Oil essential - science, technology and applications

FEA/DTA UNICAMP

ABSTRACT Aspergillus niger is a well-known fungus that has been used for many different biotransformations of organic compounds. The terpenoids include a large variety of natural hydrocarbons and their derivatives, mostly obtained from plant essential oils, but some obtained from animals or fungi. They may be acyclic or have one or more rings of various sizes, and they show a variety of biological activities that include antibacterial, antifungal, antiparasitic, antiviral, and anticancer activities. Terpenoids are classified as monoterpenoids (C10), sesquiterpenoids (C15), diterpenoids (C20), triterpenoids (C30), and others. This review summarizes experimental processes that use cultures of various A. niger strains to carry out stereoselective biochemical reactions in terpenoids, including related epoxides, lactones, N-phenylcarbamates, and saponins, to produce metabolites that may be useful as flavors and fragrances or as new experimental drug candidates. Cultures of A. niger that add hydroxyl, carbonyl, and other groups at specific positions or reduce double bonds have resulted in the production of valuable new compounds.

The monograph describes examples of the application of microbial technology for obtaining of derivatives of terpenoids. Obtaining new derivatives of terpenoids, including artemisinin derivatives with increased antimalarial activity, is an important goal of research in microbial biotechnology and medicinal chemistry.

Isophorone (3,5,5-trimethyl-2-cyclohexen-1-one), a monoterpene, and the structurally related 1,8-cineole and camphor, have demonstrated a protective effect against cancer, biological activity against a variety of microorganisms, and anti-oxidant properties. The derivatization of isophorone is, therefore, an important field of xenobiochemistry, pharmacology and toxicology. The aim of this study was to obtain derivatives of isophorone through microbial biotransformation and evaluate the biotransformation metabolites as potential antimicrobial agents. Incubation of isophorone with the fungi Alternaria alternata and Neurospora crassa afforded 4a-hydroxy- and 7-hydroxy-isophorone as transformation metabolites. The antimicrobial activities of isophorone and the metabolites were evaluated in vitro both by using agar dilution and microdilution methods. However, no significant antibacterial activity was observed when compared with those of standard substances.

Handbook of ESSENTIAL OILS Science, Technology, and Applications Handbook of ESSENTIAL OILS Science, Technology, and Applications Edited by K. Hüsnü Can Başer Gerhard Buchbauer Boca Raton London New York CRC Press is an imprint of the Taylor & Francis Group, an informa business CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2010 by Taylor and Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number: 978-1-4200-6315-8 (Hardback) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. 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CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. 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 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com 2009038020 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 Gerhard Buchbauer 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 Biotransformation of Monoterpenoids by Microorganisms, Insects, and Mammals ....................................................................................................... 585 Yoshiaki Noma and Yoshinori Asakawa Chapter 15 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 Ba şer and Chlodwig Franz vii Contents 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 Scientific 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 Ba şer 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 Gerhard Buchbauer 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 University of Messina Messina, Italy Elaine Elisabetsky Laboratory of Ethnopharmacology The Federal University of Rio Grande do Sul Porto Alegre, Brazil Micheli Figueiró Laboratory of Ethnopharmacology The Federal University of Rio Grande do Sul Porto Alegre, Brazil Chlodwig Franz Institute for Applied Botany and Pharmacognosy University of Veterinary Medicine Vienna Vienna, Austria Bob Harris SARL Essential Oil Consultants Provence, France Eva Heuberger Department of Clinical Pharmacy and Diagnostics University of Vienna Vienna, Austria Walter Jäger Department of Clinical Pharmacy and Diagnostics University of Vienna Vienna, Austria xi xii Jan Karlsen Department of Pharmaceutics University of Oslo Oslo, Norway Maria M. Kettenring Neu-Isenburg, Germany Karl-Heinz Kubeczka Untere Steigstrasse Germany Viviane de Moura Linck Laboratory of Ethnopharmacology The Federal University of Rio Grande do Sul Porto Alegre, Brazil Maria Lis-Balchin South Bank University London, United Kingdom Luigi Mondello Department of Drug-Chemical University of Messina Messina, Italy 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 Vienna, Austria Domingos Sávio Nunes Departament of Chemistry State University of Ponta Grossa Ponta Grossa, Brazil Contributors Alexander Pauli ReviewScience Zirndorf, Germany Klaus-Dieter Protzen Paul Kaders GmbH Hamburg, Germany Heinz Schilcher Immenstadt, Allgäu, Germany Erich Schmidt Nördlingen, Germany and Kurt Kitzing GmbH Wallerstein, Germany Charles Sell Givaudan UK Ltd. Ashford, Kent, England Adriana Lourenço da Silva Laboratory of Ethnopharmacology The Federal University of Rio Grande do Sul Porto Alegre, Brazil Sean V. Taylor Flavor & Extract Manufacturers Association Washington, DC Lara M. Vucemilovic Neu-Isenburg, Germany Barbara d’Acampora Zellner Department of Drug-Chemical University of Messina Messina, Italy 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, pharmacology, 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 Karl-Heinz Kubeczka CONTENTS 2.1 First Systematic Investigations ............................................................................................ 2.2 Research during the Last Half Century .............................................................................. 2.2.1 Essential Oil Preparation Techniques ...................................................................... 2.2.1.1 Industrial Processes .................................................................................. 2.2.1.2 Laboratory-Scale Techniques ................................................................... 2.2.1.3 Microsampling Techniques ...................................................................... 2.2.2 Chromatographic Separation Techniques ............................................................... 2.2.2.1 Thin-Layer Chromatography ................................................................... 2.2.2.2 Gas Chromatography ............................................................................... 2.2.2.3 Liquid Column Chromatography ............................................................. 2.2.2.4 Supercritical Fluid Chromatography ........................................................ 2.2.2.5 Countercurrent Chromatography ............................................................. 2.2.3 Hyphenated Techniques ........................................................................................... 2.2.3.1 Gas Chromatography-Mass Spectrometry ............................................... 2.2.3.2 High-Resolution Gas Chromatography-Fourier Transform Infrared Spectroscopy .............................................................................. 2.2.3.3 Gas Chromatography-Ultraviolet Spectroscopy ...................................... 2.2.3.4 Gas Chromatography-Atomic Emission Spectroscopy ............................ 2.2.3.5 Gas Chromatography-Isotope Ratio Mass Spectrometry ......................... 2.2.3.6 High-Performance Liquid Chromatography-Gas Chromatography ............ 2.2.3.7 HPLC-MS, HPLC-NMR Spectroscopy ................................................... 2.2.3.8 Supercritical Fluid Extraction-Gas Chromatography .............................. 2.2.3.9 Supercritical Fluid Chromatography-Gas Chromatography .................... 2.2.3.10 Couplings of SFC-MS and SFC-FTIR Spectroscopy .............................. 2.2.4 Identification of Multicomponent Samples without Previous Separation ............... 2.2.4.1 UV Spectroscopy ..................................................................................... 2.2.4.2 IR Spectroscopy ....................................................................................... 2.2.4.3 Mass Spectrometry ................................................................................... 2.2.4.4 13C-NMR Spectroscopy ............................................................................ References .................................................................................................................................... 3 5 5 5 5 6 11 12 12 18 20 20 21 21 23 23 24 24 24 26 26 26 27 27 27 28 28 28 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 4 Handbook of Essential Oils 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 C10H16 were known, which had been named by Kekule terpenes because of their occurrence in turpentine oil. Constituents with the molecular formulas C10H16O and C10H18O 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 6 Handbook of Essential Oils 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. History and Sources of Essential Oil Research 7 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 8 Handbook of Essential Oils 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). History and Sources of Essential Oil Research 9 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 enfleurage 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. 10 Handbook of Essential Oils 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 (<0.5 μL) and consequently SPME has no real opportunity to realize quantitative extraction. Parameters governing recovery of analytes from a sample are partitioning constants and the phase ratio between the sorbent and liquid or gaseous sample. Therefore, basing on theoretical considerations, a procedure for sorptive enrichment with the sensitivity of packed PDMS beds (Baltussen et al., 1997) has been developed for the extraction of aqueous samples using modified PDMS-coated stir bars (Baltussen et al., 1999). The stir bars were incorporated into a narrow glass tube coated with a PDMS layer of 1 mm (corresponding to 55 μL for a 10 mm length) applicable to small sample volumes. Such stir bars are commercially available under name “Twister” (Gerstel, Germany). After certain stirring time the 11 History and Sources of Essential Oil Research 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 1H-NMR spectroscopy have been available. TABLE 2.1 Techniques Applied to the Analysis of Essential Oils Chromatographic Techniques Including Two- and Multidimensional Techniques TLC CCC GC SFC LC HPLC IR NIR MS Raman 1H-NMR GC-UV GC-AES GC-IRMS HPLC-GC HPLC-MS HPLC-NMR SFE-GC SFC-GC Spectroscopic and Spectrometric Techniques UV 13C-NMR Hyphenated Techniques GC-MS GC-FTIR GC-FTIR-MS 12 Handbook of Essential Oils 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 History and Sources of Essential Oil Research 13 (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 14 Handbook of Essential Oils Monoterpenes 0 0 Sesquiterpenes Coumarins and Psoralens 25 Time (min) 50 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 Column Temperature program Carrier gas Sampling frequency Conventional GC Fast GC Ultrafast GC 30 m 0.25 mm I.D. 0.25 μm film 50–350°C 3°C/min H2 u = 36 cm/s 10 Hz 10 m 0.1 mm I.D. 0.1 μm film 50–350°C 14°C/min H2 u = 57 cm/s 20–50 Hz 10–15 m 0.1 mm I.D. 0.1 μm film 45–325°C 45–200°C/min H2 u = 120 cm/s 50–250 Hz 15 History and Sources of Essential Oil Research 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 OR6 O O R3O FIGURE 2.2 a-Glucose unit of a cyclodextrin. R2O O 16 Handbook of Essential Oils 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 Det. Injector Det. Vent Tee Valve Column FIGURE 2.3 Basic arrangement used in two-dimensional GC. Column 17 History and Sources of Essential Oil Research 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 Δp “live-T” Chiral main column ITD Oven I Oven II FID Detector precolumn : “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, GoetheUniversity of Frankfurt/Main, Germany. With permission.) 18 Handbook of Essential Oils 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 History and Sources of Essential Oil Research 19 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. 20 Handbook of Essential Oils 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 fi rst 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 History and Sources of Essential Oil Research 21 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 22 Handbook of Essential Oils 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 [C10H17] + -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+– 204 Neutral loss of m/z 96 Fragment ion 108 (Main beam) Parent ions of m/z 108 M+– 108 ×5 Daughter ions of m/z 204 30 20 40 60 96 M+– 119 148 204 161 (Main beam) 175 189 91 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.) History and Sources of Essential Oil Research 23 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. 24 Handbook of Essential Oils 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 13C/12C 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 18O/16O ratios and later 2H/1H 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 25 History and Sources of Essential Oil Research Mass spectrometer Interface MDGC CH4 He Standard H2 Ion source 90º magnet Multicolumn switching system MCS 2 Monitor detector Injector Transfer line heated Pyrolysis reactor T=1450ºC Water separator He Open split Faraday cups 2 and ampliﬁers 3 Main column Precolumn 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. 26 Handbook of Essential Oils 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 History and Sources of Essential Oil Research 27 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 28 Handbook of Essential Oils 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 13C-NMR Spectroscopy C-NMR spectroscopy is generally used for the elucidation of molecular structures of isolated chemical species. The application of 13C-NMR spectroscopy to the investigation of complex mixtures is relatively rare. However, the application of 13C-NMR spectroscopy to the analysis of 13 29 History and Sources of Essential Oil Research m/z 93 100% 50 m/z 121 m/z 152 m/z 136 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 confi rm 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 13C-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 13C-NMR technique is limited by diverse factors such as rotational sidebands, 13C-13C-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 13C-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 13C-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. 30 Handbook of Essential Oils Limonene Celery oil 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 (ppm) FIGURE 2.10 Identification of limonene in celery oil by 13C-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 13C-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). REFERENCES Adams, R.P., 1989. Identification of Essential Oils by Ion Trap Mass Spectroscopy. San Diego: Academic Press. Adams, R.P., 1995. Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. Carol Stream: Allured Publishing Corp. Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th ed. Carol Stream: Allured Publishing Corp. Amelunxen, F., T. Wahlig, and H. Arbeiter, 1969. Über den Nachweis des ätherischen Öls in isolierten Drüsenhaaren und Drüsenschuppen von Mentha piperita L. Z. Pflanzenphysiol., 61: 68–72. Arpino, P., 1990. Coupling techniques in LC/MS and SFC/MS. 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Sci., 10: 25–29. 3 Sources of Essential Oils Chlodwig Franz and Johannes Novak CONTENTS 3.1 “Essential Oil-Bearing Plants”: Attempt of a Definition ..................................................... 3.2 Phytochemical Variation ..................................................................................................... 3.2.1 Chemotaxonomy ...................................................................................................... 3.2.2 Inter- and Intraspecific Variation ............................................................................. 3.2.2.1 Lamiaceae (Labiatae) and Verbenaceae .................................................... 3.2.2.2 Asteraceae (Compositae) ............................................................................ 3.3 Identification of Source Materials ....................................................................................... 3.4 Genetic and Protein Engineering ........................................................................................ 3.5 Resources of Essential Oils: Wild Collection or Cultivation of Plants ............................... 3.5.1 Wild Collection and Sustainability .......................................................................... 3.5.2 Domestication and Systematic Cultivation .............................................................. 3.5.3 Factors Influencing the Production and Quality of Essential Oil-Bearing Plants .................................................................................... 3.5.3.1 Genetic Variation and Plant Breeding ....................................................... 3.5.3.2 Plant Breeding and Intellectual Property Rights ....................................... 3.5.3.3 Intraindividual Variation between Plant Parts and Depending on the Developmental Stage (Morpho- and Ontogenetic Variation) .................... 3.5.3.4 Environmental Influences .......................................................................... 3.5.3.5 Cultivation Measures, Contaminations, and Harvesting ........................... 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 ............................................................................... 3.6.2 ISSC-MAP: The International Standard on Sustainable Wild Collection of Medicinal and Aromatic Plants ........................................................................... 3.6.3 FairWild ................................................................................................................... 3.7 Conclusion ............................................................................................................................. References .................................................................................................................................... 3.1 39 41 41 42 42 46 52 53 54 58 59 60 61 63 65 69 69 72 72 72 73 73 73 “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 40 Handbook of Essential Oils 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 3.2.1 PHYTOCHEMICAL VARIATION 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 officinarum, Zingiberaceae), bay laurel (Laurus nobilis, Lauraceae), Japan pepper (Zanthoxylum piperitum, Rutaceae), and a number of plants of the mint family, for example, sage (S. officinalis, Salvia fruticosa, Salvia lavandulifolia), rosemary (Rosmarinus officinalis), 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). 42 Handbook of Essential Oils 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. officinalis, 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. officinalis complex (S. officinalis, 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. officinalis 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. officinalis 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. officinalis 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. officinalis. 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 43 Sources of Essential Oils Chemotypes South African sage S. stenophylla α-bisabolol δ-3-carene limonene S. repens (E)-nerolidol γ-terpinene δ-3-carene ρ-cymene α-bisabolol (E)-nerolidol S. runcinata β-phellandrene β-caryophyllene ledol (E)-nerolidol α-bisabolol (E)-nerolidol β-caryophyllene α-pinene Chemotypes European sage S. lavandulifolia S. oﬃcinalis α-pinene camphor β-thujone α-thujone camphor 1,8-cineole β-thujone camphor β-pinene 1,8-cineole caryophyllene limonene S. fruticosa 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 1,8-cineole 20 α-thujone 15 β-thujone 10 camphor α-humulene 5 ia Cz Re ec pu h bl ic Fr an ce Po rt ug al an m Ro ga ry H un A lb an ia 0 FIGURE 3.2 Composition of the essential oil of six Salvia officinalis origins. 44 Handbook of Essential Oils TABLE 3.1 Species Used Commercially in the World as Oregano Family/Species Commercial Name/s Found in Literature Labiatae Calamintha potosina Schaf. Coleus amboinicus Lour. (syn. C. aromaticus Benth) Coleus aromaticus Benth. Hedeoma floribunda 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 floribundum 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 longiflora Gray Salvia sp. Satureja thymbra L. Thymus capitatus (L.) Hoffmanns et Link (syn. Coridothymus capitatus (L.) Rchb.f.) 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, Origanum Oregano, Origanum Oregano Oregano, Origanum Oregano, Origanum Oregano, oregano cimarron, Origanum Oregano, Origanum Oregano, Origanum Oregano, Origanum Oregano, Origanum Oregano, Origanum Oregano, Origanum Oregano, Origanum Oregano Oregano, Origanum *Turkish oregano, oregano, Origanum Oregano, Origanum Oregano, Origanum Oregano, Origanum Oregano, Origanum Oregano, Origanum, oregano verde *Greek oregano, oregano, Origanum Oregano, Origanum Oregano, orenga, Oregano de Espana Oregano Oregano Oregano cabruno, oregano, Origanum *Spanish oregano, oregano, Origanum Verbenaceae Lantana citrosa (Small) Modenke Lantana glandulosissima Hayek Lantana hirsuta Mart. et Gall. Lantana involucrata L. Lantana purpurea (Jacq.) Benth. & Hook. (syn. Lippia purpurea Jacq.) Lantana trifolia L. Oregano xiu, oregano, Origanum Oregano xiu, oregano silvestre, oregano, Origanum Oreganillo del monte, oregano, Origanum Oregano, Origanum Oregano, Origanum Oregano, Origanum continued 45 Sources of Essential Oils TABLE 3.1 (continued) Species Used Commercially in the World as Oregano Family/Species Commercial Name/s Found in Literature Lantana velutina Mart.