COOH COOH 

COOH 

242 241 

240 

O 

O 

236 

HO 

COOH 

230 

O 

O 

238 

O 

235 

229 

O 

237 

O 

228 

37 

243 

227 

O 

OH 

36 

O 

OH 

OH 

34 

226 

+ 

35 

+ 

33 

+ 

33 

+ 

32 

1 

OH 

O 

245 

O 

244 

O 

OH . H 2 O 

+ 

60 

59 

COOH 

CO 2 89 COOH 

+ + 

H 2 O O O 

COOH 

COOH 

COOH 

COOH 

COOH 

O 

COOH 

OH 

COOH 

CHO 

OH 

46 

251 

250 

249 

248 

247 

246 

65 

64 

63 

62 

HO 

COOH 

HO 

CHO HO 

OH HO 

OH HO 

HO 

257 

256 

255 

253 

252 

66 

54 

HO 

FIGURE 14.202 Metabolic pathway of borneol (36), camphor (37), phellandral (64), linalool (206), and 

p-menthane (252) by microorganisms. 

254 

OH


CH 2 OH 

Fig. 202 

263 

OH 

137 

OAc 

OH 

OH 

269 

CH 2 OAc 

OH 

260 

CH 2 OH 

O 

259 268 

CH 2 OH 

CH 2 OH 

OH 

266 

CH 2 OH 

OH 

265 

294 

267 

OAc 

OH 

68 

Fig. 200 

& 201 

294 

137 

262 

273 

COOH 

CH 2 OH 

O 

206 

OH 

CH 2 COOH 

CO 2 

261 

CHO 

272 

CH 2 OH 

CHO 

258 

COOH 

CH 2 OH 

271 

275 276 

264 

CH 2 OH 

OH 

CH 2 OH 

CHO 

COOH 

COOH 

277 

281 

278 

H 2 O 

O 

CH 2 COOH 

O 

OH 

CH 2 OH 

COOH 

COOH 

279 

COOH 

COOH 

282 

283 

284 

280 

HOOC 

COOH 

CO 2 

+ 

H 2 O 

286 

285 

CH 2 OAc 

O 

CH 2 OH 

CH 2 OH 

270 

O 

HO 

299 274 

OH 

CH 2 OH 

OH 

CH 2 OH 

OH 

292 

OH 

CH 2 OH 

O 

293 

OH 

O 

298 

294 

O 

287 

291 

CH 2 OH 

OH 

300 

CH 2 OH 

CH O 

OH 

266 

CH 2 OH 

288 

COOH 

COOH 

289 

O 

OH 

O 

295 

O 

O 

290 

296 

FIGURE 14.203 Metabolic pathways of citronellal (258), geraniol (271), nerol (272), and citral (275 and 

276) by microorganisms.


OH 

HO 

OH 

HO 

373 

OH 

HO 

371 

HO 

HO 

372 

OAc 

OH 

OH 

HO 

O 

O 

O 

O 

HO 

O 

342 

HO 

O 

49-Ac 

50 

OH 

136 

132 133 

OH 

OH 

O 

O 

OH 

OH 

55 

OH 

OH 

O 

49 

O 

OH 

45 

O 

102 

O 

101 93 

O 

O 

OH 

134 

131 

O 

135 

OH 

OH 

54 

53 

HO 

48 

O 

44 

O 

O 

HO 

O 

O 

O 

O 

O 

130 

OH 

34 

68 

1 4 

+ 

HO 

51 

O 

47 

OH 

126 

OH 

OH 

HO 

O 

125 

HO 

O 

123 

O 

O 

124 

32 

+ 

33 

COOH 

52 

+ 

59 

CHO 

46 

62 54 

OH 

43 

OH 

OH 

OH 

O 

O 

122 

O 

O 

O 

127 

OH 

129 

OH 

Fig.203 

128 

65 

64 

63 

66 

FIGURE 14.204 Metabolic pathways of 1,8-cineole (122), 1,4-cineole (131), phellandrene (62), and 

carvotanacetone (47) by microorganisms.


O 

OH 

OH 

307 308 

OH 

303 

OH 

OH 

305 

302 

OH 

304 

OH 

306 

OH 

OH 

309 

FIGURE 14.205 Metabolic pathways of myrcene (302) and citronellene (309) by rat and microorganisms. 

310 

OH 

OH 

OH 

OH 

HO 

O 

O 

454 

O 

452 

OH 

453 

456 

O 

455 

OH 

OH 

5 

OH 

204 

FIGURE 14.206 Metabolic pathways of nopol (452) and nopol benzyl ether (455) by microorganisms.


Bhattacharyya, 1966; Dhavalikar et al., 1966; Ballal et al., 1967; Kayahara et al., 1973; Shima et al., 

1972). a-Terpineol (34) is also formed from linalool (206). a-Pinene (4) is metabolized by five 

pathways as follows: firstly, a-pinene (4) is metabolized to myrtenol (5), myrtenal (6), and 

myratenoic acid (7) (Shukla et al., 1968; Shukla and Bhattacharyya, 1968; Hungund et al., 1970; 

Ganapathy and Bhattacharyya, unpublished results). Myrtenal (6) is easily reduced to myrtenol 

(5) by Euglena and Dunaliella spp., (Noma et al., 1991a, 1991b; 1992d). Myrtanol (8) is metabolized 

to 3-hydroxy- (9) and 4-hydroxymyrtanol (10) (Miyazawa et al., 1994b). Secondly, a-pinene 

(4) is metabolized to verbenol (23), verbenone (24), 7-hydroxyverbenone (25), and verbanone-7-al 

(27) (Bhattacharyya et al., 1960; Prema and Bhattacharyya, 1962; Miyazawa et al., 1991d). 

Thirdly, a-pinene (4) is metabolized to thujone (28), thujoyl alcohol (29), 1-hydroxy- (30), and 

1,3-dihydroxythujone (31) (Gibbon and Pirt, 1971; Miyazawa et al., 1992a; Noma, 2000). Fourthly, 

a-pinene (4) is metabolized to sobrerol (43) and carvotanacetol (46, 1-p-menthen-2-ol) via 

a-pinene epoxide (38) and two cations (41 and 42). Sobrerol (43) is further metabolized to 

8-hydroxycarvotanacetone (44, carvonhydrate), 8-hydrocarvomenthone (45), and p-menthane-2,8- 

diol (50) (Prema and Bhattacharyya, 1962; Noma, 2007). In the metabolism of sobrerol (43), 

8-hydroxycarvotanacetone (44), and 8-hydroxycarvomenthone (45) by Aspergillus niger TBUYN-2, 

the formation of p-menthane-2,8-diol (50) is very high enantio- and diastereoselective in the 

reduction of 8-hydroxycarvomenthone (Noma, 2007). 8-Hydroxycarvotanacetone (44) is a common 

metabolite from sobrerol (43) and carvotanacetone (47). Namely, carvotanacetone (47) is 

metabolized to carvomenthone (48), carvomenthol (49), 8-hydroxycarvomenthol (50), 5-hydroxycarvotanacetone 

(51), 8-hydroxycarvotanacetone (44), 5-hydroxycarvomenthone (52), and 2,3- 

lactone (56) (Gibbon and Pirt, 1971; Gibbon et al., 1972, Noma et al., 1974a; 1985c; 1988b). 

Carvomenthone (48) is metabolized to 45, 8-hydroxycarvomenthol (50), 1-hydroxycarvomenthone 

(53), and p-menthane-1,2-diol (54) (Noma et al., 1985b, 1988b). Compound 52 is metabolized to 

6-hydroxymenthol (139), which is the common metabolite of menthol (137) (see Figure 14.201). 

Carvomenthol (49) is metabolized to 8-hydroxycarvomenthol (50) and p-menthane-2, 

9-diol (55). Finally, a-pinene (4) to borneol (36) and camphor (37) via 32 and 35 and to phellandrene 

(62) via 32 and two cations (33 and 59) as mentioned in the metabolism of b-pinene (1). 

Carvotanacetone (47) is also metabolized degradatively to 3,4-dimethylvaleric acid (177) via 56 

and 158-163 as shown in Figure 201 (Gibbon and Pirt, 1971; Gibbon et al., 1972). a-Pinene (4) is 

also metabolized to 2-(4-methyl-3-cyclohexenylidene)-propionic acid (67) (Figure 14.199). 

Metabolic pathways of limonene (68), perillyl alcohol (74), carvone (93), isopiperitenone (111), 

and piperitenone (112) are summarized in Figure 14.199. Limonene (68) is metabolized by eight 

pathways. Namely, limonene (68) is converted into a-terpineol (34) (Savithiry et al., 1997), limonene- 

1,2-epoxide (69), 1-p-menthene-9-oic acid (70), perillyl alcohol (74), 1-p-menthene-8,9-diol (79), 

isopiperitenol (110), p-mentha-1,8-diene-4-ol (80, 4-terpineol), and carveol (81) (Dhavalikar and 

Bhattacharyya, 1966; Dhavalikar et al., 1966; Bowen, 1975; Miyazawa et al., 1983; Van der Werf 

et al., 1997; Savithrry et al., 1997; Van der Werf and de Bont, 1998a, 1998b; Noma et al., 1982, 

1992d). Dihdyrocarvone (101), limonene-1,2-diol (71), 1-hydroxy-8-p-menthene-2-one (72), and 

p-mentha-2,8-diene-1-ol (73) are formed from limonene (68) via limonene epoxide (69) as intermediate. 

Limonene (68) is also metabolized via carveol (78), limonene-1,2-diol (71), carvone (93), 

1-p-menthene-6,9-diol (95), 8,9-dihydroxy-1-p-menthene (90), a-terpineol (34), 2a-hydroxy-1,8- 

cineole (125), and p-menthane-1,2,8-triol (334). Bottrospicatol (92) and 5-hydroxycarveol (94) are 

formed from cis-carveol by Streptomyces bottropensis SY-2-1 (Noma et al., 1982; Nishimura et al., 

1983a; Noma and Nishimura, 1992; Noma and Asakawa, 1992). Carveyl acetate and carveyl 

propionate (both are shown as 106) are hydrolyzed enantio- and diastereoselectively to carveol (78) 

(Oritani and Yamashita, 1980; Noma, 2000). Carvone (93) is metabolized through four pathways as 

follows: firstly, carvone (93) is reduced to carveol (81) (Noma, 1980). Secondly, it is epoxidized to 

carvone-8,9-epoxide (96), which is further metabolized to dihydrocarvone-8,9-epoxide (97), dihydrocarveol-8,9-epoxide 

(103), and menthane-2,8,9-triol (104) (Noma, 2000; Noma et al., 1980; Noma 

and Nishimura, 1982). Thirdly, 93 is hydroxylated to 5-hydroxycarvone (98), 5-hydroxydihydrocarvone


(99), and 5-hydroxydihydrocarveol (100) (Noma and Nishimura, 1982). Dihydrocarvone (101) is 

metabolized to 8-p-menthene-1,2-diol (71) via l-hydroxydihydrocarvone (72), 10-hydroxydihydrocarvone 

(106), and dihydrocarveol (102), which is metabolized to 10-hydroxydihydrocarveol (107), 

p-menthane-2,8-diol (50), dihydrocarveol-8,9-epoxide (100), p-menthane-2,8,9-triol (104), and 

dihydrobottrospicatol (105) (Noma et al., 1985a, 1985b). In the biotransformation of (+)-carvone by 

plant pathogenic fungi, Aspergillus niger Tiegh, isodihydrocarvone (101) was metabolized to 

4-hydroxyisodihydrocarvone (378) and 1-hydroxyisodihydrocarvone (72) (Noma and Asakawa, 

2008). 8,9-Epoxydihydrocarveyl acetate (109) is hydrolyzed to 8,9-epoxydihydrocarveol (103). 

Perillyl alcohol (74) is metabolized through three pathways to shisool (75), shisool-8,9-epoxide 

(76), perillyl alcohol-8,9-epoxide (77), perilladehyde (78), perillic acid (82), and 4,9-dihydroxy-1-pmenthen-7-oic 

acid (83). Perillic acid (82) is metabolized degradatively to 84–89 as shown in 

Figure 14.200 (Swamy et al., 1965; Dhavalikar and Bhattacharyya, 1966; Dhavalikar et al., 1966; 

Ballal et al., 1967; Shukla et al., 1968; Shukala and Bhattacharyya, 1968; Hungund et al., 1977; 

Kayahara et al., 1973; Shima et al., 1972). Isopiperitenol (110) is reduced to isopiperitenone (111), 

which is metabolized to 3-hydroxy- (115), 4-hydroxy- (116), 7-hydroxy- (113) and 10-hydroxyisopiperitenone 

(114), and piperitenone (112). Compounds isopiperitenone (111) and piperitenone 

(112) are isomerized to each other (Noma et al., 1992c). Furthermore, piperitenone (112) is metabolized 

to 8-hydroxypiperitone (157), 5-hydroxy- (117) and 7-hydroxypiperitenone (118). Pulegone 

(119) is metabolized to 112, 8-hydroxymenthenone (121), and 8,9-dehydromenthenone (120). 

Metabolic pathways of menthol (137), menthone (149), thymol (179), and carvacryol methyl ether 

(202) are summarized in Figure 14.201. Menthol (137) is generally hydroxylated to give 1-hydroxy- 

(138), 2-hydroxy- (140), 4-hydroxy- (141), 6-hydroxy- (139), 7-hydroxy- (143), 8-hydroxy- (142), and 

9-hydroxymenthol (144) and 1,8-dihydroxy- (146) and 7,8-dihydroxymenthol (148) (Asakawa et al., 

1991; Takahashi et al., 1994; Van der Werf et al., 1997). Racemic menthyl acetate and menthylchloroacetate 

are hydrolyzed asymmetrically by an esterase of microorganisms (Brit Patent, 1970; Moroe 

et al., 1971; Watanabe and Inagaki, 1977a, 1977b). Menthone (149) is reductively metabolized to 137 

and oxidatively metabolized to 3,7-dimethyl-6-hydroxyoctanoic acid (152), 3,7-dimethyl-6- 

oxooctanoic acid (153), 2-methyl-2,5-oxidoheptenoic acid (154), 1-hydroxymenthone (150), piperitone 

(156), 7-hydroxymenthone (151), menthone-7-al (163), menthone-7-oic acid (164), and 

7-carboxylmenthol (165) (Sawamura et al., 1974). Compound 156 is metabolized to menthone-1,2- 

diol (155) (Miyazawa et al., 1991e, 1992d,e). Compound 148 is metabolized to 6-hydroxy- (158), 

8-hydroxy- (157), and 9-hydroxypiperitone (159), piperitone-7-al (160), 7-hydroxypiperitone (161), 

and piperitone-7-oic acid (162) (Lassak et al., 1973). Compound 149 is also formed from menthenone 

(148) by hydrogenation (Mukherjee et al., 1973), which is metabolized to 6-hydroxymenthenone 

(181, 6-hydroxy-4-p-menthen-3-one). 6-hydroxymenthenone (181) is also formed from thymol 

(179) via 6-hydroxythymol (180). 6-Hydroxythymol (180) is degradatively metabolized through 

182–185 to 186, 187, and 89 (Mukherjee et al., 1974). Piperitone oxide (166) is metabolized to 

1-hydroxymenthone (150) and 4-hydroxypiperitone (167) (Lassak et al., 1973; Miyazawa et al., 

1991e). Piperitenone oxide (168) is metabolized to 1-hydroxymenthone (150), 1-hydroxypulegone 

(169), and 2,3-seco-p-menthalacetone-3-en-1-ol (170) (Lassak et al., 1973; Miyazawa et al., 1991e). 

p-Cymene (178) is metabolized to 8-hydroxy- (188) and 9-hydroxy-p-cymene (189), 2- (4-methylphenyl)-propanic 

acid (190), thymol (179), and cumin alcohol (192), which is further converted 

degradatively to p-cumin aldehyde (193), cumic acid (194), cis-2,3-dihydroxy-2,3-dihydro-p-cumic 

acid (195), 2,3-dihydroxy-p-cumic acid (197), 198–200, and 89 as shown in Figure 14.3 (Chamberlain 

and Dagley, 1968; DeFrank and Ribbons, 1977a, 1977b; Hudlicky et al., 1999; Noma, 2000). 

Compound 197 is also metabolized to 4-methyl-2-oxopentanoic acid (201) (DeFrank and 

Ribbons, 1977a). Compound 193 is easily metabolized to 192 and 194 (Noma et al., 1991a, 1992). 

Carvacrol methyl ether (202) is easily metabolized to 7-hydroxycarvacrol methyl ether (203) 

(Noma, 2000). 

Metabolic pathways of borneol (36), camphor (37), phellandral (64), linalool (206), and p-menthane 

(252) are summarized in Figure 14.202. Borneol (36) is formed from b-pinene (1), a-pinene


(4), 34, bornyl acetate (226), and camphene (229) and it is metabolized to 36, 3-hydroxy- (243), 

5-hydroxy- (235), 6-hydroxy- (228), and 9-hydroxycamphor (225), and 1,2-campholide (23). 

Compound 228 is degradatively metabolized to 6-oxocamphor (229) and 230–234, whereas 237 is 

also degradatively metabolized to 6-hydroxy-1,2-campholide (238), 6-oxo-1,2-campholide (239), 

and 240–242. 5-Hydroxycamphor (235) is metabolized to 238, 5-oxocamphor (236), and 6-oxo-1,2- 

campholide (239). Compound 243 is also metabolized to camphorquinone (244) and 2-hydroxyepicamphor 

(245) (Bradshaw et al., 1959; Conrad et al., 1961, 1965a, 1965b; Gunsalus et al., 1965; 

Chapman et al., 1966; Hartline and Gunsalus, 1971; Oritani and Yamashita, 1974). 1-p-Menthene 

(62) is formed 1 and 4 via three cations (32, 33, and 59) and metabolized to phellandrol (63) (Noma 

et al., 1991a) and p-menthane-1,2-diol (54). Compound 63 is metabolized to phellandral (64) and 

7-hydroxy-p-menthane (66). Compound 64 is furthermore metabolized degradatively to CO 2 and 

water via phellandric acid (65), 246–251, and 89 (Dhavalikar and Bhattacharyya, 1966; Dhavalikar 

et al., 1966; Bahhal et al., 1967; Shukla et al., 1968; Shukla and Bhattacharyya, 1968; Hungund 

et al., 1970). Compound 64 is also easily reduced to phellandrol (63) (Noma et al., 1991a, 1992a). 

p-Menthane (252) is metabolized via 1-hydroxy-p-menthane (253) to p-menthane-1,9-diol (254) and 

p-menthane-1,7-diol (255) (Tukamoto et al., 1974, 1975; Noma et al., 1990). Compound 255 is degradatively 

metabolized via 256–248 to CO 2 and water through the degradation pathway of phellandric 

acid (65, 246–251, and 89) as mentioned above. Linalool (206) is metabolized to a-terpineol 

(34), camphor (37), oleuropeic acid (61), 2-methyl-6-hydroxy-6-carboxy-2,7-octadiene (211), 

2-methyl-6-hydroxy-6-carboxy-7-octene (199), 5-methyl-5-vinyltetrahydro-2-furanol (215), 5-methyl- 

5-vinyltetrahydro-2-furanone (216), and malonyl ester (218). 1-Hydroxylinalool (219) is metabolized 

degradatively to 2,6-dimethyl-6-hydroxy-trans-2,7-octadienoic acid (220), 4-methyl-trans-3, 

5-hexadienoic acid (221), 4-methyl-trans-3,5-hexadienoic acid (222), 4-methyl-trans-2-hexenoic 

acid (223), and isobutyric acid (224). Compound 206 is furthermore metabolized via 213 to 61, 82, 

and 84–86 as shown in Figure 14.2 (Mizutani et al., 1971; Murakami et al., 1973; Rama Devi and 

Bhattacharyya, 1977a, 1977b; Rama Devi et al., 1977; Madyastha et al., 1977; David and Veschambre, 

1985; Miyazawa et al., 1994a, 1994b). 

Metabolic pathways of citronellol (258), citronellal (261), geraniol (271), nerol (272), citral [neral 

(275) and geranial (276)], and myrcene (302) are summarized in Figure 14.203 (Seubert and Fass, 

1964; Hayashi et al., 1968; Rama Devi and Bhattacharyya, 1977a, 1977b). Geraniol (271) is formed 

from citronellol (258), nerol (272), linalool (206), and geranyl acetate (270) and metabolized through 

10 pathways. Namely, compound 271 is hydrogenated to give citronellol (258), which is metabolized 

to 2,8-dihydroxy-2,6-dimethyl octane (260) via 6,7-epoxycitronellol (268), isopulegol (267), 

limonene (68), 3,7-dimethyloctane-1,8-diol (266) via 3,7-dimethyl-6-octene-1,8-diol (265), 267, citronellal 

(261), dihydrocitronellol (259), and nerol (272). Citronellyl acetate (269) and isopulegyl 

acetate (301) are hydrolyzed to citronellol (258) and isopulegol (267), respectively. Compound 261 

is metabolized via pulegol (263) and isopulegol (267) to menthol (137). Compound 271 and 272 are 

isomerized to each other. Compound 272 is metabolized to 271, 258, citronellic acid (262), nerol-6- 

,7-epoxide (273), and neral (275). Compound 272 is metabolized to neric acid (277). Compounds 

275 and 276 are isomerized to each other. Compound 276 is completely decomposed to CO 2 and 

water via geranic acid (278), 2,6-dimethyl-8-hydroxy-7-oxo-2-octene (279), 6-methyl-5-heptenoic 

acid (280), 7-methyl-3-oxo-6-octenoic acid (283), 6-methyl-5-heptenoic acid (284), 4-methyl-3-heptenoic 

acid (284), 4-methyl-3-pentenoic acid (285), and 3-methyl-2-butenoic acid (286). Furthermore, 

compound 271 is metabolized via 3-hydroxymethyl-2,6-octadiene-1-ol (287), 3-formyl-2,6-octadiene-1-ol 

(288), and 3-carboxy-2,6-octadiene-1-ol (289) to 3- (4-methyl-3-pentenyl)-3-butenolide 

(290). Geraniol (271) is also metabolized to 3,7-dimethyl-2,3-dihydroxy-6-octen-1-ol (292), 3,7- 

dimethyl-2-oxo-3-hydroxy-6-octen-1-ol (293), 2-methyl-6-oxo-2-heptene (294), 6-methyl-5-hepten- 

2-ol (298), 2-methyl-2-heptene-6-one-1-ol (295), and 2-methyl-g-butyrolactone (296). Furthermore, 

271 is metabolized to 7-methyl-3-oxo-6-octanoic acid (299), 7-hydroxymethyl-3-methyl-2,6-octadien-1-ol 

(291), 6,7-epoxygeraniol (274), 3,7-dimethyl-2,6-octadiene-1,8-diol (300), and 3,7- 

dimethyloctane-1,8-diol (266).


Metabolic pathways of 1,8-cineole (122), 1,4-cineole (131), phellandrene (62), carvotanacetone 

(47), and carvone (93) by micororganisms are summarized in Figure 14.204. 

1,8-Cineole (112) is biotransformed to 2-hydroxy- (125), 3-hydroxy- (123), and 9-hydroxy-1,8- 

cineole (127), 2-oxo- (126) and 3-oxo-1,8-cineole (124), lactone [128, (R)-5,5-dimethyl-4-(3¢oxobutyl)-4,5-dihydrofuran-2-(3H)-one] 

and p-hydroxytoluene (129) (MacRae et al., 1979, 

Nishimura et al., 1982; Noma and Sakai, 1984). 2-Hydroxy-1,8-cineole (125) is further converted 

into 2-oxo-1,8-cineole (126), 1,8-cineole-2-malonyl ester (130), sobrerol (43), and 8-hydroxycarvotanacetone 

(44) (Miyazawa et al., 1995b). 2-Hydroxy-1,8-cineole (125) and 2-oxo-1,8-cineole 

(126) are also biodegradated to sobrerol (43) and 8-hydroxycarvotanacetone (44), respectively. 

2-Hydroxy-1,8-cineole (125) was esterified to give malonyl ester (130). 2-Hydroxy-1,8-cineole (125) 

was formed from limonene (68) by Citrus pathogenic fungi, Penicillium digitatum (Noma and 

Asakawa 2007b). 1,4-Cineole (131) is metabolized to 2-hydroxy- (132), 3-hydroxy- (133), 8-hydroxy- 

(134), and 9-hydroxy-1,4-cineole (135). Compound 132 is also eaterified to malonyl ester 

(136) as well as 125 (Miyazawa et al., 1995b). Terpinen-4-ol (342) is metabolized to 2-hydroxy-1,4- 

cineole (132), 2-hydroxy- (372) and 7-hydroxyterpinene-4-ol (342), and p-mentane-1,2,4-triol (371) 

(Abraham et al., 1986; Noma and Asakawa, 2007a; Kumagae and Miyazawa, 1999). Phellandrene 

(62) is metabolized to carvotanacetol (46) and phellandrol (63). Carvotanacetol (46) is further 

metabolized through the metabolism of carvotanacetone (47). Phelandrol (63) is also metabolized 

to give phellandral (64), phellandric acid (65), and 7-hydroxy-p-menthane (66). Phellandric acid 

(65) is completely degradated to carbon dioxide and water as shown in Figure 14.202. 

Metabolic pathways of myrcene (302) and citronellene (309) by microorganisms and insects are 

summarized in Figure 14.205. b-Myrcene (302) was metabolized with Diplodia gossypina ATCC 

10936 (Abragam et al., 1985) to the diol (303) and a side-product (304). b-Myrcene (302) was 

metabolized with Ganoderma applanatum, Pleurotus fl abellatus, and Pleurotus sajor-caju to 

myrcenol (305) (2-methyl-6-methylene-7-octen-2-ol) and 306 (Busmann and Berger, 1994). 

b-Myrcene (302) was converted by common cutworm larvae, Spodoptera litura, to give 

myrcene-3, (10)-glycol (308) via myrcene-3,(10)-epoxide (307) (Miyazawa et al., 1998). Citronellene 

(309) was metabolized by cutworm Spodoptera litura to give 3,7-dimethyl-6-octene-1,2-diol (310) 

(Takechi and Miyazawa, 2005). Myrcene (302) is metabolized to two kinds of diols (303 and 304), 

myrcenol (305), and ocimene (306) (Seubert and Fass, 1964; Abraham et al., 1985). Citronellene 

(309) was metabolized to (310) by Spodoptera litura (Takeuchi and Miyazawa, 2005). 

Metabolic pathways of nopol (452) and nopol benzyl ether (455) by microorganisms are summarized 

in Figure 14.206. Nopol (452) is metabolized mainly to 7-hydroxyethyl-a-terpineol (453) 

by rearrangement reaction and 3-oxoverbenone (454) as minor metabolite by Aspergillus spp. 

including Aspergillus niger TBUYN-2 (Noma and Asakawa, 2006b,c). Myrtenol (5) is also metabolized 

to oleoropeic alcohol (204) by rearrangement reaction. However, nopol benzyl ether (455) was 

easily metabolized to 3-oxoverbenone (454) and 3-oxonopol-2¢,4¢-dihydroxybenzylether (456) as 

main metabolites without rearrangement reaction (Noma and Asakawa 2006c). 

14.5.2 MICROBIAL TRANSFORMATION OF TERPENOIDS AS UNIT REACTION 

Microbiological oxidation and reduction patterns of terpenoids and related compounds by fungi 

belonging to Aspergillus spp. containing Aspergillus niger TBUYN-2 and Euglena gracilis Z are 

summarized in Tables 14.18 and 14.19, respectively. Dehydrogenation of secondary alcohols to 

ketones, hydroxylation of both nonallylic and allylic carbons, oxidation of olefins to form diols and 

triols via epoxides, reduction of both saturated and a,b-unsaturated ketones and hydrogenation of 

olefin conjugated with the carbonyl group were the characteristic features in the biotransformation 

of terpenoids and related compounds by Aspergillus spp. 

Compound names: 1, b-pinene; 2, pinocarveol; 3, pinocarvone; 4, a-pinene; 5, myrtenol; 6, 

myrtenal; 7, myrtenoic acid; 8, myrtanol; 9, 3-hydroxymyrtenol; 10, 4-hydroxymyrtanol; 11, 

a-fenchol; 12, fenchone; 13, 6-hydroxyfenchone; 14, 5-hydroxyfenchone; 15, 5-oxofenchone; 16,


TABLE 14.18 

Microbiological Oxidation and Reduction Patterns of Monoterpenoids by 

Aspergillus niger TBUYN-2 

Microbiological Oxidation 

Oxidation of alcohols 

Oxidation of primary alcohols to 

aldehydes and acids 

Oxidation of secondary alcohols to 

ketones 

(-)-trans-Carveol (81a¢), (+)-transcarveol 

(81a), 

(-)-cis-carveol (81b¢), 

(+)-cis-carveol (81b), 2a-hydroxy-1,8- 

cineole (125b), 3a-hydroxy-1,8-cineole 

(123b), 3b-hydroxy-1,8-cineole (123a) 

Oxidation of aldehydes to acids 

Hydroxylation Hydroxylation of nonallylic carbon (-)-Isodihydrocarvone (101c¢), 

(-)-carvotanacetone (47¢), 

(+)-carvotanacetone (47), cis-pmenthane 

(252), 1a-hydroxy-pmenthane 

(253), 1,8-cineole (122), 

1,4-cineole (131), (+)-fenchone (12), 

(-)-fenchone (12¢), (-)-menthol (137b¢), 

(+)-Menthol (137b), (-)-neomenthol 

(137a), (+)-neomenthol (137a), 

(+)-isomenthol (137c) 

Hydroxylation of allylic carbon (-)-Isodihydrocarvone (101b), 

(+)-neodihydrocarveol (102a¢), 

(-)-dihydrocarveol (102b¢), 

(+)-dihydrocarveol (102b), (+)-limonene 

(68), (-)-limonene (68¢) 

Oxidation of olefins 

Formation of epoxides and oxides 

Formation of diols 

(+)-Neodihydrocarveol (102a¢), 

(+)-dihydrocarveol (102b), 

(-)-dihydrocarveol (102b¢), 

(+)-limonene (68), (-)-limonene (68¢) 

Lactonization 

Formation of triols 

Microbiological Reduction 

(+)-Neodihydrocarveol (102a¢) 

Reduction of aldehydes to alcohols 

Reduction of ketones to alcohols Reduction of saturated ketones (+)-Dihydrocarvone (101a¢), 

(-)-isodihydrocarvone (101b), 

(+)-carvomenthone (48a¢), 

(-)-isocarvomenthone (48b) 

Hydrogenation of olefins 

Reduction of a,b-unsaturated ketones 

Hydrogenation of olefin conjugated with 

carbonyl group 

Hydrogenation of olefin not conjugated 

with a carbonyl group 

(-)-Carvone (93¢), (+)-carvone (93), 

(-)-carvotanacetone (47¢), 

(+)-carvotanacetone (47)


TABLE 14.19 

Microbiological Oxidation, Reduction, and Another Reaction Patterns of Monoterpenoids 

by Euglena gracilis Z 

Oxidation of alcohols 

Oxidation of aldehydes to 

acids 

Hydroxylation 

Oxidation of primary alcohols to 

aldehydes and acids 

Oxidation of secondary alcohols 

to ketones 

Microbiological Oxidation 

(-)-trans-Carveol (81a¢), (+)-cis-carveol (81b), 

(+)-isoborneol (36b) 

*Diastereo- and enantioselective dehydrogenation is 

observed in carveol, borneol, and isoborneol 

Myrtenal (6), myrtanal, (-)-perillaldehyde (78), 

trans- and cis-1,2-dihydroperillaldehydes (261a and 

261b), (-)-phellandral (64), trans- and cistetrahydroperillaldehydes, 

cuminaldehyde (193), 

(+)- and (-)-citronellal (261 and 261¢) 

*Acids were obtained as minor products 

Hydroxylation of nonallylic carbon 

Hydroxylation of allylic carbon (+)-Limonene (68), (-)-limonene (68¢) 

Oxidation of olefins 

Lactonization 

Formation of epoxides and oxides 

Formation of diols 

Formation of triols 

(+)-and (-)-Neodihydrocarveol (102a¢ and a), (-)- and 

(+)-dihydrocarveol (102b¢ and b), (+)- and 

(-)-isodihydrocarveol (102c¢ and c), (+)- and 

(-)-neoisodihydrocarveol (102d¢ and d) 

Microbiological Reduction 

Reduction of aldehydes 

to alcohols 

Reduction of ketones to 

alcohols 

Hydrogenation of olefins 

Reduction of terpene aldehydes to 

terpene alcohols 

Reduction of aromatic and related 

aldehydes to alcohols 

Reduction of aliphatic aldehydes 

to alcohols 

Reduction of saturated ketones 

Reduction of a,b-unsaturated ketones 

Hydrogenation of olefin conjugated 

with carbonyl group 

Hydrogenation of olefin not 

conjugated with a carbonyl group 

Myrtenal (6), myrtanal, (-)-perillaldehyde (78), 

trans- and cis-1,2-dihydroperillaldehydes (261a and 

261b), phellandral (64), trans- and cis-1,2- 

dihydroperillaldehydes (261a and 261b), trans- and 

cis-tetrahydroperillaldehydes, cuminaldehyde (193), 

citral (275 and 276), (+)- (261) and (-)-citronellal (261¢) 

(+)-Dihydrocarvone (101a¢), (-)-isodihydrocarvone 

(101b), (+)-carvomenthone (48a¢), 

(-)-isocarvomenthone (48b), (+)-dihydrocarvone-8,9- 

epoxides (97a¢), (+)-isodihydrocarvone-8,9-epoxides 

(97b¢), (-)-dihydrocarvone-8,9-epoxides (97a) 

(-)-Carvone (93¢), (+)-carvone (93), (-)-carvotanacetone 

(47¢), (+)-carvotanacetone (47), (-)-carvone-8,9- 

epoxides (96¢), (+)-carvone-8,9-epoxides (96) 

continued



Microbiological Oxidation, Reduction, and Another Reaction Patterns of Monoterpenoids 

by Euglena gracilis Z 

Hydrolysis 

Hydrolysis Hydrolysis of ester (+)-trans- and cis-Carveyl acetates (108a and b), 

(-)-cis-carveyl acetate (108b¢), (-)-cis-carveyl 

propionate, geranyl acetate (270) 

Hydration 

Hydration of C=C bond in 

isopropenyl group to tertiary 

alcohol 

Hydration 

(+)-Neodihydrocarveol (102a¢), (-)-dihydrocarveol 

(102b¢), (+)-isodihydrocarveol (102c¢), 

(+)-neoisodihydrocarveol (102d¢) 

(-)-neodihydrocarveol (102a), (+)-dihydrocarveol 

(102b), (-)-isodihydrocarveol (102c), 

(-)-neoisodihydrocarveol (102d), trans- and 

cis-shisools (75a and 75b) 

Isomerization 

Isomerization Geraniol (271), nerol (272) 

9-hydroxyfenchone; 17, fenchone-9-al; 18, fenchone-9-oic acid; 19, fenchoquinone; 20, 2-hydroxyfenchone; 

21, 2,3-fencholide; 22, 1,2-fencholide; 23, verbenol; 24, verbenone; 25, 7-hydroxyverbenone; 

26, 7-hydroxyverbenone; 27, verbanone-4-al; 28, thujone; 29, thujoyl alcohol; 30, 

1-hydroxythujone; 31, 1,3-dihydroxythujone; 32, pinyl cation; 33, 1-p-menthene-8-cation; 34, a-terpineol; 

35, bornyl cation; 36, borneol; 37, camphor; 38, a-pinene epoxide; 39, isonovalal; 40, novalal; 

41, 2-hydroxypinyl cation; 42, 6-hydroxy-1-p-menthene-8-cation; 43, trans-sobrerol; 44, 

8-hydroxycarvotanacetone; (carvonehydrate); 45, 8-hydraocarvomenthone; 46, 1-p-menthen-2-ol; 

47, carvotanacetone; 48, carvomenthone; 49, carvomenthol; 50, 8-hydroxycarvomenthol; 51, 5-hydroxycarvotanacetone; 

52, 5-hydroxycarvomenthone; 53, 1-hydroxycarvomenthone; 54, p-menthane-1,2-diol; 

55, p-menthane-2,9-diol; 56, 2,3-lactone; 57, diepoxide; 58, 8,9-epoxy-1-p-menthanol; 

59, 1-p-menthene-4-cation; 60, terpine hydrate; 61, oleuropeic acid (8-hydroxyperillic acid); 62, 

1-p-menthene; 63, phellandrol; 64, phellandral; 65, phellandric acid; 66, 7-hydroxy-p-menthane; 67, 

2-(4-methyl-3-cyclohexenylidene)-propionic acid; 68, limonene; 69, limonene-1,2-epoxide; 70, 1-pmenthene-9-oic 

acid; 71, limonene-1,2-diol; 72, 1-hydroxy-8-p-menthene-2-one; 73, 1-hydroxy-pmenth-2,8-diene; 

74, perillyl alcohol; 75, shisool; 76, shisool-8,9-epoxide; 77, perillyl 

alcohol-8,9-epoxide; 78, perillandehyde; 79, 1-p-menthene-8,9-diol; 80, 4-hydroxy-p-menth-1,8- 

diene (4-terpineol); 81, carveol; 82, perillic acid; 83, 4,9-dihydroxy-1-p-menthene-7-oic acid; 84, 

2-hydroxy-8-p-menthen-7-oic acid; 85, 2-oxo-8-p-menthen-7-oic acid; 86, b-isopropyl pimelic acid; 

87, isopropenylglutaric acid; 88, isobutenoic acid; 89, isobutyric acid; 90, 1-p-menthene-8,9-diol; 

91, carveol-8,9-epoxide; 92, bottrospicatol; 93, carvone; 94, 5-hydroxycarveol; 95, 1-p-menthene- 

6,9-diol; 96, carvone-8,9-epoxide; 97, dihydrocarvone-8,9-epoxide; 98, 5-hydroxycarvone; 99, 

5-hydroxydihydrocarvone; 100, 5-hydroxydihydrocarveol; 101, dihydrocarvone; 102, dihydrocarveol; 

103, dihydrocarveol-8,9-epoxide; 104, p-menthane-2,8,9-triol; 105, dihydrobottrospicatol; 

106, 10-hydroxydihydrocarvone; 107, 10-hydroxydihydrocarveol; 108, carveyl acetate and carveyl 

propionate; 109, 8,9-epoxydihydrocarveyl acetate; 110, isopiperitenol; 111, isopiperitenone; 112, 

piperitenone; 113, 7-hydroxyisopiperitenone; 114, 10-hydroxyisopiperitenone; 115, 4-hydroxyisopiperitenone; 

116, 5-hydroxyisopiperitenone; 117, 5-hydroxypiperitenone; 118, 7-hydroxypiperitenone; 

119, pulegone; 120, 8,9-dehydromenthenone; 121, 8-hydroxymenthenone; 122, 1,8-cineole; 123, 

3-hydroxy1,8-cineole; 124, 3-oxo-1,8-cineole; 125, 2-hydroxy-1,8-cineole; 126, 2-oxo-1,8-cineole;


127, 9-hydroxy-1,8-cineole; 128, lactone (R)-5,5-dimethyl-4-(3¢-oxobutyl)-4,5-dihydrofuran- 

2-(3H)-one; 129, p-hydroxytoluene; 130, 1,8-cineole-2-malonyl ester; 131, 1,4-cineole; 132, 

2-hydroxy-1,4-cineole; 133, 3-hydroxy-1,4-cineole; 134, 8-hydroxy-1,4-cineole; 135, 9-hydroxy-1, 

4-cineole; 136, 1,4-cineole-2-malonyl ester; 137, menthol; 138, 1-hydroxymenthol; 139, 6-hydroxymenthol; 

140, 2-hydroxymenthol; 141, 4-hydroxymenthol; 142, 8-hydroxymenthol; 143, 7-hydroxymenthol; 

144, 9-hydroxymenthol; 145, 7,8-dihydroxymenthol; 146, 1,8-dihydroxymenthol; 147, 

3-p-menthene; 148, menthenone; 149, menthone; 150, 1-hydroxymenthone; 151, 7-hydroxymenthone; 

152, 3,7-dimethyl-6-hydroxyoctanoic acid; 153, 3,7-dimethyl-6-oxoactanoic acid; 154, 

2-methyl-2,5-oxidoheptenoic acid; 155, menthone-1,2-diol; 156, piperitone; 157, 8-hydroxypiperitone; 

158, 6-hydroxypiperitone; 159, 9-hydroxypiperitone; 160, piperitone-7-al; 161, 7-hydroxypiperitone; 

162, piperitone-7-oic acid; 163, menthone-7-al; 164, menthone-7-oic acid; 165, 

7-carboxylmenthol; 166, piperitone oxide; 167, 4-hydroxypiperitone; 168, piperitenone oxide; 169, 

1-hydroxypulegone; 170, 2,3-seco-p-menthylacetone-3-en-1-ol; 171, 2-methyl-5-isopropyl-2,5- 

hexadienoic acid; 172, 2,5,6-trimethyl-2,4-heptadienoic acid; 173, 2,5,6-trimethyl-3-heptenoic acid; 

174, 2,5,6-trimethyl-2-heptenoic acid; 175, 3-hydroxy-2,5,6-trimethyl-3-heptanoic acid; 176, 3-oxo- 

2,5,6-trimethyl-3-heptanoic acid; 177, 3,4-dimethylvaleric acid; 178, p-cymene; 179, thymol; 

180, 6-hydroxythymol 181, 6-hydroxymenthenone, 6-hydroxy-4-p-menthen-3-one; 182, 3-hydroxythymo1,4-quinol; 

183, 2-hydroxythymoquionone; 184, 2,4-dimethyl-6-oxo-3,7-dimethyl-2,4-octadienoic 

acid; 185, 2,4,6-trioxo-3,7-dimethyl octanoic acid; 186, 2-oxobutanoic acid; 187, acetic acid; 

188, 8-hydroxy-p-cymene; 189, 9-hydroxy-p-cymene; 190, 2-(4-methylphenyl)-propanoic acid; 191, 

carvacrol; 192, cumin alcohol; 193, p-cumin aldehyde; 194, cumic acid; 195, cis-2,3-dihydroxy-2, 

3-dihydro-p-cumic acid; 196, 3-hydroxycumic acid; 197, 2,3-dihydroxy-p-cumic acid; 198, 

2-hydroxy-6-oxo-7-methyl-2,4-octadien-1,3-dioic acid; 199, 2-methyl-6-hydroxy-6-carboxy-7- 

octene; 201, 4-methyl-2-oxopentanoic acid; 202, carvacrol methyl ether; 203, 7-hydroxycarvacrol 

methyl ether; 204, 8-hydroxyperillyl alcohol; 205, 8-hydroxyperillaldehyde; 206, linalool; 207, 

linalyl-6-cation; 208, linalyl-8-cation; 209, 6-hydroxymethyl linalool; 210, linalool-6-al; 211, 

2-methyl-6-hydroxy-6-carboxy-2,7-octadiene; 212, 2-methyl-6-hydroxy-7-octen-6-oic acid; 213, 

phellandric acid-8-cation; 214, 2,3-epoxylinalool; 215, 5-methyl-5-vinyltetrahydro-2-furanol; 216, 

5-methyl-5-vinyltetrahydro-2-furanone; 217, 2,2,6-trimethyl-3-hydroxy-6-vinyltetrahydropyrane; 

218, malonyl ester; 219, 1-hydroxylinalool (3,7-dimethyl-1,6-octadiene-8-ol); 220, 2,6-dimethyl- 

6-hydroxy-trans-2,7-octadienoic acid; 221, 4-methyl-trans-3,5-hexadienoic acid; 222, 4-methyltrans-3,5-hexadienoic 

acid; 223, 4-methyl-trans-2-hexenoic acid; 224, isobutyric acid; 225, 

9-hydroxycamphor; 226, bornyl acetate; 228, 6-hydroxycamphor; 229, 6-oxocamphor; 230, 

4-carboxymethyl-2,3,3-trimethylcyclopentanone; 231, 4-carboxymethyl-3,5,5-trimethyltetrahydro- 

2-pyrone; 232, isohydroxycamphoric acid; 233, isoketocamphoric acid; 234, 3,4,4-trimethyl-5-oxotrans-2-hexenoic 

acid; 235, 5-hydroxycamphor; 236, 5-oxocamphor; 237, 238, 6-hydroxy-1, 2-campholide; 

239, 6-oxo-1,2-campholide; 240, 5-carboxymethyl-3,4,4-trimethyl-2-cyclopentenone; 241, 6-carboxymethyl-4,5,5-trimethyl-5,6-dihydro-2-pyrone; 

242, 5-hydroxy-3,4,4-trimethyl-2-heptene-1,7- 

dioic acid; 243, 3-hydroxycamphor; 244, camphorquinone; 245, 2-hydroxyepicamphor; 246, 

2-hydroxy-p-menthan-7-oic acid; 247, 2-oxo-p-menthan-7-oic acid; 248, 3-isopropylheptane-1,7- 

dioic acid; 249, 3-isopropylpentane-1,5-dioic acid; 250, 4-methyl-3-oxopentanoic acid; 251, methylisopropyl 

ketone; 252, p-menthane; 253, 1-hydroxy-p-menthane; 254, p-menthane-1,9-diol; 255, 

p-menthane-1,7-diol; 256, 1-hydroxy-p-menthene-7-al; 257, 1-hydroxy-p-menthene-7-oic acid; 258, 

citronellol; 259, dihydrocitronellol; 260, 2,8-dihydroxy-2,6-dimethyl octane; 261, citronellal; 262, 

citronellic acid; 263, pulegol; 264, 7-hydroyxmethyl-6-octene-3-ol; 265, 3,7-dimethyl-6-octane-1,8- 

diol; 266, 3,7-dimethyloctane-1,8-diol; 267, isopulegol; 268, 6,7-epoxycitronellol; 269, citronellyl 

acetate; 270, geranyl acetate; 271, geraniol; 272, nerol 273, nerol-6,7-epoxide; 274, 6,7-epoxygeraniol; 

275, neral; 276, geranial; 277, neric acid; 278, geranic acid; 279, 2,6-dimethyl-8-hydroxy-7-oxo- 

2-octene; 280, 6-methyl-5-heptenoic acid; 281, 7-methyl-3-carboxymethyl-2,6-octadiene-1-oic acid; 

282, 7-methyl-3-hydroxy-3-carboxymethyl-6-octen-1-oic acid; 283, 7-methyl-3-oxo-6-octenoic 

acid; 284, 6-methyl-5-heptenoic acid; 284, 4-methyl-3-heptenoic acid; 285, 4-methyl-3-pentenoic 

acid; 286, 3-methyl-2-butenoic acid; 287, 3-hydroxymethyl-2,6-octadiene-1-ol; 288, 3-formyl-2,6-


octadiene-1-ol; 289, 3-carboxy-2,6-octadiene-1-ol; 290, 3-(4-methyl-3-pentenyl)-3-butenolide; 291, 

7-hydroxymethyl-3-methyl-2,6-octadien-1-ol; 292, 3,7-dimethyl-2,3-dihydroxy-6-octen-1-ol; 293, 

3,7-dimethyl-2-oxo-6-octene-1,3-diol; 294, 6-methyl-5-hepten-2-one; 295, 6-methyl-7-hydroxy-5- 

heptene-2-one; 296, 2-methyl-g-butyrolactone; 297, 6-methyl-5-heptenoic acid; 298, 6-methyl-5- 

hepten-2-ol; 299, 7-methyl-3-oxo-6-octanoic acid; 300, 3,7-dimethyl-2,6-octadiene-1,8-diol; 301, 

isopulegyl acetate; 302, myrcene; 303, 2-methyl-6-methylene-7-octene-2,3-diol; 304, 6-methylene- 

7-octene-2,3-diol; 305, myrcenol; 306, ocimene; 307, myrcene-3,(10)-epoxide; 308, myrcene-3,(10)- 

glycol; 309, (-)-citronellene; 309¢, (+)-citronellene; 310, (3R)-3,7-dimethyl-6-octene-1,2-diol; 310¢, 

(3S)-3,7-dimethyl-6-octene-1,2-diol; 311, (E)-3,7-dimethyl-5-octene-1,7-diol; 312, trans-rose oxide; 

313, cis-rose oxide; 314, (2Z,5E)-3,7-dimethyl-2,5-octadiene-1,7-diol; 315, (Z)-3,7-dimethyl-2,7- 

octadiene-1,6-diol; 316, (2E,6Z)-2,6-dimethyl-2,6-octadiene-1,8-diol; 317, a cyclization product; 318, 

(2E,5E)-3,7-dimethyl-2,5-octadiene-1,7-diol; 319, (E)-3,7-dimethyl-2,7-octadiene-1,6-diol; 320, 

8-hydroxynerol; 321, 10-hydroxynerol; 322, 1-hydroxy-3,7-dimethyl-2E,6E-octadienal; 323, 1-hydroxy- 

3,7-dimethyl-2E,6E-octadienoic acid; 324, 3,9-epoxy-p-menth-1-ene; 325, tetrahydrolinalool; 326, 

3,7-dimethyloctane-3,5-diol; 327, 3,7-dimethyloctane-3,7-diol; 328, 3,7-dimethyl-octane-3,8-diol; 

329, dihydromyrcenol; 330, 1,2-epoxydihydromyrcenol; 331, 3b-hydroxydihydromyrcerol; 332, 

dihydromyrcenyl acetate; 333, 1,2-dihydroxydihydromyrcenyl acetate; 334, (+)-p-menthane-1- 

b,2a,8-triol; 335, a-pinene-1,2-epoxide; 336, 3-carene; 337, 3-carene-1,2-epoxide; 338, (1R)- 

trans-isolimonene; 338, (1R,4R)-p-menth-2-ene-8,9-diol; 339, (1R,4R)-p-menth-2-ene-8,9-diol; 

340, a-Terpinene; 341, a-terpinene-7-oic acid; 342, (-)-terpinen-4-ol; 343, p-menthane-1,2,4-triol; 

344, g-terpinene; 345, g-terpinene-7-oic acid; 346, terpinolene; 347, (1R)-8-hydroxy-3-p-menthen- 

2-one; 348, (1R)-1,8-dihydroxy-3-p-menthen-2-one; 349, 6b-hydroxyfenchol; 350, 5b-hydroxyfenchol 

(a,5b,b,5a); 351, terpinolene-1,2-trans-diol; 352, terpinolene-4,8-diol; 353, terpinolene-9-ol; 

354, terpinolene-10-ol; 355, a-phellandrene; 356, a-phellandrene-7-oic acid; 357, terpinolene-7-oic 

acid; 358, thymoquinone; 359, 1,2-dihydroperillaldehyde; 360, 1,2-dihydroperillic acid; 361, 8-hydroxy-1,2-dihydroperillyl 

alcohol; 362, tetrahydroperillaldehyde (a trans, b cis); 363, tetrahydroperillic 

acid (a trans, b cis); 364, (-)-menthol monoglucoside; 365, (+)-menthol diglucoside; 366, 

(+)-isopulegol; 367, 7-hydroxy-(+)-isopulegol; 368, 10-hydroxy-(+)-isopulegol; 369, 1,2-epoxy-aterpineol; 

370, bornane-2,8-diol; 371, p-menthane-1a,2b,4b-triol; 372, 1-p-menthene-4b,6-diol; 

373, 1-p-menthene-4a,7-diol; 374, (+)-bottrospicatal; 375, 1-p-menthene-2b,8,9-triol; 376, (-)-perillyl 

alcohol monoglucoside; 377, (-)-perillyl alcohol diglucoside; 378, 4a-hydroxy-(-)-isodihydrocarvone; 

379, 2-methyl-2-cyclohexenone; 380, 2-cyclohexenone; 381, 3-methyl-2-cyclohexenone; 

382, 2-methylcyclohexanone; 383, 2-methylcyclohexanol (a, trans b, cis); 384, 4-hydroxycarvone; 

385, 2-ethyl-2-cyclohexenone; 386, 2-ethylcyclohexenone (a1R) (b1S); 387, 1-hydroxypulegone; 

388, 5-hydroxypulegone; 389, 8-hydroxymenthone; 390, 10-hydroxy-(-)-carvone; 391, 1,5,5-trimethylcyclopentane-1,4-dicarboxylic 

acid; 392, 4b-hydroxy-(-)-menthone; 393, 1a,4b-dihydroxy-(-)- 

menthone; 394, 7-hydroxy-9-carboxymenthone; 395, 7-hydroxy-1,8-cineole; 396, methyl ester 

of 2a-hydroxy-1,8-cineole; 397, ethyl ester of 2a-hydroxy-1,8-cineole; 398, n-propyl ester of 2a-hydroxy-1,8-cineole; 

399, n-butyl ester of 2a-hydroxy-1,8-cineole; 400, isopropyl ester of 2a-hydroxy- 

1,8-cineole; 401, tertiary butyl ester of 2a-hydroxy-1,8-cineole; 402, methylisopropyl ester of 

2a-hydroxy-1,8-cineole; 403, methyl tertiary butyl ester of 2a-hydroxy-1,8-cineole; 404, 2a-hydroxy-1,8-cineole 

monoglucoside (404 and 404’); 405, 2a-hydroxy-1,8-cineole diglucoside; 406, 

p-menthane-1,4-diol; 407, 1-p-menthene-4b,6-diol; 408, (-)-pinane-2a,3a-diol; 409, (-)-6b-hydroxypinene; 

410, (-)-4a,5-dihydroxypinene; 411, (-)-4a-hydroxypinen-6-one; 412, 6b,7-dihydroxyfenchol; 

413, 3-oxo-pinane; 414, 2a-hydroxy-3-pinanone; 415, 2a, 5-dihydroxy-3-pinanone; 

416, 2a,10-dihydroxy-3-pinanone; 417, trans-3-pinanol; 418, pinane-2a,3a-diol; 419, pinane-2a, 

3a, 5-triol; 420, isopinocampheol (3-pinanol); 421, pinane-1,3a-diol; 422, pinane-3a,5-diol; 423, 

pinane-3b,9-diol; 424, pinane-3b,4b,-diol; 425, 426, pinane-3a,4b-diol; 427, pinane-3a,9-diol; 

428, pinane-3a,6-diol; 429, p-menthane-2a,9-diol; 430, 2-methyl-3a-hydroxy-1-hydroxyisopropyl 

cyclohexane propane; 431, 5-hydroxy-3-pinanone; 432, 2a-methyl,3-(2-methyl-2-hydroxypropyl)- 

cyclopenta-1b-ol; 433, 3-acetoxy-2a-pinanol; 434, 8-hydroxy-a-pinene;435, 436, myrtanoic acid; 

437, camphene; 438, camphene gylcol; 439, (+)-3-carene; 440, m-mentha-4,6-dien-8-ol; 441,


m-cymen-8-ol; 442, 3-carene-9-ol; 443, 3-carene-9-carboxylic acid; 444, 3-caren-10-ol-9-carboxylic 

acid; 445, 3-carene-9,10-dicarboxylic acid; 446, (-)-cis-carane; 447, dicarboxylic acid of (-)-ciscarane; 

448, (-)-6b-hydroxypinene; 449, (-)-4a,5-dihydroxypinene; 450, (-)-4a-hydroxypinen-6- 

one; 451, 10-hydroxyverbenol; 452, (-)-nopol; 453, 7-hydroxymethyl-1-p-menthen-8-ol; 454, 

3-oxo nopol; 455, nopol benzyl ether; 456, 4-oxonopl-2¢,4¢-dihydroxybenzyl ether; 457, 7-hydroxymethyl-1-p-menthen-8-ol 

benzyl ether; 458, piperitenol; 459, thymol methyl ether; 460, 7-hydroxythymol 

methyl ether; 461, 9-hydroxythymol methyl ether; 462, 1,8-cineol-9-oic acid; 463, 

4-hydroxyphellandric acid; 464, 4-hydroxydihydrophellandric acid; 465, (+)-8-hydroxyfenchol; 466, 

(-)-9-hydroxyfenchol; 467, (+)-10-hydroxyfenchol, 468, 4a-hydroxy-6-oxo-a-pinene; 469, dihydrolinalyl 

acetate; 470, 3-hydroxycarvacrol; 471, 9-hydroxycarvacrol; 472, carvacrol-9-oic acid; 473, 

8,9-dehydrocarvacrol; 474, 8-hydroxycarvacrol; 475, 7-hydroxycarvacrol; 476, carvacrol-7-oic acid; 

477, 8,9-dihydroxycarvacrol; 478, 7,9-dihydroxycarvacrol methyl ether; 479, 7-hydroxythymol; 480, 

9-hydroxythymol; 481, thymol-7-oic acid; 482, 7,9-dihydroxythymol; 483, thymol-9-oic acid; 484, 

(1R,2R,3S,4S,5R)-3,4-pinanediol. 

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Yawata, T., M, Ogura, K. Shimoda, S. Izumi, and T. Hirata, 1998. Epoxidation of monoterpenes by the peroxidase 

from the cultured cells of Nicotiana tabacum, Proc. 42nd TEAC, pp. 142–144. 

Yonemoto, N., S. Sakamoto, T. Furuya, and H. Hamada, 2005. Preparation of (-)-perillyl alcohol oligosaccharides. 

Proc. 49th TEAC, pp. 108–110.

15 

Biotransformation of 

Sesquiterpenoids, Ionones, 

Damascones, Adamantanes, 

and Aromatic Compounds by 

Green Algae, Fungi, and 

Mammals 

Yoshinori Asakawa and Yoshiaki Noma 

CONTENTS 

15.1 Introduction ..................................................................................................................... 737 

15.2 Biotransformation of Sesquiterpenoids by Microorganisms .......................................... 738 

15.2.1 Highly Efficient Production of Nootkatone (2) from Valencene (1) .................. 738 

15.2.2 Biotransformation of Valencene (1) by Aspergillus niger 

and Aspergillus wentii ........................................................................................ 741 

15.2.3 Biotransformation of Nootkatone (2) by Aspergillus niger ............................... 743 

15.2.4 Biotransformation of Nootkatone (2) by Fusarium culmorum 

and Botryosphaeria dothidea ............................................................................ 745 

15.2.5 Biotransformation of (+)-1(10)-Aristolene (36) from the Crude Drug 

Nardostachys chinensis by Chlorella fusca, Mucor species, 

and Aspergillus niger ......................................................................................... 749 

15.2.6 Biotransformation of Various Sesquiterpenoids by Microorganisms ................ 754 

15.3 Biotransformation of Sesquiterpenoids by Mammals, Insects, 

and Cytochrome P-450 ................................................................................................... 819 

15.3.1 Animals (Rabbits) and Dosing ........................................................................... 819 

15.3.2 Sesquiterpenoids ................................................................................................ 820 

15.4 Biotransformation of Ionones, Damascones, and Adamantanes ..................................... 823 

15.5 Biotransformation of Aromatic Compounds ................................................................... 828 

References .................................................................................................................................. 835 


Recently, environment-friendly green or clean chemistry is emphasized in the field of organic and 

natural product chemistry. Noyori’s high-efficient production of (-)-menthol using (S)-BINAP-Rh 

737


catalyst is one of the most important green chemistries (Tani et al., 1982; Otsuka and Tani, 1991) 

and 1000 ton of (-)-menthol has been produced by this method in 1 year. On the other hand, 

enzymes of microorganisms and mammals are able to transform a huge variety of organic compounds, 

such as mono- sesqui-, and diterpenoids, alkaloids, steroids, antibiotics, and amino acids 

from crude drugs and spore-forming green plants to produce pharmacologically and medicinally 

valuable substances. 

Since Meyer and Neuberg (1915) studied the microbial transformation of citronellal, there are a 

great number of reports concerning biotrasformation of essential oils, terpenoids, steroids, alkaloids, 

and acetogenins using bacteria, fungi, and mammals. In 1988 Mikami (Mikami, 1988) 

reported the review article of biotransformation of terpenoids entitled “Microbial Conversion of 

Terpenoids.” Lamare and Furstoss (1990) reviewed biotransformation of more than 25 sesquiterpenoids 

by microorganisms. In this chapter, the recent advances in the biotransformation of natural 

and synthetic compounds; sesquiterpenoids, ionones, a-damascone, and adamantanes, and aromatic 

compounds, using microorganisms including algae and mammals are described. 

15.2 BIOTRANSFORMATION OF SESQUITERPENOIDS BY MICROORGANISMS 

15.2.1 HIGHLY EFFICIENT PRODUCTION OF NOOTKATONE (2) FROM VALENCENE (1) 

The most important and expensive grapefruit aroma, nootkatone (2), decreases the somatic fat ratio 

(Haze et al., 2002), and therefore its highly efficient production has been requested by the cosmetic 

and fiber industrial sectors. Previously, valencene (1) from the essential oil of Valencia orange was 

converted into nootkatone (2) by biotransformation using Enterobacter species only in 12% yield 

(Dhavlikar and Albroscheit, 1973), Rodococcus KSM-5706 in 0.5% yield with a complex mixture 

(Okuda et al., 1994), and using Cytochrome P450 (CYP450) in 20% yield with other complex 

products (Sowden et al., 2005). Nootkatone (2) was chemically synthesized from valencene (1) with 

AcOOCMe 3 in three steps and chromic acid in low yield (Wilson and Saw, 1978) and using surfacefunctionalized 

silica supported by metal catalysts such as Co 2+ , Mn 2+ , and so on with tert-butyl 

hydroperoxide in 75% yield (Salvador and Clark, 2002). However, these synthetic methods are not 

safe because they involve toxic heavy metals. An environment-friendly method for the synthesis of 

nootkatone that does not use any heavy metals such as chromium and manganese must be designed. 

The commercially available and cheap sesquiterpene hydrocarbon (+)-valencene (1) ([a] D + 84.6°, 

c = 1.0) obtained from Valencia orange oil was very efficiently converted into nootkatone (2) by 

biotransformations using Chlorella (Hashimoto et al., 2003a), Mucor species (Hashimoto et al., 

2003), Botryosphaeria dothidea, and Botryodiplodia theobromae (Furusawa et al., 2005, 2005a; 

Noma et al., 2001a). 

Chlorella fusca var. vacuolata IAMC-28 (Figure 15.1) was inoculated and cultivated while stationary 

under illumination in Noro medium MgCl 2 ◊6H 2 O (1.5 g), MgSO 4 ◊7H 2 O (0.5 g), KCl (0.2 g), 

CaCl 2 ◊2H 2 O (0.2 g), KNO 3 (1.0 g), NaHCO 3 (0.43 g), TRIS (2.45 g), K 2 HPO 4 (0.045 g), Fe-EDTA 

(3.64 mg), EDTA-2Na (1.89 mg), ZnSO 4 ◊7H 2 O (1.5 g), H 3 BO 2 (0.61 mg), CoCl 2 ◊6H 2 O (0.015 mg), 

CuSO 4 ◊5H 2 O (0.06 mg), MnCl 2 ◊4H 2 O (0.23 mg), and (NH 4 ) 6 Mo 7 O 24 ◊4H 2 O (0.38 mg), in distilled 

H 2 O 1 L (pH 8.0). Czapek-peptone medium [1.5% sucrose, 1.5% glucose, 0.5% polypeptone, 0.1% 

K 2 HPO 4 , 0.05% MgSO 4 ◊7H 2 O, 0.05% KCl, and 0.001% FeSO 4 ◊7H 2 O, in distilled water (pH 7.0)] was 

used for the biotransformation of substrate by microorganism. Aspergillus niger was isolated in our 

laboratories from soil in Osaka prefecture, and was identified according to its physiological and 

morphological characters. 

(+)-Valencene (1) (20 mg/50 mL) isolated from the essential oil of Valencia orange was added to the 

medium and biotransformed by Chlorella fusca for a further 18 days to afford nootkatone (2) [gas 

chromatography-mass spectrometry (GC-MS) peak area: 89%; isolated yield: 63%] (Figure 15.2) 

(Furusawa et al., 2005, 2005a; Noma et al., 2001a). The reduction of 2 with NaBH 4 and CeCl 3 gave

Biotransformation of Compounds by Green Algae, Fungi, and Mammals 739 

FIGURE 15.1 Chlorella fusca var. vacuolata. 

2a-hydroxyvalencene (3) in 87% yield, followed by Mitsunobu reaction with p-nitrobenzoic acid, 

triphenylphosphine, and diethyl azodicarboxylate to give nootkatol (2b-hydroxyvalencene) (4), 

possessing calcium-antagonistic activity isolated from Alpinia oxyphylla (Shoji et al., 1984) in 42% 

yield. Compounds 3 and 4 thus obtained were easily biotransformed by Chlorella fusca and 

Chlorella pyrenoidosa for only 1 day to give nootkatone (2) in good yield (80–90%), respectively. 

900,000 

26.58 

O 

800,000 

700,000 

Nootkatone (2) 

Abundance 

600,000 

500,000 

400,000 

300,000 

200,000 

100,000 

0 

Valencene (1) 

22.70 25.33 

25.88 29.89 

19.39 

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 

Time 

FIGURE 15.2 Total ion chromatogram of metabolites of valencene (1) by Chlorella fusca var. vacuolata.


TABLE 15.1 

Conversion of Valencene (1) to Nootkatone (2) by Chlorella sp. for 14 Days 

Metabolites (% of the Total in GC-MS) 

Conversion 

Chlorella sp. Valencene (1) 2a-Nootkatol (3) 2b-Nootkatol (4) Nootkatone (2) Ratio (%) 

C. fusca 11 0 0 89 89 

C. pyrenoidosa 7 0 0 93 93 

C. vulgaris 0 0 0 100 100 

The biotransformation of compound 1 was further carried out by Chlorella pyrenoidosa and 

Chlorella vulgaris (Furusawa et al., 2005, 2005a) and soil bacteria (Noma et al., 2001) to give nootkatone 

in good yield (Table 15.1). 

In the time course of the biotransformation of 1 by Chlorella pyrenoidosa, the yield of nootkatone 

(2) and nootkatol (4) without 2a-hydroxyvalencene (3) increased with the decrease in that 

of 1, and subsequently the yield of 2 increased with decrease in that of 3. In the metabolic pathway 

of valencene (1), 1 was slowly converted into nootkatol (4), and subsequently 4 was rapidly converted 

into 2, as shown in Figure 15.3. 

A fungus strain from the soil adhering to the thalloid liverwort Pallavicinia subciliata, Mucor 

species, which was inoculated and cultivated statically in Czapek-peptone medium (pH 7.0) at 30°C 

for 7 days. Compound 1 (20 mg/50 mL) was added to the medium and incubated for a further 7 days. 

2 1 

4 

10 

5 7 

Valencene (1) 

12 

11 

13 

Chlorella sp. 

slow 

Chlorella sp. 

slow 

HO 

HO 

3 4 

Fast 

Fast 

O 

Nootkatone (2) 

FIGURE 15.3 Biotransformation of valencene (1) by Chlorella species.


Nootkatone (2) was then obtained in very high yield (82%) (Furusawa et al., 2005; Noma et al., 

2001a). 

The biotransformation from 1 to 2 was also examined using the plant pathogenic fungi 

Botryosphaeria dothidea and Botryodiplodia theobromae (a total of 31 strains) separated from 

fungi infecting various types of fruit, and so on. Botryosphaeria dothidea and Botryodiplodia theobromae 

were both inoculated and cultivated while stationary in Czapek-peptone medium (pH 7.0) 

at 30°C for 7 days. The same size of the substrate 1 was added to each medium and incubated for a 

further 7 days to obtain nootkatone (42–84%) (Furusawa et al., 2005). 

The expensive grapefruit aromatic, nootkatone (2) used by cosmetic and fiber manufacturers was 

obtained in high yield by biotransformation of (+)-valencene (1), which can be cheaply obtained 

from Valencia orange, by Chlorella species, fungi such as Mucor species, Botryosphaeria dothidea, 

and Botryodiplodia theobromae. This is a very inexpensive and clean oxidation reaction, which 

does not use any heavy metals, and thus this method is expected to find applications in the industrial 

production of nootkatone. 

15.2.2 BIOTRANSFORMATION OF VALENCENE (1) BY ASPERGILLUS NIGER 

AND ASPERGILLUS WENTII 

Valencene (1) from Valencia orange oil was cultivated by Aspergillus niger in Czapek-peptone 

medium, for 5 days to afford six metabolites 5 (1.0%), 6 and 7 (13.5%), 8 (1.1%), 9 (1.5%), 10 (2.0%), 

and 11 (0.7%), respectively. Ratio of compounds 6 (11S) and 7 (11R) was determined as 1:3 by HPLC 

analysis of their thiocarbonates (12 and 13) (Noma et al., 2001a) (Figure 15.4). 

2 1 

4 

10 

5 7 

1 

12 

11 

13 

A. niger 

O 

O 

O 

5 (1.0%) 

OH 

S 

OH 

OH 

6 1:3 

7 

R 

OH 

OH 

(13.3%) 

8 (1.1%) 

OH O 

OH 

9 (1.5%) 

R 

OH 

OH 

HO 

10 (1.6%) 

OH 

OH 

O 

OH 

OH 

11 (0.7%) 

FIGURE 15.4 Biotransformation of valencene (1) by Aspergillus niger.


Compounds 8–11 could be biosynthesized by elimination of a hydroxy group of 2-hydroxyvalencenes 

(3, 4). Compound 3 was biotransformed for 5 days by Aspergillus niger to give three metabolites 

6 and 7 (6.4%), 8 (34.6%), and 9 (5.5%), respectively. Compound 4 was biotransformed for 5 days by 

Aspergillus niger to give three metabolites: 6 and 7 (21.8%), 9 (5.5%), and 10 (10.4%), respectively 


HO 

1 2 

4 

10 

5 7 

3 

12 

11 

13 

A. niger 

O 

O 

S 

OH 

OH 

OH 

OH 

6 7 8 (34.6%) 

1:3 

R 

OH 

OH 

(6.4%) 

O 

9 (5.5%) 

R 

OH 

OH 

HO 

2 1 

4 

10 

5 7 

4 

11 

13 

A. niger 

12 

O 

O 

S 

OH 

OH 

OH 

OH 

6 

1:3 

7 9 (5.5%) 

R 

O 

R 

OH 

OH 

(21.8%) 

HO 

R 

10 (10.4%) 

OH 

OH 

FIGURE 15.5 Biotransformation of 2a-hydroxyvalencene (3) and 2b-hydroxyvalencene (4) by Aspergillus 

niger.


1 

[O] 

H + H 

H + 

HO 

HO 

4H 

3 

–H 2 O 

[O] 

[O] 

[O] 

O 

9 

OH 

OH 

HO 

OH 

OH 

FIGURE 15.6 Possible pathway of biotransformation of valencene (1) by Aspergillus niger. 

10 

8 

OH 

OH 

Both ratios of 6 (11S) and 7 (11R) obtained from 3 and 4 were 1:3, respectively. From the above 

results, plausible metabolic pathways of valencene (1) and 2-hydroxyvalencene (3, 4) by Aspergillus 

niger are shown in Figure 15.6 (Noma et al., 2001a). 

Aspergillus wentii and Eurotrium purpurasens converted valencene (1) to 11,12-epoxide (14a) 

and the same diol (6, 7) (Takahashi and Miyazawa, 2005) as well as nootkatone (2) and 2a-hydroxyvalencene 

(3) (Takahashi and Miyazawa, 2006). 

Kaspera et al. (2005) reported that valencene (1) was incubated in submerged cultures of the 

ascomycete Chaetomium globosum, to give nootkatone (2), 2a-hydroxyvalencene (3), and valencene 

11,12-epoxide (14a), together with a valencene ketodiol, valencenediols, a valencene ketodiol, 

a valencene triol, or valencene epoxydiol that were detected by liquid chromatography-mass spectrocopy 

(LC-MS) spectra and GC-MS of trimethyl silyl derivatives. These metabolites are accumulated 

preferably inside the fungal cells (Figure 15.7). 

The metabolites of valencene, nootkatone (2), (3), and (14a), indicated grapefruit with sour and 

citrus with bitter odor, respectively. Nootkatone 11,12-epoxide (14) showed no volatile fragrant 

properties. 

15.2.3 BIOTRANSFORMATION OF NOOTKATONE (2) BY ASPERGILLUS NIGER 

Aspergillus niger was inoculated and cultivated rotatory (100 rpm) in Czapek-peptone medium at 

30°C for 7 days. (+)-Nootkatone (2), ([a] D + 193.5°, c = 1.0), (80 mg/200 mL), which was isolated


c 

HO 

3 

c 

HO 

1 

4 

a,c 

a,b,c 

14a 

O 

O 

O 

O 

2 

b 

5 

OH 

6,7 

OH 

OH 

b,c 

O 

OH 

O 

O 

OH 

14 

O 

FIGURE 15.7 Biotransformation of valencene (1) and nootkatone (2) by Aspergillus wentii, Epicoccum 

purpurascens, and Chaetomium globosum. 

15a 

a: Aspergillus wentii 

b: Epicoccum purpurasens 

c: Chaetomium globosum 

15b 

from the essential oil of grapefruit, was added to the medium and further cultivated for 7 days to 

obtain two metabolites, 12-hydroxy-11,12-dihydronootkatone (5) (10.6%) and C11 stereo-mixtures 

(51.5%) of nootkatone-11S,12-diol (6) and its 11R isomer (7) (11R:11S = 1:1) (Hashimoto et al., 

2000a; Noma et al., 2001a; Furusawa et al., 2003) (Figure 15.8). 

11,12-epoxide (14) obtained by epoxidation of nootkatone (2) with mCPBA was biotransformed 

by Aspergillus niger for 1 day to afford 6 and 7 (11R:11S = 1:1) in good yield (81.4%). 1-aminobenzotriazole, 

an inhibitor of CYP450, inhibited the oxidation process of 1 into compounds 5–7 (Noma 

et al., 2001a). From the above results, possible metabolic pathways of nootkatone (2) by Aspergillus 

niger might be considered as shown in Figure 15.9. 

The same substrate was incubated with Aspergillus wentii to produce diol (6, 7) and 11,12- - 

epoxide (14) (Takahashi and Miyazawa, 2005).


O 

O 

OH 

7 (47.2%) 

R 

OH 

OH 

15a (14.9%) 

O 

2 1 

4 

10 

5 7 

2 

Fusarium culmorum (7 days) 

12 

11 

13 

Aspergillus niger (5 days) 

O 

O 

O 

5 (10.6%) 

OH 

S 

OH 

OH 

6 7 

R 

OH 

OH 

(51.5%) 

O 

2 1 

4 

10 

5 7 12 

11 

2 13 

Botryosphaeria dothidea (14 days) 

O 

S 

O 

OH 

OH 

6 

3:2 

7 

R 

OH 

OH 

O 

16(20.9%) 

OH 

(54.2%) 

FIGURE 15.8 Biotransformation of nootkatone (2) by Fusarium culmorum, Aspergillus niger, and 

Botryosphaeria dothidea. 

15.2.4 BIOTRANSFORMATION OF NOOTKATONE (2) BY FUSARIUM CULMORUM 

AND BOTRYOSPHAERIA DOTHIDEA 

(+)-Nootkatone (2) was added to the same medium as mentioned above including Fusarium culmorum 

to afford nootkatone-11R,12-diol (7) (47.2%) and 9b-hydroxynootkatone (15) (14.9%) (Noma 

et al., 2001a).


O 

11 

12 

2 

Cytochrome P-450 

O 

O 

15b 

OH 

14 

O 

[H] 

[H] 

[H 2 O] 

O 

5 

12 

11 OH 

O 

6: 11S 

7: 11R 

11 12 

OH 

OH 

FIGURE 15.9 Possible pathway of biotransformation of valencene (1) by Cytochrome P-450. 

Compound 7 was stereospecifically obtained at C11 by biotransformation of 1. Purity of compound 

7 was determined as ca. 95% by high performance liquid chromatography (HPLC) analysis 

of the thiocarbonate (13). 

The biotransformation of nootkatone (2) was examined by the plant pathogenic fungus, 

Botryosphaeria dothidea separated from the fungus that infected the peach. (+)-Nootkatone (2) was 

cultivated with Botryosphaeria dothidea (Peach PP8402) for 14 days to afford nootkatone diols 

(6 and 7) (54.2%) and 7a-hydroxynootkatone (16) (20.9%). Ratio of compounds 6 and 7 was determined 

as 3:2 by HPLC analysis of the thiocarbonates (12, 13) (Noma et al., 2001a). Nootkatone 

(2) was administered into rabbits to give the same diols (6, 7) (Asakawa et al., 1986; Ishida, 2005). 

Epicoccum purpurascens also biotransformed nootkatone (2) to 5–7, 14, and 15a (Takahashi and 

Miyazawa, 2006). 

The biotransformation of 2 by Aspergillus niger and Botryosphaeria dothidea resembled to that 

of the oral administration to rabbit since the ratio of the major metabolites 11S- (6) and 11R-nootkatone- 

11,12-diol (7) was similar. It is noteworthy that the biotransformation of 2 by Fusarium culmorum 

affords stereospecifically nootkatone-11R, 12-diol (7) (Noma et al., 2001a) (Figure 15.10). 

Metabolites 3–5, 12, and 13 from (+)-nootkatone (2) and 14–17 from (+)-valencene (1) did not 

show an effective odor. 

Dihydronootkatone (17), which shows that citrus odor possesses antitermite activity, was also 

treated in Aspergillus niger to obtain 11S-mono- (18) and 11R-dihydroxylated products (19) (the ratio 

11S and 11R = 3:2). On the other hand, Aspergillus cellulosae reduced ketone group at C2 of 17 to 

give 2a- (20) (75.7%) and 2b-hydroxynootkatone (21) (0.7%) (Furusawa et al., 2003) (Figure 15.11). 

Tetrahydronootkatone (22) also shows antitermite and mosquito-repellant activity. It was incubated 

with Aspergillus niger to give two similar hydroxylated compounds (23, 13.6% and 24, 9.9%) 

to those obtained from 17 (Furusawa, 2006) (Figure 15.12). 

8,9-Dehydronootkatone (25) was incubated with Aspergillus niger to give four metabolites, a 

unique acetonide (26, 15.6%), monohydroxylated (27, 0.2%), dihydroxylated (28, 69%), and a 

carboxyl derivative (29, 0.8%) (Figure 15.13).


O 

O 

O 

5 

OH 

OH 

OH 

6 7 

OH 

OH 

8 

OH 

OH 

O 

9 

OH 

OH 

HO 

10 

OH 

OH 

O 

O 

O 

11 

OH 

OH 

12 

11 

S 

O 

O 

O 13 

11 

R 

O 

O 

O 

O 

11 12 

O 

OH 

O 

OH 

14 

O 

15 

16 

O 

OH 

O 

O 

14a 

15a 

15b OH 

FIGURE 15.10 Metabolites (5–11, 14–15b) from valencene (1) and nootkatone (2) by various microorganisms. 

When the same substrate was treated in Aspergillus sojae IFO 4389, compound 25 was converted 

to the different monohydroxylated product (30, 15.8%) from that mentioned above. Aspergillus 

cellulosae is an interesting fungus since it did not give any same products as mentioned above; 

in place, it produced trinorsesquitepene ketone (31, 6%) and nitrogen-containing aromatic compound 

(32) (Furusawa et al., 2003) (Figure 15.14). 

Mucor species also oxidized compound 25 to give three metabolites, 13-hydroxy-8,9-dehydronootkatone 

(33, 13.2%), an epoxide (34, 5.1%), and a diol (35, 19.9%) (Furusawa et al., 2003). The 

same substrate was investigated with cultured suspension cells of the liverwort, Marchantia 

polymorpha to afford 33 (Hegazy et al., 2005) (Figure 15.15). 

Although Mucor species could give nootkatone (21) from valencene (1), this fungus biotransformed 

the same substrate (25) to the same alcohol (30, 13.2%) obtained from the same starting 

compound (25) in Aspergillus sojae, a new epoxide (34, 5.1%) and a diol (35, 9.9%). 

The metabolites (3, 4, 20, 21) inhibited the growth of lettuce stem, and 3 and 4 inhibited germination 

of the same plant (Hashimoto and Asakawa, 2007). 

Valerianol (35a), from Valeriana offi cinalis whose dried rhizome is traditionally used for its 

carminative and sedative properties, was biotransformed by Mucor plumbeus, to produce three 

metabolites, a bridged ether (35b), and a triol (35c), which might be formed via C1–C10 epoxide, 

and 35d arises from double dehydration (Arantes et al., 1999). In this case, allylic oxidative compounds 

have not been found (Figure 15.16).


O 

O 

H 

A. niger 

11 

R H 

18 (4%) 

OH 

17 

O 

19 (62%) 

11S:11 R = 2:3 

11 

OH 

OH 

HO 

2 

H 

O 

H 

A. cellulosae 20 (75.7%) 

17 

HO 

2 

H 

21 (0.7%) 

FIGURE 15.11 Biotransformation of dihydronootkatone (17) by Aspergillus niger and Aspergillus cellulosae. 

O 

H 

11 

O 

H 

A. niger 

23 (13.6%) 

OH 

22 

24 (9.9%) 

FIGURE 15.12 Biotransformation of tetrahydronootkatone (22) by Aspergillus niger. 

O 

H 

11 

12 

OH 

OH


O 

A. niger 

O 

11 12 

O 

R 

O 

26 (15.6%) 

O 

27 (0.2%) 

11 

OH 

25 

O 

O 

11 

12 11 

12 

COOH 

OH 

OH 

OH 

29 (0.8%) 

28 (69.7%) 

C-11 mixtures (3:2) 

O 

A. sojae 

8 days 

O 

12 

13 

25 

OH 

30 (15.8%) 

FIGURE 15.13 Biotransformation of 8,9-dehydronootkatone (25) by Aspergillus sojae. 

15.2.5 BIOTRANSFORMATION OF (+)-1(10)-ARISTOLENE (36) FROM THE CRUDE DRUG 

NARDOSTACHYS CHINENSIS BY CHLORELLA FUSCA, MUCOR SPECIES, AND ASPERGILLUS NIGER 

The structure of sesquiterpenoid, (+)-1(10)-aristolene (= calarene) (36) from the crude drug 

Nardostachys chinensis was similar to that of nootkatone. 2-Oxo-1(10)-aristolene (38) shows 

antimelanin inducing activity and excellent citrus fragrance. On the other hand, the enantiomer 

(37) of 36 and (+)-aristolone (41) were also found in the liverworts as the natural products. In order 

O 

O 

O 

A. cellulosae 

31 (6.0%) 

25 

HO 

H 

O 

OH 

O 

NH 

O 

HO 

O 

OCH 3 

32 

FIGURE 15.14 Biotransformation of 8,9-dehydronootkatone (25) by Aspergillus cellulosae.


O 

Marchantia polymorpha cells 

25 

Mucor sp. 

O 

13 

33 (13.2%) 

12 

OH 

O 

34 (5.1%) 

FIGURE 15.15 Biotransformation of 8,9-dehydronootkatone (25) by Marchantia polymorpha and Mucor 

species. 

O 

11 

12 

11 

OH 

O 

OH 

13 

35 (19.9%) 

to obtain compound 38 and its analogues, compound 36 was incubated with Chlorella fusca var. 

vacuolata IAMC-28, Mucor species, and Aspergillus niger (Furusawa et al., 2006a) (Figure 15.17). 

Chlorella fusca was inoculated and cultivated stationary in Noro medium (pH 8.0) at 25°C for 

7 days and (+)-1(10)-aristolene (36) (20 mg/50 mL) was added to the medium and further incubated 

for 10–14 days and cultivated stationary under illumination (pH 8.0) at 25°C for 7 days to afford 

1(10)-aristolen-2-one (38, 18.7%), (-)-aristolone (39, 7.1%), and 9-hydroxy-1(10)-aristolen-2-one 

(40). Compounds 38 and 40 were found in Aristolochia species (Figure 15.18). 

Mucor species was inoculated and cultivated rotatory (100 rpm) in Czapek-peptone medium 

(pH 7.0) at 30°C for 7 days. (+)-1(10)-Aristolene (36) (100 mg/200 mL) was added to the medium and 

further for 7 days. The crude metabolites contained 38 (0.9%) and 39 (0.7%) as very minor products 

(Figure 15.19). 

O 

OH 

OH 

OH 

+H 2 O 

–H 2 O 

OH 

M. plumbeus 

35c 

OH 

–H 2 O 

OH 

O 

OH 

35a 

35b 

FIGURE 15.16 Biotransformation of valerianol (35a) by Mucor plumbeus. 

35d 

OH


1 

O 

2 

9 

10 8 

3 4 

5 6 

7 

13 

11 

14 15 

36 12 

37 38 

O 

O 

OH 

O 

39 

FIGURE 15.17 Naturally occurring aristolane sesquiterpenoids. 

40 

41 

Although Mucor species produced a large amount of nootkatone (2) from valencene (1), however, 

only poor yield of similar products as those from valencene (1) was seen in the biotransformation of 

tricyclic substrate (36). Possible biogenetic pathway of (+)-1(10)-aristolene (36) is shown in 

Figure 15.20. 

Aspergillus niger was inoculated and cultivated rotatory (100 rpm) in Czapek-peptone medium 

(pH 7.0) at 30°C for 3 days. (+)-1(10)-Aristolene (36) (100 mg/200 mL) was added to the medium and 

further for 7 days. From the crude metabolites four new metabolic products (42, 1.3%), (43, 3.2%), 

(44, 0.98%), and (45, 2.8%) were obtained in very poor yields (Figure 15.21). Possible metabolic 

pathways of 36 by Aspergillus niger are shown in Figure 15.22. 

Commercially available (+)-1(10)-aristolene (36) was treated with Diplodia gossypina and 

Bacillus megaterium. Both microorganisms converted 36 to four (46–49; 0.8%, 1.1, 0.16%, 0.38%) 

and six metabolites, (40, 50–55; 0.75%, 1.0%, 1.0%, 2.0%, 1.1%, 0.5%, 0.87%), together with 40 

(0.75%) respectively (Abraham et al., 1992) (Figure 15.23). 

1(10)-Aristolene (36) 

C. fusca 

OH 

O 

O 

O 

38 

(18.7%) 

39 

(7.1%) 

FIGURE 15.18 Biotransformation of 1(10)-aristolene (36) by Chlorella fusca. 

40 

(7.0%)


Mucor sp. 

O 

O 

36 38 (0.9%) 

39 (0.7%) 

FIGURE 15.19 Biotransformation of 1(10)-aristolene (36) by Mucor species. 

C. fusca 

HO 

C. fusca 

O 

36 

Route c 

C. fusca 

Mucor sp. 

Mucor sp. 

Route a 

Route b 

C. fusca 

Mucor sp. 

Mucor sp. 

[O] 

38 

C. fusca 

H 

OH 

OH 

O 

OH 

40 

C. fusca 

Mucor sp. 

[O] 

C. fusca 

Mucor sp. 

C. fusca 

O 

39 

FIGURE 15.20 

species. 

Possible pathway of biotransformation of 1(10)-aristolene (36) by Chlorella fusca and Mucor 

O 

CO OH 

HO 

CO OH 

36 

A. niger 

6 days 

42 (1.3%) 

HO 

43 (2.7%) 

O 

CO OH 

HO 

O 

44 (0.8%) 

FIGURE 15.21 Biotransformation of 1(10)-aristolene (36) by Aspergillus niger. 

45 (2.1%) 

O


H + 

HO 

A. niger 

[O] 

H 

36 

Route a 

Route b 

[O] 

[O] 

route c 

[O] 

O 

HO 

COOH 

HO 

COOH 

38 43 

O 

H + 

O 

H 

42 

COOH 

O 

44 

COOH 

HO 

COOH 

HO 

HO 

HO 

HO 

O 

HO 

+ 

O – 

HO 

COOH 

O 

O 

H + 

45 

FIGURE 15.22 Possible pathway of biotransformation of 1(10)-aristolene (36) by Aspergillus niger. 

O 

D. gossypina 

HO 

OH 

OH 

R 

36 46 

OH 

R 

B. megaterium 

O 

47: R=CH 2 OH 

48: R=COOH 

OH 

HO 

49 

R 

HO 

R 2 

36 50: R=α-OH 

51: R=β-OH 

40 

52: R=H 

53: R=OH 

R 1 

54: R 1 =OH,R 2 =H 

55: R 1 =H,R 2 =OH 

FIGURE 15.23 Biotransformation of 1(10)-aristolene (36) by Diplodia gossypina and Bacillus megaterium.


It is noteworthy that Chlorella and Mucor species introduce hydroxyl group at C2 of the substrate 

(36) as seen in the biotransformation of valencene (1) while Diplodia gossypina and Bacillus megaterium 

oxidizes C2, C8, C9, and/or 1,1-dimethyl group on a cyclopropane ring. Aspergillus niger 

oxidizes not only C2 but also stereoselectively oxidized one of the gem-dimethyl groups on cyclopropane 

ring. Stereoselective oxidation of one of gem-dimethyl of cyclopropane and cyclobutane 

derivatives is observed in biotransformation using mammals (see later). 

15.2.6 BIOTRANSFORMATION OF VARIOUS SESQUITERPENOIDS BY MICROORGANISMS 

Aromadendrane-type sesquiterpenoids have been found not only in higher plants but also in liverworts 

and marine sources. Three aromadendrenes (56, 57, 58) were biotransformed by Diplodia 

gossypina, Bacillus megaterium, and Mycobacterium smegmatis (Abraham et al., 1992). 

Aromadendrene (56) (800 mg) was converted by Bacillus megaterium to afford a diol (59) and a 

triol (60) of which 59 (7 mg) was the major product. The triol (60) was also obtained from the 

metabolite of (+)-(1R)-aromadendrene (56) by the plant pathogen Glomerella cingulata (Miyazawa 

et al., 1995a). allo-Aromadendrene (57) (1.2 g) was also treated in Mycobacterium smegmatis to 

afford 61 (10 mg) (Abraham et al., 1992) (Figure 15.24). 

The same substrate was also incubated with Glomerella cingulata to afford C10 epimeric triol 

(62) (Miyazawa et al., 1995a). Globulol (58) (400 mg) was treated in Mycobacterium smegmatis to 

give only a carboxylic acid (63) (210 mg). The same substrate (58) (1 g) was treated in Diplodia 

gossypina and Bacillus megaterium to give two diols, 64 (182 mg), 65 and a triol (66) from the 

former and 67–69 from the latter organism among which 64 (60 mg) was predominant (Abraham 

et al., 1992). Glomerella cingulata and Botrytis cinerea also bioconverted globulol (58) to diol (64) 

regio- and stereoselectively (Miyazawa et al., 1994) (Figures 15.25 and 15.26). 

H 

H 

OH 

OH 

H 

OH 

OH 

B. megaterium 

H 

H 

H 

OH 

56 59 

60 

H 

H 

M. smegmatis 

HO 

H 

57 61 

H 

OH 

H 

OH 

M. smegmatis 

H 

58 

63 

FIGURE 15.24 Biotransformation of aromadendrene (56), alloaromadendrene (57), and globulol (58) by 

Bacillus megaterium and Mycobacterium smegmatis. 

H 

COOH


OH 

H 

G. cingulata 

H 

O 

H 

OH 

H 

H 

H 

OH 

56 

60 

H 

G. cingulata 

H 

O 

HO 

HO 

H 

H 

H 

H 

OH 

57 

FIGURE 15.25 Biotransformation of aromadendrene (56) and alloaromadendrene (57) by Glomerella 

cingulata. 

62 

Globulol (58) (1.5 g) and 10-epiglobulol (70) (1.2 mL) were separately incubated with 

Cephalosporium aphidicola in shake culture for 6 days to give the same diol 64 (780 mg) as obtained 

from the same substrate by Bacillus megaterium mentioned above and 71 (720 mg), (Hanson 

et al., 1994). Aspergillus niger also converted globulol (58) and epiglobulol (70) to a diol (64) and 

OH 

H 

OH 

H H 

OH OH 

D. gossypina 

H 

OH 

B. megaterium 

H 

OH 

H H 

HO 

OH 

64 

65 66 

H 

58 

B. megaterium 

H 

OH 

HO 

H 

OH 

HO 

H 

OH 

H H H 

OH 

67 68 

69 

H 

OH 

A. niger 

G. cingulata 

Cephalosporium aphidicola 

H 

OH 

H 

H 

OH 

58 

FIGURE 15.26 Biotransformation of globulol (58) by various microorganisms. 

64


three 13-hydroxylated globulol (71, 72, 74) and 4a-hydroxylated product (73). The epimerization at 

C4 is very rare example (Hayashi et al., 1998). 

Ledol (75), an epimer at C1 of globulol was incubated with Glomerella cingulata to afford C13 

carboxylic acid (76) (Miyazawa et al., 1994) (Figure 15.27). 

Squamulosone (77), aromadendr-1(10)-en-9-one isolated from Hyptis verticillata (Labiatae), was 

reduced chemically to give 78–82, which were incubated with the fungus Curvularia lunata in two 

different growth media (Figure 15.28). 

From 78, two metabolites 80 and 83 were obtained. Compound 79 and 80 were metabolized to 

give ketone 81 as the sole product and 78 and 83, respectively. From compound 81, two metabolites, 

79 and 84 were obtained (Figure 15.29). From the metabolite of the substrate (82), five products 

(84–88) were isolated (Collins, Reynold, and Reese, 2002) (Figure 15.30). 

Squamulosone (77) was treated in the fungus Mucor plumbeus ATCC 4740 to give not only 

cyclopentanol derivatives (89, 90) but also C12 hydroxylated products (91–93) (Collins, Ruddock, 

et al., 2002) (Figure 15.31). 

Spathulenol (94), which is found in many essential oils, was fed by Aspergillus niger to give a 

diol (95) (Higuchi et al., 2001). Ent-10b-hydroxycyclocolorenone (96) and myli-4(15)-en-9-one 

(96a) isolated from the liverwort Mylia taylorii were incubated with Aspergillus niger IFO 4407 to 

give C10 epimeric product (97) (Hayashi et al., 1999) and 12-hydroxylated product (96b), respectively 

(Nozaki et al., 1996) (Figures 15.32 and 15.33). 

H HO 

C. aphidicola 

H HO 

A. niger 

H 

H 

OH 

70 

71 

A. niger 

H HO 

H HO 

H HO 

OH 

HO 

H 

H 

OH 

OH 

H 

OH 

72 73 74 

HO 

H 

G. cingulata 

HO 

H 

HO 

H 

H 

75 

H 

FIGURE 15.27 Biotransformation of 10-epi-glubulol (70) and ledol (75) by Cephalosporium aphidicola, 

Aspergillus niger, and Glomerella cingulata. 

CHO 

H 

76 

COOH


O 

H 

H 

OH 

77 

H 

H 

O 

78 

H 

80 

H 

OH 

C. lunata in growth medium 1 


H 

83 

FIGURE 15.28 Biotransformation of aromadendra-9-one (80) by Curvularia lunata. 

(+)-ent-Cyclocolorenone (98) [a] D - 405° (c = 8.8, EtOH), one of the major compounds isolated 

from the liverwort Plagiochila sciophila (Asakawa, 1982, 1995), was treated by Aspergillus niger to 

afford three metabolites, 9-hydroxycyclocolorenone (99, 15.9%) 12-hydroxy-(+)-cyclocolorenone 

(100, 8.9%) and a unique cyclopropane-cleaved metabolite, 6b-hydroxy-4,11-guaiadien-3-one 

(101, 35.9%), and 6b,7b-dihydroxy-4,11-guaiadien-3-one (102, trace), of which 101 was the major 

component. The enantiomer (103) [a] D + 402° (c = 8.8, EtOH) of 98 isolated from Solidago altissima 

was biotransformed by the same organism to give 13-hydroxycycolorenone (103a, 65.5%), the 

enantiomer of 100, 1b,13-dihydroxycyclocolorenone (103b, 5.0%), and its C11-epimer (103c) 

H 

OH 

H 

O 

H 

79 

H 

81 

H 

O 



H 

HO 

84 

FIGURE 15.29 Biotransformation of 10-epi-aromadendra-9-one (81) by Curvularia lunata.


OH OH 

HO HO 

OH OH 

HO 

OH 

H H H 

C. lunata 

85 

86 

87 

82 

H 

HO 

OH 

HO 

O 

H 

H 

88 

89 

FIGURE 15.30 Biotransformation of aromadendr-1(10),9-diene (82) by Curvularia lunata. 

(Furusawa et al., 2005b, 2006a). It is noteworthy that no cyclopropane-cleaved compounds from 103 

have been detected in the crude metabolites even in GC-MS analysis (Figure 15.34). 

Plagiochiline A (104) that shows potent insect antifeedant, cytotoxicity, and piscidal activity are 

very pungent 2,3-secoaromadendrane sesquiterpenoids having 1,1-dimethyl cyclopropane ring, 

isolated from the liverwort Plagiochila fruticosa. Plagiochilide (105) is the major component of this 

liverwort. In order to get more pungent component, the lactone (105, 101 mg) was incubated with 

O 

H 

M. plumbeus 

77 

HO 

O 

HO 

O 

O 

H 

H 

H 

89 

90 

91 

OH 

HO 

O 

HO 

O 

H 

H 

OH 

OH 

92 

93 

FIGURE 15.31 Biotransformation of squamulosone (77) by Mucor plumbeus.


H 

H 

OH 

OH 

A. niger 

HO 

HO 

H 

94 95 

FIGURE 15.32 Biotransformation of spathulenol (94) by Aspergillus niger. 

Aspergillus niger to give two metabolites 106 (32.5%) and 107 (9.7%). Compound 105 was incubated 

in Aspergillus niger including 1-aminobenzotriazole, the inhibitor of CYP450, to produce 

only 106, since this enzyme plays an important role in the formation of carboxylic acid (107) from 

primary alcohol (106). Unfortunately, two metabolites show nothing hot taste (Hashimoto et al., 

2003c; Furusawa et al., 2006) (Figure 15.35). 

Partheniol, 8a-hydroxybicyclogermacrene (108) isolated from Parthenium argentatum × 

Parthe nium Tometosa, was cultured in the media of Mucor circinelloides ATCC 15242 to afford 

six metabolites, a humulane (109), three maaliane- (110, 112, 113), an aromadendrane- (111), and a 

tricylohumulane triol (114), the isomer of compound (111). Compounds 110, 111, and 114 were 

isolated as their acetates (Figure 15.36). 

Compounds 110 might originate from the substrate by acidic transannular cyclization since the 

broth was pH 6.4 just before extraction (Maatooq, 2002). 

H 

A. niger 

H 

HO 

H 

HO 

H 

94 95 

O 

H 

OH 

A. niger 

O 

HO 

H 

96 97 

H 

O 

A. niger 

H 

O 

HO 

96a 

96b 

FIGURE 15.33 Biotransformation of spathulenol (94), ent-10b-hydroxycyclocolorenone (96) and myli-4- 

(15)-en-9-one (96a) by Aspergillus niger.


O 

H 

9 

OH 

O 

H 

9 

6 7 

A. niger 

99 (15.9%) 

H 

H 

H 

(+)-Cyclocolorenone (98) 

from Plagiochila sciophila 

O 

6 7 

O 

OH 

100 

6 7 

HO 

101 (35.9%) 

O 

6 7 

HO 

102 

OH 

H 

H 

HO 

H 

O 

A. niger 

O 

O 

1 

O 

(–)-Cyclocolorenone (103) 

from Solidago altissima 

103a (65.5%) 

OH 

103b (5.0%) 

OH 

HO 

103c 

FIGURE 15.34 Biotransformation of (+)-cyclocolorenone (98) and (-)-cyclocolorenone (103) by Aspergillus 

niger. 

Ac O 

O 

H 

H 

O 

Ac O 

O 

O 

H 

104 

A. niger 

O 

O 

H 

H 

A. niger 

1-aminobenzotriazole 

H 

105 

106 

OH 

Cyp-450 

O 

O 

H 

Cyp-450 

O 

O 

H 

H 

H 

HOOC 

OHC 

107 

FIGURE 15.35 Biotransformation of plagiochiline C (104) by Aspergillus niger.


OAc 

OH 

OH 

OH 

M. circinelloides 

HO 

109 110 

HO 

111 

108 

OH 

OH 

OH 

OH 

HO 

OH 

HO 

H 

OH 

HO 

112 

113 

114 

FIGURE 15.36 Biotransformation of 8a-hydroxybicyclogermacrene (108) by Mucor circinelloides. 

The same substrate (108) was incubated with the fungus Calonectria decora to afford six new 

metabolites (108a–108f). In these reactions, hydroxylation, epoxidation, and trans-annular cyclization 

were evidenced (Maatooq, 2002b) (Figure 15.37). 

ent-Maaliane-type sesquiterpene alcohol, 1a-hydroxymaaliene (115), isolated from the liverwort 

Mylia taylorii, was treated in Aspergillus niger to afford two primary alcohols (116, 117) 

(Morikawa et al., 2000). Such an oxidation pattern of 1,1-dimethyl group on the cyclopropane ring 

has been found in aromadendrane series as described above, and mammalian biotransformation of 

a monoterpene hydrocarbon, D 3 -carene (Ishida et al., 1981) (Figure 15.38). 

9(15)-Africanene (117a), a tricyclic sesquiterpene hydrocarbon isolated from marine soft corals 

of Simularia species, was biotransformed by Aspergillus niger and Rhizopus oryzae for 8 days 

to give 10a-hydroxy- (117b) and 9a,15-epoxy derivative (117c) (Venkateswarlu et al., 1999) 

(Figure 15.39). 

Germacrone (118), (+)-germacrone-4,5-epoxide (119), and curdione (120) isolated from Curcuma 

aromatica, which has been used as crude drug, was incubated with Aspergillus niger. From compound 

119 (700 mg), two naturally occurring metabolites, zedoarondiol (121) and isozedoarondiol 

(122), were obtained (Takahashi, 1994). Compound (119) was cultured in callus of Curcuma zedoaria 

and Curcuma aromatica to give the same secondary metabolites 121, 122, and 124 (Sakui 

et al., 1988) (Figures 15.40 and 15.41). 

OH 

OH 

OH 

OH 

108 

OH 

Calonectria decora 

108a 108b 108c 

OH 

OH 

OH 

O 

OH 

HO 

108d 108e 108f 

FIGURE 15.37 Biotransformation of 8a-hydroxybicyclogermacrene (108) by Calonectria decora.


OH OH O 

A. niger 

H H H 

HO 

HO 

115 116 117 

FIGURE 15.38 Biotransformation of 1a-hydroxymaaliene (115) Aspergillus niger. 

H 

A. niger 

H 

O 

H 

HO 

H 

R. oryzae 

H 

H 

117a 

117b 

117c 

FIGURE 15.39 Biotransformation of 9(15)-africanene (117a) by Aspergillus niger and Rhizopus oryzae. 

H 

OH 

O 

O 

A. niger 

O 

HO 

H 

121 

O 

HO 

H 

118 

119 

O 

O 

HO 

H 

122 

HO 

123 

FIGURE 15.40 Biotransformation of germacrone (118) by Aspergillus niger. 

O 

Curcumazedoaria and 

C. aromatica cells 

O 

HO 

H 

O 

O 

HO 

H 

118 

119 

122 

O 

HO 

FIGURE 15.41 Biotransformation of germacrone (118) by Curcuma zedoaria and Curcuma aromatica cells. 

123


O 

C. blakesleeana 

O 

p-TsOH 

O 

118 

O 

119 

C. zedoaria 

OH 

124 

HO 

H 

OH 

H 

121 

O 

HO 

OH 

H 

H 

122 

O 

O 

H 

129 

O 

C. zedoaria 

O 

C. zedoaria 

O 

O 

O 

118 

O 

125 

FIGURE 15.42 Biotransformation of germacrone (118) by Cunninghamella blakeseeana and Curcuma 

zedoaria cells. 

126 

Aspergillus niger biotransformed germacrone (118, 3g) to very unstable 3b-hydroxygermacrone 

(123), and 4,5-epoxygermacrone (119) which was further converted to two guaiane sesquiterpenoids 

(121) and (122) through trans-annular-type reaction (Takahashi, 1994). The same substrate was 

incubated in the microorganism, Cunninghamella blakeseeana to afford germacrone-4,5-epoxide 

(119) (Hikino et al., 1971) while the treatment of 118 in the callus of Curcuma zedoaria gave four 

metabolites 121, 122, 125, and 126 (Sakamoto et al., 1994) (Figure 15.42). 

The same substrate (118) was treated in plant cell cultures of Solidago altissima (Asteraceae) for 

10 days to give various hydroxylated products (121, 127, 125, 128–132) (Sakamoto et al., 1994). 

Guaiane (121) underwent further rearrangement C4–C5, cleavage and C5–C10 trans-annular cyclization 

to the bicyclic hydroxyketone (128) and diketone (129) (Sakamoto et al., 1994) (Figure 15.43). 

Curdione (120) was also treated in Aspergillus niger to afford two allylic alcohols (133, 134) and 

a spirolactone (135). Curcuma aromatica and Curcuma wenyujin produced spirolactone (135) 

which might be formed from curdione via trans-annular reaction in vivo was biotransformed to 

spirolactone diol (135) (Asakawa et al., 1991; Sakui et al., 1992) (Figure 15.44). 

Aspergillus niger also converted shiromodiol diacetate (136) isolated from Neolitsea sericea to 

2b-hydroxy derivative (137) (Nozaki et al., 1996) (Figure 15.45). 

Twenty strains of filamentous fungi and four species of bacteria were screened initially by thin 

layer chromatography (TLC) for their biotransformation capacity of curdione (120). Mucor spinosus, 

Mucor polymorphosporus, Cunninghamella elegans, and Penicillium janthinellum were found to 

be able to biotransform curdione (120) to more polar metabolites. Incubation of curdione with 

Mucor spinosus, which was most potent strain to produce metabolites, for 4 days using potato 

medium gave five metabolites (134, 134a–134d) among which compounds 134c and 134d are new 

products (Ma et al., 2006) (Figure 15.46). 

Many eudesmane-type sesquiterpenoids have been biotransformed by several fungi and various 

oxygenated metabolites obtained. 

b-Selinene (138) is ubiquitous sesquiterpene hydrocarbon of seed oil from many species of 

Apiaceae family; for example, Cryptotenia canadensis var. japonica, which is widely used as 

vegetable for Japanese soup. b-Selinene was biotransformed by plant pathogenic fungus Glomerella


O 

O 

O 

O 

H 

OH 

O 

118 

Cell culture 

(Solidago altissima) 

OH – 

O 

O 

H + 127 

HO 

H 

121 

HO 

O 

R 

130 

OH 

H 

O 

H + OH – 131 

O 

O 

H 2 O 

OH 

O 

OH 

O 

128: R=OH 

129: R=O 

HO 

H 

H 

OH 

132 

126 

FIGURE 15.43 Biotransformation of germacrone (118) by Solidago altissima cells. 

cingulata to give an epimeric mixtures (1:1) of 1b,11,12-trihydroxy product (139) (Miyazawa 

et al., 1997a). The same substrate was treated in Aspergillus wentii to give 2a,11,12-trihydroxy 

derivative (140) (Takahashi et al., 2007). 

Eudesm-11(13)-en-4,12-diol (141) was biotransformed by Aspergillus niger to give 3b-hydroxy 

derivative (142) (Hayashi et al., 1999). 

a-Cyperone (143) was fed by Collectotrichum phomoides (Lamare and Furstoss, 1990) to afford 

11,12-diol (144) and 12-manool (145) (Higuchi et al., 2001) (Figure 15.47). 

The filamentous fungi Gliocladium roseum and Exserohilum halodes were used as the bioreactors 

for 4b-hydroxyeudesmane-1,6-dione (146) isolated from Sideritis varoi subsp. cuatrecasasii. The 

former fungus transformed 146 to 7a-hydroxyl- (147), 11-hydroxy- (148), 7a,11-dihydroxy- (149), 

1a,11-dihydroxy- (150), and 1a,8a-dihydroxy derivatives (151) while Exserohilum halodes gave 

only 1a-hydoxy product (152) (Garcia-Granados et al., 2001) (Figure 15.48). 

O 

A. niger 

HO 

O 

HO 

O 

O 

120 

O 

133 

OH 

O 

134 

H 

O 

OH 

OH 

–H + O O 

H 2 

O O O 

135 

FIGURE 15.44 Biotransformation of curdione (120) by Aspergillus niger.


O 

OAc 

OAc 

A. niger 

HO 

O 

OAc 

OAc 

136 137 

FIGURE 15.45 Biotransformation of shiromodiol diacetate (136) by Aspergillus niger. 

Orabi (2000) reported that Beauvaria bassiana is the most efficient microorganism to metabolize 

plectanthone (152a) among 20 microorganisms, such as Absidia glauca, Aspergillus fl avipes, 

Beauvaria bassiana, Cladosporium resinae, Penicillium frequentans, and so on. The substrate 

(152a) was incubated with Beauvaria bassiana to give metabolites 152b (2.1%), 152c (21.2%), 152d 

(2.5%), 152e (no data), and 152f (1%) (Figure 15.49). 

(-)-a-Eudesmol (153) isolated from the liverwort Porella stephaniana was treated by Aspergillus 

cellulosae and Aspergillus niger to give 2-hydroxy (154) and 2-oxo derivatives (155), among which 

the latter product was predominantly obtained. This bioconversion was completely blocked by 1-aminobenzotriazole, 

CYP450 inhibitor. Compound 155 has been known as natural product, isolated from 

Pterocarpus santalinus (Noma et al., 1996). Biotransformation of a-eudesmol (153) isolated from the 

dried Atractylodes lancea was reinvestigated by Aspergillus niger to give 2-oxo-11,12-dihydro-aeudesmol 

(156) together with 2-hydroxy- (154), and 2-oxo-a-eudesmol (155). b-Eudesmol (157) was 

treated in Aspergillus niger, with the same culture medium to afford 2a- (158) and 2b-hydroxy-aeudesmol 

(159) and 2a,11,12-trihydroxy-b-eudesmol (160) and 2-oxo derivative (161), which was 

further isomerized to compound 162 (Noma et al., 1996, 1997) (Figure 15.50). 

Three new hydroxylated metabolites (157b–157d) along with a known 158 and (157e–157g) 

were isolated from the biotransformation reaction of a mixture of b- (157) and g-eudesmols (157a) 

by Gibberella suabinetii. The metabolites proved a super activity of the hydroxylase, dehydrogenase, 

and isomerase enzymes. The hydroxylation is a common feature; on the contrary, cyclopropyl 

ring formation like compound (158d) is very rare (Maatooq, 2002a) (Figure 15.51). 

O 

O 

O 

O 

O 

O 

134a 

134b 

O 

M. spinosus 

HO 

O 

O 

O 

O 

O 

OH 

120 

134 134c 

HO 

O 

O 

OH 

134d 

FIGURE 15.46 Biotransformation of curdione (120) by Mucor spinosus.


OH 

OH 

138 

G. cingulata 

H H O 

H 

139 

OH 

OH 

A. wentii 

HO 

H 

H 

138 140 

A. niger 

HO 

H 

OH 

HO 

HO 

H 

OH 

141 

142 

C. phomoides 

O O O 

OH 

143 144 

OH 

145 

FIGURE 15.47 Biotransformation of eudesmenes (138, 141, 143) by Aspergillus wentii, Glomerella cingulata, 

and Collectotrium phomoides. 

A furanosesquiterpene, atractylon (163) obtained from Atractylodis rhizoma was treated with 

the same fungus to yield atractylenolide III (164) possessing inhibition of increased vascular permeability 

in mice induced by acetic acid (Hashimoto et al., 2001). 

OH 

O 

R 1 

HO 

Gliocladium roseum 

O 

146 

Exserohilum halodes 

OH 

HO 

O 

R 4 

R 2 

R 3 

147: R 1 

=O, R 2 

=OH, R 3 

=R 4 

=H 

148: R 1 

=O, R 2 

=R 4 

=H, R 3 

=OH 

149: R 1 

=O, R 2 

=R 3 

=OH, R 4 

=H 

150: R 1 

=αOH, βH,R 2 

=R 4 

=H, R 3 

=OH 

151: R 1 

=αOH, βH,R 2 

=R 3 

=H, R 4 

=OH 

HO 

O 

152 

FIGURE 15.48 Biotransformation of 4b-hydroxy-eudesmane-1,6-dione (146) by Gliocladium roseum and 

Exserohilum halodes.


OAc 

OH 

O 

O 

H 

OAc 

OH 

H 

OAc 

152b 

152c 

O 

OAc 

B. bassiana 

O 

OH 

OH 

O 

OAc 

H 

OAc 

H 

OAc 

H 

OH 

OH 

152a 

152d 

152e 

OH 

O 

H 

OAc 

HO 

152f 

FIGURE 15.49 Biotransformation of eudesmenone (152a) by Beauvaria bassiana. 

The biotransformation of sesquiterpene lactones have been carried out by using different 

microorganisms. 

Costunolide (165), a very unstable sesquiterpene g-lactone, from Saussurea radix, was treated in 

Aspergillus niger to produce three dihydrocostunolides (166–168) (Clark and Hufford, 1979). 

Costunolide is easily converted into eudesmanolides (169–172) in diluted acid, thus 166–168 might 

be biotransformed after being cyclized in the medium including the microorganisms. If the crude 

A. niger HO 

A. cellulosae 

H 

OH 

H OH 

H 

153 154 155 

O 

OH 

O 

H 

156 

OH 

OH 

HO 

A. niger 

HO HO 

H 

OH 

H OH 

H 

OH H 

157 159 158 160 

OH 

OH 

O 

O 

O 

H OH 

H 

161 162 

OH 

H 

156 

OH 

OH 

FIGURE 15.50 Biotransformation of a-eudesmol (153) and b-eudesmol (157) by Aspergillus niger and 

Aspergillus cellulosae.


HO 

OH 

G. suabinetii 

H 

157b 

OH 

H 

157c 

OH 

H 

OH 

HO 

157 

H 

OH 

OH 

H 

OH 

157d 

158 

G. suabinetii 

HO 

157e 

OH 

O 

157f 

OH 

OH 

157a 

OH 

OH 

FIGURE 15.51 Biotransformation of b-eudesmol (157) and g-eudesmol (157a) by Gibberella suabinetii. 

157g 

drug including costunolide (165) is orally administered, 165 will be easily converted into 169–172 

by stomach juice (Figure 15.52). 

(+)-Costunolide (165), (+)-cnicin (172a), and (+)-salonitgenolide (172b) were incubated with 

Cunninghamella echinulata and Rhizopus oryzae. 

The former fungus converted compound 165, to four metabolites, (+)-11b,13-dihydrocostunolide 

(165a), 1b-hydroxyeudesmanolide, (+)-santamarine (166a), (+)-reynosin (166b), and (+)-1b-hydroxyarbusculin 

A (168a), which might be formed from 1b,10a-epoxide (166c). Treatment of 172a with 

Cunninghamella echinulata gave (+)-salonitenolide (172b) (Barrero et al., 1999) (Figure 15.53). 

a-Cyclocostunolide (169), b-cyclocostunolide (170), and g-cyclocostunolide (171) prepared from 

costunolide were cultivated in Aspergillus niger, respectively. From the metabolite of 169, four 

dihydro lactones (173–176) were obtained, among which sulfur-containing compound (176) was 

predominant (Figure 15.54). 

The same substrate (169) was cultivated for 3 days by Aspergillus cellulosae to afford a sole 

metabolite, 11b,13-dihydro-a-cyclocostunolide (177). Possible metabolic pathways of 169 by both 

microorganisms were shown in Figure 15.55. 

A double bond at C11–C13 of 169 was firstly reduced stereoselectively to afford 177, followed by 

oxidation at C2 to give 173, and then further oxidation occurred to furnish two hydroxyl derivatives 

(174, 175) in Aspergillus niger. The sulfide compound (176) might be formed from 175 or by Michel 

condensation of ethyl 2-hydroxy-3-mercaptopropanate, which might originate from Czapek-peptone 

medium into exomethylene group of a-cyclocostunolide (Hashimoto et al., 1999a, 2001).


O 

A. niger 

OH 

O 

O 

H 

163 164 

H 

OH 

OH 

A. niger 

H 

H 

H 

O 

O 

O 

HO 

O 

O 

O 

165 166 167 168 

O 

O 

H 

169 

O 

O 

H 

H 

O 

HO 

O 

O 

O 

O 

170 171 172 

O 

FIGURE 15.52 Biotransformation of atractylon (163) and costunolide (165) by Aspergillus niger. 

Aspergillus niger converted b-cyclocostunolide (170) to 2-oxygenated metabolites (173, 174, 

178–181) of which 173 was predominant. It is suggested that compound 173 and 174 might be formed 

during biotransformation period since metabolite media after 7 days was acidic (pH 2.7). Surprisingly, 

Aspergillus cellulose gave a sole 11b,13-dihydro-b-cyclocostunolide (182), which was abnormally 

folded in the mycelium of Aspergillus cellulosae as a crystal form after biotransformation of 170. On 

the other hand, the metabolites were normally liberated in medium outside of the mycelium of 

Aspergillus niger and Botryosphaeria dothidea (Hashimoto et al., 1999a, 2001) (Figure 15.56). 

Botryosphaeria dothidea has no stereoselectivity to reduce C11–C13 double bond of b-cyclocostunolide 

(170) since this organism gave two dihydro derivatives 182 (16.7%) and 183 (37.8%), respectively, 

as shown in Figure 15.57. 

It is noteworthy that both a- and b-cyclocostunolides were biotransformed by Aspergillus niger 

to give the sulfur-containing metabolites (176, 181). Possible biogenetic pathway of 170 is shown in 

Figure 15.58. 

When g-cyclocostunolide (171) was cultivated in Aspergillus niger to give dihydro-a-santonin 

(187, 25%) and its related C11,C13 dihydro derivatives (184–186, 188, 189) were obtained as a small 

amount. Compound 186 was recultivated for 2 days by the same organism as mentioned above to 

afford 187 (25%) and 5b-hydroxy-a-cyclocostunolide (189, 54%). Recultivation of 185 for 2 days by 

Aspergillus niger afforded compound 187 as a sole metabolite. During the biotransformation of 171, 

no sulfur-containing product was obtained. Both Aspergillus cellulosae and Botryosphaeria 

dothidea produced only dihydro-g-cyclocostunolide (184) from the substrate (171) (Hashimoto 

et al., 1999a, 2001) (Figure 15.59). 

Santonin (190) has been used as vermicide against round warm. Cunninghamella blakesleeana 

and Aspergillus niger converted 190–187 (Atta-ur Rahman et al., 1998). When 187 was fed by 

Aspergillus niger for one week to give 2b-hydroxy-1,2-dihydro-a-santonin (188, 39%) as well as 

1b-hydroxy-1,2-dihydro-a-santonin (195, 6.5%), 9b-hydroxy-1,2-dihydro-a-santonin (196, 6.9%), 

and a-santonin (190, 5.4%), which might be obtained from dehyroxylation of 188, as a minor


OH 

Cunninghamella 

echinulata 

165a 

O 

O 

H 

O 

166a 

O 

165 

O 

O 

OH 

OH 

H 

O 

166b 

O 

HO 

H 

168a 

O 

O 

H 

O 

166c 

O 

O 

O 

O 

OH 

OH 

C. echinulata 

OH 

HO 

172a 

O 

O 

FIGURE 15.53 Biotransformation of costunolide (165) and its derivative (172a) by Cunninghamella 

vechinulata and Rhizopus oryzae. 

HO 

O 

172b 

O 

component (Hashimoto et al., 2001). Compound 188 was isolated from the crude metabolite of 

g-cyclocostunolide (171) by Aspergillus niger as mentioned above (Figure 15.60). 

It was treated with Aspergillus niger for 7 days to give 191 (18.3%), 192 (2.3%), 193 (19.3%), and 

194 (3.5%) of which 193 was the major metabolite. Compound 191 was isolated from dog’s urine 

after the oral administration of 190. The structure of compound 194 was established as lumisantonin 

obtained by the photoreaction of 190. a-Santonin 190 was not converted into 1,2-dihydro 

derivative by Aspergillus niger, whereas the other strain of Aspergillus niger gave a single product, 

1,2-dihydro-a-santonin (187) (Hashimoto et al., 2001) (Figure 15.61). 

Ata and Nachtigall (2004) reported that a-santonin (190) was incubated with Rhizopus stolonifer 

to give (187a), and (183b), while with Cunninghamella bainieri, Cunninghamella echinulata, and 

Mucor plumbeus to afford the known 1,2-dihydro-a-santonin (187) (Figure 15.62). 

a-Santonin (190) and 6-epi-a-santonin (198) were cultivated in Absidia coerulea for 2 days to 

give 11b-hydroxy- (191, 71.4%) and 8a-hydroxysantonin (197, 2.0%), while 6-epi-santonin (198) 

afforded four major products (199–201, 206) and four minor analogues (202, 203–205). Asparagus 

offi cinalis also biotransformed a-santonin (190) into three eudesmanolides (187, 207, 208) and a 

guaianolide (209) in a small amount. 6-Epi-santonin (198) was also treated in the same bioreactor


1 

2 

A. cellulosae 

H 

13 3 days 

H 

O 

O 

169 O 

177 

O 

A. niger 

3 days 

O 

O 

OH 

H 

O 

173 

O 

H 

O 

174 

O 

O 

2 

H 

O 

175 

O 

O 

13 

OH 

2 

H 

O 

176 

O 

COOEt 

13 

CH 2 -S-CH 2 -CH 

OH 

FIGURE 15.54 Biotransformation of a-cyclocostunolide (169) by Aspergillus niger and Aspergillus 

cellulosae. 

O 

OH 

2 1 

H 

O 

174 

O 

2 

13 

H 

H 

O 

O 

169 O 

177 

O 

O 

H 

O 

173 

O 

HS-CH 2 -CH-COOEt 

OH 

O 

2 

H 

O 

176 

O 

13 

CH 2 -S-CH 2 -CH-COOEt 

OH 

O 

A. niger 

A. cellulosae 

2 

H 

175 

O 

O 

13 

OH 

FIGURE 15.55 Possible pathway of biotransformation of a-cyclocostunolide (169) by Aspergillus niger and 

Aspergillus cellulosae.


2 1 13 

H 

O 

170 

O 

A. niger (7 days) 

OH 

O 

2 

O 

1 

HO 

2 

H 

173 

O 

O 

H 

174 

O 

O 

H 

178 

O 

O 

HO 

OH 

HO 

2 

H O 

O 

179 

H 

180 

O 

O 

OH 

HO 

2 

H 

181 

O 

O 

13 


OH 

FIGURE 15.56 Biotransformation of b-cyclocostunolide (170) by Aspergillus niger. 

NOE 

FIGURE 15.57 

dothidea. 

6 

7 

H 11 13 

O 

170 O 

H 7 H 

H 

6 

H 11 

O 

13 

182 O 

H 

H 

O 

183 

B. dothidea (16.7%) 

A. cellulosae (78.0%) 

NaBH 4 /EtOAc (76.8%) 

Biotransformation of b-cyclocostunolide (170) by Aspergillus cellulosae and Botryosphaeria 

H 

O 

H 

NOE 

B.dothidea (37.8%) 

A.cellulosae (0.0%) 

NaBH 4 /EtOAc (1.2%)


2 1 4 

H 

11 13 

O 

170 O 

HO 

2 

H 

O 

181 

O 

13 


OH 

HO 

2 

H 

O 

180 

O 

OH 

HO 

2 

HO 

2 1 OH 

H 

11 

O 

182 O 

13 

H 

O 

178 

O 

H 

O 

179 

O 

A. niger 

A. cellulosae 

O 

4 

H 

O 

O 

O 

2 

4 

H 

O 

173 

O 

O 

OH 

1 

H 

O 

174 

O 

FIGURE 15.58 Possible pathway of biotransformation of b-cyclocostunolide (170) by Aspergillus niger and 

Aspergillus cellulosae. 

HO 

3 

5 

185 

O 

HO 

2 

O 

O 188 

O 

O 

O 

171 O 

184 

O 

11 

O 

13 

O 

3 

O 

187 

O 

A. niger 

A. cellulosae 

B. dothidea 

HO 

3 

186 

O 

O 

5 

OH 

O 

189 

O 

FIGURE 15.59 Biotransformation of g-cyclocostunolide (171) by Aspergillus niger, Aspergillus cellulosae, 

and Botryosphaeria dothidea.


A. niger 

HO 

OH 

O 

187 

O 

O 

O 188 

O 

O 

O 195 

O 

O 

OH 

O 

O 

196 O 

FIGURE 15.60 Biotransformation of dihydro-a-santonin (187) by Aspergillus niger. 

as mentioned above to give 199 and 206, the latter of which was obtained as a major metabolite 

(44.7%) (Yang et al., 2003) (Figure 15.63). 

a-Santonin (190) was incubated in the cultured cells of Nicotiana tabacum and the liverwort 

Marchantia polymorpha. Nicotiana tabacum cells gave 1,2-dihydro-a-santonin (187) (50%) for 

6 days. The latter cells also converted a-santonin to 1,2-dihydro-a-santonin, but conversion ratio 

was only 28% (Matsushima et al., 2004) (Figure 15.64). 

6-Epi-a-santonin (198) and its tetrahydro analogue (210) were also incubated with fungus 

Rhizopus nigricans to give 2a-hydroxydihydro-a-santonin (211) (Amate et al., 1991), the epimer of 

188 obtained from the biotransformation of dihydro-a-santonin (187) by Aspergillus niger 

(Hashimoto et al., 2001). The product 211 might be formed via 1,2-epoxide of 198. Compound 210 

was converted through carbonyl reduction to furnish 212 and 213 under epimerization at C4 (Amate 

et al., 1991) (Figure 15.65). 

OH – 

10 

9 

OH 

10 9 11 

O 

4 

O 

190 

O 

13 

H + O 

HO 4 

O 

O 

O 

193 

O 

O 

191 

O 

11 

O 

OH 

O 

O 

O 

O 

O 

O 

A. niger 

Dog 

O 

192 

FIGURE 15.61 Biotransformation of a-santonin (190) by Aspergillus niger and dogs. 

O 

O 

13 

OH 

O 

O 

194 

O


b,c,d 

O 

O 

187 

O 

O 

190 

O 

O 

a 

O 

O 

187a 

O 

a: Rhizopus stolonifera 

b: Cunninghamella bainieri 

c : Cunninghamella echinulata 

d: Mucor plumbeus 

FIGURE 15.62 Biotransformation of a-santonin (190) by Rhizopus stolonifera, Cunninghamella bainieri, 

Cunninghamella echinulata, and Mucor plumbeus. 

1,2,4b,5a-Tetrahydro-a-santonin (214) prepared from a-santonin (190) was treated with 

Aspergillus niger to afford six metabolites (215–220) of which 219 was the major product (21%). 

When the substrate (214) was treated with CYP450 inhibitor, 1-aminobenzotriazole, only 215 was 

obtained without its homologues, 216–220, while the C4 epimer (221) of 214 was converted by the 

same microorganism to afford a single metabolite (222) (73%). Further oxidation of 222 did not 

occur. This reason might be considered by the steric hindrance of b (axial) methyl group at C4 

(Hashimoto et al., 2001) (Figure 15.66). 

7a-Hydroxyfrullanolide (223) possessing cytotoxicity and antitumor activity, isolated from 

Sphaeranthus indicus (Asteraceae), was bioconverted by Aspergillus niger to afford 13R-dihydro 

OH 

a 

O 

187b 

O 

O 

O 

OH 

198 

O 

Absidia coerulea 

O 

O 

OH 

O 

199 

(13.4%) 

O 

OH 

HO 

O 

200 

(11.0%) 

O 

OH 

O 

201 

O 

O 

O 

202 

O 

O 

O 

205 

O 

O 

O 

HO 

O 

203 

O 

O 

204 

O 

O 

206 

CO 2 H 

OH 

Asparagus officinalis 

O 

198 

O 

O 

O 

199 

O 

O 

O 

206 

(44.7%) 

CO 2 H 

FIGURE 15.63 Biotransformation of a-epi-santonin (198) by Absidia coerulea and Asparagus offi cinalis.


O 

190 

O 

O 

Absidia coerulea 

O 

O 

191 

(71.4%) 

O 

OH 

O 

197 

(2.0%) 

O 

OH 

O 

Asparagus officinalis 

O 

O 

190 O 

Marchantia polymorpha cell 

Nicotiana tabacum 

O 

187 

O 

O 

O 

207 

H 

H 

OH 

O 

O 

O 

187 

O 

O 

O 

HO 

FIGURE 15.64 Biotransformation of 6-epi-a-santonin (190) by Absidia coerulea, Asparagus offi cinalis, 

Marchantia polymorpha, and Nicotiana tabacum. 

208 

O 

O 

O 

209 

O 

O 

derivative (224). The same substrate was also treated in Aspergillus quardilatus (wild type) to give 

13-acetyl product (225) (Atta-ur Rahman et al., 1994) (Figure 15.67). 

Incubation of (-)-frullanolide (226), obtained from the European liverwort, Frullania tamarisci 

subsp. tamarisci causes a potent allergenic contact dermatitis, was incubated by Aspergillus niger 

to give dihydrofrullanolide (227), nonallergenic compound in 31.8% yield. In this case, C11–C13 

dihydro derivative was not obtained (Hashimoto et al., 2005). 

O 

1 

2 

4 

3 

198 

O 

O 

R. nigricans 

O 

O 

O 

O 

HO 

2 1 4 

O 

211 

O 

O 

O 

R. nigricans 

H 

HO 

HO 

H H 

O 

O 

O 

210 

O 

O 

212: (4S) 

213: (4R) 

O 

FIGURE 15.65 Biotransformation of a-episantonin (198) and tetrahydrosantonin (210) by Rhizopus 

nigricans.


OH 

O 

O 

1 

2 1 

3 

4 

3 

H 

HO 

H 

O 

214 

O 

216 

O 

O 

HO 

1 

3 

H 

217 

O 

O 

HO 

H 

215 

O 

O 

HO 

HO 

2 

3 

H 

218 

O 

O 

O 

HO 

2 

3 

H 

219 

O 

O 

A. niger 

A. niger + 

Cytochrome 

P-450 inhibitor 

O 

HO 

3 2 

4 

H 

220 

O 

O 

O 

O 

2 

3 

H 

O 

O 

FIGURE 15.66 Biotransformation of 1,2,4b,5a-tetrahydro-a-santonin (214) by Aspergillus niger. 

Guaiane-type sesquiterpene hydrocarbon, (+)-g-gurjunene, (228) was treated in plant pathogenic 

fungus Glomerella cingulata to give two diols, (1S,4S,7R,10R)-5-guaien-11,13-diol (229), and 

(1S,4S,7R,10S)-5-guaien-10,11,13-triol (230) (Miyazawa et al., 1997, 1998) (Figure 15.68). 

Glomerella cingulata converted guaiol (231) and bulnesol (232) to 5,10-dihydroxy (233) and 

15-hydroxy derivative (234), respectively (Miyazawa et al., 1996) (Figure 15.69). 

When Eurotium rubrum was used as the bioreactor of guaiene (235), rotunodone (236) was 

obtained (Sugawara and Miyazawa, 2004). Guaiol (231) was also transformed by Aspergillus niger 

to give a cylopentane derivative, pancherione (237) and two dihydroxy guaiols (238, 239) (Morikawa 

et al., 2000), of which 237 was obtained from the same substrate using Eurotium rubrum for 10 days 

(Sugawara and Miyazawa, 2004; Miyazawa and Sugawara, 2006) (Figure 15.70). 

Parthenolide (240), a germacrane-type lactone, isolated from the European feverfew (Tanacetum 

parthenium) as a major constituent shows cytotoxic, antimicrobial, and antifungal, anti-inflammatory, 

antirheumatic activity, apoptosis inducing, and NF-kB and DNA binding inhibitory activity. 

This substrate was incubated with Aspergillus niger in Czapek-peptone medium for 2 days to give 

six metabolites (241, 12.3%, 242, 11.3%, 243, 13.7%, 244, 5.0%, 245, 9.6%, 246, 5.1%) (Hashimoto 

et al., 2005) (Figure 15.71). Compound 244 was a naturally occurring lactone from Michelia champaca 

(Jacobsson et al., 1995). The stereostructure of compound 243 was established by x-ray crystallographic 

analysis. 

When parthenolide (240) was treated in Aspergillus cellulosae for 5 days, two new metabolites, 

11b,13-dihydro-(247, 43.5%) and 11a,13-dihydroparthenolides (248, 1.6%) were obtained together 

with the same metabolites (241, 5.3%, 243, 11.2%, 245, 10.4%) as described above (Figure 15.72). 

Possible metabolic root of 240 has been shown in Figure 15.73 (Hashimoto et al., 2005). 

Galal et al. (1999) reported that Streptomyces fulvissimus or Rhizopus nigricans converted 

parthenolide (240) into 11a-methylparthenolide (247) in 20–30% yield while metabolite 11bhydroxyparthenolide 

(248) was obtained by incubation of 240 with Rhizopus nigricans and


O 

3 

H 

O 

221 

O 

A. niger 

3 days HO 

3 4 

H 

O 

222 

O 

4 

A. niger 

OH 

OH 

223 

O 

O 

224 

O 

O 

A. quardilatus 

225 

O 

O 

O 

OH 

A. niger 

226 

O 

O 227 

O 

O 

FIGURE 15.67 Biotransformation of C4-epimer (221) of 214, 7a-hydroxyfrullanolide (223), and frullanolide 

(226) by Aspergillus niger and Aspergillus quardilatus. 

Rhodotorula rubra. In addition to the metabolite 247, Streptomyces fulvissimus gave minor polar 

metabolite, 9b-hydroxy derivative (248a) in low yield (3%). The same metabolite (248a) was 

obtained from 247 by fermentation of Streptomyces fulvissimus as a minor constituent. Furthermore, 

14-hydroxyparthenolide (248b) was obtained from 240 and 247 as a minor component (4%) by 

Rhizopus nigricans (Figure 15.74). 

Pyrethrosin (248c), a germacranolide, was treated in the fungus Rhizopus nigricans to afford five 

metabolites (248d–248h). Pyrethrosin itself and metabolite 248e displayed cytotoxic activity 

against human malignant melanoma with IC 50 4.20 and 7.5 mg/mL, respectively. Metabolite 248h 

showed significant in vitro cytotoxic activity against human epidermoid carcinoma (KB cells) and 

against human ovary carcinoma with IC 50 < 1.1 and 8.0 mg/mL, respectively. Compounds 248f and 

H 

H 

H 

OH 

A. niger 

228 229 

OH 

OH 

230 

OH 

OH 

FIGURE 15.68 Biotransformation of (+)-g-gurjunene (228) by Glomerella cingulata.


G. cingulata 

OH 

OH 

OH 

231 233 

OH 

OH 

G. cingulata 

H 

232 

OH 

H 

234 

OH 

FIGURE 15.69 Biotransformation of guaiol (221) and bulnesol (232) by Glomerella cingulata. 

248i were active against Cryptococcus neoformans with IC 50 35.0 and 25 mg/mL, respectively while 

248a and 248g showed antifungal activity against Candida albicans with IC 50 30 and 10 mg/mL. 

Metabolites 248g and its acetate (248i), derived from 248g showed antiprotozoal activity against 

Plasmodium falciparum with IC 50 0.88 and 0.32 mg/mL, respectively without significant toxicity. 

Compound 248i also exhibited pronounced activity against the chloroquine-registant strain of 

Plasmodium falciparum with IC 50 0.38 mg/mL (Galal, 2001) (Figure 15.75). 

(-)-Dehydrocostuslactone (249), inhibitors of nitric oxide synthases and TNF-a, isolated from 

Saussurea radix, was incubated with Cunninghamella echinulata to afford (+)-11a,13-dihydrodehydrocostuslactone 

(250a). The epoxide (251) and a C11 reduced compound (250) were obtained 

by the above microorganisms (Galal, 2001). 

Eurotium rubrum 

O 

235 

236 

O 

Eurotium rubrum 

A. niger 

231 

OH 

A. niger 

237 

OH 

OH 

HO 

OH 

238 

FIGURE 15.70 Biotransformation of guaiene (235) by Eurotium rubrum and guaiol (231) by Aspergillus 

niger and Eurotium rubrum. 

OH 

OH 

239 

OH


H 

O 

H 

OH 

10 

5 

4 

O 

O 

O 

Parthenolide (240) 

A. niger 

3 days 

H 

OH 

O 

241 

(12.3%) 

H 

OH 

O 

O 

O 

242 

(11.3%) 

H 

OH 

O 

H 

HO 

O 

243 

(13.7%) 

H 

OH 

O 

H 

HO 

244 

(5.0%) 

O 

O 

H 

OH 

O 

245 

(9.6%) 

O 

H 

OH 

O 

246 

(5.1%) 

O 

FIGURE 15.71 Biotransformation of parthenolide (240) by Aspergillus niger. 

Cunninghamella echinulata and Rhizopus oryzae bioconverted 249 into C11/C13 dihydrogenated 

(250) and C10/C14 epoxidated product (251). Treatment of 252a in Cunninghamella echinulata 

and Rhizopus oryzae gave (-)-16-(1-methyl-1-propenyl) eremantholide (252b) (Galal, 2001) 

(Figure 15.76). 

The same substrate (249) was fed by Aspergillus niger for 7 days to afford four metabolites 

costuslactone (250), and their derivatives (251–253), of which 251 was the major product (28%) 

while the same substrate was cultivated with Aspergillus niger for 10 days, two minor metabolites 

(254, 255) were newly obtained in addition to 252 and 253 of which the latter lactone was predominant 

(20.7%) (Hashimoto et al., 2001) (Figure 15.77). 

When compound (249) was treated with Aspergillus niger in the presence of 1-aminobenzotriazole, 

249 was completely converted into 11b,13-dihydro derivative (250) for 3 days; however, further 

biodegradation did not occur for 10 days (Hashimoto et al., 1999, 2001). The same substrate (249) 

was cultivated with Aspergillus cellulosae IFO to furnish 11,13-dihydro- (250) (82%) for only one 

day and then the product (250) slowly oxidized into 11,13-dihydro-8b-hydroxycostuslactone (256) 

(1.6%) from 8 days (Hashimoto et al., 1999, 2001) (Figure 15.78). 

H 

H 

OH 

H 

OH 

O 

O 

O 


A. cellulosae 

5 days 

H 

OH O 

241 

(5.3%) 

FIGURE 15.72 Biotransformation of parthenolide (240) by Aspergillus cellulosae. 

O 

O 

O 

247 

(43.4%) 

O 

H 

HO 

O 

243 

(11.2%) 

O 

O 

248 

(1.6%) 

O 

O 

H 

OH O 

245 O 

(10.4%)


H 

OH 

[H] 

H 

OH 

–H 2 O 

H 

H 2 O 

10 

5 

4 O 11 13 

O 

H + 

O 


A. niger 

A. niger 

H 

OH O 

H 

O 

246 

OH 

H 

HO O 

O 

244 

Michampanolide 

[H] 

OH 

H 

O 

O 

245 

H 

OH 

10 

4 5 

H 

HO O 

243 

O 

13 

OH 

H 

O 

O 

241 

FIGURE 15.73 Possible pathway of biotransformation of parthenolide (240). 

a 

O 

O 

O 

O 

247 O 

248 

OH 

O 

b 

O 

240 

O 

O 

O 

O 

O 

O 

247 O 

248a 

O 

c 

OH 

O 

O 

248b 

O 

a 

OH 

O 

247 

O 

O 

b 

O 

O 

248b 

OH 

O 

a: Rhizopus nigricans 

b: Streptomyces fulvissimus 

c: Rhodotorula rubra 

O 

O 

248a 

O 

FIGURE 15.74 Biotransformation of parthenolide (240) and its dihydro derivative (247) by Rhizopus 

nigricans, Streptomyces fulvissimus, and Rhodotorula rubra.


OH 

OH 

O 

O 

O 

O 

O 

O 

O 

R. nigricans 

HO 

OH 

H 

OAc 

248d 

O 

O 

OH 

H 

OH 

248e 

O 

O 

OAc 

248c 

OH 

H 

OH 

248f 

HO 

OAc 

H 

OAc 

248g 

O 

O 

O 

O 

H 

H 

OAc 

HO 

H 

OAc 

248h 

248i 

FIGURE 15.75 Biotransformation of pyrethrosin (248c) by Rhizopus nigricans. 

a 

H 

H 

H 

O 

250a 

O 

H 

O 

249 

O 

b 

H 

H 

O 

a: Cunninghamella echinulata 

b: Rhizopus oryzae 

H 

O 

250 

O 

H 

O 

251 

O 

O 

O 

O 

OH 

O 

O 

O 

O 

a,b 

O 

O 

O 

O 

252a 

a: Cunninghamella echinulata 

b: Rhizopus oryzae 

252b 

FIGURE 15.76 Biotransformation of (-)-dehydrocostuslactone (249) and rearranged guaianolide (252a) by 

Cunninghamella echinulata and Rhizopus oryzae.


3 

H 

10 

14 

H 

O 

11 13 

249 

O 

A. niger 

7 days 

A. niger 

10 days 

H 

HO 

3 

H 

14 OH 

H OH 

10 

H 

O 

11 13 

H 

O 

H 

O 

250 

O 252 

O 254 

O 

H 

14 

O 

10 

HO 

H 

14 

10 

OH 

HO 

3 

H 

O 

10 

H 

O 

H 

O 

H 

O 

251 

O 

253 

O 

255 

O 

FIGURE 15.77 Biotransformation of (-)-dehydrocostuslactone (249) by Aspergillus niger. 

The lactone (249) was biodegraded by the plant pathogen Botryosphaeria dothidea for 4 days to 

give the metabolites (250) (37.8%) and 257 (8.6%) while Aspergillus niger IFO-04049 (4 days) and 

Aspergillus cellulosae for 1 day gave only 250. Thus Botryosphaeria dothidea demonstrated low 

stereoselectivity to reduce C11–C13 double bond (Hashimoto et al., 2001). Furthermore three 

Aspergillus species, Aspergillus niger IFO 4034, Aspergillus awamori IFO 4033, and Aspergillus 

terreus IFO6123 were used as bioreactors for compounds 249. Aspergillus niger IFO 4034 gave 

three products (250–252), of which 252 was predominant (56% in GC-MS). Aspergillus awamori 

IFO 4033 and Aspergillus terreus IFO 6123 converted 249 to give 250 (56% from Aspergillus 

awamori, 43% from Aspergillus terreus) and 252 (43% from Aspergillus awamori, 57% from 

Aspergillus terreus), respectively (Hashimoto et al., 2001) (Figure 15.79). 

Vernonia arborea (Asteraceae) contains zaluzanin D (258) in high content. Ten microorganisms 

were used for the biotransformation of compound 258. Botrytis cinerea converted 258 into 259 and 

260 (85:15%) and Fusarium equiseti gave 259 and 260 (33:66%). Curvularia lunata, Colletotrichum 

lindemuthianum, Alternaria alternate, and Phyllosticta capsici produced 259 as the sole metabolite 

in good yield while Sclerotinia sclerotiorumn and Rhizpctonia solani gave deactyl product (261) as 

a sole product, and 260, 262–264 among which 263 and 264 are the major products, respectively. 

Reduction of C11–C13 exocylic double bond is the common transformation of a-methylene g-butyrolactone 

(Kumari et al., 2003) (Figure 15.80).


14 

H 

10 

3 

8 

4 

6 7 

H 11 13 

O 

249 

O 

HO 

3 

H 

H 

O 

252 

O 

HO 

3 

14 

H O 

10 

H 

O 

255 O 

H 

14 

O 

10 

14 OH 

H OH 

10 

H 

H 

O 

H 

O 

H 

O 

250 

11 13 

O 

H 

O 

14 

O 

10 

254 O 

OH 

H 14 OH 

10 

H 

H 

O 

8 OH 

H 

O 

251 O 

A. niger 

A. niger + Cyt.-P450 inhibitor 

H 

O 

253 

O 

256 

O 

A. cellulosae 

FIGURE 15.78 Possible pathway of biotransformation of (-)-dehydrocostuslactone (249) by Aspergillus 

niger and Aspergillus cellulosae. 

14 

H 

H O 

10 

14 

A. niger 

H 

A. cellulosae 

13 

H 

H 

10 

A. awamori 

A. terreus 

O 11 

O 

Botryosphaeria dothidea 

O 

O 

H 

250 

251 

11 13 

O 

O 

H 

H 

249 

HO 3 

13 

H 

H 

O 

O 11 

O 

O 

252 

257 

FIGURE 15.79 Biotransformation of (-)-dehydrocostuslactone (249) by Aspergillus species and 

Botryosphaeria dothidea.


H 

H 

AcO 

HO 

H 

O 

H 

O 

H 

259 

O 260 

O 

HO 

B. cinerea 

F. equiseti 

C. lunata 

C. lindemuthianum 

B. theobromae 

F. oxysporum 

A. alternata 

P. capsici 

AcO 

H 

H 

O 

258 

O 

S. sclerotiorum 

H 

O 

261 

O 

R. solani 

H 

H 

H 

H 

O 

HO 

O 

HO 

H 

O 

H 

O 

H 

O 

H 

O 

O 

O 

O 

262 

263 

264 

FIGURE 15.80 Biotransformation of zaluzanin D (258) by various fungi. 

260 

O 

Incubation of parthenin (264a) with the fungus Beauveria bassiana in modified Richard’s 

medium gave C11–C13 reduced product (264b) in 37% yield, while C11 a-hydroxylated product 

(264c) was obtained in 32% yield from the broth of the fungus Sporotrichum pulverulentum using 

the same medium (Bhutani and Thakur, 1991) (Figure 15.81). 

Cadina-4,10(15)-dien-3-one (265) possessing insecticidal and ascaricidal activity, from Jamaican 

medicinal plant Hyptis verticillata was metabolized by Curvularia lunata ATCC 12017 in potato 

dextrose to give its 12-hydoxy- (266), 3a-hydroxycadina-4,10(15)-dien (267), and 3a-hydroxy-4,5- 

dihydrocadinenes (268) while 265 was incubated by the same fungus in peptone, yeast, and beef 

extracts and glucose medium, only 267 and 268 were obtained. Compound 267 derived synthetically 

was treated in the same fungus Curvularia lunata to afford three metabolites (269–271) 

(Collins and Reese, 2002) (Figure 15.82). 

HO HO HO 

S. pulverulentum 

B. bassiana 

O 

O 

OH 

O 

O 

O 

O 

264c 

O 

264a 

O 

264b 

O 

FIGURE 15.81 Biotransformation of parthenin (264a) by Sporotrichum pulverulentum and Beauveria 

bassiana.


O 

H 

C. lunata 

O 

H 

HO 

H 

HO 

H 

H 

H 

H 

H 

265 266 

OH 

267 268 

C. lunata 

HO 

OH 

HO 

H 

HO 

H 

H 

H 

H 

OH 

OH 

269 

270 

271 

FIGURE 15.82 Biotransformation of cadina-4,10(15)-dien-3-one (265) by Curvularia lunata. 

The incubation of the same substrate (265) in Mucor plumbeus ATCC 4740 in high iron-rich 

medium gave 270, which was obtained from Curvularia lunata mentioned above, 268, 272, 273, 

277, 278, and 279. In low iron medium, this fungus converted the same substrate 265 into three 

epoxides (274–276), a tetraol (280) with common metabolites (268, 273, 277, 278), and 271, which 

was the same metabolite used by Curvularia lunata (Collins, Reynold, and Reese, 2002). It is interesting 

to note that only epoxides were obtained from the substrate (265) by Mucor fungus in low 

iron medium (Figure 15.83). 

The same substrate (265) was incubated with the deuteromycete fungus, Beauveria bassiana, 

which is responsible for the muscaridine disease in insects, in order to obtain new functionalized 

analogues with improved biological activity. From compound 265, nine metabolites were obtained. 

O 

H 

O 

H 

O 

H 

O 

H 

H 

H 

O 

H 

M. plumbeus 

H 

R 1 R 2 

R 1 R 2 

270: R 1 =H, R 2 =OH 272: R 1 =OH, R 2 =OH 

271a: R 1 =OH, R 2 =H 273: R 1 =H, R 2 =OH 

R 1 R 2 

274: R 1 =OH, R 2 =OH 

275: R 1 =H, R 2 =OH 

H 

265 

HO 

H 

O 

HO 

H 

HO 

H 

HO 

H 

OH 

OH 

H 

H 

H 

H 

H 

OH 

276 

R 1 R 2 

268: R 1 =R 2 =H 

277: R 1 =OH, R 2 =H 

278: R 1 =H, R 2 =OH 

OH 

279 

FIGURE 15.83 Biotransformation of cadina-4,10(15)-dien-3-one (265) by Mucor plumbeus. 

280 

OH


O 

H 

O 

H 

O 

H 

H 

H 

H 

OH 

O 

H 

B. bassiana 

HO 

268a 268b 

OH 

273 

H 

H 

H 

HO 

HO 

H 

H 

H 

H 

265 

267 

268 

268c 

HO 

H 

HO 

H 

HO 

H 

H 

H 

H 

OH 

268d 

268e 

268f 

FIGURE 15.84 Biotransformation of cadina-4,10(15)-dien-3-one (265) by Beauveria bassiana. 

The insecticidal potential of the metabolites (267, 268, 268a–268f) were evaluated against Cylas 

formicarius. The metabolites (273, 268, 268d) showed enhanced activity compared with the substrate 

(265). The plant growth regulatory activity of the metabolites against radish seeds was tested. 

All the compounds showed inhibitory activity; however, their activity was less than colchicine 

(Buchanan et al., 2000) (Figure 15.84). 

Cadinane-type sesquiterpene alcohol (281) isolated from the liverwort Mylia taylorii gave a 

primary alcohol (282) by Aspergillus niger treatment (Morikawa et al., 2000) (Figure 15.85). 

Fermentation of (-)-a-bisabolol (282a) possessing anti-inflammatory activity with plant pathogenic 

fungus Glomerella cingulata for 7 days yielded oxygenated products (282b–282e) of which 

compound 282e was predominant. 3,4-Dihydroxy products (282b, 282d, 282e) could be formed by 

hydrolysis of the 3,4-epoxide from 282a and 282c (Miyazawa et al., 1995b) (Figure 15.86). 

El Sayed et al. (2002) reported microbial and chemical transformation of (S)-(+)-curcuphenol 

(282g) and curcudiol (282n), isolated from the marine sponges, Didiscus axeata. Incubation of 

compound 282g with Kluyveromyces marxianus var. lactis resulted in the isolation of six metabolites 

(3–8, 282h–282j). The same substrate was incubated with Aspergillus alliaceus to give the 

metabolites (282p, 282q, 282s) (Figure 15.87). 

Compounds 282g and 282n were treated in Rhizopus arrhizus and Rhodotorula glutinus for 

6 and 8 days to afford glucosylated metabolites, 1a-d-glucosides (282o) and 282r, respectively. The 

OH 

OH 

A. niger 

OH 

281 

282 

FIGURE 15.85 Biotransformation of cadinol (281) by Aspergillus niger.


HO 

H 

OH 

H 

O 

H 

OH 

HO 

G. cingulata 

282b 

282c 

OH 

282a 

HO 

HO 

H 

O 

HO 

HO 

H 

O 

282d 

OH 

282e 

OH 

FIGURE 15.86 Biotransformation of b-bisabolol (282a) by Glomerella cingulata. 

substrate itself showed antimicrobial activity against Candia albicans, Cryptococcus neoformans, 

and MRSA-resistant Staphylococcus aureus and Staphylococcus aureus with MIC and MFC/MBC 

ranges of 7.5–25 and 12.5–50 mg/mL, respectively. Compounds 282g and 282h also exhibited in 

vitro antimalarial activity against Plasmodium falciparium (D6 clone) and Plasmodium falciparium 

(W2 clone) of 3600 and 3800 ng/mL (selective index (S.I.) > 1.3), and 1800 (S.I. > 2.6), and 2900 

(S.I. > 1.6), respectively (El Sayed et al., 2002) (Figure 15.87). 

Artemisia annua is one of the most important Asteraceae species as antimalarial plant. There are 

many reports of microbial biotransformation of artemisinin (283), which is active antimalarial rearranged 

cadinane sesquiterpene endoperoxide, and its derivatives to give novel antimalarials with 

increased activities or differing pharmacological characteristics. 

Lee et al. (1989) reported that deoxoartemisinin (284) and its 3a-hydroxy derivative (285) were 

obtained from the metabolites of artemisinin (283) incubated with Nocardia corallina and 

Penicillium chrysogenum (Figure 15.88). 

Zhan et al. (2002) reported that incubation of artemisinin (283) with Cunninghamella echinulata 

and Aspergillus niger for 4 days at 28°C resulted in the isolation of two metabolites, 10b-hydroxyartemisinin 

(287a) and 3a-hydroxydeoxyartemisinin (285), respectively. 

Compound 283 was also biotransformed by Aspergillus niger to give four metabolites, deoxyartemisinin 

(284, 38%), 3a-hydroxydeoxyartemisinin (285, 15%), and two minor products (286, 8% 

and 287, 5%) (Hashimoto et al., 2003b). 

Artemisinin (283) was also bioconverted by Cunninghamella elegans. During this process, 

9b-hydroxyartemisinin (287b, 78.6%), 9b-hydroxy-8a-artemisinin (287c, 6.0%), 3a- hydroxydeoxoartemisinin 

(285, 5.4%), and 10b-hydroxyartemisinin (287d, 6.5%) have been formed. On the basis 

of quantitative structure-activity relationship (QSAR) and molecular modeling investigations, 9bhydoxy 

derivatization of artemisinin skeleton may yield improvement in antimalarial activity and 

may potentially serve as an efficient means of increasing water solubility (Parshikov et al., 2004) 

(Figure 15.89). 

Albicanal (288) and (-)-drimenol (289) are simple drimane sesquiterpenoids isolated from the 

liverwort, Diplophyllum serrulatum, and many other liverworts and higher plants. The latter compound 

was incubated with Mucor plumbeus and Rhizopus arrhizus. The former microorganism 

converted 289 to 6,7a-epoxy- (290), 3b-hydroxy- (291), and 6a-drimenol (292) in the yields of 2%, 

7%, and 50%, respectively. On the other hand, the latter species produced only 3b-hydroxy derivative 

(291) in 60% yield (Aranda et al., 1992) (Figure 15.90). 

(-)-Polygodial (293) possessing piscicidal, antimicrobial, and mosquito-repellant activity is the 

major pungent sesquitepene dial isolated from Polygonum hydropiper and the liverwort, Porella 

vernicosa complex. Polygodial was incubated with Aspergillus niger, however, because of its


OH OH OH 

OH 

282g 

Kluyveromyces marxianus 

OH 

HO 

OH HO 

282h 282i 282j 

OH 

OH 

OH 

OGlc 

Rhizopus arrhizus 

OH 

HO 

OHC 

HO 2 C 

H 

H 

282k 282l 282m 

OH 

282o 

OH 

OH 

282p 

OH 

OH 

Aspergillus alliaceus 

O 

282n OH 

OGlc 

Rhodotorula glutinus 

OH 

282q 

OH 

282s 

COOH 

OH 

282r 

FIGURE 15.87 Biotransformation of (S)-(+)-curcuphenol (282g) by Kluyveromyces marxianus and Rhizopus 

arrhizus and curcudiol (282n) by Aspergillus alliaceus and Rhodotorula glutinus. 

antimicrobial activity, nothing metabolite was obtained (Sekita et al., 2005). Polygodiol (295) prepared 

from polygodial (293) was also treated in the same manner as described above to afford 

3b-hyrdoxy- (297), which was isolated from Marasmius oreades as antimicrobial activity (Ayer and 

Craw, 1989) and 6a-hydroxypolygodiol (298) in 66–70% and 5–10% yields, respectively (Aranda et 

al., 1992). The same metabolite (297) was also obtained from polygodiol (295) as a sole metabolite 

from the culture broth of Aspergillus niger in Czapek-peptone medium for 3 days in 70.5% yield 

(Sekita et al., 2005), while the C9 epimeric product (296) from isopolygodial (294) was incubated 

with Mucor plumbeus to afford 3b-hydroxy- (299) and 6a-hydroxy derivative (300) in low yields,


H 

HO 

H 

a,b,c 

O 

O 

O 

H 

O 

H 

O 

H 

O 

H 

3 

4 

O 

H 

H 

10 9 

6 

7 8 

5 

O 

H 

O 

O 

284 (38%) 285 (15%) 

H 

H 

O O 

O 

Artemisinin (283) 

a: A.niger 

b: Nocardia sp. 

c: Penicillium sp. 

a 

AcO O 

H 

O 

O 

286 (8%) 

H 

HO 

O 

H 

O 

O 

287 (5%) 

H 

FIGURE 15.88 Biotransformation of artemisinin (283) by Aspergillus niger, Nocardia corallina, and 

Penicillium chrysogenum. 