&Gal. Lippia myriocephala Schlecht.&Cham. Lippia affinis 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 Oregano xiu, oregano, Origanum Oreganillo Oregano Oregano, Origanum Oregano Oreganillo, oregano montes, oregano, Origanum Oregano, Origanum Oregano, Origanum *Mexican oregano, oregano cimarron, oregano Oregano del pais, oregano, Origanum Oregano del pais, oregano, Origanum Oregano Oregano, origano del pais Oregano Oregano, Origanum Oreganillo, oregano montes, oregano, Origanum Oregano, Origanum Rubiaceae Borreria sp. Scrophulariaceae Limnophila stolonifera (Blanco) Merr. Oreganos, oregano, Origanum Oregano, Origanum Apiaceae Eryngium foetidum L. 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 46 Handbook of Essential Oils 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 2.3 0.8 n.d. n.d. 1.3 2.7 0.1 0.2 0.7 n.d. n.d. 80.6 1.3 2.8 1.9 0.3 n.d. n.d. Myrcene p-Cymene 1.8-Cineole Limonene Linalool Myrcenon Piperitone Thymol Carvacrol a-Humulene Caryophyll.-ox. Z-Dihydrocarvon/ Z-Ocimenone E-Dihydrocarvon n.d. = Not detectable; t = traces; main compounds in bold. b-Caryophyllene 1.9 6.9 0.6 0.3 1.4 n.d. n.d. 19.9 45.2 3.5 Thymol-Type Compound CarvacrolType Guatemala Fischer et al. (1996) 3.0 4.8 n.d. n.d. n.d. 1.1 5.5 2.1 0.8 0.3 n.d. n.d. 31.6 0.8 4.6 Guatemala Senatore et al. (2001) 3.3 n.d. 5.7 2.7 2.8 5.0 1.5 3.8 n.d. n.d. 6.8 1.1 8.7 Irregular Type L. graveolens n.d. t n.d. 5.0 t 2.1 t t t n.d. n.d. 7.3 71.2 9.2 El-Salvador Vernin et al. (2001) TABLE 3.2 Main Essential Oil Compounds of Lippia graveolens and L. alba According to Recent Data 4.9 1.8 13.1 0.7 6.5 t t 1.0 4.0 54.6 t n.d. n.d. 2.6 MyrcenoneType n.d. 3.0 0.6 t 1.7 t 22.8 3.2 2.4 3.2 t n.d. n.d. 1.2 CineoleType Guatemala Fischer (1998) t 1.1 0.1 0.6 0.2 0.7 14.2 43.6 1.2 n.d. 30.6 n.d. n.d. 1.0 Guatemala Senatore et al. (2001) L. alba 1.2 0.6 0.8 0.9 0.8 n.d. 1.3 2.9 55.3 n.d. n.d. n.d. n.d. 9.0 Uruguay Lorenzo (2001) Sources of Essential Oils 47 48 Handbook of Essential Oils TABLE 3.3 Main Compounds of the Essential Oil of Selected Tagetes lucida Types (in % of dm) Anethole Type (2) Substance Linalool Estragole Anethole Methyleugenol b-Caryophyllene Germacrene D Methylisoeugenol Nerolidol Spathulenol Carophyllene oxide Estragole Type (8) 0.26 11.57 73.56 1.75 0.45 2.43 1.42 0.35 0.10 0.05 Methyleugenol Type (7) Nerolidol Type (5) Mixed Type 1.01 8.68 0.52 79.80 0.45 1.90 2.00 0.31 0.12 0.45 Tr. 3.23 Tr. 17.76 2.39 Tr. Tr. 40.52 Tr. 10.34 3.68 24.28 30.17 17.09 0.88 5.41 3.88 1.24 0.23 0.53 0.69 78.02 0.75 5.50 1.66 2.89 2.78 0.32 0.16 0.27 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 70 80 60 30 20 Methyleugenol (%) 40 Estragole (%) Anethole (%) 50 60 40 20 10 0 0 1 2 3 4 7 Population 8 50 40 30 20 10 0 1 2 3 4 7 Population 8 1 2 3 4 7 Population 8 FIGURE 3.3 Variability of anethole, methyl eugenol, and methyl chavicol (estragole) in the essential oil of six Tagetes lucida—populations from Guatemala. 49 Sources of Essential Oils TABLE 3.4 Taxa within the Achillea-Millefolium-Group (Yarrow) Taxon Ploidy Level A. setacea W. et K. 2¥ Rupicoline A. aspleniifolia Vent. 2¥ (4¥) A. roseo-alba Ehrend. 2¥ A. collina Becker 4¥ A. pratensis Saukel u. Länger 4¥ 7,8-Guajanolide Artabsin-derivatives 3-Oxa-Guajanolide Artabsin-derivatives 3-Oxaguajanolide Matricinderivatives Artabsin-derivatives 3-Oxaguajanolide Matricinderivatives Matricarinderivatives 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¥ A. mill. ssp. Mill. L. 6¥ A. pannonica Scheele 8¥ (6¥) Main Compounds Guajanolidperoxide 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 Pro A Budakalaszi 210 3% 1% 1% 12% 5% Pro-chamazulene Pro-chamazulene 1,8-cineole 1,8-cineole 11% Linalool Linalool β-caryophyllene Borneol 7% 65% β-caryophyllene 95% FIGURE 3.4 Frequency distribution of proazulene individuals among two Achillea sp. populations. 50 Handbook of Essential Oils 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 CH 29 H 29 2% 2% Menemen 4% 12% 30% 29% 37% 96% 88% Bisabolol type Bisabolol oxide B type Bisabolol oxide A type Bisabolon oxide type Azulene type FIGURE 3.5 Frequency distribution of chemotypes in three varieties/populatios of chamomile [Matricaria recutita (L.) Rauschert]. 51 Sources of Essential Oils 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 Range Mean 2.5–35.2 23.2 Range Mean 6.6–31.2 21.3 Range Mean 7.6–24.2 16.8 α-Bisabolol-Type 58.8–92.1 68.8 α-Bisabololoxide A-Type 0.5–12.3 2.1 α-Bisabololoxide B-Type 0.8–6.5 2.0 Range Mean 76.3–79.2 77.8 Chamazulene-Type 5.8–8.3 7.1 α-B.-Oxide B n.d.–1.0 n.d. n.d.–3.2 n.d. 31.7–66.7 53.9 1.9–22.4 11.8 1.6–4.8 2.6 61.6–80.5 72.2 n.d.–0.8 n.d. n.d.–2.6 n.d. Source: Franz, Ch., 2000. Biodiversity and random sampling in essential oil plants. Lecture 31st ISEO, Hamburg. Main compounds in bold. mg Bisabolol 360 340 320 300 H 29 280 180 160 140 120 100 CH 29 80 60 40 5 10 15 20 25 30 35 40 45 50 n plants (individuals) 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. 52 Handbook of Essential Oils 80% 70% 60% 50% 1,8-cineole linalool trans-sabinene hydrate cis-sabinene hydrate α-terpineol α-terpinyl acetate 40% 30% 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 12C:13C 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 Sources of Essential Oils 53 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 modified 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). 54 Handbook of Essential Oils 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 dxr Mfs l-3-h — overexpress antisense cosuppress 1.7 1.6 1.7 74.7 4.3 3.6 1.2 0.4 2.1 1.8 0.4 0.1 20.5 19.6 22.7 4.1 44.5 45.6 45.2 3.0 97.8 137.9 109.7 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% (MunozBertomeu 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 monoterpene 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). 55 Sources of Essential Oils 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 Ambrette seed Amyris Angelica root Anise seed Armoise Asafoetida Basil Bay Bergamot Birch tar Buchu leaf Cade Cajuput Calamus Camphor Cananga Caraway Cardamom Carrot seed Cascarilla Cedarwood, Chinese Cedarwood, Texas Cedarwood, Virginia Celery seed Chamomile Chamomile, Roman Chenopodium Cinnamon bark, Ceylon Cinnamon bark, Chinese Cinnamon leaf Species Hibiscus abelmoschus L. Amyris balsamifera L. Angelica archangelica L. Pimpinella anisum L. Artemisia herba-alba Asso Ferula assa-foetida L. Ocimum basilicum L. Pimenta racemosa Moore Citrus aurantium L. subsp. bergamia (Risso et Poit.) Engl. Betula pendula Roth. (syn. Betula verrucosa Erhart. Betula alba sensu H.J.Coste. non L.) Agathosma betulina (Bergius) Pillans. A. crenulata (L.) Pillans Juniperus oxycedrus L. Melaleuca leucandendron L. Acorus calamus L. Cinnamomum camphora L. (Sieb.) Cananga odorata Hook. f. et Thoms. Carum carvi L. Elettaria cardamomum (L.) Maton Daucus carota L. Croton eluteria (L.) W.Wright Cupressus funebris Endl. Plant Family Used Plant Part(s) Wild Collection/ Cultivation Trade Quantitiesa Malvaceae Rutaceae Apiaceae Apiaceae Asteraceae Apiaceae Lamiaceae Myrtaceae Rutaceae Seed Wood Root Fruit Herb Resin Herb Leaf Fruit peel Cult Wild Cult Cult Cult/wild Wild Cult Cult Cult LQ LQ LQ LQ LQ LQ LQ LQ MQ Betulaceae Bark/wood Wild LQ Rutaceae Leaf Wild LQ Cupressaceae Myrtaceae Araceae Lauraceae Wood Leaf Rhizome Wood Wild Wild Cult/wild Cult LQ LQ LQ LQ Annonaceae Flower Wild LQ Apiaceae Zingiberaceae Seed Seed Cult Cult LQ LQ Apiaceae Euphorbiaceae Cupressaceae Seed Bark Wood Cult Wild Wild LQ LQ MQ Juniperus mexicana Schiede Cupressaceae Wood Wild MQ Juniperus virginiana L. Cupressaceae Wood Wild MQ Apium graveolens L. Matricaria recutita L. Anthemis nobilis L. Apiaceae Asteraceae Asteraceae Seed Flower Flower Cult Cult Cult LQ LQ LQ Chenopodium ambrosioides (L.) Gray Cinnamomum zeylanicum Nees Chenopodiaceae Seed Cult LQ Lauraceae Bark Cult LQ Cinnamomum cassia Blume Lauraceae Bark Cult LQ Cinnamomum zeylanicum Nees Lauraceae Leaf Cult LQ continued 56 Handbook of Essential Oils TABLE 3.7 (continued) 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 Citronella, Ceylon Citronella, Java Clary sage Cymbopogon nardus (L.) W. Wats. Cymbopogon winterianus Jowitt. Salvia sclarea L. Clove buds Syzygium aromaticum (L.) Merill et L.M. Perry Syzygium aromaticum (L.) Merill et L.M. Perry Coriandrum sativum L. Mentha canadensis L. (syn. M. arvensis L. f. piperascens Malinv. ex Holmes; M. arvensis L. var. glabrata. M. haplocalyx Briq.; M. sachalinensis (Briq.) Kudo) Cuminum cyminum L. Cupressus sempervirens L. Artemisia pallens Wall. Anethum graveolens L. Anethum sowa Roxb. Canarium luzonicum Miq. Eucalyptus globulus Labill. Eucalyptus citriodora Hook. Foeniculum vulgare Mill. subsp. vulgare var. vulgare Foeniculum vulgare Mill. subsp. vulgare var. dulce Abies balsamea Mill. Clove leaf Coriander Cornmint Cumin Cypress Davana Dill Dill, India Elemi Eucalyptus Eucalyptus, lemon-scented Fennel bitter Fennel sweet Fir needle, Canadian Fir needle, Siberian Gaiac Galbanum Garlic Geranium Ginger Gingergrass Grapefruit Guaiacwood Gurjum Hop Hyssop Plant Family Used Plant Part(s) Wild Collection/ Cultivation Trade Quantitiesa Poaceae Leaf Cult HQ Poaceae Lamiaceae Cult Cult HQ MQ Myrtaceae Leaf Flowering herb Leaf/bud Cult LQ Myrtaceae Leaf Cult HQ Apiaceae Lamiaceae Fruit Leaf Cult Cult LQ HQ Apiaceae Cupressaeae Asteraceae Cult Wild Cult LQ LQ LQ Apiaceae Apiaceae Burseraceae Myrtaceae Myrtaceae Fruit Leaf/twig Flowering herb Herb/fruit Fruit Resin Leaf Leaf Cult Cult Wild Cult/wild Cult/wild LQ LQ LQ HQ HQ Apiaceae Fruit Cult LQ Apiaceae Fruit Cult LQ Pinaceae Leaf/twig Wild LQ Abies sibirica Ledeb. Pinaceae Leaf/twig Wild LQ Guaiacum officinale L. Ferula galbaniflua Boiss. F. rubricaulis Boiss. Allium sativum L. Pelargonium spp. Zingiber officinale Roscoe Cymbopogon martinii (Roxb.) H. Wats var. sofia Burk Citrus × paradisi Macfad. Bulnesia sarmienti L. Dipterocarpus spp. Humulus lupulus L. Hyssopus officinalis L. Zygophyllaceae Apiaceae Resin Resin Wild Wild LQ LQ Alliaceae Geraniaceae Zingiberaceae Poaceae Bulb Leaf Rhizome Leaf Cult Cult Cult Cult/wild LQ MQ LQ Rutaceae Zygophyllaceae Dipterocarpaceae Cannabaceae Lamiaceae Fruit peel Wood Resin Flower Leaf Cult Wild Wild Cult Cult LQ MQ LQ LQ LQ continued 57 Sources of Essential Oils TABLE 3.7 (continued) 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 Juniperus communis L. Laurus nobilis L. Lavandula angustifolia Mill. × L. latifolia Medik. Lavender Lavandula angustifolia Miller Lavender, Spike Lavandula latifolia Medik. Lemon Citrus limon (L.) Burman fil. Lemongrass, Cymbopogon flexuosus Indian (Nees ex Steud.) H. Wats. Lemongrass, West Cymbopogon citratus (DC.) Indian Stapf Lime distilled Citrus aurantiifolia (Christm. et Panz.) Swingle Litsea cubeba Litsea cubeba C.H. Persoon Lovage root Levisticum officinale Koch Mandarin Citrus reticulata Blanco Marjoram Origanum majorana L. Mugwort Artemisia vulgaris L. common Mugwort, Roman Artemisia pontica L. Myrtle Myrtus communis L. Neroli Citrus aurantium L. subsp. aurantium Niaouli Melaleuca viridiflora Nutmeg Myristica fragrans Houtt. Onion Allium cepa L. Orange Citrus sinensis (L.) Osbeck Orange bitter Citrus aurantium L. Oregano Origanum spp.. Thymbra spicata L.. Coridothymus capitatus Rechb. fil.. Satureja spp. Lippia graveolens Palmarosa Cymbopogon martinii (Roxb.) H. Wats var. motia Burk Parsley seed Petroselinum crispum (Mill.) Nym. ex A.W. Hill Patchouli Pogostemon cablin (Blanco) Benth. Pennyroyal Mentha pulegium L. Pepper Piper nigrum L. Peppermint Mentha x piperita L. Petitgrain Citrus aurantium L. subsp. aurantium Pimento leaf Pimenta dioica (L.) Merr. Pine needle Pinus silvestris L.. P. nigra Arnold Pine needle, Dwarf Pinus mugo Turra Pine silvestris Pinus silvestris L. Juniper berry Laurel leaf Lavandin Plant Family Used Plant Part(s) Wild Collection/ Cultivation Trade Quantitiesa Cupressaceae Lauraceae Lamiaceae Fruit Leaf Leaf Wild Cult/wild Cult LQ LQ HQ Lamiaceae Lamiaceae Rutaceae Poaceae Leaf Flower Fruit peel Leaf Cult Cult Cult Cult MQ LQ HQ LQ Poaceae Leaf Cult LQ Rutaceae Fruit Cult HQ Lauraceae Apiaceae Rutaceae Lamiaceae Asteraceae Fruit/leaf Root Fruit peel Herb Herb Cult Cult Cult Cult Cult/wild MQ LQ MQ LQ LQ Asteraceae Myrtaceae Rutaceae Herb Leaf Flower Cult/wild Cult/wild Cult LQ LQ LQ Myrtaceae Myristicaceae Alliaceae Rutaceae Rutaceae Lamiaceae Leaf Seed Bulb Fruit peel Fruit peel Herb Cult/wild Cult Cult Cult Cult Cult/wild LQ LQ LQ HQ LQ LQ Poaceae Leaf Cult LQ Apiaceae Fruit Cult LQ Lamiaceae Leaf Cult HQ Lamiaceae Piperaceae Lamiaceae Rutaceae Herb Fruit Leaf Leaf Cult Cult Cult Cult LQ LQ HQ LQ Myrtaceae Pinaceae Pinaceae Pinaceae Fruit Leaf/twig Leaf/twig Leaf/twig Cult Wild Wild Wild LQ LQ LQ LQ continued 58 Handbook of Essential Oils TABLE 3.7 (continued) 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 Pine white Rose Rosemary Rosewood Rue Sage, Dalmatian Sage, Spanish Sage, three lobed (Greek. Turkish) Sandalwood, East Indian Sassafras, Brazilian (Ocotea cymbarum oil) Sassafras, Chinese Savory Species Pinus palustris Mill. Rosa x damascena Miller Rosmarinus officinalis L. Aniba rosaeodora Ducke Ruta graveolens L. Salvia officinalis L. Salvia lavandulifolia L. Salvia fruticosa Mill. (syn. S. triloba L.) Santalum album L. Ocotea odorifera (Vell.) Rohwer [Ocotea pretiosa (Nees) Mez.] Sassafras albidum (Nutt.) Nees. Satureja hortensis L.. Satureja montana L. Spearmint, Native Mentha spicata L. Spearmint, Scotch Mentha gracilis Sole Star anise Illicium verum Hook fil. Styrax Styrax officinalis L. Tansy Tanacetum vulgare L. Tarragon Tea tree Thyme Valerian Vetiver Wintergreen Wormwood Ylang Ylang a Artemisia dracunculus L. Melaleuca spp. Thymus vulgaris L.. T. zygis Loefl. ex L. Valeriana officinalis L. Vetiveria zizanoides (L.) Nash Gaultheria procumbens L. Artemisia absinthium L. Cananga odorata Hook. f. et Thoms. Plant Family Used Plant Part(s) Wild Collection/ Cultivation Trade Quantitiesa Pinaceae Rosaceae Lamiaceae Lauraceae Rutaceae Lamiaceae Lamiaceae Lamiaceae Leaf/twig Flower Feaf Wood Herb Herb Leaf Herb Wild Cult Cult/wild Wild Cult Cult/wild Cult Cult/wild LQ LQ LQ LQ LQ LQ LQ LQ Santalaceae Wood Wild MQ Lauraceae Wood Wild HQ Lauraceae Lamiaceae Root bark Leaf Wild Cult/wild HQ LQ Lamiaceae Lamiaceae Illiciaceae Styracaceae Asteraceae Cult Cult Cult Wild Cult/wild MQ HQ MQ LQ LQ Asteraceae Myrtaceae Lamiaceae Leaf Leaf Fruit Resin Flowering herb Herb Leaf Herb Cult Cult Cult LQ LQ LQ Valerianaceae Poaceae Ericaceae Asteraceae Annonaceae Root Root Leaf Herb Flower Cult Cult Wild Cult/wild Cult LQ MQ LQ LQ MQ HQ = High quantities (>1000 t/a); MQ = medium quantities (100–1000 t/a); LQ = low quantities (<100 t/a). 3.5.1 WILD COLLECTION AND SUSTAINABILITY Since prehistoric times mankind has gathered wild plants for different purposes, among them are aromatic, essential oil-bearing species used as culinary herbs, spices, flavoring agents, and fragrances. With increasing demand of standardized, homogeneous raw material in the industrial societies more and more wild species have been domesticated and systematically cultivated. Nevertheless, a high number of species is still collected from the wild due to the fact that • Many plants and plant products are used for the subsistence of the rural population. • Small quantities of the respective species are requested at the market only which make a systematic cultivation not profitable. Sources of Essential Oils 59 • 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). 60 Handbook of Essential Oils 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 Æ GPS to exactly localize the place 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) 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 Æ Biotechnol./in vitro Æ Biotechnol./in vitro 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. Sources of Essential Oils 61 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 62 Handbook of Essential Oils TABLE 3.9 Some Registered Cultivars of Essential Oil Plant Species Cultivar/Variety Country Year of Registration Breeding Method Achillea collina Angelica archangelica Foeniculum vulgare Lavandula officinalis SPAK VS 2 Fönicia Rapido CH FR HU FR 1994 1996 1998 1999 Crossing Recurrent pedigree Selection Polycross Levisticum officinale Matricaria recutita Amor Mabamille PL DE 2000 1995 Selection Tetraploid Ciclo-1 Lutea IT SK 2000 1995 Line breeding Tetraploid Ildikó HU 1998 Selection Landor Lemona Todd’s Mitcham Kubanskaja MSH-20 Greco Perri Cardinal Senköy CH DE USA RUS DK IT ISR ISR TR 1994 2001 1972 1980ies 2000 2000 1999 2000 1992 Selection Selection Mutation Crossing and polyploid Recurrent pedigree Synthetic Cross-breeding Cross-breeding Selection Carmeli Tavor Vulkan Carva Darpman ISR ISR GR GR DE CH TR 1999 1999 2000 2000 2002 2002 1992 Selection Selection Selfing Selfing Crossing Crossing Selection Erfo Tetrata G1 Moran Syn 1 DE DE FR ISR IT 1997 1999 1998 1998 2004 Crossing Ployploid Polycross Crossing Synthetic Varico T-16 Virginia CH DK ISR 1994 2000 2000 Selection Recurrent pedigree Selection Melissa officinalis Mentha piperita Mentha spicata Ocimum basilicum Origanum syriacum Origanum onites Origanum hirtum Origanum majorana (Majorana hortensis) Salvia officinalis Thymus vulgaris Specific Characters High in proazulene Essential oil index of roots: 180 High anethole High essential oil, high linalyl acetate High essential oil High a-bisabolol High chamazulene High a-bisabolol High essential oil, Citral A + B, linalool High essential oil High essential oil, citral Wilt resistant High essential oil, high menthol High menthol, good flavor Flavor Fusarium Resistant 5% essential oil, 60% carvacrol Carvacrol Thymol Carvacrol Carvacrol Carvacrol Carvacrol 2.5% essential oil, 55% carvacrol High essential oil, Cis-sabinene-hydrate Herb yield a-Thujone Thymol/carvacrol Thymol 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 F1 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 Sources of Essential Oils 63 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 F1 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 F1 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, 64 Handbook of Essential Oils 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 Patent Beginning of protection: registration date Restricted to “varieties” Beginning of protection: application date “Varieties” not patentable, but any other grouping of plants 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 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 Sources of Essential Oils 65 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 66 Handbook of Essential Oils 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 0.9 75 0.6 50 0.3 25 0.9 75 0.6 50 0.3 25 0.9 75 0.6 50 0.3 25 1 Myristicin 2 3 4 Apiole 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). Sources of Essential Oils 67 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 <50% in unripe seeds to over 80% in full maturity (Marotti et al., 1994). In dill weed (Anethum graveolens L.) the content on essential oil rises from 0.1% only in young sprouts to more than 1% in herb with milk ripe umbels (Gora et al., 2002). In the herb, oil a-phellandrene prevails until the beginning of flowering with up to 50%, followed by dill ether, p-cymene, and limonene. The oil from green as well as ripe umbels contains, on the other hand, mainly (S)-carvone and (R)-limonene. The flavor of dill oil changes therefore dramatically, which has to be considered when determining the harvest time for distillation. Among Compositae (Asteraceae) there are not as many results concerning ontogeny due to the fact that in general the flowers or flowering parts of the plants are harvested, for example, chamomile (Matricaria recutita), yarrow (Achillea millefolium s.l.), immortelle (Helichrysum italicum), or wormwood (Artemisia sp.) and therefore the short period between the beginning of flowering and the decay of the flowers is of interest only. In chamomile (Matricaria recutita), the flower buds show a relatively high content on essential oil between 0.8% and 1.0%, but the oil yield in this stage is rather low. From the beginning of flowering, the oil content increases until full flowering (all disc florets open) and decreases again with decay of the flower heads. At full bloom there is also the peak of (pro)chamazulene, whereas farnesene and a-bisabolol decrease from the beginning of flowering and the bisabololoxides rise (Franz et al., 1978). This was confirmed by Repčak et al. (1980). The essential oil of Tagetes minuta L. at different development stages was investigated by Worku and Bertoldi (1996). Before flower bud formation the oil content was 0.45% only, but it culminated with 1.34% at the immature seed stage. During this period cis-ocimene increased from 7.2% to 37.5% and cis-ocimenone declined from almost 40–13.1%. Little variations could be observed at cis- and trans-tagetone only. Similar results have been reported also by Chalchat et al. (1995). Also for Lippia sp. (Verbenaceae) some results are known concerning development stages (Fischer, 1998, Coronel, 2006). The oil content in the aerial parts increases from young buds (<1.0%) to fully blooming (almost 2.0%). But although quantitative variations could be observed for most components of the essential oils, the qualitative composition appeared to be constant throughout the growing season. A particular situation is given with eucalypts as they develop up to five distinct types of leaves during their lifetime, each corresponding to a certain ontogenetic stage with changing oil concentrations and compositions (Doran, 2002). Usually the oil content increases from young to matured, nonlignified leaves, and is thereafter declining until leaf lignification. Almost the same curve is valid also for the 1,8-cineole concentration in the oil. But comparing the relatively extensive literature on this topic, one may conclude that the concentration at various stages of leaf maturity is determined by a complex pattern of quantitative change in individual or groups of substances, some remaining constant, some increasing, and some decreasing. Tsiri et al. (2003) investigated the volatiles of the leaves of Eucalyptus camaldulensis over the course of a year in Greece and found a seasonal variation of the oil concentration with a peak during summer and lowest yields during winter. The constituent with highest concentration was 1,8-cineole (25.3–44.2%) regardless the time of harvest. The great variation of all oil compounds showed however no clear tendency, neither seasonal nor regarding leaf age or leaf position. Doran (2002) concluded therefore that genotypic differences outweigh any seasonal or environmental effects in eucalypts. 