H 

O 

O 

O 

H 

O 

OH 

OH 

H 

287a 

a 

H 

O 

O 

O 

H 

O 

H 

c 

H 

O 

O 

O 

H 

O 

H 

OH 

H 

O 

O 

O 

H 

O 

H 

OH 

O 

283 

b 

O 

287b 

O 

287c 

HO 

O 

O 

H 

O 

H 

H 

HO 

O 

O 

H 

O 

H 

H 

H 

O 

O 

O 

H 

O 

OH 

H 

O 

O 

O 

285 a: Cunninghamella echinulata 

b: Aspergillusniger 

c: Cunningham ellaelegans 

285 

287d 

FIGURE 15.89 Biotransformation of artemisinin (283) by Cunninghamella echinulata, Cunninghamella 

elegans, and Aspergillus niger.


CHO 

H 

288 

OH OH OH OH 

H 

Mucor plumbeus 

H 

O 

HO 

H 

H 

OH 

289 

290 

(2%) 

Rhizopus arrhizus* 

291 

(7%) 

(60%)* 

292 

(50%) 

FIGURE 15.90 Biotransformation of drimenol (289) by Mucor plumbeus and Rhizopus arrhizus. 

7% and 13% (Aranda et al., 1992). Drim-9a-hydroxy-11b,12-diacetoxy-7-ene (301) derived from 

polygodiol (295) was treated in the same manner as described above to yield its 3b-hydroxy derivative 

(302, 42%) (Sekita et al., 2005) (Figures 15.91 and 15.92). 

Cinnamodial (303) from the Malagasy medicinal plant, Cinnamosma fragrans, was also treated 

in the same medium including Aspergillus niger to furnish three metabolites, respectively in very 

law yields (304, 2.2%; 305, 0.05%; and 306, 0.62%). Compound 305 and 306 are naturally occurring 

cinnamosmolide, possessing cytotoxicity and antimicrobial activity, and fragrolide. In this 

case, the introduction of 3b-hydroxy group was not observed (Sekita et al., 2006) (Figure 15.93). 

Naturally occurring rare drimane sesquiterpenoids (307–314) were biosynthesized by the fungus 

Cryptoporus volvatus with isocitric acids. Among these compounds, in particular, cryptoporic 

acid E (312) possesses antitumor promoter, anticolon cancer, and very strong super oxide anion 

radical scavenging activities (Asakawa et al., 1992). When the fresh fungus allowed standing in 

moisture condition, olive fungus Paecilomyces varioti grows on the surface of the fruit body of this 

293 

CHO 

CHO 

LiAlH 4 

295 

OH 

M. plumbeus* 

OH R. arrhizus* 

A. niger** 

HO 

297 

(66–70%)* 

(70.5%)** 

OH 

OH 

OH 

298 

(5–10%)* 

OH 

OH 

CHO 

CHO 

OH 

OH M. plumbeus 

OH 

OH 

OH 

OH 

294 

296 

FIGURE 15.91 Biotransformation of polygodiol (295) by Mucor plumbeus, Rhizopus arrhizus, and 

Aspergillus niger. 

HO 

299 

(7%) 

OH 

300 

(13%)


OAc 

OH 

OAc 

A. niger 

OAc 

OH 

OAc 

3 days 

HO 

301 

302 (47.2%) 

FIGURE 15.92 Biotransformation of drim-9a-hydroxy-11,12-diacetoxy-7-ene (301) by Aspergillus niger. 

fungus. 2 kg of the fresh fungus was infected by Cryptoporus volvatus for 1 month, followed by the 

extraction of methanol to give the crude extract, then purification using silica gel and Sephadex 

LH-20 to give five metabolites (316, 318–321), which were not found in the fresh fungus (Takahashi 

et al., 1993a). Compound 318 was also isolated from the liverworts, Bazzania and Diplophyllum 

species (Asakawa, 1982, 1995) (Figure 15.94). 

Liverworts produce a large number of enantiomeric mono-, sesqui-, and diterpenoids to those 

found in higher plants and lipophilic aromatic compounds. It is also noteworthy that some liverworts 

produce both normal and its enantiomers. The more interesting phenomenon in the chemistry 

of liverworts is that the different species in the same genus, for example, Frullania tamarisci subsp. 

tamarisci and Frullania dilatata produce totally enantiomeric terpenoids. Various sesqui- and 

diterpenoids, bibenzyls, and bisbibenzyls isolated from several liverworts show characteristic 

fragrant odor, intensely hot and bitter taste, muscle relaxing, antimicrobial, antifungal, allergenic 

contact dermatitis, antitumor, insect antifeedant, superoxide anion release inhibitory, piscicidal, and 

neurotrophic activity (Asakawa, 1982, 1990, 1995, 1999, 2007, 2008; Asakawa and Ludwiczuk, 

2008). In order to obtain the different kind of biologically active products and to compare the 

metabolites of both normal and enantiomers of terpenoids, several secondary metabolites of specific 

liverworts were biotransformed by Penicillium sclerotiorum, Aspergillus niger, and Aspergillus 

cellulosae. 

(-)-Cuparene (322) and (-)-2-hydroxycuparene (323) have been isolated from the liverworts, 

Bazzania pompeana and Marchantia polymorpha, while its enantiomer (+)-cuparene (324) and 

(+)-2-hydroxycuparene (325) from the higher plant, Biota orientalis and the liverwort Jungermannia 

rosulans. (R)-(-)-a-Cuparenone (326) and grimaldone (327) demonstrate intense flagrance. In 

order to obtain such compounds from both cuparene and its hydroxy compounds, both enatiomers 

mentioned above were cultivated with Aspergillus niger (Hashimoto et al., 2001a) (Figure 15.95). 

From (-)-cuparene (322), five metabolites (328–332) all of which contained cyclopentanediols or 

hydroxycyclopentanones were obtained. An aryl methyl group was also oxidized to give primary 

alcohol, which was further oxidized to afford carboxylic acids (329–331) (Hashimoto et al., 2001a) 

(Figure 15.96). 

From (+)-cuparene, six metabolites (333–338) were obtained. These are structurally very similar 

to those found in the metabolites of (-)-cuparene, except for the presence of an acetonide (336), but 

they are not identical. All metabolites possess benzoic acid moiety. 

11 

CHO 

OH 

9 CHO 

12 

6 

A. niger 

4 days 

HO 

HO 

9 

6 

O 

11 

12 

HO 

O 

O 

11 O 12 

O 

9 

8 

6 

OAc 

OAc 

OAc 

O 

Cinnamodial (303) 

304 (2.2%) 

305 (0.05%) 

Cinnamosmolide 

306 (0.20%) 

Fragrolide 

FIGURE 15.93 Biotransformation of cinnamodial (303) by Aspergillus niger.


O 

O 

O 

O 

COOMe COOMe COOH 

COOMe CO COOH 

O 

COOMe COOMe COOH 

COOMe COOH COOH 

H 

Cryptoporic acid A 

(307) 

H 

COOH COOR 

COOMe 

H 

OH 

Cryptoporic acid B (308) 

H 

OH 

315 

O 

R=Me Cryptoporic acid C (309) 

R=H Cryptoporic acid F (310) 

OH 

OH 

O 

O 



H 319 

OH 

H 

Albicanol (318) 

H 

Cryptoporic acid H 

(314) 

H 

OH 

316 

OH 

O 

COOMe CO COOH 

O 

H 

O 

COOH CO COOMe 

O 

Cryptoporic acid D (311) 

H 

O 

COOH CO COOH 

O 

O 

COOH CO COOH 

O 

317 

H 

OH 

COOH COOR COOMe 

O 

COOMe CO COOH 

O 

O 

R=Me Cryptoporic acid E (312) 

R=H Cryptoporic acid G (313) 

HO 

H 

COOH 

320 

OH 

H 321 

COOH 

FIGURE 15.94 Biotransformation of cryptoporic acids (307–317, 316) by Paecilomyces varioti. 

The possible biogenetic pathways of (+)-cuparene (324) has been proposed in Figure 15.97. 

Unfortunately, none of the metabolites show strong mossy odor (Hashimoto et al., 2006). The 

presence of an acetonide in the metabolites has also been seen in those of dehydronootkatone (25) 

(Furusawa et al., 2003) (Figure 15.98). 

The liverwort Herbertus adancus, Herbertus sakuraii, and Mastigophora diclados produce 

(-)-herbertene, the C3 methyl isomer of cuparene, with its hydroxy derivatives, for example, herbertanediol 

(339), which shows NO production inhibitory activity (Harinantenaina et al., 2007) and 

herbertenol (342). Treatment of compound (339) in Penicillium sclerotiorum in Czapek-polypeptone 

medium gave two dimeric products, mastigophorene A (340) and mastigophorene B (341), which 

showed neurotrophic activity (Harinantenaina et al., 2005). 

When (-)-herbertenol (342) was biotransformed for 1 week by the same fungus, no metabolic 

product was obtained; however, five oxygenated metabolites (344–348) were obtained from its 

methyl ether (343). The possible metabolic pathway is shown in Figure 15.99. Except for the presence 

of the ether (348), the metabolites from 342 resemble those found in (-)- and (+)-cuparene 

(Hashimoto et al., 2006) (Figures 15.100 and 15.101). 

Maalioxide (349), mp 65–66°, [a] D 

21 

−34.4°, obtained from the liverwort, Plagiochila sciophila 

was inoculated and cultivated rotatory (100 rpm) in Czapek-peptone medium (pH 7.0) at 30°C for 

2 days. (-)-Maalioxide (349) (100 mg/200 mL) was added to the medium and further cultivated for 

2 days to afford three metabolites, 1b-hydroxy-(350), 1b,9b-dihydroxy- (351), and 1b,12-dihydroxymaalioxides 

(352), of which 351 was predominant (53.6%). When the same substrate was cultured 

with Aspergillus cellulosae in the same medium for 9 days, 7b-hydroxymaalioxide (353) was 

obtained as a sole product in 30% yield. The same substrate (349) was also incubated with the 

fungus Mucor plumbeus to obtain a new metabolite, 9b-hydroxymaalioxide (354), together with 

two known hydroxylated products (350, 353) (Wang et al., 2006). 

Maalioxide (349) was oxidized by m-chloroperbenzoic acid to give a very small amount of 

353 (1.2%), together with 2a-hydroxy- (355, 2%) and 8a-hydroxymaalioxide (356, 1.5%), which


(–)-Cuparene (322) 

Bazzania pompeana 

Marchantia polymorpha 

S 

(+)-Cuparene (324) 

Jungermannia rosulans 

Biota orientalis (higher plant) 

R 

HO 

HO 

(–)–2-Hydroxycuparene (323) (+)–2-Hydroxycuparene (325) 

Bazzania pompeana 

Marchantia polymorpha 

Jungermannia rosulans 

Biota orientalis (higher plant) 

326 

O 

O 

327 

FIGURE 15.95 Naturally occurring cuparene sesquiterpenoids (322–327). 

HO 

OH 

OH 

HOOC 

OH 

OH 

328 329 

(–)-Cuparene (322) 

A. niger 

HOOC 

HO 

OH 

HOOC 

O 

330 331 

O 

HO 

OH 

332 

OH 

FIGURE 15.96 Biotransformation of (-)-cuparene (322) by Aspergillus niger.


O 

HOOC 

HOOC 

333 

334 

COOH 

O 

A. niger 

HOOC 

O 

HOOC 

O 


335 

OH 

336 

HO 

HOOC 

O 

HOOC 

O 

337 

338 

FIGURE 15.97 Biotransformation of (+)-cuparene (324) by Aspergillus niger. 

have not been obtained in the metabolite of 349 in Aspergillus niger and Aspergillus cellulosae 

(Tori et al., 1990) (Figure 15.102). 

Plagiochila sciophila is one of the most important liverworts, since it produces bicyclohumulenone 

(357), which possesses strong mossy note and is expected to manufacture compounding 

perfume. In order to obtain much more strong scent, 357 was treated in Aspergillus niger for 4 days 

to give 4a,10b-dihydroxybicyclohumulenone (358, 27.4%) and bicyclohumurenone-12-oic acid 

(359). An epoxide (360) prepared by m-chloroperbenzoic acid was further treated in the same 

fungus as described above to give 10b-hydoxy derivative (361, 23.4%). Unfortunately, these metabolites 

possess only faint mossy odor (Hashimoto et al., 2003c) (Figure 15.103). 

The liverwort Reboulia hemisphaerica biosynthesizes cyclomyltaylanoids like 362 and also 

ent-1a-hydroxy-b-chamigrene (367). Biotransformation of cyclomyltaylan-5-ol (362) in the same 

15 

9 

10 

[O] 

HO 

HOOC 

O 


[O] 

335 

–H 2 

O 

HOOC 

O 

16 

O 

HOOC 

HOOC 

OH 

O 

336 

333 

[O] 

337 

[O] 

HOOC 

OH 

OH 

[O] 

HOOC 

[O] 

OH 

HOOC 

O 

FIGURE 15.98 Possible pathway of biotransformation of (+)-cuparene (324) by Aspergillus niger.


HO OH HO OH 

Penicillium sclerotiorum 

Mastigophorene A (340) 

HO 

OH 

(–)-Herbertenediol (339) 

HO OH HO OH 

Mastigophorene B (341) 

FIGURE 15.99 Biotransformation of (-)-herbertenediol (339) by Penicillium sclerotiorum. 

medium including Aspergillus niger gave four metabolites, 9b-hydroxy- (363, 27%), 9b,15- 

dihydroxy- (364, 1.7%), 10b-hydroxy- (365, 10.3%), and 9b,15-dihydroxy derivative (366, 12.6%). 

In this case, the stereospecificity of alcohol was observed, but the regiospecificity of alcohol moiety 

was not seen in this substrate (Furusawa et al., 2005b, 2006b) (Figure 15.104). 

The biotransformation of spirostructural terpenoids was not carried out. Ent-1a-hydroxy-bchamigrene 

(367) was inoculated in the same manner as described above to give three new metabolites 

(368–370), of which 370 was the major product (46.2% in isolated yield). The hydroxylation of 

vinyl methyl group has been known to be very common in the case of microbial and mammalian 

biotransformation (Furusawa et al., 2005, 2006) (Figure 15.105). 

A. niger 

No metabolites 

OH 

(–)–α-Herbertenol (342) 

HOOC 

OH 

HO 

(Me) 2 SO 4 

OMe 

O 

OMe 

OH 

HOOC 

344(159 mg) 

HO 

345 

A. niger 

OMe 

(-)-Methoxy-α-herbertene 

(343) 

1 week 

OMe 

346 

OH 

O 

OMe 

347 

OH 

OH 

O 

348 

FIGURE 15.100 Biotransformation of (-)-methoxy-a-herbertene (343) by Aspergillus niger.


15 

OMe 

9 

13 

10 

[O] 

HO 

OMe 

[O] 

HO 

OM e 

OH 

(-)-Methoxy-α-herbertene 

(343) 

HO 

[O] 

347 

OH 

OMe 

345 

HOOC 

HOOC 

[O] 

HOOC 

HOOC 

OH 

[O] 

O 

–CH 3 

HOOC 

OMe 

[O] 

10 

O 

13 

OH 

[O] 

OMe 

346 

O 

CH 2 OH 

O 

348 

OH 

OMe 

344 

O 

FIGURE 15.101 Possible pathway of biotransformation of (-)-methoxy-a-herbertene (343) by Aspergillus 

niger. 

b-Barbatene (= gymnomitrene) (4), a ubiquitous sesquiterpene hydrocarbon, from liverwort like 

Plagiochila sciophila and many others. Jungermanniales liverworts was treated in the same manner 

using Aspergillus niger for 1 day gave a triol, 4b,9b,10b-trihydoxy-b-barbatene (27, 8%) (Hashimoto 

et al., 2003c). 

Pinguisane sesquiterpenoids have been isolated from the Jungermanniales, Metzgeriales, and 

Marchantiales. In particular, the Lejeuneaceae and Porellaceae are rich sources of this unique type 

of sesquiterpenoids. One of the major furanosesquitepene (373) was biodegradated by Aspergillus 

niger to afford primary alcohol (375), which might be formed from 374 as shown in Figure 15.106 

(Lahlou et al., 2000) (Figure 15.107). 

In order to obtain more pharmacologically active compounds, the secondary metabolites from 

crude drugs and animals, for example, nardosinone (376) isolated from the crude drug, Nardostachys 

chinensis, which has been used for headache, stomachache, and diuresis possesses antimalarial 

activity. Hinesol (384), possessing spasmolytic activity, obtained from Atractylodis lanceae rhizoma, 

animal perfume (-)-ambrox (391) from ambergris were biotransformed by Aspergillus niger, 

Aspergillus cellulosae, Botryosphaeria dothidea, and so on. 

Nardosinone (376) was incubated in the same medium including Aspergillus niger as described 

above for 1 day to give six metabolites (377, 45%; 378, 3%; 379, 2%; 380, 5%; 381, 6%; and 382, 

3%). Compounds 380–382 are unique trinorsesquiterpenoids although their yields are very poor. 

Compound 380 might be formed by the similar manner to that of phenol from cumene (383) (Figure 

15.108) (Hashimoto et al., 2003b) (Figure 15.109).


HO 

OH 

OH 

H 

H 

O O O 

H 

mCPBA 

355 

(2.0%) 

353 

(1.2%) 

356 

(1.5%) 

OH OH OH OH 

A. niger 

H 

H H H 

O O O O 

OH 

M. plumbeus 

349 350 

351 352 

(6.2%) (53.6%) (11.0%) 

OH 

OH 

A. cellulosae 

OH 

H H H 

O O O 

350 353 354 

FIGURE 15.102 Biotransformation of maalioxide (349) by Aspergillus niger, Aspergillus cellulosae, and 

Mucor plumbeus. 

From hinesol (384), two allylic alcohols (386, 387) and their oxygenated derivative (385), and 

three unique metabolites (388–390) having oxirane ring were obtained. The metabolic pathway is 

very similar to that of oral administration of hinesol since the same metabolites (395–387) were 

obtained from the urine of rabbits (Hashimoto et al., 1998, 1999b, 2001) (Figure 15.110). 

To obtain a large amount of ambrox (391), a deterrence, labda-12,14-dien-7a,8-diol obtained 

from the liverwort, Porella pettottetiana as a major component, was chemically converted into 

O 

H 

4 2 

10 

1 12 

A. niger 

HO 

O 

H 

OH 

O 

H 

CO 2 H 

357 358 

(27.4%) 

mCPBA 

359 

(6.1%) 

O 

O 

360 

H 

A. niger 

FIGURE 15.103 Biotransformation of bicyclohumulenone (357) by Aspergillus niger. 

O 

3 2 

O 

10 

361 

(23.4%) 

H 

OH


HO 

HO 

9 

OH 

5 

9 

10 

15 

A. niger 

363 (27.3%) 

OH 

364 (1.7%) 

O 

362 

5 

OH 

HO 

9 

OH 

HO 

365 (10.3%) 

OH 

366 (12.6%) 

OH 

FIGURE 15.104 Biotransformation of cyclomyltaylan-5-ol (362) by Aspergillus niger. 

OH 

9 

8 

1 

15 

367 

A. niger 

HO 

9 

OH 

1 

8 

OH 

1 

8 

OH 

1 

15 

OH 

HO 

HO 

368 (4.8%) 369 (25.0%) 370 (46.2%) 

FIGURE 15.105 Biotransformation of ent-1a-hydroxy-b-chamigrene (367) by Aspergillus niger. 

H 

H 

A. niger 

HO 

OH 

FIGURE 15.106 Biotransformation of b-barbatene (371) by Aspergillus niger. 

HO 

371 372


–H 2 O +H 2 O 

O 

OH 

373 

A. niger 

HO 

O 

OH 

H + 

373a 

O 

O 

373c 

H 

O 

HO 

O 

373b 

HO 

O 

OHC 

HO 

HO 

O 

HO 

O 

O 

374 

375 

FIGURE 15.107 Biotransformation of pinguisanol (373) by Aspergillus niger. 

(-)-ambrox via six steps in relatively high yield (Hashimoto et al., 1998a). Ambrox was added to 

Czapek-peptone medium including Aspergillus niger, for 4 days, followed by chromatography of the 

crude extract to afford four oxygenated products (392–395), among which the carboxylic acid (393, 

52.4%) is the major product (Hashimoto et al., 2001) (Figure 15.111). 

O 

O 

7 

OH 

6 

7 

OH 

11 

OH 

11 

377 (45%) 

378a (3%) 

O 

6 7 

O 

O 

A. niger 

1 day 

O 

H 

10 

7 

OH 

O 

6 

OH 

Nardosinone (376) 

379 (2%) 

380 (5%) 

HO 

2 

H 

O 

6 7 

O 

HO 

2 

H 

O 

6 7 

O 

381 

(6%) 

FIGURE 15.108 Biotransformation of nardosinone (376) by Aspergillus niger. 

382 

(3%)


O O H OH 

H 3 O + 

6 7 O 2 

O 6 

OH – 

Cumene (383) 

O 

O 

O 

11 O 

11 O OH 

OH 

(CH 3 ) 2 CO 

Nardosinone (376) 

380 

FIGURE 15.109 Possible pathway of biotransformation of nardosinone (376) to trinornardosinone (380) by 

Aspergillus niger. 

When ambrox (391) was biotransformed by Aspergillus niger for 9 days in the presence of 

1-aminobenzotriazole, an inhibitor of CYP450, compounds 396 and 397 were obtained instead of 

the metabolites (392–395), which were obtained by incubation of ambrox in the absence of the 

inhibitor. Ambrox was cultivated by Aspergillus cellulosae for 4 days in the same medium to afford 

H 

OH 

Hinesol (384) 

A. niger 

Rabbit 

A. niger 

O 

O 

385 

H 

OH 

HO 

388 

H 

OH 

HO 

386 

H 

OH 

HO 

389 

O 

H 

OH 

O 

HO 

OH 

H 

387 

HO 

390 

FIGURE 15.110 Biotransformation of hinesol (384) by Aspergillus niger. 

O 

H 

OH


O 

H 

(–)-Ambrox (391) 

A. niger 

at 30°C for 4 days 

O 

O 

HO 

3 

H 

OH 

392 

HO 

H 

18 

COOH 

393 (52.4%) 

HO 

3 

H 

8 

OH 

OH 

O 

OH 

OH 

H 

394 

395 

FIGURE 15.111 Biotransformation of (-)-ambrox (391) by Aspergillus niger. 

C1 oxygenated products (398 and 399), the former of which was the major product (41.3%) 


The metabolite pathways of ambrox are quite different between Aspergillus niger and Aspergillus 

cellulosae. Oxidation at C1 occurred in Aspergillus cellulosae to afford 398 and 399, which was 

also afforded by John’s oxidation of 398, while oxidation at C3 and C18 and ether cleavage between 

C8 and C12 occurred in Aspergillus niger to give 392–395. Ether cleavage seen in Aspergillus niger 

is very rare. 

Fragrance of the metabolites (392–395) and 7a-hydroxy-(-)-ambrox (400) and 7-oxo-(-)-ambrox 

(401) obtained from labdane diterpene diol were estimated. Only 399 demonstrated a similar 

odor to ambrox (391) (Hashimoto et al., 2001) (Figure 15.113). 

(-)-Ambrox (391) was also microbiotransformed with Fusarium lini to give mono-, di-, and 

trihydroxylated metabolites (401a–401d), while incubation of the same substrate with Rhizopus 

stolonifera afforded two metabolites (394, 396), which were obtained from 391 by Aspergillus niger 

as mentioned above, together with 397 and 401e (Choudhary et al., 2004) (Figure 15.114). 

The sclareolide (402) which is C12 oxo derivative of ambrox was incubated with Mucor plumbeus 

to afford three metabolites 3b-hydroxy- (403, 7.9%), 1b-hydroxy- (404, 2.5%), and 3-ketosclareolide 

(405, 7.9%) (Aranda et al., 1991) (Figure 15.115). 

Aspergillus niger in the same medium as mentioned above converted sclareolide (402) into two 

new metabolites (406, 407), together with known compounds (403, 405), of which 3b-hydroxysclareolide 

(403) is preferentially obtained (Hashimoto et al., 2007) (Figure 15.116). 

From the metabolites of sclareolide (402) incubated with Curvularia lunata and Aspergillus 

niger, five oxidized compounds, (403, 404, 405, 405a, 405b) were obtained. Fermentation of 402 

with Gibberella fujikuroii afforded (403, 404, 405, 405a). The metabolites, 403 and 405a were


O 

H 

(–)-Ambrox 

Aspergillus niger 

A. niger + 1-Aminobenzotriazole 

at 30°C for 4 days 

O 

HO 

H 

396 

O 

O 

HO 

H 

OH 

O 

H 

OH 

OH 

397 

392 

HO 

H 

394 

OH 

O 

OH 

HO 

H 

COOH 

393 

O 

H 

395 

FIGURE 15.112 Possible pathway of biotransformation of (-)-ambrox (391) by Aspergillus niger. 

formed from the same substrate by the incubation of Fusarium lini. No microbial transformation of 

402 was observed with Pleurotus ostreatus (Atta-ur Rahman et al., 1997) (Figure 15.117). 

Compound 391 treated in Curvularia lunata gave metabolites 401e and 396, while Cunninghamella 

elegans yielded compounds 401e and 396 and (+)-sclareolide (402) (Figure 15.113). The metabolites 

(401a–401e, 396) from 391 do not release any effective aroma when compared to 391. Compound 

394 showed a strong sweet odor quite different from the amber-like odor (Choudhary et al., 2004). 

Sclareolide (402) exhibited phytotoxic and cytotoxic activity against several human cancer cell 

lines. Cunninghamella elegans gave new oxidized metabolites (403, 404, 405a, 405c, 405d, 405e), 

resulting from the enantioselective hydroxylation. Metabolites 403, 404, and 405a have been known 

as earlier as biotransformed products of 402 by many different fungi and have shown cytotoxicity 

against various human cancer cell lines. The metabolites (403, 404, and 405a) indicated significant 

phytotoxicity at higher dose against Lemna minior L. (Choudhary et al., 2004) (Figure 15.117). 

Ambrox (391) and sclareolide (402) were incubated with the fungus Cephalosporium aphidicola for 

10 days in shake culture to give 3b-hydroxy- (396), 3b,6b-dihydroxy- (401g), 3b,12-dihydroxy- (401h),


O 

H 

(–)-Ambrox (391) 

A. cellulosae 

4 days 

OH 

1 

O 

CrO 3 - H 2 SO 4 

O 

1 

O 

H 

H 

398 (41.3%) 399 (1.6%) 

O 

O 

H 

OH 

H 

O 

400 

FIGURE 15.113 Biotransformation of (-)-ambrox (391) by Aspergillus cellulosae. 

and sclareolide 3b,6b-diol (401f), and 3b-hydroxy- (403), 3-keto- (405), and sclareolide 3b,6b-diol 

(401f), respectively (Hanson and Truneh, 1996) (Figure 15.118). 

Zerumbone (408), which is easily isolated from the wild ginger, Zingiber zerumbet and its epoxide 

(409) were incubated with Fusarium culmorum and Aspergillus niger in Czapek-peptone medium, 

respectively. The former fungus gave (1R,2R)-(+)-2,3-dihydrozerumbol (410) stereospecifically via 

either 2,3-dihydrozerumbone (408a) or zerumbol (408b) or both and accumulated 410 in the mycelium. 

The facile production of optically active 410 will lead a useful material of woody fragrance, 

namely 2,3-dihydrozerumbone. Aspergillus niger biotransformed 408 via epoxide (409) to several 

metabolites containing zerumbone-6,7-diol as a main product. The same fungus converted the epoxide 

(409) into three major metabolites containing (2R,6S,7S,10R,11S)-1-oxo7,9-dihydroxyisodaucane 

(413) via dihydro derivatives (411, 412). However, Aspergillus niger biotransformed 409 only into 

412 in the presence of CYP450 inhibitor, 1-aminobenzotriazole (Noma et al., 2002). 

The same substrate was incubated in the Aspergillus niger, Aspergillus orysae, Candia rugosa, 

Candia tropicalis, Mucor mucedo, Bacillus subtilis, and Schizosaccharomyces pombe; however, 

any metabolites have been obtained. All microbes except for the last organism, zerumbone epoxide 

(409), prepared by mCPBA, bioconverted into two diastereoisomers, 2R,6S,7S-dihydro- (411) and 

2R,6R,7R-derivative (412), whose ratio was determined by gas chromatography (GC) and their 

enantio-excess was over 99% (Nishida and Kawai, 2007) (Figure 15.119). 

Several microorganisms and a few mammals (see later) for the biotransformation of (+)-cedrol 

(414) which is widely distributed in the cedar essential oils were used. Plant pathogenic fungus 

Glomerella cingulata converted cedrol (414) into three diols (415–417) and 2a-hydroxycedrene 

(418) (Miyazawa et al., 1995). The same substrate (414) was incubated with Aspergillus niger to give 

416 and 417 together with a cyclopentanone derivative (419) (Higuchi et al., 2001). Human skin 

microbial flora, Staphylococcus epidermidis also converted (+)-cedrol into 2a-hydroxycedrol (415) 

(Itsuzaki et al., 2002) (Figure 15.120). 

401


OH 

O 

HO 

OH 

O 

H 

H 

H 

O 

a 

401a 

OH 

O 

401b 

HO 

OH 

O 

O 

391 

HO 

H 

OH 

401c 

H 

OH 

401d 

c,d 

O 

OH 

OH 

HO 

H 

HO 

H 

O 

b 

396 

394 

O 

H 

O 

O 

391 

O 

H 

397 

HO 

H 

OH 

401e 

a: Fusarium lini 

b: Rhizopus strolonifera 

c: Curvlaria lanata 

d: Cunninghamella elegans 

c,d 

FIGURE 15.114 Biotransformation of (-)-ambrox (391) by Fusarium lini and Rhizopus stronifera. 

Cephalosporium aphidicola bioconverted cedrol (414) into 417 (Hanson and Nasir, 1993). On 

the other hand, Corynesphora cassiicola produced 419 in addition to 417 (Abraham et al., 1987). It 

is noteworthy that Botrytis cinerea that damages many flowers, fruits and vegetables biotransformed 

cedrol into different metabolites (420–422) from those mentioned above (Aleu et al., 1999). 

4a-Hydroxycedrol (424) was obtained from the metabolite of cedrol acetate (423) by using 

Glomerella cingulata (Matsui et al., 1999) (Figure 15.121). 

Patchouli alcohol (425) was treated in Botrytis cinerea to give three metabolites two tertiary 

alcohols (426, 427), four secondary alcohols (428, 430, 430a), and two primary alcohols (430b, 

O 

O 

O 

OH 

O 

M. plumbeus 

O 

O 

3 H 

H 

H 

H 

HO 

O 

18 

Sclareolide (402) 

403 404 405 

FIGURE 15.115 Biotransformation of (+)-sclareolide (402) by Mucor plumbeus. 

O 

O


O 

O 

O 

3 

18 

402 

O 

A. niger 

HO 

O 

[O] 

3 3 

O 

403 

404 

O 

[O] 

[O] 

O 

O 

HO 

3 

O 

[O] 

3 

18 

O 

18 

OH 

OH 

O 

406 

407 

FIGURE 15.116 Biotransformation of (+)-sclareolide (402) by Aspergillus niger. 

430c) of which compounds 425, 427, and 428 are the major metabolites (Aleu et al., 1999) while 

plant pathogenic fungus Glomerella cingulata converted the same substrate to 5-hydroxy- (426) and 

5,8-dihydroxy derivative (429) (Figure 15.122). 

In order to confirm the formation of 429 from 426, the latter product was reincubated in the same 

medium including Glomerella cingulata to afford 429 (Miyazawa et al., 1997b) (Figure 15.123). 

Patchouli acetate (431) was also treated in the same medium to give 426 and 429 (Matsui and 

Miyazawa, 2000). 5-Hydroxy-a-patchoulene (432) was incubated with Glomerella cingulata to 

afford 1a-hydroxy derivative (426) (Miyazawa et al., 1998a). 

(-)-a-Longipinene (433) was treated with Aspergillus niger to afford 12-hydroxylated product 

(434) (Sakata et al., 2007). 

Ginsenol (435), which was obtained from the essential oil of Panax ginseng, was incubated with 

Botrytis cinerea to afford four secondary alcohols (436–439) and two cyclohexanone derivatives 

(440) from 437 and 441 from 438 or 439. Some of the oxygenated products were considered as potential 

antifungal agents to control Botrytis cinerea (Aleu et al., 1999a) (Figures 15.124 and 15.125). 

(+)-Isolongifolene-9-one (442), which was isolated from some cedar trees was treated in 

Glomerella cingulata for 15 days to afford two primary alcohols (443, 444) and a secondary alcohol 

(445) (Sakata and Miyazawa, 2006) (Figure 15.126). 

Choudhary et al. (2005) reported that fermentation of (-)-isolongifolol (445a) with Fusarium lini 

resulted in the isolation of three metabolites, 10-oxo- (445b), 10a-hydroxy- (445c), and 9ahydroxyisolngifolol 

(445d). Then the same substrate was incubated with Aspergillus niger to yield 

the products 445c and 445d. Both 445c and 445d showed inhibitory activity against 

butylcholinesterease enzyme in a concentration-dependent manner with IC 50 13.6 and 299.5 mM, 

respectively (Figure 15.127). 

(+)-Cycloisolongifol-5b-ol (445e) was fermented with Cunninghamella elegans to afford three 

oxygenated metabolites: 11-oxo- (445f), 3b-hydroxy- (445g), and 3b,11a-dihydroxy derivative 

(445h) (Choudhary et al., 2006a). (Figure 15.128). 

A daucane-type sesquiterpene derivative, lancerroldiol p-hydroxybenzoate (446) was hydrogenated 

with cultured suspension cells of the liverwort, Marchantia polymorpha to give 3,4- 

dihydrolancerodiol (447) (Hegazy et al., 2005) (Figure 15.129). 

Widdrane sesquiterpene alcohol (448) was incubated with Aspergillus niger to give an oxo and 

an oxy derivatives (449, 450) (Hayashi et al., 1999) (Figure 15.130).


O 

O 

HO 

H 

OH 

405f 

O 

O 

a,b 

g 

g 

O 

O 

O 

O 

g 

OH 

O 

O 

H 

d,e,f 

HO 

H 

402 403 404 

405 

O 

H 

H 

a,d,e,f 

OH 

O 

O 

OH 

O 

O 

HO 

H 

HO 

H 

c 

405a 

O 

O 

OH 

405b 

O 

O 

OH 

O 

O 

a: Curvularia lunata 

b: Mucro plumbeus 

c: Cunnighamella elegans 

d: Aspergillus niger 

e: Gibberella fujikuroii 

f: Fusarium lini 

(only 403, 405a) 

g: Cephalosporium 

HO 

aphidicola 

O 

H 

H 

404 405 

O 

O HO 

HO 

O 

O 

H 

405a 

O 

O 

H 

HO 

H 

HO 

H 

405c 

405d 

405e 

FIGURE 15.117 Biotransformation of (+)-sclareolide (402) by various fungi. 

(-)-b-Caryophyllene (451), one of the ubiquitous sesquiterpene hydrocarbons found not only in 

higher plants but also in liverworts, was biotransformed by Pseudomonas cruciviae, Diplodia gossypina, 

and Chaetomium cochlioides (Lamare and Furstoss, 1990). Pseudomonas cruciviae gave a 

ketoalcohol (452) (Devi, 1979), while the latter two species produced the 14-hydroxy-5,6-epoxide 

(454), its carboxylic (455), and 3a-hydroxy- (456) and norcaryophyllene alcohol (457), all of which 

might be formed from caryophyllene C5,C6-epoxide (453). Oxidation pattern of (-)-b-caryophyllene 

by the fungi is very similar to that by mammals (see later) (Figure 15.131). 

Fermentation of (-)-b-caryophyllene (451) with Diplodia grossypina afforded 14 different 

metabolites (453–457j), among which 14-hydroxy-5,6-epoxide (454) and the corresponding acid


O 

O 

O 

a 

HO 

H 

396 

OH 

HO 

H 

OH 

401g 

O 

H 

O 

O 

391 

a: Cephalosporium aphidocola 

HO 

H 

401h 

HO 

H 

OH 

401f 

FIGURE 15.118 Biotransformation of (-)-ambrox (391) by Cephalosporium aphidicola. 

(455) were the major metabolites. Compound 457j is structurally very rare and found in Poronia 

punctata. The main reaction path is epoxidation at C5, C6 as mentioned above and selective hydroxylation 

at C4 (Abraham et al., 1990) (Figure 15.132). 

(-)-b-Caryophyllene epoxide (453) was incubated with Cephalosporium aphidicola for 6 days 

to afford two metabolites (457l, 457m) while Macrophomina phaseolina biotransformed the same 

substrate to 14- (454) and 15-hydroxy derivatives (457k). The same substrate was treated in 

Aspergillus niger, Gibberella fujikuroii, and Rhizopus stolonifera, for 8 days and Fusarium lini for 

10 days to afford the metabolites 457n, 457o, 457p and 457q, and 457r, respectively. All metabolites 

were estimated for butyrylcholine esterase inhibitory activity and compound 457k was found to 

show potency similar activity to galanthamine HBr (IC 50 10.9 versus 8.5 mM) (Choudhary 

et al., 2006) (Figure 15.133). 

O 

OH 

O 

1 2 

10 

9 

aa 

3 

7 6 

408a 

OH 

O 

410 

b 

O 

HO 

O 

H 

408 

O 

mPCBA 

408b 

O 

O 

O 

409 

HO 

O 

411 

b + 1-aminobenzotriazole 

413 

O 

409 

b-g 

O 

411 

O 

412 

a: Fusarium culmorum 

b: Aspergillus niger 

c: Aspergillus orizae 

d: Candida rugosa 

e: Candida tropicalis 

f: Mucor mucedo 

g: Bacillus subtilis 

FIGURE 15.119 Biotransformation of zerumbone (408) by various fungi.


a 

c 

HO 

OH 

a 

HO 

HO 

H 

415 

H 

418 

H 

414 

OH 

a 

b,f 

H 

416 

OH 

a: Glomerella cingulata 


c: Staphylococcus epidermidis 

d: Cephalosporium aphidicola 

e: Corynespora cassiicola 

f: Botrytis cinerea 

a,b 

d,e 

HO 

OH 

H 

417 

e 

O 

OH 

H 

419 

FIGURE 15.120 Biotransformation of cedrol (414) by various fungi. 

HO 

HO 

OH 

OH 

OH 

B. cinerea 

H 

420 

HO 

H 

421 

H 

414 

HO 

OH 

H 

422 

OAc G. cingulata 

OAc 

H 

423 

HO 

H 

424 

FIGURE 15.121 Biotransformation of cedrol (414) by Botrytis cinerea and Glomerella cingulata.


HO 

HO 

HO 

OH 

OH 

426 

H 

427 

OH 

H 

429 

HO 

a,b 

HO 

OH 

HO 

OH 

HO 

HO 

H 

425 

H 

428 

OH 

H 

430 

H 

430a 

HO 

HO 

HO 

a: B. cinerea 

b: G. cingulata 

FIGURE 15.122 Biotransformation of patchoulol (425) by Botrytis cinerea. 

H 

430b 

H 

430c 

OH 

RO 

HO 

G. cingulata G. cingulata 

HO 

OH 

H 

425: R=H 

431: R=Ac 

OH 

OH 

426 429 

OH 

432 

FIGURE 15.123 Biotransformation of patchoulol (425) by Glomerella cingulata. 

OH 

A. niger 

433 434 

FIGURE 15.124 Biotransformation of a-longipinene (433) by Aspergillus niger.


OH 

10 

9 

6 

8 

B. cinerea 

HO 

OH 

10 

OH OH 

9 

OH HO 

8 

OH 

6 

OH 

435 

436 437 438 439 

OH O OH 8 

O 

9 

6 

OH 

440 441 

FIGURE 15.125 Biotransformation of ginsenol (435) by Botrytis cinerea. 

OH 

OH 

O O O O 

G. cingulata 

OH 

442 

443 444 

445 

FIGURE 15.126 Biotransformation of (+)-isolongifolene-9-one (442) by Glomerella cingulata. 

Fusarium lini 

O 

OH 

OH 

445a 

OH OH OH OH 

445b 445c 445d 


FIGURE 15.127 Biotransformation of (-)-isolongifolol (445a) by Aspergillus niger and Fusarium lini. 

OH 

OH 

C. elegans 

OH OH OH OH 

O 

HO 

445e 

445f 445g 445h 

FIGURE 15.128 Biotransformation of (+)-cycloisolongifol-5b-ol (445e) by Cunninghamella elegans.


O 

O 

Marchantia polymorpha cell 

HO 

O 

OH 

HO 

O 

OH 

O 

O 

446 

447 

FIGURE 15.129 Biotransformation of lancerodiol p-hydroxybenzoate (446) by Marchantia polymorpha cells. 

OH A. niger 

OH OH 

O 

HO 

448 449 450 

FIGURE 15.130 Biotransformation of widdrol (448) by Aspergillus niger. 

15 

14 

H 

10 8 7 6 

9 

1 5 

11 

2 3 4 

H 

13 

1) Pseudomonus ceuciviae 

2) Diplodia gossypina 

3) Chaetominum cochlioides 

HO 

H O 

12 

451 

452 

H 

OH 

H 

O 

O 

H 

H 

453 454 

D. gossypina 

C. cochlioides 

OH 

H 

HOOC 

H 

OH 

H 

O 

O 

O 

H 

H 

H 

OH 

457 455 

(5%) 

456 

(12%) 

FIGURE 15.131 Biotransformation of (-)-b-caryophyllene (451) by Pseudomonas ceuciviae, Diplodia 

gossypina, and Chaetomium cochlioides.


H 

HO 

H 

HOOC 

H 

O 

O 

O 

H 

H 

453 454 455 

H 

OH H 

OH 

H 

OH 

H 

15 

14 

H 

5 

2 

H 

11 

451 

6 

Diplodia gossypina 

H 

OH 

H 

H 

OH 

H 

O 

O 

O 

H 

OH 

H 

457 457a 

OH 

457b 

OH 

OH 

OH H 

H 

O 

O 

HO 

O 

H 

H 

457c O 

OH 457d 

OH 

457e 

OH 

OH 

H 

H 

H 

O 

OH 

H 

O 

O 

O 

H 

H 

457f OH 457g OH 457h 

OH 

H 

H 

O 

OH 

OHC 

OH 

457i 

457j 

FIGURE 15.132 Biotransformation of (-)-b-caryophyllene (451) by Diplodia gossypina. 

The fermentation of (-)-b-caryophyllene oxide (453) using Botrytis cinerea and the isolation of 

the metabolites were carried out by Duran et al. (1999) Kobuson (457w) was obtained with fourteen 

products (457s–457u, 457x). Diepoxides 457t and 457u could be the precursors of epimeric alcohols 

457q and 457y obtained through reductive opening of the C2,C11-epoxide. The major reaction 

paths are stereoselective epoxidation and introduction of hydroxyl group at C3. Compound 457ae 

has a caryolane skeleton (Figure 15.134). 

When isoprobotryan-9a-ol (458) produced from isocaryophyllene was incubated with Botrytis 

cinerea, it was hydroxylated at tertiary methyl groups to give three primary alcohols (459–461) 

(Aleu et al., 2002) (Figure 15.135). 

Acyclic sesquiterpenoids, racemic cis-nerolidol (462), and nerylacetone (463) were treated by the 

plant pathogenic fungus, Glomerella cingulata (Miyazawa et al., 1995a). From the former substrate, 

a triol (464) was obtained as the major product. The latter was bioconverted to give the two methyl 

ketones (465, 467) and a triol (468), among which 465 was the predominant. The C10,C11 diols 

(464, 465) might be formed from both epoxides of the substrates, followed by the hydration although 

no C10,C11-epoxides were detected (Figure 15.136).


a 

OH 

H 

H 

15 

14 

H 

H 

2 

6 

5 

11 

453 

O 

H 

H 

O HO 

H 

454 457k 

H 

OH 

O 

a: M. phaseolina 

b: C. aphidicola 

b 

H 

457l 

OH 

HO 

OH 

457m 

a 

H 

OH 

HO 

457n 

OH 

b 

H 

OH 

15 

14 

H 

H 

11 

2 

6 

5 

453 

O 

a: A. niger 

b: G. fujikuroii 

c: R. stolonifera 

d: F. lini 

c 

HO 

HO 

H 

H 

O 

OH 

457o 

O 

457p 

HO 

H 

H 

457q 

O 

d 

H 

OH 

O 

HO 

H 

457r 

FIGURE 15.133 Biotransformation of (-)-b-caryophyllene epoxide (453) by various fungi. 

Racemic trans-nerolidol (469) was also treated in the same fungus to afford w-2 hydroxylated 

product (471) and C10,C11 hydroxylated compounds (472) as seen in racemic cis-nerolidol (462) 

(Miyazawa et al., 1996a) (Figure 15.137). 

12-Hydroxy-trans-nerolidol (472a) is an important precursor in the synthesis of interesting flavor 

of a-sinensal. Hrdlicka et al. (2004) reported the biotransformation of trans-(469) and cis-nerolidol


15 

14 

H 

H 

5 6 B. cinerea 

O 

2 3 

H 

H 

11 453 457s 

OH 

OH 

H 

H 

454 

O 

B. cinerea 

H 

H 

H 

HO 

H 

O 

O 

O 

H 

H 

457k 

O 

457t 

O 

457u 

H 

H 

H 

O 

O 

O 

H 

O 

H 

O 

457v 

H 

H 

O 

457w 

H 

O 

H 

457x 

O 

O 

O 

HO 

H 

457y 

HO 

H 

457q 

H 

HO 

CHO 

457z 

H 

H 

H 

O 

O 

O 

H 

H 

OH 

457aa 

H 

HO 

H 

457ab 

H 

HO 

457ac 

H 

HO 

O 

457ad 

OH 

H 

HO 

O 

457ae 

FIGURE 15.134 Biotransformation of (-)-b-caryophyllene epoxide (453) by Botrytis cinerea. 

(462) and cis-/trans-mixture of nerolidol using repeated batch culture of Aspergillus niger grown in 

computer-controlled bioreactors. Trans-nerolidol (469) gave 472a and 472 and cis-isomer (462) 

afforded 464a and 464. From a mixture of cis- and trans-nerolidol, 12-hydroxy-trans-neroridol 

472a (8%) was obtained in postexponential phase at high dissolved oxygen. At low dissolved oxygen 

condition, the mixture gave 472a (7%) and 464a (6%) (Figure 15.138). 

From geranyl acetone (470) incubated with Glomerella cingulata, four products (473–477) were 

formed. It is noteworthy that the major compounds from both substrates (469, 470) were w–2


HO 

H 

458 

B. cinerea 

HO 

H 

HO 

HO 

H 

HO 

H 

OH 

OH 

459 460 461 

FIGURE 15.135 Biotransformation of isoprobotryan-9a-ol (458) by Botrytis cinerea. 

hydroxylated products, but not C10,C11 dihydroxylated products as seen in cis-nerolidol (462) and 

nerylacetone (463) (Miyazawa et al., 1995c) (Figure 15.136). 

The same fungus bioconverted (2E,6E)-farnesol (478) to four products, w-2 hydroxylated product 

(479), which was further oxidized to give C10,C11 dihydroxylated compound (480) and 5-hydroxy 

derivative (481), followed by isomerization at C2,C3 double bond to afford a triol (482) 

(Miyazawa et al., 1996b) (Figure 15.140). 

The same substrate was bioconverted by Aspergillus niger to afford two metabolites, 10,11- 

dihydroxy- (480) and 5,13-hydroxy derivative (480a) (Madyastha and Gururaja, 1993). 

The same fungus also converted (2Z,6Z)-farnesol (483) to three hydroxylated products: 10,11- 

dihydroxy-(2Z,6Z)- (484), 10,11-dihydroxy (2E,6Z)-farnesol (485), and (5Z)-9,10-dihydroxy-6,10- 

dimethyl-5-undecen-2-one (486) (Nankai et al., 1996) (Figure 15.140). 

12 10 8 

11 9 

13 462 

7 

6 

5 

4 

OH G. cingulata 

3 1 

2 

HO 

OH 

464 

OH 

OH 

O 

HO 

465 

463 

O 

G. cingulata 

466 

OH 

HO 

OH 

468 

OH 

O 

HO 

467 

FIGURE 15.136 Biotransformation of cis-nerolidol (462) and cis-geranyl acetone (463) by Glomerella 

cingulata.


469 

OH 

HO 

G. cingulata 

OH 

471 

OH 

HO 

472 

OH 

OH 

G. cingulata 

HO 

473 

O 

HO 

OH 

476 

OH 

470 

O 

474 

OH 

HO 

475 

O 

HO 

477 

OH 

FIGURE 15.137 Biotransformation of trans-nerolidol (469) and trans-geranyl acetone (470) by Glomerella 

cingulata. 

OH 

OH 

OH 

462 

A. niger 

OH 

464a 

OH 

HO 

464 

OH 

OH 

A. niger 

OH 

472a 

OH 

469 

HO 

OH 

FIGURE 15.138 Biotransformation of cis- (462) and trans-nerolidol (469) by Aspergillus niger.


a 

HO 

479 

OH 

HO 

481 

OH 

OH 

2E,6E-farnesol (478) 

OH 

a,b 

HO 

OH 

480 

OH 

HO 

482 

OH 

OH 

a: Cytochrome P-450 


b 

HO 

480a 

OH 

OH 

FIGURE 15.139 Biotransformation of 2E,6E-farnesol (478) by Cytochrome P-450 and Aspergillus niger. 

HO 

OH 

OH 

12 10 

11 

13 

5 

478 

1 

OH 

A. niger 

480 

OH 

OH 

HO 

480a 

FIGURE 15.140 Biotransformation of 2E,6E-farnesol (478) by Aspergillus niger. 

OH 

HO 

OH 

484 

OH 

OH 

A. niger 

HO 

OH 

2E,6Z-farnesol (483) 

485 

OH 

O 

HO 

FIGURE 15.141 Biotransformation of 2Z,6Z-farnesol (478) by Aspergillus niger. 

486 

O OH 

A. niger 

O OH 

HO 

487 488 

FIGURE 15.142 Biotransformation of 9-oxo-trans-nerolidol (487) by Aspergillus niger.


OH 

a 

OH 

O OH O OH O OH 

488a 488b 488c 

b,c 

OH 

O 

OH 

488e 

a: Aspergillus niger 

b: Cephalsporium aphidicola 

c: Neurospora crassa 

O 

OH 

488d 

OH 

FIGURE 15.143 Biotransformation of diisophorone (488a) by Aspergillus niger, Cephalosporium aphidicola, 

and Neurospora crassa. 

A linear sesquiterpene 9-oxonerolidol (487) was treated in Aspergillus niger to give w-1 hydroxylated 

product (488) (Higuchi et al., 2001) (Figure 15.142). 

Racemic diisophorone (488a) dissolved in ethanol was incubated with the Czapek–Dox medium 

of Aspergillus niger to afford 8a- (488b), 10b- (488c), and 17-hydroxydiisophorone (488d) (Kiran 

et al., 2004). 

On the other hand, the same substrate was fed with Nicotiana crassa and Cephalosporium 

aphidicola to afford only 8b-hydroxydiisophorone (488e) in 20% and 10% yield, respectively 

(Kiran et al., 2005) (Figure 15.143). 

From the metabolites of 5b,6b-dihydroxypresilphiperfolane 2b-angelate (488f) using the fungus 

Mucor ramannianus, 2,3-epoxyangeloyloxy derivative (488g) was obtained (Orabi, 2001) 

(Figure 15.144). 

15.3 BIOTRANSFORMATION OF SESQUITERPENOIDS BY MAMMALS, 

INSECTS, AND CYTOCHROME P-450 

15.3.1 ANIMALS (RABBITS) AND DOSING 

Six male albino rabbits (2–3 kg) were starved for 2 days before experiment. Monoterpene were 

suspended in water (100 mL) containing polysorbate 80 (0.1 g) and were homogenized well. This 

solution (20 mL) was administered to each rabbit through a stomach tube followed by water (20 mL). 

This dose of sesquiterpenoids corresponds to 400–700 mg/kg. Rabbits were housed in stainless 

steal metabolism cages and were allowed rabbit food and water ad libitum. The urine was collected 

daily for 3 days after drug administration and stored at 0–5∞C until the time of analysis. The urine 

was centrifuged to remove feces and hairs at 0∞C and the supernatant was used for the experiments. 

O 

O 

488f 

OH 

OH 

M. ramannianus 

H 

O 

O 

O 

488g 

OH 

OH 

FIGURE 15.144 Biotransformation of 5b,6b-dihydroxypresilphiperfolane 2b-angelate (488f) by Mucor 

ramannianus.


The urine was adjusted to pH 4.6 with acetate buffer and incubated with b-glucuronidase-arylsufatase 

(3 mL/100 mL of fresh urine) at 37∞C for 48 h, followed by continuous ether extraction for 48 h. 

The ether extracts were washed with 5% NaHCO 3 and 5% NaOH to remove the acidic and phenolic 

components, respectively. The ether extract was dried over MgSO 4 , followed by evaporation of the 

solvent to give the neutral crude metabolites (Ishida et al., 1981). 

15.3.2 SESQUITERPENOIDS 

Wild rabbits (hair) and deer damage the young leaves of Chamaecyparis obtusa, one of the most 

important furniture and house-constructing tree in Japan. The essential oil of the leaves contains a 

large amount of (-)-longifolene (489). Longifolene (36 g) was administered to 18 of rabbits to obtain 

the metabolites (3.7 g) from which an aldehyde (490) (35.5%) was isolated as pure state. In the 

metabolism of terpenoids having an exomethylene group, glycol formation was often found, but in 

the case of longifolene such as a diol was not formed. Introduction of an aldehydes group in biotrans 

formation is very remarkable. Stereoselective hydroxylation of the gem dimethyl group on a sevenmembered 

ring is first time (Ishida et al., 1982) (Figure 15.145). 

(-)-b-Caryophyllene (451) is one of the ubiquitous sesquiterpene hydrocarbons in plant kingdom 

and the main component of beer hops and clove oil, and is being used as a culinary ingredient and as 

a cosmetic in soaps and fragrances. (-)-b-Caryophyllene also has cytotoxic against breast carcinoma 

cells and its epoxide is toxic to Planaria worms. It contains unique 1,1-dimethylcyclobutane skeleton. 

(-)-b-Caryophyllene (3 g) was treated in the same manner as described above to afford the crude 

metabolite (2.27 g) from which (10S)-14-hydroxycaryophyllene-5,6-oxide (491) (80%) and a diol (492) 

were obtained (Asakawa et al., 1981). Later, compound (491) was isolated from the Polish mushroom, 

Lactarius camphorates (Basidiomycetes) as a natural product (Daniewski et al., 1981). 14-Hyroxy-bcallyophyllene 

and 1-hydroxy-8-keto-b-caryophyllene have been found in Asteraceae and Pseudomonas 

species, respectively. In order to confirm that caryophyllene epoxide (453) is the intermediate of both 

metabolites, it was treated in the same manner as described above to give the same metabolites (491) 

and (492), of which 491 was predominant (Asakawa et al., 1981, 1986) (Figure 15.146). 

The grapefruit aroma, (+)-nootkatone (2) was administered into rabbits to give 11,12-diol (6, 7). 

The same metabolism has been found in that of biotransformation of nootkatone by microorganisms 

as mentioned in the previous paragraph. Compounds (6, 7) were isolated from the urine of hypertensive 

subjects and named urodiolenone. The endogenous production of 6, 7 seem to occur interdentally 

from the administrative manner of nootkatone or grapefruit. Synthetic racemic nootkatone 

epoxide (14) was incubated with rabbit-liver microsomes to give 11,12-diol (6, 7) (Ishida, 2005). 

Thus, the role of the epoxide was clearly confirmed as an intermediate of nootkatone (2). 

(+)-ent-Cyclocolorenone (98) and its enantiomer (103) were biotransformed by Aspergillus 

species to give cyclopropane-cleaved metabolites as described in the previous paragraph. 

In order to compare the metabolites between mammals and microorganisms, the essential oil 

(2 g/rabbit) containing (-)-cyclocolorenone (103) obtained from Solidago altissima was administered 

in rabbits to obtain two metabolites; 9b-hydroxycyclocolorenone (493) and 10-hydroxycyclocolorenone 

(494) (Asakawa et al., 1986). 10-Hydroxyaromadendrane-type compounds are well 

HO 

Rabbit 

H H 

O 

CHO 

489 

FIGURE 15.145 Biotransformation of longifolene (489) by rabbit. 

490 

CHO


H 

H 

O 

O 

H 

H 

O 

H 

OH 

OH 

492 

15 

O 

OH 

14 

H 8 7 13 

H 

H 

6 

6 

11 9 

Rabbit 

1 5 O 

453 

5 

10 

2 4 

H 

H 

3 

OH 

12 451 14 H 

491 

O 

H 

FIGURE 15.146 Biotransformation of (-)-b-caryophyllene (451) by rabbit. 

known as the natural products. No oxygenated compound of cyclopropane ring was found in the 

metabolites of cyclocolorenone in rabbit (Figure 15.147). 

From the metabolites of elemol (495) possessing the same partial structures of monoterpene 

hydrocarbon, myrcene, and nootkatone, one primary alcohol (496) was obtained from rabbit urine 

after the administration of 495 (Asakawa et al., 1986) (Figure 15.148). 

Components of cedar wood such as cedrol (414) and cedrene shorten the sleeping time of mice. 

In order to search for a relationship between scent, olfaction, and detoxifying enzyme induction, 

(+)-cedrol (414) was administered to rabbits and dogs. From the metabolites from rabbits, two 

C3 hydroxylated products (418 and 497) and a diol (415 or 416), which might be formed after the 

hydrogenation of double bond. Dogs converted cedrol (414) into the different metabolite products, 

H 

H 

OH 

H 

OH 

O 

Rabbit 

O 

O 

103 

493 

494 

FIGURE 15.147 Biotransformation of (+)-ent-cyclocolorenone (101) by rabbit. 

Rabbit 

H 

OH 

HO 

495 

FIGURE 15.148 Biotransformation of elemol (495) by rabbit. 

H 

496 

OH


3 

2 

OH Rabbit 

HO 

3 

OH 

HO 

HO 

H 

H 

H 

H 

414 

Dog 415 or 417 

418 

497 

HO 15 

HO 

OH 

OH 

OH 

2 

H 

H 

HO H 

HO H 

415 416 

498 499 

FIGURE 15.149 Biotransformation of cedrol (414) by rabbits or dogs. 

COOH 

OH 

C2 (498) and C2/C14 hydroxylated products (499), together with the same C3 (415) and C15 hydroxylated 

products (416) as those found in the metabolites of microorganisms and rabbits. The above 

species-specific metabolism is very remarkable (Bang and Ourisson, 1975). 

The microorganisms, Cephalosporium aphidicoda, Corynespora cassicoda, Botrytis cinerea, 

and Glomerella cingulata also biotransformed cedrol to various C2, C3, C4, C6, and C15 hydroxylated 

products as shown in the previous paragraph. The microbial metabolism of cedrol resembles 

that of mammals (Figure 15.149). 

Patchouli alcohol (425) with fungi static properties is one of the important essential oils in perfumery 

industry. Rabbits and dogs gave two oxidative products (500, 501) and one norpatchoulen- 

1-one (502) possessing a characteristic odor. Plant pathogen, Botrytis cinerea causes many diseases 

for vegetables and flowers. This pathogen gave totally different five metabolites (426–430) from 

those found in the urine metabolites of mammals as described above (Bang et al., 1975) 

(Figure 15.150). 

Sandalwood oil contains mainly a-santalol (503) and b-santalol. Rabbits converted a-santalol to 

three diastereomeric primary alcohols (504–506) and dogs did carboxylic acid (507) (Zundel, 1976) 

(Figure 15.151). 

(2E,6E)-Farnesol (478) was treated in cockroach Cytochrome P-450 (CYP4C7) to form regionand 

diastereospecifically w-hydroxylated at the C12 methyl group to the corresponding diol (508) 

with 10E-configuratation (Sutherland et al., 1998) (Figure 15.152). 

Juvenile hormone III (509) was also treated in cockroach CYP4C7 to the corresponding 12-hydroxylated 

product (510) (Sutherland et al., 1998). 

The African locust converted the same substrate (509) into a 7-hydroxy product (511) and a 

13-hydroxylated product (512). It is noteworthy that the African locust and cockroach showed clear 

species specificity for introduction of oxygen function (Darrouzet et al., 1997). 

HO 

Rabbit 

HO 

HO 

HO 

or Dog 

H 

15 

H 

HOOC H 

H 

OH 

425 500 501 502 

FIGURE 15.150 Biotransformation of patchouli alcohol (425) by rabbits or dogs.


OH 

15 

504 

OH 

10 

14 

15 

OH 

Rabbit 

14 

OH 

OH 

503 

Dog 

505 

13 

OH 

10 

COOH 

OH 

506 

507 

FIGURE 15.151 Biotransformation of santalol (503) by rabbits or dogs. 

15.4 BIOTRANSFORMATION OF IONONES, DAMASCONES, 

AND ADAMANTANES 

Racemic a-ionone (513) was converted to 4-hydroxy-a-ionone (514), which was further dehydrogenated 

to 4-oxo-a-ionone (515) by Chlorella ellipsoidea IAMC-27 and Chlorella vulgaris IAMC- 

209. a-Ionone (513) was reduced preferentially to a-ionol (516) by Chlorella sorokiniana and 

Chlorella salina (Noma et al. 1997a). 

a-Ionol (516) was oxidized by Chlorella pyrenoidosa to afford 4-hydroxy-a-ionol (524). The 

same substrate was fed by the same microorganism and Aspergillus niger to furnish a-ionone (513) 

(Noma and Asakawa, 1998) (Figure 15.153). 

13 

12 

11 

10 

7 

2 

478 

1 

OH 

CYP450 

(CYP4C7) 

HO 

12 

508 

2 

OH 

13 

O 

12 

11 

10 

7 

2 

509 

O 

CYP450 

OMe 

(CYP4C7) 

O 

HO 

12 

R 1 

12' 

O 

R 2 

12 

510 

7 

511: R 1 =H, R 2 =OH 

512: R 1 =OH, R 2 =H 

O 

O 

OMe 

OMe 

FIGURE 15.152 Biotransformation of 2E,6E-farnesol (478) by cockroach Cytochrome P-450 and 10,11- 

epoxyfarnesic acid methyl ester (509) by African locust Cytochrome P-450.


A. cellulosae 

C. pyrenoidosa 

O O 

C. salina 

12 

OH 

13 

C. sorokiniana 

7 

D. tertiolocta 

E. gracilis 5 6 1 H. anomala 

8 9 10 

A. sojae 4 2 

522 3 11 513 A. niger 

516 


A. niger 

E. gracilis 

C. ellipsoidea 



C. vulgaris 

OH O OH 

O 

O 

OH 

523 HO 

514 

HO 

524 

A. niger 

B. dothidea 

C. ellipsoidea 




D. tertiolecta 

E. gracilis 

O 

O 

H. anomala 

OH 

A. niger 

Rh. minuta 


A. cellulosae 

A. niger 

O 

A. sojae 

O 

520 515 

517 

A. niger 

A. sojae 

A. cellulosae, A sojae 

B. dothidea, E. gracilis 

D. tertiolecta, Rh. minuta 

FIGURE 15.153 Biotransformation of a-ionone (513) by various microorganisms. 

O 


B. dothidea 


O 

E. gracilis 

O 

521 O 

O 

518 

519 

OH 

4-Oxo-a-ionone (515), which is one of the major product of a-ionone (513) by Aspergillus Niger, 

was transformed reductively by Hansenula anomala, Rhodotorula minuta, Dunaliella tertiolecta, 

Euglena gracilis, Chlorella pyrenoidosa C28 and other eight kinds of Chlorella species, 

Botryosphaeria dothidea, Aspergillus cellulosae IFO 4040 and Aspergillus sojae IFO 4389 to give 

4-oxo-a-ionol (517), 4-oxo-7,8-dihydro-a-ionone (518), and 4-oxo-7,8-dihydro-a-ionol (519). 

Compound 515 was also oxidized by Aspergillus niger and Aspergillus sojae to give 1-hydroxy-4-oxoa-ionone 

(520) and 7,11-oxido-4-oxo-7,8-dihydro-a-ionone (521). C7–C8 Double bond of a-ionone 

(513), 4-oxo a-ionone (515), and 4-oxo-a-ionol (517) were easily reduced to their corresponding 

dihydro products (522, 518, 519), respectively, by Euglena, Aspergillus, Botrysphaeria, and Chlorella 

species. The metabolite (522) was further reduced to 523 by Euglena gracilis (Noma et al., 1998). 

Biotransformation of (+)-1R-a-ionone (513a), [a] D +386.5∞; 99% ee and (-)-1S-a-ionone (513a¢), 

[a] D -361.6∞, 98% ee, which were obtained by optical resolution of racemic a-ionone (513), was fed 

by Aspergillus niger for 4 days in Czapek-peptone medium. From (513a), 4a-hydroxy-a-ionone 

(514a), 4b-hydroxy-a-ionone (514b), and 4-oxo-a-ionone (515a) were obtained, while from compound 

513a¢, the enantiomers (514a¢, 514b¢, 515a¢) of the metabolites from 513a were obtained; 

however, the difference of their yields were observed. In case of 513a, 4a-hydroxy-a-ionone (514a) 

was obtained as the major product, while 515a¢ was predominantly obtained from 513a¢. This oxidation 

was inhibited by 1-aminobenzotriazole, thus CYP-450 is contributed to this oxidation process 


a-Damascone (525) was incubated with Aspergillus niger and Aspergillus terreus, in Czapekpeptone 

medium to give cis- (525) and trans-3-hydroxy-a-damascones (527) and 3-oxo-a-damscone


O O O O 

1R 

513a 

A. niger 

HO HO O 

514a 

(46.2%) 

514b 

(23.1%) 

515a 

(15.6%) 

O O O O 

1S 

A. niger 

HO 

HO 

O 

513a' 

514a' 

(17.0%) 

514b' 

(6.5%) 

515a' 

(69.2%) 

FIGURE 15.154 Biotransformation of (1R)-a-ionone (513a) and (1S)-a-ionone (513a¢) by Aspergillus 

niger. 

(528), while the latter Aspergillus species afforded 3-oxo-8,9-dihydro-a-damascone (529). The 

hydroxylation process of 525–527 was inhibited by CYP-450 inhibitor. Hansenula anomala reduced 

a-damascone (525) to a-damascol (530). Cis- (526) and trans-4-hydroxy-a-damascone (527) were 

fed by Chlorella pyrenoidosa in Noro medium to give 4-oxodamascone (528) (Noma et al., 2001a) 

(Figure 15.155). 

b-Damascone (531) was also treated in Aspergillus niger to afford 5-hydroxy-b-damascone 

(532), 3-hydroxy-b-damascone (533), 5-oxo- (534), 3-oxo-b-damscone (535), and 3-oxo-1, 9-dihydroxy-1,2-dihydro-b-damascone 

(536) as the minor components. In case of Aspergillus terreus, 

3-hydroxy-8,9-dihydro-b-damascone (537) was also obtained (Figure 15.156). 

Adamantane derivatives have been used as many medicinal drugs. In order to obtain the drugs, 

adamantanes were incubated by many microorganisms, such as Aspergillus niger, Aspergillus 

awamori, Aspergillus cellulosae, Aspergillus fumigatus, Aspergillus sojae, Aspergillus terreus, 

O 

A. niger 

CYP450 inhibitor 

12 13 O 

9 

5 6 1 7 8 

4 2 

10 H. anomala 

3 11 

525 

A. niger 

CYP450 inhibitor 

X 

530 

OH 

O 

HO 

526 


O 


HO 

527 

O 

A. terreus 

O 

528 

O 

529 

FIGURE 15.155 Biotransformation of a-damascone (525) by various microorganisms.


HO 

O 

A. niger 

O 

532 

531 

O 

O 

O 

O 

O 

534 

A. niger 

OH 

533 

A. terreus 

OH 

537 

O 

OH 

A. niger 

OH 

535 

O 

536 

O 

FIGURE 15.156 Biotransformation of b-damascone (531) by Aspergillus niger and Aspergillus terreus. 

Botryospherica dothidea, Chlorella pyrenoidosa IMCC-28, Chlorella sorokiriana, Fusarium 

culmurorum, Euglena glacilis, and Hansenula anomala (Figure 15.157). 

Adamantane (538) was incubated with Aspergillus niger, Aspergillus Cellulosea, and 

Botryosphaeria dothidea in Czapek-peptone medium. The same substrate was also treated in 

Chlorella pyrenoidosa in Noro medium. Compound 538 was converted into both 1-hydroxy- (539) 

and 9a-hydroxyadamantane (540) by all four microorganisms, followed by oxidation oxidized 

to give 1,9a-dihydroxyadamantanol (541) by Aspergillus Niger, which was further oxidized to 1-hydroxyadamantane-9-one 

(542), which was reduced to afford 1,9b-hydroxyadamantane (544). 

Aspergillus niger gave the metabolite (541) as the major product in 80% yield. Aspergillus cellulosae 

converted 538 to 539 and 540 in the ratio of 81:19. Chlorella pyrenoidosa gave 539, 540 and 

adamantane-9-one (543) in the ratio 74:16:10. 4a-Adamantanol (540) was directly converted by 

Chlorella pyrenoidosa, Aspergillus niger, and Aspergillus cellulosae to afford 543, which was also 

reduced to 9a-adamantanol (540) by Aspergillus niger. The biotransformation of adamantane, however, 

did not occur by the microorganisms: Hansenula anomala, Chlorella sorokiriana, Dunaliella 

tertiolecta, and Euglena glacilis (Noma et al., 1999). 

OH O OH 

a a a 

a 

HO HO HO HO 

539 541 542 

544 

a-d 

a 

8 OH 

7 9 

10 

4 3 a-d 

a 

5 

a 

6 

1 2 

538 540 543 

O 

a: A. niger 

b: A. cellulosae 

c: B. dothidea 

d: C. pyrenoidosa 

FIGURE 15.157 Biotransformation of adamantane (538) by various microorganisms.


Adamantanes (538–543, 542) were also incubated with various fungi including with Fusarium 

culmurorum. 1-Hydroxyadamantane-9-one (542) was reduced stereoselectively to 541 by Aspergillus 

niger, Aspergillus cellulosae, Botryosphaeria dothidea, and Fusarium culmurorum. On the 

contrary, Fusarium culmurorum reduced 541–542. Aspergillus cellulosae and Botryosphaeria 

dothidea bioconverted 542 to 1,9b-hydroxyadamantane (544) stereoselectively. Adamantane-9-one 

(543) was treated by Aspergillus niger to give nonsteroselectively 545–547 that were further converted 

into diketone (548, 549) and a diol (550). It is noteworthy that oxidation and reduction reactions 

were observed between ketoalcohol (547) and diols (551, 552). The same phenomenon was 

also seen between 546 and 553. The latter diol was also oxidized by Aspergillus niger to furnish 

diketone (549) (Noma et al., 2001b, 2003). Direct hydroxylation at C3 of 1-hydroxyadamantane- 

9-one (542) was seen in the incubation of 539 with Aspergillus niger. 

4-Adamantanone (543) showed promotion effect of cell division of the fungus, while 1-adamantanol 

(539) and adamantane-9-one (543) inhibited germination of lettuce seed. 1-Hydroxyadamantane- 

9-one (542) inhibited the elongation of root of lettuce while and adamantane-1,4-diol (544) and 

adamantane itself (538) promoted root elongation (Noma et al., 1999, 2001b) (Figure 15.158). 

Stereoselective reduction of racemic bicycle[3.3.1]nonane-2,6-dione (555a, 555a¢) was carried 

out by Aspergillus awamori, Aspergillus fumigatus, Aspergillus cellulosae, Aspergillus 

sojae, Aspergillus terreus, Aspergillus niger, Botryosphaeria dothidea, and Fusarium culmorum 

in Czapek-peptone, Hansenula anomala in yeast, Euglena glacilis in Hunter, and Dunaliella 

tertiolecta in Noro medium, respectively. All microorganisms reduced 555 and 555a¢ to give 

a-e 

a 

OH 

OH 

538 HO 539 

HO 554 

a 

a-d 

OH 

OH 

a: A. niger 

b: A. cellulosae 

c: C. pyrenoidosa 

d: B. dothidea 

e: F. curmurorum 

540 

543 

a 

O 

a 

a 

HO 

HO 

HO 

HO 

HO 

e 

542 

545 

546 

547 

FIGURE 15.158 Biotransformation of adamantane (538) and adamantane-9-one by various microorganisms. 

541 

a,b,d 

O 

O 

O 

O 

a 

a,b,d 

a 

a 

a 

a 

HO 

a 

O 

O 

a 

HO 

a 

a 

544 

550 

548 

a 

a 

549 

a 

OH 

O 

O 

HO 

a 

HO 

HO 

HO 

552 

546 

553 

551 

OH 

O 

OH 

OH


O 

555 

O 


B. dothidea 

F. curmurorum 

H. anomala 

E. gracilis 


OH 

O 

556 

OH 

557 

OH 

O 

HO 

HO 

O 

HO 

O 

555a 556a 557a 

Piridium dichromate 

FIGURE 15.159 Biotransformation of bicyclo[3.3.1]nonane-2,6-dione (555a, 555a¢) by various 

microorganisms. 

corresponding monoalcohol (556, 556a) and optical-active (-)-diol (557a) ([a] D -71.8∞ in the case 

of Aspergillus terreus), which was formed by enantioselective reduction of racemic monool, namely 

556 and 556a (Noma et al., 2003) (Figure 15.159). 

15.5 BIOTRANSFORMATION OF AROMATIC COMPOUNDS 

Essential oils contain aromatic compounds, such as p-cymene, carvacrol, thymol, vanillin, cinnamaldehyde, 

eugenol, chavicol, safrole, and asarone (558), among others. 

Takahashi (1994) reported that simple aromatic compounds, propylbenzene, hexylbenene, decylbenzene, 

o- and p-hydroxypropiophenones, p-methoxypropiophenone, 4-hexylresorcinol, and methyl 

4-hexylresorcionol were incubated with Aspergillus niger. From hexyl- and decylbenzenes, w1-hydroxylated 

products were obtained, whereas from propylbenzene, w2 hydroxylated metabolites 

were obtained (Takahashi, 1994). 

Asarone (558) and dihydroeugenol (562) were not biotransformed by Aspergillus niger. However, 

dihydroasarone (559) and methyldihyroeugenol (563) were biotransformed by the same fungus to 

produce a small amount of 2-hydroxy (560, 561) and 2-oxo derivatives (564, 565), respectively. The 

chirality at C2 was determined to be R and S mixtures (1:2) by the modified Mosher method 

(Takahashi, 1994) (Figure 15.160). 

Chlorella species are excellent microalgae as oxidation bioreactors as mentioned earlier. 

Treatment of monoterpene aldehydes and related aldehydes were reduced to the corresponding primary 

alcohols, indicating that these green algae possess reductase. 

A microalgae, Euglena gracillis Z. also contains reductase. The following aromatic aldehydes 

were treated in this organism. Benzaldehyde, 2-cyanobenzaldehyde, o-, m-, and p-anisaldehyde, 

salicylaldehyde, o-, m-, and p-tolualdehyde, o-chlorobenzaldehyde, p-hydroxybenzaldehyde, o-nitro-, 

m-, and p-nitrobenaldeyde, 3-cyanobenzaldehyde, vanillin, isovanillin, o-vanillin, nicotine 

aldehyde, 3-phenylpropionaldehyde, ethyl vanillin. Veratraldehyde, 3-nitrosalicylaldehde, penylacetaldehyde, 

and 2-phenylproanaldehyde gave their corresponding primary alcohols. 2-Cyanobenzaldehyde 

gave its corresponding alcohol with phthalate. m- and p-Chlorobenzaldehyde gave its 

corresponding alcohols and m- and p-chlorobenzoic acids. o-Phthaldehyde and p-phthalate and isoand 

terephthaldehydes gave their corresponding monoalohols and dialcohols. When cinnamaldehyde 

and a-methyl cinnamaldehyde were incubated in Euglena gracilis, cinnamyl alcohol and 

3-phenylpropanol, and 2-methylcinnamyl alcohol, and 2-methyo-3-phenylpropanol were obtained


OMe 

OMe 

MeO 

558 

OMe 

OMe 

OMe 

OMe 

OMe 

OMe 

A. niger 

MeO 

MeO 

OH 

MeO 

O 

559 

560 

561 

OH 

OMe 

562 

OMe 

OMe 

OMe 

OMe 

OMe 

OMe 

A. niger 

OH 

O 

FIGURE 15.160 

niger. 