68 Handbook of Essential Oils 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. Sources of Essential Oils 69 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. Terpinene4-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 70 Handbook of Essential Oils 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 officinalis 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 14C-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 <1.0%, in apple mint, linalool and neryl acetate decreased while myrcene, linalyl acetate, and linalyl propionate increased. Further problems to be taken into consideration in plant production are contaminations with heavy metals, damages caused by pests and diseases, and residues of plant protection products. The most important toxic heavy metals Cd, Hg, Pb, and Zn, but also Cu, Ni, and Mn may influence the plant growth severely and by that way also the essential oil, as they may act as cofactors in the plant enzyme system. But as contaminants, they remain in the plant residue after distillation (Zheljazkov et al., 1996, 1997). Some plant species, for example, yarrow and chamomile accumulate heavy metals to a greater extent. This is, however, problematic for using the crude drug or for deposition of distillation wastes mainly. The same is valid for the microbial contamination of the 71 Sources of Essential Oils 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. Starting material Species, variety, cultivar chemotype, resistances spec. characters (e.g. “usability value”) Cultivation Growing site, crop rotation, production technique and maintenance, fertilization, plant protection, spec. methods (e.g. organic production), harvest time and technique Stepwise quality control Safety Prevention against intoxications Storage Storehouse conditions, packaging FIGURE 3.10 Post harvest handling Transport from ﬁeld, washing, drying, processing Main items of “good agricultural practices (GAP)” for medicinal and aromatic plants. 72 Handbook of Essential Oils 3.6 3.6.1 INTERNATIONAL STANDARDS FOR WILD COLLECTION AND CULTIVATION GA(C)P: GUIDELINES FOR GOOD AGRICULTURAL (AND COLLECTION) PRACTICE MEDICINAL AND AROMATIC PLANTS OF 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, Sources of Essential Oils 73 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. REFERENCES Agnaniet, H., C. Menut, and J.M. Bessière, 2004. Aromatic plants of tropical Africa XLIX: Chemical composition of essential oils of the leaf and rhizome of Aframomum giganteum K. Schum from Gabun. Flavour Fragr. J., 19: 205–209. Akhila, A. and M. Rani, 2002. Chemical constituents and essential oil biogenesis in Vetiveria zizanioides. In Vetiveria—The genus Vetiveria, M. Maffei (ed.), pp. 73–109. New York: Taylor & Francis. Aiello, N., F. Scartezzini, C. Vender, L. D’Andrea, and A. Albasini, 2001. 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Carol Stream: Allured Publishing. 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 84 Handbook of Essential Oils 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 Orange oils Cornmint oil Lemon oils Eucalyptus oils Peppermint oil Clove leaf oil Citronella oil Spearmint oils Cedarwood oils Litsea cubeba oil Patchouli oil Lavandin oil Grosso Corymbia Citriodora Production in Metric Tons (2008) Main Production Countries 51000 32000 9200 4000 3300 1800 1800 1800 1650 1200 1200 1100 1000 USA, Brasil, Argentina India, China, Argentina Argentina, Italy, Spain China, India, Australia, South Africa India, USA, China Indonesia, Madagascar China, Sri Lanka USA, China USA, China China Indonesia, India France 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. 85 Production of Essential Oils EO production worldwide quantities figures 2008 (partly esteemed) South Africa; 1.0% Marocco; 0.1% Indonesia; 1.9% Hungary; 0.1% Vietnam; 0.1% Others; 10.0% India; 25.8% USA; 16.8% France; 1.0% Argentina; 4.9% China; 9.0% Australia; 0.6% Egypt; 0.1% Brasil; 28.6% 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, 86 Handbook of Essential Oils 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 parviflora, 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. Eucalyptus 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 dissitiflora. 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 Production of Essential Oils 87 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. 88 Handbook of Essential Oils 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 Production of Essential Oils 89 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 90 Handbook of Essential Oils 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. Production of Essential Oils 4.1.8 91 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 officinalis, 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 92 Handbook of Essential Oils 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 officinalis (“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 93 Production of Essential Oils 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 Limonene 0.9–1.0 0.2–0.4 0.9–1.1 0.3–0.5 Ocimene cis 0.3–0.5 0.4–0.6 Ocimene trans 0.5–0.7 0.8–1.0 0.5–0.7 1.4–1.6 Copaene alpha 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 94 Handbook of Essential Oils 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 95 Production of Essential Oils 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 Lime oil Lime oil distilled Pericarp; fruit juice or crushed fruits Citrus aurantium L., syn. Citrus amara Link, syn. Citrus bigaradia Loisel, syn. Citrus vulgaris Risso Bitter orange oil Neroli oil, Bitter orange petitgrain oil Flower, pericarp, leaf, and twigs with sometimes little green fruits Citrus bergamia (Risso et Poit.), Citrus aurantium L. subsp. bergamia (Wight et Arnott) Engler Bergamot oil Bergamot petitgrain oil Pericarp, leaf, and twigs with sometimes little green fruits Citrus hystrix DC., syn. Citrus torosa Blanco Kaffir lime oil, Combava Kaffir leaves oil Pericarp, leaves Citrus latifolia Tanaka Lime oil Persian type Citrus limon (L.) Burm. f. Lemon oil Lemon petitgrain oil Flower, pericarp, leaf, and twigs with sometimes little green fruits Citrus reticulate Blanco syn. Citrus nobilis Andrews Mandarin oil Mandarin petitgrain oil Flower, pericarp, leaf, and twigs with sometimes little green fruits Citrus sinensis (L.) Osbeck, Citrus djalonis A. Chevalier Sweet orange oil Pericarp Citrus × paradisi Macfad. Grapefruit oil Pericarp Pericarp 96 Handbook of Essential Oils 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. Production of Essential Oils 97 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. 98 Handbook of Essential Oils 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. Production of Essential Oils 99 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 = rA + rB (r is the total vapor pressure of the system). 100 Handbook of Essential Oils 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 (TM) lies below the boiling temperatures (TA and TB) 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 oil P = oil N water Pwater where Noil stands for the number of moles of the oil in the vapor phase and Nwater 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. Production of Essential Oils 101 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. 102 Handbook of Essential Oils 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 m3 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 103 Production of Essential Oils Vapor + Oil Biomass Separation bottoms Condenser Steam Oil Back to steam generator FIGURE 4.10 Florentine ﬂask Water 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/m3. 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. 104 FIGURE 4.12 Handbook of Essential Oils 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. Production of Essential Oils FIGURE 4.14 105 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. 106 FIGURE 4.16 Handbook of Essential Oils 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. Oil Water Oil Oil heavier than water FIGURE 4.17 Two varieties of Florentine flasks. Water Oil lighter than water Production of Essential Oils FIGURE 4.18 107 (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. 108 Handbook of Essential Oils 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. Production of Essential Oils FIGURE 4.21 109 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. 110 FIGURE 4.23 Handbook of Essential Oils 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 m3. 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 Production of Essential Oils 111 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. 112 Handbook of Essential Oils 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 m3 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 Production of Essential Oils 113 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). 114 Handbook of Essential Oils 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. Production of Essential Oils 115 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 116 Handbook of Essential Oils 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 Production of Essential Oils 117 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 Cinnamomum 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 118 Handbook of Essential Oils 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. 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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 5.2 5.3 5.4 5.5 Introduction ....................................................................................................................... Basic Biosynthetic Pathways ............................................................................................. Polyketides and Lipids ....................................................................................................... Shikimic Acid Derivatives ................................................................................................ Terpenoids ......................................................................................................................... 5.5.1 Hemiterpenoids ..................................................................................................... 5.5.2 Monoterpenoids ..................................................................................................... 5.5.3 Sesquiterpenoids .................................................................................................... 5.6 Synthesis of Essential Oil Components ............................................................................. References .................................................................................................................................. 5.1 121 121 123 126 129 131 131 135 140 149 INTRODUCTION 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 121 122 Handbook of Essential Oils 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-1,4dihydropyridine. 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 O P = OPO32– Carbon dioxide + Water + Sunlight Glucose Green plants Glycolysis O OH Lignans Coumarins Flavonoids OP CO2– HO OH Phosphoenolpyruvate 1 OH Shikimic acid 4 Polyketides Lipids O O CoA Acetyl coenzyme A Terpenoids OH HO Mevalonic acid 2 FIGURE 5.1 OH General pattern of biosynthesis of secondary metabolites. 3 123 Chemistry of Essential Oils 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 O –O2C CoA 2 O CoA R CoA R CoA CO2– 5 –CO2 O R O TPNH CoA R –H2O CoA etc. FIGURE 5.2 Polyketide and lipid biosynthesis. OH R O O DPNH CoA R O CoA etc. 124 Handbook of Essential Oils O O – O OH O O –H2O Enolise OH OH O O HO O O OH O O OH HO O O O 8 7 O 6 HO O O OH OH O OH HO Cl OH OH 9 10 O O 11 O HO OH OH O O 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. HO [O] R R' R R' H O O R FIGURE 5.4 + R' R Fragmentation of polyunsaturated fats to give aldehydes. R' 125 Chemistry of Essential Oils 13 OH 12 O 14 16 O O 15 O O 18 O 17 O O O O 20 19 O 21 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 F1a (25) and also for methyl jasmonate O O H OH OH HO O O O O 22 O 23 O O HO OH OH O 25 24 O O HO HO OH O O O 27 FIGURE 5.6 Biosynthesis of prostaglandins and jasmines. O 26 126 Handbook of Essential Oils (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 CO H 2 HO OH TPN+ O O OP HO HO CO2H CO2H O O OP Erythrose-4 phosphate 28 OP OH OH OH H TPN Heptulosonic acid monophosphate HO CO2H CO2H OH OH OH OH CO2H O CO2H CO2H TPNH HO OH O OH O OH OH OH OH Shikimic acid 4 5-Dehydroshikimic acid 5-Dehydroquinic acid FIGURE 5.7 Biosynthesis of shikimic acid. HO OH OH 127 Chemistry of Essential Oils O O OH OH OH CO2H O CO2H O HO O OH OH OH 4 30 29 CO2H CO2H NH2 CO2H 31 OH CO2H CO2H CO2H 32 O O OH O 37 FIGURE 5.8 36 OH CO2H OH OH 35 OH 34 33 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, CO2H CO2H OH 40 38 NH2 HO 43 O O O O O NH2 OH 39 CO2H O 44 41 O OH O 42 FIGURE 5.9 N H Hydroxy- and aminobenzoic acid derivatives. O 45 128 Handbook of Essential Oils 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 N H 47 46 O OH 49 48 O O O O 50 O 51 O O 52 FIGURE 5.10 Some shikimate essential oil components. 53 O 129 Chemistry of Essential Oils O HO O 53 O O 54 56 O 57 O 55 O O 58 O O 59 HO O O O O O HO O 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 terpenoids. 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. 130 Handbook of Essential Oils OH 63 64 62 65 OH FIGURE 5.12 Isoprene units in some common terpenoids. Head Head Tail Tail FIGURE 5.13 Head-to-tail coupling of two isoprene units. O OH Mn2+ HO OH 3 _base O 66 O _base O PP 66 68 PP PP 67 H O PP O Mg2+ 66 H 67 O PP PP etc. 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. 131 Chemistry of Essential Oils 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 77 72 + 65 + 76 74 75 OH OH O 80 79 + 78 FIGURE 5.15 Formation of monoterpenoid skeletons. O + 81 82 132 Handbook of Essential Oils 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), 86 70 84 85 73 87 88 65 76 77 FIGURE 5.16 Some of the more common terpenoid hydrocarbons. 83 89 133 Chemistry of Essential Oils 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 fistulosa 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-1,7,7trimethylbicyclo[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 FIGURE 5.17 Key acyclic monoterpenoid alcohols. 91 92 134 Handbook of Essential Oils OH OH OH 74 93 OH 94 OH 95 OH OH 96 79 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 98 FIGURE 5.19 Some monoterpenoid ethers. O 99 O 100 135 Chemistry of Essential Oils O O O O 101 102 104 103 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 105 106 107 O 108 O O O 109 O 110 FIGURE 5.21 Some monoterpenoid ketones. 80 82 136 Handbook of Essential Oils 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 + OH + 128 112 127 115 + + + 124 + 114 121 + + + 129 116 HO 122 HO 123 + OH 130 125 + + 117 119 + + + 131 OH 126 120 FIGURE 5.22 Some biosynthetic pathways from (Z,E)-farnesol. 118 + 137 Chemistry of Essential Oils (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 + 132 135 133 O + 142 134 136 OH + + 138 141 OH 144 + HO 143 O 139 140 O OH 137 145 FIGURE 5.23 Some biosynthetic pathways from (E,E)-farnesol. 138 Handbook of Essential Oils 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 113 111 OH 147 FIGURE 5.24 Some sesquiterpenoid alcohols. 139 Chemistry of Essential Oils 146 148 150 149 FIGURE 5.25 Some sesquiterpenoid hydrocarbons. 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 152 151 O 153 HO HO O 154 FIGURE 5.26 155 Components of vetiver, patchouli, and grapefruit. 156 140 Handbook of Essential Oils 157 O O 158 159 O 163 164 162 161 160 O O O O 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 141 Chemistry of Essential Oils HO OH 169 OH O O 170 O 171 O 172 FIGURE 5.28 Iripallidal and the irones. 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 142 Handbook of Essential Oils OH O Rapeseed oil 173 O3 OH OH 174 O O + O O 175 H O 177 Castor oil OH O O 175 + 176 1) cyclopentanone/base 2) H+ 3) H2/cat. 4) RCO3H H+ 179 O OH 178 O O O FIGURE 5.29 Some natural feedstocks for synthesis of lipids and polyketides. 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 O 56 O 57 O O HO HO HO 54 53 O FIGURE 5.30 Shikimates from eugenol and safrole. O O O 59 55 O O O O 60 61 143 Chemistry of Essential Oils OH OH O2 OH [O] 182 183 cat. 184 O2 cat. cat. 185 180 O By-product AlCl3 [O] O OH 47 H2 / cat. 186 O H+ 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. 144 Handbook of Essential Oils O 52 O 53 OH OH 183 O 38 OH OH 184 OH OH 188 OH + HO HO 190 189 OH O O OH O O O 192 HO 191 193 O O HO O 55 O O O O 61 50 O O 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 145 Chemistry of Essential Oils R O Cl 181 O RCO2Na 199 Cl2/R . or hn 198 H2O [O] OH O OH O 194 195 + 196 O OH O O 48 197 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 O OH 202 201 FIGURE 5.34 Synthesis of methyl anthranilate. NH2 41 146 Handbook of Essential Oils O OH OH O O H H H 205 203 204 73 105 O O 135 206 FIGURE 5.35 Partial synthesis of terpenoids from natural extracts. 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 O 89 74 80 OH 65 OH OH OH 63 23 208 207 158 FIGURE 5.36 Products from a-pinene. OH O O 71 91 147 Chemistry of Essential Oils OH 63 76 70 O 94 OH 209 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 O OH OH H2/cat. Base 211 213 212 O 214 210 O H+ O OH Base 71 216 FIGURE 5.38 Citral from acetylene and acetone. 215 148 Handbook of Essential Oils 220 H 218 OH Pd O OH 217 221 223 71 O O O O FIGURE 5.39 O 1) Ag:SiO2/O2 2) Pd 219 222 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 70 O N 209 224 225 Mentha arvensis OH 226 OH 97 FIGURE 5.40 Competing routes to l-menthol. OH 227 OH 94 Chemistry of Essential Oils 149 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 Significance. 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. 150 Handbook of Essential Oils 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 ....................................................................................................................... 6.2 Classical Analytical Techniques ........................................................................................ 6.3 Modern Analytical Techniques ......................................................................................... 6.3.1 Use of GC and Linear Retention Indices in Essential Oils Analysis .................... 6.3.2 Gas Chromatography-Mass Spectrometry ............................................................. 6.3.3 Fast GC for Essential Oil Analysis ........................................................................ 6.3.4 Gas Chromatography-Olfactometry for the Assessment of Odor-Active Components of Essential Oils ................................................................................ 6.3.5 Gas Chromatographic Enantiomer Characterization of Essential Oils ................. 6.3.6 LC and Liquid Chromatography Hyphenated to MS in the Analysis of Essential Oils ................................................................................................ 6.3.7 Multidimensional Gas Chromatographic Techniques ........................................... 6.3.8 Multidimensional Liquid Chromatographic Techniques ....................................... 6.3.9 On-Line Coupled Liquid Chromatography-Gas Chromatography (LC-GC) ........ 6.4 General Considerations on Essential Oil Analysis ............................................................ References .................................................................................................................................. 151 152 156 157 158 159 162 164 165 167 174 176 177 177 6.1 INTRODUCTION 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 151 152 Handbook of Essential Oils 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), enfleurage, 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 153 Analysis of Essential Oils 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]D20, 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: aT [a]Tl = c ◊ll , 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]D20, 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: sin i N = , sin e 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, 154 Handbook of Essential Oils 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. Analysis of Essential Oils 155 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 156 Handbook of Essential Oils 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 (Rf ) value for each spot, which is defined as Rf = ZS , Z 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 Rf value is characteristic for any given compound on the same stationary phase using the identical mobile phase. Hence, known Rf 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 Analysis of Essential Oils 157 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 158 Handbook of Essential Oils 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 IT ), 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) Analysis of Essential Oils 159 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 160 Handbook of Essential Oils (a) Inten.(×10,000) 1.00 93 0.75 0.50 0.25 91 77 41 80 65 69 53 40 50 60 70 80 90 100 (b) Inten.(×10,000) 1.00 136 121 105 0.00 110 120 130 m/z 93 0.75 0.50 91 77 0.25 41 65 53 69 136 80 0.00 40 50 60 70 80 (c) Inten.(×10,000) 1.00 90 100 110 120 130 m/z 121 93 0.75 0.50 121 107 107 41 53 0.25 67 79 81 161 133 136 147 189 204 0.00 50 75 100 125 (d) Inten.(×10,000) 1.00 0.25 175 200 m/z 121 0.75 0.50 150 93 41 107 53 67 79 81 133 136 147 161 189 204 0.00 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 161 Analysis of Essential Oils 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 df column) and fast (10 m ¥ 0.10 mm I.D., 0.10 mm df 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 11 6 21 32 9 20,000 19 Intensity 15,000 27 22 1 13 10,000 26 5000 8 7 10 12 3 0 Wh = 126 ms 0 12 16 14 24 15 30 28 31 25 29 24 23 18 17 20 Wh = 192 ms Wh = 180 ms 36 48 Seconds 35 34 36 33 60 72 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.) 162 Handbook of Essential Oils 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 (C8–C14), b-sinensal, and linalool presented the major CHARM responses. On the other hand, AEDA has 163 Analysis of Essential Oils Nootkatone β-Sinensal 2E, 4E-Decadienal Dodecanal Geranial Nerol Geraniol 100 Linalool Octanol Terpenen-4-ol 2E-Decenal Neral Response (mV) 200 Citronellal 300 Nonanal Limonene 400 Octanal 500 Decanal 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 0 400 800 1200 6 8 10 12 14 16 20 18 Time (min) 22 24 26 28 30 32 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.) 164 Handbook of Essential Oils (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 affi rmed 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 Analysis of Essential Oils 165 enantiomeric pairs; resolution (RS) can be improved by optimizing the gas linear velocity, a factor of high importance in cases of difficult enantiomer separation. Satisfactory resolution requires RS ≥ 1, and baseline resolution is obtained when RS ≥ 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]. 166 Handbook of Essential Oils Inten.(×10,000) 1.00 41 0.75 69 (3S)-(–)-β-citronellol 55 H 81 HO 95 0.50 0.25 109 123 138 ﬂoral, geranium-like odour 156 0.00 40 50 60 Inten.(×10,000) 1.00 41 70 80 90 LRI 1343 100 110 120 130 140 150 m/z 69 (3R)-(+)-β-citronellol 0.75 55 H 81 95 0.50 0.25 OH 109 123 138 citronella oil like odour 156 0.00 40 FIGURE 6.4 50 60 70 80 90 LRI 1346 100 110 120 130 140 150 m/z 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 167 Analysis of Essential Oils 1.60 8 1.40 1.20 1.00 AU 0.80 0.60 i.s. 0.40 9 11 3 0.20 0.00 5 4 7 10 20.00 Minutes 12 13 14 15 16 17 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 168 Handbook of Essential Oils 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 (1D) 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 1D and a CSP capillary in the second dimension (2D). 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 1D and 2D, 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 169 Analysis of Essential Oils 0.04 0.02 0.00 0 0.06 Volts Linalool Volts 0.06 0.08 Terpinen-4-ol α-Terpineol 0.08 0.10 Limonene 0.10 Sabinene β-Pinene (a) 0.04 0.02 25 0.00 50 Minutes (b) 0.10 0.10 * 0.08 0.08 * Volts 0.06 Volts 0.06 * 0.04 0.04 * 0.02 0.02 * * 0.010 0.006 0.002 15 0.022 (–)-α-Terpineol (+)-α-Terpineol 0.018 0.014 Volts 0.014 (–)-Linalol (+)-Linalol Volts 0.018 (+)-Limonene 0.022 0.026 (–)-Limonene 0.026 (+)-β-Pinene (–)-β-Pinene (–)-Sabinene (c) 0.00 50 Cut 1 Cut 2 Cut 3 Cut 4 Cut 5 (+)-Terpinen-4-ol (–)-Terpinen-4-ol 0 (+)-Sabinene 0.00 0.010 0.006 0.002 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.) 170 Handbook of Essential Oils 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 2D; retention time windows of eight peaks were defined. The eight peaks (camphene, b-pinene, sabinene, limonene, camphor, isoborneol, borneol, terpinen4-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 2D analysis can only be restricted to a few regions UV(×10,000) Chromatogram 2.5 9 7 5 4 E 1 2.0 8 1.5 A C 2+3 1.0 D B 6 0.5 0.0 1.5 2.0 2.5 3.0 3.5 UV(×10,000) Chromatogram (+)2 (–)1 (+)1 (–)2 4.0 4.0 4.5 5.0 5.5 min (–)7 (+)5 (–)5 3.0 (+)4 (+)9 2.0 (–)4 1.0 (–)9 (–)6 (+)6 (+)7 (+/–)3 (–)8 (+)8 0.0 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 min 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 2D 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.) 171 Analysis of Essential Oils 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 1D, onto the 2D 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 1D, and a polar column is used as the fast 2D 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 1D 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 <100 sample components. The GC ¥ GC system used in this research was composed of a GC equipped with a thermal modulation unit (Zoex Corporation, Lincoln, Nebraska, USA), illustrated in Figure 6.8. The thermal modulation cycle is a three-step process, involving sample accumulation, focusing, and acceleration stages. The column set used was composed of a nonpolar column (1 m ¥ 100 mm I.D., 3.5 mm df ) in the 1D and one of intermediate polarity (2 m ¥ 100 mm I.D., 0.5 mm df ) in the 2D, connected by a press fit. A two- to threefold increase in separation power was obtained for the GC ¥ GC analyses of both mint oils; peppermint essential oil was found to contain 89 identifiable peaks by GC ¥ GC compared to 30 peaks in GC-MS, while in the spearmint oil, 68 peaks were detected by GC ¥ GC and 28 by means of GC-MS. The simple alignment of the 2D retention times of the investigated oils revealed that both have 52 components in common, as opposed to 18 matches by monodimensional GC. GC ¥ GC analyses of essential oils of high complexity have also been carried out, such as of vetiver oil (Vetiver zizanoides) [117]. The work was performed on a gas chromatographic system (6890GC, Agilent Technologies, Santa Clara, USA) retrofitted to a longitudinal modulated cryogenic system (LMCS, Chromatography Concepts, Doncaster, Australia). In Figure 6.9, a schematic diagram of the Rotator shaft Phase termination A From injector To detector x1 B x2 FIGURE 6.8 Top-view scheme of the thermal modulator. The arrows marked as x1 and x 2 indicate the length of the modulator tube. The rotating heater is in position A prior to each modulation cycle. Its counterclockwise movement (from position A to B) over the modulator tube produces the thermal modulation. (From Dimandja, J.M.D. et al., 2000. J. High Res.Chromatogr., 23: 208–214. With permission.) 172 Handbook of Essential Oils Injector Modulation device Detector Support guides CO2, N2 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 1D column (25 m ¥ 0.25 mm I.D., 0.25 mm df ) connected to a 50% phenyl–50% dimethylpolysiloxane 2D column (0.8 m ¥ 0.1 mm I.D., 0.1 mm df ), 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 2D 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 2D 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 2D 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 1D, but due to their wide range of component polarities these are found to spread throughout a broader region of the 2D plane. According to the authors, by using GC ¥ GC the detection of subtle 173 Analysis of Essential Oils (a) 80 70 60 50 Response / pA 40 30 (b) 20 80 (expanded scale) 60 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 Y 4.00 3.50 S 3.00 2.50 2.00 1.50 M 1.00 Z 0.50 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 1D; M: monoterpene hydrocarbons, S: sesquiterpene hydrocarbons. (From Shellie, R. et al., 2002. J. Chromatogr. A, 970: 225–234. With permission.) 174 Handbook of Essential Oils differences in the analyses of lavender essential oils from different cultivars should be simplified, since it could be based on a 2D 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 1D 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 2D 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 2D 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 175 Analysis of Essential Oils LC column in the first and two conventional columns in the 2D. 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 1D and a monolithic C18 column in the 2D 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 1D. Under optimized LC conditions, the high degree of orthogonality between the NP and RP systems tested, resulted in increased 2D 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 C40 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 1D and 2D, respectively. Free carotenoids in orange essential oil and juice (after saponification), were identified by combining the two-dimensional retention data 1.000 0.875 5 0.750 2 0.625 8 4 0.500 7 3 0.375 9 1 0.250 6 0.125 0 5 10 15 20 25 min 10 30 11 35 40 45 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.) 176 Handbook of Essential Oils 120.0 UV at 450 nm Mono-ols tR (RP), sec 106.7 93.4 80.0 14 Hydrocarbons 10 13 2 9 1 8 7 15 12 41 25 33 36 43 40 27 32 49 23 28 30 19 21 42 53 17 20 53.4 26.7 Di-ol di-epoxides 18 66.7 40.0 Di-ol mono-epoxides Mono-ol mono-epoxides Di-ols 16 54 56 62 57 Tri-ols + Tri-ol mono-epoxides 61 64 68 35 38 45 60 44 52 51 55 31 22 59 50 26 63 34 66 Di-ol mono-epoxides 70 69 73 75 13.4 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 tR (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 1D 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 2D [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. Analysis of Essential Oils 177 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. 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Comprehensive two-dimensional gas chromatography in the analysis of volatile samples of natural origin: A multidisciplinary approach to evaluate the influence of second dimension column coated with mixed stationary phases on system orthogonality. J. Chromatogr. A, 1132: 268. 123. Shellie, R., P. Marriott, and P. Morrison, 2004. Comprehensive two-dimensional gas chromatography with flame ionization and time-of-flight mass spectrometry detection: Qualitative and quantitative analysis of West Australian sandalwood oil. J. Chromatogr. Sci., 42: 417. 124. Özel, M.Z., F. Gogus, and A.C. Lewis, 2003. Subcritical water extraction of essential oils from Thymbra spicata. Food Chem., 82: 381. 125. Özel, M.Z., F. Gogus, J.F. Hamilton, and A.C. Lewis, 2004. The essential oil of Pistacia vera L. at various temperatures of direct thermal desorption using comprehensive gas chromatography coupled with time-of-flight mass spectrometry. Chromatographia, 60: 79. 126. Roberts, M.T., J.P. Dufour, and A.C. Lewis, 2004. Application of comprehensive multidimensional gas chromatography combined with time-of-flight mass spectrometry (GC ¥ GC-TOFMS) for high resolution analysis of hop essential oil. J. Sep. Sci., 27: 473. 127. Özel, M.Z., F. Göğüş, and A.C. Lewis, 2006. Determination of Teucrium chamaedrys volatiles by using direct thermal desorption–comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry. J. Chromatogr. A, 1114: 164. 128. Özel, M.Z., F. Gogus, and A.C. Lewis, 2006. Comparison of direct thermal desorption with water distillation and superheated water extraction for the analysis of volatile components of Rosa damascena Mill. Using GC ¥ GC-TOF/MS. Anal. Chim. Acta, 566: 172. 129. Eyres, G., P.J. Marriott, and J.P. Dufour, 2007. The combination of gas chromatography–olfactometry and multidimensional gas chromatography for the characterisation of essential oils. J. Chromatogr. A, 150: 70. 130. Ma, C., H. Wang, X. Lu, H. Li, B. Liu, and G. Xu, 2007. Analysis of Artemisia annua L. volatile oil by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry. J. Chromatogr. A, 1150: 50. 131. Zhu, S., X. Lu, Y. Qiu, T. Pang, H. Kong, C. Wu, and G. Xu, 2007. Determination of retention indices in constant inlet pressure mode and conversion among different column temperature conditions in comprehensive two-dimensional gas chromatography. J. Chromatogr. A, 1150: 28. 132. Dugo, P., M.D. Fernandez, A. Cotroneo, and G. Dugo, 2006. Optimization of a comprehensive twodimensional normal-phase and reversed phase-liquid chromatography system. J. Chromatogr. Sci., 44: 1. 133. Dugo, P., O. Favoino, R. Luppino, G. Dugo, and Mondello, L., 2004. Comprehensive two-dimensional normal-phase (adsorption)-reversed-phase liquid chromatography. Anal. Chem., 73: 2525–2530. 134. François, I.D., A. Villiers, and P. Sandra, 2006. Considerations on the possibilities and limitations of comprehensive normal phase-reversed phase liquid chromatography (NPLC ¥ RPLC). J. Sep. Sci., 29: 492. 135. Dugo, P., M. Herrero, T. Kumm, D. Giuffrida, G. Dugo, and L. Mondello, Comprehensive normalphase ¥ reversed-phase liquid chromatography coupled to photodiode array and mass spectrometry detectors for the analysis of free carotenoids and carotenoid esters from mandarin. J. Chromatogr. A, 2008, in press. Analysis of Essential Oils 183 136. Dugo, P., V. Škeříková, T. Kumm, A. Trozzi, P. Jandera, and L. Mondello, 2006. Elucidation of carotenoid patterns in citrus products by means of comprehensive normal-phase × reversed-phase liquid chromatography. Anal. Chem., 78: 7743–7750. 137. Grob, K., 1987. On-line coupled HPLC-HRGC. In Proc. 8th Int. Symp. on Capillary Chromatography. Italy: Riva del Garda. 138. Mondello, L., K.D. Bartle, G. Dugo, and P. Dugo, 1994. Automated HPLC-HRGC: A powerful method for essential oils analysis. Part III. Aliphatic and terpene aldehydes of orange oil. J. High Resol. Chromatogr., 17: 312. 139. Mondello, L., P. Dugo, K.D. Bartle, G. Dugo, and A. Cotroneo, 1995. Automated HPLC-HRGC: A powerful method for essential oils analysis. Part V. Identification of terpene hydrocarbons of bergamot, lemon, mandarin, sweet orange, bitter orange, grapefruit, clementine and Mexican lime oils by coupled HPLCHRGC-MS(ITD). Flavour Fragr. J., 10: 33. 140. Dugo, G., A. Verzera, A. Trozzi, A. Cotroneo, L. Mondello, and K.D. Bartle, 1994. Automated HPLCHRGC: A powerful method for essential oils analysis. Part I. Investigation on enantiomeric distribution of monoterpene alcohols of lemon and mandarin essential oils. Essenz. Deriv. Agrum., 64: 35. 141. Dugo, G., A. Verzera, A. Cotroneo, I. Stagno d’Alcontres, L. Mondello, and K.D. Bartle, 1994. Automated HPLC-HRGC: A powerful method for essential oil analysis. Part II. Determination of the enantiomeric distribution of linalool in sweet orange, bitter orange and mandarin essential oils. Flavour Fragr. J., 9: 99. 142. Mondello, L., P. Dugo, K.D. Bartle, B. Frere, and G. Dugo, 1994. On-line high performance liquid chromatography coupled with high resolution gas chromatography and mass spectrometry (HPLC-HRGC-MS) for the analysis of complex mixtures containing highly volatile compounds. Chromatographia, 39: 529. 143. Mondello, L., P. Dugo, G. Dugo, and K.D. Bartle, 1996. On-line HPLC-HRGC-MS for the analysis of natural complex mixtures. J. Chromatogr. Sci., 34: 174. 7 Safety Evaluation of Essential Oils: A Constituent-Based Approach Timothy B. Adams and Sean V. Taylor CONTENTS 7.1 Introduction ....................................................................................................................... 7.2 Constituent-Based Evaluation of an Essential Oil ............................................................. 7.3 Scope of Essential Oils Used in Food ............................................................................... 7.3.1 Plant Sources ......................................................................................................... 7.3.2 Processing of Essential Oils for Flavor Functions ................................................ 7.3.3 Chemical Composition and Congeneric Groups ................................................... 7.3.4 Chemical Assay Requirements and Chemical Description of Essential Oil ......... 7.3.4.1 Intake of the Essential Oil ....................................................................... 7.3.4.2 Analytical Limits on Constituent Identification ...................................... 7.3.4.3 Intake of Congeneric Groups .................................................................. 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 ....................................... 7.4.2 Safety of Constituents and Congeneric Groups in Essential Oils ......................... 7.5 The Guide and Example for the Safety Evaluation of Essential Oils ............................... 7.5.1 Introduction ........................................................................................................... 7.5.2 Elements of the Guide for the Safety Evaluation of the Essential Oil .................. 7.5.2.1 Introduction ............................................................................................. 7.5.2.2 Prioritization of Essential Oil According to Presence in Food ............... 7.5.2.3 Organization of Chemical Data: Congeneric Groups and Classes of Toxicity .................................................................................. 7.6 Summary ........................................................................................................................... References .................................................................................................................................. 186 187 187 187 188 188 190 191 192 192 193 193 193 194 195 195 196 196 196 197 205 205 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 185 186 Handbook of Essential Oils 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. 7.1 INTRODUCTION 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 188 Handbook of Essential Oils 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, 189 Safety Evaluation of Essential Oils 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 Menthyl acetate OH Menthol OH O OH 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. 190 Handbook of Essential Oils 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. Safety Evaluation of Essential Oils 191 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 <1.5 mg/d, there should be no requirement to identify and quantify that constituent. Determining the level to which the constituents of an essential oil should be identified depends on estimates of intake of food or flavor additives. These estimates are traditionally calculated using a “volume-based” or a “menu-census” approach. A volume-based approach assumes that the total annual volume of use of a substance reported by an industry is distributed over a portion of the population consuming that substance. A menu-census approach is based on the concentration of the substance (essential oil) added to each flavor, the amount of flavor added to each food category, the portion of food consumed daily, and the total of all exposures across all food types. Although the latter is quite accurate for food additives consumed at higher levels in a wide variety of food such as food emulsifiers, the former method provides an efficient and conservative approximation of intake, if a fraction of the total population is assumed to consume all of the substance. For the World Health Organization (WHO) and the U.S. Food and Drug Administration (U.S. FDA), intake is calculated using a method known as the per capita intake (PCI ¥ 10) method (Rulis et al., 1984; Woods and Doull, 1991). The PCI ¥ 10 method assumes that only 10% of the population consumes the total annual reported volume of use of a flavor ingredient. This approximation provides a practical and cost-effective approach to the estimation of intake for flavoring substances. The annual volumes of flavoring agents are relatively easy to obtain by industry-wide surveys, which can be performed on a regular basis to account for changes in food trends and flavor consumption. The most recent poundage survey of U.S. flavor producers was collected in 2005 and published by the Flavor and Extract Manufacturers (FEMA) in 2007 (Adams et al., 2007). Similar surveys were conducted in recent years in Europe (EFFA, 2005) and Japan (JFFMA, 2002). Calculation of intake using the PCI ¥ 10 method has been shown to result in conservative estimates of intake and this is appropriate for safety evaluation. Over the last three decades, two comprehensive studies of flavor intake have been undertaken. One involved a detailed dietary analysis (DDA) of a panel of 12,000 consumers who recorded all foods that they consumed over a 14-day period, and the flavoring ingredients in each food were estimated by experienced flavorists to estimate intake of each flavoring substance (Hall, 1976; Hall and Ford, 1999). The other study utilized a robust full stochastic model (FSM) to estimate intake of flavoring ingredients by typical consumers in the United Kingdom (Lambe et al., 2002). The results of the data-intensive DDA method and the model-based FSM support the use of PCI data as a conservative estimate of intake. With regard to essential oils, the PCI ¥ 10 method provides overestimates of intake for oils that are widely distributed in food. The large annual volume of use reported for essential oils such as orange oil, lemon oil, and peppermint oil indicates widespread use in a large variety of foods resulting in consumption of these oils by significantly more than 10% of the population. Citrus flavor is pervasive in a multitude of foods and beverages. Therefore, for selected high-volume essential oils, a simple PCI rather than a per capita ¥ 10 intake may be more appropriate. However, the intake of the congeneric groups and the group of unidentified constituents for these high-volume oils is still estimated by the PCI ¥ 10 method. 192 Handbook of Essential Oils 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 <1.5 mg/d will not need to be identified. For instance, if the annual volume of use of coriander oil in the USA is 10,000 kg, then the estimated daily PCI of the oil is 10,000 kg/yr ¥ 109 mg/ kg = 978 mg coriander oil/person/d. 365 d/yr ¥ 28,000,000 persons Based on the intake of coriander oil (978 μg/d), any constituent present at >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 Safety Evaluation of Essential Oils 193 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 7.4.1 SAFETY CONSIDERATIONS FOR ESSENTIAL OILS, CONSTITUENTS, AND CONGENERIC GROUPS 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 194 Handbook of Essential Oils 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. Safety Evaluation of Essential Oils 195 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 7.5.1 THE GUIDE AND EXAMPLE FOR THE SAFETY EVALUATION OF ESSENTIAL OILS 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 196 Handbook of Essential Oils 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 197 Safety Evaluation of Essential Oils 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 and 100% 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. 