563 

564 

Biotransformation of dihydroasarone (559) and methyldihydro eugenol (563) by Aspergillus 

in good yield. Euglena gracilis could convert acetophenone to 2-phenylethanol; however, its enantioexcess 

is very poor (10%) (Takahashi, 1994). 

Raspberry ketone (566) and zingerone (574) are the major components of raspberry (Rubus 

idaeus) and ginger (Zingeber offi cinale) and these are used as food additive and spice. Two 

substrates were incubated with the Phytolacca americana cultured cells for 3 days to produce 

two secondary alcohols (567, 568) as well as five glucosides (569–572) from 566, and a secondary 

alcohol (576) and four glycoside products (575, 577–579) from 574. In the case of raspberry 

ketone, phenolic hydroxyl group was preferably glycosylated after the reduction of carbonyl group 

of the substrate occurred. It is interesting to note that one more hydroxyl group was introduced 

into the benzene ring to give 568, which were further glycosylated one of the phenolic hydroxyl 

groups and no glycocide of the secondary alcohol at C2 were obtained (Figure 15.161). 

On the other hand, zingerone (574) was converted into 576, followed by glycosylation to give 

both glucosides (577, 578) of phenolic and secondary hydroxyl groups and a diglucoside (579) of 

both phenolic and secondary hydroxyl group in the molecule. It is the first report on the introduction 

of individual glucose residues onto both phenolic and secondary hydroxyl groups by cultured plant 

cells (Shimoda et al., 2007) (Figure 15.162). 

Thymol (580), carvacrol (583), and eugenol (586) were glucosylated by glycosyl transferase of 

cell-cultured Eucalyptus perriniana to each glucoside (581, 3%; 584, 5%; 587, 7%) and gentiobioside 

565


HO 

GlcO 

Raspberry (566) 

O 

569 O 

HO 

GlcO 

HO 

567 OH 

570 OH 

571 OGlc 

HO 

GlcO 

HO 

HO 

568 OH 

HO 

572 OH 

GlcO 

573 OH 

FIGURE 15.161 Biotransformation of raspberry ketone (566) by Phytolacca americana cells. 

(582, 87%; 585, 56%; 588, 58%). The yield of thymol glycosides was 1.5 times higher than that of 

carvacrol and 4 times higher than that of eugenol. Such glycosylation is useful to obtain higher 

water-soluble products from natural and commercially available secondary metabolites for food 

additives and cosmetic fields (Shimoda et al., 2006) (Figure 15.163). 

Hinokitiol (589), which is easily obtained from cell suspension cultures of Thujopsis dolabrata 

and possesses potent antimicrobial activity, was incubated with cultured cells of Eucalyptus 

perriniana for 7 days to give its monoglucosides (590, 591, 32%) and gentiobiosides (592, 593) 

(Furuya et al., 1997, Hamada et al., 1998) (Figure 15.164). 

(-)-Nopol benzyl ether (594) was smoothly biotransformed by Aspergillus niger, Aspergillus 

cellulosae, Aspergillus sojae, Aspergillus Usami, and Penicillium species in Czapek-peptone 

medium to give (-)-4-oxonopol-2¢,4¢-dihydroxybenzyl ether (595, 23% in the case of Aspergillus 

niger), which demonstrated antioxidant activity (ID 50 30.23 m/M), together with a small amount of 

nopol (6.3% in Aspergillus niger). This is very rare direct introduction of oxygen function on the 

phenyl ring (Noma and Asakawa, 2006) (Figure 15.165). 

HO 

H 3 CO 

Gingerone (574) O 

GlcO 

H 3 CO 

575 O 

HO 

GlcO 

HO 

GlcO 

H 3 CO H 3 CO H 3 CO 

H 3 CO 

576 OH 

577 OH 

578 OGlc 

FIGURE 15.162 Biotransformation of zingerone (574) by Phytolacca americana cells. 

579 

OGlc


HO 

580 

HO 

HO 

OH 

O 

O 

OH 

581 

HO 

HO 

OH 

O 

O 

OH 

HO 

HO 

O O 

OH 

582 

HO 

HO 

HO 

OH 

O 

O 

OH 

HO 

HO 

OH 

O 

O 

OH 

HO 

HO 

O 

O 

OH 

583 

584 

585 

HO 

OCH 3 

HO 

HO 

OH 

O 

O 

OH 

OCH 3 

HO 

HO 

OH 

O 

O 

OH 

HO 

HO 

O 

OH 

O 

OCH3 

586 

587 

FIGURE 15.163 Biotransformation of thymol (580), carvacrol (583), and eugenol (586) by Eucalyptus 

perriniana cells. 

588 

O 

HO 

Hinokitiol (589) 

E. perriniana 

O 

HO 

HO 

O 

(a) 

(b) 

HO 

HO 

CH 2 OH 

O 

O 

OH 

O 

HO 

HO 

CH 2 OH 

O 

O 

CH 

OH 

2 

HO O 

HO O 

OH 

O 

FIGURE 15.164 Biotransformation of hinokitiol (589) by Eucalyptus perriniana cells. 

HO 

HO 

590 

CH 2 OH 

O O 

O 

HO 

591 

HO 

HO 

CH 2 OH 

O 

O 

HO CH 2 

HO 

HO O 

O 

HO 

593 

592 

O 

O 

A. niger 

A. cellulosae 

A. sojae 

A. usami 

Penicillium sp. KCPYN 

HO 

O 

OH 

(–)-Nopol benzylether (594) 

595 (23%) 

FIGURE 15.165 Biotransformation of nopol benzylether (531) by Aspergillus and Penicillium species.


MeO 

HO 

N 

H 

O 

597 

(60.9%) 

8 

9 

11 

OH 

O 

MeO 

A. niger 

MeO 

N 

HO 

H 

HO 

Capsaicin (596) 

N 

H 

O 

598 

(16.0%) 

11 

OH 

MeO 

HO 

N 

H 

O 

599 

(13.6%) 

7 

COOH 

FIGURE 15.166 Biotransformation of capsaicin (596) by Aspergillus niger. 

Capsicum annuum contains capsaicin (596) and its homologues having an alkylvanillylamides 

possess various interesting biological activities such as anti-inflammation, antioxidant, saliva, and 

stomach juice inducing activity, analgesic, antigenotoxic, antimutagenic, anticarcinogenic, antirheunatoid 

arthritis, diabetic neuropathy, and used as food additives. On the other hand, because of 

potent pungency and irritation on skin and mucous membrane, it has not yet been permitted as 

medicinal drug. In order to reduce this typical pungency and application of nonpungent capsaicin 

metabolites to the crude drug, capsaicin (596) (600 mg) including 30% of dihydrocapsaicin (600) 

was incubated in Czapek-peptone medium including Aspergillus niger for 7 days to give three 

metabolites, w1-hydroxylated capsaicin (597, 60.9%), 8,9-dihydro-w1-hydroxycapsaicin (598, 16%), 

and a carboxylic acid (599, 13.6%). All of the metabolites do not show pungency (Figure 15.166). 

Dihydrocapsaicin (600) was also treated in the same manner as described above to afford 

w1-hydroxydihyrocpsaicin (598, 80.9%) in high yield and the carboxylic acid (599, 5.0%). Capsaicin 

itself showed carbachol-induced contraction of 60% in the bronchus at a concentration of 1 m mol/L. 

11-Hydroxycapsicin (85) retained this activity of 60% at a concentration of 30 m mol/L. 

Dihydrocapsicin (600) showed the same activity of contraction in the bronchus, at the same concentration 

as that used in capsaicin. However, the activity of contraction in the bronchus of 11-hydroxy 

derivative (598) showed weaker (50% at 30 mmol/L) than that of the substrate. Since both 

metabolites (597 and 598) are tasteless, these products might be valuable for the crude drug 

although the contraction in the bronchus is weak. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radicalscavenging 

activity test of capsaicin and dihydrocapsaicin derivatives was carried out. 11-Hydroxycapsaicin 

(597), 11-dihydrocapsaicin (598), and capsaicin (596) showed higher activity than 

(±)-a-tocopherol and 11-dihydroxycapsaicin (598) displayed the strong scavenging activity (IC 50 

50 mmol/L) (Hashimoto, Asakawa, unpublished results) (Figure 15.167). 

Shimoda et al. (2007a) reported the bioconversion of capsaicin (596) and 8-nordihydrocapsaicin 

(601) by the cultured cell of Catharathus roseus to give more water-soluble capsaicin derivatives. 

From capsaicin, three glycosides, capsaicin 4-O-b-d-glucopyranoside (602), which was one of the 

capsaicinoids in the fruit of Capsicum and showed 1/100 weaker pungency than capsaicin, 

4-O-(6- O-b-d-xylopyranosyl)-b-d-glucoside (603) and 4-O-(6-O-a-l-arbinosyl)-b-d-glucopyranoside 

(604) were obtained. 8-Nor-dihydrocapsaicin (601) was also incubated with the same cultured 

cell to afford the similar products (605–607) all of which reduced their pungency and enhanced 

water solubility. Since many synthetic capsaicin glycosides possess remarkable pharmacological 

activity, such as decrease of liver and serum lipids, the present products will be used for valuable 

prodrugs (Figure 15.168).


MeO 

HO 

O 

N 

H 

Dihydrocapsaicin (600) 


O 

MeO 

HO 

N 

H 

598 

OH 

O 

MeO 

COOH 

N 

H 599 

HO 

FIGURE 15.167 Biotransformation of dihydrocapsaicin (600) by Aspergillus niger. 

HO 

H 3 CO 

H 

N 

596 O 

OH 

HO O O 

HO OH 

H 3 CO 

H 

N 

602 O 

HO O 

O 

HO 

OH O 

HO O 

HO OH 

H 3 CO 

OH 

O 

O 

HO OH O 

HO O 

HO OH 

H 3 CO 

H N 

603 O 

H 

N 

604 O 

HO 

H 3 CO 

H 

N 

601 O 605 O 

HO O 

O 

HO OH O 

HO OH HO 

O 

HO OH 

O O 

H 

H 3 CO 

HO OH N 

H 3 CO 

OH 

O 

O 

HO OH O 

HO 

O 

HO OH 

H 3 CO 

FIGURE 15.168 Biotransformation of capsaicin (596) and 8-nor-dihydrocapsaicin (601) by Catharanthus 

roseus cells. 

H 

N 

606 O 

H 

N 

607 O


Zingiber offi cinale contains various sesquiterpenoids and pungent aromatic compounds such as 

6-shogaol (608) and 6-gingerol (613) and their pungent compounds that possess cardio tonic and 

sedative activity. 6-Shogaol (608) was incubated with Aspergillus niger in Czapek-peptone medium 

for 2 days to afford w1-hydroxy-6-shagaol (609, 9.9%), which was further converted to 8-hydroxy 

derivative (610, 16.1%), a g-lactone (611, 22.4%), and 3-methoxy-4-hydroxyphenylacetic acid (612, 

48.5%) (Figure 15.169). 

MeO 

HO 

O 

6 

7 

6-Shagol (608) 


2 days 

MeO 

HO 

O 

6 

8 

7 

609 (9.9%) 

OH 

MeO 

HO 

COOH 

9 

612 (48.5%) 

MeO 

HO 

O 

O 5 

8 

611 (22.4%) 

MeO 

HO 

OH 

8 

610 (16.1%) 

5 

OH 

FIGURE 15.169 Biotransformation of 6-shogaol (608) by Aspergillus niger. 

MeO 

HO 

O OH 

6 

6-Gingerol (613) 

1 

A. niger 

A. niger 

MeO 

O 

OH 

6 

OH 

1 

MeO 

O 

OH 

6 

OH 

2 

HO 

614 (39.8%) 

HO 

615 (19.9%) 

MeO 

HO 

O OH 

6 

616 (14.5%) 

3 

COOH 

MeO 

HO 

O 

6 

617 (14.5%) 

1 

OH 

MeO 

O 

O 

6 

3 

O 

MeO 

OH 

8 

O 

O 

HO 

618 (16.9%) HO 

619 (21.1%) 

FIGURE 15.170 Biotransformation of 6-gingerol (613) by Aspergillus niger.


6-Gingerol (613) (1 g) was treated in the same condition as mentioned above to yield six metabolites, 

w1-hydroxy-6-gingerol (614, 39.8%), its carboxylic derivative (616, 14.5%), a g-lactone (618) 

(16.9%) that might be formed from (616), its 8-hydoxy-g-lactone (619, 12.1%), w2-hydroxy-6-gingerol 

(615, 19.9%), and 6-deoxy-gingerol (617, 14.5%) (Takahashi et al., 1993). 

The metabolic pathway of 6-gingerol (613) resembles that of 6-shagaol (608). That of 6-shogaol 

and dihydrocapsaicin (600) is also similar since both substrates gave carboxylic acids as the final 

metabolites (Takahashi et al., 1993) (Figure 15.170). 

In conclusion, a number of sesquiterpenoids were biotransformed by various fungi and mammals 

to afford many metabolites, several of which showed antimicrobial and antifungal, antiobesity, 

cytotoxic, neurotrophic, and enzyme inhibitory activity. Microorganisms introduce oxygen atom at 

allylic position to give secondary hydroxyl and keto groups. Double bond is also oxidized to give 

epoxide, followed by hydrolysis to afford a diol. These reactions precede stereo- and regiospecifically. 

Even at nonactivated carbon atom, oxidation reaction occurs to give primary alcohol. Some 

fungi like Aspergillus niger cleave the cyclopropane ring with a 1,1-dimethyl group. It is noteworthy 

that Aspergillus niger and Aspergillus cellulosae produce the totally different metabolites from the 

same substrates. Some fungi occurs reduction of carbonyl group, oxidation of aryl methyl group, 

phenyl coupling, and cyclization of a 10-membered ring sesquiterpenoids to give C6/C6- and C5/ 

C7-cyclic or spiro compounds. Cytochrome P-450 is responsible for the introduction of oxygen 

function into the substrates. 

The present methods are very useful for the production of medicinal and agricultural drugs as 

well as fragrant components from commercially available cheap, natural, and unnatural terpenoids 

or a large amount of terpenoids from higher medicinal plants and spore-forming plants like liverworts 

and fungi. 

The methodology discussed in this chapter is a very simple one-step reaction in water, nonhazard, 

and very cheap, and it gives many valuable metabolites possessing different properties from 

those of the substrates. 

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16 

Industrial Uses of 


W. S. Brud 

CONTENTS 

16.1 Introduction ..................................................................................................................... 843 

16.2 The History ..................................................................................................................... 843 

16.3 Fragrances ....................................................................................................................... 844 

16.4 Flavors ............................................................................................................................. 845 

16.5 Production and Consumption .......................................................................................... 846 

16.6 Changing Trends ............................................................................................................. 849 

16.7 Conclusions ..................................................................................................................... 853 

Acknowledgments ...................................................................................................................... 853 

References ................................................................................................................................. 853 

Further Reading ......................................................................................................................... 853 

Web Sites .................................................................................................................................... 853 


The period when essential oils were used first on an industrial scale is difficult to identify. The nineteenth 

century is generally regarded as the commencement of the modern phase of industrial application 

of essential oils. However, the large-scale usage of essential oils dates back to ancient Egypt. 

In 1480 bc, Queen Hatshepsut of Egypt sent an expedition to the country of Punt (now Somalia) to 

collect fragrant plants, oils, and resins as ingredients for perfumes, medicaments, and flavors and 

for the mummification of bodies. Precious fragrances have been found in many Egyptian archeological 

excavations, as a symbol of wealth and social position. 

If significant international trade of essential oil-based products is the criterion for industrial use, 

“Queen of Hungary Water” was the first alcoholic perfume in history. This fragrance, based on rosemary 

essential oil distillate, was created in the mid-fourteenth century for the Polish-born Queen 

Elisabeth of Hungary. Following a special presentation to King Charles V, The Wise of France in 1350, 

it became popular in all medieval European courts. The beginning of the eighteenth century saw the 

introduction of “Eau de Cologne,” based on bergamot and other citrus oils, which remains widely 

used to this day. This fresh citrus fragrance was the creation of Jean Maria Farina, a descendant of 

Italian perfumers who came to France with Catherine de Medici and settled in Grasse in the sixteenth 

century. According to the city of Cologne archives, Jean Maria Farina and Karl Hieronymus Farina, in 

1749, established factory (Fabriek) of this water, which sounds very “industrial.” The “Kölnisch 

Wasser” became the first unisex fragrance rather than one simply for men, known and used all over 

Europe, and it has been repeated subsequently in innumerable countertypes as a fragrance for men. 

843


16.2 THE HISTORY 

The history of production of essential oils dates back to ca. 3500 bc when the oldest known water distillation 

equipment for essential oils was employed, and may be seen today in the Texila museum in 

Pakistan. Ancient India, China, and Egypt were the locations where essential oils were produced and 

widely used as medicaments, flavors, and fragrances. Perfumes came to Europe most probably from the 

East at the time of the crusades, and perfumery was accorded a professional status by the approval of a 

French guild of perfumers in Grasse by King Philippe August in 1190. For centuries, Grasse remained 

the center of world perfumery and was also the home of the first ever officially registered essential oilsproducing 

company—Antoine Chiris—in 1768. (It is worth noting that not much later, in 1798, the first 

American essential oil company—Dodge and Olcott Inc.—was established in New York.) 

About 150 years earlier, in 1620, an Englishman, named Yardley, obtained a concession from 

King Charles I to manufacture soap for the London area. Details of this event are sparse, other than 

the high fee paid by Yardley for this privilege. Importantly, however, Yardley’s soap was perfumed 

with English lavender, which remains the Yardley trademark today, and it was probably the first 

case of use of an essential oil as a fragrance in large-scale soap production. 

The use of essential oils as food ingredients has a history dating back to ancient times. There are 

many examples of the use of citrus and other squeezed (manually or mechanically expressed) oils 

for sweets and desserts in ancient Egypt, Greece, and the Roman Empire. Numerous references 

exist to flavored ice creams in the courts of the Roman Emperor Nero and of China. The reintroduction 

of recipes in Europe is attributed to Marco Polo on his return from traveling to China. In other 

stories, Catherine de Medici introduced ice creams in France, whereas Charles I of England served 

the first dessert in the form of frozen cream. Ice was used for freezing drinks and food in many civilizations 

and the Eastern practice of using spices and spice essential oils both as flavoring ingredients 

and as food conservation agents was adopted centuries ago in Europe. 

Whatever may be regarded as the date of their industrial production, essential oils, together with 

a range of related products—pomades, tinctures, resins, absolutes, extracts, distillates, concretes, 

and so on—were the only ingredients of flavor and fragrance products until the late nineteenth century. 

At this stage, the growth in consumption of essential oils as odoriferous and flavoring materials 

stimulated the emergence of a great number of manufacturers in France, the United Kingdom, 

Germany, Switzerland, and the United States (Table 16.1). 

The rapid development of the fragrance and flavor industry in the nineteenth century was generally 

based on essential oils and related natural products. In 1876, however, Haarman and Reimer 

started the first production of synthetic aroma chemicals—vanillin, then coumarin, anisaldehyde, 

heliotropin, and terpineol. Although aroma chemicals made a revolution in fragrances with top 

discoveries in the twentieth century, for many decades both flavors and fragrances were manufactured 

with constituents of natural origin, the majority of which were essential oils. 

16.3 FRAGRANCES 

The main reason for the expansion of the essential oils industry and the growing demand for products 

was the development of the food, soap, and cosmetics industries. Today’s multinational companies, 

the main users of fragrances and flavors, have evolved directly from the developments during 

the mid-nineteenth century. 

In 1806, William Colgate opened his first store for soaps, candles, and laundry starch on Dutch 

Street in New York. In 1864, B.J. Johnson in Milwaukee started the production of soap, which came 

to be known as Palmolive from 1898. In 1866, Colgate launched its first perfumed soaps and 

perfumes. In 1873, Colgate launched toothpaste in a glass jug on the market and in the tube first in 

1896. In 1926, two soap manufacturers—Palmolive and Peet—merged to create Palmolive–Peet, 

which 2 years later merged with Colgate to establish the Colgate–Palmolive–Peet company (renamed 

as the Colgate–Palmolive Company in 1953).

Industrial Uses of Essential Oils 845 

TABLE 16.1 

The First Industrial Manufacturers of Essential Oils, Flavors, 

and Fragrances 

Company Name Country Established 

Antoine Chiris France (Grasse) 1768 

Cavallier Freres France (Grasse) 1784 

Dodge & Olcott Inc. USA (New York) 1798 

Roure Bertrand Fils and Justin Dupont France (Grasse) 1820 

Schimmel & Co. Germany (Leipzig) 1829 

J. Mero-Boyveau France (Grasse) 1832 

Stafford Allen and Sons United Kingdom (London) 1833 

Robertet et Cie France (Grasse) 1850 

W.J. Bush United Kingdom (London) 1851 

Payan-Bertrand et Cie France (Grasse) 1854 

A. Boake Roberts United Kingdom (London) 1865 

Fritsche-Schimmel Co USA (New York) 1871 

V. Mane et Fils France (Grasse) 1871 

Haarman&Reimer Germany (Holzminden) 1874 

R.C. Treatt Co. United Kingdom (Bury) 1886 

N.V. Polak und Schwartz Holland (Zaandam) 1889 

Ogawa and Co. Japan (Osaka) 1893 

Firmenich and Cie Switzerland (Geneve) 1895 

Givaudan S.A. Switzerland (Geneve) 1895 

Maschmeijer Aromatics Holland (Amsterdam) 1900 

Note: Companies continuing to operate under their original name are printed in bold. 

In October 1837, William Procter and James Gamble signed a formal partnership agreement to 

develop their production and marketing of soaps (Gamble) and candles (Procter). “Palm oil,” “rosin,” 

“toilet,” and “shaving” soaps were listed in their advertisements. An “oleine” soap was described as 

having a violet odor. Only 22 years later, Procter & Gamble (P&G) sales reached 1 million dollars. 

In 1879, a fine but inexpensive “ivory” white toilet soap was offered to the market with all purpose 

applications as a toilet and laundry product. In 1890, P&G was selling more than 30 different soaps. 

The story of a third player started in 1890 when William Hesket Lever created his concept of the 

Sunlight Soap, which revolutionized the idea of cleanliness and hygiene in Victorian Britain. 

The very beginning of twentieth century marked the next big event when the young French 

chemist Eugene Schueller prepared his first hair color in 1907 and established what is now L’Oreal. 

These were the flagships in hundreds of emerging (and disappearing by fusions, takeovers, or bankruptcy) 

manufacturers of perfumes, cosmetics, toiletries, detergents, household chemicals, and 

related products, the majority of which were and are perfumed with essential oils. 

16.4 FLAVORS 

Over the same time period, another group of users of essential oils entered the markets. In 1790, 

the term “soda water” for carbon dioxide saturated water as a new drink appeared for the first time in the 

United States and in 1810, the first U.S. patent was issued for the manufacture of imitations of natural 

gaseous mineral waters. Only 9 years later the “soda fountain” was patented by Samuel Fahnestock. In 

1833, carbonated lemonade flavored with lemon juice and citric acid was on sale in England. In 1835, 

the first bottled soda water appeared in the United States. It is, however, interesting that the first flavored 

sparkling drink—Ginger Ale—was created in Ireland in 1851. The milestones in flavored soft drinks


appeared 30 years later: 1881—the first cola-flavored drink in the United States; 1885—Dr Pepper 

was invented by Charles Aderton in Waco, Texas; 1886—Coca-Cola by Dr John S. Pemberton in Atlanta, 

Georgia; and in 1898—Pepsi-Cola, created by Caleb Bradham, known from 1893 as “Brad’s Drink.” 

Dr Pepper was advertised as the king of beverages, free from caffeine (which was added to it later 

on), was flavored with black cherry artificial flavor, and was first sold in the Old Corner Drug Store 

owned by Wade Morrison. Its market success and position as one of the most popular U.S. soft drinks 

started by a presentation during the St Louis World’s Fair, where some other important flavor-consuming 

products—ice cream cones, hot dog rolls, and hamburger buns—were also shown. All of them remain 

major users of natural flavors based on essential oils. Hundred years later after the merger with 

another famous lemon–lime drink 7UP in 1986, it finally became a part of Cadbury. 

Dr. John Pemberton was a pharmacist and he mixed up a combination of lime, cinnamon, coca 

leaves, and cola to make the flavor for his famous drink, first as a remedy against headache 

(Pemberton French Wine Coca) and then reformulated according to the prohibition law and used it 

to add taste to soda water from his “soda fountain.” The unique name and logo was created by his 

bookkeeper Frank Robinson and Coca-Cola was advertised as a delicious, exhilarating, refreshing 

and invigorating temperance drink. Interestingly, the first year of sales resulted in $20 loss, as the 

cost of the flavor syrup used for the drink was higher than the total sales of $50. In 1887, another 

pharmacist, Asa Candler, bought the idea and with aggressive marketing in 10 years introduced his 

drink all over the United States and Canada by selling syrup to other companies licensed to manufacture 

and retail the drink. Until 1905, Coca-Cola was known as a tonic drink and contained the 

extract of cocaine and cola nuts and with the flavoring of lime and sugar. 

Like Pemberton, Caleb Bradham was a pharmacist and in his drugstore, he offered soda water 

from his “soda fountain.” To promote sales, he flavored the soda with sugar, vanilla, pepsin, cola, 

and “rare oils”—obviously the essential oils of lemon and lime—and started selling it as a cure for 

dyspepsia, “Brad’s Drink” than Pepsi-Cola. 

The development of the soft drinks industry is of great importance because it is a major consumer 

of essential oils, especially those of citrus origin. It is enough to say that nowadays, according to their 

web pages, only Coca-Cola-produced beverages are consumed worldwide in a quantity exceeding 

1 billion drinks per day. If we consider that the average content of the appropriate essential oil in the 

final drink is about 0.001–0.002%, and the standard drink is ca. 0.3 l (300 g), we approach a daily 

consumption of essential oils by this company alone at the level of 3–6 tons per day, which gives an 

annual usage well over 2000 tons. Although all other brands of the food industry use substantial 

quantities of essential oils in ice creams, confectionary, bakery, and a variety of fast foods (where 

spice oils are used), these together use less oils than the beverage manufacturers. 

There is one special range of products that can be situated between the food and cosmetic–toiletries 

industry sectors and it is a big consumer of essential oils, especially of all kinds of mint, eucalyptus, 

and some other herbal and fruity oils. These are oral care products, chewing gums, and all 

kinds of mouth refreshing confectioneries. As mentioned above, toothpastes appeared on the market 

in the late nineteenth century in the the United States. Chewing gums or the custom of chewing certain 

plant secretions were known to the ancient Greeks (e.g., mastic tree resin) and to ancient Mayans 

(e.g., sapodilla tree gum). Chewing gum, as we know it now, started in America around 1850 when 

John B. Curtis introduced flavored chewing gum, which was first patented in 1859 by William 

Semple. In 1892, William Wrigley used chewing gum as a free gift with sales of baking powder in 

his business in Chicago and very soon he realized that chewing gum has real potential. In 1893, Juicy 

Fruit gum came into market and was followed in the same year by Wrigley’s Spearmint; today, both 

products are known and consumed worldwide and their names are global trademarks. 

16.5 PRODUCTION AND CONSUMPTION 

This brief and certainly incomplete look into the history of industrial usage of essential oils as 

flavor and fragrance ingredients shows that the real industrial scale of flavor and fragrance industry


developed in the second half of the nineteenth century together with transformation of “manufacture” 

into “industry.” 

There are no reliable data on the scale of consumption of essential oils in specific products. On 

the basis of different sources, it can be estimated that the world market for the flavors and fragrances 

has a value of 10–12 billion euro, being equally shared by each group of products. It is very difficult 

to estimate the usage of essential oils in each of the groups. More oils are used in flavors than 

in fragrances which today are mainly based on aroma chemicals, especially in large volume 

compounds used in detergents and household products. Table 16.2 presents estimated data on world 

consumption of major essential oils (each used over 500 tons per annum). 

TABLE 16.2 

Estimated World Consumption of the Major Essential Oils 

Oil Name 

Consumption 

(tons) 

Approximate 

Value (€ million) a 

Major Applications b 

Orange 50,000 275 Soft drinks, sweets, fragrances 

Cornmint 

(Mentha arvensis) c 25,000 265 Oral care, chewing gum, confectionery, 

fragrances, menthol crystals 

Peppermint 4500 120 Oral care, chewing gum, confectionery, 

liquors, tobacco, fragrances 

Eucalyptus 

(Eucalyptus globulus) 

4000 22 Oral care, chewing gum, confectionery, 

pharmaceuticals, fragrances 

Lemon 3500 21 Soft drinks, sweets, diary, fragrances, 

household chemicals 

Citronella 3000 33 Perfumery, toiletries, household chemicals 

Eucalyptus 

(Eucalyptus citriodora) 

2100 10 Confectionery, oral care, chewing gum, 

pharmaceuticals, fragrances 

Clove leaf 2000 24 Condiments, sweets, pharmaceuticals, 

tobacco, toiletries, household chemicals 

Spearmint 

2000 46 Oral care, chewing gum, confectionery 

(Mentha spicata) 

Cedarwood (Virginia) 1500 22 Perfumery, toiletries, household chemicals 

Lime 1500 66 Soft drinks, sweets, diary, fragrances 

Lavandin 1000 15 Perfumery, cosmetics, toiletries 

Litsea cubeba 1000 20 Citral for soft drinks, fragrances 

Cedarwood (China) 800 11 Perfumery, toiletries, household chemicals 

Camphor 700 3 Pharmaceuticals 

Coriander 700 40 Condiments, pickles, processed food, 

fragrances 

Grapefruit 700 9 Soft drinks, fragrances 

Star anise 700 7 Liquors, sweets, bakery, household 

chemicals 

Patchouli 600 69 Perfumery, cosmetics, toiletries 

Basil 500 12 Condiments, processed food, perfumery, 

toiletries 

Mandarine 500 30 Soft drinks, sweets, liquors, 

perfumery, toiletries 

a 

Based on average prices offered in 2007. 

b 

Almost all of the major oils are used in alternative medicine. 

c 

Main source of natural menthol.


The following oils are used in quantities between 100 and 500 tons per annum: bergamot, cassia, 

cinnamon leaf, clary sage, dill, geranium, lemon petitgrain, lemongrass, petitgrain, pine, rosemary, 

tea tree, and vetivert. It must be emphasized that most of the figures given above on the production 

volume are probably underestimates because no reliable data are available on the domestic consumption 

of essential oils in major producing countries, such as China, India, and Indonesia. 

Therefore quantities presented in various sources are sometimes very different. For example, consumption 

of Mentha arvensis is given as 5000 and 25,000 tons per annum. The lower one probably 

relates to the direct usage of the oil, the higher includes the oil used for the production of menthol 

crystals. In Table 16.2, the highest available figures are presented. Considering the above and general 

figures for flavors and fragrances, it can be estimated that the total value of essential oils used 

worldwide is somewhere between 2 and 3 billion euro. Price fluctuations (e.g., the patchouli oil price 

jump in mid-2007) and many other unpredictable changes cause any estimation of essential oils 

consumption value to be very risky and disputable. The figures given in the table are based on average 

trade offers. Table 16.2 does not include turpentine, which is sometimes added into essential oils 

data. Being used mainly as a chemical solvent or a raw material in the aroma chemicals industry, it 

has no practical application as an essential oil, except in some household chemicals. 

As noted earlier, the largest world consumer of essential oils is the flavor industry, especially for soft 

drinks. However, this is limited to a few essential oils, mainly citrus (orange, lemon, grapefruit, mandarin, 

lime), ginger, cinnamon, clove, and peppermint. Similar oils are used in confectionery, bakery, 

desserts, and dairy products, although the range of oils may be wider and include some fruity products 

and spices. The spicy oils are widely used in numerous salted chips, which are commonly consumed 

along with beverages and long drinks. Also, the alcoholic beverage industry is a substantial user of 

essential oils; for example, anis in numerous specialties of the Mediterranean region; herbal oils in 

liqueurs; ginger in ginger beer; peppermint in mint liquor; and in many other flavored alcohols. 

Next in importance to beverages in the food sector are the sweet, dairy, confectionery, dessert 

(fresh and powdered), sweet bakery, and cream manufacturing sector, for which the main oils used 

are citrus, cinnamon, clove, ginger, and anis. Many other oils are used in an enormous range of very 

different products in this category. 

The fast food and processed food industries are also substantial users of essential oils, although 

the main demand is for spicy and herbal flavors. Important oils here are coriander (especially popular 

in the United States), pepper, pimento, laurel, cardamom, ginger, basil, oregano, dill, and fennel, 

which are added to the spices with the aim of strengthening and standardizing the flavor. 

The major users of essential oils are the big compounders—companies that emerged from the 

historical manufacturers of essential oils and fragrances and flavors and new ones established by 

various deals between old players in the market or, like International Flavors and Fragrances (IFF), 

were created by talented managers who left their parent companies and started on their own. Today’s 

big 10 are listed in Table 16.3. 

Out of the 20 companies listed in Table 16.1, seven were located in France but by 2007, out of 

10 largest, only two are from France. Also, only four of today’s big 10 are over a century old with 

two leaders—Givaudan and Firmenich—from Switzerland and Mane and Robertet from France. 

The flavor and fragrance industry is the one where the majority of oils are introduced into appropriate 

flavor and fragrance compositions. Created by flavorists and perfumers, an elite of professionals 

in the industry, the compositions, complicated mixtures of natural and nature identical ingredients 

for flavoring, and natural and synthetic components for fragrances, are offered to end users. The 

latter are the manufacturers of millions of very different products from luxurious “haute couture” 

perfumes, and top-class-flavored liquors and chocolate pralines through cosmetics, household 

chemicals, sauces, condiments, cleaning products, air fresheners, and aroma marketing. 

It is important to emphasize that a very wide range of essential oils are used in alternative or 

“natural” medicine with aromatherapy—treatment of many ailments with the use of essential oils 

as bioactive ingredients—being the leading outlet for the oils and products in which they are applied 

as major active components. The ideas of aromatherapy from a niche area dominated by lovers of


TABLE 16.3 

Leading Producers of Flavors and Fragrances 

Position Company Name (Headquarters) Sales in Million (€) a 

1 Givaudan S.A. (Vernier, Switzerland) 2550 

2 Firmenich S.A. (Geneve, Switzerland) 1620 

3 International Flavors and Fragrances (New York, USA) 1500 

4 Symrise AG (Holzminden, Germany) 1160 

5 Takasago International Corporation (Tokyo, Japan) 680 

6 Sensient Technologies Flavors&Fragrances (Milwaukee, USA) 400 

7 T. Hasegawa Co. Ltd (Tokyo, Japan) 280 

8 Mane S.A. (Le Bar-sur-Loup, France) 260 

9 Frutarom Industries Ltd (Haifa, Israel) 220 

10 Robertet S.A. (Grasse, France) 210 

a 

Estimated data based on web pages of the companies, various reports, and journals. 

nature and some kind of magic, although based on very old and clinically proved experience, came 

into mass production appearing as an advertising “hit” in many products including global ranges. 

Examples include Colgate–Palmolive liquid soaps, a variety of shampoos, body lotions, creams, 

and so on by many other producers, and fabric softeners emphasizing the benefits to users’ mood 

and condition from the odors of essential oils (and other fragrant ingredients) remaining on fabrics. 

Aromatherapy and “natural” products, where essential oils are emphasized as “the natural” ingredients, 

are a very fast developing segment of the industry and this is a return to what was a common 

practice in ancient and medieval times. 

16.6 CHANGING TRENDS 

Until the second half of the nineteenth century, formulas of perfumes and flavors (although much 

less data are available on flavoring products in history) were based on essential oils and some other 

naturals (musk, civet, amber, resins, pomades, tinctures, extracts, etc.). Now, some 150 years later, 

old formulations are being taken out of historical books and are advertised as the “back to nature” 

trend. Perfumery handbooks published until the early twentieth century listed essential oils, and 

none or only one or two aroma chemicals (or isolates from essential oils). A very good illustration 

of the changes that affected the formulation of perfumes in the twentieth century is a comparison of 

rose fragrance as recorded in perfumery handbooks. Dr Heinrich Hirzel in his Die Toiletten Chemie 

(1892, p. 384) gave the following formula for high-quality white rose perfume: 

400 g of rose extract 

200 g of violet extract 

150 g of acacia extract 

100 g of jasmine extract 

120 g of iris infusion 

25 g of musk tincture 

5 g of rose oil 

10 drops of patchouli oil. 

Felix Cola’s milestone work Le Livre de Parfumeur (1931, p. 192) recorded a white rose formula 

containing only 1% of rose oil, 2% of rose absolute, 7.5% other oils, and aroma chemicals.


Rose Blanche 

Rose oil 

Rose absolute 

Patchouli oil 

Bergamot oil 

Linalool 

Benzyl acetate 

Phenylethyl acetate 

Citronellol 

Geraniol 

Phenylethyl alcohol 

10 g 

20 g 

25 g 

50 g 

60 g 

7 g 

75 g 

185 g 

200 g 

300 g 

In the mid-twentieth century, perfumers were educated to consider chemicals as the most convenient, 

stable, and useful ingredients for fragrance compositions. Several rose fragrance formulas 

with less than 2% rose oil or absolute can be found in F.V. Wells and M. Billot’s Perfumery 

Technology, (1975), and rose fragrance without any natural rose product is nothing curious in a 

contemporary perfumers’ notebook. However, looking through descriptions of new fragrances 

launched in the last few years, one can observe a very strong tendency to emphasize the presence 

of natural ingredients—oils, resinoids, and absolutes—in the fragrant mixture. The “back to 

nature” trend creates another area for essential oils usage in many products. 

A very fast growing group of cosmetics and related products today are the so-called organic products. 

These are based on plant ingredients obtained from wild harvesting or from “organic cultivation” 

and which are free of pesticides, herbicides, synthetic fertilizers, and other chemicals widely 

used in agriculture. According to different sources, sales of “organic” products in 2007 will reach 

4–5 billion U.S. dollars. The same “organic raw materials” are becoming more and more popular in 

the food industry, which in consequence will increase the consumption of “organic flavors” based on 

“organic essential oils.” “Organic” certificates, available in many countries (in principle for agricultural 

products, although they are institutions that also certify cosmetics and related products), are 

product passports to a higher price level and selective shops or departments in supermarkets. The 

importance of that segment of essential oils consumption can be illustrated by comparison of the average 

prices for standard essential oils as listed in Table 16.4 and the same oils claimed as “organic.” 

The consumption of essential oils in perfumed products varies according to the product (Table 16.5): 

from a very high level in perfumes (due to the high concentration of fragrance compounds in perfumes 

and the high content of natural ingredients in perfume fragrances) and in a wide range of 

“natural” cosmetics and toiletries to relatively low levels in detergents and household chemicals, in 

which fragrances are based on readily available low-priced aroma chemicals. However, it must be 

emphasized that although the concentration of essential oils in detergents and related products is low, 

the large volume sales of these consumer products result in substantial consumption of the oils. 

Average values given for fragrance dosage in products and for the content of oils in fragrances are 

based on literature data and private communications from the manufacturers. It should be noted that 

in many cases the actual figures for individual products can be significantly different. “Eau Savage” 

from Dior is a very good example: analytical data indicate a content of essential oils (mainly bergamot) 

of over 70%. Toothpastes are exceptional in that the content of essential oils in the flavor is in 

some cases nearly 100% (mainly peppermint, spearmint cooled with natural menthol). 

While the average dosage of fragrances in the final product can be very high, flavors in food 

products are used in very low dosages, well below 1%. The high consumption of essential oils by 

this sector results from the large volume of sales of flavored foods. Average dosages of flavors and 

the content of essential oils in the flavors are given in Table 16.6. 

As in the case of fragrances, the average figures given in Table 16.6 vary in practice in 

individual cases, both in the flavor content in the product and much more in the essential oils


TABLE 16.4 

Prices of Selected Standard and “Organic” Essential Oils 

Oil Name Standard Quality (€/kg) a Organic Quality (€/kg) a 

Orange 5.50 35 

Cornmint (M. arvensis) 10.50 50 

Peppermint 27.00 100 

Eucalyptus (E. globulus) 5.50 26 

Lemon 6.00 30 

Citronella 11.00 23 

Eucalyptus (E. citriodora) 5.00 34 

Clove leaf 12.00 60 

Spearmint (M. spicata) 23.00 40 

Cedarwood (Virginia) 15.00 58 

Lime 44.00 92 

Lavandin 15.00 36 

Litsea cubeba 20.00 44 

Cedarwood (China) 14.00 53 

Camphor 4.50 24 

Coriander 57.00 143 

Grapefruit 13.00 170 

Patchouli 115.00 250 

a 

Average prices based on commercial offers in 2007. 

TABLE 16.5 

Average Dosage of Fragrances in Consumer Products and Content of Essential Oils 

in Fragrance Compounds 

Position 

Product 

Average Dosage of 

Fragrance Compound 

in Product (%) 

Average Content 

of Essential Oils in 

Fragrance (%) 

1 Perfumes 10.0–25.0 5–30 a 

2 Toilet waters 3.0–8.0 5–50 a 

3 Skin care cosmetics 0.1–0.6 0 –10 

4 Deodorants (inclusive deoparfum) 0.5–5.0 0 –10 

5 Shampoos 0.3–2.0 0 –5 

6 Body cleansing products (liquid soaps) 0.5–3.0 0 –5 

7 Bath preparations 0.5–6.0 0 –10 

8 Soaps 0.5–3.0 0 –5 

9 Toothpastes 0.5–2.5 10 –50 b 

10 Air fresheners 0.5–30.0 0 –20 

11 Washing powders and liquids 0.1–0.5 0 –5 

12 Fabric softeners 0.1–0.5 0 –10 

13 Home care chemicals 0.5–5.0 0 –5 

14 Technical products 0.1–0.5 0 –5 

15 Aromatherapy and organic products 0.1–0.5 100 

a 

b 

Traditional perfumery products contained more natural oils than modern ones. 

Mainly mint oils.


TABLE 16.6 

Average Content of Flavors in Food Products and of Essential Oils in Flavors 

Position 

Food Products 

Flavor Dosage in 

Food Product (%) 

Essential Oils Content 

in Flavor (%) 

1 Alcoholic beverages 0.05–0.15 3–100 

2 Soft drinks 0.10–0.15 2–5 

3 Sweets (confectionery, chocolate, etc.) 0.15–0.25 1–100 

4 Bakery (cakes, biscuits, etc.) 0.10–0.25 1–50 

5 Ice creams 0.10–0.30 2–100 

6 Diary products, desserts 0.05–0.25 1–50 

7 Meat and fish products (also canned) 0.10–0.25 10–20 

8 Sauces, ketchup, condiments 0.10–0.50 2–10 

9 Food concentrates 0.10–0.50 1–25 

10 Snacks 0.10–0.15 2–20 

percentage in the flavor. Again “natural” or “organic” products contain only essential oils, since it 

is unacceptable to include any synthetic aroma chemicals or so-called nature identical food flavors. 

It should be noted that a substantial number of flavorings are oleoresins: products that are a combination 

of essential oils and other plant-derived ingredients, which are especially common in hot 

spices (pepper, chili, pimento, etc.) containing organoleptically important pungent components 

that do not distill in steam. This group of oleoresin products must be included in the total consumption 

of essential oils. 

For many years after World War II, aroma chemicals were considered the future for fragrance 

chemistry and there was strong, if unsuccessful, pressure by the manufacturers to get approval for 

the wide introduction of synthetics (especially those regarded as “nature identical”) in food flavors. 

The very fast development of production and usage of aroma chemicals caused increasing concern 

over safety issues for the human health and for the environment. One by one certain products were 

found harmful either for human health (e.g., nitro musks) or for nature. This resulted in wide research 

on the safety of the chemicals and the development of new safe synthetics. Concurrently, the attention 

of perfumers and producers turned in the direction of essential oils, which as derived from 

natural sources and known and used for centuries were generally considered safe. According to 

recent research, however, this belief is not entirely true and some, fortunately very few, oils and 

other fragrance products obtained from plants have been found dangerous, and their use has been 

banned or restricted. However, these are exceptional cases and the majority of essential oils are 

found safe both for use on the human body as cosmetics and related products as well as for consumption 

as food ingredients. 

It is important to appreciate that the market for “natural,” “organic,” and “ecological” products 

both in body care and food industries has changed from a niche area to a boom in recent years with 

the growth exceeding 30% per annum. The estimated value of sales for “organic” cosmetics and 

toiletries is 600–800 million euro in Europe, the United States, and Japan and will grow steadily 

together with organic foods. This creates a very sound future for the essential oils industry, which 

as such or as isolates derived from the oils will be widely used for fragrance compounds in cosmetic 

and related products as well as for flavors. 

Furthermore, the modernization of agricultural techniques and the growth of plantation areas 

result in better economical factors for the production of essential oil-bearing plants, creating workplaces 

in developing countries of Southeast Asia, Africa, and South America as well as further 

development of modern farms in the United States and Europe (Mediterranean area, Balkans). 

Despite some regulatory restrictions (EU, REACH, FDA, etc.), essential oils are and will have an


important and growing share in the fragrance and flavor industry. The same will be true for the 

usage of essential oils and other products of medicinal plants in pharmaceutical products. It is well 

known that the big pharmaceutical companies invest substantial resources in studies of folk and 

traditional medicine as well as in research on biologically active constituents of plant origin. Both 

of these areas cover applications of essential oils. The same is observed in cosmetic and toiletries 

using essential oils as active healing ingredients. 

16.7 CONCLUSIONS 

It can be concluded that the industrial use of essential oils is a very promising area and that regular 

growth shall be observed in future. Much research work will be undertaken both on the safety of 

existing products and on development of new oil-bearing plants that are used locally in different 

regions of the world both as healing agents and as food flavorings. Both directions are equally 

important. Global exchange of tastes and customs shall not lead to unification by Coca-Cola or 

McDonalds. With all the positive aspects of these products, there are many local specialties that can 

become world property, like basil-oregano-flavored pizza, curry dishes, spicy kebab, or the universal 

and always fashionable Eau de Cologne. With the growth of the usage of the commonly known 

essential oils, new ones coming from exotic flowers of the Amazon jungle or from Indian Ayurveda 

books can add new benefits to the flavor and fragrance industry. 

ACKNOWLEDGMENTS 

The author is most grateful to K.D. Protzen of Paul Kaders GmbH and Dr C. Green for their help 

and assistance in preparation of this chapter. 

REFERENCES 

Cola, F., 1931. Le Livre du Parfumeur. Paris: Casterman. 

Hirzel, H., 1892. Die Toiletten Chemie. Leipzig, Germany: J.J. Weber Verlag. 

FURTHER READING 

Dorland, W.F. and J.A. Rogers Jr., 1977. The Fragrance and Flavor Industry. New Jersey: V.E. Dorland. 

Lawrence, B.M., 2000, Essential Oils 1995–2000. Wheaton, IL: Allured Publishing. 



Wells, F.V. and M. Billot, 1981. Perfumery Technology. London: E. Horwood Ltd. 

WEB SITES 

American Beverage Association: http://www.ameribev.org 

The Coca-Cola Company: http://www.thecoca-colacompany.com/heritage/ourheritage.html 

Colgate-Palmolive: http://www.colgate.com/app/Colgate/US/Corp/History/1806.cvsp 

Pepsi Cola History: http://www.solarnavigator.net/sponsorship/pepsi_cola.htm 

Procter & Gamble: http://www.pg.com/company/who_we_are/ourhistory.shtml 

Unilever: http://www.unilever.com/aboutus

17 

Encapsulation and Other 

Programmed Release 

Techniques for Essential Oils 

and Volatile Terpenes 

Jan Karlsen 

CONTENTS 

17.1 Introduction ..................................................................................................................... 855 

17.2 Controlled Release of Volatiles ....................................................................................... 856 

17.3 Use of Hydrophilic Polymers .......................................................................................... 858 

17.4 Alginate ........................................................................................................................... 859 

17.5 Stabilization of Essential Oil Constituents ..................................................................... 859 

17.6 Controlled Release of Volatiles from Nonvolatile Precursors ........................................ 859 

17.7 Cyclodextrin Complexation of Volatiles ......................................................................... 860 

17.8 Concluding Remarks ....................................................................................................... 860 

References .................................................................................................................................. 860 


In order to widen the applications of volatiles (essential oils), it is necessary to lower the volatility 

of the compounds to obtain a longer shelf life of products made with these compounds. By lowering 

the volatility, one can also imagine a possibility to better test the biological effects of these compounds. 

The encapsulation processes are means by which a liquid essential oil is enclosed in a carrier 

matrix to provide a dry, free-flowing powder. However, for the prolonged effect of volatile 

compounds many other techniques are used, where methods are copied from other fields of research 

when one wants to control the release of active ingredients. 

To lower the volatility, one needs to encapsulate the volatiles into a polymer matrix, utilize complex 

formation, use the covalent bonding to a matrix—to mention a few techniques. We therefore 

need to formulate the volatiles and take many of these techniques from areas where controlled 

release formulations have already been in use for many years. The area with the maximum number 

of applications of controlled release formulations is the field of drug delivery. In this area of research, 

there exists a large number of publications as well as a large number of patents where one can find 

inspiration for the formulation of programmed release of volatiles (Deasy, 1984). 

So far in the area of volatile terpenes/essential oils, we have seen a large number of investigations 

that focused on plant selection, volatiles isolation techniques, separation of the volatiles isolated, 

identification of isolated compounds, and the biochemical formation of terpenes. The formulation 

855


of volatiles into products has been seen as an area of industrial research. This has naturally led to a 

large number of patents but very few scientific publications on the formulation of essential oils and 

lower terpenes. 

The idea of this chapter is to give an introduction to the area of making a controlled release 

product of volatiles and, in particular, of essential oils and their constituents. 

17.2 CONTROLLED RELEASE OF VOLATILES 

The main interest of volatiles encapsulation is the possibility to extend the biological effect of the 

compounds. For essential oils, we want to prolong the activity by lowering the evaporation of 

the volatile compounds. During the last 10 years, there are not many publications on this topic in the 

scientific literature but there are quite a number of patents that describe the various ways of prolonging 

the effect of volatiles (Porzio, 2008; Sair and Sair, 1980a; Sair, 1980b; Zasypkin and Porzio, 

2004; Fulger and Popplewell, 1997, 1998; McIver, 2005). One reason for this fact is that the prolonging 

of the effect of volatile compounds is regarded as so close to practical applications and therefore 

the area of industrial research where the results will be bonded in patent applications. However, 

there are signs that this idea is changing. In order to lower the volatility, thus prolonging the effect 

of essential oils and terpenes, we have to look into another area of scientific research. In the field of 

drug delivery, many techniques have been studied for the controlled delivery of active molecules. 

The reasons for controlled release (encapsulation of volatiles) may be the following: 

Changing the impact of fragrance and flavors 

Adding fragrance to textiles 

Stabilizing specific compounds 

Tailoring the fragrance to the intended use of a product 

Lowering the volatility and thereby prolonging the shelf life of a fragrance product. 

The slow or controlled release of volatiles is achieved by: 

Encapsulation 

Solution/dispersion in a polymer matrix 

Complex formation 

Covalent bonding to another molecule or matrix. 

For essential oils and lower terpenes, the following techniques can be utilized depending on the 

volatiles and the intended use of the final product: 

Microcapsule production 

Microparticle production 

Melt extrusion 

Melt injection 

Complex formation 

Liposomes 

Micelles 

Covalent bonding to a matrix 

Combination of nanocapsules into larger microcapsules. 

Since the making of one of the above-mentioned type of products and techniques will influence 

the activity toward the human biological membranes in one way or the other. Therefore, the relevant 

sizes of biological units are listed in Table 17.1 and the average sizes of units produced in consumer 

products, where volatile compounds are involved are listed in Table 17.2.

Encapsulation and Other Programmed Release Techniques 857 

TABLE 17.1 

Size Diameters of Biological Entities 

Human blood cells 

Bacteria 

Human cell nucleus 

Nanoparticles that can cross biomembranes 

Virus 

Hemoglobin molecule 

Nanoparticle that can cross blood–brain barrier 

DNA helix 

Water molecule 

7000–8000 nm 

800–2000 nm 

1000 nm 

60 nm 

17–300 nm 

3.3 nm 

4 nm 

3 nm 

0.3 nm 

TABLE 17.2 

Average Size of Formulation Units in nm (Sizes 

below 150 nm may be Invisible to the Naked Eye) 

Solutions 0.1 

Micellar solutions 0.5 

Macromolecular solutions 0.5 

Microemulsions 5–20 

Liposomes SUV 20–150 

Nanospheres 100–500 

Nanocapsules 100–500 

Liposomes LUV 200–500 

Liposomes MLV 200–1000 

Microcapsules 5000–30,000 

Simple emulsions 500–5000 

Multiple emulsions 10,000–100,000 

Abbreviations: SUV is small unilamellar vesicles, LUV is large unilamellar 

vesicles, and MUV is multilamellar vesicles. 

Since the introduction of the encapsulation of volatiles (essential oils/lower terpenes), the number 

of applications has multiplied. Encapsulation of volatiles gives us a more predictable and longlasting 

effect of the products. The areas of applications are large and the industry of essential oils 

and terpenes foresee many prospects for microencapsulated products. 

Application markets of encapsulated essential oils and terpenes are: 

Medicine 

Food, household items, and personal care 

Biotechnology 

Pharmaceuticals 

Electronics 

Photography 

Chemical industry 

Textile industry 

Cosmetics.


It is therefore easy to understand that the encapsulation procedures will open up a much larger 

market for essential oil/terpene products. Experience from all the areas mentioned above can be 

applied to the study of volatile compounds in products. 

In the area of essential oils and lower terpenes, simple encapsulation procedures from the area of 

drug delivery are applied. The essential oils or single active constituents are mixed with a hydrophilic 

polymer and spray-dried using a commercial spray-drier. Depending on whether we have an 

emulsion or a solution of the volatile fraction in the polymer, we obtain monolithic particles or a 

normal microcapsule. 

The most usual polymers used for encapsulation are: 

Oligosaccharides from a-amylase 

Modified starches from maize, cassava, rice, and potato 

Acacia gum 

Gum arabic 

Alginate 

Chitosan. 

Many different emulsifiers are used to solubilize the essential oils totally or partly, prior to the 

encapsulation procedure. This can result in a monolithic particle or a usual capsule, where the 

essential oil is surrounded by a hydrophilic coating. When the mixture of an essential oil and a 

hydrophilic polymer is achieved, the application of a spray-drying procedure of the resulting mixture 

will result in the formation of microcapsules. The techniques for achieving an encapsulated 

product in high efficiency will depend on many technical parameters and can be found in the patent 

literature. Normally a mixture of essential oil:hydrophilic polymer (4:1) can be used, but this will 

also depend on the type of equipment used. The reader is advised to refer to the parameters given 

for the polymer used in the experiment. To achieve the encapsulated product, a mixture of low pressure 

and temperature is used in the spray-drying equipment and a loss of essential oil/volatiles is 

inevitable. However, a recovery of more than 70% can be achieved by carefully monitoring the 

production conditions. 

17.3 USE OF HYDROPHILIC POLYMERS 

In product development, one tends to use cheap derivatives of starches or other low-grade quality 

polymers. Early studies with protein-based polymers such as gelatine, gelatine derivatives, 

soy proteins, and milk-derived proteins gave reasonable technical quality of the products. 

However, even if these materials show stable emulsification properties with essential oils, they 

have some unwanted side effects in products. We have seen that a more careful control of the 

polymer used can result in real high-tech products, where the predictability of the release of the 

volatiles can be assured like a programmed release of drug molecules in drug delivery devices. 

The polymer quality to be used will, of course, depend on the intention of the final product. In 

the cosmetic industry, where one is looking for an essential oil product, free-flowing and dry, to 

mix with a semisolid or a solid matrix, the use of simple starch derivatives will be very good. 

For other applications, where the release of the volatiles needs to be controlled or predicted more 

accurately, it is recommended that a more thorough selection of a well-characterized polymer is 

done. One example of a very good and controllable polymer is alginate. This polymer is available 

in many qualities and can be tailored to any controlled release product. The chemistry of 

alginate is briefly discussed below as this discussion will allow the reader to decide whether to 

opt for an alginate of technical quality or, if a high-tech product is the aim, to choose a better 

characterized hydrocolloid.


17.4 ALGINATE 

Alginates are naturally occurring polycarbohydrates consisting of the monomers a-l-glucuronic 

acid (G) and b-d-mannuronic acid (M). The relative amounts of these two building blocks will 

influence the total chemistry of the polymer. The linear polymer is water soluble due to its polarity. 

Today the alginate can be produced by the bacteria that allow us to control the composition of the 

monomers (G/M ratio). The chemical composition of the alginate is dependent on the origin of the 

raw material. The marine species display seasonal differences in the composition and different parts 

of the plant produce different alginates. Alginates may undergo epimerization to obtain the preferred 

chemical composition. This composition (G/M ratio) will determine the diffusion rate through 

the swollen alginate gel, which surrounds the encapsulated essential oils (Elias, 1997; Amsden, 

1998a, 1998b; Ogston, 1973). An important structure parameter is also the distribution of the carboxyl 

groups along the polymer chain. The molecular weight of the polymer is equally important, 

and molecular weights between 12,000 and 250,000 are readily available in the market. The alginate 

polymer can form a swollen gel by hydrophobic interaction or by cross-linking with divalent 

ions like calcium. The G/M ratio determines the swelling rate and therefore also the release of 

encapsulated compounds. The diffusion of different substances has been studied and references can 

be made to essential oil encapsulation. The size of alginate capsules can also vary from 100 μm or 

more down to the nanometer range depending on the production procedure chosen (Draget et al., 

1994, 1997; Donati et al., 2005; Tønnesen and Karlsen, 2002; Shilpa, 2003). 

17.5 STABILIZATION OF ESSENTIAL OIL CONSTITUENTS 

The encapsulation of essential oils in a hydrophilic polymer may stabilize the constituents of the oil 

but a better technique for this purpose will often be to use cyclodextrins in the encapsulation process. 

The use of cyclodextrins will lead to a complexation of the single compounds, which will again 

stabilize the complexed molecule. Complex formation with cyclodextrins is often used in drug 

delivery to promote solubility of lipophilic compounds; however, in the case of volatiles containing 

compounds that may oxidize, the complex formation will definitely prolong the shelf life of the 

finished product. A good review of the flavor encapsulation advantages is given by Risch and 

Reineccius (1988). The most important aspect of essential oil encapsulation in a hydrophilic polymer 

is that the volatility is lowered. Lowering the volatility will result in longer shelf life of products 

and a better stability of the finished product in this respect. 

17.6 CONTROLLED RELEASE OF VOLATILES FROM 

NONVOLATILE PRECURSORS 

The limited effect of volatiles for olfactive perception has led to the development of encapsulated 

volatiles and also to the development of covalent-bonded fragrance molecules to matrices. In this 

way, molecules release their fragrance components by the cleavage of the covalent bond. Mild reaction 

conditions met in practical life initiated by light, pH, hydrolysis, temperature, oxygen, and 

enzymes may release the flavors. The production of “profragrances” is a very active field for the 

industry and has led to numerous patents. The plants producing essential oils have invented means 

by which the volatiles are produced, stored, and released into the atmosphere at predestined times 

related to the environmental factors. The making of a profragrance involves mimicking these natural 

procedures into flavor products. However, we are simplifying the process by using only one 

parameter in this release process, that is, the splitting of a covalent bond. In theory, the making of a 

long-lasting biological impact and the breakdown of a constituent are contradictory reactions. 

However, in practice the use of a covalent bond and thereafter the control of the splitting of this 

bond by parameters such as light, humidity, temperature, and so on can be built into suitable flavor


and fragrance products. Naturally, this technique using covalent bonding is only applicable to single 

essential oil constituents but constitutes a follow-up of essential oil encapsulation (Herrmann, 2004, 

2007; Powell, 2006). 

17.7 CYCLODEXTRIN COMPLEXATION OF VOLATILES 

Cyclodextrin molecules are modified carbohydrates that have been used for many years to modify 

the solubility properties of drug molecules by complexation. The cyclodextrin can also be applied 

to volatiles to protect them against the environmental hazards and thus prolong the shelf life of these 

compounds. Cyclodextrin complexation will also modify the volatility of the essential oils and prolong 

the bioactivity. The cyclodextrins will give a molecular encapsulation by the complexation 

reaction with volatile molecules. The complexation of the volatiles with cyclodextrin may improve 

the heat stability, improve the stability toward oxygen, and improve the stability against light (Szente, 

1988). A significant lowering of the volatility has been observed for the complexation with essential 

oils (Risch and Reineccius, 1988). The complexation of essential oils by the use of cyclodextrins 

will also result in increased heat stability. This is in contrast with the stability of volatiles that have 

been adsorbed on a polymer matrix. The use of cyclodextrins can protect the volatiles against 

Loss of volatiles upon storage of a finished product 

Light-induced instability 

Heat decomposition 

Production of free-flowing “dry” powders 

Oxidation. 

17.8 CONCLUDING REMARKS 

The encapsulation/complexation of essential oils, volatiles, or single oil constituents will result in a 

remarkable lowering of the volatility, stabilize the constituents, improve the shelf life of finished 

products, and prolong the biological activity. The control of these parameters will depend on the 

nature of the volatiles to be encapsulated. Most of the literature on the encapsulation of volatiles is 

found in the patent literature. Techniques described in the literature allow the user of essential oils 

to choose the polymer matrix in which to encapsulate an essential oil according to the use of the 

finished product. The effect of controlled delivery of flavor and fragrance molecules opens up large 

areas of applications, which previously were limited due to the volatility of the essential oils and 

their constituents. The encapsulation or lowering the volatility of compounds like essential oil 

ingredients will allow for more relevant studies of the biological effects of volatile compounds. 

REFERENCES 

Amsden, B., 1998a. Solute diffusion within hydrogels. Macromolecules, 31: 8382–8395. 

Amsden, B., 1998b. Solute diffusion in hydrogels. Polym. Gels Networks, 6: 13–43. 

Deasy, P.B., 1984. Microencapsulation and Related Drug Processes. New York: Marcel Dekker. 

Donati, I., S. Holtan, Y.A. Morch, M. Borgogna, M. Dentini, and G. Skjåk-Bræk, 2005. New hypothesis on the 

role of alternating sequences in calcium-alginate gels. Biomacromolecules, 6: 1031–1040. 

Draget, K.I., G. Skjåk-Bræk, B.E. Christensen, O. Gåserød, and O. Smidsrød, 1997. Swelling and partial solubization 

of alginic acid beads in acids. Carbohydr. Polym., 29: 209–215. 

Draget, K.I., G. Skjåk-Bræk, and O. Smidsrød, 1994. Alginic acid gels; the effect of alginate chemical composition 

and molecular weight. Carbohydr. Polym., 25: 31–38. 

Elias, H.-G., 1997. An Introduction to Polymer Science. Weinheim, Germany: VCH. 

Fulger, C. and M. Popplewell, 1997. Flavour encapsulation. U.S. Patent 5.601.845. 

Fulger, C. and M. Popplewell, 1998. Flavour encapsulation. U.S. Patent 5.792.505. 

Herrmann, A., 2004. Photochemical fragrance delivery systems based on the norrish type II reaction—a review. 

Spectrum, 17: 10–13 and 19.


Herrmann, A., 2007. Controlled release of volatiles under mild reaction conditions: From nature to everyday 

products. Angew. Chem. Int. Ed., 46: 5836–5863. 

McIver, B., 2005. Encapsulation of flavour and/or fragrance composition. U.S. Patent 6.932.982. 

Ogston, A.G., B.N. Preston, and J.D. Wells, 1973. On the transport of compact particles through solutions of 

chain polymers. Proc. R. Soc. (London) A, 333: 297–309. 

Porzio, M., 2008. Melt extrusion and melt injection. Perfumer Flavorist, 33: 48–53. 

Powell, K., J. Benkhoff, W. Fischer, and K. Fritsche, 2006. Secret sensations: Novel functionalities triggered by 

light—Part II: Photolatent fragrances. Eur. Coat. J., 9: 40–49. 

Risch, S.J. and G.A. Reineccius, 1988. Flavor Encapsulation. ACS Symposium Series 370, American Chemical 

Society, Washington. 

Sair, L., 1980b. Food supplement concentrate in a dense glass house extrudate. U.S. Patent 4.232.047. 

Sair, L. and R. Sair, 1980a. Encapsulation of active agents as microdispersions in homogeneous natural 

polymers. U.S. Patent 4.230.687. 

Shilpa, A., S.S. Agarwal, and A.R. Ray, 2003. Controlled delivery of drugs from alginate matrix. Macromol. 

Sci. Polym. Rev., C, 43: 187–221. 

Szente, L. and J. Szejtli, 1988. Stabilization of flavors by cyclodextrins. In: Flavor Encapsulation, S.J. Risch 

(ed.), pp. 148–157. ACS Symposium Series 370, Washington DC: American Chemical Society. 

Tønnesen, H.H. and J. Karlsen, 2002. Alginate in drug delivery systems. Drug Dev. Ind. Pharm., 28: 

621–630. 

Zasypkin, D. and M. Porzio, 2004. Glass encapsulation of flavours with chemically modified starch blends. 

J. Microencapsulation, 21: 385–397.

18 

Aroma-Vital Cuisine 

Healthy and Delightful 

Consumption by the 

Use of Essential Oils 

Maria M. Kettenring and Lara-M. Vucemilovic-Geeganage 

CONTENTS 

18.1 Basic Principles of the Aroma-Vital Cuisine .................................................................. 864 

18.1.1 The Heart of Culinary Arts is Based on Exquisite Ingredients and 

an Accomplished Rounding ............................................................................... 864 

18.1.2 Quality Criteria and Specifics that have to be Adhered to, while 

Handling Essential Oils for Food Preparation ................................................... 864 

18.1.3 Storage ............................................................................................................... 865 

18.1.4 Quantity ............................................................................................................. 865 

18.1.5 Emulsifiers and Forms of Administering .......................................................... 865 

18.1.6 To Add Spice with Natural Aromas in a Balanced Way .................................... 865 

18.1.7 Essential Oils are able to Lift Our Spirits as well ............................................. 866 

18.2 A Small Culinary Trip: Aroma-Vital Cuisine Recipes and Introduction ....................... 866 

Your nourishment ought to be your remedies and your medicaments shall be your food. 

—Hippocrates 

Certainly, the value of our nutrition, in terms of nutritional physiology, is not only conditioned by its 

nutrient and calorie contents. Moreover, also health-conscious and constitutional eating habits 

require an adequate preparation of meals as well as an appropriate form of presentation. Early 

sophisticated civilizations and their health doctrines, like that of the Traditional Chinese Medicine 

(TCM), Ayurveda in Southeast Asia, and for instance the medical schools during the ancient Greek 

period examined individuals and their reaction on life circumstances, habits, nutrition, and 

substances, to contribute to a long-lasting health. To support a person’s balance the aim was to 

develop a conscious way of using the senses and a balanced sensory perception. 

Thus fragrances are a kind of soul food, as the information of scents can be perceived in every section 

of our self, physical, energetic as well as intellectual, from a holistic point of view. Adding spice 

with essential oils according to the Aroma-Vital cuisine combines sensuality with sanative potential. 

People across continents and cultures have experimented with the healing virtues of “nature’s 

bouquet” or just simply tried to enhance the flavor and vitality of their meals. The ancient Egyptian 

civilization reverted to an elaborated dinner ceremony by using the efficacy of essential oils to get 

863


the participants in the mood for the meal. Before the food was served, heated chalices with scented 

fats, enriched with a variety of herbs and spices were provided, not only to spread pleasant smells, 

rather as a kind of odorous aperitif to activate ones saliva to prepare for digestion. Meals that have 

been enriched with essential oils or expressed oils, rebound to a conscious awareness of consuming 

food, are well-nigh comparable, like going on a culinary expedition. This fare is perceived as a 

composition of tastes, which is not only tastefully ingenious, but also might be able to raise the 

food’s virtue. 

In this regard the entropy rather than the potency of the condiment is significant. The abundance 

of nuances, the art of adding flavor on the cusp of being noticeable, becomes more important than 

giving aroma officiously. The scents hovering above the meals, almost like a slight breeze, compound 

the food’s own natural flavor in a subtle manner. “Less is more” is the economic approach 

which in this context is indicative. 

The sensation of satiety is taking place early on. Due to this desire to savor to the fullest, the taste 

is excited and leads to longer chewing. This in turn activates a-amylase (amylolytic enzyme, already 

working in the oral cavity). Conditionally on the high bioavailability, especially of the monoterpenes, 

which are significant and available in the paring of citrus fruits and some kind of herbs, in a 

sense the Aroma-Vital cuisine shows aspects of the salutary genesis (Salutogenese). The savoriness 

of the food, pleasant smell, and appetizing appearance plays a prominent role here, at last the appetite 

regulates between physiological needs and pleasure and thus variety and vitally enhanced meals 

are in demand. 

18.1 BASIC PRINCIPLES OF THE AROMA-VITAL CUISINE 

18.1.1 THE HEART OF CULINARY ARTS IS BASED ON EXQUISITE INGREDIENTS 

AND AN ACCOMPLISHED ROUNDING 

Natural aromas, from blossoms, herbs, seeds, and spices, extracted in artificial pure essential oils, 

delicately accompany the elaborate cuisine. They are not supposed to supersede fresh herbs, rather 

complementing them. If, however, herbs are not available, natural essences are delightfully suited to 

add nuances. They are giving impetus to and are flexible assistants for preparing last-minute menus. 

One should use this rich source to compile a first-aid assortment of condiments or even a mobile 

spice rack. 

18.1.2 QUALITY CRITERIA AND SPECIFICS THAT HAVE TO BE ADHERED TO, 

WHILE HANDLING ESSENTIAL OILS FOR FOOD PREPARATION 

The regional legal regulations of the food chemical codex or the local food legislation might differ 

and if one is going to use essential oils professionally, one has to be firm with them, but still there 

are certain basics that deserve attention and lead to a safe and healthy way of practicing this subtle 

culinary art. 

For cooking, solely 100% pure essential oils from controlled organic cultivation should be used. 

Oils that are not available of controlled organic origin, particularly those that are cold-pressed, 

a residue check should be guaranteed by the manufacturer to ensure that the product does not 

contain harmful amounts of pesticides. The label should not only contain name, contents, and 

quantity but also 

• Latin definition 

• Country of origin 

• Description of used plant parts 

• Used method of extraction

Aroma-Vital Cuisine 865 

• Date of expiry 

• If the oil has been thinned, the exact ratio of mixture 

• If solvents have been used, they should be mentioned. 

For the Aroma-Vital cuisine, the only acceptable solvent would be alcohol. As the oil is used in 

very small and thinned concentrations it would not be harmful to children. Less qualitative oils 

from industrial origin sometimes might even contain other substances. It should be indicated that 

natural flavorings used in food production should be pure and free of animal by-products such as 

gelatin or glycerin, which has been obtained by saponification of animal fat. 

18.1.3 STORAGE 

Essential oils are very sensible to the disposure of light, air and temperature; therefore they should 

be stored adequately. In this way, long-lasting essential oils keep their aroma as well as their ingredients 

and might even develop their bouquet. Foods or processed foods with essential oils may not 

be stored in tin boxes. Very important: essential oils should be kept away from children. 

18.1.4 QUANTITY 

The internal use of essential oils has to be practiced carefully. This subtle art is an amazing tool, but 

swallowed in too huge amounts, they are bad for one’s health. One should never add the pure 

concentrate of essential oils to foods; it should not be forgotten that 1 drop is often comparable to a 

huge amount of plant material. Therefore, they ought to be always thinned and the dilution should 

be used teaspoon by teaspoon. 

18.1.5 EMULSIFIERS AND FORMS OF ADMINISTERING 

Essential oils are not water soluble; therefore, emulsifiers are necessary to spread their aroma, they 

are for example 

1. Basic oils, special oils, or macerated oils 

2. Butter, milk, curd, egg yolk, and mayonnaise 

3. Alcohol and vinegar 

4. Syrups, molasses, honeys, treacles, and sugars 

5. Salt 

6. Tofu, soy sauce and tamarind sauce 

7. Avocado, lemon juice, and coconut 

8. Sesame seeds, sunflower seeds, almonds, and walnuts. 

On the basis of these emulsifiers and a mixture of essential oils, a variety of “culinary assistants” 

can be conjured up: spiced oils, spiced butter or mayonnaises, spiced alcohols, spiced syrups, spiced 

sauces, or even spiced salts. These blends can be prepared in advance and stored to use them for 

everyday meals. Another nice variation is the use of hydrolates (a partial extract of plant material 

extracted by distillation) such as rose water, for food preparation. 

18.1.6 TO ADD SPICE WITH NATURAL AROMAS IN A BALANCED WAY 

To know how food and essential oils interact is a great help to create a harmonic assembly of foods, 

which is nourishing us from a holistic point of view. In this manner, the sun-pervaded seed oils of 

anise, bay, dill, fennel, or caraway might be able to aerate the earthy corm- and root-vegetable. 

Salads can be enhanced and prepared to be more digestive by adding pure natural essential oils such 

as thyme, rosemary, and clementine to the marinade, or another rather Asian variation would be to 

add ginger, pepper, and lemon grass.


18.1.7 ESSENTIAL OILS ARE ABLE TO LIFT OUR SPIRITS AS WELL 

A condiment ensemble of orange, vanilla extract, cacao extract, and rose for example, is able to support 

soul foods such as milk rice, milk shakes, and desserts in their attitude to supply security and 

confidence. 

18.2 A SMALL CULINARY TRIP: AROMA-VITAL CUISINE 

RECIPES AND INTRODUCTION 

TABLE 18.1 

Basic Spice Rack of Essential Oils: How to Prepare Essential Oil Mixtures and Essential 

Oil Seasonings 

Basic Essential Oils 

Mixtures 

Emulsifier 

Seasonings 

EURO ASIA 

Recipes Example 

Lime (Citrus aurantiifolia) 5 drops 1. Oil 50 mL sesame oil Asian style 

2. Dairy prod. 50 mL mayonnaise Eggs 

Coriander seed (coriandrum sativum) 1 drop 3. Vinegar 50 mL rice vinegar Sushi 

4. Sweetener 50 mL agave syrup Chutney 

Ginger (Zingiber offi cinalis) 2 drops 5. Salt 50 mg sea-salt Spice 

6. Tofu and co 50 mL soy sauce Marinated fried tofu 

Lemongras (Cymbopogon citratus) 1 drop 7. Vegetables 50 mL coconut milk Rice and curry 

and fruits 

8. Nuts and seeds 50 mg sesame seeds Spice 

Green pepper (Piper nigrum) 1 drop 

O SOLE MIO 

1. 50 mL olive oil Pasta 

Thyme linalool (Thymus vulgaris) 1 drop 2. 50 mL egg yolk Omelette 

3. 50 mL balmy vinegar Salad 

Rosemary cineole 

1/2 drop 4. 50 mL honey Cuisine Provencal 

(Rosmarinus offi cinalis) 

5. 50 mg sea-salt Spice 

Clementine (Citrus deliciosa) 5 drops 6. 50 mg tofu Grilled tofu 

7. 50 mg avocado Guacamole 

8. Pesto 

CAPRI 

Orange (Citrus sinensis) 5 drops 1. 50 mL hazelnut oil Desserts 

2. 50 mL buttermilk Drink 

Lemon (Citrus limon) 3 drops 3. 50 mL cider vinegar Salad 

4. 100 mL maple syrup Desserts 

5. 50 mg sea-salt Spice 

6. 50 mL apple vinegar Fruit salad 

7. 50 mg avocado Sauce 

8. 50 mg walnuts Cakes 

BERGAMOT-GRAND MANIER 

Grapefruit (Citrus paradisi) 5 drops 1. 50 mL walnut oil Salad 

2. 50 mg butter Cake 

continued



Basic Spice Rack of Essential Oils: How to Prepare Essential Oil Mixtures and Essential 

Oil Seasonings 

Basic Essential Oils 

Mixtures 

Emulsifier 

Seasonings 

Recipes Example 

Orange (Citrus sinensis) 5 drops 3. 1 L white vine Beverage 

4. 50 mg raw sugar Sweets 

Limon (Citrus limon) 2 drops 5. 50 mg sea-salt Spice 

6. 50 mL tamarind sauce Thai cuisine 

Bergamot (Citrus bergamia) 2 drops 7. 50 mL lemon juice Drink 

8. 50 mg pumpkin seeds Soup 

MAGIC ORANGE 

Orange (Citrus sinensis) 5 drops 1. 50 mL almond oil Sweets 

2. 50 mg goat cheese Oriental 

Vanillaextract (Vanilla planifolia) 3 drops 3. 50 mL raspberry vinegar Fruit salad 

or balsamic vinegar 

4. 100 mL honey/treacle Sweets 

Kakaoextract (Theobroma cacao) 3 drops 5. — 

6. 50 mL seitan tofu Oriental 

Rose (Rosa damascena) 1/2 drop 7. 50 mg bananas Desserts 

8. 50 mg almonds Spice 

CLARY SAGE AND BERGAMOT 

Clary sage (Salvia sclarea) 2 drops Spice 

Bergamot (Citrus bergamia) 5 drops 5. 50 g sea-salt 

Peppermint (Mentha piperita) 

Lavender (Lavandula offi cinalis) 

PEPPERMINT 

Rather less—2 4. 100 mL maple syrup Drink 

drops per 

100 mL/mg 

LAVENDER 

Rather less—2 4. 100 mL honey Cuisine Provencal 

drops per 

100 mL/mg 

MENU 

BASICS 

Crispy Coconut Flakes (Flexible Asian Spice Variation) 

Gomasio (Sesame Sea-Salt Spice) 

Honey Provencal 

BEVERAGES 

Aroma Shake with Herbs 

Earl Grey at His Best 

Lara’s Jamu 

Rose-Cider 

Syrup Mint-Orange


ENTREES 

a. Soups: 

Peppermint Heaven 

Perky Pumpkin Soup 

b. Salads: 

Melon-Plum Purple Radish Salad 

Salad with Goat Cheese and Ricotta 

APPETIZER AND FINGER FOOD 

Crudities—Flavored Crispy Raw Vegetables 

Maria’s Dip 

Tapenade 

Tofu Aromanaise 

Vegetable Skewer 

MAIN COURSE 

Celery—Lemon Grass Patties 

Chèvre Chaude-Goat Cheese “Provence” with Pineapple 

Crispy Wild Rice-Chapatis 

Mango–Dates–Orange Chutney 

Prawns Bergamot 

DESSERT, CAKES, AND BAKED GOODS 

Apple Cake Rose 

Chocolate Fruits and Leaves 

Homemade Fresh Berry Jelly 

Rose Semifreddo 

Sweet Florentines 

(Chocolate should not be heated up more than 40°. Essential oils are best at 40° as well.) 