327,494 kg/yr ¥ 109 mg/ kg = 3204 mg/person/d. 365 days/yr ¥ 280 ¥ 106 persons 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. 198 Handbook of Essential Oils 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. 199 Safety Evaluation of Essential Oils 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 well-established 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 a 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 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. 200 Handbook of Essential Oils 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 105.” 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 <10,000 lb/yr (Hall and Oser, 1968), this alone implies intakes commonly many orders of magnitude below the no-effect level. Nonadditivity thus can often be assumed. As is customary in the evaluation of any substance, high-end data for exposure (consumption) are used, and multiple other conservatisms are employed to guard against underestimation of possible risk. All of these apply to complex mixtures as well as to individual substances. 7.5.2.3.1 Cornmint Oil Congeneric Groups In cornmint oil, the principal congeneric group is composed of terpene alicyclic secondary alcohols, ketones, and related esters, as represented by the presence of (−)-menthol, (−)-menthone, Safety Evaluation of Essential Oils 201 (+)-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 commercial cornmint oil, are considered separately in the guide. In this case, the daily PCI of 64 μg/person/d (1.07 μg/kg bw/d) does not exceed the 90 μg/d threshold for Class III. However, a 90-day study on pulegone (NTP, 2002) showed a NOAEL (9.375 mg/kg bw/d) that is approximately 8700 times the intake of pulegone and its metabolites as constituents of cornmint oil. Also, in a 28-day study with peppermint oil (Mentha piperita) containing approximately 4% pulegone and menthofuran, a NOAEL of 200 mg/kg bw/d for male rats and a NOAEL of 400 mg/kg bw/d for female rats were established that corresponds to a NOAEL of 8 mg/kg bw/d for pulegone and menthofuran (Serota, 1990). In a 90-day study with a mixture of Mentha piperita and Mentha arvensis oils (Splindler and Madsen, 1992; Smith et al., 1996), a NOAEL of 100 mg/kg bw/d was established that corresponds to a NOAEL of 4 mg/kg bw/d for pulegone and menthofuran. The only other congeneric group that accounts 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 (<3%) in the finished commercial oil. Using the 8% figure to determine a conservative estimate of intake, the intake of terpene hydrocarbons is 256 μg/person/d (4.27 μg/kg bw/d). This group is predominantly metabolized by cytochrome P450-catalyzed hydroxylation, conjugation, and excretion (Ishida et al., 1981; Madyastha and Srivatsan, 1987; Crowell et al., 1994; Poon et al., 1996; Vigushin et al., 1998; Miyazawa et al., 2002). The daily PCI of 256 μg/person/d is less than the exposure threshold (1800 μg/person/d) for Structural Class I. Although no additional data would be required to complete the evaluation of this group, NOAELs (300 mg/kg bw/d) from long-term studies (NTP, 1990) on principal members of this group are orders of magnitude greater than the daily PCI (“eaters only”) of terpene hydrocarbons (0.025 mg/ kg bw/d). Therefore, all congeneric groups in cornmint oil are considered safe for use when consumed in cornmint oil. Aliphatic terpene hydrocarbon (e.g., limonene, pinene) Secondary alicyclic saturated and unsaturated alcohol/ketone/ ketal/ester (e.g., menthol, menthone, isomenthone, menthyl acetate) Congeneric Group Assignment I (1800) II (540) Structural Class (TTC) (µg/Person/d) Steps 2 and 3: Constituent Identification/Congeneric Grouping/Structural Class Assignment 8 95 High % From Multiple Commercial Samples 256 3044 Intake (µg/ Person/d) Step 4: Oil Composition by Congeneric Group, Resulting Intake TABLE 7.2 Safety Evaluation of Cornmint Oil, Mentha arvensisa 1. w-Oxidation to yield polar hydroxy and carboxy metabolites excreted as glucuronic acid conjugates 2. w-Oxidation of the side chain substituents to yield various polyols and hydroxy acids and excreted as glucuronic acid conjugates 1. Glucuronic acid conjugation of the alcohol followed by excretion in the urine Metabolism Pathways Yes, 256 μg /person/d, <1800 μg/ person/d No, 3044 μg/person/d >540 μg/ person/d Intake of Congeneric Group or Total of Unidentified Constituents Group < TTC for Class? Additive Interactions Among Congeneric Groups? NOEL of 400,000 μg/kg bw/d for menthone (28-day gavage study in rats) (Madsen et al., 1986) Not required NOEL of None anticipated 600,000 μg/ kg bw/d for menthol (103-week dietary study in mice) (NCI, 1979) Relevant Toxicity Data if Intake of Group >TTC Steps 5,6,7,8: Metabolism/Toxicology Data/Evaluation 202 Handbook of Essential Oils III (90) 2 64 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) 1. Reduction to yield menthone or isomenthone, followed by hydroxylation of ring or side chain positions and then conjugation with glucuronic acid a Note: In Steps 2 and 3, individual constituents and their assignment to structural classes are not shown. Based on daily PCI of 3204 μg/person/d for cornmint oil, as determined in Step 1. 2-Isopropylidene cyclohexanone and metabolites (e.g., pulegone) Yes, 64 μg/person/d <90 μg/ person/d Not required. But NOAEL of 9375 μg/kg bw/d for pulegone (90-day gavage study in rats) (NTP, 2002) Safety Evaluation of Essential Oils 203 204 Handbook of Essential Oils H H H Hydrolysis OAc Isopulegyl acetate H Reduction Isomerization O OH Isopulegol Isopulegone O 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. Safety Evaluation of Essential Oils 7.6 205 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. 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Res., 26: 93–98. 8 Metabolism of Terpenoids in Animal Models and Humans Walter Jäger CONTENTS 8.1 Introduction ....................................................................................................................... 8.2 Metabolism of Monoterpenes ............................................................................................ 8.2.1 Camphene .............................................................................................................. 8.2.2 Camphor ................................................................................................................ 8.2.3 Carvacrol ............................................................................................................... 8.2.4 Carvone ................................................................................................................. 8.2.5 1,4-Cineole ............................................................................................................ 8.2.6 1,8-Cineole ............................................................................................................ 8.2.7 Citral ...................................................................................................................... 8.2.8 Citronellal .............................................................................................................. 8.2.9 Fenchone ................................................................................................................ 8.2.10 Geraniol ................................................................................................................. 8.2.11 Limonene ............................................................................................................... 8.2.12 Linalool .................................................................................................................. 8.2.13 Linalyl Acetate ...................................................................................................... 8.2.14 Menthol .................................................................................................................. 8.2.15 Myrcene ................................................................................................................. 8.2.16 Pinene .................................................................................................................... 8.2.17 Pulegone ................................................................................................................ 8.2.18 a-Terpineol ............................................................................................................ 8.2.19 a- and b-Thujone ................................................................................................... 8.2.20 Thymol .................................................................................................................. 8.3 Metabolism of Sesquiterpenes .......................................................................................... 8.3.1 Caryophyllene ........................................................................................................ 8.3.2 Farnesol ................................................................................................................. 8.3.3 Longifolene ............................................................................................................ 8.3.4 Patchouli Alcohol .................................................................................................. References .................................................................................................................................. 8.1 209 210 210 210 212 213 213 214 216 217 217 218 218 219 221 221 223 223 225 225 225 226 227 227 227 229 230 232 INTRODUCTION 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, 209 210 Handbook of Essential Oils 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 officinalis). 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 211 Metabolism of Terpenoids in Animal Models and Humans Rabbit in vivo + O O Camphene Rabbit in vivo + OH OH OH OH OH 10-Hydroxytricyclene Camphene-2,10-glycol Camphene-2,10-glycol HO HO HO 3-Hydroxytricyclene 6-exo-Hydroxycamphene 7-Hydroxycamphene 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.) Human, rat, rabbit, dog in vitro O Rabbit in vivo HO O + + O OH H HO H OH Camphor Rabbit in vitro/in vivo 3-endo-Hydroxycamphor not in vitro human HO H O O 5-endo-Hydroxycamphor not in vitro human 5-exo-Hydroxycamphor not in vivo rabbit O H O O Borneol Isoborneol 2,3-Bornanedione 2,5-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.) 212 Handbook of Essential Oils (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 metabolism. 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 Rat in vivo OH OH OH 2,3-Dihydroxy-p-cymene OH 2-(3-Hydroxy-4-methylphenyl)propan-2-ol Carvacrol Rat in vivo Rat in vivo CH2OH OH OH CH2OH 2-(3-Hydroxy-4-methylphenyl)propan-1-ol 2-Hydroxymethyl-5-(1-methylethyl)phenol COOH OH OH CH2OH OH COOH 2-(3-Hydroxy-4-methylphenyl)propionic acid 2-Hydroxymethyl-4-(1-methylethyl)benzoic acid CH2OH 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 Mensch und Säugetier: Bedeutung für die pharmazeutische Praxis. MPharm. diploma thesis, University of Vienna, Austria.) 213 Metabolism of Terpenoids in Animal Models and Humans 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 (Cmax) 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-8en-9-ol, and 1,4-cineole-9-carboxylic acid (Asakawa et al., 1988). Using rat and human liver H L-Carvone O-glu OH O H 4L,6D-Carveol H 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.). 214 Handbook of Essential Oils OH OH Dihydrocarveol Carveol Human in vivo Carveol-glucuronid Human in vivo O O Human in vivo Human in vivo O Human in vivo OH HO 10-Hydroxycarvone OH Carvone Uroterpenolone Human in vivo O O O OH Carvonic acid O 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 215 Metabolism of Terpenoids in Animal Models and Humans O O HO OH Rabbit in vivo OH 3,8-Dihydroxy-1,4-cineole O O O OH Rabbit in vivo OH OH 8,9-Dihydroxy-1,4-cineole 1,4-Cineole-8-en-9-ol 1,4-Cineole O O Human, rat in vitro OH OH COOH 9-Hydroxy-1,4-cineole 1,4-Cineole-9-carboxylic acid O 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 in Mensch und Säugetier: Bedeutung für die pharmazeutische Praxis. MPharm. diploma thesis, University of Vienna, Austria.) Human in vivo/vitro HO 2-Hydroxy-1,8-cineole Human in vivo/in vitro O Rat in vitro Rat in vitro 1,8-Cineole O HO 3-Hydroxy-1,8-cineole Rat in vitro CH2OH 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.) 216 Handbook of Essential Oils 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-dimethyl2,6-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 CHO Rat in vivo CHO CHO CHO CHO CHO OH OH (E) (Z) (E) Citral (Z) (E) 8-Hydroxy-3,7-dimethyl-2,6-octadienal (Z) 3,7-Dimethyl-2,6-octadienedial Rat in vivo COOH COOH COOH COOH COOH COOH (Z) (E) (E) (Z) 3,7-Dimethyl-2,6-octadienedioic acid E-3,8-Dimethyl-2,6-octadienedioic acid OH OH OH COOH COOH COOH COOH COOH OH (E) (Z) OH 3,8-Dihydroxy-3,7-dimethyl-6-octenoic acid (E) 3-Hydroxy-3,7-dimethyl-6-octenedioic acid COOH (Z) 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.) 217 Metabolism of Terpenoids in Animal Models and Humans 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-dimethyl2,6-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 officinalis (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) CO2H + + OH OH OH OH (–)-trans-menthane-3,8-diol (+)-cis-Menthane-3,8-diol + OH CO2H (–)-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.) 218 Handbook of Essential Oils HO Human in vivo + H O 6-exo-Hydroxyfenchone H HO O 6-endo-Hydroxyfenchone Human in vivo 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 3,7dimethyl-2,6-octenedioic acid (Hildebrandt’s acid) or directly oxidized to geranic acid and 3hydroxycitronelic 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 219 Metabolism of Terpenoids in Animal Models and Humans OH Rat in vivo Rat in vivo Rabbit in vitro Geraniol COOH OH OH 8-Hydroxygeraniol Geranic acid Rat in vivo OH COOH OH COOH COOH 8-Carboxygeraniol COOH 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 Mensch und Säugetier: Bedeutung für die pharmazeutische Praxis. MPharm. diploma thesis, University of Vienna, Austria.) 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). 220 Handbook of Essential Oils CH2OH Perillyl alcohol trans-Carveol OH Human in vivo OH Limonene-1,2-diol Carvone Human, rabbit, guinea pig, dog in vitro Human, rabbit, guinea pig, dog in vitro HO OH Rabbit, O guinea pig, dog in vitro HO Rat in vitro O Human in vivo OH 1,2-Glycol 1,2-Epoxid OH OH Human Limonene-8,9-diol in vivo (Uroterpenol) Rat in vitro Rat in vitro O-Glu Limonene-8,9-diolglucuronide Human in vivo Rat in vitro O-Glu OH O Limonene-10-ol D-Limonene 8,9-Epoxid Human, rat in vivo Human in vivo Limonene-10-olglucuronide Rat in vitro Rat in vitro CH2OH COOH COO-Glu Human in vivo OH OH 8,9-Glycol HOOC Isomer of perillic acid (p-Mentha-1,8-dien-carboxylic acid) Perillyl alcohol Perillic acid Human, rat in vivo COO-Glu COO-Glu Perillic acid-glucuronide Rat in vitro COOH COOH Human in vivo + cis-Dihydroperillic acidglucuronide + trans-Dihydroperillic acid- cis-Dihydroperillic acid trans-Dihydroperillic acid glucuronide 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.) 221 Metabolism of Terpenoids in Animal Models and Humans O-Glu Linalool-glucuronic acid Rat in vivo OH OH OH Rat in vivo OH O 8-Carboxylinalool OH OH Rat in vivo OH 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 in Mensch und Säugetier: Bedeutung für die pharmazeutische Praxis. MPharm. diploma thesis, University of Vienna, Austria.) 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 OH O O Linalyl acetate Rat in vitro 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 Mensch und Säugetier: Bedeutung für die pharmazeutische Praxis. MPharm. diploma thesis, University of Vienna, Austria.) 222 Handbook of Essential Oils 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 OH COOH OH OH COOH 3-Hydroxy-p-menthane-9-carboxylic acid p-Menthane-3,7-diol 3-Hydroxy-p-menthane-7-carboxylic acid Rat in vivo COOH Rat in vivo Rat in vitro/in vivo Rat in vivo OH OH OH OH OH 3,8-Dihydroxy-7-carboxylic acid Menthol p-Menthane-3,9-diol Rat in vitro/vivo Human/rat in vivo OH Rat in vivo Glucuronide Rat in vivo OH Rat in vitro/in vivo OH OH Glucuronide COOH p-Menthane-3,8-diol OH p-Menthane-3,7,8-triol COOH OH OH 3,8-Dihydroxy-p-menthane-7-carboxylic acid O O 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.) 223 Metabolism of Terpenoids in Animal Models and Humans 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 OH O OH O OH OH Rabbit in vivo Myrcene-3(10)-glycol 3-Hydroxymyrcene-10-carboxylic acid O OH OH Rabbit in vivo O OH Myrcene Myrcene-1,2-glycol OH 2-Hydroxymyrcene-1-carboxylic acid Rabbit in vivo OH CH2OH 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.) 224 Handbook of Essential Oils 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 officinalis) 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 CH2OH Rabbit in vivo COOH Rabbit in vivo + OH α-Pinene trans-Verbenol Human in vivo Myrtenol Myrtenic acid Human in vivo CH2OH OH cis-Verbenol Human in vivo trans-Pinocarveol 10-Pinanol Rabbit in vivo Rabbit in vivo O β-Pinene Rabbit in vivo CH2OH OH OH α-Terpineol 1-p-Menthene-7,8-diol 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.) Metabolism of Terpenoids in Animal Models and Humans 225 a- and b-pinene in humans leads to the formation of trans- and cis-verbenol, 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 226 Handbook of Essential Oils Human in vivo O O O Human in vivo Pulegone Human in vivo Menthofuran OH 10-Hydroxypulegone O OH O Menthone Pulegol 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 fistulosa 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 hydroxylation was only a minor reaction (Figure 8.21). 227 Metabolism of Terpenoids in Animal Models and Humans O OH OH Rat in vitro OH OH p-Menthane-1,2,8-triol Rat in vitro OH CH2OH COOH COOH α-Terpineol OH p-Menth-1-ene-7,8-diol OH Oleuropeic acid OH 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 (Figure 8.22). 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 228 Handbook of Essential Oils Glucuronide Glucuronide Glucuronide Glucuronide HO O O OH OH 2-Hydroxy-α-thujon 2-Hydroxy-β-thujon Human, rat, mouse in vitro Rat, mouse in vivo OH O 4-Hydroxy-α-thujon Human, rat, mouse in vitro Rat, mouse in vivo O 4-Hydroxy-β-thujon Human, rat, mouse in vitro Rat, mouse Human, rat, mouse in vivo in vitro Rat, mouse in vivo OH Rabbit, in vivo O Mouse, in vitro/ in vivo Rat, mouse in vivo O Human, rat, mouse in vitro OH 8-Hydroxy-α-thujon 8-Hydroxy-β-thujon Rat, mouse in vivo α-Thujon β-Thujon Human, rat, mouse in vitro Rat, mouse in vivo O Human, rat, mouse in vitro Human, rat, mouse in vitro Rat, mouse in vivo Rat, mouse in vivo Human, rat, mouse O in vitro Thujol (R) Neothujol (S) OH O 10-Hydroxy-α-thujon 10-Hydroxy-β-thujon O Glucuronide OH 7,8-Dehydro-α-thujon 7,8-Dehydro-β-thujon 7-Hydroxy-α-thujon 7-Hydroxy-β-thujon 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.) 229 Metabolism of Terpenoids in Animal Models and Humans HO OH OH 2,5-Dihydroxy-p-cymene Thymol OH OH OH 2-(2-Hydroxy-4-methylphenyl)propan-1-ol OH 5-Hydroxymethyl-2-(1-methylethyl)phenol OH COOH OH OH OH COOH 2-(2-Hydroxy-4-methylphenyl)propionic acid OH 3-Hydroxy-4-(1-methylethyl)bezoic acid 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). 230 Handbook of Essential Oils H H (–)-Caryophyllene Rabbit in vivo Rabbit in vivo O H HO H H H (–)-Caryophyllene-5,6-oxide O O OH H H OH Caryophyllene-5,6-oxide-2,12-diol HO H H (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). 231 Metabolism of Terpenoids in Animal Models and Humans O O HO HO OH OH Hydroxyfarnesyl-glucuronide Human in vitro OH COOH Human, rabbit, rat in vitro HO OH Farnesol Hydroxyfarnesol Human in vitro 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; Jahrmann, R., 2007. Metabolismus von Monterpenen und Sesquiterpenen in Mensch und Säugetier: Bedeutung für die pharmazeutische Praxis. MPharm. diploma thesis, University of Vienna, Austria.) Rabbit in vivo O Longifolene HO CHO 14-Hydroxyisolongifolaldehyde CHO 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 Mensch und Säugetier: Bedeutung für die pharmazeutische Praxis. MPharm. diploma thesis, University of Vienna, Austria.) 232 HO Handbook of Essential Oils Rabbit in vivo HO HO OH Patchouli alcohol Yielding diol HO COOH 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.) REFERENCES Asakawa, Y., T. Ishida, M. Toyota, and T. Takemoto, 1986. Terpenoid biotransformation in mammals IV. 