AROMA-VITAL CUISINE RECIPES 

BASICS 

Crispy Coconut Flakes (Flexible Asian Spice Variation) 

Nice with Asian flavored dishes or sweet baked goods. 

Ingredients: 

– 50 g dried coconut flakes 

– 10 drops EURO ASIA intermixture (spicy variation) or 10 drops MAGIC ORANGE intermixture 

(sweet variation) 

– 1 preserving jar. 

Preparation: Roast the coconut flakes in a frying pan. Lightly scatter the chosen essential oils into 

the empty jar. Spread the oil well, then fill in the roasted coconut rasps and shake it well. 

Gomasio (A Sesame Sea-Salt Spice) 

Gomasio is a secret of the Middle Eastern cuisine, which completes your spice rack and gives a 

subtle salty flavor to the dish. Nice to combine with soy sauce, fresh thyme leaves, or cumin.


Ingredients: 

– 50 g sesame seeds 

– 1 teaspoon EURO ASIA seasoning salt no. 5 

– 1 preserving jar. 

Preparation: Roast the sesame seeds in a frying pan, then mix the seeds with the salt in a mortar. 

Crush them lightly with a pestle to release the flavor. Fill into a preserving jar and shake it well. If 

necessary add a few more drops of EURO ASIA intermixture. 

Honey Provencal 

A great basic for the cuisine Provencal. 

Ingredients: 

– 100 ml acacia honey 

– 5 drops O SOLE MIO intermixture 

– 2 drops LAVENDER pure essential oil 

– 1 drop EURO ASIA intermixture 

– 1 drop CLARY SAGE AND BERGAMOT intermixture. 

Preparation: Emulsify the ingredients well. Use the honey to brush grilled vegetables, tofu, 

goat and sheep cheese, or to season gratins, to add a fabulous distinctly French flavor to a 

simple dish. 

BEVERAGES 

Aroma Shake with Herbs 

This green fruity flavored cleansing juice certainly is a great rejuvenator. 

Ingredients: 

– 500 mL organic buttermilk 

– 100 mL organic soy milk 

– 5 tablespoons sprouts (alfalfa, adzuki bean sprouts, and cress) 

– 100 mL carrot juice 

– 3 drops CAPRI intermixture 

– 2 drops EURO ASIA intermixture 

– 1 tablespoon maple syrup 

– 1 tablespoon parsley finely chopped. 

Preparation: Pour the buttermilk and soy milk in a blender and process for a few minutes until 

combined. Add the carrot juice, then emulsify the essential oils with the maple syrup and stir it 

into the mixture. Fill into iced tall glasses and serve chilled. A decorative idea is to dive the top 

of the glasses into lemon juice and then into the finely chopped parsley, before filling in the 

shake. 

Earl Grey at His Best 

Ingredients: 

– 1 preserving jar (100 g capacity) 

– 100 g Darjeeling tea “first flush” 

– 10 drops BERGAMOT basic essential oil.


Preparation: Lightly scatter the “BERGAMOT” basic essential oil into the empty jar. Add the tea, 

close the jar and shake it well. Repeat the procedure to shake the jug for the next 5–10 days; then 

this incredible sort of flavored tea will be ready to serve. 

Lara’s Jamu 

Jamu is a kind of herbal tonic from Southeast Asia. Every country and family has their own recipes. 

This one is a tasty booster for the immune system. 

Ingredients: 

– The rind of two limes in thin shreds 

– Juice of two limes 

– 2 tablespoons freshly grated ginger 

– 1 handful fresh or dried nettle 

– 50 ml maple treacle 

– 2 teaspoons curcuma powder 

– 500 ml water 

– 750 ml of sparkling water (optional) 

– 5 drops EURO ASIA intermixture 

– 2 drops PEPPERMINT basic essential oil 

– 3 drops CAPRI intermixture. 

Courtesy of Subash J. Geeganage. 

Preparation: Boil the mixture of lime, ginger, and nettle with 500 ml water for 10 min; then let it 

cool down a bit to be able to sieve it later into decorative chalices. Mix the curcuma powder with 

fresh lime juice and the EURO ASIA basic essential oil and stir it into the herbal mixture. Now the 

maple treacle mixed with PEPPERMINT basic essential oil will be stirred in as a sweetener. Serve 

hot or chilled with sparkling water, fresh mint sprigs, and sliced lime. 

Rose-Cider 

Refreshing and inspiring. 

Ingredients: 

– 1 L cider 

– 1/2 drop rose basic essential oil or 1 tablespoon organic rose water.


Preparation: Stir in the rose oil or rose water. Serve cold. 

Syrup Mint-Orange 

A refreshing hot summer drink. 

Ingredients: 

– 50 mL PEPPERMINT seasoning syrup no. 4 


Preparation: Simply mix the ingredients and you have a refreshing basic syrup, which can be used 

for drinks, baked goods, to pour it into soda water, tea juices, or even into ice cubes. To serve, 

garnish the drinks with some fresh peppermint leaves. 

ENTREES 

Soups 

Peppermint Heaven 

Ingredients: 

– 500 mL vegetable stock 

– Fresh peppermint leaves for decoration 

– 2–3 drops PEPPERMINT basic essential oil 

– 1 drop BERGAMOT basic essential oil 

– 150 mL cream 

– O SOLE MIO salt no. 5 or regular salt to season to taste. 

Preparation: Whip the cream; then add the basic essential oils to it. Meanwhile boil the vegetable 

stock; then stir in the cream. Ladle into soup bowls to serve and garnish each with a little bit 

whipped cream and fresh mint leaves. 

Perky Pumpkin Soup 

Warm and spicy—the perfect autumn dinner. 

Ingredients: 


– 1 large onion, finely chopped 

– 2 carrots, sliced finely 

– 1 tablespoon pumpkin seed-oil or butter 

– 500 g peeled pumpkin, finely chopped into cubes 

– 200 mL vegetable stock 


– 1 teaspoon curry powder 

– 1 tablespoon EURO ASIA seasoning oil no. 1 

– Fresh coriander to garnish 

– 1 tablespoon CAPRI seasoning salt no. 5 

– A little bit sherry. 

Preparation: Heat the pumpkin seed oil in a saucepan. Add the onion and carrots and cook over 

moderate heat until it softens. Stir in the pumpkin pieces and cook until the pumpkin is soft. Process 

the mixture in a blender and pour it to the pan. Stir in the vegetable stock and cream and season with 

the essential oils, salt, and sherry. Ladle into warm soup bowls and garnish each with some fresh 

coriander leaves.


Salads 

Melon-Plum Purple Radish Salad 

A refreshing hot summer party dish. 

Ingredients: 

– 1 mid-size watermelon or 2 Galia melons 

– 1 handful radishes rinsed and chopped 

– 1 bell pepper rinsed and sliced 

– 3 pears rinsed and chopped 

– Juice of 1 lemon 

– 1 tablespoon CAPRI or O SOLE MIO seasoning oil no. 1 

– 250 g sour cream 

– 150 g curd 

– Salt 

– Freshly ground black pepper 

– Some fresh summer herbs like thyme, cress or lemon balm. 

Courtesy of Ulla Mayer-Raichle. 

Preparation: Half the melon in a zigzag manner, separate the halves, remove the seeds from the 

melon halves, and use a melon baller to scoop out even-sized balls. Place the half of the melon balls, 

radishes, bell pepper, and pears in a large salad bowl and marinade the salad with lemon juice. Then 

store the melon halves and the salad in the fridge for at least half an hour. Meanwhile mix the 

seasoning oil of your choice with sour cream and curd and season with salt and pepper. Stir the 

mixture into the salad carefully and fill the salad into the melon halves. Garnish them with herbs 

and some of the extra melon balls. 

Salad with Goat Cheese and Ricotta 

A refreshing companion for spicy foods. 

Ingredients: 

– 1 red bell pepper rinsed, sliced 

– 1 green bell pepper rinsed, sliced 

– 1 scallion, chopped 

– 1 head salad greens (Aragula, Sorrel, Dandelion, etc.), rinsed, dried, and chopped.


For the salad dressing: 

– 1 drop O SOLE MIO intermixture 


– 4 tablespoons dark olive oil 


– Sea-salt 

– 100 g goat cheese or ricotta, chopped 

– 1/2 handful fresh eatable spring blossoms (daisies, primroses, etc.), rinsed 

– 2 handfuls fresh herbs of your choice (coriander, parsley, basil, etc.), rinsed 

– Roasted sesame. 

Preparation: Emulsify the essential oil intermixtures with the olive oil; add the lemon juice and season 

with salt. Place the dressing in a large bowl, marinade the cheese, and add the salad leaves, bell 

peppers, and scallion. Mix well and garnish with the herbs and blossoms and the roasted sesame. 

APPETIZER AND FINGER FOOD 

Crudities—Flavored Crispy Raw Vegetables 

Simple and delicious. 

Ingredients: 

– 750 g vegetables well rinsed and cut into crudities (radishes, scallions, chicory, carrots, etc.) 



Preparation: Emulsify the CAPRI intermixture into the lemon juice, fill it into a spray flacon, and 

spread it on top of the sliced vegetables. Serve with dip and breadsticks or baguette. 

Maria’s Dip 

Ingredients: 


– 1 tablespoon creme fraiche 

– 1/2 teaspoon salt 

– 250 g sour cream. 

Preparation: Emulsify the CAPRI essential oil intermixture into the creme fraiche. Stir in the salt 

and sour cream until combined. Ready to serve with bread, toast, and for example, the flavored 

crudities. 

Tapenade 

An Italian secret simple to make and perfect for dipping or seasoning. 

Ingredients: 

For the olives: 

– 200 g pitted green or black olives, rinsed and halved 

– 100 mL dark olive oil 

– 1 handful fresh rosemary 

– 10 drops O SOLE MIO intermixture.


For the tapenade: 

– 60 g capers 

– 1 crushed garlic clove 

– Freshly ground black pepper. 

Preparation: Marinate the olives in a mixture of olive oil, rosemary, and O SOLE MIO intermixture 

for at least 1 h. Place the olives, capers, and garlic in a food processor or blender and process 

until combined. Gradually add the flavored marinade and blend to a coarse paste; season with 

pepper. Keep stored in the fridge for up to 1 week. 

Tofu Aromanaise 

Served with the veggie skewers—a truly impressive dinner party dish. 

Ingredients: 

– 200 g organic pure tofu or smoked tofu 

– 3 tablespoons sunflower oil 

– 2 tablespoons EURO ASIA seasoning oil no. 1 

– EURO ASIA seasoning salt no. 5 

– A few chives. 

Preparation: Put the tofu in a blender and process it until the tofu is smooth. Transfer the creamy 

tofu to a bowl and stir in the sunflower oil very slowly, then add the EURO ASIA seasoning oil, and 

season with EURO ASIA salt. Garnish the top with chopped chives. Serve cold. 

Veggie Skewers 

A tasty idea for your next barbecue. 

Ingredients: 

– 20 skewers 

– 1000 g fresh young vegetables 

(tomatoes, fennel, eggplants, carrots, bell peppers, scallions, etc.). 

For the marinade: 

– 5 tablespoons dark olive oil 

– 3 tablespoons either O SOLE MIO or EURO ASIA seasoning oil no. 1 

– Freshly grounded pepper 

– 1 handful fresh chopped herbs (basil, thyme, parsley, etc.) or dried herbs. 

Courtesy of Ulla Mayer-Raichle.


Preparation: Prepare the vegetables and cut them into cubes. Mix all the marinade ingredients in a 

shallow dish and add the vegetable cubes. Spoon the marinade over the vegetables and leave to 

marinate in the fridge for at least 1 h. Then thread the cubes onto skewers. Brush with the marinade 

and broil or grill until golden, turning occasionally. Serve with baguette, tofu aromannaise tapenade, 

or any other dip. 

MAIN COURSE 

Celery Lemon Grass Patties 

Delicious, little, and flexible to combine. 

Ingredients: 

– 1–2 large celery 

– 250 mL liquid vegetable stock 

– 1 organic free range egg 

– 4 lemon slices 

– 1 pinch of BERGAMOT CLARY SAGE no. 5. 

Asian variation: 

– 3 tablespoons coconut flakes 

– 2 tablespoons EURO ASIA seasoning no. 1 

– Coconut oil or roasted sesame oil to fry. 

Mediterranean variation: 

– 2 tablespoons O SOLE MIO seasoning no. 1 

– 3 tablespoons sesame seeds 

– Soy oil to fry. 

Preparation: Blanche the washed and sliced celery roots in the vegetable stock. Choose your 

favorite cookie cutter, like heart or star, and cut them out of the blanched celery. Whisk the egg 

and stir in the essential oil variation of your choice. Marinate the celery stars and hearts, then coat 

them with coconut flakes or sesame seeds and fry them until they have a delicious golden brown 

color. To serve, top them with a small amount of the essential oil seasoning. They are great to 

accompany salads, baked potatoes with sour cream, and other vegetarian dishes or if you prefer, 

beef creations. 

Chévre Chaude-Goat Cheese “Provence” with Pineapple 

Ingredients: 

– 4 slices of fresh pineapple 

– 1 tablespoon sunflower oil or butter or ghee 

– 1 teaspoon “O SOLE MIO honey” no. 4 

– 1 tablespoon CAPRI honey no. 4 

– 2 tablespoons honey PROVENCAL (basics) 

– 2–3 small goat or sheep cheese 

– A little bit fresh or dried thyme to garnish 

– Sour cream 

– Salad or Parma ham (optional).



Preparation: Halve the pineapple slices and fry them on both sides. Lower the heat and top them 

with CAPRI honey. Preheat the oven to 180°C. Halve the cheese and place them on top of each of 

the two pineapple slices. Drop a little bit of honey PROVENCAL on each portion and bake it shortly 

until the cheese starts to caramelize. Serve immediately with the rest of the aromatized honeys 

dispersed on the surface, fresh herbs above, the sour cream on top, and with Parma ham or fresh 

salad aside. 

Crispy Wild Rice-Chapatis 

Ingredients: 

– 200 g wild rice 

– 400–500 mL warm water 

– 1 laurel leaf 

– 1 small onion or 3 scallions, finely chopped 

– 1 teaspoon EURO ASIA seasoning oil no. 1 

– 1 tablespoon EURO ASIA seasoning soy sauce no. 6 

– 2 organic or free range eggs 

– Curry powder 

– Lemon juice as you like 

– Around 2 tablespoons oil or ghee to fry. 

Preparation: Steam the wild rice briefly, then fill it up with the rest of the warm water, and add the 

laurel leave. Cook it for another 15–20 min, then turn the heat down and stir in the EURO ASIA 

seasoning oil no. 1. Cover it, leave it and let it chill until firm. Then stir all ingredients into the wild 

rice. Divide the mixture into walnut-sized balls; then flatten them slightly. Heat the oil or ghee in a 

pan and fry the chapatis until golden brown on each side. Drain on paper towels and serve at once. 

These crispy wild rice-chapatis taste delicious with steamed vegetables and dips or even salads. 

They are ideal as a snack or a nice idea for the next picnic. 

Mango–Dates–Orange Chutney 

A spice dip-trip to Asia. 

Ingredients: 

For 1000 g you need 

– 250 g organic well-scrubbed oranges (e.g., sweet and juicy sorts like Valencia) 

– 250 g onions


– 250 g sliced mangoes 

– 350 mL acacia honey 

– If this is not available choose any other treacle or honeys, which is neutral in taste and of 

organic origin 

– 50 mL maple syrup 

– 2 teaspoons CAPRI essential oil seasoning salt no. 5 

– A little bit of chile powder or 1 fresh chile pepper 

– 350 mL cider vinegar 

– 250 mg chopped dates 

– 50 mL of either EURO ASIA 

– or MAGIC ORANGE essential oil seasoning vinegar no. 3 

– 2 tablespoons CAPRI essential oil seasoning syrup no. 4 

– 5 drops pure EURO ASIA condiment intermixture. 

Preparation: Remove long, thin shreds of orange rind, using a grater (zester). Scrape it firmly along 

the surface of the fruit. Remove the white layer of the oranges; then slice the oranges and remove 

the pits. Finely chop the onions. Peel the mangoes and cut them into small chunks. Mix honey, 

syrup, chile powder, and vinegar with 1 teaspoon of the CAPRI salt no. 5 and boil it in a huge saucepan 

until the honey melts, stir it well. Add mangoes, onions, dates, oranges, and the half of the 

shredded orange rind. Then lower the heat and let it simmer for 1 h, until the mixture has formed a 

thick mass. Stir in the rest of the shredded orange rind and the chosen essential oil vinegar no. 3. 

Then emulsify the pure EURO ASIA condiment intermixture into the CAPRI syrup no. 4 and stir 

it in the chutney. Use the rest of the CAPRI salt no. 5 to add spice. Fill the mixture into sterilized 

warm preserving jars, store them cold and dark. Nice to serve with the Chévre chaude or the crispy 

wild rice-chapatis and veggie skewers. 

Prawns Bergamot 

Ingredients: 

– 500 g large prawns 

Marinade: 

– 5 drops pure CAPRI essential oil intermixture 

– 1 small onion 

– 1/2 crushed garlic clove 

– 1 handful flat leaf parsley 

– 3 scallions 

– Juice of a lemon 

– 2 drops pure BERGAMOT essential oil 

– 1/2 teaspoon fennel seed 

– 6 tablespoons olive oil 

– Salt and fresh pepper 

– 3 tablespoons BERGAMOT–GRAND MANIER vine no. 3. 

Preparation: Prepare and wash the prawns as usual. Slice the onions and garlic, chop the parsley 

finely and cut the scallions into quarters. Take a teaspoon of lemon juice and emulsify the essential 

oils in it and mix in the rest of the ingredients. Let the prawns soak in the marinade and keep 

it in the fridge for 1 h. Then separate the prawns from the marinade; filter the marinade and keep 

the parts separately. Fry the prawns inside of the liquid parts of the marinade, then add the rest.


Stir it well for another minute, season with salt, pepper, and BERGAMOT vine and let it simmer 

slowly. Nice to serve with baguette or the crispy wild rice chapatis and vegetables like green 

asparagus tips. 

DESSERT, CAKES, AND BAKED GOODS 

Apple Cake Rose 

This classic combination is an apples favorite destiny. Suited even for diabetics. 

Ingredients: 

– 250 g spelt flour 

– 120 g finely sliced cold butter 

– 1 organic or free range egg 

– 1 tablespoon CAPRI essential oil seasoning no. 1 

– Salt 

– 50–100 mL warm water 

– 1000 g sweet ripe apples 

– Juice of a half lemon 

– 1 tablespoon organic rose water. 

For the topping: 

– 250 ml cream 

– 1 egg yolk of an organic or free range egg 

– 5–7 drops MAGIC ORANGE pure seasoning intermixture 

– 1 tablespoon organic rose water 

– 50 g sliced almonds to garnish the top of the cake. 

Preparation: Sift the flour, butter, egg, warm water, and the CAPRI seasoning no. 1 into a large 

mixing bowl. Mix everything together until combined; then store the cake mixture in the fridge for 

a half hour. In the meanwhile, peel and core the apples, slice them into wedges, and slice the 

wedges thinly. Combine lemon juice with rose water and splash it over the apples. For the topping, 

beat the egg yolk with the cream and the pure essential oil intermixture MAGIC ORANGE. Then 

pour the cake mixture into the prepared pan, smooth the surface, then make a shallow hollow in a 

ring around the edge of the mixture. Arrange the apple slices on top of the cake mixture. Pour the 

topping carefully above the apple slices and garnish the sliced almonds above. Cover the cake with 

aluminum foil. Bake for 30–40 min, until firm and the mixture comes away from the side of the 

pan. Lower the heat, remove the foil, and bake it for another 5 min. Serve warm. 

Chocolate Fruits and Leaves 

A delicious way to consume your favorite fruits, dried fruits, nuts, or even leaves like rose leaves. 

Ingredients: 

– 250 g organic chocolate couverture (bitter chocolate) 

– 5 drops MAGIC ORANGE or BERGAMOT–GRAND MANIER or CAPRI intermixture – 

or 2–3 drops PEPPERMINT, LAVENDER, or GINGER pure basic essential oil, depending 

on your taste—spicy, mint, or fruity.



Preparation: Warm up the chocolate couverture until you have a creamy consistency. Stir in 

your choice of basic essential oils or intermixture. Dive in the fruits, and let them dry. Serve 

chilled. 

Homemade Fresh Berry Jelly 

Ingredients: 

– 500 gm mixed berries 

– (blue berries; rasp berries; red, white, and black currant; black berries; strawberries, cranberries, 

cherries) 

– 100 mL water 

– 1 tablespoon agar or 2 tablespoons kuzu or sago (binding agent) 

– 1–2 tablespoons cold water 

– 12 drops MAGIC ORANGE intermixture 

– 3 tablespoons maple syrup. 

Preparation: Take the clean fruits and boil them in the water. Stir the binding agent into the small 

amount of cold water, then add it to the warm fruits and let them boil for another 3–5 min before 

you lower the heat, then leave the mixture to cool. Emulsify the essential oils intermixture with the 

maple honey; then stir it into the jelly. Serve cool with fresh berries or a spoonful of whipped cream 

with mint leaves. 

Rose Semifreddo 

Romantic and delicate aromatic dessert. 

Ingredients: 

– 150 g creme fraiche 

– 75 g low fat quark 

– 100 mL acacia honey 

– 1 tablespoon rose water 

– Rose leaves from 2 roses (organic farming) 

– 2 tablespoons cognac


– Nonalcoholic alternative—1 drop pure MAGIC ORANGE intermixture in 2 tablespoons 

maple syrup 

– 150 mL whipped cream 

– 1 drop of pure MAGIC ORANGE intermixture. 

Preparation: Place the creme fraiche and the quark in a bowl and cream together. Keep some rose 

leaves for decoration aside, process the rest of the leaves in a food processor until smooth, then 

transfer them into the bowl; add the acacia honey and stir to mix. Whisk in the rose water and 

either the cognac or the MAGIC ORANGE maple syrup. Fold in the whipped cream and the pure 

MAGIC ORANGE intermixture gently, being careful not to over mix. Pour the mixture into some 

small plastic containers, cover and freeze until the ice is firm. Transfer the ice to the refrigerator 

about 20 min before serving to allow it to soften a little. Serve in scoops decorated with rose leaves 

and berries. 

Sweet Florentine 

Sweet almond munchies. 

Ingredients: 

– 500 g butter 

– 200 g sugar 

– 2 packages organic bourbon vanilla sugar 


– 300 g sliced almonds 

– 30 g spelt or wheat grain 

– 15–20 drops MAGIC ORANGE or CAPRI intermixture emulsified in 1 tablespoon maple 

treacle 

– 100 g chocolate couverture with 5–8 drops CAPRI or MAGIC ORANGE intermixture. 

Preparation: Caramelize the sugar, then stir in the bourbon vanilla, butter until the sugar has been 

melted, then stir in almonds and flour. Preheat the oven to 180°C, then spoon the mixture on a 

baking tray and bake for 10 min. Do not worry, it is in their nature to melt. To serve, just cut them 

into diamonds after cooling down and remove them from the pan. Dive them halfway into the 

chocolate couverture only (the lower smooth side) then let them dry. Serve chilled or iced. 

RÉSUMÉ 

Aroma-vital cuisine is an aspect of aroma culture and therefore an art and cultivation of using the 

senses especially taste and smell.

19 

Essential Oils Used in 

Veterinary Medicine 

K. Hüsnü Can Başer and Chlodwig Franz 

CONTENTS 

19.1 Introduction ..................................................................................................................... 881 

19.2 Oils Attracting Animals .................................................................................................. 883 

19.3 Oils Repelling Animals .................................................................................................. 883 

19.4 Oils against Pests ............................................................................................................ 884 

19.4.1 Insecticidal, Pest Repellent, and Antiparasitic Oils .......................................... 884 

19.4.2 Fleas and Ticks .................................................................................................. 884 

19.4.3 Mosquitoes ......................................................................................................... 884 

19.4.4 Moths ................................................................................................................. 885 

19.4.5 Aphids, Caterpillars, and Whiteflies ................................................................. 885 

19.4.5.1 Garlic Oil ........................................................................................... 885 

19.4.6 Ear Mites ........................................................................................................... 885 

19.4.7 Antiparasitic ...................................................................................................... 885 

19.5 Essential Oils used in Animal Feed ................................................................................ 886 

19.5.1 Ruminants .......................................................................................................... 886 

19.5.2 Poultry ............................................................................................................... 887 

19.5.2.1 Studies with CRINA Poultry ............................................................. 887 

19.5.2.2 Studies with Herbromix ..................................................................... 888 

19.5.3 Pigs .................................................................................................................... 889 

19.6 Essential Oils used in Treating Diseases in Animals ..................................................... 890 

References .................................................................................................................................. 891 


Essential oils are volatile constituents of aromatic plants. These liquid oils are generally complex 

mixtures of terpenoid and/or nonterpenoid compounds. Mono-, sesqui-, and sometimes diterpenoids, 

phenylpropanoids, fatty acids and their fragments, benzenoids, and so on may occur in various 

essential oils (Baser and Demirci, 2007). 

Except for citrus oils obtained by cold pressing, all other essential oils are obtained by distillation. 

Products obtained by solvent extraction or supercritical fluid extraction are not technically 

considered as essential oils (Baser, 1995). 

Essential oils are used in perfumery, food flavoring, pharmaceuticals, and sources of 

aromachemicals. 

Essential oils exhibit a wide range of biological activities and 31 essential oils have monographs 

in the latest edition of the European Pharmacopoeia (Table 19.1). 

881


TABLE 19.1 

Essential Oil Monographs in the European Pharmacopoeia (6.5 Edition, 2009) 

English Name Latin Name Plant Name 

Anise oil Anisi aetheroleum Pimpinella anisum L. fruits 

Bitter-fennel fruit oil Foeniculi amari fructus aetheroleum Foeniculum vulgare Miller subsp. vulgare var. 

vulgare 

Bitter-fennel herb oil Foeniculi amari herba aetheroleum Foeniculum vulgare Miller subsp. vulgare var. 

vulgare 

Caraway oil Carvi aetheroleum Carum carvi L. 

Cassia oil Cinnamomi cassiae aetheroleum Cinnamomum cassia Blume (Cinnamomum 

aromaticum Nees) 

Cinnamon bark oil, Ceylon Cinnamomi zeylanici corticis aetheroleum Cinnamomum zeylanicum Nees 

Cinnamon leaf oil, Ceylon Cinnamomi zeylanici folium aetheroleum Cinnamomum verum J.S. Presl. 

Citronella oil Citronellae aetheroleum Cymbopogon winterianus Jowitt 

Clarysage oil Salviae sclareae aetheroleum Salvia sclarea L. 

Clove oil Caryophylli aetheroleum Syzigium aromaticum (L.) Merill et L.M. 

Perry (Eugenia caryophyllus C.S. Spreng. 

Bull. et Harr 

Coriander oil Coriandri aetheroleum Coriandrum sativum L. 

Dwarf pine oil Pini pumilionis aetheroleum Pinus mugo Turra. 

Eucalyptus oil Eucalypti aetheroleum Eucalyptus globulus Labill. 

Juniper oil Juniperi aetheroleum Juniperus communis L. meyvesi 

Lavender oil Lavandulae aetheroleum Lavandula angustifolia P. Mill. (Lavandula 

offi cinalis Chaix.) 

Lemon oil Limonis aetheroleum Citrus limon (L.) Burman fil. 

Mandarin oil Citri reticulatae aetheroleum Citrus reticulata Blanco 

Matricaria oil Matricariae aetheroleum Matricaria recutita L. (Chamomilla recutita 

(L.) Ranschert) 

Mint oil, partly 

dementholized 

Neroli oil (formerly 

bitter-orange flower oil) 

Menthae arvensis aetheroleum 

partim mentholi privum 

Neroli aetheroleum (formerly Aurantii 

amari fl oris aetheroleum) 

Mentha canadensis L. (Mentha arvensis 

L. var. glabrata (Benth.) Fern, Mentha 

arvensis L. var. piperascens Malinv. ex 

Holmes) Japanese mint 

Citrus aurantium L. subsp. aurantium (Citrus 

aurantium L. subsp. amara Engl.) 

Nutmeg oil Myristicae fragrantis aetheroleum Myristica fragrans Houtt. 

Peppermint oil Menthae piperitae aetheroleum Mentha × piperita L. 

Pine silvestris oil Pini silvestris aetheroleum Pinus silvestris L. 

Rosemary oil Rosmarini aetheroleum Rosmarinus offi cinalis L. 

Spanish sage oil Salviae lavandulifoliae aetheroleum Salvia lavandulifolia Vahl. 

Spike lavender oil Spicae aetheroleum Lavandula latifolia Medik. 

Star anise oil Anisi stellati aetheroleum Illicium verum Hooker fil. 

Sweet orange oil Aurantii dulcis aetheroleum Citrus sinensis (L.) Osbeck (Citrus aurantium 

L. var. dulcis L.) 

Tea tree oil Melaleucae aetheroleum Melaleuca alternifolia (Maiden et Betch) 

Cheel, Melaleuca linariifolia Smith, 

Melaleuca dissitifl ora F. Mueller and other 

species 

Thyme oil Thymi aetheroleum Thymus vulgaris L., T. zygis L. 

Turpentine oil, Pinus 

pinaster type 

Terebinthini aetheroleum ab pinum 

pinastrum 

Pinus pinaster Aiton. 

(Maritime pine)

Essential Oils Used in Veterinary Medicine 883 

Antimicrobial activities of many essential oils are well documented (Bakkali et al., 2008). Such 

oils may be used singly or in combination with one or more oils. For the sake of synergism this may 

be necessary. 

Although many are generally regarded as safe (GRAS), essential oils are generally not recommended 

for internal use. However, their much diluted forms (e.g., hydrosols) obtained during oil 

distillation as a by-product may be taken orally. 

Topical applications of some essential oils (e.g., oregano and lavender) in wounds and burns 

bring about fast recovery without leaving any sign of cicatrix. By inhalation, several essential oils 

act as a mood changer and have effect especially on respiratory conditions. 

Several essential oils (e.g., citronella oil) have been used as pest repellents or as insecticides and 

such uses are frequently encountered in veterinary applications. 

In recent years, especially after the ban on the use of antibiotics in animal feed in the European 

Union since January 2006, essential oils have emerged as a potential alternative to antibiotics in 

animal feed. 

Essential oils used in veterinary medicine may be classified as follows: 

1. Oils attracting animals 

2. Oils repelling animals 

3. Insecticidal, pest repellent, and antiparasitic oils 

4. Oils used in animal feed 

5. Oils used in treating diseases in animals. 

19.2 OILS ATTRACTING ANIMALS 

Valeriana oils (and valerianic and isovalerianic acids) and nepeta oils (and nepetalactones) are wellknown 

feline-attractant oils. Their odor attracts male cats. 

Douglas fir oil and its monoterpenes have been claimed to attract deer and wild boar (Buchbauer 

et al., 1994). 

Dogs are normally drawn to floral oils and usually choose to take these by inhalation only. 

Monoter pene-rich oils are usually too strong for dogs, with the exception of bergamot, Citrus bergamia. 

Cats also usually select only floral oils for inhalation. Cats do not have metabolic mechanism to 

break down essential oils due to the lack of enzyme glucuronidase. Therefore, they should not be 

taken by mouth and should not be generally applied topically (http://www.ingraham.co.uk). 

19.3 OILS REPELLING ANIMALS 

Peppermint oil (Mentha piperita) repels mice. It can be applied under the sink in the kitchen or applied 

in staples to prevent mice annoying horses and livestock. A few drops of peppermint oil in a bucket of 

water used to scrub out a stall and sprinkling a few drops around the perimeter and directly on straw 

or bedding is said to eliminate or severely curtail the habitation of mice (Anonymous, 2001). 

A patent (United States Patent 4961929) claims that a mixture of methyl salicylate, birch oil, 

wintergreen oil, eucalyptus oil, pine oil, and pine-needle oil repels dogs. 

Another patent (United States Patent 4735803) claims the same using lemon oil and a-terpinyl 

methyl ether. 

Another similar formulation (United States Patent 4847292) claims that a mixture of citronellyl 

nitrile, citronellol, a-terpinyl methyl ether, and lemon oil repels dogs. 

A mixture of black pepper and capsicum oils and the oleoresin of rosemary is claimed to repel 

animals (United States Patent 6159474). 

Citronella oil repels cats and dogs (Moschetti, 2003). 

Repellents alleged to repel cats include allyl isothiocyanate (oil of mustard), amyl acetate, anethole, 

capsaicin, cinnamaldehyde, citral, citronella, citrus oil, eucalyptus oil, geranium oil, lavender


oil, lemongrass oil, menthol, methyl nonyl ketone, methyl salicylate, naphthalene, nicotine, 

paradichlorobenzene, and thymol. Oil of mustard, cinnamaldehyde, and methyl nonyl ketone are 

said to be the most potent. 

Essential oils comprised of 10 g/L solutions of cedarwood, cinnamon, sage, juniper berry, lavender, 

and rosemary, each were potent snake irritants. Brown tree snakes exposed to a 2-s burst of 

aerosol of these oils exhibited prolonged, violent undirected locomotory behavior. In contrast, exposure 

to a 10 g L -1 concentration of ginger oil aerosol caused snakes to locomote, but in a deliberate, 

directed manner. The 10 g/L solutions delivered as aerosols of m-anisaldehyde, trans-anethole, 

1,8- cineole, cinnamaldehyde, citral, ethyl phenylacetate, eugenol, geranyl acetate, or methyl salicylate 

acted as potent irritants for brown tree snakes (Boiga irregularis) (Clark and Shivik, 2002). 

19.4 OILS AGAINST PESTS 

19.4.1 INSECTICIDAL, PEST REPELLENT, AND ANTIPARASITIC OILS 

The essential oil of bergamot (Citrus bergamia), anise (Pimpinella anisum), sage (Salvia offi cinalis), 

tea tree (Melaleuca alternifolia), geranium (Pelargonium sp.), peppermint (Mentha piperita), thyme 

(Thymus vulgaris), hyssop (Hyssopus offi cinalis), rosemary (Rosmarinus offi cinalis), and white clover 

(Trifolium repens) can be used to control certain pests on plants. They have been shown to 

reduce the number of eggs laid and the amount of feeding damage by certain insects, particularly 

lepidopteran caterpillars. Sprays made from Tansy (Tanacetum vulgare) have demonstrated a repellent 

effect on imported cabbageworm on cabbage, reducing the number of eggs laid on the plants. 

Teas made from wormwood (Artemisia absinthium) or nasturtiums (Nasturtium spp.) are reputed to 

repel aphids from fruit trees, and sprays made from ground or blended catnip (Nepeta cataria), 

chives (Allium schoenoprasum), feverfew (Tanacetum parthenium), marigolds (Calendula, Tagetes, 

and Chrysanthemum spp.), or rue (Ruta graveolens) have also been used by gardeners against pests 

that feed on leaves (Moschetti, 2003). 

19.4.2 FLEAS AND TICKS 

Dogs, cats, and horses are plagued by fleas and ticks. One to two drops of citronella or lemongrass 

oils added to the shampoo will repel these pests. Alternatively, 4–5 drops of cedarwood oil and pine 

oil is added to a bowl of warm water and a bristle hair brush is soaked with this solution to brush the 

pet down with it. Eggs and parasites gathered in the brush are rinsed out. This is repeated several 

times. This solution can be used similarly for livestock after adding citronella and lemon grass oils 

to this mixture. 

Flea collar can be prepared by a mixture of cedarwood (Juniperus virginiana), lavender 

(Lavandula angustifolia), citronella (Cymbopogon winterianus (Java)), thyme oils, and 4–5 garlic 

(Allium sativum) capsules. This mixture is thinned with a teaspoonful of ethanol and soaked with a 

collar or a cotton scarf. This is good for 30 days (Anonymous, 2001). 

Ticks can be removed by applying 1 drop of cinnamon or peppermint oil on Q-tip by swabbing 

on it. 

Carvacrol-rich oil (64%) of Origanum onites and carvacrol was found to be effective against the 

tick Rhipicephalus turanicus. Pure carvacrol killed all the ticks following 6 h of exposure, while 

25% and higher concentrations of the oil were effective in killing the ticks by the 24-h posttreatment 

(Coskun et al., 2008). 

19.4.3 MOSQUITOES 

Catnip oil (Nepeta cataria) containing nepetalactones can be used effectively as a mosquito repellent. 

It is said to be 10 times more effective than DEET (Moschetti, 2003). Juniperus communis berry oil


is a very good mosquito repellent. Ocimum volatile oils including camphor, 1,8-cineole, methyl 

eugenol, limonene, myrcene, and thymol strongly repelled mosquitoes (Regnault-Roger, 1997). 

Citronella oil repels mosquitoes, biting insects, and fleas. 

Essential oils of Zingiber offi cinale and Rosmarinus offi cinalis were found to be ovicidal and 

repellent, respectively, toward three mosquito species (Prajapati et al., 2005). Root oil of Angelica 

sinensis and ligustilide was found to be mosquito repellent (Wedge et al., 2009). 

19.4.4 MOTHS 

Cedarwood oil is used in mothproofing. A large number of patents have been assigned to the preservation 

of cloths from moths and beetles: Application of a solution containing clove (Syzigum 

aromaticum) essential oil on woolen cloth; filter paper containing Juniperus rigida oil, and tablets 

of p-dichlorobenzene mixed with essential oils to be placed in wardrobe. 

19.4.5 APHIDS, CATERPILLARS, AND WHITEFLIES 

19.4.5.1 Garlic Oil 

Essential oils effective in insect pest control (Regnault-Roger, 1997). 

19.4.6 EAR MITES 

Peppermint oil is applied to a Q-tip and swabbed inside of the ear. 

19.4.7 ANTIPARASITIC 

A patent (United States Patent 6800294) on an antiparasitic formulation comprising eucalyptus oil 

(Eucalyptus globulus), cajeput oil (Melaleuca cajeputi), lemongrass oil, clove bud oil (Syzigium 

aromaticum), peppermint oil (Mentha piperita), piperonyl, and piperonyl butoxide. The formulation 

can be used for treating an animal body, in the manufacture of a medicament for treating ectoparasitic 

infestation of an animal, or for repelling parasites. 

Two essential oils derived from Lavandula angustifolia and Lavandula ¥ intermedia were investigated 

for any antiparasitic activity against the human protozoal pathogens Giardia duodenalis and 

Trichomonas vaginalis and the fish pathogen Hexamita infl ata, all of which have significant infection 

and economic impacts. The study has demonstrated that low (£1%) concentrations of Lavandula 

angustifolia and Lavandula × intermedia oil can completely eliminate Trichomonas vaginalis, 

Giardia duodenalis, and Hexamita infl ata in vitro. At 0.1% concentration, Lavandula angustifolia 

oil was found to be slightly more effective than Lavandula × intermedia oil against Giardia duodenalis 

and Hexamita infl ata (Moon et al., 2006). 

The antiparasitic properties of essential oils from Artemisia absinthium, Artemisia annua, and 

Artemisia scoparia were tested on intestinal parasites, Hymenolepis nana, Lambli intestinalis, 

Syphacia obvelata, and Trichocephalus muris [Trichuris muris]. Infested white mice were injected 

with 0.01 ml/g of the essential oils (6%) twice a day for 3 days. The effectiveness of the essential oils 

was observed in 70–90% of the tested animals (Chobanov et al., 2004). 

Parasites, such as head lice and scabies, as well as internal parasites, are repelled by oregano oil 

(86% carvacrol). The oil can be added to soaps, shampoos, and diluted in olive oil for topical applications. 

By taking a few drops daily under the tongue, one can gain protection from waterborne 

parasites, such as Cryptosporidium and Giardia. Internal dosages also are effective in killing parasites 

in the body (http://curingherbs.com/wild_oregano_oil.htm) (Foster, 2002). 

Essential oils from Pinus halepensis, Pinus brutia, Pinus pinaster, Pinus pinea, and Cedrus 

atlantica were tested for molluscicidal activity against Bulinus truncatus. The oil from Cedrus


atlantica was found the most active (LC 50 = 0.47 ppm). Among their main constituents, a-pinene, 

b-pinene, and myrcene exhibited potent molluscicidal activity (LC 50 = 0.49; 0.54, and 0.56 ppm, 

respectively). These findings have important application of natural products in combating schistosomiasis 

(Lahlou, 2003). 

Origanum essential oils have exhibited differential degrees of protection against myxosporean 

infections in gilthead and sharpsnout sea bream tested in land-based experimental facilities 

(Athanassopoulou et al., 2004a, 2004b). 

19.5 ESSENTIAL OILS USED IN ANIMAL FEED 

Essential oils can be used in feed as appetite stimulant, stimulant of saliva production, gastric and 

pancreatic juices production enhancer, and antimicrobial and antioxidant to improve broiler performance. 

Antimicrobial effects of essential oils are well documented. Essential oils due to their potent 

nature should be used as low as possible levels in animal nutrition. Otherwise, they can lead to feed 

intake reduction, gastrointestinal (GIT) microflora disturbance, or accumulation in animal tissues 

and products. Odor and taste of essential oils may contribute to feed refusal; however, encapsulation 

of essential oils could solve this problem (Gauthier, 2005). 

Generally, Gram-positive bacteria are considered more sensitive to essential oils than Gramnegative 

bacteria because of their less complex membrane structure (Lis-Balchin, 2003). 

Carvacrol, the main constituent of oregano oils, is a powerful antimicrobial agent (Baser, 2008). 

It asserts its effect through the biological membranes of bacteria. It acts through inducing a sharp 

reduction of the intercellular ATP pool through the reduction of ATP synthesis and increased hydrolysis. 

Reduction of the membrane potential (transmembrane electrical potential), which is the driving 

force of ATP synthesis, makes the membrane more permeable to protons. A high level of 

carvacrol (1 mM) decreases the internal pH of bacteria from 7.1 to 5.8 related to ion gradients across 

the cell membrane. 1 mM of carvacrol reduces the internal potassium (K) level of bacteria from 

12 mmol/mg of cell protein to 0.99 mmol/mg in 5 min. K plays a role in the activation of cytoplasmic 

enzymes and in maintaining osmotic pressure and in the regulation of cytoplasmic pH. K efflux 

is a solid indication of membrane damage (Ultee et al., 1999). 

It has been shown that the mode of action of oregano oils is related to an impairment of a variety of 

enzyme systems, mainly involved in the production of energy and the synthesis of structural components. 

Leakage of ions, ATP, and amino acids also explain the mode of action. Potassium and phosphate 

ion concentrations are affected at levels below the MIC concentration (Lambert et al., 2001). 

19.5.1 RUMINANTS 

A recent review compiled information on botanicals including essential oils used in ruminant health 

and productivity (Rochfort et al., 2008). Unfortunately, there are few reports on the effects of essential 

oils and natural aromachemicals on ruminants. It was demonstrated that the consumption of 

terpene volatiles such as camphor and a-pinene in “tarbush” (Flourencia cernua) effected feed 

intake in sheep (Estell et al., 1998). In vitro and in vivo antimicrobial activities of essential oils have 

been demonstrated in ruminants (Cardozo, 2005; Elgayyar et al., 2001; Moreira et al., 2005; Wallace 

et al., 2002). Synergistic antinematodal effects of essential oils and lipids were demonstrated 

(Ghisalberti, 2002). Other nematocidal volatiles reported are as follows: benzyl isothiocyanate 

(goat), ascaridole (goat and sheep) (Githiori et al., 2006; Ghisalberti, 2002), geraniol, eugenol 

(Githiori et al., 2006; Chitwood, 2002), and menthol, 1,8-cineole (Chitwood, 2002). 

Methylsalicylate, the main component of the essential oil of Gaultheria procumbens 

(Wintergreen), is topically used as emulsion in cattle, horses, sheep, goats, and poultry in the treatment 

of muscular and articular pain. The recommended dose is 600 mg/kg bw twice a day. The 

duration of treatment is usually less than 1 week (EMEA, 1999). It is included in Annex II of


Council Regulation (EEC) N. 2377/90 as a substance that does not need an MRL level. Gaultheria 

procumbens should not to be used as flavoring in pet food since salicylates are toxic to dogs and 

cats. As cats metabolize salicylates much more slowly than other species, they are more likely to be 

overdosed. Use of methylsalicylate in combination with anticoagulants such as warfarin can result 

in adverse interactions and bleedings (Chow et al., 1989; Ramanathan, 1995; Tam et al., 1995; Yip 

et al., 1990). 

The essential oil of Lavandula angustifolia (Lavandulae aetheroleum) is used in veterinary 

medicinal products for topical use together with other plant extracts or essential oils for antiseptic 

and healing purposes. The product is used in horses, cattle, sheep, goats, rabbits, and poultry. It is 

included in Annex II of Council Regulation (EEC) N. 2377/90 as a substance that does not need an 

MRL level (EMEA, 1999; Franz et al., 2005). 

The outcomes of in vitro studies investigating the potential of Pimpinella anisum essential oil as 

a feed additive to improve nutrient use in ruminants are inconclusive, and more and larger preferably 

in vivo studies are necessary for evaluation of efficacy (Franz et al., 2005). 

Carvacrol, carvone, cinnamaldehyde, cinnamon oil, clove bud oil, eugenol, and oregano oil have 

resulted in a 30–50% reduction in ammonia N concentration in diluted ruminal fluid with a 50:50 

forage concentrate diet during the 24-h incubation (Busquet et al., 2006). 

Carvacrol has been suggested as a potential modulator of ruminal fermentation (Garcia et al., 

2007). 

19.5.2 POULTRY 

19.5.2.1 Studies with CRINA Poultry 

Dietary addition of essential oils in a commercial blend (CRINA ® Poultry) showed a decreased 

Escherichia coli population in ileo-cecal digesta of broiler chickens. Furthermore, in high doses, a 

significant increase in certain digestive enzyme activities of the pancreas and intestine was observed 

in broiler chickens (Jang et al., 2007). 

In another study, CRINA Poultry was shown to control the colonization of the intestine of broilers 

with Clostridium perfringens and the stimulation of animal growth was put down to this development 

(Losa, 2001). 

Commercial essential oil blends CRINA Poultry and CRINA Alternate were tested in broilers 

infected with viable oocysts of mixed Eimeria spp. It was concluded that these essential oil blends 

may serve as an alternative to antibiotics and/or ionophores in mixed Eimeria infections in noncocci-vaccinated 

broilers, but no benefit of essential oil supplementation was observed for vaccinated 

broilers against coccidia (Oviedo-Rondon et al., 2006). 

19.5.2.1.1 Other Studies 

Supplementation of 200 ppm essential oil mixture (EOM) that included oregano, clove, and anise 

oils (no species name or composition given!) in broiler diets was said to significantly improve the 

daily live weight gain and feed conversion ratio (FCR) during a growing period of 5 weeks (Ertas 

et al., 2006). Similar results were obtained with 400 mg/kg anise oil (composition not known!) 

(Ciftci et al., 2005). 

A total of 50 and 100 mg/kg of feed of oregano oil * were tested on broilers. No growth- promoting 

effect was observed. At 100 mg/kg of feed, antioxidant effect was detected on chicken tissues 

(Botsoglou et al., 2002a). 

Positive results were also reported for oregano oil added in poultry feed (Bassett, 2000). 

* Oregano essential oil was in the form of a powder called Orego-Stim. This product contains 5% oregano essential oil 

(Ecopharm Hellas, SA, Kilkis, Greece) and 95% natural feed grade inert carrier. The oil of Origanum vulgare subsp. 

hirtum used in this product contains 85% carvacrol + thymol.


Antioxidant activities of rosemary and sage oils on lipid oxidation of broiler meat have been 

shown. Following dietary administration of rosemary and sage oils to the live birds, a significant 

inhibition of lipid peroxidation was reported in chicken meat stored for 9 days (Lopez-Bote 

et al., 1998). A dietary supplementation of oregano essential oil (300 mg/kg) showed a positive 

effect on the performance of broiler chickens experimentally infected with Eimeria tenella. 

Throughout the experimental period of 42 days, oregano essential oil exerted an anticoccidial effect 

against Eimeria tenella, which was, however, lower than that exhibited by lasalocid. Supplementation 

with dietary oregano oil to Eimeria tenella-infected chickens resulted in body weight gains and feed 

conversion ratios not differing from the noninfected group, but higher than those of the infected 

control group and lower than those of chickens treated with the anticoccidial lasalocid (Giannenas 

et al., 2003). 

Inclusion of oregano oil at 0.005% and 0.01% in chicken diets for 38 days resulted in a significant 

antioxidant effect in raw and cooked breast and thigh muscle stored up to 9 days in refrigerator 

(Botsoglou et al., 2002b). 

Oregano oil (55% carvacrol) exhibited a strong bactericidal effect against lactobacilli and following 

the oral administration of the oil MIC values of amicain, apramycin, and streptomycinand 

neomyc against Escherichia coli strains increased (Horosova et al., 2006). 

An in vitro assay measuring the antimicrobial activity of essential oils of Coridothymus capitatus, 

Satureja montana, Thymus mastichina, Thymus zygis, and Origanum vulgare was carried out 

against poultry origin strains of Escherichia coli, Salmonella enteritidis, and Salmonella essen, and 

pig origin strains of enterotoxigenic Escherichia coli (ETEC), Salmonella choleraesuis, and 

Salmonella typhimurium. Origanum vulgare (MIC £ 1% v/v) oil showed the highest antimicrobial 

activity against the four strains of Salmonella. It was followed by Thymus zygis oil (MIC £ 2% v/v). 

Thymus mastichina oil inhibited all the microorganisms at the highest concentration, 4% (v/v). 

Monoterpenic phenols carvacrol and thymol showed higher inhibitory capacity than the monoterpenic 

alcohol linalool. The results confirmed potential application of such oils in the treatment and 

prevention of poultry and pig diseases caused by salmonella (Penalver et al., 2005). 

In another study, groups of male, 1-day-old Lohmann broilers were given maize–soya bean meal 

diets, with oils extracted from thyme, mace, and caraway or coriander, garlic, and onion (0, 20, 40, 

and 80 mg/kg) for 6 weeks. The average daily gain and FCR were not different between the broilers 

fed with the different oils; meat was not tainted with flavor or smell of the oils (Vogt and Rauch, 

1991). 

19.5.2.2 Studies with Herbromix 

Essential oils from oregano herb (Origanum onites), laurel leaf (Laurus nobilis), sage leaf (Salvia 

fruticosa), fennel fruit (Foeniculum vulgare), myrtle leaf (Myrtus communis), and citrus peel (rich 

in limonene) were mixed and formulated as feed additive after encapsulation. It is marketed in 

Turkey as poultry feed under the name Herbromix ® . 

The following three in vivo experiments with this product were recently accomplished. 

19.5.2.2.1 In Vivo Experiment 1 

In this study, 1250 sexed 1-day-old broiler chicks obtained from a commercial hatchery were randomly 

divided into five treatment groups of 250 birds each (negative control, antibiotic, and essential 

oil combination (EOC) at three levels). Each treatment group was further subdivided into five 

replicates of 50 birds (25 males and 25 females) per replicate. Commercial EOC at three different 

levels (24, 48, and 72 mg) and antibiotic (10 mg avilamycin) per kg were added to the basal diet. 

There were significant effects of dietary treatments on body weight, feed intake (except at day 42), 

FCR, and carcass yield at 21 and 42 days. Body weights were significantly different between the 

treatments. Birds fed on diet containing 48 mg essential oil/kg being the highest and this treatment 

was followed by chicks fed on the diet containing 72 mg essential oil/kg, antibiotic, negative control, 

and 24 mg essential oil/kg at day 42.


Supplementation with 48 mg EOC/kg to the broiler diet significantly improved the body weight 

gain, FCR, and carcass yield compared to other dietary treatments on 42 days of age. EOC may be 

considered as a potential growth promoter in the future of the new era, which agrees with producer 

needs for increased performance and today’s consumer demands for environment-friendly broiler 

production. The EOC can be used cost effectively when its cost is compared with antibiotics and 

other commercially available products in the market. 


In this study, 1250 sexed 1-day-old broiler chicks were randomly divided into five treatment groups 

of 250 birds each (negative control, organic acid, probiotic, and EOC at two levels). Each treatment 

group was further subdivided into five replicates of 50 birds (25 males and 25 females) per replicate. 

The oils in the EOC were extracted from different herbs growing in Turkey. The organic acid at 

2.5 g/kg diet, the probiotic at 1 g/kg diet, and the EOC at 36 and 48 mg/kg diet were added to the 

basal diet. 

The results obtained from this study indicated that the inclusion of 48 mg EOC/kg broiler diet 

significantly improved the body weight gain, FCR, and carcass yield of broilers compared to organic 

acid and probiotic treatments after a growing period of 42 days. The EOC may be considered as a 

potential growth promoter like organic acids and probiotics for environment-friendly broiler 

production. 


The aim of the present study was to examine the effect of essential oils and breeder age on growth 

performance and some internal organs weight of broilers. A total of 1008 unsexed 1-day-old broiler 

chicks (Ross-308) originating from young (30 weeks) and older (80 weeks) breeder flocks were 

randomly divided into three treatment groups of 336 birds each, consisting of control and two 

EOMs at a level of 24 and 48 mg/kg diet. There were no significant effects of dietary treatments on 

body weight gain of broilers at days 21 and 42. 

On the other hand, there were significant differences on the feed intake at days 21 and 42. The 

addition of 24 or 48 mg/kg EOM to the diet reduced significantly the feed intake compared to the 

control. The groups fed with the added EOM had significantly better FCR than the control at days 

21 and 42. Although, there was no significant effect of broiler breeder age on body weight gain at 

day 21, significant differences were observed on body weight gain at 42 days of age. Broilers originating 

from young breeder flock had significantly higher body weight gain than those originating 

from old breeder flock at 42 days of age. No difference was noticed for carcass yield, liver, pancreas, 

proventriculus, gizzard, and small intestine weight. Supplementation with EOM to the diet in both 

levels significantly decreased mortality at days 21 and 42. 

The results indicated that the Herbromix may be considered as a potential growth promoter. 

However, more trials are needed to determine the effect of essential oil supplementation to diet on 

the performance of broilers with regard to variable management conditions including different 

stress factors, essential oils and their optimal dietary inclusion levels, active substances of oils, 

dietary ingredients, and nutrient density (Cabuk et al., 2006a, 2006b; Alcicek et al., 2003, 2004; 

Bozkurt and Baser, 2002a, 2002b). 

19.5.3 PIGS 

CRINA ® Pigs was tested on pigs. The results for the first 21-day period showed that males grew 

faster, ate less, and exhibited superior FCR compared to females. Although female carcass weight 

was higher, males had a significantly lower carcass fat than females (Losa, 2001). 

The addition of fennel (Foeniculum vulgare) and caraway (Carum carvi) oils was not found 

beneficial for weaned piglets. In feed choice conditions, fennel oil caused feed aversion (Schoene 

et al., 2006).


Oregano oil was found to be beneficial for piglets (Molnar and Bilkei, 2005). 

In a preliminary investigation, the effects of low-level dietary inclusion of rosemary, garlic, and 

oregano oils on pig performance and pork quality were carried out. Unfortunately, no information 

on the species from which the oils were obtained and their composition existed in the paper. The 

pigs appeared to prefer the garlic-treated diet, and the feed intake and the average daily gain were 

significantly increased although no difference in the feed efficiency was observed. Carcass and meat 

quality attributes were unchanged, although a slight reduction of lipid oxidation was noted in 

oregano-fed pork. Since the composition of the oils is not clear, it is not possible to evaluate the 

results (Janz et al., 2007). 

A study revealed that the inclusion of essential oil of oregano in pigs’ diet significantly 

improved the average daily weight gain and FCR of the pigs. Pigs fed with the essential oils had 

higher carcass weight, dressing percentage, and carcass length than those fed with the basal and 

antibiotic- supplemented diet. The pigs that received the essential oil supplementation had a significantly 

lower fat thickness. Also lean meat and ham portions from these pigs were significantly 

higher. Therefore, the use of Origanum essential oil as feed additive improves the growth of pigs 

and has greater positive effects on carcass composition than antibiotics (Onibala et al., 2001). 

Ropadiar ® , an essential oil of the oregano plant, was supplemented in the diet of weaning pigs 

as alternative for antimicrobial growth promoters (AMGPs), observing its efficacy on the performance 

of the piglets. Ropadiar liquid contains 10% oregano oil and has been designed to be added 

to water. Compared to the negative control (without AMGP), Ropadiar ® improved performance 

only during the first 14 days after weaning. Based on the results of this trial, it cannot be argued 

about the usefulness of Ropadiar ® as an alternative for AMGP in diets of weanling pigs. However, 

its addition in prestarter diets could improve performance of these animals (Krimpen and 

Binnendijk, 2001). 

The objective of another trial was to ascertain the effect on nutrient digestibilities and N-balance, 

as well as on parameters of microbial activity in the gastrointestinal tract of weaned pigs after adding 

oregano oil to the feed. The apparent digestibility of crude nutrients (except fiber) and the 

N-balance of the weaned piglets in this study were not influenced by feeding piglets restrictively 

with this feed additive. By direct microbiological methods, no influence of the additive on the gut 

flora could be found (Moller, 2001). 

The inclusion of essential oil of spices in the pigs’ diet significantly improved the average daily 

weight gain and FCR of the pigs in Groups 3, 4, and 5, as compared to Groups 1 and 2 (P < 0.01). 

Furthermore, pigs fed with the essential oils had higher carcass weight (P < 0.01), dressing percentage 

(P < 0.01), and carcass length (P < 0.01) than those fed with the basal and antibiotic-supplemented 

diet. In Groups 3, 4, and 5, backfat thickness was significantly lower than those in Groups 1 

and 2. Moreover, lean meat and ham portions from pigs in Groups 3, 4, and 5 were significantly 

higher than those from pigs in Groups 1 and 2. In conclusion, the use of essential oils as feed additives 

improves the growth of pigs and has greater positive effects on carcass composition than antibiotics 

(Onibala et al., 2001). 

19.6 ESSENTIAL OILS USED IN TREATING DISEASES IN ANIMALS 

There is scarce scientific information on the use of essential oils in treating diseases in animals. 

Generally, the oils used in treating diseases in humans are also recommended for animals. 

Internet literature is abound with valid and/or suspicious information in this issue. We have tried 

to compile relevant information using the reachable resources. The information may not be concise 

or comprehensive but should be seen as an effort to combine the available information in a short 

period of time. 

The oil of Ocimum basilicum has been reported as an expectorant in animals. The combined oils 

of Ocimum micranthum and Chenopodium ambrosioides is claimed to treat stomach ache and colic 

in animals (http://www.ansci.cornell.edu/plants/medicinal/basil.html).


Bad breath as a result of gum disease and bacterial buildup on the teeth of pets can be treated by 

brushing their teeth with a mixture of a couple of tablespoons of baking soda, 1 drop of clove oil and 

1 drop of aniseed oil. Lavender, myrrh, and clove oils can also be directly applied to their gums. 

For wounds, abscesses, and burns, lavender and tea tree oils are used by topical application. Skin 

rashes can be treated with tea tree, lavender, and chamomile oils. 

Earache of pets can be healed by dripping a mixture of lavender, chamomile, and tea tree oils 

(1 drop each) dissolved in a teaspoonful of grapeseed or olive oil in the infected ears. 

Hoof rot in livestock can be treated with a hot compress made up of 10 drops of chamomile, 15 drops 

of thyme, and 5 drops of melissa oils diluted in about 100 ml of vegetable oil (e.g., grapeseed oil). 

Intestinal worms of horses can be expelled by applying 3–4 drops of thyme oil and tansy leaves 

to each feed. Melissa oil can be added to feed to increase milk production of both cows and goats 

(http://scentsnsensibility.com/newsletter/Apr0601.htm). 

Aromatic plants such as Pimpinella isaurica, Pimpinella aurea, and Pimpinella corymbosa are 

used as animal feed to increase milk secretion in Turkey (Tabanca et al., 2003). 

To calm horses, chamomile oil is added to their feed. Pneumonia in young elephants caused by 

Klebsiella is claimed to be healed by Lippia javanica oil. Rose and yarrow oils bring about emotional 

release in donkeys by licking them. Wounds in horses are treated with Achillea millefolium 

oil; sweet itch is treated with peppermint oil. Matricaria recutita and Achillea millefolium oils are 

used to heal the skin with inflammatory conditions (Anonymous, 2008). 

A study evaluated the effect of dietary oregano etheric oils as nonspecific immunostimulating 

agents in growth-retarded, low-weight growing-finishing pigs. A group of pigs were fed with commercial 

fattening diet supplemented with 3000 ppm oregano additive (Oregpig ® , Pecs, Hungary), 

composed of dried leaf and flower of Origanum vulgare, enriched with 500 g/kg cold-pressed 

essential oils of the leaf and flower of Origanum vulgare, and containing 60 g carvacrol and 55 g 

thymol/kg. Dietary oregano improved growth in growth-retarded growing-finishing pigs and had 

nonspecific immunostimulatory effects on porcine immune cells (Walter and Bilkei, 2004). 

Menthol is often used as a repellent against insects and in lotions to cool legs (especially for 

horses) (Franz et al., 2005). 

Milk cows become restless and aggressive each time a group of cows are separated and regrouped. 

This can last a few days putting cows in more stress resulting in a drop in milk production. Two 

Auburn University scientists could solve this problem by spraying anise oil (Pimpinella anisum) on 

the cows. Treated animals could not distinguish any differences among the cows in new or old 

groupings. They were mellower and kept their milk production up. Among many other oils tested 

but only anise seemed to work (Anonymous, 1990). 

Essential oils have been found effective in honeybee diseases (Ozkirim, 2006; Ozkirim et al., 

2007). 

In this review, we tried to give you an insight into the use of essential oils in animal health and 

nutrition. Due to the paucity of research in this important area there is not much to report. Most 

information on usage exists in the form of not-so-well-qualified reports. We hope that this rather 

preliminary report can be of use as a starting point for more comprehensive reports. 

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with Eimeria tenella. Arch. Anim. Nutr., 57(2): 99–106. 

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gastrointestinal helminthes in livestock with emphasis on small ruminants. Vet. Parasitol., 139: 308–320. 

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20 

Trade of Essential Oils 

Hugo Bovill 

The essential oil industry is highly complex and fragmented. There are at least 100 different producing 

countries, as can be seen from the map Essential Oils of the World (Figure 20.1). Many of these 

producing countries have been active in these materials for many decades. They are often involved 

in essential oils due to historical colonization, for example, clove oil from Madagascar has traditionally 

been purchased via France, nutmeg from Indonesia through Holland, and West Indian and 

Chinese products through Hong Kong and the United Kingdom. The main markets for essential oils 

are the United States (New Jersey), Germany, the United Kingdom, Japan, and France (Paris and 

Grasse). Within each producing country, there is often a long supply chain starting with the small 

peasant artisanal producer, producing just a few kilos, who then sells it to a collector who visits 

different producers and purchases the different lots that are then bulked together to form an export 

lot, which is then often exported by a firm based in the main capital or main seaport of that country. 

This exporter is equipped with the knowledge of international shipping regulations, in particular for 

hazardous goods, which applies to many essential oils. They also are able to quote in US$ or Euros, 

which is often not possible for small local producers (Figure 20.2). 

Producers of essential oils can vary from the very large, such as an orange juice factory where 

orange oil is a by-product, down to a small geranium distiller (Figures 20.3 and 20.4). 

The business is commenced by sending type samples that are examples of the production from 

the supplier and should be typical of the production that can be made going forward. Lot samples 

are normally provided to the purchaser in the foreign country to enable them to chemically analyze 

the quality organoleptically both on odor and flavor. It is essential that the qualities remain constant 

as differing qualities are not acceptable and there is normally no such thing as a “better” quality; it 

is either the same or it is not good. This is the key to building close relationships between suppliers 

in the country of origin and the purchaser. 

Many suppliers try to improve their processes by adapting their equipment and modernizing. In 

Paraguay, petitgrain distillers replaced wooden stills with stainless steel stills on the advice of overseas 

aid noncommercial organizations (NCOs). This led to a change in quality and the declining 

usage of petitgrain oil. The quality issues made customers unhappy, and in fact the Paraguayan 

distillers reverted back to their traditional wooden stills (Figure 20.5). 

Market information, as provided by the processor, is essential to developing long-term relationships. 

To enable the producer to understand market pricing, he should appreciate that when receiving 

more enquiries for an oil, it is likely that the price is moving upward and it is by these signs of 

demand that he can establish that there are potential shortages in the market (Figure 20.6). 

Producers and dealers exporting oil should be prepared to commit to carry inventory to ensure 

carryover and adequate delivery reliability. It is important to note that with climate change, weather 

and market conditions are becoming increasingly important, and prior to planting, advice should be 

sought from the buyer as to their intentions, for short, medium, and long term. Long- and mediumterm 

contracts are unusual and it is becoming increasingly common for flavor and fragrance companies 

not to commit over 1 year but to buy hand to mouth and purely give estimated volume needs 

going forward. This strengthens the role of the essential oil dealers, of whom there are very few 

remaining in the main trading centers of the world, such as the United States, France, the United 

Kingdom, Germany, and Japan. 

895


FIGURE 20.1 (See color fold-out insert at the back of the book) World map showing production centers of essential oils. Courtesy of Treatt PLC.

Trade of Essential Oils 897 

Distillers in country of 

origin 

Merchant 

Essential oil factory 

Food & beverage 

manufacturers 

& 

health/household 

Essential oils 

FIGURE 20.2 Flowchart showing the supply chain from distiller to finished product. 

FIGURE 20.3 South American orange juice factory. (Photograph by kind permission of Sucocitrico 

Cutrale Ltd.) 

FIGURE 20.4 

Copper Still in East Africa.


FIGURE 20.5 Petitgrain still. 

FIGURE 20.6 Market information.


To quote from Marketing Essential Oils (n.d.) by W.A. Ennever of R.C. Treatt & Co. Ltd, London 

in the 1960s, “The dealer serves as a buffer between these two interests (producer and essential oil 

merchant house) by purchasing and carrying stocks of oils for his own account and risk when the 

producer and/or merchant house is unable to wait for the user’s demand and hold stocks until the 

latter is ready to purchase. The risk of market fluctuations to the essential oil dealer or merchant in 

this practice, is quite considerable, but naturally, is reduced by his knowledge and experience of the 

trade. He is equipped to handle large or small quantities and a range of qualities, as a buyer or seller. 

Thus through the dealer’s participation, the producer has a larger number of outlets for his production 

and the user can be reasonably certain of finding supplies of the oils required when he considers 

it necessary to purchase.” The dealer is aware of world markets and potential shortages that other 

producers may not be aware of, as these are happening in different continents. They can also have 

the knowledge of increasing demand and movements in consumer tastes. 

Some essential oils are produced for their chemical constituents, whereas most are produced for 

their aromatic parts, and it is important that suppliers understand what is expected of them by their 

customer, whether it is chemical constituents naturally occurring or whether it is the aroma and flavor. 

Examples of this are turpentine oil, litsea cubeba oil, sassafras oil, clove leaf oil, and coriander oil. 

There is greater demand for ethical supplies, but it should be borne in mind that these surprisingly 

often do not receive a premium and when entering the essential oil industry it is important to 

note that it is not always the highest priced oils that give the best return as these are often those that 

are the most popular for new entrants to produce. Before entering into production of an essential oil, 

it is important to fully verify the market. It may be that there is good supply locally of the herb, for 

example, but maybe this is for a traditional purpose such as local medicinal use, producing local 

foodstuffs, or liqueurs. 

Origins are constantly changing and moving, as can be seen from the following: peppermint oil 

Mitcham production went from England to the United States; mint came from China, then went to 

Brazil and Paraguay, back to China and now to India. 

Within the essential oil market, there are generally four different types of buyers: aromatherapy, 

the flavor and fragrance industries, and dealers. Many of these can be contacted through agents who 

would not pay for the goods themselves but would take a nominal commission of, say, 5%. The end 

users range from aromatherapists selling very small volumes of high, fine quality, natural essential 

oils to flavor and fragrance companies, and in a few cases, consumer product companies. The main 

markets are the essential oils dealers, of which there are probably 10 or 20 major companies remaining 

in the world, some of which are also involved in the manufacture of flavors or fragrances. To 

avoid conflicts of interest, it is perhaps better to work with those who concentrate solely on raw 

materials. Several of these companies have been established for many years and have a good trading 

history. Some information about them can be gained from their websites, but without meeting them 

in person, it is not easy to establish their credentials. 

Conditions of trade are normally done on a FOB or a CIF basis, and the price should be given 

before samples are sent. With each sample, a Material Safety Data Sheet (MSDS), a Child Labor 

Certificate, and a Certificate of Analysis should be sent. It should be noted that the drums should be 

sealed and that the sample should be fully topped with nitrogen or be full to ensure that there is no 

oxygen present, in order to make sure that oxidation is avoided. The sample bottles should be made 

from glass and not from plastic to avoid contamination by phthalates. The lots should be bulked 

before sampling and a flashpoint test should be obtained to guarantee that it is within the law to send 

the sample by mail or by air freight with the correct labeling. 

Many customers are able to give advice on production, but dealers in particular are best placed 

to advise. To enable contact with such dealers, it is worthwhile attending international meetings 

such as the International Federation of Essential Oils and Aroma Trades (IFEAT) annual conference 

or reading the Perfumer and Flavorist magazine, which gives full details of brokers, dealers, 

and essential oil suppliers. There is no reference site that is 100% reliable in pricing for essential


oils; this information should be gained by working with a variety of buyers, and from this a knowledge 

of the market can be acquired. 

The essential oil industry is very traditional and even though there have been changes in analytical 

methods and demands, the knowledge required in 1950 by buyers such as Mr Ennever of Treatt 

(as can be seen from his quotation earlier in this chapter) are not too different from today. There 

is greater demand for organically certified, Kosher, Halal, and other standards. The market can 

change far quicker now than in the past, thanks to the worldwide web. Producers are often their 

own worst enemies and can destroy their own successful markets by communicating with their 

neighboring farmers, thereby encouraging them to enter the market. This can depress prices as a 

result of increased supply, but on the other hand, it can sometimes be in the interest of a sole 

producer to have other producers participating in the supply, to ensure guarantees of supply and to 

lower the costs of production, which in turn encourages buyers to use the oil. Oils such as patchouli 

and grapefruit have had significant changes in price, as can be seen in the price graphs in Figures 20.7 

through 20.9. 

These price movements have reduced demand as major buyers of these products have had to look 

for alternatives to replace them as they are unable to cope with the massively increased prices from 

US$10 to US$100 for grapefruit and from US$12.5 to US$70 per kilo for peppermint oil. It can be 

seen, therefore, that stable pricing can lead to increased demand. Unstable pricing can lead to the 

death of essential oils. This is an important reason for holding inventory so that producers can enter 

into long-term associations with essential oil buyers to ensure good relationships. 

FIGURE 20.7 

US$/kg 

120.00 

100.00 

80.00 

60.00 

40.00 

20.00 

0.00 

Price graph of grapefruit oil. 

DATE 

1-Jun-75 

1-Jan-85 

1-Jan-90 

1-Jan-95 

01-Jan-05 

01-Apr-05 

01-Aug-05 

01-Mar-06 

Florida grapefruit oil 

Average US$30.36 

01-Aug-06 

01-Dec-06 

01-Feb-07 

01-Jun-07 

01-Nov-07 

01-Jan-08 

01-Apr-08 

01-Nov-08 

US$/kg 

80 

70 

60 

50 

40 

30 

Yearly average price for piperita 

peppermint oil 

20 

10 

0 

1992 1994 1996 1998 2000 2002 2004 2006 2008 


FIGURE 20.8 

Price graph of peppermint oil (piperita).


US$/kg 

12 

Yearly average price for 

demontholised peppermint oil 

10 

8 

6 

4 

2 

0 

1997 1999 2001 2003 2005 2007 2009 


FIGURE 20.9 

Price graph of peppermint oil (arvensis). 

In the 1970s, there was considerable fraud of millions of dollars, caused by the shipment of 

essential oils from Indonesia to the major buyers. The oils were in fact water, despite analysis certificates 

from Indonesian Government laboratories showing them to be the named essential oil. Payment 

had been made by letters of credit and this fraudulent practice has discouraged buyers from opening 

letters of credit to suppliers today. Terms of trade should normally be cash against shipping documents 

or payment after receipt and quality control of goods. 

The United States produces import statistics for essential oils and these can often be useful 

sources of information, and the European Union (EU) also has such statistics. The EU statistics 

cover a wide range of essential oils in each tariff; therefore the information is very vague and should 

not be used to make decisions. These statistics give no clues as to the quality of the product and it is 

that which can determine the price. The production of essential oils, as can be seen in the quotation 

by V.A. Beckley OBE, MC, Senior Agricultural Chemist, Kenya, who said during a meeting in 1931 

in Nairobi, is perhaps more chancy than most farming propositions; it most certainly requires more 

attention and supervision than most, and, with certain rare exceptions, does not pay much more 

highly is still valid to this day, despite this being said in 1935. 

The essential oil industry is a very small, tightly knit circle of traders, dealers, producers and 

consumers, and apart from some notable exceptions there is a very strong trade ethos. As it is a relatively 

small industry in terms of global commodities, statistics are not produced and it is by relationships 

with customers that information becomes available. Much that is on the Internet is misleading 

as it is for small quantities or is often written by consultants, and this information can be rapidly 

out-of-date as prices can move extremely quickly in either direction.

21 

Storage and Transport 

of Essential Oils 


CONTENTS 

21.1 Marketing of Essential Oils: The Fragrant Gold of Nature Postulates Passion, 

Experience, and Knowledge ............................................................................................ 903 

21.2 The Impact and Consequences on the Classification of Essential Oils as 

Natural but Chemical Substances ................................................................................... 905 

21.3 Dangerous Substances and Dangerous Goods ................................................................ 907 

21.4 Packaging of Dangerous Goods ...................................................................................... 909 

21.5 Labeling .......................................................................................................................... 910 

21.6 List of Regulations for the Consideration of Doing Business in the EU ........................ 913 

References .................................................................................................................................. 915 

21.1 MARKETING OF ESSENTIAL OILS: THE FRAGRANT GOLD OF NATURE 

POSTULATES PASSION, EXPERIENCE, AND KNOWLEDGE 

The trade of essential oils is affected more and more by Legal regulations related to Safety Aspects. 

The knowledge and the compliance with these superseding regulations have today become a Conditio 

Sine Qua Non (precondition) to ensure trouble-free global business relation as far as regulatory requirements 

are concerned as these requirements often may adversely affect usual commercial aspects. 

When placing essential oils on the market in the EU for use as flavors and fragrances in foods, 

animal feed, cosmetic pharmaceuticals, aromatherapy, and so on, among others, the following regulations 

have to be observed (Dueshop, 2008): 

– Council Directive 79/831/EEC—Dangerous Substances (see Section 21.6) 

– Dangerous Preparations 99/45/EEC 

– EU Flavouring Directive No. 88/388/EEC and a new EU flavor regulation in the stage of 

announcement 

– Novel Food Directive No. 258/97/EEC 

– Labelling Directive 2000/13/EC—food allergens 

– EU Food Regulation No. 178/2002/EU 

– New Pesticide Provisions—Regulation No. 396/2005/EU 

– New EU Cosmetic Regulations Amending Directive No. 76/768/EEC—restrictions and 

bans (see Table 21.2 at the end of this chapter) 

– Detergent Use Regulation No. 648/2004/EC 

– EU Pharmaceutical Legislation—GMP aspects 

– Biocide Use Directive No. 98/8/EEC 

– Dangerous Substance Directive DSD 67/584/EEC 

903


Essential oils are agro-based products that are generally manufactured by or collected from 

small individual producers. A large-scale production would require capital investment, which is 

rarely attracted as investors evidently realize the problems that no quick return of money is ensured 

because of too many factors influencing the market negatively like the dependency on weather conditions 

affecting the size of a crop over the whole vegetation period, competing crops challenging 

the acreage, a keen global competition striving for market shares, and narrow margins that do not 

compensate the involved risks. These aggravating factors also have an impact on the trade of essential 

oils. 

A major part of the essential oil industry and the trade of these articles are dominated by smallscale 

and medium-sized family enterprises as only entrepreneurs with passion, a personal engagement 

and a persistent dedication as well as a long-standing experience nerve themselves to stay 

successfully in this business of the liquid gold of nature. 

Success in the field of essential oils depends on enthusiasm and hard work, on a broad knowledge 

of the market situation, in spending a lot of time and cost to investigate new ideas of state-of-the-art 

conditions of processing raw materials that affect yield and quality and the return of investment, the 

adherence to comply with ever changing administrative regulations. 

Essential oils are natural substances mainly obtained from vegetable raw materials either by 

distillation with water or steam or by mechanical process (expression) from the epicarp of citrus 

fruits. They are concentrated fragrance and flavor materials of complex composition, in general 

volatile alcohols, aldehydes, ethers, esters, ketones, hydrocarbons, and phenols of the group of 

mono- and sesquiterpenes or phenylpropanes as well as nonvolatile lactones. 

A definition of the term essential oils and related fragrance/aromatic substances is given in the ISO- 

Norm 9235 Aromatic Natural Raw Materials (International Standard Organization, Geneva, 1997). 

Because of their composition essential oils are classified by regulatory authorities in the EU as 

“Natural” but also as “Chemical Substances” (Dueshop, 2007). 

The classification of chemical Substances is laid down in the Council Directive 67/548 and subsequent 

amendments but in particular in Council Directive 79/831/EEC of 18-09-1979. This 6th 

amendment is the basis of all existing regulations for dangerous/hazardous chemicals as it earmarked 

the beginning of a new era. 

The topic REACH will not be covered in this chapter because of its complexity and too many 

open questions and answers respectively at the time of this writing. I hope, however, that in exchange 

a brief introduction to the historic development of the existing regulatory framework can be of help 

to understand the Safety Aspects, which are the background of the actual regulations as well as the 

forthcoming impediments in connection with REACH. 

REACH is the abbreviation for Registration, Evaluation, Authorization of Chemicals. It is another 

impeding Regulation in Europe—the consistent continuation of the existing rules to satisfy the EU 

administration of a perfect system to safeguard absolute security to protect humans and the environment 

regarding the use of chemicals within the EU. 

For the trade, that is, the industry as well as importers and dealers of essential oils, REACH is a 

heavy burden demanding, already in the forefront, an unbelievable amount of time to clarify questions 

regarding the required product information for an appropriate registration of the so-called 

natural complex substances (NCS). 

Before the publication of Directive 79/831/EEC only a few people were aware of the aftermath 

of a centralized European administration. Regulations regarding transport of dangerous goods 

were adhered—the trade of essential oils was well aware of the risk of flammability of many of the 

oils but most people, however, were caught more or less unprepared with regard to the new classification 

that natural essential oils have to be considered as “chemicals.” The new Directive with 

its detailed regulations came as a surprise. It terminated the familiar view that essential oils 

because of their natural origin (and the fact they were used for centuries in medicines, flavors, and 

fragrances) could continue to exist as a special group of natural products like a sleeping beauty in 

the reality of a hostile world of administrative regulations. Now, all of a sudden it caused essential

Storage and Transport of Essential Oils 905 

oils to be considered as chemical substances of which a major part had to be classified as hazardous 

“chemical” substances. 

21.2 THE IMPACT AND CONSEQUENCES ON THE CLASSIFICATION OF 

ESSENTIAL OILS AS NATURAL BUT CHEMICAL SUBSTANCES 

The bell for the new era sounded when chemical substances in use within the EC during a reference 

period of 10 years had to be notified for European Inventory of Existing Commercial Chemical 

Substances (EINECS). 

At that time EINECS enabled the EC administration not only to dispose of, for the first time, a 

survey of all chemical substances that had been in use in the EC between January 1, 1971 and 

September 18, 1981, but also to distinguish between “known substances” and “new substances.” 

“KNOWN” substances are all chemicals notified for EINECS, whereas all chemical substances 

that were not notified (and subsequently registered as “known substances” in EINECS) are considered 

by the EU administration as “new chemicals.” 

EINECS is a “closed list”—“New” chemical substances to be placed on the market in the EU 

after the deadline of September 18, 1981, therefore have to be notified for the European List of 

Notified Chemical Substances (ELINCS), the list complementing EINECS. 

NEW chemical substances can be placed on—and used in—the market of the EU only after 

clearance according to uniform EC standards by competent (national) authorities. Thus, from the 

beginning, all potential risks of a (new) chemical substance are ascertained for a proper labeling for 

handling to avoid risks for humans as well as to protect the environment. 

“Known” chemical substances (notified for EINECS) enjoyed, in a transitional phase, temporary 

exemption from the obligation to furnish the same safety data required for new chemical substances. 

Based on the experience gathered during their use, for quite a while it was assumed (Dueshop, 

2007) that the temporary continuation of their use could be tolerated according to the hitherto used 

older standards of safety—and in view of the fact that a short-term clearance of approximately 

100,000 chemical substances registered in EINECS could not be effected overnight. 

Because these products have been notified for EINECS and therefore known to the regulative 

agencies in the EC, they are screened step by step either depending on their potential risk or according 

to the volumes produced or imported respectively to make sure that the known substances also 

comply with the new safety standards according to the following volume bands: 


But ATTENTION—the CAS number is an identification number for a chemical substance allotted 

by a private enterprise in the United States and must not be confused with the EINECS registration 

number. 

EINECS and ELINCS numbers are registration numbers allocated by the EU administration, 

that is, ECB/JRC at ISPRA. 

CAS numbers are assigned by the (private) CAS organization in the United States with the 

purpose of identification of (defined) chemical substances. A CAS number is allocated to a new 

(defined) chemical substance only after thorough examination of the product as per IUPAC Rules 

by the CAS organization to make sure that irrespective of different chemical descriptions and/or 

coined names that have been given to a product, a substance can be clearly related by the allocated 

CAS number according to the (CAS) principle “one substance—one number.” 

Using the CAS number system to register also chemical substances in EINECS that are not 

defined chemicals, the problem had to be sorted out how to register, for example, essential oils as 

they are products of complex composition. It was therefore necessary to extend the CAS system for 

this reason to allot a CAS number also to the so-called UVCBs, that is, substances that have been 

summed up under this abbreviation as substances of “unknown or variable composition, complex 

reaction products, and biological materials.” 

Essential oils are eventually registered as NCS by their botanical origin as for example: 

Lavender oil: Lavender—Lavandula angustifolia ext. 

EINECS registration no. 289-995-2—CAS no. (Einecs) 90063-37-9 extractives and their physically 

modified derivatives such as tinctures, concretes, absolutes, essential oils, terpenes, terpenefree 

fractions, distillates, and residues from Lavandula angustifolia—Labiatae (Lamiaceae) 

Lavender oil: Lavender—Lavandula angustifolia ext. 

EINECS registration no. 283-994-0—CAS no. (Einecs) 84776-65-8 extractives . . . from 

Lavandula angustifolia angustifolia— Labiatae (Lamiaceae) 

Lavender concrete/absolute: Lavender—Lavandula angustifolia ext. 



fractions, distillates, and residues from Lavandula angustifolia—Labiatae (Lamiaceae) 

Lavandin oil: Lavandula hybrida ext. 



fractions, distillates, and residues from Lavandula hybrida—Labiatae (Lamiaceae) 

Lavandin oil abrialis: Lavandula hybrida abrial ext. 

EINECS registration no. 297-384-7—CAS no. (Einecs) 93455-96-0 extractives and . . . from 

Lavandula hybrida abrial—Labiatae (Lamiaceae) 

Lavandin oil grosso: Lavandula hybrida grosso ext. 

EINECS registration no. 297-385-2—CAS no. (Einecs) 93455-97-1 extractives and . . . from 

Lavandula hybrida grosso—Labiatae (Lamiaceae). 

Since essential oils are registered as extractives under their botanical origin, concretes/ absolutes 

and other natural extractives of the same botanical origin have the same EINECS and CAS numbers 

as the essential oil. 

When checking an EINECS number it is important to investigate in the official original documentation 

as in the secondary literature there exist too many inaccuracies.


TABLE 21.1 

Examples of Different CAS-Numbers used in USA and EINECS in EU 

CAS No. USA CAS No. EINECS EC Registration No. 

Eucalyptus oil 8000-48-4 84625-32-1 283-406-2 

Eucalyptus globulus Lab.—Myrtaceae 

Lavender oil 8000-28-0 90063-37-9 289-995-2 

Lavandula angustifolia—Labiatae 

Lavandula angustifolia angustifolia—Labiatae 84776-65-8 283-994-0 

Lemon oil 8008-56-8 8028-48-6 284-515-8 

84929-31-7 284-515-8 

Citrus limon L.—Rutaceae 

Orange oil 8008-52-9 8028-48-6 232-433-8 

Citrus sinensis—Rutaceae 

Peppermint oil 8006-90-4 98306-02-6 308-770-2 

Mentha piperita L.—Lamiaceae 

Manuka oil tairawhiti — 223749-44-8 425-630-7 

Leptospermum scoparium—Myrtaceae 

(ELINCS) 

Due to the lack of rules for an uniform classification of UVCBs (as an example the correct identification 

of the botanical origin of an essential oil), it happened that against the principles of the 

CAS organization in some cases several CAS numbers had been allocated to essential oils of the 

same denomination and in addition: 

– An older CAS number allocated for an (earlier) registration of the product in the USA. 

– A new CAS number allocated for registration in the EC for EINECS/ELINCS, respectively. 

Once again, a CAS number does not mean that the product is registered in the European 

EINECS—the CAS number is just an identification number of a chemical substance allotted upon 

request by the (private) CAS organization. 

Table 21.1 is exemplifying the allocation of several CAS numbers for the same essential oils but 

in connection with EINECS only the CAS number (EINECS) is of relevance. 

Manuka Oil from New Zealand is the first (and only) essential oil that had to be notified for ELINCS 

as a new chemical substance after the Council Directive 79/831/EEC became effective on September 18, 

1981 (Dueshop, 2007). It is quoted here only for the sake of completeness and curiosity. 

This brief reflection on the background of EINECS and ELINCS is made as an introduction of 

the actual situation with regard to safety requirements and to alert new players in the field of essential 

oils to make sure that before intending to place a fragrance or flavor raw material on the European 

market they check whether or not this product is listed in EINECS or ELINCS respectively or is 

marketed in compliance with the Regulations of REACH for new substances. Placing of chemical 

substances in the states of the EU that are not meeting these requirements is a breach of law that can 

even be prosecuted as an offense with a penalty or a fine up to euros 100,000. 

21.3 DANGEROUS SUBSTANCES AND DANGEROUS GOODS 

There is a significant difference between the similar sounding words and regulations regarding 

DANGEROUS SUBSTANCES and DANGEROUS (HAZARDOUS) GOODS. 

Both regulations are targeted to protect humans and the environment, but the term “Dangerous 

Substance” refers to the risks connected with the properties of the substance, that is, the potential 

risk of a direct contact with the product during production, packaging, and use.


Dangerous substance 

Dangerous good 

Production 

Packaging 

Storage 

Use (blending) 

Road 

Rail 

Ship 

Air 

Handling 

Transport 

FIGURE 21.1 Interrelationship between dangerous substances and dangerous goods. (Friendly permission, 

Paul Kaders, Hamburg.) 

Dangerous Substances are chemicals that fall into the categories quoted in article 2 of the 

already mentioned Council Directive 79/831/EEC—the 6th amendment of Directive 67/548. They 

are categorized as 

Explosive 

Oxidizing 

Flammable (extremely flammable—highly flammable—flammable) 

Toxicity (very toxic—toxic—harmful) 

Corrosive (corrosive—irritant) 

Dangerous for the environment (ecotoxicity) 

Carcinogenic—teratogenic—mutagenic (CMR). 

To protect humans and the environment—but principally the workers using them—articles that 

fall in these categories have to be classified as “Dangerous Substances” and labeled as per the 

subsequent Dangerous Substances Directive. 

The term Dangerous Goods refer to dangerous substances properly packed and labeled for 

storage and transport by road, rail, sea, or air (Figure 21.1). 

As per the rules and recommendations developed by a UN Committee of Experts regarding the 

transport of dangerous goods or substances they are defined as articles or substances that are capable 

of posing a risk to health, safety, property, or the environment. 

Dangerous goods are classified into the following groups (classes of relevance for essential oils 

have been marked in bold font): 

Class 1: Explosives 

Class 2: Gases 

Class 3: Flammable liquids 

Class 4: Flammable solids 

Class 5: Oxidizing substances and organic peroxides 

Class 6: Toxic and infectious substances—eventually “poison” 

Class 7: Radioactive material 

Class 8: Corrosives 

Class 9: Miscellaneous dangerous goods. 

21.4 PACKAGING OF DANGEROUS GOODS 

Dangerous goods must be transported in UN-approved packaging, which has been tested for sufficient 

stability and graded in the packing groups (PGs) I, II, and III. 

PG III (low risk)—Suitable for dangerous goods having a low-risk classification only. 

PG III corresponds to the UN packing code “Z”


PG II (medium risk)—This type of packing matches the requirements for most of the essential 

oils. 

PG II corresponds to UN packing code “Y”—PG II includes PG III. 

PG I (high risk) corresponds to UN packing code “X”. 

PG I includes PG II and III—this type of packing has the highest stability. 

All dangerous goods have to be packed in the so-called UN-approved packing. 

Essential oils that are classified as dangerous goods and shipped in bulk, that is, drum lots for 

example, will only be accepted for transport if they are packed in UN-approved iron drums. These 

drums with a bunghole for example bear the following UN code: 

UN 1A1/Y/1.4/150/(06)/(NL)/(VL824) 

This specification reveals the following details: 

1A1 

Steel drum—nonremovable head 

Y 

PG II 

1.4 Maximum relative density at which the packing has been tested 

150 Test pressure 

(0.6) Year of manufacture 

(NL) 

State (country) 

(VL 123) 

Code number of manufacturer 

The potential risks of dangerous substances or goods respectively have to be declared in the 

relevant transport documentation. In addition to this information, also warning labels have to be 

used on the packages to alert workers regarding the nature of the goods they are handling. 

The aim of dangerous goods regulations is not only to protect persons occupied with the conveyance 

of dangerous substances but to also serve, for example, fire brigades, who in case of an accident 

or fire are called and have to be aware of the risks. 

In this connection, a few words are due on the so-called UN/ID numbers for dangerous goods. These 

UN numbers are assigned to dangerous goods according to their hazard classification and composition. 

These UN (hazard identification) numbers should not be confused with the number of UN packaging. 

UN numbers are listed in all regulations for transport of dangerous goods and are identical for 

all types of transport. 

Approximately 170 essential oils have to be classified as dangerous substances/goods. According 

to their composition, the following UN numbers have been assigned to these oils: 

65 UN no. 1169—extracts, aromatic, and liquid 

52 UN no. 3082—environmentally hazardous substance, liquid, n.o.s. 

14 UN no. 1272—pine oil(s) 

6 UN no. 1992—flammable liquid, toxic, n.o.s. 

6 UN no. 2810—toxic liquid organic n.o.s. 

5 UN no. 2319—terpene hydrocarbons 

and others are distributed among the UN nos. 2811 (3), 2924 (3), 1545 (2), 1130 (1), 1197 (1), 1201 

(1), 1299 (1), 1990 (1), and 3077 (1). 

Details can be found in EFFA’s Code of Practice (CoP, 2008, et seq.), which is described later on. 

Consignments of dangerous substances (and dangerous goods respectively) must be accompanied 

by a so-called Material Safety Data Sheet. For this purpose, the International Standard


Organization (ISO) has developed a standard form that—divided into 16 headings—provides basically 

information on 

Name of the supplier 

Name and identification of the substance/preparation 

Composition/components of the article 

Hazard identification 

First aid measures 

Fire fighting measures 

Accidental release measures 

Ecological information 

Transport information 

Regulatory information and so on 

to inform users and forwarders about the risks in connection with the chemical substance. 

Not only producers but also suppliers have the responsibility that the MSDS Form (material 

safety datasheet) is properly completed. 

21.5 LABELING 

IFRA and IOFI have regularly informed their members as well as stakeholders in the industry and 

trade for more than five decades about potential health risks that have been assessed for natural and 

synthetic raw materials used in flavors and fragrances in research and tests. 

Since a couple of years ago, the European Association of the Flavour and Fragrance Industry in 

Europe (EFFA) has been publishing a Code of Practice (CoP, 2008) with recommendations regarding 

a proper classification and labeling of aromatic chemicals and essential oils. 

This “CoP” is complementing the information of IFRA and IOFI. It is continuously updated by 

experts of the industry and the trade by the Hazard Communication Working Group (HCWG) and 

furnishes for the disposal of people all over the world occupied in handling essential oils and aromatic 

chemicals; an up-to-date recommendation for a proper classification and labeling of hazardous 

fragrance and flavor raw materials (Protzen, 1989). 

The actual version of this CoP 2009 is available on the internet free of charge from the homepage 

of EFFA: http://www.effa.be/. 

Because of the compiled state-of-the-art expertise, EFFA’s CoP has almost obtained in practice 

the quality of an official documentation. Therefore not only the trade but also the port and transport 

authorities who are in charge of controlling the compliance of safety regulations for transport of 

dangerous goods are today referring to this guideline (Protzen, 1998). 

For approximately. 1200 aromatic chemicals used in the flavor and fragrance industry and 220 

commercially used essential oils as well as information on 60 natural extracts like absolutes and 

resinoids, the CoP contains a guideline detailing information on 

EC registration number 

CAS number relevant in the EC/EU 

CAS number relevant in the USA 

Commercial name 

Content of hydrocarbons (%) 

Warning labels 

UN Transport Regulations (dangerous goods class, required class of packing group class, 

appropriate UN number)


R (Risk) phrases 

S (Safety) phrases. 

Before the Council Directive 79/831/EEC was issued, flammability of essential oils was considered 

the main danger emanating from these articles. Today’s knowledge of potential risks of essential 

oils is extended. As a precaution very rigid safety regulations that consider extreme conditions 

often exceeding empirical and practical experience require that from the 220 essential oils listed in 

the CoP 2008, approximately 70%, that is, 170 essential oils, are classified as dangerous Substances 

and therefore must be labeled accordingly for storage, use, and transport, as for example: 

Xn 

N 

Xi 

Gesundheitsschãdlich 

Harmful 

Umweltgefãhrlich 

Dangerous for the 

environment 

Reizend 

Irritant 

The following warning labels cover the majority of risks: 

190 Xn Harmful—a St. Andrew’s Cross (Xn) 

174 N Dangerous for the environment 

60 Xi Irritant—a St. Andrew’s Cross (Xi) 

12 T Toxic—a skull and cross-bones (T) 

3 C Corrosive—the symbol showing the damaging effect of an acid 

In addition to this information also R-phases and R labels must be used on the packaging. A list 

that explains the meaning of R + S phrases required for labeling essential oils as per the CoP is 

enclosed for further perusal. 

A statistical evaluation of the R (Risk) labels to be used is shown in the following differentiation 

to have a better and detailed idea of the potential risks: 

205 R-43 May cause sensation by skin contact 

158 R-65 Harmful—may cause lung damage if swallowed 

103 R-51/53 Toxic to aquatic organisms—may cause long-term adverse 

effects on the aquatic environment 

95 R-38 Harmful if swallowed 

88 R-50/53 Very toxic to aquatic organisms—may cause long-term 

adverse effects on the aquatic environment 

80 R-10 Flammable 

40 R-52/53 Harmful to aquatic organisms—may cause long-term adverse 

effects on the aquatic environment 

38 R-22 Harmful if swallowed.


Flammability as a major risk of essential oils is today outnumbered by the potential risks emanating 

from these concentrated fragrances and flavors causing harm to the skin, to the health risk if 

swallowed, and their ecotoxicity. 

The cumulative frequency of occurrence reveals that the majority of essential oils have to be 

handled with care and workers should use a protection particularly when a contact of these concentrated 

volatile natural fragrance and flavor materials with the skin is possible. 

Special care and attention should be given when handling essential oils labeled with 

R-50/53 Very toxic to aquatic organisms—may cause long-term adverse effects 

on the aquatic environment 

R-34 Causes burns (oils containing thymol) 

R-45 May cause cancer (oils containing safrol) 

R-68 Possible risk of irreversible effects 

Safety starts at the point of production but in the chain of supply each party involved is directly 

responsible for proper handling, that is, declaration and labeling of goods. In Europe, a special 

transport police is in the ports and on the roads intensifying the controls for correct declaration, 

packaging and labeling of dangerous goods and heavy fines are imposed: 

Risk phrases applicable for storage and transport of essential oil—data as per EFFA CoP 2008: 

R-10 Flammable 

R-20 Harmful by inhalation 

R-21 Harmful in contact with the skin 

R-22 Harmful if swallowed 

R-23 Toxic by inhalation 

R-24 Toxic in contact with the skin 

R-25 Toxic if swallowed 

R-26 Very toxic by inhalation 

R-27 Very toxic in contact with the skin 

R-34 Causes burns 

R-36 Irritating to eyes 

R-37 Irritating to the respiratory system 

R-38 Irritating to the skin 

R-41 Risk of serious damage to eyes 

R-43 May cause sensation by skin contact 

R-45 May cause cancer 

R-65 Harmful—may cause lung damage if swallowed 

R-66 Repeated exposure may cause skin dryness or cracking 

R-68 Possible risk of irreversible effects 

R-21/22 Harmful in contact with skin and if swallowed 

R-36/38 Irritating to eyes and skin 

R-50/53 Very toxic to aquatic organisms—may cause long-term adverse effects on the 

aquatic environment 

R-51/53 Toxic to aquatic organisms—may cause long-term adverse effects on the 


R-52/53 Harmful to aquatic organisms—may cause long-term adverse effects on the 


R-68/22 Harmful—possible risk of irreversible effects if swallowed


21.6 LIST OF REGULATIONS FOR THE CONSIDERATION 

OF DOING BUSINESS IN THE EU 

• Council Directive 79/831/EEC of 18 September 1979 amending for the sixth time Directive 

67/548/EEC on the approximation of the laws, regulations and administrative provisions 

relating to the classification, packaging and labelling of dangerous substances 

OJ L 259, 15.10.1979, p. 10–28 (DA, DE, EN, FR, IT, NL) 

• Directive 1999/45/EC of the European Parliament and of the Council of 31 May 1999 

concerning the approximation of the laws, regulations and administrative provisions of 

the Member States relating to the classification, packaging and labelling of dangerous 

preparations 

OJ L 200, 30.7.1999, p. 1–68 (ES, DA, DE, EL, EN, FR, IT, NL, PT, FI, SV) 

• Council Directive 88/388/EEC of 22 June 1988 on the approximation of the laws of the 

Member States relating to flavourings for use in foodstuffs and to source materials for their 

production 

OJ L 184, 15.7.1988, p. 61–66 (ES, DA, DE, EL, EN, FR, IT, NL, PT) 

• Regulation (EC) No 258/97 of the European Parliament and of the Council of 27 January 

1997 concerning novel foods and novel food ingredients 


• Directive 2000/13/EC of the European Parliament and of the Council of 20 March 2000 

on the approximation of the laws of the Member States relating to the labelling, presentation 

and advertising of foodstuffs 


• Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 

January 2002 laying down the general principles and requirements of food law, establishing 

the European Food Safety Authority and laying down procedures in matters of 

food safety 


• Regulation (EC) No 396/2005 of the European Parliament and of the Council of 

23 February 2005 on maximum residue levels of pesticides in or on food and feed of 

plant and animal origin and amending Council Directive 91/414/EECText with EEA 

relevance. 

OJ L 70, 16.3.2005, p. 1–16 (ES, CS, DA, DE, ET, EL, EN, FR, IT, LV, LT, HU, MT, NL, 

PL, PT, SK, SL, FI, SV) 

• Regulation (EC) No 648/2004 of the European Parliament and of the Council of 31 March 

2004 on detergents (Text with EEA relevance) 


• Directive 98/8/EC of the European Parliament and of the Council of 16 February 1998 

concerning the placing of biocidal products on the market 


• Council Directive 67/548/EEC of 27 June 1967 on the approximation of laws, regulations 

and administrative provisions relating to the classification, packaging and labelling of dangerous 

substances 

OJ 196, 16.8.1967, p. 1–98 (DE, FR, IT, NL) English special edition: Series I Chapter 1967 

P. 0234 

• Council Directive 76/768/EEC of 27 July 1976 on the approximation of the laws of the 

Member States relating to cosmetic products 

(OJ L 262, 27.9.1976, p. 169) 

For amendments see Table 21.2.


TABLE 21.2 

COUNCIL DIRECTIVE 

of 27 July 1976 

on the approximation of the laws of the Member States relating to cosmetic products 

(76/768/EEC) 

(OJ L 262, 27.9.1976, p. 169) 

Official Journal 

Amended by No Page Date 

M1 Council Directive 79/661/EEC of 24 July 1979 L 192 35 31.7.1979 

M2 Commission Directive 82/147/EEC of 11 February 1982 L 63 26 6.3.1982 

M3 Council Directive 82/368/EEC of 17 May 1982 L 167 1 15.6.1982 

M4 Commission Directive 83/191/EEC of 30 March 1983 L 109 25 26.4.1983 

M5 Commission Directive 83/341/EEC of 29 June 1983 L 188 15 13.7.1983 

M6 Commission Directive 83/496/EEC of 22 September 1983 L 275 20 8.10.1983 

M7 Council Directive 83/574/EEC of 26 October 1983 L 332 38 28.11.1983 

M8 Commission Directive 84/415/EEC of 18 July 1984 L 228 31 25.8.1984 

M9 Commission Directive 85/391/EEC of 16 July 1985 L 224 40 22.8.1985 





M14 Council Directive 88/667/EEC of 21 December 1988 L 382 46 31.12.1988 


M16 Council Directive 89/679/EEC of 21 December 1989 L 398 25 30.12.1989 




M20 Commission Directive 92/86/EEC of 21 October 1992 L 325 18 11.11.1992 

M21 Council Directive 93/35/EEC of 14 June 1993 L 151 32 23.6.1993 

M22 Commission Directive 93/47/EEC of 22 June 1993 L 203 24 13.8.1993 

M23 Commission Directive 94/32/EC of 29 June 1994 L 181 31 15.7.1994 

M24 Commission Directive 95/34/EC of 10 July 1995 L 167 19 18.7.1995 


M26 Commission Directive 97/1/EC of 10 January 1997 L 16 85 18.1.1997 

M27 Commission Directive 97/18/EC of 17 April l997 L 114 43 1.5.1997 


M29 Commission Directive 98/16/EC of 5 March 1998 L 77 44 14.3.1998 

M30 Commission Directive 98/62/EC of 3 September 1998 L 253 20 15.9.1998 

M31 Commission Directive 2000/6/EC of 29 February 2000 L 56 42 1.3.2000 



M34 Commission Directive 2002/34/EC of 15 April 2002 L 102 19 18.4.2002 



M37 Directive 2003/15/EC of the European Parliament and of the Council L 66 26 11.3.2003 

of 27 February 2003 


continued



Official Journal 

Amended by No Page Date 









M47 Commission Directive 2005/80/EC of 21 November 2005 L 303 32 22.11.2005 






M53 Commission Directive 2007/53/EC of 29 August 2007 L 226 19 30.8.2007 

M54 Commission Directive 2007/54/EC of 29 August 2007 L 226 21 30.8.2007 

M55 Commission Directive 2007/67/EC of 22 November 2007 L 305 22 23.11.2007 




M59 Commission Directive 2008/123/EC of 18 December 2008 L 340 71 19.12.2008 

M60 Directive 2008/112/EC of the European Parliament and of the Council L 345 68 23.12.2008 

of 16 December 2008 


Amended by 

A1 Act of Accession of Greece L 291 17 19.11.1979 

A2 Act of Accession of Spain and Portugal L 302 23 15.11.1985 

Corrected by 

C1 

C2 

C3 

C4 

C5 

C6 

C7 

C8 

C9 

C10 

Corrigendum, OJ L 255, 25.9.1984, p. 28 (84/415/EEC) 



Corrigendum, OJ L 273, 25.10.1994, p. 38 (94/32/EC) 







REFERENCES 

EFFA Code of Practice 2008, 2008. The European Flavour & Fragrance Association. Accessed October 2008 

from http://www.effa.be/ 

Dueshop, L., 2007. Personal communications.


Dueshop, L., 2008. Personal communications. 

Protzen, K-D., 1989. Guideline for Classifi cation and Labelling of Essential Oils for Transport and Handling 

distributed during IFEAT CONFERENCE, London. 

Protzen, K-D., 1998. Transportation/Safety Regulations Update, International Conference on Essential Oils 

and Aromas, November 8–12, 1998, London.

22 

Recent EU Legislation on 

Flavors and Fragrances and 

Its Impact on Essential Oils 

Jan C.R. Demyttenaere 

CONTENTS 

22.1 Introduction ............................................................................................................... 917 

22.2 Cosmetic and Detergent Legislation and Allergen Labeling ...................................... 918 

22.2.1 History and Background ................................................................................ 918 

22.2.2 Cosmetic Directive and its Seventh Amendment ........................................... 920 

22.2.3 Impact on Extracts and Essential Oils and Aromatic Natural 

Raw Materials ............................................................................................... 921 

22.2.4 Recent Data on Sensitization to Fragrances .................................................. 922 

22.2.5 First Amendment of the Detergent Regulation and Allergen Labeling .......... 925 

22.3 Current Flavouring Directive and Future Flavouring Regulation: Impact on 

Essential Oils ............................................................................................................ 926 

22.3.1 Current Flavouring Directive 88/388/EC ...................................................... 927 

22.3.1.1 Maximum Levels of “Biologically Active Substances” .................. 927 

22.3.1.2 Definition of “Natural” ................................................................... 927 

22.3.2 Future Flavouring Regulation ....................................................................... 928 

22.3.2.1 Maximum Levels of “Biologically Active Substances” .................. 929 

22.3.2.2 Definition of “Natural” ................................................................... 933 

22.4 Hazard Classification and Labeling of Flavors and Fragrances ................................ 935 

22.5 Conclusion ................................................................................................................ 939 

References ......................................................................................................................... 947 


In the last years, several new European Regulations and Directives have been adopted or announced 

in relation to flavors and fragrances. As essential oils and extracts are very important ingredients for 

flavoring and fragrance applications, these new regulations will have a major impact on the trade 

and use in commerce of these essential oils and extracts. 

This chapter will focus on some pieces of legislation that are of major importance for the Flavour 

and Fragrance (F&F) Industry, such as the Cosmetic Directive 76/768/EC and especially its Seventh 

Amendment (2003/15/EC) and the first amendment of the Detergent Regulation (June 2006), which 

make the labeling of 26 specific fragrance ingredients (the so-called 26 “alleged” allergens) 

mandatory: the presence of these materials above the given threshold has to be declared irrespective 

of the way they are added (as such or as being part of “complex ingredients” such as extracts and 

essential oils). 

917


Some attention will be paid to the new Flavouring Regulation (part of the so-called Food 

Improvement Agents Package) that will replace the current Flavouring Directive 88/388/EEC and 

that is currently under discussion at the EU Commission, EU Parliament and Council levels. 

Also the issue of hazard classification and labeling of dangerous substances and preparations, 

and essential oils containing hazardous components will be addressed and some examples will be 

given. This relates to the recent publication of the Commission Directive 2006/8/EC amending the 

Dangerous Preparations Directive 1999/45/EC. 

22.2 COSMETIC AND DETERGENT LEGISLATION AND ALLERGEN LABELING 

22.2.1 HISTORY AND BACKGROUND 

In recent years (the late 1980s and 1990s), there has been a scientific debate on the safety of fragrance 

(perfumery) ingredients. Dermatologists have highlighted the risk of contact allergy from 

fragrance ingredients (Santussi et al., 1987; Becker et al., 1994), and actions to prevent the disease 

have been requested (Frosch et al., 1995; Larsen et al., 1996; SCCNFP 0017/98). 

As a result of this and in response to a question from a Member State (MS) and members of the 

European parliament, the Scientific Committee on Cosmetic Products and Non-Food Products 

Intended for Consumers (SCCNFP) has been asked by DG Enterprise (EU Commission) to respond 

to the following mandate in relation to the safety of fragrance ingredients and to answer (among 

others) the following questions: 

• It is proposed that all known fragrance allergens are labeled on cosmetics if used in the 

products. Does the SCCNFP agree to this proposal? If so, 

– Which chemicals fall under this classification? 

– Is there a maximum concentration of each chemical permissible without the requirement 

for labeling? 

• Restrictions are proposed for the three most common fragrance allergens (cinnamic aldehyde, 

isoeugenol, and hydroxycitronellal). Does the SCCNFP agree to restriction on the 

use of common fragrance allergens (Annex III listing)? If so 

– Which fragrance materials should be subject to restrictions? 

– What are the conditions for restrictions (maximum concentration, fields of applications, 

etc.)? 

Other questions were related to industry-restricted and industry-prohibited substances. 

In its Interim position on Fragrance allergy SCCNFP/0202/99 adopted at the SCCNFP session 

of June 23, 1999, the SCCNFP already stated: “Contact allergy to fragrance substances is an important 

clinical problem. Up to 10% of individuals with eczema are allergic to fragrance substances and 

possibly 1–2% of the general population.” 

In the same Interim position, SCCNFP considered that the mandate from the European Commission 

could be usefully divided into the following two sections: 

1. Identification of those fragrance ingredients that are of concern as allergens for the 

consumer. Recommendations on informing the consumer of the presence of important 

allergens to permit the consumer with a known fragrance allergy as a means to avoid contact 

with an allergen. An opinion as to whether such an identification can be related to 

concentrations present in a product when elicitation levels are known. 

2. An opinion on the adoption of industry-prohibited substances into Annex 2 and adoption 

of industry-restricted substances into Annex 3. Consideration as to whether the concentration 

limits or other restrictions suggested by industry can be supported or need to be 

changed if there is such an inclusion in Annex 22.3. Whether there are additional substances 

that should be subject to inclusion in an annex.

Recent EU Legislation on Flavors and Fragrances 919 

Taking into account the importance and enormity of the mandate, it was concluded that the first 

section should be considered initially. 

As a result, the SCCNFP published, as a follow-up to the Interim position, its opinion 

SCCNFP/0017/98 (adopted by the SCCNFP during the plenary session of December 8, 1999) 

entitled “Fragrance allergy in consumers—A review of the problem: Analysis of the need for appropriate 

consumer information and Identification of consumer allergens.” 

This opinion relates to the first section mentioned above and consists of 

– A critical review of the problem of fragrance allergy in consumers. 

– Identification of those fragrance ingredients that are well recognized as consumer 

allergens. 

– An opinion as to whether such identification can be related to concentrations present in a 

product when elicitation levels are known. 

Allergy to natural ingredients (such as oakmoss) was not addressed in this opinion but was 

analyzed separately (see SCCNFP opinion of October 24, 2000). 

It was the opinion of the SCCNFP that 

– Fragrance ingredients have to be considered as an important cause of contact allergy. 

– Based on criteria restricted to dermatological data reflecting the clinical experience, it was 

possible to identify 24 fragrance ingredients, which correspond to the most frequently 

recognized allergens. Thirteen of these have been reported more frequently; these are 

well-recognized contact allergens in consumers and are thus of most concern; 11 others are 

less well documented. 

In the opinion (SCCNFP/0017/98), two lists were given: a List A with 13 fragrance chemicals, 

which according to existing knowledge, are most frequently reported and well-recognized consumer 

allergens, and a List B with 11 fragrance chemicals, which are less frequently reported and 

thus less documented as consumer allergens. 

Tables 22.1 and 22.2 review the substances of Lists A and B. 

In addition, the SCCNFP stated in its opinion that information should be provided to consumers 

about the known presence in cosmetic products of fragrance ingredients with a well-recognized 

potential to cause contact allergy: “Information regarding these fragrance chemicals should be 

given to consumers if deliberately added to a fragrance formulation either in the form of a chemical 

or as an identified constituent of an ingredient.” 

TABLE 22.1 

List A (SCCNFP/0017/98)—13 Most Frequently Reported Allergens (CAS No.) 

Amyl cinnamal (122-40-7) Amylcinnamyl alcohol (101-85-9) 

Benzyl alcohol (100-51-6) Benzyl salicylate (118-58-1) 

Cinnamyl alcohol (104-54-1) Cinnamal (104-55-2) 

Citral (5392-40-5) Coumarin (91-64-5) 

Eugenol (97-53-0) Geraniol (106-24-1) 

Hydroxycitronellal (107-75-5) Hydroxymethylpentylcyclohexene-carboxaldehyde (HMPCC) (31906-04-4) 

Isoeugenol (97-54-1) 

Note: Substances highlighted in bold are naturally occurring fragrance materials and the other substances are synthetic 

fragrance ingredients that are not known to occur in nature.


TABLE 22.2 

List B (SCCNFP/0017/98)—11 Less Frequently Reported Allergens (CAS No.) 

Anisyl alcohol (105-13-5) Benzyl benzoate (120-51-4) 

Benzyl cinnamate (103-41-3) Citronellol (106-22-9) 

Farnesol (4602-84-0) Hexyl cinnamaldehyde (101-86-0) 

Lilial (80-54-6) d-Limonene (5989-27-5) 

Linalool (78-70-6) Methyl heptine carbonate (111-12-6) 

3-Methyl-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one (=alpha-iso-methylionone) (127-51-5) 

Note: Substances highlighted in bold are naturally occurring fragrance materials and the other substances are synthetic 

fragrance ingredients that are not known to occur in nature. 

Additionally, as mentioned above, also two natural ingredients, oakmoss and tree moss extracts, 

were addressed in a separate SCCNFP opinion (adopted during the 14th plenary meeting of October 24, 

2000). 

These two natural mosses are identified as follows: oakmoss extracts derived from the lichen, 

Evernia prunastri (L.) Arch. (Usneaceae), growing primarily on oak trees, and tree moss extracts 

derived from a mixture of lichens, mainly Evernia furfuracea (L.) Arch. (Usneaceae) growing on 

Pinus species. 

Oakmoss extract has CAS no. 90028-68-5 and EINECS no. 289-861-3. 

Tree moss extract has CAS no. 90028-67-4 and EINECS no. 289-860-8. 

The term “labeling” comes from the EU Commission (DG Enterprise) and whether a fragrance 

ingredient should be labeled or not is a Risk Manager’s decision. In its memorandum of 2001 

(SCCNFP/0450/01), the SCCNFP (being the Risk Assessor) clearly states that “because of the lack 

of dose/elicitation data for these substances, the SCCNFP has been unable to provide recommendations 

on levels above which the information to the consumer would be necessary.” Nevertheless, 

SCCNFP mentions in its memorandum that it is “aware that for practical risk management reasons 

there is a need for threshold levels for the provision of information.” There is a proposal that for 

leave-on products, this threshold level should be 10 ppm in the finished cosmetic product, whereas 

for rinse-off products, the SCCNFP would consider a working level 10 times higher than that 

recommended for leave-on products to be reasonable, being 100 ppm. 

22.2.2 COSMETIC DIRECTIVE AND ITS SEVENTH AMENDMENT 

The EU Commission has implemented the above-mentioned SCCNFP opinions in the 7th Amendment 

of the Cosmetic Directive 76/768/EC (2003/15/EC) by adding the following restrictions [limitations 

and requirements (for labeling)] to 26 fragrance substances in Annex III, Part 1: “The 

presence of the substance must be indicated in the list of ingredients referred to in Article 6(1)(g) 

when its concentration exceeds 0.001% in leave-on products and 0.01% in rinse-off products.” 

However, no further restrictions (such as maximum authorized concentrations in the finished 

cosmetic products), except the labeling requirements were introduced at that time. 

This means that the presence of any of the 26 alleged allergens (sensitizers) must be indicated 

(labeled) in the list of ingredients on the packaging of the finished cosmetic products when its concentration 

exceeds 10 ppm (leave-on products) or 100 ppm (rinse-off products), according to Art. 6.1(g) of 

the Cosmetic Directive, 7th Amendment. 

However, it is important to note here that the Fragrance Industry is self-regulating by issuing the 

International Fragrance Association Code of Practice (IFRA CoP), which is published by the IFRA. 

This CoP consists of Standards (the so-called IFRA Standards) for the fragrance ingredients with 

certain restrictions/limitations and in some cases bans to which the International Fragrance Industry 

should comply. The last amendment of the IFRA CoP is the 44th Amendment. The IFRA CoP and


its 44th Amendment and the IFRA Standards can be found on the homepage of the International 

Fragrance Association: www.ifraorg.org. 

22.2.3 IMPACT ON EXTRACTS AND ESSENTIAL OILS AND AROMATIC NATURAL RAW MATERIALS 

The mandatory labeling requirement for the 26 alleged allergens is irrespective of the source of the 

allergen or the way by which it has been introduced in the final cosmetic product. In other words, 

the presence of these materials above the given threshold has to be declared irrespective of the way 

they are added (as such or as being part of “complex ingredients” such as extracts and essential oils). 

This means that the use of essential oils containing them in formulations may lead to the presence 

of such allergens and the labeling requirement will apply. 

Sixteen of the 24 alleged allergenic substances are naturally occurring (see substances indicated 

in bold in Tables 22.1 and 22.2), the other eight substances are synthetic fragrance ingredients that 

do not occur in nature as far as known. 

The structures of the 16 naturally occurring allergenic substances are depicted in Figures 22.1 

and 22.2. 

The remaining two alleged allergens are aromatic natural raw materials by themselves: oakmoss 

(E. prunastri) and tree moss (E. furfuracea). 

According to the current knowledge of the F&F Industry, these 16 allergens occur in about 180 

natural raw materials (extracts and essential oils) (EFFA CoP, 2007). 

A list of aromatic natural raw materials containing any of the 16 naturally occurring sensitizers 

and their presence (if >0.1%) or concentration can be found in Annex 22.1 to this chapter—this is 

based on earlier internal communication (2004) of the F&F industries related to a former version of 

the EFFA CoP. 

One of the key challenges for the Fragrance Industry is the analysis and identification of the 16 

naturally occurring allergens in the natural raw materials (extracts and essential oils) and fragrance 

compounds (mixtures and preparations). To address this work, the Fragrance Industry has established 

an Analytical Working Group of IFRA where methods of analysis are developed. A recommended 

method of analysis for gas chromatography-mass spectrometry (GC-MS) quantification of 

suspected allergens in fragrance compounds has been published by this group in 2003 (Chaintreau 

et al., 2003). Some further work on the investigation of the GC-MS determination of allergens 

(GC-MS quantification of allergens in fragrances and data treatment strategies and method performances) 

was published more recently by the same group (Chaintreau et al., 2007). 

OH 

OH 

O 

c/t 

O 

Benzyl alcohol Cinnamyl alcohol Cinnamaldehyde 

Citral (neral + geranial) 

OH 

OH 

O 

O 

O 

O 

O 

O 

c/t 

OH 

Eugenol c/t-Isoeugenol Benzyl salicylate Coumarin 

FIGURE 22.1 Structures of the 16 naturally occurring alleged allergenic substances (part 1).


O 

O 

OH 

OH 

O 

Benzyl cinnamate Geraniol Linalool Anisyl alcohol 

O 

OH 

O 

OH 

OH 

Benzyl benzoate Farnesol Citronellol D-Limonene 

FIGURE 22.2 Structures of the 16 naturally occurring alleged allergenic substances (part 2). 

22.2.4 RECENT DATA ON SENSITIZATION TO FRAGRANCES 

Recently a new study on the sensitization to the 26 fragrance ingredients (24 single substances and 

two natural extracts) that have to be labeled according to the European Regulation was published by 

the group of Schnuch et al. (2007). This study was part of the multicenter project Information 

Network of Departments of Dermatology (IVDK) (Schnuch et al., 1997, 2004). The aim was to 

study the frequency of sensitization to these 26 alleged allergenic fragrances, in particular the actual 

frequencies of contact allergy to these 26 fragrances. To test this, the fragrance ingredients were 

patch tested in consecutive, unselected patients (in total 21,325 patients) by the IVDK network 

during a 2-year period, consisting of four periods of 6 months. The number of patients tested with 

each of the fragrance substances ranged from 1658 to 4238. 

The frequency of sensitization was expressed by the proportion of patients reacting allergic (% pos.), 

that is, the number of allergic patients compared to the number of patients tested (n pos./n tested, %) 

and the frequency of allergic reactions was then standardized for age and sex. 

The “allergenic” fragrances were divided into three groups, depending on the frequency of 

sensitization, based on the 95% confidence interval (CI). 

The first group of ingredients with the upper CI > 1.0% could be regarded as important allergens 

and was called Group I. This group includes the two natural extracts, oakmoss and tree moss, and 

the substances HMPCC, hydroxycitronellal, isoeugenol, cinnamic acid, and farnesol. 

Another group of ingredients with an upper CI between 0.5% and 1.0% was found to be clearly 

allergenic but less important in terms of sensitization frequency (Group II). This group comprises 

cinnamic alcohol, citral, citronellol, geraniol, eugenol, coumarin, lilial, amyl-cinnamic alcohol, and 

benzyl cinnamate. 

On the other hand, the third group (Group III) comprises substances that have turned out to be 

(extremely) rare sensitizers in this study, or which in other instances may even be considered as 

nonsensitizers, according to the authors. This group with an upper CI of less than 0.5% contains 

10 materials: benzyl alcohol, linalool, methylheptin carbonate, a-amyl-cinnamic aldehyde, a-hexylcinnamic 

aldehyde, limonene, benzyl salicylate, g-methylionone, benzyl benzoate, and anisyl alcohol. 

It was further concluded that sensitization to allergens of the first group is significantly more frequent 

than sensitization to allergens of the third group. 

Regarding Group III it is also worth noting that some molecules are not allergens as such, but 

only turn into allergens after substantial oxidation, for example, limonene and linalool (Karlberg 

and Dooms-Goossens, 1992; Karlberg et al., 1992; Hagvall and Karlberg, 2006).


It is interesting to note that there is a difference in the classification of the allergens (reported 

frequency) according to the opinion of the SCCP (SCCP/0017/98) and the classification in groups by 

Schnuch et al. For example one substance that is an important allergen according to the study of 

Schnuch (Group I), farnesol, is according to SCCP “less frequently reported.” The same applies to 

two substances of Group II that are according to SCCP “less frequently reported,” namely citronellol 

and benzyl cinnamate. On the other hand, two materials that are according to the study of 

Schnuch (extremely) rare sensitizers (Group III) are according to SCCP “frequently reported,” 

namely benzyl alcohol and benzyl salicylate. A comparison of the classifications is given in 

Table 22.3. Differences in classification according to the two sources are highlighted in bold. 

It is important to focus in some more depth on the allergenic potential of the natural ingredients, 

oakmoss and tree moss. In contrast to oakmoss, which is known to be a potent sensitizer since a long 

time ago, tree moss had not been systematically tested in cosmetic patch test series in the past, and 

the study by Schnuch et al. (2007) is claimed to be the first study in which tree moss was tested in 

a larger population. In this study, tree moss was found to be the most frequent allergen. Earlier study 

reports had already identified atranol and chloroatranol (degradation products of atranorin and chloroatranorin) 

as the most potent allergens (Johansen et al., 2003, 2006). The chemical structures of 

atranol and chloroatranol are depicted in Figure 22.3. 

TABLE 22.3 

Classification of Alleged Allergens According to SC Opinion (Frequently or Less 

Frequently Reported) 

Name 

Tree moss extract 1 

Oakmoss extract 1 

Frequency 

(SCCNFP/0017/98) 

(Except for the Mosses 

(SCCNFP opinion of October 

24, 2000; SCCP 

(SCCP/00847/04)) Group (Schnuch et al., 2007) 

SCCNFP/0202/99 and 

SCCP/00847/04: potent 

SCCNFP/0202/99 and 

SCCP/00847/04: potent 

Frequency of 

Sensitization a 

Group I 2.4 

Group I 2.0 

Isoeugenol Frequently reported Group I 1.1 

Cinnamal Frequently reported Group I 1.0 

Farnesol Less frequently reported Group I 0.9 

Cinnamyl alcohol Frequently reported Group II 0.6 

Citral Frequently reported Group II 0.6 

Citronellol Less frequently reported Group II 0.5 

Eugenol Frequently reported Group II 0.4 

Coumarin Frequently reported Group II 0.4 

Geraniol Frequently reported Group II 0.4 

Benzyl cinnamate Less frequently reported Group II 0.3 

Benzyl alcohol Frequently reported Group III 0.3 

Linalool Less frequently reported Group III 0.2 

Benzyl salicylate Frequently reported Group III 0.1 

d-Limonene Less frequently reported Group III 0.1 

Anisyl alcohol Less frequently reported Group III 0.0 

Benzyl benzoate Less frequently reported Group III 0.0 

Source: SCCNFP opinions versus the publication by Schnuch et al., 2007. 

a 

Frequency of sensitization (%) = n test /n pos. (standardized for age and sex).


Cl 

HO 

OH 

HO 

OH 

Atranol 

O 

O 

Chloroatranol 

FIGURE 22.3 Structures of atranol and chloroatranol, most potent allergens in oakmoss and tree moss. 

This also seems to be in line with the opinion of the SCCP (SCCP/00847/04) on atranol and chloroatranol 

present in natural extracts (e.g., oakmoss and tree moss extract) where both constituents 

were regarded as very potent allergens. Because chloroatranol was shown to cause elicitation of reactions 

by repeated open exposure at the ppm level (0.0005%) and at the ppb level on patch testing (50% 

elicit at 0.000015%), the SCCP concluded that “chloro-atranol and atranol should not be present in 

cosmetic products.” 

As a result, today the Fragrance Industry is producing oakmoss and tree moss with reduced levels 

of atranol and chloroatranol (i.e., oakmoss and tree moss absolutes treated for the selective 

removal of atranol and chloroatranol). 

The authors concluded in their paper that the study again emphasizes the need for a “different 

look” on fragrances as contact allergens, which is a confirmation of previous findings (Schnuch 

et al., 2002). The authors propose a differentiated evaluation of ingredients of each group for overall 

evaluation, considering not only frequency of sensitization, but also the amount of exposure 

or use, as well as allergenic potency—also exposure to (highly) oxidized materials could be taken 

into account. 

In particular, for Group I substances the authors agree that a regulation in terms of restrictions 

(or even ban) and labeling is needed, whereas for Group II substances labeling alone may be adequate 

enough for the purpose of prevention. But according to the authors, for some of the ingredients 

of Group III, neither restrictions nor labeling seems justified. 

Thus, the authors express their opinion, justified by the findings of this study, that the Commission 

Decision on the labeling of all 26 “alleged allergens” should be revised. 

The Fragrance Industry in turn has taken note of this study and comes to the same conclusion. 

Based on this, the Industry would now like to propose a pragmatic and different approach for the 

three Groups of “alleged fragrance ingredients”: for example for Group III materials, Industry 

would advocate no labeling requirements and only restrictions where needed based on scientifically 

justified concern and for Group II and Group I materials, the Industry would propose appropriate 

and adequate measures based on scientific data. The Industry would like to avoid overregulation 

and overlabeling for alleged sensitizers. 

Also the Commission took note of the publication of this study and as a consequence DG 

Enterprise sent a mandate to the SCCP with a request for an updated scientific opinion on the 

fragrance substances hydroxycitronellal (CAS 107-75-5), isoeugenol (CAS 97-54-1), and d-limonene 

(CAS 5989-27-5). 

Currently, the presence of hydroxycitronellal and isoeugenol needs to be labeled in the final cosmetic 

product according to Annex III, Part 1 of the Cosmetic Directive (Entries 72 and 73, respectively). 

However, in the future, restrictions to these fragrance ingredients may be proposed, because the 

Commission is considering a maximum concentration of 1.0% of hydroxycitronellal and of 0.02% 

of cis- and trans-isoeugenol (or their sum) in finished cosmetic products (except oral care products). 

DG Enterprise has asked SCCP its opinion whether they consider these concentrations to be safe for


consumers when used in cosmetic products taking into account the scientific data provided. In fact 

such restrictions would be more in line with the self-regulating policy and principles of the Fragrance 

Industry, as applied through the IFRA CoP and its Standards, as explained above. 

Regarding limonene, DG Enterprise has asked SCCP to re-evaluate the level of peroxides for the 

limonenes in cosmetic products. In parallel, the Fragrance Industry through the Research Institute 

for Fragrance Materials (RIFM) is conducting some local lymph node assay (LLNA) work on 

limonene and some other key materials for a better scientific substantiation of the maximum peroxide 

level. RIFM is planning to test limonene with different (low) levels of peroxide to determine the 

EC3 value (equivalent to the human NOEL). This is a project with the University of Göteburg, 

Sweden (Professor. A.-T. Karlberg). Some of the goals of this research are to investigate the fundamental 

scientific basis of the auto-oxidation of four important structurally related fragrance 

ingredients (e.g., limonene) and one essential oil; to look more closely at the sensitization potential 

of limonene (and hence to challenge the current sensitization hazard classification of R43 of 

limonene, which itself is not a sensitizer, and essential oils rich in limonene such as orange oil); and 

to challenge the sensitization hazard classification of other essential oils, containing another important 

fragrance ingredient, labeled as allergen, namely linalool. This is possible if it can be demonstrated 

that linalool oxidizes differently in an essential oil as compared to the pure compound. 

The impact of hazard classification (e.g., R43 Risk Phrase) of fragrance ingredients on the classification 

of essential oils containing them will be discussed in more detail in the section on Hazard 

Classification and Labeling. 

The impact of allergen labeling requirements on essential oils is very important and a different 

approach of the regulators toward labeling based on the new scientific data available could be very 

high. If for example no further labeling requirements would apply to Group III materials (which 

include the following six naturally occurring substances: benzyl alcohol, linalool, benzyl salicylate, 

d-limonene, anisyl alcohol, and benzyl benzoate), the number of affected natural raw materials 

(extract and essential oils) would be reduced from about 180 to only 80 extracts/essential oils that 

would need to be taken into account for labeling purposes (see Annex 22.2 to this chapter), according 

to the EFFA CoP. This would also have a favorable impact on the analytical burden: much less 

essential oils would have to be analyzed for the presence and concentration of the allergens; also the 

number of target analytes (allergens) would be reduced from 16 to 10 naturally occurring ones. 

The issue on allergen labeling can also have detrimental business impact as some customers 

(clients) of the fragrance industry (being the cosmetic and detergent industry) are requesting 

fragrances (i.e., perfume mixtures and preparations) that are “allergen-free.” This would mean that 

the suppliers of essential oils would need to produce “allergen-free” essential oils and extracts, that 

is, natural materials that do not contain any of the 16 naturally occurring “alleged allergens.” This 

is of course practically impossible. Moreover, generally producing extracts and essential oils without 

or even with reduced levels of the 16 naturally occurring “allergens” (which are very important 

fragrance constituents by themselves), for example, by selectively removing them, would have a 

very high and negative impact on the organoleptic and sensory properties of the essential oils and 

hence on the fine fragrances and perfumes containing them. As mentioned above, only in particular 

cases (e.g., for oakmoss and tree moss extracts, which are of high importance to the perfumer 

but also very potent allergens) the Industry is successfully producing new qualities with reduced 

levels of allergens (in casu atranol and chlorotranol) to reduce the sensitization potential of the 

mosses. It is worthwhile to mention here that a considerable number of IFRA Standards (IFRA 

prohibited materials) are part of the Cosmetics Directive (banned materials under Annex II of the 

Cosmetics Directive). 

22.2.5 FIRST AMENDMENT OF THE DETERGENT REGULATION AND ALLERGEN LABELING 

In line with the 7th Amendment of the Cosmetic Directive 76/768/EEC as just discussed above in 

the previous paragraph, the first amendment of the Detergent Regulation (from June 2006) makes


the labeling of the 26 alleged “allergenic” materials mandatory: the presence of these materials 

above the given threshold has to be declared irrespective of the way they are added (as such or as 

being part of “complex ingredients” such as essential oils). 

This first amendment is the Commission Regulation (EC) No. 907/2006 (20/06/06) amending 

Regulation (EC) No. 648/2004 on detergents. The recital (whereas) (4) of this regulation states the 

following: 

(4) There is a requirement to declare allergenic fragrances if they are added in the form of pure substances. 

However there is no requirement to declare them if they are added as constituents of complex 

ingredients such as essential oils or perfumes. To ensure better transparency to the consumer, allergenic 

fragrances in detergents should be declared irrespective of the way they are added to the detergent. 

The threshold for labeling is defined as 0.01% by weight (100 ppm), according to the adaptation 

of Annex VII (for labeling and ingredient data sheet) as follows: 

If added at concentrations exceeding 0.01% by weight, the allergenic fragrances that appear on the list 

of substances in Annex III, Part 1 to Directive 76/768/EEC, as a result of its amendment by Directive 

2003/15/EC of the European Parliament and of the Council to include the allergenic perfume ingredients 

from the list fi rst established by the Scientifi c Committee on Cosmetics and Non-food Products 

(SCCNFP) in its opinion SCCNFP/0017/98, shall be listed using the nomenclature of that Directive, as 

shall any other allergenic fragrances that are subsequently added to Annex III, Part 1 to Directive 

76/768/EEC by adaptation of that Annex to technical progress. 

This text for Annex VII is written in such a way to ensure that the presence of the 26 alleged 

fragrance materials above the given threshold has to be declared irrespective of the way they are 

added (i.e., as such or as being part of “complex ingredients” such as essential oils). 

In that way according to the first amendment of the Detergent Regulation, the same rules apply 

for detergents as for cosmetic end products for the requirements of allergen labeling. 

22.3 CURRENT FLAVOURING DIRECTIVE AND FUTURE FLAVOURING 

REGULATION: IMPACT ON ESSENTIAL OILS 

In the European Union for flavorings, the current Flavouring Directive 88/388/EC still applies. 

This is the Council Directive of June 22, 1988, on the approximation of the laws of the MS relating 

to flavorings for use in foodstuffs and to source materials for their production, as published in the 

Official Journal on 15/07/88 (OJ L 184, p. 61). It has been amended once by the Commission 

Directive 91/71/EEC of 16/01/91 (OJ L 42, p. 25, 15/02/91). As this is a Directive, it is up to the EU 

MS to take the necessary measures to ensure that flavorings may not be marketed or used if they 

do not comply with the rules laid down in this Directive, as stated in Art. 3 of this Directive. 

However since recent years (around 2002) a Proposal for a new Regulation of the European 

Parliament and of the Council on flavorings and certain food ingredients with flavoring properties 

for use in and on foods is under discussion. The last version of the Commission Proposal that was 

the basis for further discussions and Amendments from EU Parliament was issued in July 2006. 

As many essential oils and extracts either contain flavoring substances or are regarded as “food 

ingredients with flavoring properties,” this new Flavouring Regulation will have an impact on essential 

oils and their use as flavoring ingredients for food products. Extracts and essential oils contain 

certain constituents (substances) that according to this regulation “should not be added as such to 

food” or to which maximum levels apply. They are often referred to as “biologically active 

substances” or “active principles.” Especially the application of maximum levels of these substances 

will have an impact on how and when extracts, essential oils but also herbs and spices may or can 

be applied to food. Also the definitions for “natural” have drastically changed. The difference


between the current Directive 88/388/EC and the future Flavour Regulation will be outlined in the 

next paragraphs. 

22.3.1 CURRENT FLAVOURING DIRECTIVE 88/388/EC 

22.3.1.1 Maximum Levels of “Biologically Active Substances” 

In the current Flavouring Directive 88/388/EC, Annex II sets maximum levels (limits) for certain 

substances obtained from flavorings and other food ingredients with flavoring properties in foodstuffs 

as consumed in which flavorings have been used. Art. 4 (c) stipulates that 

(c) the use of fl avourings and of other food ingredients with fl avouring properties does not result in the 

presence of substances listed in Annex II in quantities greater than those specifi ed therein. 

The limits apply to Foodstuffs and Beverages (mg/kg) — exceptions apply: for example, alcoholic 

beverages and confectionaries. In Table 22.4, the maximum levels (without the exceptions) for 

these substances for foodstuffs in general and beverages are given. A more detailed table (with all 

the exceptions) is given in Annex 22.3 to this chapter. 

This means that for essential oils, extracts, complex mixtures containing these “biologically 

active substances” (e.g., nutmeg, cinnamon, peppermint, and sage oils) and when added to food and 

flavorings, maximum levels apply. The same applies to herbs and spices containing these “biologically 

active substances” as herbs and spices are also “food ingredients with flavoring properties.” 

22.3.1.2 Definition of “Natural” 

Also important is how the current Flavouring Directive addresses “naturalness” of flavors and how 

“natural” is defined for the purpose of labeling. This is stipulated by Art. 9a.2 (amending the original 

Art. 9.2 of 88/388/EC by 91/71/EEC): 

2. the word ‘natural’, or any other word having substantially the same meaning, may be used only for 

fl avourings in which the fl avouring component contains exclusively fl avouring substances as defi ned 

in Article 1 (2) (b) (i) and/or fl avouring preparations as defi ned in Article 1 (2) (c). If the sales description 

of the fl avourings contains a reference to a foodstuff or a fl avouring source, the word ‘natural’ or 

any other word having substantially the same meaning, may not be used unless the fl avouring component 

has been isolated by appropriate physical processes, enzymatic or microbiological processes 

or traditional food-preparation processes solely or almost solely from the foodstuff or the fl avouring 

source concerned. 

TABLE 22.4 

Annex II of 88/388/EC—Maximum Levels (mg/kg) for Certain 

Substances in Foodstuffs and Beverages 

Substance 

Foodstuffs and 

Beverage Substance Foodstuffs Beverages 

Agaric acid 20 Aloin 0.1 0.1 

b-Asarone 0.1 Berberine 0.1 0.1 

Coumarin 2 Hydrocyanic acid 1 1 

Hypericine 0.1 Pulegone 25 100 

Quassine 5 Safrole and isosafrole 1 1 

Santonin 0.1 Thujone (a and b) 0.5 0.5


How a “natural flavoring substance” can be obtained is thus defined in Art. 1.2 (b) (i): 

(b) ‘fl avouring substance’ means a defi ned chemical substance with fl avouring properties which is 

obtained: 

(i) by appropriate physical processes (including distillation and solvent extraction) or enzymatic or 

microbiological processes from material of vegetable or animal origin either in the raw state or after 

processing for human consumption by traditional food-preparation processes (including drying, 

torrefaction and fermentation), 

How a “flavoring preparation” can be obtained is defined in Art. 1.2 (c): 

(c) ‘fl avouring preparation’ means a product, other than the substances defi ned in (b) (i), whether concentrated 

or not, with fl avouring properties, which is obtained by appropriate physical processes 

(including distillation and solvent extraction) or by enzymatic or microbiological processes from material 

of vegetable or animal origin, either in the raw state or after processing for human consumption 

by traditional food-preparation processes (including drying, torrefaction and fermentation); 

The above means that a “flavoring preparation” is by default always “natural” and that extracts 

and essential oils (obtained by appropriate physical processes such as distillation and solvent extraction) 

from material of vegetable origin (e.g., plant material) can be considered as “flavoring preparation” 

and thus “natural.” 

22.3.2 FUTURE FLAVOURING REGULATION 

As mentioned above, a Proposal for a new Flavouring Regulation is under discussion since the last 

years at three levels: the EU Commission, the EU Parliament, and the Council (MS-level). The full 

title of this proposal is Proposal for a Regulation of the European Parliament and of the Council 

on fl avourings and certain food ingredients with fl avouring properties for use in foods and amending 

Council Regulation (EEC) No. 1576/89, Council Regulation (EEC) No. 1601/91, Regulation 

(EC) No. 2232/96 and Directive 2000/13/EC. The last (amended) proposal from the Commission 

dates from 24/10/2007 (Commission Directive 91/71/EEC). 

With this new Regulation, the former Council Directive 88/388/EEC of June 22, 1988, as well as 

its amendment Directive 91/71/EEC and the Commission Decision 88/389/EEC will be repealed. 

This Flavouring Regulation is part of a larger package, called the “Food Improvement Agents 

Package” (FIAP), comprising the Flavouring, Additives and Enzymes Regulation and the Common 

Authorisation Procedure. The drafting of the entire package started at Commission level, has 

undergone a tremendous amount of amendments, as issued and adopted by the European 

Parliament, and was at the time of the preparation of the manuscript for this chapter under discussion 

with three parties: the EU Commission, the EU Parliament, and the Council under Portuguese 

Presidency. The last chance for the parties to come to a political agreement and to reach a common 

position under the first Reading was in December 2007. However, a common position could not be 

reached by the end of 2007 and there was a second Reading (Plenary session) in July 2008: under 

Slovenian Presidency. 

For a long time (at the time of the preparation of the manuscript of this chapter) there was not one 

final document but two major draft versions: the last amended Commission Proposal of 24/10/2007 

(Commission Directive 91/71/EEC) and the last Council Proposal for Political Agreement of 

10/12/2007 (Commission Directive 93/21/EEC), which were the basis for discussion for the Council 

meeting (Agriculture and Fisheries) on December 17–18, 2007. The major differences between the 

two versions (Commission Proposal and Council Proposal) available at the end of 2007 (at the time 

of the preparation of this manuscript), and the impact on essential oils will be outlined below. As a 

final Proposal of the European Parliament and the Council had just come available shortly before 

submission of this manuscript for publication (Council Proposal, July 15, 2008), also this final


Council Proposal will be discussed briefly, in order to be as much as possible up-to-date. Meanwhile 

at the time of the publication of this book, the final version of the new Flavouring Regulation has 

been published in the Official Journal on 31 December 2008 (OJ L 354, 31.12.2008, p. 34): Regulation 

(EC) No 1334/2008. In essence this Regulation as published is the same as the final Council Proposal 

which was published on July 15, 2008. It has entered into force on January 20, 2009 and will apply 

as from January 20, 2011. As of this application date, the current Flavouring Directive 88/388/EEC 

will be repealed. 

22.3.2.1 Maximum Levels of “Biologically Active Substances” 

Apart from the fact that the current Directive 88/388/EC will turn into a Regulation, there are many 

changes that will have an impact on how essential oils and extracts will be used as source of flavors. 

The most important issue is how the so-called biologically active substances are addressed. 

This is addressed by Art. 5 of the Draft Council Proposal (Art. 6 of Commission Proposal): 

“Presence of certain substances,” which refers to Annex III with the same title. Both Council and 

Commission proposals clearly state in the first paragraph that “Substances listed in Part A of Annex 

III shall not be added as such to food.” 

However, when it comes to the levels of these substances coming from the use of flavorings and 

food ingredients with flavoring properties (such as extracts, essential oils, herbs, and spices), the 

Commission and Council proposals differ slightly. 

Art. 6.2 in the Commission Proposal reads as follows: 

2. Maximum levels of certain substances, naturally present in fl avourings and food ingredients with 

fl avouring properties, in the compound foods listed in Part B of Annex III shall not be exceeded as a 

result of the use of fl avourings and food ingredients with fl avouring properties in and on those foods. 

The maximum levels shall apply to the compound foods as offered ready for consumption or as 

prepared according to the instructions of the manufacturer. 

Art. 5.2 in the Council Proposal (December 10, 2007) reads as follows: 

2. Without prejudice to Council Regulation No. 1576/89 maximum levels of certain substances, naturally 

present in fl avourings and/or food ingredients with fl avouring properties, in the compound foods 

listed in Part B of Annex III shall not be exceeded as a result of the use of fl avourings and/or food 

ingredients with fl avouring properties in and on those foods. 

The maximum levels of the substances set out in Annex III apply to foods as marketed, unless 

otherwise stated. By way of derogation from this principle, for dried and/or concentrated foods which 

need to be reconstituted the maximum levels apply to the food as reconstituted according to the instructions 

on the label, taking into account the minimum dilution factor. 

The wording in the latest Council Proposal of July 15 (Art. 6) is essentially the same as the wording 

of the Council Proposal of December 10 (Art. 5). 

This means that maximum levels of these substances also apply when the substances come from 

any type of food ingredients with flavoring properties; the only difference between the Commission 

Proposal and the Council proposals is that in the Council proposals an exception is given to dried 

and/or concentrated foods that can have higher levels before they are diluted/reconstituted. Upon 

dilution/reconstitution, the normal maximum levels apply again. 

The main difference between the current Flavouring Directive 88/388 and the future Flavouring 

Regulation is that in the Directive 88/388 there is only one list (Annex II) of substances to which the 

maximum levels apply—all those substances may not be added as such to food. In contrast, in the 

future Flavouring Regulation, the Annex III is split into two parts: Part A with “Substances which 

may not be added as such to food” and Part B establishing “Maximum levels of certain substances, 

naturally present in flavourings and food ingredients with flavouring properties, in certain compound 

food as consumed to which flavourings and/or food ingredients with flavouring properties 

have been added.”


TABLE 22.5 

Annex III, Part A: Substances that May Not be Added As Such to Food 

Agaric Acid Aloin Capsaicin 

1,2-Benzopyrone, coumarin Hypericine b-Asarone 

1-Allyl-4-methoxybenzene, estragole Hydrocyanic acid Menthofuran 

4-Allyl-1,2-dimethoxybenzene, methyleugenol Pulegone Quassin 

1-Allyl-3,4-methylene dioxy benzene, safrole Teucrin A Thujone (a and b) 

Note: Substances in bold are those that are in Part A, Annex III of both the Council and Commission 

proposals—aloin and coumarin are not included in Annex III, Part A of the Commission proposal. 

Part A contains 15 substances (according to the Council proposals) or 13 substances (according 

to the Commission Proposal—aloin and coumarin are not in), whereas Part B contains 11 substances 

(according to the Council proposals) or 10 substances (according to the Commission Proposal— 

coumarin is not in). 

Table 22.5 lists the Substances of Part A of Annex III “which may not be added as such to food” 

and Table 22.6 lists the 11 substances of Part B with their respectively maximum levels in the various 

compound foods according to the Council proposals. 

There are some major differences between the Part B of Annex III in the Council proposals and 

the list in the Commission Proposal: 

– As mentioned above, maximum levels for coumarin are only set in the Council proposals 

and not in the Commission Proposal. 

– For Teucrin A different levels are set in the Council proposals for different compound 

foods, whereas in the Commission Proposal only for one category, namely alcoholic 

beverages, a maximum level of 2 mg/kg applies. 

– Regarding the chemical names, in the Commission Proposal no trivial name is given for 

1-allyl-4-methoxybenzene (estragol) and 4-allyl-1,2-dimethoxy-benzene (methyleugenol) 

in contrast to the Council Proposal (synonyms given). 

– But the most important and major difference is the statement in the Council Proposal of 

December 10 on top of the table, which is not in the Commission Proposal, which reads as 

follows: 

“These maximum levels shall not apply to compound foods which are prepared and consumed 

on the same site, contain no added flavourings and contain only herbs and spices as food ingredients 

with flavouring properties.” 

This statement is to allow the unrestricted use of herbs and spices to foods that are prepared and 

consumed on the same site, for example, restaurants and catering services. 

However, in the latest Council Proposal of July 15, this statement has disappeared. 

Instead another footnote has been introduced applying to three of the substances that are marked 

with an asterisk (*): estragol, safrole, and methyl eugenol. This footnote reads as follows (Council 

Proposal, July 15): 

*The maximum levels shall not apply where a compound food contains no added fl avourings and the 

only food ingredients with fl avouring properties which have been added are fresh, dried or frozen 

herbs and spices. After consultation with the Member States and the Authority, based on data made 

available by the Member States and on the newest scientifi c information, and taking into account the


TABLE 22.6 

Maximum Levels of Certain Substances, Naturally Present in Flavorings and Food 

Ingredients with Flavoring Properties, in Certain Compound Foods Consumed to which 

Flavorings and/or Food Ingredients with Flavoring Properties have been Added 

Name of the Substance 

Compound Food in which the Presence of the 

Substance is Restricted 

Maximum Level 

(mg/kg) 

b-Asarone Alcoholic beverages 1.0 

1-Allyl-4-methoxybenzene, Dairy products 50 

estragol 

Processed fruits, vegetables (including mushrooms, fungi, 

50 

roots, tubers, pulses, and legumes), nuts, and seeds 

Fish products 50 

Nonalcoholic beverages 10 

Hydrocyanic acid Nougat, marzipan, or its substitutes or similar products 50 

Canned stone fruits 5 

Alcoholic beverages 35 


Mint/peppermint containing confectionery, except micro breath 500 

freshening confectionery 

Micro breath freshening confectionery 3000 

Chewing gum 1000 

Mint/peppermint containing alcoholic beverages 200 

4-Allyl-1,2-dimethoxy-benzene, Dairy products 20 

methyleugenol 

Meat preparations and meat products, including poultry 

15 

and game 

Fish preparations and fish products 10 

Soups and sauces 60 

Ready-to-eat savouries 20 


Pulegone 

Mint/peppermint containing confectionery, except 

250 

micro breath freshening confectionery 

Micro breath freshening confectionery 2000 

Chewing gum 350 

Mint/peppermint containing nonalcoholic beverages 20 

Mint/peppermint containing alcoholic beverages 100 

Quassin Nonalcoholic beverages 0.5 

Bakery wares 1 

Alcoholic beverages 1.5 

1-Allyl-3,4-methylene dioxy Meat preparations and meat products, including poultry 

15 

benzene, safrole 

and game 

Fish preparations and fish products 15 

Soups and sauces 25 


Teucrin A Bitter-tasting spirit drinks or bitter a 5 

Liqueurs b with a bitter taste 5 

Other alcoholic beverages 2 

Thujone (a and b) 

Alcoholic beverages, except those produced from 

10 

Artemisia species 

Alcoholic beverages produced from Artemisia species 35 

Nonalcoholic beverages produced from Artemisia species 0.5 

continued



Maximum Levels of Certain Substances, Naturally Present in Flavorings and Food 

Ingredients with Flavoring Properties, in Certain Compound Foods Consumed to which 

Flavorings and/or Food Ingredients with Flavoring Properties have been Added 

Name of the Substance 

Compound Food in which the Presence of the 

Substance is Restricted 

Maximum Level 

(mg/kg) 

Coumarin 

Traditional and/or seasonal bakery ware containing 

50 

cinnamon in the labeling 

“Breakfast cereals” including muesli 20 

Fine bakery ware with exception of traditional and/or 

15 

seasonal bakery ware containing cinnamon in the labeling 

Desserts 5 

a 

As defined by Article 1.4 (p) of EC Regulation 1576/89. 

b 

As defined by Article 1.4 (r) of EC Regulation 1576/89. 

use of herbs and spices and natural fl avouring preparations, the Commission, if appropriate, proposes 

amendments to this derogation. 

This means that the maximum levels do not apply to estragol, safrole, and methyl eugenol when 

only fresh, dried, or frozen herbs and spices are added! However when “food ingredients with flavoring 

properties” such as essential oils are added, or when essential oils and/or other flavorings are 

added in combination with herbs and spices, the levels do apply. 

It is anticipated that nothing will change anymore in Art. 6 of the Council Proposal of July 15 

until the adoption of the final text and that also the Annex III will remain as it is now. However, as 

stipulated in the footnote under the Annex III (applying to the three substances with an asterisk) 

amendments to the current derogations for herbs and spices can be expected. 

It is also important to note that according to Art. 30 of the Flavouring Regulation (Entry into 

Force), which will only apply 24 months after its Entry into Force, Art. 22 shall apply from the date 

of its Entry into Force. Art. 22 concerns the Amendments to Annexes II through V. This means that 

if the Flavouring Regulation would be adopted by the end of 2008 and hence would apply end of 

2010, the Annexes can be amended immediately, if necessary. 

Whereas the Art. 6 of the Council Proposal of July 15 discussed above (i.e., Art. 6 of the 

Commission Proposal) relates to “certain substances,” Art. 7 of the Council Proposal of July 15 

(i.e., Art. 7 of the Commission Proposal) relates to “Use of certain source materials,” which is 

even more important in relation to herbs, spices, extracts, and essential oils. This article refers to 

Annex IV of the Regulation, which is a new annex that was not in the current Flavouring Directive 

88/388/EC entitled “List of source materials to which restrictions apply for their use in the production 

of flavourings and food ingredients with flavouring properties.” This Annex IV consists 

of two parts: 

– Part A: Source materials that shall not be used for the production of flavorings and food 

ingredients with flavoring properties. 

– Part B: Conditions of use for flavorings and food ingredients with flavoring properties 

produced from certain source materials. 

The complete Annex IV according to the current Draft proposals (there is no difference between 

the Draft Council and Commission proposals regarding the content of Annex IV) is given in 

Annex 22.4 to this chapter.


Art. 7 of the Council Proposal of July 15 (Art. 7 of the Commission Proposal) stipulates the 

following: 

1. Source materials listed in Part A of Annex IV shall not be used for the production of 

fl avourings and/or food ingredients with fl avouring properties. 

2. Flavourings and/or food ingredients with fl avouring properties produced from source 

materials listed in Part B of Annex IV may only be used under the conditions indicated in 

that Annex. 

With the exception of the fact that “and/or” in “flavourings and/or food ingredients” in both 

paragraphs in the Council Proposal is replaced by “and” (“flavourings and food ingredients”) in the 

Commission Proposal, the remainder of the article is exactly the same. It is anticipated that nothing 

will change anymore on this article and the related Annex IV for the final version of the Flavouring 

Regulation. 

22.3.2.2 Definition of “Natural” 

Regarding “naturalness” of flavors and how “natural” is defined for the purpose of labeling, the situation 

has drastically changed since the current Flavouring Directive 88/388/EC. 

For example today according to 88/388/EC there are three categories of flavoring substances: 

natural, nature-identical (NI), and artificial. However, with the new Flavouring Regulation there 

will be only two categories: natural and not natural, meaning that the difference between the former 

categories NI and artificial will disappear and these two will merge in one category of “synthetic 

flavorings.” 

Also the position of the Council is clearly different from the position of the Commission. In both 

proposals, “natural flavoring substance” is defined by Art. 3.2 (c). 

This definition according to the Commission proposals (December 10 and July 15) reads as 

follows: 

(c) ‘natural fl avouring substance’ shall mean a fl avouring substance obtained by appropriate physical, 

enzymatic or microbiological processes from material of vegetable, animal or microbiological origin 

either in the raw state or after processing for human consumption by one or more of the traditional 

food preparation processes listed in Annex II. 

This definition according to the Council Proposal reads as follows: 

(c)‘natural fl avouring substance’ shall mean a fl avouring substance obtained by appropriate physical, 

enzymatic or microbiological processes from material of vegetable, animal or microbiological origin 

either in the raw state or after processing for human consumption by one or more of the traditional 

food preparation processes listed in Annex II; Natural fl avouring substances correspond to substances 

that are naturally present and have been identifi ed in nature; 

Important is the additional line, according to the Council proposals, stating that a substance has 

to be identified in nature before it can be regarded as “natural,” so it is not only sufficient to produce 

it “in a natural way”: it has to be identical to something that is present in nature. This is to avoid the 

problem that arises when an enzymatic or microbial process is developed by which a flavoring substance 

can be produced “by enzymatic or microbial processes from material of vegetable origin” (i.e., natural 

source materials) that up to then has never been identified in nature (and is not naturally occurring), 

such as ethylvanillin, then such a material would be labeled as a “natural flavoring substance.” 