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Birnbaum, 1990. Metabolism of citral, an a,b-unsaturated aldehyde, in male F344 rats. Drug Metab. Dispos., 18: 886–875. Engel, W., 2001. In vivo studies on the metabolism of the monoterpenes S-(+)- and R-(-)-carvon in humans using the metabolism of ingestion-correlated amounts (MICA) approach. J. Agric. Food Chem., 49: 4069–4075. Engel, W., 2003. In vivo studies on the metabolism of the monoterpene pulegone in humans using the metabolism of ingestion-correlated amounts (MICA) approach: Explanation for the toxicity differences between (S)-(-)- and (R)-(+)-pulegon. J. Agric. Food Chem., 51: 6589–6597. Eriksson, K. and J.O. Levin, 1996. Gas chromatographic-mass spectrometric identification of metabolites from a-pinene in human urine after occupational exposure to sawing fumes. J. Chromatogr. B, 677: 85–98. Gelal, A., P. Jacob, L. Yu, and N.L. Benowitz, 1999. Disposition kinetics and effects of menthol. Clin. Pharmacol. Ther., 66: 128–235. Gyoubu, K. and M. Miyazawa, 2007. In vitro metabolism of (-)-camphor using human liver microsomes and CYP2A6. Biol. Pharm. Bull., 30: 230–233. Metabolism of Terpenoids in Animal Models and Humans 233 Hall, R.L. and B.L. Oser, 1965. Recent progress in the consideration of flavoring ingredients under the foodadditives amendment III GRAS substances. Food Technol., 19: 253–271. Höld, K.M., N.S. Sirisoma, T. Ikeda, T. Narahashi, and J.E. Casida, 2000. a-Thujone (the active component of absinthe): g-Aminobutyric acid type A receptor modulation and metabolic detoxification. Environ. Chem. Toxicol., 97: 3826–3831. Höld, K.M., N.S. Sirisoma, and J.E. Casida, 2001. Detoxification of a-and b-thujones (the active ingredients of absinthe): Site specificity and species differences in cytochrome P450 oxidation in vitro and in vivo. Chem. Res. Toxicol., 14: 589–595. Ishida, T., 2005. Biotransformation of terpenoids by mammals, microorganisms, and plant-cultured cells. Chem. Biodivers., 2: 569–590. Ishida, T., Y. Asakawa, T. Takemoto, and T. Aratani, 1979. Terpenoid biotransformation in mammals II: Biotransformation of dl-camphene in rabbits. J. Pharm. Sci., 68: 928–930. Ishida, T., Y. Asakawa, T. Takemoto, and T. Aratani, 1981. Terpenoid biotransformation in mammals III: Biotransformation of a-pinene, b-pinene, pinane, 3-carene, carane, myrcene, and p-cymene in rabbits. J. Pharm. Sci., 70: 406–415. Ishida, T., M. Toyota, and Y. Asakawa, 1989. Terpenoid biotransformation in mammals. V. Metabolism of (+)-citronellal, (+/-)-7-hydroxycitronellal, citral, (-)-perillaldehyd, (-)-myrtenal, cuminaldehyde, thujone, and (+/-)-carvone in rabbits. Xenobiotica, 19: 843–855. Jäger, W., M. Mayer, P. Platzer, G. Resnicek, H. Dietrich, and G. Buchbauer, 2000. Stereoselective metabolism of the monoterpene carvone by rat and human liver microsomes. J. Pharm. Pharmacol., 52: 191–197. Jäger, W., M. Mayer, G. Resnicek, and G. Buchbauer, 2001. Percutaneose absorbtion of the monoterpene carvone: Implication of stereoselective metabolism on blood. J. Pharm. Pharmacol., 53: 637–642. Jahrmann, R., 2007. Metabolismus von Monterpenen und Sesquiterpenen in Mensch und Säugetier: Bedeutung für die pharmazeutische Praxis. MPharm. diploma thesis, University of Vienna, Austria. Leibmann, K.C. and E. Ortiz, 1972. Mammalian metabolism of terpenoids, I. Reduction and hydroxylation of camphor and related compounds. Drug Metab. Dispos., 1: 543–551. Madyastha, K.M. and C.P. Raj, 1992. Metabolic fate of menthofuran in rats. Drug Metab. Dispos., 20: 295–301. Madyastha, K.M. and V. Srivatsan, 1988a. Biotransformations of a-terpineol in the rat: Its effects on the liver microsomal cytochrome P450 system. Environ. Contam. Toxicol., 41: 17–25. Madyastha, K.M. and V. Srivatsan, 1988b. Studies on the metabolism of l-menthol in rats. Drug Metab. Dispos., 16: 765–772. Mendes, A.C., M.M. Caldeira, C. Silva, S.C. Burgess, M.E. Merritt, F. Gomes, C. Barosa et al., 2006. Hepatic UDP-glucose 13C isotopomers from [U-13C] glucose: A sample analysis by 13C NMR of urinary menthol glucuronide. Magn. Reson. Med., 56: 1121–1125. Miyazawa, M. and K. Gyoubu, 2006. Metabolism of (+)-fenchon by CYP2A6 and CYP2B6 in human liver microsomes. Biol. Pharm. Bull., 29: 2354–2358. Miyazawa, M., M. Shindo, and T. Shimada, 2001a. Roles of cytochrome P450 3A enzymes in the 2-hydroxylation of 1,4-cineole, a monoterpene cyclic ether, by rat and human liver microsomes. Xenobiotica, 31: 713–723. Miyazawa, M., M. Shindo, and T. Shimada, 2001b. Oxidation of 1,8-cineole, monoterpene cyclic ether originated from Eucalyptus polybractea, by cytochrome P450 3A enzymes in rat and human liver microsomes. Drug Metab. Dispos., 29: 200–205. Miyazawa, M., M. Shindo, and T. Shimada, 2002. Metabolism of (+)- and (-)-limonene to respective carveols and perillyl alcohols by CYP2C9 and CYP2C19 in human liver microsomes. Drug Metab. Dispos., 30: 602–607. Mühlbauer, R.C., A. Lozano, S. Palacio, A. Reinli, and R. Felix, 2003. Common herbs, essential oils, and monoterpenes potently modulate bone metabolism. Bone, 32: 372–380. Robertson, J.S. and M. Hussain, 1969. Metabolism of camphors and related compounds. Biochem. J., 113: 57–65. Shimada, T., M. Shindo, and M. Miyazawa, 2002. Species differences in the metabolism of R-(-)- and S-(+)limonenes and their metabolites, carveols and carvones, by cytochrome P450 enzymes in liver microsomes of mice, rats, guinea pigs, rabbits, dogs, monkeys, and humans. Drug Metab. Pharmacokinetics, 17: 507–515. Spatzenegger, M. and W. Jäger, 1995. Clinical importance of hepatic cytochrome P450 in drug metabolism. Drug Metab. Rev., 27: 397–417. 234 Handbook of Essential Oils Spichinger M., R.C. Mühlbauer, and R. Brenneisen, 2004. Determination of menthol in plasma and urine of rats and human by headspace solid phase microextraction and gas chromatography-mass spectrometry. J. Chromatogr. B, 799: 111–117. Staines, A.G., P. Sindelpar, M.W.H. Coughtrie, and B. Burchell, 2004. Farnesol is glucuonidated in human liver, kidney and intestine in vitro, and is novel substrate for UGT2B7 and UGT1A1. Biochem. J., 384: 637–645. Wichtel, M., 2002. Teedrogen und Phytopharmaka. Stuttgart: Wissenschaftliche Verlagsgesellschaft. Yamaguchi, T., J. Caldwell, and P.B. Farmer, 1994. Metabolic fate of [3H]-l-menthol in the rat. Drug Metab. Dispos., 22: 616–624. 9 Biological Activities of Essential Oils Gerhard Buchbauer CONTENTS 9.1 Introduction ........................................................................................................................ 9.1.1 Anticancer Properties ............................................................................................. 9.1.2 Antinociceptive Effects .......................................................................................... 9.1.3 Antiviral Activities ................................................................................................. 9.1.4 Antiphlogistic Activity ........................................................................................... 9.1.5 Penetration Enhancement ....................................................................................... 9.1.6 Antioxidative Properties ......................................................................................... 9.1.6.1 Reactive Oxygen Species ......................................................................... 9.1.6.2 Antioxidants ............................................................................................. 9.1.7 Test Methods ........................................................................................................... 9.1.7.1 Free Radical Scavenging Assay ............................................................... 9.1.7.2 b-Carotene Bleaching Test ....................................................................... 9.1.7.3 Deoxyribose Assay ................................................................................... 9.1.7.4 TBA Test .................................................................................................. 9.1.7.5 Xanthine–Xanthine Oxidase Assay ......................................................... 9.1.7.6 Linoleic Acid Assay ................................................................................. References ................................................................................................................................... 235 236 239 244 246 254 256 256 256 257 257 257 258 258 258 258 273 9.1 INTRODUCTION 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 235 236 Handbook of Essential Oils 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 officinalis 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. 238 Handbook of Essential Oils Balsam fir oil was active against all tumor cell lines with GI50 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 (GI50 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 (ED50 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). Biological Activities of Essential Oils 239 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 (IC50) 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, * Adorjan, M., 2007. Part of her master thesis, University of Vienna. 240 Handbook of Essential Oils 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-flick 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 241 Biological Activities of Essential Oils 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 multiflora 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 multiflora 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 Control 5 mg/kg 80 70 Essential oil 60 900 mg/kg p.o. 50 40 500 mg/kg i.p 30 Morphine 5 mg/kg 20 10 FIGURE 9.1 in in 0m 12 5m m in 10 90 m in 75 m in 60 m in 45 m in 30 15 m in 0 Antinociceptive activity of Zataria multiflora in tail-flick test in rats. 242 Handbook of Essential Oils 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. Biological Activities of Essential Oils 243 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). 244 9.1.3 Handbook of Essential Oils 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 IC50 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 (CC50) of 112 mg/mL, which leads to a CC50/IC50 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% (TC50) at very low concentrations. In the plaque-reduction assay an IC50 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%). * Adorjan, M., 2007. Part of her master thesis, University of Vienna. Biological Activities of Essential Oils 245 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 (VC50) 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 VC50 values of 60 and 150 ppm, respectively. The antiviral activity of the EO of the Lamiaceae Melissa officinalis 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 officinalis 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 penetration 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. 246 Handbook of Essential Oils 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% (VC50) 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 IC50 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 IC50 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 IC50 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 (IC50: 120 ± 1.2 mg/mL, resp. 130 ± 0.4 mg/mL) and against Herpes simplex virus type 1 (IC50: 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 * Adorjan, M., 2007. Part of her master thesis, University of Vienna. Biological Activities of Essential Oils 247 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 248 Handbook of Essential Oils (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 CO2 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 fischeri 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 fischeri 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 Biological Activities of Essential Oils 249 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 IC50 value of 72 mg/mL. Both enzymes were inhibited by only one EO that of Ocimum gratissimum with IC50 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 IC50 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. 250 Handbook of Essential Oils 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 LD50 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 251 Biological Activities of Essential Oils 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-2picrylhydrazyl (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) Etodolac (50 mg/kg) EC-I (0.025 mL/kg) EC-II (0.050 mL/kg) EC-III (0.100 mL/kg) EC-IV (0.200 mL/kg) 95.7 43.4 46.5 90.2 66.9 82.8 252 Handbook of Essential Oils 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 (IC50 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. Biological Activities of Essential Oils 253 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-methylbutyrate (7.9%) and isoamyl valerate (6.5%). The anti-inflammatory activity was evaluated using the 5-lipoxygenase enzyme and Eriocephalus dinteri achieved best results (IC50: 35 mg/mL). Wang and Zhu (2006) studied the anti-inflammatory effect of ginger oil (Zingiber officinale, 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 254 Handbook of Essential Oils 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. * Adorjan, M., 2007. Part of her master thesis, University of Vienna. 255 Biological Activities of Essential Oils 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 permeation 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 × 10−7 256 Handbook of Essential Oils 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. 257 Biological Activities of Essential Oils TABLE 9.3 Reactive Oxygen Species Species O2-• HO2• H2O2 HO• RO• ROO• Name Superoxide radical Perhydroxyl Hydrogen peroxide Hydroxyl radical R-oxyl radical 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). 258 Handbook of Essential Oils 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 Fe3+ /EDTA/H2O2 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 O2• and H2O2 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 O2•. 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 Biological Activities of Essential Oils 259 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 IC50 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 IC50 value of 0.90 ± 0.05 mg/mL and the EO an IC50 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 IC50 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 260 Handbook of Essential Oils 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 Biological Activities of Essential Oils 261 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 (<17%), germacrene D (up to 15%), p-cymene, and ascaridol. In the used test method the EO or a-tocopherol, the reference compound, are added to a solution of AAPH (= 2,2-azobis (2-amidinopropane)-dihydrochloride) and luminol. Chemoluminescence intensities of both blank and assay were monitored and the percentage of inhibition was calculated. The EO showed a high antioxidative activity that is similar to the activity of a-tocopherol (Masotti et al., 2003). Also Artemisia abyssinica and Artemisia afra (African wormwood)—due to the content of a-terpineol—showed an inhibition in the deoxyribose assay (Burits et al., 2001). A Canadian author team investigated seven wild collected Artemisia species from Western Canada, determined the composition of the EOs from their aerial parts, and found—interestingly—that these oils exerted only weak antioxidant activities in the b-carotene/linoleate model and DPPH test (Lopes-Lutz et al., 2008). The GC-MS analysis of the EO of the herbal parts of Achillea biebersteinii Afan (yarrow, Asteraceae), which were collected during the anthesis, furnished a high number of oxygenated monoterpenes, such as piperitone (34.9%), 1,8-cineole (13.0%), and camphor (8.8%) as the main compounds. The antioxidative activity has been assessed by four different methods: free radical scavenging assay with DPPH, hydroxyl radical scavenging with the deoxyribose assay, inhibition of the lipid peroxidation with the TBA test, and inhibition of the superoxide radicals xanthine–xanthine assay. Curcumin, ascorbic acid, and BHT were taken as positive control. Difficulties arising from the water-insoluble parts of the extracts render the spectroscopic measuring negatively. On this account, only the water-soluble parts of the extract could be determined for antioxidative action. The results of the tests showed that the EO possesses a better capacity in the donation of the hydrogen atoms or electrons than curcumin and BHT, but there was no difference in the action to ascorbic acid (Sökmen et al., 2004). Also hydroxyl radical scavenging and the lipid peroxidation inhibition were more effective than curcumin. Due to these results the EO of Achillea biebersteinii could be a valuable raw material for natural antioxidant additives. Also the EO of Achillea millefolium ssp. millefolium (common yarrow) in which 1,8-cineole and a-terpineol are major constituents shows a strong antioxidative activity. This was assessed by the deoxyribose assay, the xanthine–xanthine assay, and the DPPH test (Candan et al., 2003). Also the inhibition of the lipid peroxide generation was measured. The results of these tests were compared with the antioxidative activity of three wellknown and often used antioxidants, namely BHT, curcumin, and ascorbic acid. On account of the scavenging activity for the stable free radical DPPH with an IC50 value of 1.56 mg/ml, this oil showed a better antioxidative capacity than the just aforementioned well-known antioxidants. Other Achillea species wild samples of Achillea ligustica (Ligurian yarrow), collected in different parts of Sardinia, are also characterized by the content of 1,8-cineole as one of the main compounds of the EO with an average value of about 2.9%. In the DPPH assay the EO shows an interesting antioxidative activity that can be used for applying such oils as nutraceutical products or as additives in food industry (Tuberoso et al., 2005). Helichrysum (strawflower, Asteraceae) species are traditionally used for the treatment of wounds, infections, and respiratory conditions (Lourens et al., 2004). In the inflammatory process free radicals adsorb on the phagocyte cells, so the antioxidative activity of the extract and the EOs of different Helichrysum species were examined. Therefore, the plant material of Helichrysum dasyanthum, Helichrysum excisum, and Helichrysum petiolare were collected and the EO obtained by hydrodistillation. The GC-MS analysis showed that 1,8-cineole (20–34%), p-cymene (6–10%), and a-pinene (3–17%) are principal components of the EO. The EOs of all three different species show an antioxidative activity in the DPPH assay, but it has to be noticed that the extract, which was obtained by a cold 262 Handbook of Essential Oils 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 CCl4, 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 C18-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 H2O2, 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 Biological Activities of Essential Oils 263 the reaction between hydrogen peroxide and Fe2+ (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). 264 Handbook of Essential Oils 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 94.44 ± 0.05 84.60 ± 0.04 A-tocopherol Trolox 4.28 ± 0.5 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 officinalis (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 LC50 value of Salvia repens EO (comprising about 22% b-caryophyllene) rests with more than 100.0 mg/mL or Salvia multicaulis which showed an IC50 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 Cu2+ -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, Biological Activities of Essential Oils 265 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 IC50 value of 14.9 mL/mL in the DPPH assay, a 50% inhibition in the deoxyribose assay at 0.4 mL/mL, and an IC50 value of 0.20 mL/mL by inhibition of the lipid peroxidation. On the other hand, pure a-pinene showed an IC50 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, FeCl3 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 H2O2, whereas Fe2+ 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 IC50 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 266 Handbook of Essential Oils 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 floribundum 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 IC50 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). Biological Activities of Essential Oils 267 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 268 Handbook of Essential Oils 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 (IC50 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 Fe2+ 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 Biological Activities of Essential Oils 269 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 multiflora (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 multiflora 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 officinalis 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 IC50 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 270 Handbook of Essential Oils 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 O2•− 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 FeCl3 is added to the sample, both the EO and eugenol showed an antioxidative activity. The EO and eugenol were able to chelate the Fe3+ 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 IC50 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 Biological Activities of Essential Oils 271 time of 60 min an IC50 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 IC50 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 FeIII tripyridyltriazine from a colorless FeIII 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 FeIII 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 officinalis 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 IC50 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 272 Handbook of Essential Oils 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 IC50 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 IC50 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 (IC50 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 (IC50 value 0.161 mg/mL against 2.35 g/mL); however, the efficiency of ascorbic acid and trolox was higher with IC50 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 IC50 values of 12.48 and 11.88 mg/mL, respectively. The inhibition power was better than ascorbic acid with an IC50 value of 18.63 mg/mL. The antioxidative activity of BHT was higher than the activity of the EO with an IC50 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 Fe2+ chelating activity, whereas especially Hedychium spicatum also showed a complete different DPPH radical scavenging profile than the samples from the other species. 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Nutr., 125(11): 2763–2767. Zeytinoglu, H., Z. Incesu, and K.H.C. Baser, 2003. Inhibition of DNA synthesis by carvacrol in mouse myoblast cells bearing a human N-RAS oncogene. Phytomedicine, 10(4): 292–299. 