More important than the definition of “natural” as such (Art. 3.2 (c)), however, is how “appropriate 

physical process” is defined. 

Annex II to the Flavouring Regulation gives a list of “traditional food preparation processes” by 

which (natural) flavoring substances and natural flavoring preparations are obtained (in the title of


Annex II in the Commission Proposal the wording “natural flavouring preparations” is used, which 

is confusing since flavoring preparations are, as per definition, natural, see below). The full list of 

traditional food preparation processes is given in Annex 22.5 to this chapter. 

The definition of “appropriate physical process” is different between the two proposals and is 

described in Art. 3.2 (k). 

This definition according to the Commission Proposal reads as follows: 

(k) ‘appropriate physical process’ shall mean a physical process which does not intentionally modify 

the chemical nature of the components of the fl avouring, without prejudice to the listing of traditional 

food preparation processes in Annex II, and does not involve the use of singlet oxygen, ozone, inorganic 

catalysts, metal catalysts, organometallic reagents and/or UV radiation. 

This definition according to the Council Proposal (December 10, 2007) reads as follows: 

(k) ‘appropriate physical process’ shall mean a physical process which does not intentionally modify the 

chemical nature of the components of the fl avouring and does not involve among others the use of singlet 

oxygen, ozone, inorganic catalysts, metal catalysts, organometallic reagents and/or UV radiation. 

It can be noted that the definition according to the Council Proposal does no longer refer to the 

processes listed in Annex II. In contrast to the Commission Proposal where all traditional food preparation 

processes as listed in Annex II also fall under the definition of “appropriate physical processes” 

(cf. the wording “without prejudice to the listing of º ”), according to the Council Proposal only 

processes that do not intentionally modify the chemical nature of the components of the flavoring are 

considered to be “appropriate physical processes” in order to obtain a “natural flavouring substance.” 

This means that distillation and certain extraction techniques that do modify the chemical nature 

of the components are not regarded as “appropriate physical processes” for obtaining natural flavoring 

substances. 

Fortunately thanks to very strong and effective, successful lobbying by the European Flavour 

Industry, this has been rectified and an amendment to this definition (Art. 3.2 (k)) has been accepted 

by Council, Commission, and European Parliament. 

According to the latest Council Proposal (July 15, 2008), this definition reads as follows: 

(k) “appropriate physical process” shall mean a physical process which does not intentionally modify 

the chemical nature of the components of the fl avouring, without prejudice to the listing of traditional 

food preparation processes in Annex II, and does not involve, inter alia, the use of singlet oxygen, 

ozone, inorganic catalysts, metal catalysts, organometallic reagents and/or UV radiation. 

This definition again refers to the Annex II, which means that all processes listed in Annex II 

also fall again under the definition of “appropriate physical processes.” 

When looking at the situation for “flavouring preparations” (such as essential oils and extracts), 

the wording of the Commission Proposal is slightly different than that of the Council Proposal. 

According to Art. 16.2 of the Council Proposal (July 15) and Art. 15.2 of the Commission Proposal, 

the term “natural” may be used for the description of a flavoring if the flavoring component comprises 

only flavoring preparations, which means that a “flavoring preparation” can be regarded as 

“natural” by definition. In other words, there is no such thing as a “synthetic flavoring preparation.” 

The definition for “flavouring preparation” according to the Commission Proposal reads as follows 

(Art. 3.2 (d)): 

(d) ‘fl avouring preparation’ shall mean a product, other than a fl avouring substance, obtained from: 

(i) food by appropriate physical, enzymatic or microbiological processes either in the raw state 

of the material or after processing for human consumption by one or more of the traditional 

food preparation processes listed in Annex II and/or appropriate physical processes;


and/or 

(ii) material of vegetable, animal or microbiological origin, other than food, obtained by one or 

more of the traditional food preparation processes listed in Annex II and/or appropriate physical, 

enzymatic or microbiological processes; 

The definition for “flavouring preparation” according to the Council proposals (versions 

December 10 and July 15 being identical) reads as follows (Art. 3.2(d)): 

(d) ‘fl avouring preparation’ shall mean a product, other than a fl avouring substance, obtained from: 

and/or 

(i) food by appropriate physical, enzymatic or microbiological processes either in the raw state 

of the material or after processing for human consumption by one or more of the traditional 

food preparation processes listed in Annex II; 

(ii) material of vegetable, animal or microbiological origin, other than food, by appropriate 

physical, enzymatic or microbiological processes, the material being taken as such or prepared 

by one or more of the traditional food preparation processes listed in Annex II; 

Although the wording of Commission and Council differ on this definition, it could be concluded 

that according to the latter definition (according to Council Proposal) essential oils and extracts 

obtained from plant material (material of vegetable origin) prepared by distillation (which is a traditional 

food preparation process listed in Annex II) followed by an appropriate physical process 

can be considered as a “flavoring preparation” and thus natural as long as the chemical nature of the 

components is not intentionally modified during the physical process. 

However, it can also be argued that if the physical process after the distillation (e.g., extraction, 

drying, evaporation, condensation, dilution, etc.) intentionally modifies the chemistry of the components 

(which is often the case), then the end product can no longer be regarded as fl a vor i ng prep a ra - 

tion and thus natural, according to the Council Proposal. 

In that respect, the wording of the Commission Proposal (part (ii)) is much broader and less 

ambiguous and will not lead to these restrictions. According to the Commission Proposal, an extract, 

essential oil, absolute or concrete, … obtained from material of vegetable origin (flower, root, fruit, 

etc.) by one or more of the processes listed in Annex II complies with the definition of “flavoring 

preparation” and can thus be regarded as a “natural.” 

However, it is anticipated that the latest version, being the Council Proposal of July 15, 2008, 

is the blueprint of the final Flavouring Regulation, and will be the text as it will most probably be 

adopted by the end of the year and published. Entry into force of the new EU Flavouring Regulation 

will be on the twentieth day following that of its publication in the Official Journal of the European 

Union. It is anticipated that this will be at the earliest by the end of 2008 or the beginning of 2009. 

As stated above, it shall apply 24 months after the entry into force of this Regulation. 

22.4 HAZARD CLASSIFICATION AND LABELING OF FLAVORS 

AND FRAGRANCES 

This section describes the rules for hazard classification and labeling of F&F substances and preparations, 

including natural raw materials, such as extracts and essential oils, containing hazardous 

constituents, according to the EU regulations. 

For trade of F&F (including pure substances and mixtures or preparations thereof and natural 

raw materials) within the European Union, certain rules apply within the European Industry which 

are established by the European Flavour and Fragrance Association Code of Practice (EFFA CoP). 

The following general considerations are taken over from the Introductory note to the EFFA CoP, 

which is published yearly on the EFFA website: www.effa.be. It should be noted that the most recent


version of the EFFA CoP (version of 2009) for the first time also takes into account the Globally 

Harmonized System of Classification and Labeling of Chemicals (UN-GHS). This GHS has now 

also been implemented in the EU with the Publication of the EU-GHS Regulation (so-called “CLP 

Regulation”: Classification, Labelling and Packaging of substances and mixtures) [Regulation (EC) 

No. 1336/2008, OJ L 354, 31.12.2008, p. 60]. It has entered into force on 20 January 2009 and the 

current Directive 67/548/EEC (DSD) and Directive 1999/45/EC (DPD) shall be repealed with effect 

from 1 June 2015. However, Annex I of the DSD has already been repealed and transferred into 

Annex VI of the EU-CLP Regulation, with exception of the last two technical adaptations (ATP 30 

and ATP 31) to the DSD. 

However, the following section on classification on labeling is still based on the currently applicable 

DSD and DPD. 

Within the European Union, substances and preparations have to be classified and, if dangerous 

according to criteria laid down in the regulations, have to be labeled according to certain rules. The 

classification and labeling of substances are either prescribed in Annex I to the Dangerous Substances 

Directive 67/548/EEC (DSD) or have to be done by the supplier using the criteria of Annex VI of 

this Directive. For preparations, like F&F compounds, it is done according to the Dangerous 

Preparations Directive 1999/45/EC (DPD). 

Several substances of interest to the fragrance and flavor industry are mentioned in Annex I of 

the DSD. They are included in the respective attachments to the EFFA CoP with their Annex I 

number next to their CAS and EU numbers. The label mentioned in the attachment has to be used 

in the MS of the European Union. 

Special emphasis is put on Classification of aspiration hazard (Xn; R65) of both substances that 

can easily reach the lungs upon ingestion and cause lung damage (substances with low viscosity and 

low surface tension) and mixtures/preparations with a high hydrocarbon (HC) content and low kinematic 

viscosity that will pose the same hazard. 

Based on measurement results for a number of natural raw materials (e.g., extracts and essential 

oils) with HC contents between 10% and 90+% and on similar measurements of some F&F 

compounds, a dedicated Working Group of the F&F Industry has come to the conclusion that in 

practice, substances and preparations containing more than 10% of HC(s) fall within the criteria for 

viscosity and surface tension. 

Therefore, the European F&F Industry through its EFFA CoP recommends 

• To determine the HC content of substances (supplier information, analysis) and preparations 

(including extracts and essential oils) (calculation) and to classify as Xn; R65 if more 

than 10% HC is present. 

• That nonclassification should only be possible if viscosity and/or surface tension measurement 

results are available for a specific substance or preparation (including extracts and 

essential oils). 

In addition, classification and labeling of skin sensitizers is addressed in the CoP: the issue on 

skin sensitization (and labeling of the alleged allergens for the purpose of the Cosmetic Directive, 

7th Amendment) has been in depth discussed in the first section. However, it should be noted here 

that classification of substances and essential oils or extracts as sensitizers (R43) has nothing to do 

with the requirement to label the 26 alleged allergens on the final cosmetic products according to 

the Cosmetic Directive (7th Amendment). 

Following the EFFA CoP, skin sensitizers are labeled Xi, R43. According to the CoP, it is recommended 

to use the administrative limit concentration of 1% when classifying preparations (including 

extracts and essential oils) containing them in all cases, unless a different threshold is laid down 

in Annex I to the DSD. 

In the EFFA CoP, special attention is paid to the hazard classification and labeling of natural raw 

materials, referred to as “natural complex substances” (NCSs) in the CoP. The terminology Natural


Complex Substance is used because in some cases the natural raw material (the complex) is regarded 

as a single substance, rather than a complex mixture. 

NCSs (e.g., essential oils, and extracts from botanical and animal sources) require special procedures 

due to the fact that they might have quite different chemical compositions (and therefore 

hazard classifications) under the same designation. This may occur even when this differentiates 

between species, cultivars and chemotypes and different production procedures (e.g., absolutes, 

resinoids, and distilled oils). 

There are two ways of classifying and labeling NCSs such as extracts and essential oils: either 

based on the data known and available on the natural raw material as such (NCS is regarded as a 

single “substance”) or based on the hazardous constituents they are composed of (NCS is regarded 

as a complex mixture). 

In the first case, an NCS may be classified on the basis of the data obtained by testing the NCS. 

The test results of an NCS, even if containing classified constituents, are evaluated in accordance 

with the DSD. The health and environmental hazard classifications derived following this approach 

are quality dependent, which is also indicated in the EFFA CoP. 

In the second case, for grades of NCSs and for endpoints for which reliable test data are lacking, 

the EU’s Labelling Guide (Annex VI to the DSD) incorporates a requirement introduced by 

Commission Directive 93/21/EEC, whereby the hazard classification of complex substances shall 

be evaluated on the basis of levels of their known chemical constituents. Where knowledge about 

constituents exists, for example, on substances limited as per Annex II of Directive 88/388/EC 

(the so-called biologically active substances—see above) or on substances with sensitizing, toxic, 

harmful, corrosive, and environmentally hazardous properties, the classification and labeling of 

these NCSs according to the requirements of the European Union should follow the rules for preparations 

(= mixtures) as prescribed by the DPD. 

One dedicated section of the EFFA CoP also provides a list with the composition of the NCSs 

(extracts, essential oils, concretes, absolutes, etc.) in terms of the presence (content in %) of hazardous 

constituents and HCs in the NCSs that have to be taken into account for the classification and 

labeling of the NCSs or a preparation containing these NCSs, based on the DPD. 

F&F compounds that are preparations (i.e., compounded mixtures, formulations, or compositions) 

should be classified and labeled according to the EU’s DPD 1999/45/EC and its articles 

6 and 7. 

In practice, test data on the flavor or fragrance compounds (preparations) are not available or 

collected. Therefore the classification of these preparations should be based on the chemical 

composition and should include the contributions of hazardous substances present as constituents in 

the NCSs present in the formulation. This is another reason why the composition of the NCSs in 

terms of presence of the hazardous constituents is also part of the EFFA CoP. 

Examples of important constituents to take into account for classifi cation: 

– Sensitizers (R43) Æ NCSs (essential oils and extracts) to be classified as R43 if the content 

(%) of the sensitizer (if one) or the content of their sum (if more than one) is greater than or 

equal to 1%. 

– CMRs (carcinogenic, mutagenic, and reprotoxic materials: R45; R46; R68) Æ NCSs 

to be classified as CMR if the content of the CMR substance(s) is greater than or equal 

to 0.1%. 

The final classification and labeling of an essential oil can be totally different depending on the 

approach used for the classification, either based on data on the essential oil as such (the first case 

described above) or based on the hazardous constituents in the essential oil (the second case 

described above). This is illustrated below with two examples: orange oil, containing mainly 

limonene [which is classified as both a sensitizer (R43) and very toxic for the environment (R50/53)], 

and nutmeg oil, containing safrole which is a CMR (T; R45-22-68).


TABLE 22.7 

Composition of Orange Oil with Main Hazardous Constituents 

Constituent Concentration (%) Classification 

d-Limonene 96.2 R10-38-43-50/53 

Linalool 0.5 Not classified 

Citral 0.2 R38-43 

Orange oil: Table 22.7 below depicts the composition of orange oil with the classification of the 

main constituents. 

Resulting classification of orange oil: 

• Classification and labeling “as a single substance” (based on data on the oil as such): 

Xn 

R10 

R65 

Harmful 

Flammable 

Harmful: may cause lung damage if swallowed 

• Classification based on its constituents: 

R10 

Flammable 

R38 

Irritating to skin 

R43 

May cause sensitization by skin contact 

R50/53 Very toxic to aquatic organisms (environment) 

R65 


• Labeling (pictograms) based on its constituents: 

Xn: 

Harmful 

N: dangerous for the environment 

Nutmeg oil: Table 22.8 below depicts the composition of nutmeg oil with the classification of the 

main constituents. 

Resulting classification of nutmeg oil: 

• Classification and labeling “as a single substance” (based on data on the oil as such): 

Xn 

R10 

R65 

Harmful 

Flammable 


TABLE 22.8 

Composition of Nutmeg Oil with Main Hazardous Constituents 

Constituent Concentration (%) Labeling and Classification 

Pinenes 40 Xi; R43, N; R50/53 

Limonene 7 Xi; R38-43, N; R50/53 

Safrole 2 T; R45, Xn; R22-68 

Isoeugenol 1 Xn; R21/22, Xi; R36/38-43


• Classification based on its constituents: 

R10 

Flammable 

R43 

May cause sensitization by skin contact 

R45 

May cause cancer 

R50/53 Very toxic to aquatic organisms 

(environment) 

R65 

Harmful: may cause lung damage if 

swallowed 

R68 

Possible risk of irreversible effects 

• Labeling (pictograms) based on its constituents: 

T: Toxic (CMR) 

N: Dangerous for the environment 

So with these examples, it can be demonstrated that the final Classification and Labeling (C&L) 

of essential oils will change significantly depending on the approach used: based on existing data 

(for the various endpoints) on the essential oil as such or based on the hazardous constituents. It 

should however be underlined that according to the rules of the EFFA CoP, C&L of natural raw 

materials or NCSs can only be done for the endpoints for which data (on the NCS as such) are available 

(e.g., skin irritation, sensitization, environmental toxicity, etc.)—if no data are available, then 

the constituents must be taken into account for the classification for these endpoints. 


As described and outlined above, several new European Regulations and Directives have been 

adopted during the last years, and other regulations are currently under discussion and will soon 

enter into force in relation to flavors and fragrances and cosmetics. NCSs or raw materials (such as 

essential oils and extracts) are very important ingredients for flavoring and fragrance applications. 

As a result, these new regulations will have a major impact on the trade and use in commerce of 

these essential oils and extracts, in particular on labeling issues, as has been demonstrated with the 

7th Amendment of the Cosmetic Directive (labeling of alleged allergens) and the coming new 

Flavouring Regulation (labeling of “natural”). The labeling issue is especially important because of 

its impact on consumer behavior: consumers do not want to buy cosmetic end products that are 

labeled with potentially allergenic ingredients. Overlabeling should always be avoided. Therefore it 

is essential to lobby for a pragmatic approach toward allergen labeling and to advocate no labeling 

requirements for fragrance ingredients that are proven to be (extremely) rare sensitizers or no sensitizers 

at all, especially if these are naturally occurring constituents of a wide variety of essential oils 

and extracts. With respect to food, consumers prefer natural flavorings to synthetic ones. Good and 

pragmatic definitions in the Flavouring Regulation that will soon replace the current Flavouring 

Directive are essential to ensure that all natural raw materials such as essential oils and extracts can 

be labeled as natural.


ANNEX 22.1 

Aromatic Natural Raw Materials Containing Any of the 16 Naturally Occurring Alleged Aensitizers 

Benzyl 

Alcohol 

Benzyl 

Salicylate 

Cinnamyl 

Alcohol 

Cinnamal 

Citral 

Coumarin 

Eugenol 

Geraniol 

Isoeugenol 

Anisyl 

Alcohol 

Benzyl 

Benzoate 

Benzyl 

Cinnamate 

Citronellol 

Farnesol 

Limonene 

Linalool 

Total % 

Aromatic Natural Raw 

Materials Type 

Ambrette * * * * * * * * * * * * * 5 * 1 6 

Angelica root * * * * * * * * * * * * * * 18 0.3 18.3 

Angelica seed * * * * * * * * * * * * * * * * 0 

Star Anise * * * * * * * * * * * * * * 3 1.5 4.5 

Anise * * * * * * * * * * * * * * 2 0.1 2.1 

Armoise * * * * * * * * * * * * 0.2 * 2 * 2.2 

Basil Linalol * * * * * * 15 0.2 * * * * 0.3 * 1 62 78.5 

Basil Me-chavicol * * * * * * 0.5 * * * * * * * 1 1.1 2.6 

Bay * * * * * * 56 * * * * * * * 4 3 63 

Benzoin note 1 * * * * * * * * * * 0.2 0.8 * * * * 1 

Bergamot (s) cold press * * * * 0.7 * * * * * * * * * 45 15 60.7 

Bergamot bergapten-free * * * * 0.7 * * * * * * * * * 45 15 60.7 

Bergamot distilled * * * 0.4 * * * * * * * * * 40 40 80.4 

Bitter orange * * * * 0.1 * * * * * * * * * 95 0.2 95.3 

Buchu (s) * * * * * * * * * * * * * * 30 0.5 30.5 

Cabreuva * * * * * * * * * * * * * 3 * * 3 

Cajuput * * * * * * * 0.4 * * * * * * 10 3.6 14 

Camphor * * * * * * * * * * * * * * 25 0.5 25.5 

Cananga * 3 * * * * 0.7 1.5 * * 5 * * 2 * 3 15.2 

Caraway * * * * * * * * * * * * * * 45 * 45 

Cardamom * * * * 0.6 * * 1.2 * * * * * * 4 4 9.8 

Carrot * * * * * * * 2 * * * * * * 3 2 7 

Cascarilla * * * * * * 0.3 * * * * * * * 5 5 10.3 

Cassia * * 1 90 * 4 0.5 * * * 1 * * * 0.1 * 96.6 

Cedarwood (s) * * * * * * * * * * * * * * * * 0 

Celery * * * * * * * * * * * * * * 79 0.1 79.1 

Chamomile Roman * * * * * * * 0.7 * * * * 0.7 * 5 0.8 7.2


Chamomile Blue * * * * * * * * * * * * * * 1 0.4 1.4 

Cinnamon bark * * 0.2 75 * 0.3 6 * * * 1.5 * * * 1 6 90 

Cinnamon leaf * * 1 3 * 0.3 85 * * * 4 * * * * 4 97.3 

Cistus (Rockrose) * * * * * * * 1.2 * * * * * * 4 0.5 5.7 

Sri Lanka Citronella * * * * 1.1 * * 23 * * * * 8.5 * 12 1 45.6 

Java Citronella * * * * 1.3 * 1 25 * * * * 14 1 5 1.5 48.8 

Clary Sage (s) * * * * * * * 2.2 * * * * * * 1 24 27.2 

Clove (s) * * * * * * 92 * 0.5 * * * * * * * 92.5 

Copaiba * * * * * * * * * * * * * * * * 0 

Coriander fruit * * * * * * * 3 * * * * * * 5 78 86 

Coriander leaf * * * * * * * 0.4 * * * * * * 1 25 26.4 

Corn mint Arvensis * * * * * * * * * * * * * * 7 * 7 

Cubeb * * * * * * * * * * * * * * 1 1.2 2.2 

Cypress (s) * * * * * * * * * * * * * * 14 0.8 14.8 

Davana * * * * * * 0.1 * * * * * * * 0.1 1 1.2 

Dill (s) * * * * * * * * * * * * * * 45 4 49 

Elemi * * * * * * * * * * * * * * 72 * 72 

Estragon Tarragon * * * * * * 0.2 * * * * * * * 6 * 6.2 

Eucalyptus globulus * * * * * * * * * * * * * * 8 * 8 

Eucalyptus dives * * * * * * * * * * * * * * 1 1 2 

Eucalyptus citriodora * * * * * * * * * * * * 0.7 * 1 0.3 2 

Fennel Bitter * * * * * * * * * * * * * * 35 * 35 

Fennel Sweet * * * * * * * * * * * * * * 4 * 4 

Galbanum * * * * * * * * * * * * * * 3.4 * 3.4 

Geranium N. Africa * * * * 1.5 * * 18 * * * * 36 * 1 8.5 65 

Geranium Bourbon * * * * 1.5 * * 20 * * * * 26 * 1 11 59.5 

Geranium China * * * * 1.2 * * 12 * * * * 43 * 1 4.5 61.7 

Ginger (s) * * * * 0.7 * * * * * * * * * 2 0.6 3.3 

Grapefruit (s) * * * * 0.15 * * * * * * * * * 95 0.1 95.25 

Guaiac wood * * * * * * * * * * * * * * * * 0 

Gurjun * * * * * * * * * * * * * * * * 0 

Hay note 1 * * * * * 8 * * * * * * * * * * 8 

Ho (s) * * * * * * * 0.4 * * * * * * 0.2 90 90.6 

Hop * * * * * * * 0.2 * * * * * * 1 0.6 1.8 

continued


ANNEX 22.1 (continued) 

Benzyl 

Alcohol 

Benzyl 

Salicylate 

Cinnamyl 

Alcohol 

Cinnamal 

Citral 

Coumarin 

Eugenol 

Geraniol 

Isoeugenol 

Anisyl 

Alcohol 

Benzyl 

Benzoate 

Benzyl 

Cinnamate 

Citronellol 

Farnesol 

Limonene 

Linalool 

Total % 



Hyssop * * * * * * * * * * * * * * 4 * 4 

Immortelle * * * * * * * * * * * 0.2 * * 3.8 6 10 

Juniper berry * * * * * * * * * * * * * * 20 0.2 20.2 

Labdanum * * * * * * * * * * * * * * 4 * 4 

Laurel (s) * * * * * * 2 0.3 * * * * * * 5 11 18.3 

Lavandin (s) * * * * * 0.1 * 0.6 * * * * * * 2 38 40.7 

Lavandin absolute * * * * * 5 * 1.5 * * * * * * 1 42 49.5 

Lavender absolute * * * * * 8 * 1.5 * * * * * * 0.3 42 51.8 

Lavender (s) * * * * * * * 1.1 * * * * * * 1 45 47.1 

Spike lavender * * * * * 0.2 0.1 0.2 * * * * 0.3 * 3 50 53.8 

Lemon (s) * * * * 3 * * 0.2 * * * * * * 73 0.3 76.5 

Lemongrass (s) * * * * 90 * 0.3 7 * * * * 0.8 * 4 2 100 

Lime (s) distilled * * * * 3 * * 0.4 * * * * * * 50 0.2 53.6 

Lime (s) cold press * * * * 6.5 * * 0.4 * * * * * * 55 0.2 62.1 

Litsea cubeba 

Mace 

* * * * 78 * * 1.5 * * * * 1.5 * 15 3 99 

* * * * * * 1.1 * 0.2 * * * * * 4 0.4 5.7 

Mandarin (s) * * * * * * * * * * * * * * 75 0.3 75.3 

Marjoram sweet * * * * * * * 0.3 * * * * * * 5 25 30.3 

Marjoram wild Spanish * * * * * * * * * * * * * * 6 48 54 

Mentha citrata (s) * * * * * * * 2 * * * * * * 1 50 53 

Myrrh * * * * * * * * * * * * * * * * 0 

Myrtle 

Neroli 

Niaouli 

Nutmeg 

* * * * * 0.2 0.7 0.8 * * * * 0.3 * 12 2 16 

* * * * 0.3 * * 3.5 * * * * * 4 18 44 69.8 

* * * * * * * * * * * * * * 10 0.2 10.2 

* * * * * * 0.6 0.4 1 * * * 0.2 * 7 0.4 9.6 

Oakmoss note 1 * * * * * * * * * * * * * * * * 0 

Olibanum note 1 * * * * * * * * * * * * * * 18.5 * 18.5 

Opoponax note 1 * * * * * * * * * * * * * * * * 0


Sweet orange (s) * * * * 0.1 * * * * * * * * * 95 0.4 95.5 

Origanum Spanish * * * * 0.5 * 0.3 * 0.3 * * * * * 1 15 17.1 

Palmarosa (s) * * * * 1 * * 85 * * * * * 1.2 1 4 92.2 

Parsley (s) * * * * * * * * * * * * * * 5 * 5 

Patchouli (s) * * * * * * * * * * * * * * * * 0 

Pepper (s) Black * * * * * * * * * * * * * * 23 1 24 

Peppermint Piperita * * * * * * * * * * * * * * 3 0.4 3.4 

Peru oil note 1 4.5 * * * * * * * * * 78 9 * * * * 91.5 

Bitter orange petitgrain * * * * 0.3 * * 4 * * * * * * 6 30 40.3 

Lemon petitgrain * * * * 26 * * 3.5 * * * * * * 42 2.5 74 

Mandarin petitgrain * * * * 0.1 * * * * * * * * * 11 0.7 11.8 

Bergamot petitgrain * * * * 1 * * 4 * * * * * * 1.5 30 36.5 

Petitgrain Paraguay * * * * 1 * * 4.5 * * * * * * 2 30 37.5 

Pimento (s) * * * * * * 85 * 1 * * * * * 2 0.5 88.5 

Pine (s) * * * * * * * * * * * * * * 6 0.2 6.2 

Rose oil Bulgaria * * * * 1 * 1.5 22 * * * * 34 1 * 1.4 60.9 

Rose oil Maroc * * * * 1 * 1.5 47 * * * * 20 1 * 1.4 71.9 

Rose oil China * * * * 1.5 * 1.5 18.3 * * * * 31 1 * 1.3 54.6 

Rose oil (s) Turkey * * * * 2 * 1.5 20 * * * * 49 1 * 1.3 74.8 

Rosemary (s) * * * * * * * * * * * * * * 6 0.8 6.8 

Rosewood 

* * * * * * * 2.5 * * 1.6 * * * 1 90 95.1 

Sage (s) * * * * * * * 0.4 * * * * * * 5 9 14.4 

Sandalwood * * * * * * * * * * * * * * * * 0 

Schinus molle (s) * * * * * * * * * * * * * * 15 0.5 15.5 

Spearmint (s) * * * * * * * * * * * * * * 24 0.2 24.2 

Spikenard * * * * * * * * * * * * * * * * 0 

Styrax resin note 1 0.1 * 3.5 * * * * * * * * 1.5 * * * * 5.1 

Styrax oil * * 54 1.1 * * * * * * * * * * * * 55.1 

Tagete (s) * * * * * * 0.2 * * * * * 3 * 9 0.5 12.7 

Tangerine (s) * * * * * * * * * * * * * * 95 0.5 95.5 

Tarragon Estragon * * * * * * 0.2 * * * * * * * 6 * 6.2 

Tea tree * * * * * * * * * * * * * * 4 * 4 

Red thyme Spanish * * * * 0.5 * * 0.5 * * * * * * 1 6.5 8.5 

Tolu note 1 0.5 * 0.6 1.7 * * 0.1 * * * 37.5 4.6 * * * * 45 

continued



Benzyl 

Alcohol 

Benzyl 

Salicylate 

Cinnamyl 

Alcohol 

Cinnamal 

Citral 

Coumarin 

Eugenol 

Geraniol 

Isoeugenol 

Anisyl 

Alcohol 

Benzyl 

Benzoate 

Benzyl 

Cinnamate 

Citronellol 

Farnesol 

Limonene 

Linalool 

Total % 



Tonka * * * * * 65 * * * * * * * * * * 65 

Treemoss note1 * * * * * * * * * * * * * * * * 0 

Turpentine (s) * * * * * * * * * * * * * * 7 * 7 

Valerian * * * * * * * * * * * * * * 2 * 2 

Vetiver (s) * * * * * * * * * * * * * * * * 0 

Ylang extra super 0.5 3.5 * * * * 0.5 0.7 0.5 * 6 * * 2 * 13 26.7 

Ylang extra (s) 0.5 4 * * * * 0.5 3 0.5 * 8 * * 3 * 24 43.5 

Ylang I (s) 0.5 4 * * * * 0.5 2.6 0.5 * 9.2 * * 3 * 19 39.3 

Ylang II (s) 0.5 4 * * * * 0.5 2.4 0.5 * 10 * * 4 * 9.5 31.4 

Ylang III (s) 0.5 5 * * * * 0.5 0.8 0.5 * 8.5 * * 4 * 40 59.8 

Legend * = < 0.1% % = Actual value ISO standardized essential oils, INCI chemical names used in this list 

Source: Internal communication (2004) regarding former version of EFFA CoP.


ANNEX 22.2 

Natural Raw Materials (Natural Complex Substances) Containing the 10 Naturally 

Occurring Alleged Allergens of Groups I and II According to Schnuch et al. (2007) 

Ambrette Clove stem Linaloe wood Rose absolute 

Armoise (A. alba) Coriander leaf/herb Litsea cubebe Rose, Bulg 

Armoise (A. vulgar.) Coriander seed Mace Rose, China 

Artemisia Deertongue leaf abs Melissa (lem. balm) Rose, Maroc 

Basil, linal. type Euc. Citriodora Mentha citrata Rose, Turkey 

Bay Flouve Myrtle Rosewood 

Cabreuva Geranium, Bourbon Neroli Sandalwood Aus. (S. spicatum) 

Cananga Geranium, Chin. Nutmeg Snakeroot 

Cardamon Geranium, N. Afr. Orange flower abs Styrax 

Carnation abs Hay abs Osmanthus abs Thyme, wild (T. serpyllum) 

Carrot seed Hyacinth absolute Palmarosa Tolu abs 

Cassia Labdanum Peru balsam oil Tonka abs 

Cassie abs (Acacia) Laurel (Sweet bay) Peru balsam resinoid Tuberose abs 

Cinnamon bark Lavandin abs Petit grain, bergamot Turmeric 

Cinnamon leaf Lavender Petit grain, lemon Verbena abs 

Citronella Ceylon Lavender abs Petit grain, orange Verbena oil 

Citronella Java Lemon Petit grain, Paraguay Ylang extra sup. 

Clary sage Lemongrass Pimento berry Ylang extra, Com. (Mad.) 

Clove bud Lime, dist Pimento leaf Ylang, Com., (Mad.) 

Clove leaf Lime, expr. Rhodinol 

Source: EFFA-CoP. 

ANNEX 22.3 

Maximum Limits for Certain Substances Obtained from Flavorings and other Food 

Ingredients with Flavoring Properties Present in Foodstuffs as Consumed in which 

Flavorings have been Used (Annex II of 88/388/EC) 

Substances 

Foodstuffs 

(mg/kg) 

Beverages 

(mg/kg) 

Exceptions and/or Special Restrictions 

Agaric acid ( 1 ) 20 20 100 mg/kg in alcoholic beverages and foodstuffs 

containing mushrooms 

Aloin ( 1 ) 0.1 0.1 50 mg/kg in alcoholic beverages 

b-Asarone ( 1 ) 0.1 0.1 1 mg/kg in alcoholic beverages and seasonings used in snack foods 

Berberine ( 1 ) 0.1 0.1 10 mg/kg in alcoholic beverages 

Coumarin ( 1 ) 2 2 10 mg/kg in certain types of caramel confectionery 

50 mg/kg in chewing gum 

10 mg/kg in alcoholic beverages 

Hydrocyanic acid ( 1 ) 1 1 50 mg/kg in nougat, marzipan or its substitutes or similar products 

1 mg/% volume of alcohol in alcoholic beverages 

5 mg/kg in canned stone fruit 

Hypericine ( 1 ) 0.1 0.1 10 mg/kg in alcoholic beverages 

1 mg/kg in confectionery 

continued



Maximum Limits for Certain Substances Obtained from Flavorings and other Food 

Ingredients with Flavoring Properties Present in Foodstuffs as Consumed in which 

Flavorings have been Used (Annex II of 88/388/EC) 

Substances 

Foodstuffs 

(mg/kg) 

Beverages 

(mg/kg) 

Exceptions and/or Special Restrictions 

Pulegone ( 1 ) 25 100 250 mg/kg in mint or peppermint-flavored beverages 

350 mg/kg in mint confectionery 

Quassine ( 1 ) 5 5 10 mg/kg in confectionery in pastille form 

50 mg/kg in alcoholic beverages 

Safrole and 

isosafrole ( 1 ) 

1 1 2 mg/kg in alcoholic beverages with not more than 25% volume 

of alcohol 

5 mg/kg in alcoholic beverages with more than 25% volume of alcohol 

15 mg/kg in foodstuffs containing mace and nutmeg 

Santonin ( 1 ) 0.1 0.1 1 mg/kg in alcoholic beverages with more than 25% volume of alcohol 

Thuyone (a and b) ( 1 ) 0.5 0.5 5 mg/kg in alcoholic beverages with not more than 25% volume 

of alcohol 

10 mg/kg in alcoholic beverages with more than 25% volume of alcohol 

25 mg/kg in foodstuffs containing preparations based on sage 

35 mg/kg in bitters 

( 1 ) May not be added as such to foodstuffs or to flavorings. May be present in a foodstuff either naturally or following the 

addition of flavorings prepared from natural raw materials 

ANNEX 22.4 

List of Source Materials to which Restrictions Apply for Their Use in the Production of 

Flavorings and Food Ingredients with Flavoring Properties (Annex IV of Draft Flavouring 

Regulation, According to Council Proposal, December 10, 2007) 

Part A: 

Source materials which shall not be used for the production of flavorings and food ingredients 

with flavoring properties 

Source Material 

Latin Name 

Tetraploid form of Acorus calamus 

Common Name 

Tetraploid form of Calamus 

Part B: Conditions of use for flavorings and food ingredients with flavoring properties produced from certain 

source materials 

Source Material 

Latin Name 

Quassia amara L. and 

Picrasma excelsa (Sw) 

Laricifomes offi cinales 

(Vill.: Fr) Kotl. et Pouz or 

Fomes offi cinalis 

Hypericum perforatum 

Teucrium chamaedrys 

Common Name 

Quassia 

White agaric mushroom 

St Johns wort 

Wall germander 

Conditions of Use 

Flavorings and food ingredients with flavoring properties 

produced from the source material may only be used for 

the production of beverages and bakery wares 

Flavorings and food ingredients with flavoring properties 

produced from the source material may only be used for 

the production of alcoholic beverages


ANNEX 22.5 

List of Traditional Food Preparation Processes (Annex II of Draft Flavouring 

Regulation, According to Council Proposal) 

Chopping 

Heating, cooking, baking, frying (up to 240°C at 

atmospheric pressure) and pressure cooking (up to 120°C) 

Cutting 

Drying 

Evaporation 

Fermentation 

Grinding 

Infusion 

Microbiological processes 

Peeling 

Pressing 

Roasting /grilling 

Steeping 

Cooling 

Coating 

Distillation/rectification 

Emulsification 

Extraction, including solvent extraction in 

accordance with Directive 88/344/EEC 

Filtration 

Maceration 

Mixing 

Percolation 

Refrigeration/freezing 

Squeezing 

REFERENCES 

7th Amendment of the Cosmetic Directive: Directive 2003/15/EC of the European Parliament and of the 

Council of February 27, 2003 (OJ L 66/26, 11.3.2003). 

18th ATP of DSD: Commission Directive 93/21/EEC of April 27, 1993 adapting to technical progress for the 

18th time Council Directive 67/548/EEC on the Approximation of the laws, regulations and administrative 

provisions relating to the classification, packaging and labelling of dangerous substances (OJ L 110, 

4.5.1993, p. 20). 

88/389/EEC: Council Decision of June 22, 1988 on the establishment, by the Commission, of an inventory of 

the source materials and substances used in the preparation of flavourings (OJ L 184, 15.7.1988, p. 67). 

Becker, K., E. Temesvari, and I. Nemeth, 1994. Patch testing with fragrance mix and its constituents in a 

Hungarian population. Cont. Dermat., 30: 185–186. 

Chaintreau, A. et al., 2003. GC-MS quantification of fragrance compounds suspected to cause skin reactions. 

1. J. Agric. Food Chem., 51: 6398–6403. 

Chaintreau, A. et al., 2007. GC-MS quantification of suspected volatile allergens in fragrances. 2. Data treatment 

strategies and method performances. J. Agric. Food Chem., 55: 25–31. 

Commission Directive 2006/8/EC of January 23, 2006 (OJ L 19/12, 24.1.2006). 

Commission Directive 91/71/EEC of January 16, 1991 completing Council Directive 88/388/EEC on the 

approximation of the laws of the Member States relating to flavourings for use in foodstuffs and to source 

materials for their production (OJ L 42, 15.2.1991, p. 25). 

Cosmetic Directive: Council Directive 76/768/EEC of July 27, 1976 on the approximation of the laws of the 

Member States relating to cosmetic products (OJ L 262, 27.7.1976, p. 169). 

Council Proposal, July 15, 2008. Proposal for a Regulation of the European Parliament and of the Council on 

flavourings and certain food ingredients with flavouring properties for use in and on foods and amending 

Council Regulations (EEC) No. 1576/89 and (EEC) No. 1601/91, Regulation (EC) No. 2232/96 and 

Directive 2000/13/EC – Outcome of the European Parliament’s second reading (Strasbourg, 7 to 10 July 

2008), Brussels, 15 July 2008. 11479/08, CODEC 922, DENLEG 86. 

Dangerous Preparations Directive: Directive 1999/45/EC of the European Parliament and of the Council of 

May 31, 1999 concerning the approximation of the laws, regulations and administrative provisions of 

the Member States relating to the classification, packaging and labeling of dangerous preparations (OJ 

L 200, 30.7.1999, p. 1). 

Dangerous Substances Directive: Council Directive 67/548/EEC of June 27, 1967 on the approximation of 

laws, regulations and administrative provisions relating to the classification, packaging and labelling 

of dangerous substances (OJ P 196, 16.8.1967, p. 1).


EFFA Code of Practice (EFFA CoP): Home page of the European Flavour & Fragrance Association (EFFA): 

www.effa.be 

First amendment of the Detergent Regulation: Commission Regulation (EC) No. 907/2006 of June 20, 2006 

(OJ L 168/5, 21.6.2006). 

Flavouring Regulation of December 31, 2008. Regulation (EC) No. 1334/2008 of the European Parliament and 

of the Council of 16 December 2008 on flavourings and certain food ingredients with flavouring properties 

for use in and on foods and amending Council Regulation (EEC) No. 1601/91, Regulations (EC) No. 

2232/96 and (EC) No. 110/2008 and Directive 2000/13/EC – OJ L 354, 31.12.2008, p. 34. 

Frosch, P.J., B. Pilz, K.E. Andersen, et al., 1995. Patch testing with fragrances : Results of a multicenter study 

of the European Environmental and Contact Dermatitis Research Group with 48 frequently used 

constituents of perfumes. Cont. Dermat., 33: 333–342. 

Hagvall, L. and A.-T. Karlberg, 2006. Air exposure turns common fragrance terpenes into strong allegens. Book 

of Abstracts, 37th Int. Symp. on Essential Oils (ISEO 2006), Grasse, September 10–13, PL-2. 

IFRA Code of Practice (IFRA CoP): Home page of the International Fragrance Association (IFRA): www. 

ifraorg.org 

Johansen, J.D., K.E. Andersen, C. Svedman, et al., 2003. Chloroatranol, an extremely potent allergen hidden in 

perfumes: A doseresponse elicitation study. Cont. Dermat., 49: 180–184. 

Johansen, J.D., G. Bernard, E. Gimenez-Arnau, J.P. Lepoittevin, M. Bruze, and K.E. Andersen, 2006. 

Comparison of elicitation potential of chloroatranol and atranol—2 allergens in oak moss absolute. Cont. 

Dermat., 54: 192–195. 

Karlberg, A.-T. et al., 1992. Air oxidation of d-limonene (the citrus solvent) creates potent allergens. Cont. 

Dermat., 26: 332–340. 

Karlberg, A.-T. and A. Dooms-Goossens, 1992. Contact allergy to oxidized d-limonene among dermatitis 

patients. Cont. Dermat., 36: 201–206. 

Larsen, W., H. Nakayama, M. Lindberg, et al., 1996. Fragrance contact dermatitis. A worldwide multicentre 

investigation (Part I). Am. J. Cont. Dermat., 7: 77–83. 

Memorandum on the SCCNFP Opinion concerning Fragrance Allergy in Consumers adopted by the SCCNFP 

during the 16th Plenary Meeting of March 13, 2001. 

Proposal for a Regulation of the European Parliament and of the Council on flavourings and certain food ingredients 

with flavouring properties for use in and on foods (COM(2006) 427 final. DG Sanco, Brussels, 

28.7.2006). Current Flavouring Directive: Council Directive 88/388/EC of June 22, 1988 on the approximation 

of the laws of the Member States relating to flavourings for use in foodstuffs and to source 

materials for their production (OJ L 184, 15.7.1988, p. 61). 

Regulation (EC) No. 1336/2008 of the European Parliament and of the Council of 16 December 2008 amending 

Regulation (EC) No. 648/2004 in order to adapt it to Regulation (EC) 1272/2008 on classification, 

labelling and packaging of substances and mixtures. OJ L 354, 31.12.2008, p. 60. 

Regulation (EC) No. 648/2004 of the European Parliament and of the Council of March 31, 2004 on detergents 

(OJ L 104, 8.4.2004, p. 1). 

SCCNFP Opinion concerning Oakmoss/Treemoss Extracts and Appropriate Consumer Information adopted by 

the SCCNFP during the 14th plenary meeting of October 24, 2000. 

SCCNFP/0017/98 Final of December 1999: SCCNFP opinion concerning: Fragrance allergy in consumers—a 

review of the problem: Analysis of the need for appropriate consumer information and identification of 

consumer allergens. 

SCCNFP/0202/99: SCCNFP Opinion concerning the interim position on fragrance allergy—adopted by the 

Scientific Committee on Cosmetic Products and Non-food Products intended for Consumers during the 

plenary session of June 23, 1999. 

SCCP/00847/04: SCCP Opinion on atranol and chloroatranol present in natural extracts (e.g., oak moss and 

tree moss extract), adopted by the SCCP during the 2nd plenary meeting of December 7, 2004. 

Santussi, B., A. Cristaudo, C. Cannistraci, and M. Picardo, 1987. Contact dermatitis to fragrances. Cont. 

Dermat., 16: 93–95. 

Schnuch, A., J. Geier, W. Uter, et al., 1997. National rates and regional differences in sensitization to allergens 

of the standard series. Population adjusted frequencies of sensitization (PAFS) in 40,000 patients from a 

multicenter study (IVDK). Cont. Dermat., 37: 200–209. 

Schnuch, A., J. Geier, W. Uter, and P.J. Frosch, 2002. Another look at allergies to fragrances: Frequencies of sensitization 

to the fragrance mix and its constituents. Results from the IVDK. Exogenous Dermatol., 1: 231–237. 

Schnuch, A., H. Lessmann, J. Geier, P.J. Frosch, and W. Uter, 2004. Contact allergy to fragrances: Frequencies 

of sensitization from 1996 to 2002. Results of the IVDK. Cont. Dermat., 50: 65–76. 

Schnuch, A., W. Uter, J. Geire, H. Lessmann, and P.J. Frosch, 2007. Sensitization to 26 fragrances to be labelled 

according to current European regulation. Cont. Dermat., 57: 1–10.

Index 

Note: (Alphanumeric/numeric character) indicates figure subset serial number. 

A 

A-549 (human lung carcinoma cell line), 237, 239 

Abiatae, 906 

Abies balsamea, 237 

Absidia coerulea, 770, 775, 776 

Absidia glauca, 765 

Absolutes, 844, 937 

Acanthus-leaved thistle, 253 

Acetic acid-induced writhing test, 240, 242 

2,3-di-O-Acetyl-6-O-tert-butyldimethylsilylβ-cyclodextrin, 

17 

Achillea biebersteinii, 261 

Achillea ligustica, 261 

Achillea millefolium, 891 

Achillea schischkinii, 243 

Achillea sp., 48, 494 

ACHN, 237 

Acorus gramineus Solander (Acoraceae), 300, 302 

Actinobacillus actinomycetemcomitans, 320 

Activated charcoal, 9 

Acyclic monoterpene 

metabolic pathways of, 587 

alcohols, 588 

aldehydes, 588 

hydrocarbons, 587 

Acyclic monoterpenoids, 587 

Adamantanes, biotransformation of, 823–828 

Addind spice 

with natural aromas, in balanced way, 865 

Adenosine triphosphate, 122 

Aderton, Charles, 846 

ADT. See Agar diffusion test (ADT) 

Aeolanthus suaveolens, 301 

Afan, 261 

Aframomum giganteum, 65 

African brasil, Lamiaceae, 249 

9(15)-Africanene (117a) biotransformation 

by Aapergillus niger, 762 

by Rhizopus oryzae, 762 

African juniper, 264 

African pepper, 262 

African sage, 264 

African wormwood, 261 

Agar diffusion test (ADT), 354 

anise oil, 356–359 

bitter fennel, 365–369 

caraway oil, 372–376 

cassia oil, 380 

Ceylon cinnamon bark oil, 383–386 

Ceylon cinnamon leaf oil, 390 

citronella oil, 393–396 

clary sage oil, 400 

clove leaf oil, 404–408 

coriander oil, 414–418 

dwarf pine oil, 421 

eucalyptus oil, 423–425 

juniper oil, 429–430 

lavender oil, 434–436 

lemon oil, 442–446 

mandarin oil, 451–453 

matricaria oil, 455–457 

mint oil, 461–463 

neroli oil, 465–466 

peppermint oil, 474–477 

Pinus sylvestris oil, 482–483 

rosemary oil, 487–492 

star anise oil, 500–501 

sweet orange oil, 503–507 

tea tree oil, 512 

thyme oils, 521–524 

Agathosma, 252 

Agathosma collina, 252 

Agricultural crop establishment, 92–94 

Ajowan, 267 

Ajwain, 267 

Alambique Vial Gattefossé, 88 

Aldehydes, 904 

formation of, 124 

Alembic à bain-marie, 88 

Alertness and attention, fragrances influences 

on, 290–293 

Alganite, 858, 859 

Algerian oregano, 266 

Alleged allergens,classification of, 923 

Allergenic fragrances, 922 

Allergen labelling issue, 925 

Allergies, 562 

Allium sativum, 884 

Allium schoenoprasum, 884 

Alloaromadendrene (57) bitrasformation 

by Bacillus megaterium, 754 

by Glomerella cingulata, 755 

by Mycobacterium smegmatis, 754 

4-Allyl-1,2-dimethoxy-benzene, 930 

1-Allyl-4-methoxybenzene, 930 

Allyl isothiocyanate, 304, 883 

Almond, 561 

Almonella enteritidis, 888 

Aloin, 930 

Alopecia areata, 575 

Aloysia gratissima, 245 

α-amylase, 864 

α-amyl-cinnamic aldehyde, 922 

α-and β-thujone, 225–226, 228 

α-bisabolol, 138, 272 

949

950 Index 

α block, 285 

α-cadinol, 239 

α-copaene, 250 

α-cyclocostunolide (169), 771 

α-damascone (525), 825 

α-episantonin (198), 775, 776 

α-eudesmol (153), 767 

α-eudesmol, 306 

α-farnesene, 238 

trans,trans-α-farnesene, 238 

α-fenchol, 709 

α-glucose unit, of cyclodextrin, 15 

α-hexylcinnamic aldehyde, 922 

α-humulene, 239, 250 

α-ionone (513), 824 

α-keto-γ-methiolbutyric acid, 262 

α-longipinene (433), 806, 810 

α-longipinene, 253 

α-phellandrene, 238, 616 

α-pinene, 129, 145–146, 237, 253, 254, 259, 261, 

262, 264, 265, 303, 587, 677, 709, 717 

α rhythm, 285 

α-santonin (190), 774, 775 

6-epi-α-santonin (190), 776 

α-terpinene, 262, 265, 305, 616 

α-terpineol, 225, 227, 247, 254, 262, 264, 270, 305, 594, 

629, 709, 717 

α-terpinolene, 247, 259 

α-terpinyl methyl ether, 883 

α-tocopherol, 259 

α, β-unsaturated ketone, 648, 700 

Alpha waves, 285 

Alpinia oxyphylla, 739 

Alpinia zerumbet (Pers.), 242 

Alternaria alternate, 783 

Alzheimer’s syndrome, 553 

Amber, 849 

Ambrox (391), 803 

(-)-Ambrox (391), 802, 803, 804 

o-Aminobenzoic acid, 127 

1-Aminocylopropane-1-carboxylic acid, 262 

Amomum, 254 

Amyl acetate, 883 

Anacetum vulgare, 884 

Analysis of essential oils, 151–152 

classical analytical techniques, 152–156 

general considerations, 177 

modern analytical techniques, 156–157 

fast GC for essential oil analysis, 159–162 

GC and linear retention indices, use of, 157–158 

GC enantiomer characterization, 164–165 

GC-mass spectrometry, 158–159 

GC-olfactometry for the assessment of 

odor-active components of essential 

oils, 162–164 

LC and LC-MS, 165–167 

MDGC techniques, 167–174 

MDLC techniques, 174–176 

on-line LC-GC, 176–177 

Analytical Working Group, of IFRA, 921 

Anethole, 128, 272, 883 

trans-Anethole, 884, 305 

Angelica sinensis, 885 

Animal models, EOs effects in, 299 

Anisaldehyde, 128, 844 

m-Anisaldehyde, 884 

Anise, 884 

Anise oil, 534 

inhibitory data 

obtained in agar diffusion test, 356–359 

obtained in dilution test, 360–363 

obtained in vapor phase test, 364 

Anisyl alcohol, 922, 925 

Anonaceae, 238, 262 

Anthranilic acid, 127 

Anti-inflammation, 338–339 

Antimicrobial growth promoters, 890 

Antinociception, function of, 239 

Antitussive, 334 

Antoine Chiris, 844 

Aphids, 885 

Apiaceae, 65, 263, 264, 267, 272 

Apis mellifera, 253 

Apoptotic cell death, 238 

Apothecaries, 556 

Appetizer and finger food recipes 

crudities, 873 

Maria’s Dip, 873 

Tapenade, 873–874 

Tofu Aromanaise, 874 

Veggie Skewers, 874–875 

Apple Cake Rose, 878 

Appropriate physical process, 934 

Apricot kernel, 561 

Aqueous essence, 114 

Aquifoliaceae, 270 

Arachidonic acid, 125 

Aristolane sesquiterpenoids, 751 

(+)-1(10)-Aristolene (36), 749–754 

1(10)-Aristolene (36) biotransformation 

by Aspergillus niger, 752, 753 


by Chlorella fusca, 751 

by Diplodia gossypina, 753 

by Mucor species, 752 

Aristolochia mollissima, 239 

Aristolochia species, 750 

Aristolochiaceae, 239, 243 

Aromachology, 551 

Aroma shake, with Herbs, 869 

Aromadendr-1(10),9-diene (82) biotransformation 

by Curvularia lunata, 758 

Aromadendra-9-one (80) biotransformation 

by Curvularia lunata, 757 

10-epi-Aromadendra-9-one (81) by Curvularia 

lunata, 757 

Aromadendrene (56) biotransformation 




Aromatherapy, 551, 549 

blending of, 561 

chemical agents, 575 

clinical studies, 569–570 

common fragrance components, 567–568 

cosmetologic, precursors of, 554–555 

definition, 550–551 

false claims, 563

Index 951 

historical background, 553–554 

incense, in Ancient Egypt, 554 

perfumed oil producing method, 555 

internal usage, 561–562 

massage, 559–560 

medicinal uses, 555 

middle ages, 556 

modern perfumery, 557 

past clinical studies, 571–575 

perfume and cosmetics, 554–555 

placebo effect, 564 

practices, 557–558 

methods, 558 

psychophysiology, 563–564 

recent clinical studies, 570 

in dementia, 570–571 

safety issue in, 565 

scientific evidence, 551–553 

selected toxicities, 568–569 

synthetic components, 562 

therapeutic claims, 562–563 

toxicity 

in humans, 566–567 

young children, 568 

in UK and US, 550 

Aromatic compounds, biotransformation of, 828–835 

Aromatic plants, 85 

as sedatives or stimulants, 297–299 

Aromatology, 551 

Aroma-Vital Cuisine recipies, 868 

addind apice 

with natural aromas, in balanced way, 865 

appetizer and finger food 

crudities, 873 

Maria’s dip, 873 




basics 

Crispy Coconut Flakes (Flexible Asian Spice 

Variation), 868 

Gomasio (A Sesame Sea-Salt Spice), 868–869 

Honey Provencal, 869 

beverages 

Aroma Shake, with Herbs, 869 

Earl Grey, at His Best, 869–870 

Lara’s Jamu, 870 

Rose-Cider, 870–871 

Syrup Mint-Orange, 871 

culinary arts 

on Exquisite Ingredients and Accomplished 

Rounding, 864 

culinary trip, 866 

dessert, cakes and baked goods 


Chocolate Fruits and Leaves, 878–879 

Homemade Fresh Berry Jelly, 879 

Rose Semifreddo, 879–880 

Sweet Florentine, 880 

emulsifiers and administering forms, 865 

entrees 

salads, 872–873 

soups, 871 

essential oils, to lift spirits, 866 

main course 

Celery Lemon Grass Patties, 875 

Chévre Chaude-Goat Cheese “Provence”, with 

Pineapple, 875–876 

Crispy Wild Rice-Chapatis, 876 

Mango–Dates–Orange Chutney, 876–877 

Prawns Bergamot, 877–878 

quality criteria and specifics 

while Handling Essential Oils, for Food 

Preparation, 864–865 

quantity, 865 

recipies, 868 

résumé, 880 

storage, 865 

Arousal, 284 

Artemisia, 260 

Artemisia absinthium, 260, 884, 885 

Artemisia abyssinica, 261 

Artemisia afra, 261 

Artemisia annua, 788, 885 

Artemisia arborescens L., 246 

Artemisia douglasiana, 245 

Artemisia dracunculus, 329 

Artemisia molinieri, 261 

Artemisia santonicum, 260, 261 

Artemisia scoparia, 885 

Artemisia spicigera, 260, 261 

Artemisia vulgaris, 92 

Artemisinin (283), 788, 790 

Arvensis, 901 

Asarone (558), 828 

Ascaridol, 261 

Asclepiadaceae, 245 

Ascorbic acid, 256 

Asiasari radix, 243 

Asparagus offi cinalis, 770, 775, 776 

Aspergillus alliaceus, 787, 789 

Aspergillus awamori IFO 4033, 783 

Aspergillus cellulosae, 607, 746, 747, 765, 767, 768, 771, 

772, 773, 780, 792, 824 

Aspergillus fl avipes, 765 

Aspergillus niger, 587, 720, 721, 738, 741, 756, 765, 767, 

768, 783, 769, 771, 772, 773, 777, 778, 779, 

780, 788, 790, 791, 792, 802, 804, 808, 810, 

812, 817, 818, 824 

Aspergillus orysae, 804 

Aspergillus quardilatus, 776, 778 

Aspergillus sojae, 690, 747 

Aspergillus spp., 720 

Aspergillus terreus, 783, 824 

Aspergillus usami, 690 

Aspergillus wentii, 743, 764, 766 

Aspirin®, 243 

Asteraceae, 46–52, 237, 243, 245, 246, 253, 260, 261 

Asthmaweed, 248 

Atractylodes lancea, 765, 797 

Atractylodis rhizoma, 766 

atractylon (163), 769 

Atranol, 123, 923 

structures of, 924 

Attentional functions, fragrances influences on, 290–293 

Attenuated reflection IR spectroscopy, 28 

Ayurveda, 863 

Azoricus thyme, 259

952 Index 

B 

B16F10 (mouse cell line), 237 

Bacilli Chalmette Guerin, 252 

Bacillus megaterium, 751, 754, 755 

Bacillus subtilis, 804 

Backhousia citriodora, 318, 320 

Baill, 240 

Balm of Gilhead, 554 

Balsam fir oil, 237, 238 

Balsamodendron myrrha, 554 

Balsamodendron opobalsamum, 554 

Bark oils, 91 

Barton, D. H. R., 4 

Basic recipes 

crispy coconut flakes (flexible asian spice 

variation), 868 

gomasio (a sesame sea-salt spice), 

868–869 

honey provencal, 869 

Basil, 270, 563 

Bazzania species, 792 

Bazzania pompeana, 792 

Beauvaria bassiana, 765, 767, 785, 786, 787 

Beckley, V.A., 901 

Bel-7402, 237 

Benzene, as a feedstock for shikimates, 143 

p-Benzoquinone, 243 

Benzydamine hydrochloride, 322 

Benzyl alcohol, 922, 923, 925 

Benzyl benzoate, 922, 925 

Benzyl cinnamate, 923 

Benzyl salicylate, 922, 923, 925 

Bergamot oil, 17, 291, 848, 884 

Bergapten, 128 

Berlocque dermatitis, 567 

Berthelot, M., 4 

β-acoradiene, 250 

β-asarone, 298, 305 

β-barbatene (371), 799 

β-bisabolene, 272 

β-bisabolol (282a), 788 

β-carotene bleaching test, 257, 259, 264 

β-carotene, 256 

β-caryophyllene, 238, 239, 250, 253, 265, 

268, 269 

(-)-β-caryophyllene, 821 

(-)-β-caryophyllene (451), 819 

(-)-β-caryophyllene epoxide (453), 814 

β-citronellol, 236 

β-cyclocostunolide (170), 772, 773 

β-damascone (531), 825 

β-d-mannuronic acid (M), 859 

β-elemene, 239 

β-eudesmene, 306 

β-eudesmol (157), 767, 768 

β-eudesmol, 299, 305 

β-pinene, 147, 253, 680, 709 

β rhythm, 285 

β-selinene (138), 763 

Betula alba, 331 

Beverage recipes 

Aroma Shake, with Herbs, 869 





BHA. See Butylated hydroxyanisole (BHA) 

BHT. See Butylated hydroxytoluene (BHT) 

Bicuculline, 243 

Bicyclic monoterpene, 677 

alcohol, 690 

aldehyde, 689 

ketones, 700 

Bicyclogermacrene, 250 

Bicyclohumulenone (357), 798 

Billot, M., 850 

BIOLANDES, 111 

Biologically active substances, 926 

Bio-oils, 116 

Biosynthetic pathways, 121–123 

Biota orientalis, 792 

Birch oil, 883 

(E)-g-Bisabolene, 239 

Bitter fennel, 534 



obtained in dilution test, 370 


Black cumin, 237, 240, 268 

Black cumin seeds, 251 

Black sage, 253 

Blue gum eucalyptus, 249 

Blue mint bush, 260 

Blue mountain sage, 264 

Blume syn., 270 

Bog myrtle, 238 

Boiga irregularis, 884 

Boiss, 241 

Borage seed, 561 

Boraginaceae, 253 

Borago offi cinalis, 561 

Borneol, 305, 696, 713, 717 

Bornyl acetate, 304, 719 

Boswellia carterii, 554 

Boswellia thurifera, 554 

Botanical source, of the essential oil, 117 

Botryodiplodia theobromae, 738, 741 

Botryosphaeria dothidea, 738, 741, 746, 769, 772, 773, 

783, 784, 824 

Botrytis cinerea, 594, 595, 754, 783, 805, 806, 810, 811, 

815, 816, 821 

Bottrospicatol, 717 

Bradham, Caleb, 846 

Brain imaging techniques, 296 

British Pharmacopoeia, 353 

Bronchoalveolar lavage, 251 

Bronchodilation and airway hyperresponsiveness, 

335–336 

Brown process, 98 

Buddleja cordobensis, 246 

Bulinus truncates, 885 

Bulnesol (232), 779 

Burseraceae, 247, 554 

Bush distillation device, 100 

Butylated hydroxyanisole (BHA), 257, 266, 270 

Butylated hydroxytoluene (BHT), 257, 267, 268, 269, 270, 

273, 274

Index 953 

C 

C4-epimer (221), 778 

Caco-2, 237 

Cadbury, 846 

Cadina-4,10(15)-dien-3-one (265), 786, 787 

Cadinol (281), 787 

Caespititius, 259 

Cajeput, 254, 552, 885 

Calamenene, 250 

Calendula, 561, 884 

Calendula offi cinalis, 561 

Calonectria decora, 761 

Camphene, 210, 237, 259, 719 

(±)-Camphene, 686 

1,2-Campholide, 719 

Camphor thyme, 259 

Camphor tree, 250 

Camphor, 4, 210–212, 261, 552, 713, 717, 885 

Camphorweed, 245 

Candia albicans, 788 

Candia rugosa, 804 

Candia tropicalis, 804 

Candida albicans, 779 

Candida treatments, 562 

Candler, Asa, 846 

Cape ivy, 237 

Capitate trichomes, 40 

Capsaicin, 243, 883 

Capsaicin (596), 832, 833 

Capsicum annuum, 832 

Caraway oil, 534, 889 





Carbocation, 131 

Carbonated lemonade flavoured, with lemon juice, 845 

Carbowax 20M, 17 

Cardamom, 250, 254 

3-Carene, 688 

Carlina acanthifolia, 253 

Carotenoids, 139, 140 

Carrageenin edema test, 240 

Carum carvi, 268, 325, 889 

Carum nigrum, 268 

Carvacrol, 212, 238, 260, 268, 272, 306, 884, 886, 887 

Carvacrol (583), 831 

Carvacrol methyl ether, 633, 712 

Carveol, 236, 634, 717 

Carvomenthol, 646, 717 

Carvone, 213, 304, 711, 717, 720, 887 

Carvone-8,9-epoxide, 717 

Carvonhydrate, 717 

Carvotanacetol, 717, 720 

Carvotanacetone, 709, 712, 715, 717, 720 

Caryophyllaceae, 248 

Caryophyllene, 227, 229, 238, 250 

Caryophyllene oxide, 244, 250, 306 

Casaeria sylvestris Sw., 250 

CAS number, 906, 907 

Caspase-3 activity, 238 

Cassia oil, 848 


obtained in agar diffusion test, 380 



Catalase, 256 

Caterpillars, 885 

Catharanthus roseus, 595, 833 

Catherine de Medici, 843, 844 

Catnip, 884 

Cats, 883 

Causae et Curae, 556 

CCC. See Countercurrent chromatography (CCC) 

Cedar of libanon, 246 

Cedarwood, 884, 885 

Cedrol (414), 809, 821 

Cedrus atlantica, 575, 885 

Cedrus deodara, 246 

Cedrus libani A., 246 

Cedus atlantica, 886 


Celery oil, limonene identification 

by 13 C-NMR spectroscopy, 30 

Celite, 9 

Central nervous system, essential oils effects in, 281 

activation and arousal, 284 

fragrances and EOs 

on brain potentials indicative of arousal, 285–290 

on cognitive functions, 290–296 

psychopharmacology of essential oils, 297 

in animal models, 299 

aromatic plants, as sedatives or stimulants, 

297–299 

mechanism of action, 302–306 

Cephalosporium aphidicoda, 755, 803, 805, 808, 821 

Ceylon cinnamon bark oil, 534 










Chaemomelum nobile, 329 

Chaetomium cochlioides, 807, 812 

Chaetomium globosum, 743 

Chamaecyparis obtusa, 820 

Chamaemelum nobile, 331 

Chamomile, 67, 891 

Charcoal, activated, 9 

Charles, King V, 843 

CHARM (combined hedonic aroma response 

method), 162 

Chemistry of essential oils, 121 

basic biosynthetic pathways, 121–123 

polyketides and lipids, 123–126 

shikimic acid derivatives, 126–129 

synthesis, 140–149 

terpenoids, 129–130 

hemiterpenoids, 131 

monoterpenoids, 131–135 

sesquiterpenoids, 135–140 

Chemotaxonomy, 41–42 

Chemotherapeutic agents, 239

954 Index 

Chenopodium ambrosioides, 890 

Chévre Chaude-Goat Cheese “Provence”, with Pineapple, 

875–876 

Chewing gum, 846 

Chinese eucalyptus oils, 86 

Chiral GC, 13–15 

Chives, 884 

Chloratranol, 123 

Chlorella ellipsoidea, 823 

Chlorella fusca, 738, 739, 750 

Chlorella pyrenoidosa, 739, 740, 823, 824, 825 

Chlorella salina, 823 

Chlorella sorokiniana, 823 

Chlorella vulgaris, 740, 823 

Chlorhexamed®, 321 

Chloroatranol, 923 

structures of, 924 


Chromatographic separation techniques, 11 

countercurrent chromatography, 20 

droplet countercurrent chromatography, 20 

rotation locular countercurrent chromatography, 

20–21 

gas chromatography, 12 

chiral GC, 13–15 

comprehensive multidimensional GC, 18 

fast and ultrafast GC, 13 

two-dimensional GC, 15–18 

liquid column chromatography, 18 

high-performance liquid column 

chromatography, 19 

preseparation, of essential oils, 18–19 

supercritical fluid chromatography, 20 

thin-layer chromatography, 12 

Chrysanthemum boreale, 238 

Chrysanthemum sibiricum, 248 

Chrysanthemum spp., 884 

Cidreira, 297 

p-Cimen-8-ol, 247 

Cineole, 133–134 

1,4-Cineole, 213–214, 261, 715, 720 

1,8-Cineole, 214, 216, 244, 247, 250, 254, 260, 261, 262, 

293, 298, 303, 563, 715, 720, 884, 885 

1,8-Cineole-2-malonyl ester, 720 

Cinnamaldehyde, 272, 883, 884, 887 

Cinnamic acid, 128, 922 

Cinnamodial (303), 792 

Cinnamomum camphora, 331 

Cinnamomum camphora ct 1,8-cineole, 320 

Cinnamomum osmophloeum, 250 

Cinnamomum verum, 270 

Cinnamomum zeylanicum, 65, 270, 317 

Cinnamon, 65, 254, 270, 848, 887 

Cinnamon leaf, 848 

Cinnamosma fragrans, 791 

Cinnamyl tiglate, 248 

Circulatory distillation apparatus, 5 

Cistus ladaniferus, 554 

Citral, 147–148, 216–217, 304, 588, 590, 714, 883, 884 

Citronella, 200, 217, 271, 306, 588, 714, 883, 884 

Citronella oil, 113 





(+)- and (-)-Citronellal, 588 

Citronellene, 588, 717, 720 

Citronellic acid, 590 

Citronellol, 133, 249, 306, 588, 883, 923 

(+)- and (-)-Citronellol, 588 

Citronellyl acetate 

fermentation of, 594 

Citronellyl formate, 249 

Citronellyl nitrile, 883 

Citrus aurantium, 263, 318 

Citrus bergamia, 883, 884 

Citrus EOs, 297 

Citrus fruit 

oil cells of, 96 

parts of, 96 

Citrus limon, 322, 331 

Citrus oil, 65, 91, 883 

Citrus pathogenic fungi, 608, 720 

Citrus peel, 95, 96, 888 

Citrus sinensis, 236, 331 

Citrus sinesis, 263 

Citrus yuko, 263 

Citrus-based aromas, 301 

Civet, 849 

Cladosporium resinae, 765 

Cladosporium sp., 608 

Clary sage oil, 534, 848 





Classical analytical techniques, 152–156 

Climate, 89–90 

Clinical aromatherapy, 555 

Clobetasol propionate, 254 

Cloning, 94–95 

Clostridium perfringens, 887 

Clove, 250, 848 

Clove buds, 89, 885, 887 

Clove leaf oil, 534, 899 





from Madagascar, 895 

CLP Regulation, 936 

13 

C-NMR spectroscopy, 28–30 

CNV. See Contingent negative variation (CNV) 

CO25 (N-ras transformed mouse myoblast 

cell line), 237 

Coca-Cola, 846 

Cocos nucifera, 561 

Code of Practice (CoP), 909, 910 

Coenzyme-A, 122 

Cold expression, 95 

Cold-pressed lemon-peel oil, 27 

Cold pressing, 5 

Colgate, William, 844 

Colgate–Palmolive Company, 844 

Colgate–Palmolive liquid soaps, 849 

Colgate–Palmolive–Peet company, 844 

Collectotrichum phomoides, 764 

Collectotrium phomoides, 766

Index 955 

Colletotrichum lindemuthianum, 783 

Colpermin®, 327 

Combined hedonic aroma response method. See CHARM 

(combined hedonic aroma response method) 

Commercial essential oil extraction methods, 95 

expression, 95–99 

steam distillation, 99–117 

Commiphora myrrha, 554 

Commiphora opobalsamum, 554 

Commission Directive 93/21/EEC, 928 

Commission Proposal, 928, 929, 932, 933 

appropriate physical process, 934 

flavouring preparation, 934–935 

natural flavoring substance, 933 

Compositae (Asteraceae), 67 

Compounds isopiperitenone, 718 

Comprehensive liquid chromatography (LC × LC), 174 

Comprehensive multidimensional GC, 18 

Comprehensive two-dimensional gas chromatography 

(GC × GC), 171, 172–174 

Comunelles, 94 

Concentrated oil, 114 

Concolina, 97 

Concretes, 844 

Constituent-based evaluation, 187 

Contaminations, 70 

Contingent negative variation (CNV), 289–290 

Conyza bonariensis, 248 

Copper still, in East Africa, 897 

Cordia verbenacea, 253, 253 

Coriander oil, 535, 899 





Corido thyme, 260 

Coridothymus capitatus, 888 

Cornmint oil, 221 

safety evaluation of, 197, 200–204 

Corynesphora cassiicola, 805, 821 

Corynespora cassiicola, 607 

Cosmetic and Detergent Legislation and Allergen 

Labeling, 918–920 

Cosmetic Directive 

and its Seventh Amendment, 920–921 

Extracts and Essential Oils and Aromatic Natural 

Raw Materials 

impacts on, 921–922 

First amendment of, 925–926 

Sensitization to Fragrances, Data on, 922–925 

Cosmetic Directive 

and its Seventh Amendment, 920–921 

Cosmetics, 568 

Costunolide (165), 767, 769, 770 

Coumarin, 844, 930 

Council Directive 79/831/EEC, 911 

Council proposal, 928, 929, 932 

appropriate physical process, 934 

natural flavoring substance, 933 

flavouring preparation, 935 

Countercurrent chromatography (CCC), 20 

droplet countercurrent chromatography (DCCC), 20, 21 

rotation locular countercurrent chromatography 

(RLCC), 20–21 

Countries producing essential oils, 84–85 

COX-2. See Cyclooxygenase-2 (COX-2) 

CPE. See Cytopathic effect (CPE) 

Creeping sage, 264 

Creeping thyme, 260 

CRINA ® Pigs, 889 

CRINA Poultry Study, 887–888 

Crispy Coconut Flakes (Flexible Asian Spice Variation), 868 


Crithmum maritimum, 264 

Croton cajucara, 247 

Croton fl avens L., 239 

Croton nepetaefolius, 240, 337 

Croton sonderianus, 243 

Croton urucurana, 271, 272 

Crudities, 873 

Cryogenic trapping, 8 

Cryptococcus neoformans, 779, 788 

Cryptoporic acids (307–317, 316), 793 

Cryptoporus volvatus, 791, 792 

Cryptosporidium, 885 

Cryptotaenia japonica, 272 

Cryptotenia canadensis, 763 

CT-26 (different human cancer cell lines), 237 

Culinary arts 

on exquisite ingredients and accomplished 

rounding, 864 

Cultivation measures, 69 

Cumin, 263 

Cuminaldehyde, 621 

Cuminum cyminum, 263 

Cunninghamella bainieri, 770, 775 

Cunninghamella blakeseeana, 763 

Cunninghamella blakesleeana, 769 

Cunninghamella echinulata, 768, 770, 775, 779, 780, 782, 

788, 790 

Cunninghamella elegans, 763, 788, 790, 803, 806, 811 

Cunninghamella vechinulata, 770 

(-)-Cuparene (322), 794 

(+)-Cuparene (324), 795 

Cupressaceae, 246, 264 

Cupressus sempervirens, 331 

Curcudiol (282n), 789 

(S)-(+)-Curcuphenol (282g), 789 

Curcuma aromatica, 761, 763 

Curcuma domestica Val., 248 

Curcuma longa L., 27, 267 

Curcuma wenyujin, 763 

Curcuma zedoaria, 761, 763 

Curcumin, 261 

Curdione (120), 764 

Current Flavouring Directive 88/388/EC 

“Biologically Active Substances”, Levels of, 927 

“Natural”, Definition of, 927–928 

Current Flavouring Directive and Future Flavouring 

Regulation 

Impact, on Essential Oils, 926–927 

Current Flavouring Directive 88/388/EC 

“Biologically Active Substances”, Levels of, 927 


Future Flavouring Regulation, 

“Biologically Active Substances”, Levels of, 

929–933 

“Natural”, Definition of, 933–935

956 Index 

Curtis, John B., 846 

Curvularia lunata, 756, 783, 785, 786, 802, 803 

Cyanobacterium, 596, 610 

Cyclic monoterpene epoxide, 670 

Cyclic monoterpenoids 

metabolic pathways of, 603 

(+)-Cyclocolorenone (98) biotransformation 

by Aspergillus niger, 760 

(-)-Cyclocolorenone (103) biotransformation 


Cyclodextrin complexation 

of volatiles, 860 

Cyclodextrin derivatives, 14, 16 

Cyclodextrin, α-Glucose unit of, 15 

Cyclodextrins, 859 

(+)-Cycloisolongifol-5β-ol (445e) biotransformation 

by Cunninghamella elegans, 813, 818 

Cyclomyltaylan-5-ol (362), 799 

Cyclooxygenase-2 (COX-2), 242 

Cylas formicarius, 787 

Cymbopogon citratus, 240 

Cymbopogon fl exuosa, 569 

Cymbopogon giganteus, 249 

Cymbopogon winteranus, 218 

Cymbopogon winterianus, 884 

Cymbopogon winterianus Jowitt (Poaceae), 300 

p-Cymene, 132, 247, 251, 253, 261, 262, 265, 268, 272, 

305, 712 

Cynanchum stauntonii, 245 

Cyperaceae, 239 

Cyperus rotundus, 239 

Cytochrome P-450 biotransformation 

by sesquiterpenoids, 819 

Cytopathic effect (CPE), 245 

D 

DAB 6. See “Deutsches Arzneibuch 6” (DAB 6) 

Dadai, 263 

Damascones, biotransformation of, 823–828 

Dangerous goods, 908 

dangerous substance and, 907–908 

interrelation between, 908 

packing of, 908–910 

Dangerous substance, 908 

consignments of, 909 

and dangerous goods, 907–908 

interrelation between, 908 

Danio rerio, 568 

Dark opal basil, 269 

Daucus carota, 320 

DB-Wax, 16 

DCCC. See Droplet countercurrent chromatography 

(DCCC) 

De Materia Medica, 555 

Deans-type pressure balancing, 17 

Decongestants, 552 

DEET, 884 

Dehydroaromadendrene, 237 

8,9-Dehydromenthenone, 718 

8,9-Dehydronootkatone (25) biotransformation 

by Aspergillus cellulosae, 749 

by Aspergillus sojae, 749 

by Marchantia polymorpha, 750 

(-)-Dehydrocostuslactone (249), 782, 783, 784 

Delayed-type hypersensitivity, 253 

Δ-3-Carene, 304 

Demotic medical papyri, 555 

DEN-2, 245, 246 

1-Deoxy-d-xylulose-5-phosphate synthase gene, 54 

Deoxyribose assay, 258, 265 

Deoxyxylulose phosphate reductoisomerase, 54 

Dermasport®, 331 

Dermatophagoides farina, 317 

Dermatophagoides pteronyssinus, 317 

Dessert, cakes and baked good recipes 






Desynchronization, 285 

Detergent Regulation, First Amendment, 925–926 

“Deutsches Arzneibuch 6” (DAB 6), 353 

Devil in the bush, 268 

Dextran edema test, 240 

Diabetes, 562 

Dicots, families of, 41 

Didiscus axeata, 787 

Diepoxide, 709 

Diethyl nitrosamine, 236 

Die Toiletten Chemie, 849 

Different human cancer cell lines. See CT-26 (different 

human cancer cell lines) 

Differential scanning calorimetry, 254 

Dihydro-α-santonin (187), 774 

Dihdyrocarvone, 717 

Dihydroasarone (559), 829 

Dihydrobottrospicatol, 718 

Dihydrocapsaicin (600), 832, 833 

8-nor-Dihydrocapsaicin (601), 833 

Dihydrocarveol, 639, 718 

Dihydrocarveol-8,9-epoxide, 717, 718 

Dihydrocarvone, 718 

Dihydrocarvone-8,9-epoxide, 717 

Dihydrocitronellol, 590 

Dihydrofolic acid, 237 

Dihydromycenol, 602 

Dihydronootkatone (17), 748 

Dihydronootkatone (17), biotransformation of, 748 

1,2-Dihydrophellandral, 620 

4,9-Dihydroxy-1-pmenthen-7-oic acid, 718 

8,9-Dihydroxy-1-p-menthene, 717 

2,9-Dihydroxy-3-pinanone, 709 

6,7-Dihydroxyfenchol, 709 

2,5-Dihydroxy-pinanone, 709 

1,3-Dihydroxythujone, 717 

Diisophorone (488a), 819 

DIL. See Dilution test (DIL) 

Dill, 67, 848 

Dillapiol, 247 

Dilution test (DIL), 354–355 


bitter fennel, 370 




Ceylon cinnamon leaf oil, 391

Index 957 


clary sage oil, 401–402 








mandarin oil, 454 


neroli oil, 467 

nutmeg oil, 469–471 





star anise oil, 502 


tea tree oil, 513–519 


3,7-Dimethyl-1,7-octanediol, 590 

3,7-Dimethyl-1-propargylxanthine (DMPX), 242 

(R)-5,5-Dimethyl-4-(3′-oxobutyl)-4,5-dihydrofuran-2- 

(3H)-one, 720 

3,7-Dimethyl-6-octene-1,2-diol, 720 

Dimethyl sulfoxide (DMSO), 264 

3,4-Dimethylvaleric acid, 717 

2,4-Dinitrochlorobenzene (DNCB), 253 

Diol, 720 

Dior, 850 

Dioscorides, 555 

1,1-Diphenyl-2-picrylhydrazyl, 251, 257 

2,2-Diphenyl-1-picrylhydrazyl (DPPH), 257, 262, 265, 

266, 270, 271 

Diplodia gossypina, 587, 607, 720, 751, 754, 807, 813 

Diplophyllum species, 792 

Diplophyllum serrulatum, 788 

1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), 242 

Directive 1999/45/EC, 936 

Directive 67/548/EEC, 936 

Distillates, 844 

Distillation, 99, 103, 116 

art of, 87 

extraction apparatus, 7 

solvent extraction, 6 

Distillation chimie fine aroma process, 110 

Distilled oils, 937 

Distinctness, uniformity, and stability, 64 

DLD-1 (human colon adenocarcinoma cell line), 237 

DMPX. See 3,7-Dimethyl-1-propargylxanthine (DMPX) 

DMSO. See Dimethyl sulfoxide (DMSO) 

DNA, 52–53 

DNA-barcoding, 53 

DNCB. See 2,4-Dinitrochlorobenzene (DNCB) 

Dodge and Olcott Inc., 844 

Dogs, 883 

Domestication and systematic cultivation, 59–60 

Douglas fir oil, 883 

DPCPX. See 1,3-Dipropyl-8-cyclopentylxanthine 

(DPCPX) 

DPN/DPNH, 122 

DPPH. See 2,2-Diphenyl-1-picrylhydrazyl (DPPH) 

Dr Pepper, 846 

Dragon’s blood, 271 

drim-9α-Hydroxy-11,12-diacetoxy-7-ene (301), 792 

Drimenol (289), 791 

Dromiceius novaehol-landiae, 561 

Droplet countercurrent chromatography 

(DCCC), 20, 21 

Dry-column chromatography, 18 

Dry distillation, 112 

Dumas, M. J., 3 

Dunaliella sp., 709 

Dunaliella tertiolecta, 592, 824 

Dwarf pine oil, 535 





E 

Ear Mites, 885 


Eau de Cologne, 843 

Eau Savage, 850 

EC-I, 250 

EC-II, 250 

EC-III, 250 

EC-IV, 250 

ECB/JRC (the European Chemical Bureau/Joint Research 

Centre of the European Commission), 905 

EFFA CoP. See European Flavour and Fragrance 

Association Code of Practice (EFFA CoP) 

EI-mass spectrum, of essential oil, 29 

Eimeria, 887 

Eimeria tenella, 888 

Eimeria furfuracea, 921 

Eimeria prunastri, 921 

Electroencephalogram, 285 

Electron-capture detector, 13 

Elemol (495), 821 

Elettaria cardamomum, 254, 329, 333 

Elionurus elegans, 272 

Emulsifiers and Administering Forms, 865 

Emu oil, 561 

Enantioselective GC, 15, 164 

Enantioselective multidimensional gas chromatography 

(Es-MDGC), 165, 168, 196 

Encapsulation, 855 

of essential oils 

in hydrophilic polymer, 859 

procedures, 858 

polymer used for, 858 

of volatiles, 857 

Enfleurage, 555 

Ennever, W.A., 899 

Enterobacter, 738 

Enteroplant®, 325 

Enterotoxigenic Escherichia coli, 888 

ent-1α-Hydroxy-β-chamigrene (367), 799 

Entrée recipes 

salads, 872–873 

soups, 871 

Entry into Force, 932 

Enzymatic antioxidants, 256 

Epicoccum purpurascens, 746

958 Index 

Epidermal carcinoma, 239 

Epidermophyton fl occosum, 319 

Epi-nepetalactone, 305 

Epling, 245 

8,9-Epoxy-1-p-menthanol, 709 

2,3-Epoxycitronellol, 593 

8,9-Epoxydihydrocarveol, 718 

8,9-Epoxydihydrocarveyl acetate, 718 

6,7-Epoxygeraniol, 593 

6,7-Epoxynerol, 593 

Eppendorf MicroDistiller®, 8 

Eremanthus erythropappus, 243, 253 

Eriocephalus dinteri, 253 

Eriocephalus ericoides, 253 

Eriocephalus klinghardtensis, 253 

Eriocephalus luederitzianus, 253 

Eriocephalus merxmuelleri, 253 

Eriocephalus pinnatus, 253 

Eriocephalus scariosus, 253 

Escherichia coli, 888 

Es-MDGC. See Enantioselective multidimensional gas 

chromatography (Es-MDGC) 

Essence oil, 114 

Essential oil-bearing plants, 39 

Essential oil combination, 888 

Essential oil mixture, 887 

Essential oil of wine yeast, 86 

Essential oils, phytotherapeutic uses of, 315 

acaricidal activity, 316–317 

allergic rhinitis, 342 

anticarcinogenic, 317 

antimicrobial 

antibacterial, 317–318 

antifungal, 318–319 

antiviral, 319–320 

atopic dermatitis, microflora controlling 

in, 323 

fungating wounds, odor management 

for, 323 

oral cavity, microbes of, 320–322 

functional dyspepsia, 325–326 

gastroesophageal refl ux, 325 

hepatic and renal stones, dissolution of 

gall and biliary tract stones, 323–324 

renal stones, 325 

hyperlipoproteinemia, 326–327 

irritable bowel syndrome, 327–328 

nausea, 329 

pain relief, 329 

dysmenorrhea, 330 

headache, 330 

infantile colic, 331 

joint physiotherapy, 331 

nipple pain, 331 

osteoarthritis, 331 

postherpetic neuralgia, 331–332 

postoperative pain, 332 

prostatitis, 332 

pruritis, 332 

pediculicidal activity, 332–333 

recurrent aphthous stomatitis, 333 

respiratory tract 

1,8-cineole, 336–342 

menthol, 333–336 

snoring, 342 

swallowing dysfunction, 342–343 

Esters, 904 

determination, 155 

Estragol, 930, 931 

Estragole, 128 

Ethers, 904 

Ethyl everninate, 123 

Ethyl phenylacetate, 884 

Ethylalcohol, 250 

Etodolac, 250 

Eucalyptus camaldulensis, 67 

Eucalyptus citriodora, 249 

Eucalyptus globules, 331, 333 

Eucalyptus globulus, 249, 265, 317, 552, 885 

Eucalyptus, 265 

Eucalyptus oil, 535, 883 





Eucalyptus paucifl ora, 319 

Eucalyptus perriniana, 829, 830, 831 

Eucalyptus radiate, 320 

Eucalyptus tereticornis, 249, 337 

EU Commission, 920, 928 

Eudesmenes (138, 141, 143), 766 

Eudesmenone (152a), 767 

Eugenia brasiliensis, 250 

Eugenia caryophyllata Thunb (Myrtaceae), 300 

Eugenia caryophyllata, 238, 250, 251 

Eugenia involucrate, 250 

Eugenia jambolana, 250 

Eugenol, 238, 250, 269, 298, 303, 884, 887 

Eugenol (586), 831 

Eugenia oil, 254 

Eugenyl acetate, 250 

Euglena gracilis, 824, 829 

Euglena gracilis Z, 591, 722–723 

Euglena sp., 709 

EU legislation, on flavors and fragrances 

cosmetic and detergent legislation and allergen 

labeling, 918–920 

aromatic natural raw materials, impacts on, 

921–922 

cosmetic directive and its seventh amendment, 

920–921 

first amendment of, 925–926 

sensitization to fragrances, data on, 922–925 

current flavouring directive and future flavouring 

regulation 

current flavouring directive 88/388/ec, 927–928 

future flavouring regulation, 928–935 

impact, on essential oils, 926–927 

hazard classification and labeling of, 935–939 

EU Parliament, 928 

Eupatorium patens, 245 

Euphorbiaceae, 239, 240, 243, 271 

European Association of the Flavour and Fragrance 

Industry, 910 

European Chemical Bureau/Joint Research Centre of the 

European Commission. See ECB/JRC 

(European Chemical Bureau/Joint Research 

Centre of the European Commission)

Index 959 

European Flavour and Fragrance Association Code of 

Practice (EFFA CoP), 909, 925, 935–936 

recommendations, 936 

European Flavour Industry, 934 

European Inventory of Existing Commercial Chemical 

Substances, 905, 907 

used in EU, 907 

European List of Notified Chemical Substances, 905, 907 

European Pharmacopoeia, 353 

essential oil monographs in, 882 

Eurotium rubrum, 777, 779 

Eurotrium purpurasens, 743 

Evaporation residue, determination of, 154 

Expression, 95–99, 555 

Exserohilum halodes, 764, 766 

Extracts, 844, 849 

F 

F&F Industry, 921 

Fabriek, 843 

Fahnestock, Samuel, 845 

FairWild, 73 

False bottom apparatus, 101 

Farina, Jean Maria, 843 

Farnesol, 136, 137, 138, 227, 231, 236, 922 

2E,6E-Farnesol (478), 823 

2Z,6Z-Farnesol (478), 828 

trans-Farnesyl acetate, 238 

Fast and ultrafast GC, 13 

Fast GC for essential oil analysis, 159–162 

Fe iii tripyridyltriazine, 271 

Felix Cola, 849 

Fenchol, 697, 709 

Fenchone, 217–218, 563, 709 

Fenchoquinone, 709 

Fenchyl acetate, 697 

Fennel oil, 889 

Fennel flower, 268 

Fennel fruit, 888 

Ferric-nitrilo-acetate, 236 

Ferric reducing antioxidant power (FRAP), 271 

Ferula galbanifl ua, 554 

Ferulic acid derivatives, 129 

Fetal calf serum, 247 

Feverfew, 884 

Ficoidaceae, 259 

FID. See Flame ionization detector (FID) 

Flacourtiaceae, 250 

Flame ionization detector (FID), 12 

Flammability, 912 

Flavine, 256 

Flavor and Extract Manufacturers Association, 565 

Flavoring preparation, 928 

Flavors, 845–846 

contents, in food products 

and essential oils, 852 

industrial manufactures of, 845 

Flavors and fragrances 

leading producers of, 849 

Flavouring Directive 88/388/EC, 926 

Flavoring preparation, 934 

Flavoring Regulation, 935 

Fleas and Ticks, 884 

Floral water, 102 

Florentine flask, 106 

oil and muddy water in, 107 

Flourencia cernua, 886 

Foeniculum vulgare, 329, 330, 331, 888, 889 

Folded oil, 114 

Food, scope of essential oils in, 187 

chemical assay requirements and chemical 

description, 190–193 

chemical composition and congeneric groups, 

188–190 

flavor functions, processing for, 188 

plant sources, 187–188 

Food Improvement Agents Package, 928 

Food machinery corporation-in-Line, 98 

Foodstuffs and beverages 

substances in, 927 

Forest red gum, 249 

Formalin, 243 

Formalin test, 240 

4β-Hydroxy-eudesmane-1,6-dione (146), 766 


Fourier transform infrared spectrometer, 23 

Fragrance and flavor industry 

development of, 844 

Fragrance industry, 923 

Fragrances, 568, 844–845 

adverse reactions to, 566 

dosage, in consumer products 

and essential oil contents, 851 

industrial manufactures of, 845 

Fragrances and EOs influence, on brain potentials 

indicative of arousal, 285 

contingent negative variation, 289–290 

spontaneous electroencephalogram activity, 285–289 

Frankincense, 554 

Franz diffusion cells, 254 

FRAP. See Ferric reducing antioxidant power (FRAP) 

Free Radical Scavenging Assay, 257 

Free radicals, 256 

French Pharmacopoeia X, 353 

Fruit oils, 91 

Frullania dilatata, 792 

Frullania tamarisci, 776, 792 

Frullanolide (226), 778 

Fusarium culmorum, 804 

Fusarium equiseti, 783 

Fusarium lini, 803, 811 

Fusobacterium nucleatum, 320, 321 

Future Flavouring Regulation 

“Biologically Active Substances”, Levels of, 929–933 


G 

GABA. See γ-Aminobutyric acid (GABA) 

Galbanum, 554 

Galangal oil, 254 

γ-aminobutyric acid (GABA), 302 

type A antagonist, 243 

γ-cyclocostunolide (171), 773 

γ-eudesmol (157a), 768 

(+)-γ-gurjunene (228), 778 

γ-interferon, and IL-4, 249

960 Index 

γ-methylionone, 922 

γ-selinene, 272 

γ-terpinene, 253, 262, 264, 265, 616 

Gamble, James, 845 

Ganoderma applanatum, 587, 720 

Garlic oil, 885 

Gas chromatographic enantiomer characterization, 

164–165 

Gas chromatography (GC), 12, 157–158 

fast and ultrafast GC, 13 

chiral GC, 13–15 

comprehensive multidimensional GC, 18 

two-dimensional GC, 15–18 

Gas chromatography-atomic emission spectroscopy 

(GC-AES), 24 

Gas chromatography-combustion IRMS device 

(GC-C-IRMS), 24 

Gas chromatography-Fourier transform infrared 

(GC-FTIR), 13, 24 

Gas chromatography-isotope ratio mass spectrometry 

(GC-IRMS), 24 

Gas chromatography-mass spectrometry (GC-MS), 6, 

21–23, 158–159, 238, 261, 262, 921 

Gas chromatography-olfactometry systems, 162–164 

Gas chromatography-pyrolysis-IRMS (GC-P-IRMS), 24 

Gas chromatography-ultraviolet spectroscopy (GC-UV), 

23, 24 

Gaultheria procumbens, 331 

Gaultheria procumbens, 886, 887 

GC. See Gas chromatography (GC) 

GC × GC. See Comprehensive two-dimensional gas 

chromatography (GC × GC) 

GC-AES. See Gas chromatography-atomic emission 

spectroscopy (GC-AES) 

GC-C-IRMS. See Gas chromatography-combustion 

IRMS device (GC-C-IRMS) 

GC-FTIR. See Gas chromatography-Fourier transform 

infrared (GC-FTIR) 

GC-IRMS. See Gas chromatography-isotope ratio mass 

spectrometry (GC-IRMS) 

GC-MS. See Gas chromatography-mass spectrometry 

(GC-MS) 

GC-P-IRMS. See Gas chromatography-pyrolysis-IRMS 

(GC-P-IRMS) 

GC-UV. See Gas chromatography-ultraviolet 

spectroscopy (GC-UV) 

Generally regarded as safe, 883 

Genetic engineering, 53–54 

Genetic variation and plant breeding, 61–63 

artificially generated new variability, 63 

extended variability, 61–63 

natural variability exploitation, 61 

Geraniaceae, 249 

Geranial, 271 

Geranic acid, 588 

Geraniol, 133, 218, 219, 236, 237, 249, 304, 306, 

588, 714 

Geranium oil, 848, 883, 884 

Geranium species, 551 

Geranyl acetate, 249, 884 

cis-Geranyl acetone (463) biotransformation 


trans-Geranyl acetone (470) 


Germacrene B, 250 

Germacrene D, 250, 261, 268 

Germacrone, 239 

Germacrone (118) biotransformation, 761, 763 


by Curcuma aromatica, 762 

by Curcuma zedoaria, 762 

by Solidago altissima cells, 764 

Giardia duodenalis, 885 

Giardia, 885 

Gibberella fujikuroii, 802, 808 

Gibberella suabinetii, 765, 768 

Gil-Av, 14 

Ginger, 848 

Ginger Ale, 845 

6-Gingerol (613), 834 

Ginsenol (435), 813 

biotransformation by Botrytis cinerea, 811 

Glibenclamide, 243 

Gliocladium roseum, 764, 766 

Global demand, for essential oil, 115 

Globally Harmonized System of Classification 

and Labeling of Chemicals, 936 

Globulol (58) biotransformation, 755 



Globulol, 250 

Glomerella cingulata, 594, 612, 754, 755, 763–764, 766, 

777, 779, 787, 804, 805, 806, 811, 816, 817, 821 

10-epi-Glubulol (70) biotransformation 


by Cephalosporium aphidicola, 756 


Glutathione, 256 

Glutathione peroxidase, 256 

Glutathione S-transferase, 237 

Glycine soya, 561 

Gomasio (A Sesame Sea-Salt Spice), 868–869 

Gram-negative bacteria, 886 

Gram-positive bacteria, 886 

Grapefruit leaf oil, 65 

Grapefruit, 848 

Grapeseed, 561 

Grapeseed oil, 891 

Grasse, 844 

Grossulariaceae, 272 

Group I, 922 

Grumichama, 250 

Guadeloupe, 239 

Guaianolide (252a), 782 

Guaiazulene, 138 

Guaicwood oil, 138 

Guaiene (235), 779 

Guaiol (221), 779 

Guaiol (231), 779 

Guava, 250 

Guayava, 265 

Gum turpentine, oil of, 86 

Gynaecological papyrus, 555 

H 

Haemoglobin, 256 

Hairy fingerleaf, 249

Index 961 

Halogenated hydrocarbons and heavy metals, 

determination of, 154 

Hansenula anomala, 824, 825 

Harvest, timing of, 91–92 

Harvesting, 71 

Hassk, 272 

Hazard classification and labeling, of flavors and 

fragrance, 935–939 

Hazard Communication Working Group, 910 

Headspace sorptive extraction, 10–11 

Headspace techniques, 8, 88 

dynamic method, 8–10 

static method, 8 

Heart-cutting technique, 15 

Hedeoma pulegoides, 225 

Hedychium, 272 

Hela, 237, 239 

Helianthus annuus, 561 

Helichrysum dasyanthum, 249, 261 

Helichrysum excisum, 249, 261 

Helichrysum felinum, 249 

Helichrysum italicum, 320 

Helichrysum petiolare, 249, 261 

Helicobacter pylori, 253, 254, 326 

Heliotropin, 844 

Hellandrene, 720 

Hemagglutination valence-reduction test, 245 

hemiterpenoids, 130, 131 

Hep G2, 237, 239 

Hep-2 (human laryngeal cancer cell line), 237 

Hepatic lipid peroxides, 237 

Herb oil, 65 

(-)-Herbertenediol (339), 796 

Herbertus adancus, 793 

Herbertus sakuraii, 793 

Herbromix Study, 888–889 

in vivo experiments, 888–889 

Herpes simplex virus, 244 

Heterothalamus alienus, 246 

Heterotheca latifolia, 245 

Hexamita infl ata, 885 

High performance liquid chromatography (HPLC), 19, 

254 

separation of essential oils, 19 

High-performance liquid chromatography-gas 

chromatography (HPLC-GC), 24–26 

High-pressure valve injector, 26 

High-resolution gas chromatography (HRGC), 6 

High-resolution gas chromatography-Fourier transform 

infrared spectroscopy, 23 

High-speed centrifugal countercurrent chromatography, 

21 

Hinesol (384), 801 

Hinokitiol, 306 

Hinokitiol (589), 831 

Hirzel, Heinrich Dr, 849 

Historical origins, of essential oil production, 86 

History and sources, of essential oil research, 3 

chromatographic separation techniques, 11 

countercurrent chromatography, 20–21 

gas chromatography, 12–18 

liquid column chromatography, 18–19 

supercritical fluid chromatography, 20 

thin-layer chromatography, 12 

first systematic investigations, 3–5 

hyphenated techniques, 21 

gas chromatography-atomic emission 

spectroscopy, 24 

gas chromatography-isotope ratio mass 

spectrometry, 24 

gas chromatography-mass spectrometry, 21–23 

gas chromatography-ultraviolet spectroscopy, 23 

high-performance liquid chromatography-gas 

chromatography, 24–26 

high-resolution gas chromatography-Fourier 

transform infrared spectroscopy, 23 

HPLC-MS, HPLC-NMR spectroscopy, 26 

SFC-MS AND SFC-FTIR spectroscopy, couplings 

of, 27 

supercritical fluid chromatography-gas 


supercritical fluid extraction-gas chromatography, 

26 

multicomponent samples, identification of, 27 

13 


IR apectroscopy, 28 

mass spectrometry, 28 

UV Spectroscopy, 27–28 

preparation techniques, 5 

industrial processes, 5 

laboratory-scale techniques, 5–6 

microsampling techniques, 6–11 

HL-60 (human promyelocytic leukemia cell line), 237, 

238 

Holmskiodiopsis, 249 


Honey Provencal, 869 

Hops oregano, 266 

Hot-plate test, 240, 242 

Hot steam, 88–89 

HPLC. See High performance liquid chromatography 

(HPLC) 

HPLC-GC. See High-performance liquid 

chromatography-gas chromatography 

(HPLC-GC) 

HRGC. See High-resolution gas chromatography (HRGC) 

Human colon adenocarcinoma cell line. See DLD-1 

(human colon adenocarcinoma cell line) 

Human lung carcinoma cell line. See A-549 (human lung 

carcinoma cell line) 

Human melanoma cell line. See M14 WT (human 

melanoma cell line) 

Human promyelocytic leukemia cell line. See HL-60 

(human promyelocytic leukemia cell line) 

Humidity, 89 

Hybrid breeding, 62 

Hydrocarbons, 904 

8-Hydrocarvomenthone, 717 

Hydrodistillation, 100, 103, 116 

Hydroperoxy-octadeca-dienoic acid isomers, 258 

Hydrophilic polymers, 858 

Hydroquinone, 256 

Hydrosols, 883 

8α-Hydroxybicyclogermacrene (108) biotransformation 

by Calonectria decora, 761 

by Mucor circinelloides, 761 

6-Hydroxy-1,2-campholide, 719 

2-Hydroxy-1,4-cineole, 720

962 Index 

9-Hydroxy-1,4-cineole, 720 



6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic 

acid, 257 

1-Hydroxy-3,7-dimethyl-(2E,6E)-octadienal, 596 

1-Hydroxy-3,7-dimethyl-(2E,6E)-octadienoic acid, 596 

1-Hydroxy-3,7-dimethyl-6-octen-2-one, 590 

1-Hydroxy-8-p-menthene-2-one (72), 717 

3-Hydroxy isopiperitenone, 718 

o-Hydroxybenzoic acid, 127 

3-Hydroxy-camphene, 719 



5-Hydroxycamphor, 719 

9-Hydroxycamphor, 719 

5-Hydroxycarveol, 717 

8-Hydroxycarvomenthol, 717 

5-Hydroxycarvone, 717 

8-Hydroxycarvotanacetone, 717, 720 

2-Hydroxycineole, 720 



Hydroxycitronellal, 922, 924 

5-Hydroxydihydrocarveol, 718 

10-Hydroxydihydrocarveol, 718 

1-Hydroxydihydrocarvone, 718 



7-Hydroxyethyl-α-terpineol, 720 

6-Hydroxyfenchol, 709 



4-Hydroxyisopiperitenone, 718 

7-Hydroxyisopiperitenone, 718 

Hydroxyl radical scavenging, 261 

8-Hydroxymenthenone, 718 

6-Hydroxymenthol, 717 

3-Hydroxymyrtanol, 717 

4-Hydroxymyrtanol, 717 

10-Hydroxynerol, 596 

8-Hydroxynerol, 596 

7-Hydroxypiperitenone, 718 

5-Hydroxypiperitone, 718 

8-Hydroxypiperitone, 718 

7-Hydroxy-p-menthane, 720 

7-Hydroxyterpinene-4-ol, 720 

1-Hydroxythujone, 717 

p-Hydroxytoluene, 720 

7-Hydroxyverbenone, 717 

Hymenolepis nana, 885 

Hyphenated techniques, 21 

GC-atomic emission spectroscopy, 24 

GC-isotope ratio mass spectrometry, 24 

GC-mass spectrometry, 21–23 

GC-ultraviolet spectroscopy, 23 

high-resolution GC-Fourier transform infrared 

spectroscopy, 23 

HPLC-GC, 24–26 

HPLC-MS, HPLC-NMR spectroscopy, 26 

SFC-MS AND SFC-FTIR spectroscopy, couplings of, 

27 

SFE-gas chromatography, 26–27 

supercritical fluid extraction-gas chromatography, 26 

Hyptis verticillata, 756, 785 

Hyssop, 884 

Hyssopus offi cinalis, 884 

I 

IFRA. See International Fragrance Research Association 

(IFRA) 

Illegal marketing, in EU 

Not Registered with EINECS and ELINCS 

Nor Compliance with Reach Regulations, 

905–907 

Illex paraguariensis, 270 

Indole, 128 

Indomethacin, 250 

Industrial uses, of essential oils, 843 

changing trends, 849–853 

flavors, 845–846 

fragrances, 844–845 

history, 844 

production and consumptions, 846–849 

Inducible nitric oxide synthetase (inos), 242 

Industrial manufactures 

of essential oils, flavors, and fragrances, 845 

Industrial processes, in essential oil preparation, 5 

Information Network of Departments of 

Dermatology, 922 

Inhalation influence, of number of Eos, 291 

Inhibiory effect, 245 

Insect stress and microorganisms, 90 

Insecticidal, Pest Repellent, and Antiparasitic Oils, 884 

Insects biotransformation, sesquiterpenoids, 819 

Interleukin, 246 

International Federation of Essential Oils and Aroma 

Trades, 899 

International Fragrance Association Code of 

Practice, 920 

International Fragrance Research Association 

(IFRA), 567 

international standard on sustainable wild collection of 

medicinal and aromatic plants (ISSC-MAP), 

59, 72–73 

International Standard Organization (ISO), 910 

In vitro antimicrobial activities, of essential oils, 

353–354, 537–540 

agar diffusion test (ADT), 354 


bitter fennel, 365–369 














mandarin oil, 451–453 


mint oil, 461–463

Index 963 

neroli oil, 465–466 




star anise oil, 500–501 




dilution test (DIL), 354–355 








clary sage oil, 401–402 


















tea tree oil, 513–519 


vapor phase test (VPT), 355 

anise oil, 364 


caraway oil, 379 




citronella oil, 399 


clove leaf oil, 413 

coriander oil, 420 


eucalyptus oil, 428 

juniper oil, 433 


lemon oil, 450 


matricaria oil, 460 

mint oil, 464 


nutmeg oil, 473 

peppermint oil, 481 

Pinus sylvestris oil, 486 



sweet orange oil, 511 



p-Iodonitrophenyltetrazolium violet, 354 

IOFI, 910 

Ionones, biotransformation of, 823–828 

IR spectroscopy, 28 

ISO. See International Standard Organization (ISO) 

Isoeugenol, 129, 305, 563, 696, 922, 924 

Isolimonene, 614 

(+)-Isolongifolene-9-one (442), 806 

biotransformation by Glomerella cingulata, 811 

(-)-Isolongifolol (445a), 818 

(+)-Isomenthol, 627 

ISO-Norm 9235 Aromatic Natural Raw Materials, 904 

Isopinocampheol (3-Pinanol), 693 

Isopiperitenol, 643, 717, 718 

Isopiperitenone, 711, 717, 718 

Isoprobotryan-9 α-ol (458), 813 

biotransformation by Botrytis cinerea, 816 

Isopulegol, 305, 627 

Isovalerianic acids, 883 

ISSC-MAP. See International standard on sustainable 

wild collection of medicinal and aromatic 

plants (ISSC-MAP) 

J 

J774 (mouse monocytic cell line), 237, 247 

J774.A1, 242 

Jamzad, 252 

Jasmine, 563 

biosynthesis of, 125 

Jasmine fragrance, 288, 290, 292, 294 

trans-Jasminlactone, 304 

Jasminum sambac, 40 

Jatamansi oil, 569 

Java, 884 

Java citronella, 70 

Java grass, 300 

Johnson, B.J., 844 

Jojoba, 561 

Juicy Fruit gum, 846 

Jungermannia rosulans, 792 

Junin virus (JUNV), 244, 245, 246 

Juniper oil, 117, 353 





Juniperus communis, 884 

Juniperus procera, 264 

Juniperus rigida, 885 

Juniperus virginiana, 884 

JUNV. See Junin virus (JUNV) 

Jura Chalk of the Haute Provence, 90 

K 

K562 (human erythroleucemic cell line), 237 

Kaneh, 250 

Kekule terpenes, 4 

Keshary–Chien diffusion cells, 254, 255 

Ketodiol, 590 

Ketones, 904 

Klonemax ® , 324 

Kluyveromyces marxianus, 787, 789

964 Index 

KNOWN, 905 

Kölnisch Wasser, 843 

Kunyit, 248 

L 

L1210 leukemia cell, 239 

Labdanum, 554 

Labeling, 910–912 

Laboratory-scale techniques, 5–6 

Lactarius camphorates, 820 

Lactarius graveolens, 46 

Lactone, 720 

Lambli intestinalis, 885 

Lamiaceae (Labiatae), 40, 42–46, 237, 238, 239, 240, 241, 

243, 245, 252, 258, 259, 264, 265, 267, 268, 

269, 272, 906 

Lancerodiol p-hydroxybenzoate (446) biotransformation 

by Marchantia polymorpha cells, 812 


Larvae of Cécidomye, 90 

Lauraceae, 239, 241, 246, 249, 250, 270 

Laurel leaf, 888 

Lauric acid, 256 

Laurus nobilis, 241, 246, 249, 270, 271, 320, 888 

Lavandin oil, 101, 906 

Lavandin oil grosso, 906 

Lavandula angustifolia, 323, 556, 575, 884, 885, 887 

Lavandula angustifolia angustifolia, 906 

Lavandula angustifolia ext., 906 

Lavandula hybrida, 241 

Lavandula hybrida ext., 906 

Lavandula hybrida grosso ext., 906 

Lavandula offi cinalis, 320, 330 

Lavandulae aetheroleum, 887 

Lavender, 90, 93, 94, 883, 884, 906 

Lavender fragrance, 288, 290, 292, 294 

Lavender oil, 300, 302, 535, 883, 906 




obtained in vapor phase test, 440–441 

LC. See Liquid chromatography (LC) 

LC × LC. See Comprehensive liquid chromatography 

(LC × LC) 

Ldengue virus type 2, 244 

Learning and memory, fragrances and EOs on, 293–295 

Least basil, 270 

Ledol (75) biotransformation, 756 


by Cephalosporium aphidicola, 756 


Ledum groelandicum, 320 

Le Livre de Parfumeur, 849 

Lemna minior L., 803 

Lemon, 848 

Lemon balm, Lamiaceae, 271 

Lemon bee balm, 268 

Lemon eucalyptus, 249 

Lemongrass, 240, 848 

Lemongrass oil, 884, 885 

Lemon–lime drink 7UP, 846 

Lemon oil, 28, 302, 535, 883 





Lemon petitgrain, 848 

Leptospermum scoparium, 246, 320 

Lever, William Hesket, 845 

Lichen resinoids, 569 

Licorice plant, 249 

Ligularia fi scheri var. Spiciformis, 248 

Ligularia, 248 

Ligurian yarrow, 261 

Lilac, 255 

Lime oil, conventional and fast-GC separation of, 14 

Limonene, 218–219, 220, 238, 247, 248, 259, 264, 270, 

304, 568–569, 595, 603, 711, 717, 720, 885, 922 

d-Limonene, 236, 254, 924, 925 

S-(–)-Limonene, 306 

Limonene epoxide, 717 

Limonene-1,2-diol, 717 

Limonene-1,2-epoxide, 717 

Linalool, 68, 133, 219, 221, 236, 242, 249, 269, 270, 271, 

272, 293, 298, 301, 303, 569, 594, 596, 709, 

713, 717, 922, 925 

(−)-Linalool, 242, 243 

(R)-(–)-Linalool, 304 

(S)-(+)-Linalool, 304 

Linalool and linalyl acetate, 248 

Linalyl acetate, 221, 306, 596 

Linear retention indices in essential oils analysis, 157–158 

Linfluenza virus A3, 244 

Linoleic acid assay, 258–272 

Linoleic acid model system, 271 

Lipid peroxidation inhibition, 261 

Lipopolysaccharide, 242 

5-Lipoxygenase, 251 

Lippia alba, 46, 47, 298 

Lippia graveolens, 47 

Lippia javanica, 891 

Lippia junelliana, 245 

Lippia multifl ora, 317, 321 

Lippia origanoides, 318 

Lippia sidoides, 253, 322 

Lippia sp., 67 

Lippia turbinate, 245 

Liquid chromatography (LC), 18, 165–167 

high-performance liquid column chromatography, 19 

preseparation, of essential oils, 18–19 

Liquid wax jojoba oil, 255 

Liquidambar orientalis, 554 

Lisbon lemon, 263 

Litsea cubeba oil, 899 

Live-T-connection, 17 

Ljunin virus, 244 

Local lymph node assay, 925 

Location, of essential oil cells, 91 

Locus ceruleus, noradrenergic projections 

from, 284 

Longifolene, 229, 231 

Longifolene (489), 820 

Longitudinal modulated cryogenic system, 172 

L’Oreal, 845 

Lycopine, 256 

Lynderia strychnifolia, 239 

Lysogenic cavities, 40

Index 965 

M 

M14 WT (human melanoma cell line), 237 

M4BEU, 237 

Macadamia integrifolia, 561 

Macadamia nut, 561 

Maceration, 555 

Macrophomina phaseolina, 808 

Main course recipes 


Chévre Chaude-Goat Cheese “Provence”, with 

Pineapple, 875–876 




Majorana hortensis, 8 

Malonyl ester, 720 

Mammals, sesquiterpenoids by 

animals (rabbits) and dosing, 819–820 


Mandarin oil, 535, 848 






Manuka oil, 246 

Marasmius oreades, 789 

Marchantia polymorpha, 747, 774, 776, 792, 806, 812 

Marcusson device, 6 

Maria’s Dip, 873 

Marigolds, 884 

Marjoram, 69 

Market information, 898 

Marketing, of essential oils, 905 

Mass spectrometry, 28 

Massage 

pain relief, 564 

techniques, 559 

Mastic thyme, 259 

Mastic tree resin, 846 

Mastigophora diclados, 793 

Mate, 270 

Material Safety Data Sheet, 899, 909 

Matricaria oil, 535 





Matricaria recutita, 50, 67, 891 

Maximal electroshock, 300 

MCF-7, 237 

MCF7 adrr, 239 

MCF7 WT, 239 

MDGC. See Multidimensional gas chromatography 

(MDGC) 

MDGC-C/P-IRMS device, 25 

MDLC. See Multidimensional liquid chromatographic 

techniques (MDLC) 

Medicines and Healthcare Products Regulatory Agency, 

569 

Megabac®, 323 

Megaleion, 555 

Melaleuca, 244, 262 

Melaleuca alternifolia, 238, 247, 262, 317, 318, 320, 322, 

323, 884 

Melaleuca armillaris, 244, 262 

Melaleuca cajeputi, 885 

Melaleuca ericifolia, 244, 569 

Melaleuca leucadendron, 244, 331 

Melaleuca quinquenervia, 320 

Melaleuca rosalina, 569 

Melaleuca styphelioides, 244 

Melaleuca oil of, 86 

Meleguetta pepper, 262 

Melissa, 254 

Melissa offi cinalis, 92 

Melissa offi cinalis L., 237, 245, 271 

Melissa oil, 570–571, 891 

melting and congealing points, estimation of, 154 

Memory and learning, fragrances and EOs on, 293–295 

p-Mentane-1,2,4-triol, 720 

p-Mentha-1,8-diene-4-ol, 717 

p-Mentha-2,8-diene-1-ol, 717 

Mentha aquatica, 8, 258 

Mentha arvensis, 318, 848 

p-Menthane, 614, 713 

p-Menthane-1,2,8-triol, 717 

p-Menthane-1,2-diol, 709 

p-Menthane-2,8,9-triol, 718 

p-Menthane-2,8-diol, 717, 718 

p-Menthane-2,9-diol, 717 

Mentha piperita, 8, 189, 245, 258, 318, 322, 883, 884, 885 

Mentha pulegium, 225 

Mentha spicata, 318 

Mentha X piperita, 329 

trans-p-2,8-Menthadien-1-ol, 251 

Cis-p-2,8-Menthadien-1-ol, 251 

trans-p-1(7),8-Menthadien-2-ol, 251 

Cis-p-11:(7),8-Menthadien-2-ol, 251 

1-p-Menthen-2-ol, 709, 717 

1-p-Menthene, 614 



8-p-Menthene-1,2-diol, 718 



1-p-Menthene-8-cation, 709 

1-p-Menthene-9-oic acid, 717 

Menthol, 221–222, 236, 333–334, 590, 621, 712, 884, 891 

l-Menthol, 148–149 

Menthone, 712 

Menu 

appetizers and finger food, 868 

basics, 867 

beverages, 867 

dessert, cakes and baked goods, 868 

entrees, 868 

main course, 868 

Meridol®, 321 

Mesopotamian art of distillation, 87 

Methicillin-Resistant Staphylococcus aureus, 318 

Methotrexate, 237 

(-)-Methoxy-α-herbertene (343), 796, 797 

2,5-di-O-Methylacetophenone, 250 

3-Methyl-2-butenoic acid, 590 

2-(4-Methyl-3-cyclohexenylidene)-propionic acid, 717 

2,6-Methyl-3-pentyl-b-cyclodextrin, 16

966 Index 

2,6-Methyl-3-pentyl-β-cyclodextrin, 15 

6-Methyl-5-heptenoic acid, 590 

2-Methyl-6-methylene-7-octen-2-ol, 720 

Methyl 3-methylorsellinate, 123 

Methyl anthranilate, synthesis of, 145 

Methylchavicol, 304 

20-Methylcholanthrene, 237 

N-Methyl-d-aspartic acid receptors, 242 

Methyldihydro eugenol (563), 829 

Methyl eugenol, 243, 244, 305, 885, 930, 931 

Methylheptin carbonate, 922 

Methyl jasmonate, 304 

Methyl nonyl ketone, 884 

2-Methylpropanal, 272 

Methyl salicylate, 883, 884, 886 

Mexican oregano, 46 

MIC. See Minimum inhibitory concentration (MIC) 

Michelia champaca, 777 

Microdistillation, 6–7 

Microhydrodistillation device, 8 

Microorganisms, sesquiterpenoids by, 738–754 

Microorganisms and insect stress, 90 

Microsampling techniques, 6 

direct sampling, from secretory structures, 7–8 

headspace techniques, 8 

dynamic method, 8–10 

static method, 8 

microdistillation, 6–7 

solid-phase microextraction, 10 

stir bar sorptive extraction and headspace sorptive 

extraction, 10–11 

Microsporum canis, 319 

Microsporum gypseum, 319 

Microsporum nanum, 319 

Microwave-assisted hydrodistillation methods, 112 

Migraine headaches, 566 

Minimum inhibitory concentration (MIC), 355 

Minthostachys verticillata, 245 

Mint oil, 535 




Mintoil®, 327 

Mint plant, 272 

Mitomycin C, 238 

Modern analytical techniques, 156–157 

fast GC for essential oil analysis, 159–162 

gas chromatographic enantiomer characterization, 

164–165 

gas chromatography-mass spectrometry, 158–159 

gas chromatography-olfactometry for odor-active 

components assessment, 162–164 

GC and linear retention indices, use of, 157–158 

LC and liquid chromatography hyphenated to MS, 

165–167 

multidimensional gas chromatographic techniques, 

167–174 

multidimensional liquid chromatographic techniques, 

174–176 

on-line coupled liquid chromatography-gas 

chromatography (LC-GC), 176–177 

Modern perfumery, 557 

Molecular markers, 53 

Monarda citriodora, 268 

Monarda didyma L., 268 

Monarda fi stulosa, 133 

Monocyclic monoterpene alcohol, 621 

Monocyclic monoterpene aldehyde, 619 

Monocyclic monoterpene hydrocarbon, 603 

Monocyclic monoterpene ketone, 648 

Monomers α-l-glucuronic acid (G), 859 

Monoterpene alcohol linalool, 265 

Monoterpene aldehydes, 134 

Monoterpene ester, 248 

Monoterpenes metabolism 

α-and β-thujone, 225–226, 228 

α-terpineol, 225, 226 

camphene, 210 

camphor, 210–212 

carvacrol, 212 

carvone, 213 

1,4-cineole, 213–214 

1,8-cineole, 214–216 

citral, 216–217 

citronellal, 217 

fenchone, 217–218 

geraniol, 218 

limonene, 218–219, 220 

linalool, 219, 221 

linalyl acetate, 221 


myrcene, 223 

pinene, 223–225 

pulegone, 225 

thymol, 226 

Monoterpenes, 41, 236 

Monoterpenic phenol, 238 

Monoterpenoid alcohols, 133 

Monoterpenoid ketones, 134–135 

Monoterpenoids, 130, 131–135 

Monoterpenoids, biotransformation of, 585 

insects, 585, 716 

mammals, 585, 712, 716 

metabolic pathways 

acyclic monoterpenoids, 587 

bicyclic monoterpenoids, 677 

cyclic monoterpenoids, 603 

microbial transformation 

by unit reactions, 720–726 

microorganisms 

alcohols and aldehydes, 588–590 

hydrocarbons, 587–588 

metabolic pathways, 709–720 

Monterey Pine, 265 

Moraceae, 238 

Morpho- and ontogenetic variation, 65–69 

Morrison, Wade, 846 

Mosla chinensis, 245 

Mosquitoes, 884 

Moths, 884–885 

Mouse plasmocytoma. See SP2/0 (mouse plasmocytoma) 

MS. See Mass spectrometry (MS) 

Mucolytic and Mucociliary Effects, 337–338 

Mucor, 738 

Mucor circinelloides, 759 

Mucor mucedo, 804 

Mucor plumbeus, 747, 756, 770, 775, 786, 

788, 789, 791, 802

Index 967 

Mucor polymorphosporus, 763 

Mucor ramannianus, 819 

Mucor spinosus, 763 

Mugwort, 245, 261 

Multicomponent samples, identification of, 27 

13 


IR spectroscopy, 28 

mass spectrometry, 28 

UV spectroscopy, 27–28 

Multidimensional gas chromatography (MDGC), 18, 

167–174 

Multidimensional liquid chromatographic techniques 

(MDLC), 174–176 

Multidrug resistance, 239 

Murine leukemia (P388), 239 

Musk, 849 

Mycobacterium smegmatis, 754, 755 

Myli-4-(15)-en-9-one (96a) biotransformation 


Mylia taylorii, 756, 761, 787 

Myoporum crassifolium, 138 

Myratenoic acid, 717 

Myrcene, 223, 238, 304, 587–588, 717, 720, 885 

Myrcene-3,(10)-epoxide, 720 

Myrcenol, 720 

Myrica gale L., 238 

Myricaceae, 238 

Myristic acid, 256 

Myrrh, 554 

Myrtaceae, 238, 239, 246, 247, 249, 250, 258, 262, 265 

Myrtanal, 689 

Myrtanol, 691, 717 

Myrte, 254 

Myrtenal, 689, 709, 717 

Myrtenic acid, 709 

Myrtenol, 690, 709, 717, 720 

Myrtle, 258 

Myrtle leaf, 888 

Myrtol, 262 

Myrtus communis, 258, 265, 888 

N 

NADP/NADPH, 122 

Naloxone, 242 

Naphthalene, 884 

Nardosinone (376), 800, 801 

Nardostachys chinensis, 749, 797 

Nasal decongestant, 334 

Nasturtium spp, 884 

Nasturtiums, 884 

Natural, definition of, 927–928, 933–935 

Natural aromas, 864 

Natural complex substances, 904, 928, 933, 936–937 

Naturally occurring alleged allergenic substances 

structures of, 921–922 

Nature-identical, 933 

Near infrared (NIR), 12 

Negro pepper, 262 

Neomenthol, 627 

Nepalese lemongrass, 569 

Nepeta cataria, 884 

Nepetalactones, 883 

Nepeta oils, 883 

Neral, 271 

Nerol, 588, 590, 714 

Nerolidol, 236, 237 

cis-Nerolidol (462), 813, 814, 816 

biotransformation by Glomerella cingulata, 816 

trans-Nerolidol (469), 821, 822, 824 

Neroli oil, 536 





Neuro-2a (mouse neuroblastoma), 237, 247 

Neurospora crassa, 819 

Neurotoxic aromachemicals, 568 

Niaouli oil, 254 

Nicotiana tabacum, 613, 774, 776 

Nicotine, 884 

Nigella sativa, 237, 240, 268 

NIR. See Near infrared (NIR) 

NIR-FT-Raman spectroscopy, 28 

Nitric oxide, 242 

Nitrogen-containing compounds, 13 

Nitrogen–phosphorus detector, 13 

Nitro musks, 852 

Nitro triazolium blue, 258 

Nocardia corallina, 788, 790 

Noncommercial organizations, 895 

Nonenzymatic antioxidants, 256, 257 

Nonenzymatic lipid peroxidation, 265 

Nonvolatile precursors, volatile controlled release from, 

859–860 

Nootkatone (2) biotransformation 

by Aspergillus niger, 743–745 

by Fusarium culmorum and botryosphaeria 

dothidea, 745–749 

Nootkatone (2) production from valencene (1), 738–741 

Nopol, 699, 720 

Nopol benzylether, 699, 720 

Nopol benzylether (531), 831 

Nor-sesquiterpene ketone khusimone, 21 

Novak, Johannes, 91 

N-ras-Oncogene, 238 

N-ras transformed mouse myoblast cell line. See CO25 

(N-ras transformed mouse myoblast cell line) 

Nuclear-factor-κ-B (human mouth epidermal carcinoma 

cell line), 237 

Nuclear magnetic resonance, 117 

Nutmeg, 89 

Nutmeg oil, 536 

composition, with main hazardous constituents, 938 





O 

Oakmoss, 921, 922, 923 

fragrance industry, 924 

Obovata, 249 

Ocimene, 259, 720 

trans-Ocimene, 305 

allo-Ocimene, 305 

Ocimum, 46

968 Index 

Ocimum basilicum, 219, 239, 269, 270, 298, 890 

Ocimum gratissimum, 249, 317, 318 

Ocimum micranthum, 243, 270, 890 

Ocimum volatile oils, 885 

Ocotea bofo, 263, 264 

Ocotea pretiosa, 569 

(Z)-9-Octadecenamide, 272 

Odor pleasantness, 287 

Odor, sex-specific effect of, 281 

Odor-active components, assessment of, 162–164 

Odorants, 283 

psychoactive effects, 290 

Odorous fatty oils, 88 

Oenothera biennis, 561 

Oil-laden steam, 111 

Oil of Geranium ISO/DIS 4730, 86 

Oils against pests 

antiparasitic, 885–886 

aphids, caterpillars, and whiteflies, 885 

ear mites, 885 

fleas and ticks, 884 

insecticidal, pest repellent, and antiparasitic oils, 884 

mosquitoes, 884 

moths, 884–885 

Oils attracting animals, 883 

Oils in animal feed 

pigs, 889–890 

poultry, 887–889 

ruminants, 886–887 

Oils repelling animals, 883–884 

Oils Treating Diseases, in Animals, 890–891 

Old Corner Drug Store, 846 

Olea europaea, 561 

Oleine, 845 

Oleoresins, 852 

Oleoropeic alcohol, 720 

Oleuropeic acid, 709 

Olfaction, 282 

Olibanum americanum, 554 

Olive, 561 

Omum, 267 

1α-Hydroxymaaliene (115) biotransformation 


On-line coupled liquid chromatography-gas 


On-site/container distillation, 107 

Open mouth loading, 102 

Opoponax chironium, 554 

Opoponax, 554 

Orange, 848 

Orange oil, 254 

hazardous constituents, 938 

Orange peels, 297 

Orange terpenes, 563 

Oregano, 43, 44–45, 68, 883 

Oregano herb, 888 

Oregano oil, 45, 887, 888, 890 

Origanum, 45, 886 

Origanum acutidens, 266 

Origanum fl oribundum, 266 

Origanum glandulosum, 266 

Origanum majorana, 45, 266 

Origanum marjorana, 333 

Origanum onites, 45, 238, 316, 884, 888 

Origanum syriacum L., 265 

Origanum vulgare, 40, 888, 891 

Orris distillation, 107 

Ovalbumin, 251 

Oxidative stress, 256 

2-Oxo,3-hydroxygeraniol, 590 

6-Oxo-1,2-campholide, 719 

2-Oxo-1,8-cineole, 720 

3-Oxo-1,8-cineole, 720 

5-Oxocamphor, 719 

6-Oxocamphor, 719 

2-Oxo-cineole, 720 

3-Oxonopol-2′,4′-dihydroxybenzylether, 720 

9-Oxo-trans-nerolidol (487), 818 

3-Oxoverbenone, 720 

Oxygen radicals, 256 

Oxygen-containing compounds, 13 

P 

P388 (murine leukemia cell line), 237 

Padova System, 110 

Paecilomyces varioti, 791, 793 

Paeonia moutan, 250 

Pallavicinia subciliata, 740 

Palmolive, 844 

Palmolive–Peet, 844 

Panax ginseng, 806 

Papyrus Ebers, 555 

Papyrus Edwin Smith, 555 

Papyrus Hearst, 555 

Paranix®, 333 

Parasites, 885 

Parthe nium Tometosa, 759 

Parthenin (264a), 785 

Parthenium argentatum, 759 

Parthenolide (240), 780, 781 

Patchouli, 563, 900 

Patchouli alcohol, 230, 232 

Patchouli oil, 139 

Patchoulol (425) biotransformation 

by Botrytis cinerea, 810 

PBQ-induced abdominal constriction test, 240 

PC-3, 237 

PCI X 10 method, 191 

Peet, 844 

Pelargonium, 331, 551, 884 

Pelargonium graveolens, 249 

Pellatrici method, 97, 98 

Peltate glands, 40 

Pemberton French Wine Coca, 846 

Pemberton, John S., 846 

Penicillium chrysogenum, 788, 790 

Penicillium digitatum, 590, 594, 609, 720 

Penicillium frequentans, 765 

Penicillium janthinellum, 763 

Penicillium sclerotiorum, 792, 793, 796 

2,3-Pentyl-6-methyl-β- and -γ-cyclodextrin, 15 

Pentylenetetrazole-induced convulsions, 299 

Peppermint, 53, 54, 68, 69, 258, 291, 294, 552, 848 

Peppermint essential oil 

medical examinations, 328–329 

Peppermint herb oil, 68 

Peppermint oil, 221, 255, 536, 883, 885

Index 969 





price graph of, 900, 901 

Pepsi-Cola, 846 

Perforated sieve-like plates, 102 

Perfume, 554–555 

methods of producing, 555 

Perfumery Technology, 850 

Perilla frutescens, 46 

Perilladehyde, 718 

Perillaldehyde, 272, 619, 709 

Perillic acid, 709, 718 

Perillyl alcohol, 236, 645, 711, 717, 718 

Perillyl alcohol-8,9-epoxide, 718 

Peritoneal leukocytes (ptls), 248 

Perkin Elmer gas chromatograph, 12 

Petitgrain still, 898 

Petitgrain, 848 

Phelandrol, 720 

Phellandral, 620, 713, 720 

Phellandrene, 715, 717, 720 

Phellandric acid, 720 

Phellandrol, 720 

Phellodendron sp., 65 

Phenol, 143, 144 

Phenotypic variation, in essential oils, 61 

2-Phenylethanol, 128 

2-Phenyl ethyl alcohol, 304 

Phenylpropanes, 904 

Philippe, King, 844 

phosphoenolpyruvate, 122 

Photosensitivity, 568 

Phototoxicity, 568 

Phyllosticta capsici, 783 

Physiological serum, 250 

Phytochemical variation 

chemotaxonomy, 41–42 

inter- and intraspecific variation, 42–52 

Phytol, 238 

Phytolacca americana, 829, 830 

Pigs, 889–890 

Pimpinella anisum, 884, 887, 891 

Pimpinella aurea, 891 

Pimpinella corymbosa, 891 

Pimpinella isaurica, 891 

Pinaceae, 237, 246, 265 

Pinane-2,3-diol, 692, 709 

Pine oil, 883 

Pine, 848 

Pinene, 223–225 

Pine-needle oil, 883 

Pinguisanol (373), 800 

Pinimenthol®, 342 

Pinocarveol, 691 

Pinocarvone, 65 

Pinus brutia, 885 

Pinus halepensis, 885 

Pinus longifolia, 229 

Pinus pinaster, 885 

Pinus pinea, 885 

Pinus radiate, 265 






Pinyl cation, 709 

Pipe bundle condenser, 103 

Piperaceae, 265 

Piper crassinervium, 265 

Piperita, 900 

Piperitenol, 643 

Piperitenone, 711, 717, 718 

Piperonyl, 885 

Pipe-stem tree, 249 

Pityrosporum ovale, 319 

Placebo effect, 291 

Plagiochila fruticosa, 758 

Plagiochila sciophila, 757, 793, 795, 797 

Plagiochilide (105), 758 

Plagiochiline A (104), 758 

plagiochiline C (104) biotransformation 


Plant breeding 

and genetic variation. See Genetic variation and 

plant breeding 

and intellectual property rights, 63 

plant protection (plant patents), 64–65 

plant variety protection, 64 

Plant material production, factors influencing, 60–71 

cultivation measures, contaminations, and 

harvesting, 69–71 

environmental influences, 69 

genetic variation and plant breeding, 61–63 

intraindividual variation, between plant parts, 65–69 

plant breeding and intellectual property rights, 63–65 

Plant nutrition and fertilizing, 70 

Plants, 89, 90 

Plant sources, 187–188 

Plaque-reduction assay, 244 

Plasmodium falciparium, 788 

Plasmodium falciparum, 779 

Pleasant odors, 292, 296 

Pleurotus fl abellatus, 587, 720 

Pleurotus ostreatus, 803 

Pleurotus sajor-caju, 587, 720 

Poaceae Cymbopogon giganteus, 251 

Poaceae, 240, 249 

Pogostemon cablin, 323 

Polyanthes tuberose, 40 

Polydimethylsiloxane, 10 

Polygodial, 788 

Polygodiol (295), 791 

Polygonum hydropiper, 788 

Polyketides and lipids, 123–126 

Pomades, 844, 849 

Porella pettottetiana, 798 

Porella stephaniana, 765 

Porella vernicosa, 788 

Poronia punctata, 808 

Porophyllum ruderale, 248 

Porphyromonas gingivalis, 320, 321 

Portuguese Presidency Council, 928 

Poultry, 887–889 

CRINA Poultry Study, 887–888 

Herbromix Study, 888–889

970 Index 

Powis castle, 246 


Preparation, of essential oils, 5, 18–19 

industrial processes, 5 

laboratory-scale techniques, 5–6 

microsampling techniques, 6 

direct sampling, from secretory 

structures, 7–8 

headspace techniques, 8–10 

microdistillation, 6–7 

solid-phase microextraction, 10 

stir bar sorptive extraction and headspace sorptive 

extraction, 10–11 

Primrose, 561 

Processing of essential oils for flavor functions, 188 

Procter, William, 845 

Procter & Gamble (P&G), 845 

Production, of essential oils, 83, 84, 88–89 

agricultural crop establishment, 92–94 

biomass used, 91 

climate, 89–90 

commercial essential oil extraction methods, 95 

expression, 95–99 

harvest, timing of, 91–92 

insect stress and microorganisms, 90 

location, of oil cells, 91 

seed and clones, propagation from, 94–95 

soil quality and preparation, 90 

steam distillation, 99–117 

water stress and drought, 90 

Profragrances, 859 

Programmed temperature vaporization injector, 11 

Propionibacterium acnes, 317, 318 

Prostaglandin E2, 242 

Prostaglandins, biosynthesis of, 125 

Protein engineering, 54 

Protium, 247 

Protium grandifolium, 247 

Protium hebetatum, 247 

Protium heptaphyllum, 247 

Protium lewellyni, 247 

Protium strumosum, 247 

Proton NMR spectroscopy, 28 

Prunus amygdalus var. dulcis, 561 

Prunus armeniaca, 561 

Pseudoisoeugenyl esters, 66 

Pseudomonas aeruginosa, 590 

Pseudomonas ceuciviae, 812 

Pseudomonas citronellolis, 589, 590 

Pseudomonas cruciviae, 807 

Pseudomonas incognita, 590, 591 

Pseudomonas mendocina, 590 

Pseudorabies virus (prv), 244 

Psidium guajava, 239, 250, 265 

Psidium widgrenianum, 250 

Psychopharmacology of essential oils, 297 

in animal models, 299 

aromatic plants, as sedatives or stimulants, 297–299 

mechanism of action, 302–306 

Pterocarpus santalinus, 765 

Pulegone, 225, 226, 306, 718 

Pulmonary function, 339 

Pyrethrosin (248c), 782 

Pyridine, 304 

Q 

Qualitative analysis of an essential oil, 29 

Quality Criteria and Specifics 

while Handling Essential Oils, for Food Preparation, 

864–865 

Queen Hatshepsut, 843 

“Queen of Hungary Water”, 843 

Quinone transferase, 237 

R 

R43 Risk Phrase, 925 

Random amplification of polymorphic DNA, 43 

Ranunculaceae, 237, 240, 251, 268 

Rapeseed oil, 141 

Raschig rings, 113 

Raspberry ketone (566), 830 

Rastrello, 96 

Rattus norvegicus, 248 

RAW 264.7 cells, 248 

R.C. Treatt & Co. Ltd, 899 

REACH (Registration, Evaluation, Authorization of 

Chemicals), 904 

Reactive Oxygen Species, 257 

Reboulia hemisphaerica, 795 

Refractive index, determination of, 153 

Registered cultivars, of essential oil plant, 62 

Registration, Evaluation, Authorization of Chemicals. 

See REACH (Registration, Evaluation, 

Authorization of Chemicals) 

Repair enzymes, 256, 257 

Research Institute for Fragrance Materials, 566, 925 

Resinoids, 937 

Resins, 844, 849 

Resources of essential oils, 54 

domestication and systematic cultivation, 59–60 

plant material production, factors influencing, 60–71 

wild collection and sustainability, 58–59 

Respiratory drive and respiratory comfort, 335 

Reticular activating system, 283, 284 

Reverchon “Grosso”, 241 

Rf values, 156 

Rhipicephalus turanicus, 884 

Rhizopus arrhizus, 787, 788, 789, 791 

Rhizopus nigricans, 774, 776, 777, 778, 781, 782 

Rhizopus oryzae, 761, 768, 770, 780, 782 

Rhizopus stolonifer, 770, 775 

Rhizopus stolonifera, 802, 808 

Rhizpctonia solani, 783 

Rhodotorula glutinus, 787, 789 

Rhodotorula minuta, 824 

Rhodotorula rubra, 778, 781 

Rhodotorula sp., 608 

Ribes nigrum L., 272 

RLCC. See Rotation locular countercurrent 

chromatography (RLCC) 

Robinson, Frank, 846 

Rock samphire, 264 

Root essential oil, 65–66 

Ropadiar ® , 890 

Rosa centifolia, 330 

Rosa mosqueta, 561 

Rosa spp., 40

Index 971 

Rose alcohols, 133 


Rose geranium, 249 

Rose hip seed, 561 

Rosemary, 255, 269, 294, 552, 563, 848, 884 

Rosemary oil, 536 





Rose oil, 10, 300 


Rosewood oils, 86 

Rosmarinus offi cinalis, 70, 264, 269, 320, 333, 575, 

884, 885 

Rotation locular countercurrent chromatography 

(RLCC), 20–21 

Rowachol, 325 

Rowatinex, 325 

RP-18 HPLC, 19 

Rubus idaeus, 829 

Rue, 884 

Ruminants, 886–887 

Rutaceae, 95, 236, 252, 263 

Ruta graveolens, 884 

S 

Sacaca, Euphorbiaceae, 247 

Safety evaluation of essential oils, 185–187 

constituent-based evaluation, 187 

constituents, and congeneric groups, safety 

considerations for, 193–195 

food, scope in, 187 

chemical assay requirements and chemical 

description, 190–193 

chemical composition and congeneric groups, 

188–190 

flavor functions, processing for, 188 

plant sources, 187–188 

guide and example, 195–204 

Safrol, 306 

Safrole, 930, 931 

Sage, 42, 884 

Sage leaf, 888 

St Louis World’s Fair, 846 

Salads, 872–873 

Salicylic acid, 127 

Salinity, 70 

Salmonella, 888 

Salmonella choleraesuis, 888 

Salmonella essen, 888 

Salmonella typhimurium, 888 

Salt stress, 70 

Salvia africana-caerulea, 252 

Salvia africana-lutea, 252 

Salvia chamelaeagnea, 252 

Salvia fruticosa, 42, 264, 888 

Salvia. fruticosa, 42 

Salvia L., 42, 264 

Salvia lanceolata, 252 

Salvia offi cinalis, 42, 43, 884 

Salvia repens, 264 

Salvia runcinata, 264 

Salvia sclarea, 330 

Salvia stenophylla, 264 

Sandalwood, 554 

Santalaceae, 244 

Santalol (503), 823 

Santalum album, 244, 554 

Santolina triene, 250 

Sapodilla tree gum, 846 

Sarcoptes scabiei, 317 

Sassafras, 569 

Sassafras oil, 899 

Saturated ketone, 667, 701 

Satureja hortensis, 267 

Satureja hortensis, 240, 241 

Satureja khuzestanica, 252, 333 

Satureja montana L., 267, 268 

Satureja thymbra, 241 

Saussurea, 779 

Saussurea radix, 767 

Savory, 267, 268 

Scavangers, 256 

Schizogenic oil ducts, 40 

Schizosaccharomyces pombe, 804 

Schueller, Eugene, 845 

Scientific Committee on Cosmetic Products and 

Non-Food Products Intended for 

Consumers, 918–920 

Sclareolide (402), 803 

(+)-Sclareolide (402), 805, 806 

Sclerotinia sclerotiorumn, 783 

Scodella method, 97 

Scrophulariaceae, 246 

Sea purslane, 259 

Sea wormwood, 260 

Secretory structures, direct sampling from, 7–8 

Seed and clones, propagation from, 94–95 

Selected standard price, and organic essential oils, 851 

Selective ion monitoring, 21 

Semmler, F. W., 4 

Semple, William, 846 

Senecio mikanioides O., 237 

Sensory memory, 293 

Sesquiterpene-less essential oils, 114 

Sesquiterpenes, 41, 130, 135–140, 250, 820–823, 904 

by cytochrome p-450, 819 

by insects, 819 

by mammals, 819 

by microorganisms, 738 

Sesquiterpenes metabolism 

caryophyllene, 227, 229 

farnesol, 227, 229, 231 

longifolene, 229, 231 

patchouli alcohol, 230 

Sesuvium portulacastrum, 259 

7α-Hydroxyfrullanolide (223), 778 

Seventh Amendment of Cosmetic Directive, 920–921 

SFC. See Supercritical fluid chromatography (SFC) 

SFC-MS and SFC-FTIR spectroscopy, couplings of, 27 

Sfumatrici methods, 97 

Shell ginger, 242 

Shikimic acid derivatives, 126–129 

Shiromodiol diacetate (136), 765 

Shisool, 718 

Shisool-8,9-epoxide, 718

972 Index 

6-Shogaol (608), 834 

Sideritis varoi, 764 

Silica gel, 19 

Silver pipestem tree, 249 

Simmondsia californica, 561 

Sister-chromatid-exchange assay, 238 

Sluggish wormwood, 260 

Sobrerol, 709, 717, 720 

Soda water, 845 

Sodium lauryl sulphate, 256 

Soil quality and preparation, 90 

Soledum®, 336 

Solidago altissima, 757, 763, 820 

Solid-phase microextraction, 6, 10, 17 

Solubility test, 153–154 

Solvent vapor exit, 25 

Šorm, F., 4 

Soups, 871 

Sources, of essential oils, 39 

genetic and protein engineering, 53–54 

identification, of source materials, 52–53 

international standards, for wild collection and 

cultivation 

FairWild, 73 

GA(C)P, 72 

ISSC-MAP, 72–73 

phytochemical variation 

chemotaxonomy, 41–42 

inter- and intraspecific variation, 42–52 

resources of essential oils, 54 

domestication and systematic cultivation, 59–60 

plant material production, factors influencing, 

60–71 

wild collection and sustainability, 58–59 

South American orange juice factory, 897 

Soya bean, 561 

SP2/0 (mouse plasmocytoma), 237, 247 

Span 80, 256 

Spathulenol, 250 

Spathulenol (94), 756 

biotransformation by Aspergillus niger, 759 

(−)Spathulenol, 244 

Special sfumatrici method, 97 

Spectrophotometric assay, 257 

Sphaeranthus indicus, 775 

Spice rack, of essential oils 

oil mixture and oil seasoning, preparation of, 

866–867 

Spiked thyme, 269 

Spike lavender, 556 

Spodoptera litura, 596, 720 

Spodptera litura, 610 

Spontaneous electroencephalogram activity, 285–289 

Sporotrichum pulverulentum, 785 

Squamulosone (77), 756 

biotransformation by Mucor plumbeus, 758 

Staphylococcus aureus, 323, 788 

Staphylococcus epidermidis, 323, 804 

Star anise oil, 536 





Steam distillation, 99–117 

Stir bar sorptive extraction, 10–11 

Storage and transport, of essential oils 

dangerous substance and dangerous goods, 907–908 

illegal marketing, in EU, 905–907 

labeling, 910–912 

marketing, 903–905 

packing of dangerous goods, 908–910 

Strawberry everlasting, 249 

Strawflower, 261 

Streblus asper Lour., 238 

Streptococcus mutans, 320 

Streptomyces bottropensis, 717 

Streptomyces fulvissimus, 777, 778, 781 

Streptomyces ikutamanensis, 593 

Styrax, 554 

Styrax offi cinalis, 554 

Styrene monomer/propylene oxide process, 142 

Subambient trapping technique, 23 

Substances 

in flavorings and food ingredients, 931–932 

not added, to food, 930 

Sugandha kokila oil, 569 

Summer savory, 240, 267 

Sunflower, 561 

Supercritical fluid chromatography (SFC), 20 

Supercritical fluid chromatography-gas chromatography, 

26–27 

Supercritical fluid extraction-gas chromatography, 26 

Superficial cells, 91 

Superoxide anions, 258 

Superoxide dismutase, 256 

Superoxide radical, 256 

Supply chain flowchart 

from distiller to finished products, 897 

Suprathreshold fragrances, 295 

Sweet basil, 269 


Sweet orange oil, 536 





Synthesis of essential oil components, 140–149 

Syphacia obvelata, 885 

Syrian oregano, 265 


Syzygium aromaticum, 271, 331, 885 

T 

Tagetes, 46, 48, 567, 884 

Tagetes lucida, 48 

Tagetes minuta L., 67 

Tagetes oil, 569 

Tail-fl ick test, 240 

Tanacetum parthenium, 777, 884 

Tanaka, 263 

Tansy, 884 


Tarbush, 886 

Tarocco, 263 

Tarocco orange, 263 

TAS procedure, 8 

TCD. See Thermal conductivity detector (TCD)

Index 973 

Tea tree, 238, 244, 262, 536, 848 





Temperature, 89 

Terpene alcohols, 593 

Terpene und Campher, 4 

Terpene-less essential oils, 114 

Terpenes, 4, 151 

Terpenoids, 129–130 

hemiterpenoids, 131 

monoterpenoids, 131–135 


Terpenoids metabolism, in animal models and humans, 

209–210 

monoterpenes 

α- and β-thujone, 225–226, 228 

α-terpineol, 225, 226 

camphene, 210 

camphor, 210–212 

carvacrol, 212 

carvone, 213 

1,4-cineole, 213–214 

1,8-cineole, 214–216 

citral, 216–217 

citronellal, 217 

fenchone, 217–218 

geraniol, 218 

limonene, 218–219, 220 

linalool, 219, 221 

linalyl acetate, 221 


myrcene, 223 

pinene, 223–225 

pulegone, 225 

thymol, 226 

sesquiterpenes 

caryophyllene, 227, 229 

longifolene, 229, 231 

patchouli alcohol, 230 

Terpentine-4-ol, 65 

Terpine hydrate, 709 

Terpinen-4-ol, 244, 247, 255, 720 

(-)-Terpinen-4-ol, 631 

Terpineol, 844 

4-Terpineol, 717 

Terpinolene, 267, 616 

Tessaria absinthioides, 245 

Tessaria, 245 

12-Tetradecanoylphorbol 13-acetate, 253 

1,2,4b,5α-Tetrahydro-α-santonin (214), 777 

Tetrahydronootkatone (22) biotransformation 


Tetrahydrosantonin (210), 776 

Teucrin A, 930 

Teucrium marum, 272 

Teucrium polium L., 241 

Texarome, 110 

Texila museum, 844 

Thermal conductivity detector (TCD), 12 

Thermal modulator, 171 

θ rhythm, 285 

Thin-layer chromatography (TLC), 12, 155–156, 257 

Thiobarbituric acid reactive substances, 258, 266, 269, 

270, 272 

Thresholds of toxicological concern, 198 

Thuja orientalis, 246 

Thujone, 305, 709, 717 

Thujopsene, 250 

Thujopsis dolabrata, 830 

Thujoyl alcohol, 717 

Thymbra spicata, 269 

Thyme oils, 259, 536, 884 





Thymol, 226, 229, 260, 268, 304, 631, 712, 885 

Thymol (580), 831 

Thymol methyl ether, 631 

Thymoquinone, 237, 240, 252, 268 

Thymus, 259 

Thymus camphorates, 259 

Thymus capitata, 260 

Thymus mastichina, 259 

Thymus pectinatus, 259, 260 

Thymus porlock, 258, 269 

Thymus serpyllus, 260 

Thymus spathulifolius, 260 

Thymus vulgaris, 26, 52, 260, 263, 267, 270, 272, 320, 

575, 884 

Thymus zygis, 259, 268, 888 

Tiger Balm, 552 

Tinctures, 844, 849 

Tinea pedis, 576 

TLC. See Thin-layer chromatography (TLC) 

TNF-α. See Tumor necrosis factor-α (TNF-α), 246 


Toluene, 143, 145 

Toluene-ethyl-acetate, 257 

Toothpastes, 850 

TPN/TPNH, 122 

Trachyspermum ammi, 267 

Traditional Chinese Medicine, 298, 863 

Transgenic or genetically modified organisms, 53 

Trazodone hydrochloride, 255 

Tree moss, 921, 922, 923, 924 

Treibs, W., 4 

Trichocephalus muris, 885 

Trichomonas vaginalis, 885 

Trichophyton mentagrophytes, 319 

Trichophyton rubrum, 319 

Trichophyton tonsurans, 319 

Trichophyton violaceum, 319 

Trichuris muris, 885 

Trifolium repens, 884 

Trigeminal system, 283 

Trinornardosinone (380), 801 

2,3,5-Triphenyltetrazolium chloride, 354 

Triticum vulgare, 561 

TRPM8, 334 

Tumor necrosis factor-α (TNF-α), 246 

Turbo distillation, 105 

Turmeric, 267 

Turpentine, 145, 552 

Turpentine oil, 536, 899 

Tween 80, 256

974 Index 

Twister, 10 

2α-Hydroxy-1,8-cineole, 717 

2-β-Pinene, 259 

2ent-10β-Hydroxycyclocolorenone (96) transformation 

Two-dimensional GC, 15–18 

U 

Ultrafast module-GC, 161 

Ultraviolet spectroscopy, 5, 27–28 

Umbelliferae, 65 

UN-approved packing, 909 

UN/ID numbers, 909 

United States Patent 4735803, 883 

United States Patent 4847292, 883 

Unpleasant fragrances, 295 

UVCB, 905, 906, 907 

V 

Valencene (1) biotransformation 

by Aspergillus niger, 741–743 

by Aspergillus wentii, 741–743 

Valeriana offi cinalis, 747 

Valeriana oils, 883 

Valerianol (35a) biotransforamtion 

by Mucor plumbeus, 750 

Valia–Chien horizontal diffusion cells, 254 

Vanillin, 129, 844 

Vapor phase test (VPT), 355 

anise oil, 364 


caraway oil, 379 




citronella oil, 399 


clove leaf oil, 413 

coriander oil, 420 


eucalyptus oil, 428 

juniper oil, 433 


lemon oil, 450 


matricaria oil, 460 

mint oil, 464 


nutmeg oil, 473 

peppermint oil, 481 




sweet orange oil, 511 



Vaporub®, 341 


Veillonella sp., 321 

Verbanone-7-al, 717 

Verbenaceae, 42–46, 245, 249 

Verbenol, 699, 709, 717 

Verbenone, 717 

Vermouth, 260 

Vernonia arborea, 783 

Vero, 244 

Veterinary medicine, essential oils use in 

in animal feed 

pigs, 889–890 

poultry, 887–889 

ruminants, 886–887 

attracting animals, 883 

against pests 

antiparasitic, 885–886 

aphids, caterpillars, and whiteflies, 885 

ear mites, 885 

fleas and ticks, 884 

insecticidal, pest repellent, and antiparasitic oils, 

884 

mosquitoes, 884 

moths, 884–885 

repelling animals, 883–884 

treating diseases, in animals, 890–891 

Veterinary papyrus, 555 

Vetiver oil, 21, 139 

GC-EIMS-MS of khusimone of, 22 

Vetiveria zizanioides, 40 

Vetivert, 848 

Vicks VapoRub, 552 

Vigilance, 290, 291 

Viridiflorene, 239 

Virucidal effect, 245 

Visual information processing, physico-chemical odorant 

properties on, 292 

Vitamin C, 256 

Vitamin E, 262 

Vitex obovata, 249 

Vitex pooara, 249 

Vitex rehmanni, 249 

Vitex zeyheri, 249 

Vitis vinifera, 561 

Volatile alcohols, 904 

Volatile encapsulation, 856 

introduction of, 857 

Volatile oils, 88 

Volatiles 

alganite, 859 

control release of, 856–858 

from non-volatile precursors, 859–860 

cyclodextrin complexation of, 860 

essential oil constituents, stabilization of, 859 

hydrophilic polymers, use of, 858 

Von Baeyer, A., 4 

VPT. See Vapor phase test (VPT) 

W 

Wagner, G., 4 

Wagner–Meerwein rearrangement, 4 

Wallach, O., 4 

Waterberg poora-berry, 249 

Water stress and drought, 90 

Wells, F.V., 850 

Wheatgerm, 561 

Whitebrush, 245 

White clover, 884 

Whiteflies, 885

Index 975 

Widdrol (448) biotransformation 


Wild coffee, 250 

Wild collection and cultivation, international 

standards for 

FairWild, 73 

GA(C)P, 72 

ISSC-MAP, 72–73 

Wild collection and sustainability, 58–59 

Wilmsii, 249 

Wintergreen, 886 

Wintergreen oil, 883 

The Wise of France, 843 

Wood oils, 91 

Woodward, R. B., 5 

Woodward rules, 5 

World consumption, of major essential oils, 847 

World map, of essential oils, 896 

Wormwood, 260, 884 

Wrigley, William, 846 

Wrigley’s Spearmint, 846 

Writhings, 240 

X 

Xanthine, 258 

Xanthine–xanthine assay, 258, 262 

Xenopus laevis, 568 

X-ray diffraction, 254 

Xylopia aethiopica, 238 

Y 

Yardley, 844 

Yarrow, 261 

Yellow balsam, 239 

Yellow rot of lavandin, 70 

Yerba porosa, 248 

Ylang complete, 105 

Ylang, 255 

Ylang-Ylang, 104, 563 

Z 

Zaluzanin D (258), 785 

Zataria, 241 

Zataria, Lamiaceae, 269 

Zataria multifl ora, 241, 269, 319, 333 

Zerumbone (408), 804, 808 

Zinc selenide (ZnSe), 23 

Zingeber offi cinale, 829, 834 

Zingerone (574), 830 

Zingiberaceae, 65, 242, 248, 253, 254, 267, 272 

Zingiber offi cinale, 253, 329, 331, 885 

Zingiber zerumbet, 804 

Ziziphora clinopodioides, 260

FIGURE 4.3 Lavender drying on the field.

FIGURE 4.4 Parts of a citrus fruit.

FIGURE 4.5 “Pellatrici method.” The spiked Archimedes screw with lemons, washed with water. 

FIGURE 4.6 “Brown” process. A battery of eight juice squeezers waiting for fruits.

FIGURE 4.7 FMC extractor. 

FIGURE 4.18 Oil and muddy water in the Florentine flask.

Handbook of Essential Oils: Science, Technology, and Applications

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