10 Effects of Essential Oils in the Central Nervous System CONTENTS 10.1 Central Nervous System Effects of Essential Oils in Humans ....................................... Eva Heuberger 10.1.1 Introduction ........................................................................................................ 10.1.2 Activation and Arousal: Definition and Neuroanatomical Considerations ....... 10.1.3 Influence of EOs and Fragrances on Brain Potentials Indicative of Arousal .... 10.1.3.1 Spontaneous Electroencephalogram Activity ................................... 10.1.3.2 Contingent Negative Variation .......................................................... 10.1.4 Effects of EOs and Fragrances on Selected Basic and Higher Cognitive Functions ........................................................................ 10.1.4.1 Alertness and Attention ..................................................................... 10.1.4.2 Learning and Memory ....................................................................... 10.1.4.3 Other Cognitive Tasks ....................................................................... 10.1.5 Conclusions ........................................................................................................ 10.2 Psychopharmacology of Essential Oils ........................................................................... Domingos Sávio Nunes, Viviane de Moura Linck, Adriana Lourenço da Silva, Micheli Figueiró, and Elaine Elisabetsky 10.2.1 Aromatic Plants Used in Traditional Medical Systems as Sedatives or Stimulants ................................................................................. 10.2.2 Effects of EOs in Animal Models ...................................................................... 10.2.2.1 Effects of Individual Components ..................................................... 10.2.2.2 Effects of Inhaled EOs ...................................................................... 10.2.3 Mechanism of Action Underlying Psychopharmacological Effects of EOs ...... References .................................................................................................................................. 10.1 281 281 284 285 285 289 290 290 293 295 296 297 297 299 301 301 302 306 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 281 282 Handbook of Essential Oils 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 olf mos los OB tos OT AON Tu OpT L G PIR-FR AM MB CP EA PIR-TP 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.) 283 Effects of Essential Oils in the Central Nervous System 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 Basal ganglia Cortex Hypothalamus Thalamus Midbrain Raphé Nuclei Amygdala Cerebellum Pons Medulla Cell bodies Locus ceruleus 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.) 284 Handbook of Essential Oils 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 AM S CS LDT ADT MDT AH RF SC SMP CC PH SR T 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.) Effects of Essential Oils in the Central Nervous System 285 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 286 Handbook of Essential Oils 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 fi ndings 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 Effects of Essential Oils in the Central Nervous System 287 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 288 Handbook of Essential Oils 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 Effects of Essential Oils in the Central Nervous System 289 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 densiflora 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 290 Handbook of Essential Oils 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 Effects of Essential Oils in the Central Nervous System 291 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 292 Handbook of Essential Oils 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 Effects of Essential Oils in the Central Nervous System 293 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 294 Handbook of Essential Oils 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 Effects of Essential Oils in the Central Nervous System 295 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 (H2S), a trigeminal stimulus, that is, carbon dioxide (CO2), 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 CO2 decreased it, and only CO2, 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. 296 Handbook of Essential Oils 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, Effects of Essential Oils in the Central Nervous System 297 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 Domingos Sávio Nunes, Viviane de Moura Linck, Adriana Lourenço da Silva, Micheli Figueiró, and Elaine Elisabetsky 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 SEDATIVES OR STIMULANTS AS 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 298 Handbook of Essential Oils 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 Effects of Essential Oils in the Central Nervous System 299 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 Ca2+ 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 Ca2+ 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 300 Handbook of Essential Oils 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 anticonvulsant 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). Effects of Essential Oils in the Central Nervous System 301 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 officinalis L. (Lamiaceae), Rosmarinus officinalis 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 302 Handbook of Essential Oils 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 303 Effects of Essential Oils in the Central Nervous System cultured neurons were also reported for Acorus gramineus EO, apparently attained through the blockade of NMDA receptors given that this oil inhibits [3H]-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 Ca2+-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-[3H]-glutamate binding (Elisabetsky et al., 1999), and also shows a dose-dependent noncompetitive inhibition of [3H]-MK801 binding (IC50 =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 Ca2+ 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 [3H]-muscinol binding (Brum, 2001a) suggesting that, if existing at all, linalool modulation of the GABAergic system is not mediated by GABAA receptors. Potentially relevant to the pharmacology profile of linalool (3), patch-clamp techniques demonstrated that the monoterpene (no isomer specified) suppressed the Ca2+ 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 M2, dopamine D2, adenosine A1 and A2A, 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) MeO HO HO Linalool (3) Eugenol (4) 304 Handbook of Essential Oils O O O O O Methyl jasmonate (5) trans-Jasminlactone (6) HO HO H3C H3C (R)-(-)-linalool (7) (S)-(+)-linalool (8) O D-3-Carene (9) O Bornyl acetate (10) NCS Allyl isothiocyanate (12) N Pyridine (11) OH 2-Phenyl ethyl alcohol (13) Limonene (14) O Myrcene (15) Citral (16) O MeO Methylchavicol (18) Carvone (17) OH OH Geraniol (20) Thymol (19) 305 Effects of Essential Oils in the Central Nervous System a-Terpinene (22) p-Cymene (21) O MeO O trans-Anethole (24) Epi-nepetalactone (23) trans-Ocimene (25) MeO allo-Ocimene (26) MeO MeO MeO Methyl eugenol (27) OMe b-Asarone (28) H OH OH H Isoborneol (30) Borneol (29) OH OH a-Terpineol (32) Isopulegol (31) O OH Thujone (33) b-Eudesmol (34) 306 Handbook of Essential Oils OH O a-Eudesmol (35) Caryophyllene oxide (36) O Citronellal (38) b-Eudesmene (37) OH OH Geraniol (39) Citronellol (40) HO Carvacrol (41) O O O Pulegone (42) CH3COO Linalyl acetate (44) Safrol (43) O S-(-)-limonene (45) HO Hinokitiol (46) REFERENCES Almeida, R.N., S.C. 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Acaricidal activity ........................................................................................................... Anticarcinogenic ............................................................................................................. Antimicrobial .................................................................................................................. 11.4.1 Antibacterial .................................................................................................... 11.4.1.1 Methicillin-Resistant Staphylococcus aureus ............................... 11.4.2 Antifungal ........................................................................................................ 11.4.3 Antiviral .......................................................................................................... 11.4.4 Microbes of the Oral Cavity ............................................................................ 11.4.4.1 Activity of Listerine against Plaque and/or Gingivitis ................... 11.4.4.2 Antiviral Listerine ........................................................................... 11.4.4.3 Activity of Essential Oils ................................................................ 11.4.5 Controlling Microflora in Atopic Dermatitis .................................................. 11.4.6 Odor Management for Fungating Wounds ...................................................... Dissolution of Hepatic and Renal Stones ........................................................................ 11.5.1 Gall and Biliary Tract Stones .......................................................................... 11.5.2 Renal Stones .................................................................................................... Functional Dyspepsia ...................................................................................................... Gastroesophageal reflux .................................................................................................. Hyperlipoproteinemia ..................................................................................................... Irritable Bowel Syndrome ............................................................................................... Medical Examinations .................................................................................................... Nausea ............................................................................................................................. Pain Relief ....................................................................................................................... 11.12.1 Dysmenorrhea .................................................................................................. 11.12.2 Headache ......................................................................................................... 11.12.3 Infantile Colic .................................................................................................. 11.12.4 Joint Physiotherapy .......................................................................................... 11.12.5 Nipple Pain ...................................................................................................... 11.12.6 Osteoarthritis ................................................................................................... 11.12.7 Postherpetic Neuralgia .................................................................................... 11.12.8 Postoperative Pain ........................................................................................... 11.12.9 Prostatitis ......................................................................................................... 11.12.10 Pruritis ............................................................................................................. Pediculicidal Activity ...................................................................................................... Recurrent Aphthous Stomatitis ....................................................................................... 316 316 317 317 317 318 318 319 320 321 321 321 323 323 323 323 325 325 326 326 327 328 329 329 330 330 331 331 331 331 331 332 332 332 332 333 315 316 Handbook of Essential Oils 11.15 Respiratory Tract ............................................................................................................. 11.15.1 Menthol ............................................................................................................ 11.15.1.1 Antitussive ....................................................................................... 11.15.1.2 Nasal Decongestant ......................................................................... 11.15.1.3 Inhibition of Respiratory Drive and Respiratory Comfort ................................................................ 11.15.1.4 Bronchodilation and Airway Hyperresponsiveness ........................ 11.15.1.5 Summary ......................................................................................... 11.15.2 1,8-Cineole ...................................................................................................... 11.15.2.1 Antimicrobial .................................................................................. 11.15.2.2 Antitussive ....................................................................................... 11.15.2.3 Bronchodilation ............................................................................... 11.15.2.4 Mucolytic and Mucociliary Effects ................................................ 11.15.2.5 Anti-Inflammatory Activity ............................................................ 11.15.2.6 Pulmonary Function ........................................................................ 11.15.2.7 Summary ......................................................................................... 11.15.3 Treatment with Blends Containing both Menthol and 1,8-Cineole ................ 11.16 Allergic Rhinitis .............................................................................................................. 11.17 Snoring ............................................................................................................................ 11.18 Swallowing Dysfunction ................................................................................................. 11.19 Conclusion ....................................................................................................................... References .................................................................................................................................. 11.1 333 333 334 334 335 335 336 336 336 337 337 337 338 339 341 341 342 342 342 343 343 INTRODUCTION 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 multiflora 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). 318 Handbook of Essential Oils 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). Phytotherapeutic Uses of Essential Oils 319 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, butenafi ne 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 pauciflora had a strong fungicidal activity against Epidermophyton floccosum, 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 multiflora 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 multiflora 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 multiflora 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 multiflora 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. 320 Handbook of Essential Oils 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 officinalis, and Rosmarinus officinalis against Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, and Streptococcus mutans. The essential oils inhibited all of the test bacteria, acting bactericidally except for Lavandula officinalis. 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. Phytotherapeutic Uses of Essential Oils 321 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 multiflora was first examined in vitro for antimicrobial activity against ATCC strains and clinical isolates of the buccal flora. A significant 322 Handbook of Essential Oils 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). Phytotherapeutic Uses of Essential Oils 323 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 324 Handbook of Essential Oils TABLE 11.1 Composition of Rowachol and Rowatinex Declared by the Manufacturers Component a-pinene b-pinene Camphene 1,8-cineole Fenchone Menthone Borneol Menthol anethole Rowachol Rowatinex 20.0 37.0 5.0 9.0 8.0 3.0 — 9.0 8.0 48.0 — 22.0 4.0 6.0 — 15.0 — 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. Phytotherapeutic Uses of Essential Oils 325 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-HT4 agonist that stimulates upper gastrointestinal tract motility, over a 4-week period. 326 Handbook of Essential Oils 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). Phytotherapeutic Uses of Essential Oils 327 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. 328 Handbook of Essential Oils TABLE 11.2 Examples of Clinical Trials of Peppermint Oil in the Treatment of Irritable Bowel Syndrome Patients 18 29 25 35 110 42 178 57 Treatment 0.2–0.4 mL thrice daily for 3 weeks 0.2–0.4 mL thrice daily for 2 weeks 0.2 mL thrice daily for 2 weeks One Colpermin thrice daily for 24 weeks One Colpermin 3–4 times daily for 2 weeks 1–2 Colpermin thrice daily for 2 weeks Two Mintoil thrice daily for 3 months Two Mintoil twice daily for 4 weeks Outcome Superior to placebo in relieving abdominal symptoms Superior to placebo in relieving abdominal symptoms No significant change in symptoms as compared to placebo Effective in relieving symptoms 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 Reference Rees et al. (1979) Dew et al. (1984) Lawson et al. (1988) Shaw et al. (1991) 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). Phytotherapeutic Uses of Essential Oils 329 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 officinale 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 officinale, 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 officinale, 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. 330 11.12.1 Handbook of Essential Oils 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 E2 (PGE2)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 officinalis, 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). Phytotherapeutic Uses of Essential Oils 11.12.3 331 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 officinale (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 332 Handbook of Essential Oils 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 Phytotherapeutic Uses of Essential Oils 333 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 multiflora, 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 multiflora 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. 334 Handbook of Essential Oils 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. Phytotherapeutic Uses of Essential Oils 335 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 336 Handbook of Essential Oils 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. 2. 3. 4. 5. Antitussive at low concentration. Increases the sensation of nasal airflow giving the impression of decongestion. No physical decongestant activity. Depresses ventilation and the respiratory drive at comparatively higher concentration. 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. Phytotherapeutic Uses of Essential Oils 337 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 Ca2+ 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, 338 Handbook of Essential Oils 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 B4 (LTB4), PGE2, 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 B2, and LTB4 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 Phytotherapeutic Uses of Essential Oils 339 samples were taken from which monocytes were collected and stimulated ex vivo for LTB4 and PGE2 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 LTB4 and PGE2 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. 340 Handbook of Essential Oils Table 11.3 Summary of Human Trials Demonstrating the Beneficial Effects of 1,8-Cineole in Various Respiratory Conditions Patients 11 10 Treatment Cineole inhalation, 8 days Cineole 3 ¥ 200 mg daily, 3 days 32 Cineole 3 ¥ 200 mg daily, 12 weeks 60 Vaporub 3 min 676 Gelomyrtol 4 ¥ 300 mg daily, 14 days 9 Cineole inhalation, 8 days Cineole 3 ¥ 200 mg daily, 7 days Gelomyrtol 3 ¥ 300 mg daily, 6 months 100 246 Outcome Reference Asthma Objective rise in expiratory peak flow found. Subjective experiences of illness significantly improved LTB4 and PGE2 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 Juergens et al. (1998c) Acute bronchitis Decreased breathing frequency, suggesting “easier breathing” Coughing, sputum consistency, well-being, bronchial hyperreactivity, and associated symptoms all improved similarly by Gelomyrtol, Ambroxol, and Cefuroxime Berger et al. (1978a) Mattys et al. (2000) Chronic bronchitis Objective rise in expiratory peak flow found. Subjective experiences of illness significantly improved All lung function parameters significantly improved Reduced acute exacerbations, reduced requirement for antibiotics, reduced treatment times when antibiotics taken. Well-being significantly improved COPD Improved mucociliary clearance Grimm (1987) Juergens et al. (2003) Grimm (1987) Mahlo (1990) Meister et al. (1999) 20 Gelomyrtol 4 ¥ 0.3 g daily, 7 days 20 Gelomyrtol 1 ¥ 0.3 g daily, 14 days All parameters relating to coughing improved. Sputum volume increased Ulmer and Schött (1991) 12 Cineole 4 ¥ 200 mg, 4 days Significant improvement of mucociliary clearance Dorow (1989) 51 including 16 asthmatics Cineole 3 ¥ 200 mg daily, 8 weeks Significant improvement in airway resistance (21%), positive effects on sputum output and dyspnea 30 Cineole 1 ¥ 200 mg daily, 4 days 29 Cineole 3 ¥ 200 mg daily, 7 days Significant improvements in lung functions of FVC and FEV1 (Ambroxol and cineole equieffective), significant increase in ciliary beat frequency All lung function parameters, peak flow and dyspnea improved from day 1 onward Habich and Repges (1994) Kaspar et al. (1994) 24 Eucalyptus oil (9% of a mixture) 331 Gelomyrtol 300 mg, 6 days Cineole 3 ¥ 200 mg daily, 7 days 152 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 Dorow et al. (1987) Wittmann et al. (1998) Cohen and Dressler (1982) Federspil et al. (1997) Kehrl et al. (2004) Phytotherapeutic Uses of Essential Oils 341 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. 2. 3. 4. 5. 6. 7. Antimicrobial Antitussive Bronchodilatory Mucolytic Ciliary transport promotion Anti-inflammatory 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 342 Handbook of Essential Oils 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. Phytotherapeutic Uses of Essential Oils 343 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). 11.19 CONCLUSION 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. 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Med., 182(1): 33–38. 12 In Vitro Antimicrobial Activities of Essential Oils Monographed in the European Pharmacopoeia 6th Edition Alexander Pauli and Heinz Schilcher CONTENTS 12.1 Introduction ..................................................................................................................... 12.1.1 Agar Diffusion Test (ADT) ................................................................................ 12.1.2 Dilution Test (DIL) ............................................................................................. 12.1.3 Vapor Phase Test (VP) ....................................................................................... 12.2 Results ............................................................................................................................. 12.3 Discussion ....................................................................................................................... References .................................................................................................................................. 353 354 354 355 534 537 540 12.1 INTRODUCTION 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 353 354 Handbook of Essential Oils 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 CO2 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 fi rst 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 (MICair) 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 MICair 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; MICair, 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. TSA, 24 h, 42°C Bac- Bac- Bac- Bac- Escherichia coli Escherichia coli Escherichia coli Escherichia coli Bac- Bac- Pseudomonas aeruginosa Proteus vulgaris Bac- Bac- Proteus sp. Pseudomonas aeruginosa Bac- Neisseria perflava Proteus vulgaris Bac- Bac- Moraxella sp. Bac- Bac- Klebsiella pneumoniae Klebsiella sp. Bac- Bac- Escherichia coli Bac- Bac- Escherichia coli Klebsiella aerogenes 6, 2500 Cited, 18 h, 37°C Bac- Escherichia coli Flavobacterium suaveolens 15, 2500 Cited Bac- Cited NA, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C Cited NA, 24 h, 37°C ISA, 48 h, 25°C Cited ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C NA, 18 h, 37°C TSA, 24 h, 35°C ISA, 48 h, 25°C NA, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C 4 (h), 10,000 15, 2500 —, sd —, sd 4 (h), 10,000 15, 2500 —, sd 4 (h), 10,000 15, 2500 4 (h), 10,000 10 (h), 100,000 4 (h), 10,000 6 (h), pure 4 (h), 25,000 4 (h), 10,000 10 (h), 100,000 —, sd 4 (h), 10,000 5000 on agar Erwinia carotovora NA, 24 h, 37°C ISA, 48 h, 25°C 4 (h), 25,000 4 (h), 10,000 Bac- ISA, 48 h, 25°C Bac- Bac- Citrobacter freundii 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 —, sd 4 (h), 10,000 Disk Size (mm), Quantity (µg) Enterococcus faecalis Bac- Campylobacter jejuni ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C Test Parameters Enterobacter aerogenes Bac- Bac- Bac- Aeromonas hydrophila Beneckea natriegens Bac- Alcaligenes faecalis Bac- Acinetobacter calcoaceticus MO Class Aerobacter aerogenes Microorganism TABLE 12.1 Inhibitory Data of Anise Oil Obtained in the Agar Diffusion Test 0 0 1.5 0 1 1 0 0 0 0 6 11 4.5 7 1 0 0 0 0 0 0 0 4.5 0 0 6 0 0 Inhibition Zone (mm) Pizsolitto et al. (1975) Maruzzella and Lichtenstein (1956) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Pizsolitto et al. (1975) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Pizsolitto et al. (1975) Deans and Ritchie (1987) Narasimha Rao and Nigam (1970) Deans and Ritchie (1987) Yousef and Tawil (1980) Smith-Palmer et al. (1998) Janssen et al. (1986) Pizsolitto et al. (1975) Deans and Ritchie (1987) Narasimha Rao and Nigam (1970) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Di Pasqua et al. (2005) Deans and Ritchie (1987) Deans and Ritchie (1987) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Ref. 356 Handbook of Essential Oils Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Lactobacillus delbrueckii Lactobacillus plantarum Lactobacillus plantarum Lactococcus garvieae Lactococcus lactis subsp. lactis Leuconostoc cremoris 4 (h), 10,000 ISA, 48 h, 25°C Bac+ Bac+ Brochotrix thermosphacta Bac+ Bac+ Brochotrix thermosphacta Clostridium sporogenes Bac+ Corynebacterium diphtheriae 4 (h), 10,000 ISA, 48 h, 25°C Bac+ Brevibacterium linens ISA, 48 h, 25°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C NA, 18 h, 37°C —, sd 4 (h), 10,000 5000 on agar 5000 on agar 5000 on agar 4 (h), 10,000 5000 on agar 10 (h), 100,000 5000 on agar 4 (h), 10,000 6 (h), pure 6, 2500 Bacillus subtilis NA, 24 h, 37°C Cited, 18 h, 37°C Bac+ 4 (h), 10,000 15, 2500 —, sd 4 (h), 10,000 10 (h), 100,000 —, sd 4 (h), 10,000 10 (h), 100,000 Bac+ ISA, 48 h, 25°C Cited NA, 24 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C 4 (h), 10,000 15, 2500 Bacillus subtilis Bac+ ISA, 48 h, 25°C Cited Bacillus subtilis Bac+ Bac- Shigella sp. Vibrio cholerae Bacillus subtilis Bac- Serratia sp. Bacillus sp. Bac- Bac- Serratia marcescens Bac- Bac- Serratia marcescens Bac+ Bac- Salmonella typhi Bacillus mesentericus 15, 2500 Cited Bac- Yersinia enterocolitica 15, 2500 Cited Bac- Salmonella sp. 5000 on agar 4 (h), 25,000 Salmonella pullorum TSA, 24 h, 35°C NA, 24 h, 37°C 6, 2500 Bac- Cited, 18 h, 37°C 4 (h), 10,000 6 (h), pure Bac- Bac- Pseudomonas aeruginosa ISA, 48 h, 25°C NA, 18 h, 37°C Salmonella enteritidis Bac- Pseudomonas sp. Bac- Pseudomonas aeruginosa Pseudomonas aeruginosa 0 5 0 0 0 0 0 0 0 5.5 0 9 12.5 8 4 0 1 3 0 17 1 1 1 0 16 0 0 4.5 0 6.7 0 Deans and Ritchie (1987) Di Pasqua et al. (2005) Di Pasqua et al. (2005) Di Pasqua et al. (2005) Deans and Ritchie (1987) Di Pasqua et al. (2005) continued Narasimha Rao and Nigam (1970) Deans and Ritchie (1987) Deans and Ritchie (1987) Di Pasqua et al. (2005) Deans and Ritchie (1987) Yousef and Tawil (1980) Janssen et al. (1986) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Pizsolitto et al. (1975) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Narasimha Rao and Nigam (1970) Pizsolitto et al. (1975) Pizsolitto et al. (1975) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Narasimha Rao and Nigam (1970) Pizsolitto et al. (1975) Deans and Ritchie (1987) Smith-Palmer et al. (1998) Di Pasqua et al. (2005) Janssen et al. (1986) Yousef and Tawil (1980) Deans and Ritchie (1987) In Vitro Antimicrobial Activities of Essential Oils 357 Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Streptococcus viridans Streptomyces venezuelae Alternaria porri Alternaria solani Aspergillus flavus Aspergillus fumigatus Aspergillus niger Aspergillus niger Aspergillus niger Bac+ Bac+ Streptococcus faecalis Staphylococcus epidermidis Streptococcus sp. Bac+ Bac+ Staphylococcus epidermidis Bac+ Bac+ Bac+ Staphylococcus aureus Staphylococcus aureus Bac+ Staphylococcus aureus Staphylococcus aureus Bac+ Bac+ Staphylococcus aureus Staphylococcus aureus Bac+ Staphylococcus aureus —, sd NA, 24 h, 37°C Bac+ Bac+ Sarcina lutea 4 (h), 10,000 6 (h), pure ISA, 48 h, 25°C NA, 18 h, 37°C Bac+ Bac+ Micrococcus luteus Mycobacterium phlei Staphylococcus aureus 4 (h), 25,000 TSA, 24 h, 35°C Bac+ PDA, 72 h, 28°C SMA, 2–7 d, 20°C Cited SMA, 2–7 d, 20°C PDA, 48 h, 28°C SMA, 2–7 d, 20°C Cited SMA, 2–7 d, 20°C Cited NA, 24 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C Cited NA, 18 h, 37°C Cited, 18 h, 37°C TSA, 24 h, 35°C Cited NA, 24 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C NA, 24 h, 37°C 4 (h), 10,000 5, 5000 6.35, sd —, pure 6.35, sd 5, 5000 6.35, sd —, pure 6.35, sd 15, 2500 10 (h), 100,000 4 (h), 10,000 10 (h), 100,000 15, 2500 6 (h), pure 6, 2500 4 (h), 25,000 15, 2500 5000 on agar 4 (h), 10,000 10 (h), 100,000 —, sd 5000 on agar Listeria monocytogenes NA, 24 h, 37°C ISA, 48 h, 25°C Disk Size (mm), Quantity (µg) Bac+ Test Parameters Bac+ MO Class Listeria monocytogenes (continued) Listeria monocytogenes Microorganism TABLE 12.1 35 9 28 9 0 13 27 7 0 16 0 12 4 14.5 8.3 4 1 0 0 0 0 0 18 0 4 0 0 Inhibition Zone (mm) Pawar and Thaker (2007) Maruzzella and Liguori (1958) Gangrade et al. (1991) Maruzzella and Liguori (1958) Pawar and Thaker (2006) Maruzzella and Liguori (1958) Gangrade et al. (1991) Maruzzella and Liguori (1958) Pizsolitto et al. (1975) Narasimha Rao and Nigam (1970) Deans and Ritchie (1987) Narasimha Rao and Nigam (1970) Pizsolitto et al. (1975) Yousef and Tawil (1980) Janssen et al. (1986) Smith-Palmer et al. (1998) Pizsolitto et al. (1975) Di Pasqua et al. (2005) Deans and Ritchie (1987) Narasimha Rao and Nigam (1970) Maruzzella and Lichtenstein (1956) Maruzzella and Lichtenstein (1956) Yousef and Tawil (1980) Deans and Ritchie (1987) Smith-Palmer et al. (1998) Di Pasqua et al. (2005) Lis-Balchin et al. (1998) Ref. 358 Handbook of Essential Oils a Fungicidal. Aspergillus niger Fusarium oxysporum Fusarium oxysporum f.sp. cicer Geotrichum sp. Helminthosporium sativum Microsporum canis Microsporum gypseum Mucor mucedo Mucor sp. Nigrospora panici Penicillium chrysogenum Penicillium digitatum Rhizopus nigricans Rhizopus sp. Trichophyton mentagrophytes Trichophyton rubrum Candida albicans Candida albicans Candida albicans Candida albicans Candida glabrata Candida krusei Candida krusei Candida parapsilosis Candida pseudotropicalis Candida tropicalis Candida tropicalis Cryptococcus neoformans Cryptococcus rhodobenhani Saccharomyces cerevisiae Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast SDA, 8 d, 30°C Cited PDA, 72 h, 28°C SDA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 2–7 d, 20°C SDA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SDA, 2–7 d, 20°C SDA, 2–7 d, 20°C SMA, 2–7 d, 20°C Cited, 18 h, 37°C SDA, 18 h, 30°C SDA, 2–7 d, 20°C SDA, 2–7 d, 20°C SDA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 2–7 d, 20°C SDA, 2–7 d, 20°C SDA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C 6 (h), pure —, pure 5, 5000 5 (h), 60,000 6.35, sd 5 (h), 60,000 5 (h), 60,000 6.35, sd 6 (h), pure 6.35, sd 6 (h), pure 6.35, sd 6.35, sd 6 (h), pure 5 (h), 60,000 5 (h), 60,000 6.35, sd 6, 2500 6 (h), pure 5 (h), 60,000 5 (h), 60,000 5 (h), 60,000 6.35, sd 5 (h), 60,000 5 (h), 60,000 5 (h), 60,000 6.35, sd 6.35, sd 6.35, sd 6.35, sd 2 9 0 0 a a 0 30 a 60 32 15 17 10 27 21 6 12 12 60 15 3 13 23 25 2 12 15 29 21 Yousef and Tawil (1980) Gangrade et al. (1991) Pawar and Thaker (2007) Kosalec et al. (2005) Maruzzella and Liguori (1958) Kosalec et al. (2005) Kosalec et al. (2005) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Kosalec et al. (2005) Kosalec et al. (2005) Maruzzella and Liguori (1958) Janssen et al. (1986) Yousef and Tawil (1980) Kosalec et al. (2005) Kosalec et al. (2005) Kosalec et al. (2005) Maruzzella and Liguori (1958) Kosalec et al. (2005) Kosalec et al. (2005) Kosalec et al. (2005) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) In Vitro Antimicrobial Activities of Essential Oils 359 62.5 MHB, DMSO, 24 h, 37°C BacBacBacBac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Salmonella typhimurium Salmonella typhimurium Bacillus cereus Bacillus subtilis Brochotrix thermosphacta Listeria monocytogenes Mycobacterium phlei Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Alternaria alternata Alternaria tenuissima >10,000 250 MHB, DMSO, 24 h, 37°C Bac- Salmonella typhi MHB, DMSO, 24 h, 37°C Cited Cited NB, Tween 20, 18 h, 37°C MHB, DMSO, 24 h, 37°C TSB, 24 h, 35°C NB, Tween 20, 18 h, 37°C TSB, 24 h, 35°C M17, 24 h, 20°C NB, Tween 20, 18 h, 37°C TSB, 24 h, 37°C MHB, DMSO, 24 h, 37°C TSB, 24 h, 35°C NB, Tween 20, 18 h, 37°C 100% inh. 600 100% inh. 600 25,000 >10,000 125 >10,000 200 >10,000 1600 >10,000 500 >50,000 >500 62.5 Salmonella enteritidis >500 125 Bac- MHB, DMSO, 24 h, 37°C MHB, DMSO, 24 h, 37°C MHB, DMSO, 24 h, 37°C 294.7–589.4 Bac- Bac- Klebsiella pneumoniae Cited, 20 h, 37°C NB, Tween 20, 18 h, 37°C Pseudomonas aeruginosa Bac- Helicobacter pylori >500 50,000 Pseudomonas aeruginosa Bac- Escherichia coli MHB, DMSO, 24 h, 37°C >10,000 Bac- Bac- Escherichia coli TSB, 24 h, 37°C >10,000 >10,000 Bac- Bac- Escherichia coli TSB, 24 h, 35°C TSB, 24 h, 42°C MIC (µg/mL) Proteus mirabilis Bac- Test Parameters Proteus vulgaris Bac- Escherichia coli MO Class Campylobacter jejuni Microorganism TABLE 12.2 Inhibitory Data of Anise Oil Obtained in the Dilution Test Shukla and Tripathi (1987) Shukla and Tripathi (1987) Yousef and Tawil (1980) Al-Bayati (2008) Smith-Palmer et al. (1998) Yousef and Tawil (1980) Smith-Palmer et al. (1998) Di Pasqua et al. (2005) Yousef and Tawil (1980) Al-Bayati (2008) Al-Bayati (2008) Di Pasqua et al. (2005) Al-Bayati (2008) Smith-Palmer et al. (1998) Yousef and Tawil (1980) Al-Bayati (2008) Al-Bayati (2008) Al-Bayati (2008) Al-Bayati (2008) Weseler et al. (2005) Yousef and Tawil (1980) Al-Bayati (2008) Di Pasqua et al. (2005) Smith-Palmer et al. (1998) Smith-Palmer et al. (1998) Ref. 360 Handbook of Essential Oils Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Aspergillus awamorii Aspergillus flavus Aspergillus flavus Aspergillus flavus Aspergillus fumigatus Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus ochraceus Aspergillus ochraceus Aspergillus ochraceus Aspergillus oryzae Aspergillus parasiticus Aspergillus parasiticus Aspergillus parasiticus Aspergillus parasiticus Aspergillus sydowi Aspergillus tamari Aspergillus terreus Botryodiplodia theobromae Cephalosporium sacchari Ceratocystis paradoxa Cladosporium herbarum Colletotrichum capsici Curvularia lunata Curvularia lunata Curvularia pallescens Epicoccum nigrum Epidermophyton floccosum Cited PDA, 7–14 d, 28°C PDA, 6–8 h 20°C, spore germ. inh. Cited Cited Cited Cited OA, EtOH, 3 d, 20°C OA, EtOH, 3 d, 20°C Cited Cited Cited OA, EtOH, 3 d, 20°C Cited Cited SA, Tween 80, 21 d, 20°C PDA, 7–14 d, 28°C PDA, 6–8 h 20°C, spore germ. inh. Cited Cited NB, Tween 20, 8 d, 30°C YES broth, 10 d Cited PDA, 7–14 d, 28°C YES broth, 10 d Cited PDA, 5 d, 27°C Cited PDA, 5 d, 27°C <300 >1000 100% inh. 600 500 50–100 100% inh. 600 100% inh. 600 100% inh. 600 100% inh. 600 20,000 1000 100% inh. 600 100% inh. 600 100% inh. 600 4000 100% inh. 600 100% inh. 600 >1000 500 50–100 100% inh. 600 100% inh. 600 1600 83% inh. 10,000 100% inh. 600 500 82% inh. 10,000 250 100% inh. 600 continued Shukla and Tripathi (1987) Soliman and Badeaa (2002) Thompson (1986) Shukla and Tripathi (1987) Shukla and Tripathi (1987) Shukla and Tripathi (1987) Shukla and Tripathi (1987) Narasimba Rao et al. (1971) Narasimba Rao et al. (1971) Shukla and Tripathi (1987) Shukla and Tripathi (1987) Shukla and Tripathi (1987) Narasimba Rao et al. (1971) Shukla and Tripathi (1987) Shukla and Tripathi (1987) Janssen et al. (1988) Soliman and Badeaa (2002) Thompson (1986) Shukla and Tripathi (1987) Shukla and Tripathi (1987) Yousef and Tawil (1980) Lis-Balchin et al. (1998) Shukla and Tripathi (1987) Soliman and Badeaa (2002) Lis-Balchin et al. (1998) Okazaki and Oshima (1953) Thompson and Cannon (1986) Shukla and Tripathi (1987) Thompson and Cannon (1986) In Vitro Antimicrobial Activities of Essential Oils 361 Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Mucor mucedo Mucor racemosus Mucor racemosus f. racemosus Mucor sp. Nigrospora oryzae Penicillium chrysogenum Penicillium chrysogenum Penicillium chrysogenum Penicillium citrinum Physalospora tucumanensis Phytophthora capsici MO Class Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi (continued) Fusarium accuminatum Fusarium culmorum Fusarium equisiti Fusarium moniliforme Fusarium moniliforme Fusarium moniliforme Fusarium moniliforme var. subglutinans Fusarium oxysporum Fusarium semitectum Fusarium udum Geotrichum sp. Helminthosporium sacchari Macrophomina phaesoli Microsporum canis Microsporum gypseum Mucor hiemalis Microorganism TABLE 12.2 NB, Tween 20, 8 d, 30°C Cited Cited NB, Tween 20, 8 d, 30°C Cited Cited OA, EtOH, 3 d, 20°C PDA, 7 d, 23.5°C Cited PDA, 5 d, 27°C PDA, 5 d, 27°C Cited YES broth, 10 d Cited Cited PDA, 7–14 d, 28°C PDA, 7 d, 23.5°C OA, EtOH, 3 d, 20°C Cited Cited Cited SGB, 3–7 d, 25°C OA, EtOH, 3 d, 20°C Cited SGB, 3–7 d, 25°C SGB, 3–7 d, 25°C PDA, 5 d, 27°C Test Parameters >1000 400 100% inh. 600 100% inh. 600 1600 250 100% inh. 600 2000 36% inh. 10,000 >1000 250 >1000 100% inh. 600 69% inh. 10,000 100% inh. 600 100% inh. 600 500 52% inh. 10,000 2000 100% inh. 600 100% inh. 600 100% inh. 600 15,600 4000 100% inh. 600 1000 2000 MIC (µg/mL) Yousef and Tawil (1980) Shukla and Tripathi (1987) Shukla and Tripathi (1987) Yousef and Tawil (1980) Okazaki and Oshima (1953) Shukla and Tripathi (1987) Narasimba Rao et al. (1971) Mueller-Ribeau et al. (1995) Okazaki and Oshima (1953) Thompson and Cannon (1986) Thompson and Cannon (1986) Shukla and Tripathi (1987) Lis-Balchin et al. (1998) Shukla and Tripathi (1987) Shukla and Tripathi (1987) Soliman and Badeaa (2002) Mueller-Ribeau et al. (1995) Narasimba Rao et al. (1971) Shukla and Tripathi (1987) Shukla and Tripathi (1987) Shukla and Tripathi (1987) Kosalec et al. (2005) Narasimba Rao et al. (1971) Shukla and Tripathi (1987) Kosalec et al. (2005) Kosalec et al. (2005) Thompson and Cannon (1986) Ref. 362 Handbook of Essential Oils Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Yeast Rhizoctonia solani Rhizopus 66-81-2 Rhizopus arrhizus Rhizopus chinensis Rhizopus circinans Rhizopus japonicus Rhizopus kazanensis Rhizopus nigricans Rhizopus oryzae Rhizopus pymacus Rhizopus sp. Rhizopus stolonifer Rhizopus tritici Sclerotina sclerotiorum Sclerotium rolfsii Trichophyton mentagrophytes Trichophyton mentagrophytes Trichophyton rubrum Trichophyton rubrum Candida albicans Candida albicans Candida glabrata Candida krusei Candida parapsilosis Candida pseudotropicalis Candida tropicalis SGB, 3–7 d, 25°C SGB, 3–7 d, 25°C NB, Tween 20, 18 h, 37°C SGB, 3–7 d, 25°C SGB, 3–7 d, 25°C SGB, 3–7 d, 25°C SGB, 3–7 d, 25°C SGB, 3–7 d, 25°C PDA, 7 d, 23.5°C OA, EtOH, 6 d, 20°C SA, Tween 80, 21 d, 20°C SGB, 3–7 d, 25°C SA, Tween 80, 21 d, 20°C PDA, 5 d, 27°C NB, Tween 20, 8 d, 30°C PDA, 5 d, 27°C PDA, 5 d, 27°C Cited PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 7 d, 23.5°C PDA, 5 d, 27°C <300 2000 10,000 800 1000 2000 1000 1000 1000 >1000 100% inh. 10,000 2000 300–625 7800 >1000 >1000 3200 >1000 >1000 100% inh. 600 >1000 >1000 >1000 >1000 >1000 100% inh. 10,000 Kosalec et al. (2005) Kosalec et al. (2005) Yousef and Tawil (1980) Kosalec et al. (2005) Kosalec et al. (2005) Kosalec et al. (2005) Kosalec et al. (2005) Kosalec et al. (2005) Mueller-Ribeau et al. (1995) Narasimba Rao et al. (1971) Janssen et al. (1988) Kosalec et al. (2005) Janssen et al. (1988) Thompson and Cannon (1986) Yousef and Tawil (1980) Thompson and Cannon (1986) Thompson and Cannon (1986) Shukla and Tripathi (1987) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Mueller-Ribeau et al. (1995) Thompson and Cannon (1986) In Vitro Antimicrobial Activities of Essential Oils 363 WFA, 42 d, 25°C WFA, 42 d, 25°C Bac- Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Salmonella typhi Salmonella typhi Bacillus megaterium Bacillus subtilis var. aterrimus Corynebacterium diphtheriae Mycobacterium avium Staphylococcus aureus Staphylococcus aureus Streptococcus faecalis Streptococcus faecalis Streptococcus pyogenes Fungi Fungi Fungi Fungi Fungi Fungi Yeast Aspergillus niger Eurotium amstelodami Eurotium herbarum Eurotium repens Eurotium rubrum Penicillium corylophilum Candida albicans Aspergillus flavus Bac- Neisseria sp. NA, 24 h, 37°C WFA, 42 d, 25°C WFA, 42 d, 25°C WFA, 42 d, 25°C WFA, 42 d, 25°C WFA, 42 d, 25°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Bac- Escherichia coli Test Parameters MO Class Microorganism TABLE 12.3 Inhibitory Data of Anise Oil Obtained in the Vapor Phase Test ~20,000 Disk, 50,000 Disk, 50,000 Disk, 50,000 Disk, 50,000 Disk, 50,000 Disk, 50,000 ~20,000 Disk, 50,000 ~20,000 6.35, sd ~20,000 6.35, sd ~20,000 6.35, sd ~20,000 6.35, sd ~20,000 6.35, sd ~20,000 ~20,000 Quantity (µg) + + +++ +++ +++ +++ +++ +++ + +++ ++ +++ +++ +++ + +++ ++ +++ ++ + +++ Activity Kellner and Kober (1954) Guynot et al. (2003) Guynot et al. (2003) Guynot et al. (2003) Guynot et al. (2003) Guynot et al. (2003) Guynot et al. (2003) Guynot et al. (2003) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Ref. 364 Handbook of Essential Oils BacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBac- Aeromonas hydrophila Agrobacterium tumefaciens Alcaligenes faecalis Beneckea natriegens Burkholderia gladioli pv. agaricicola Citrobacter freundii Enterobacter aerogenes Enterobacter aerogenes Erwinia carotovora Erwinia carotovora subsp. atroseptica Erwinia carotovora subsp. carotovora Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Flavobacterium suaveolens Klebsiella pneumoniae Moraxella sp. Proteus vulgaris Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas agarici Pseudomonas chichorii MO Class Acinetobacter calcoaceticus Microorganism KBA, 48 h, 25°C KBA, 48 h, 25°C MHA, 48 h, 27°C NA, 18 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C WA, 48 h, 25°C MHA, 48 h, 27°C NA, 18 h, 37°C NA, 18 h, 37°C ISA, 48 h, 25°C WA, 48 h, 25°C WA, 48 h, 25°C ISA, 48 h, 25°C MHA, 48 h, 27°C ISA, 48 h, 25°C ISA, 48 h, 25°C WA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C WA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C Test Parameters TABLE 12.4 Inhibitory Data of Bitter Fennel Obtained in the Agar Diffusion Test 6, 8000 6, 8000 6, 15,000 6 (h), pure 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 6, 8000 5 (h), -30,000 6, 15,000 6 (h), pure 4 (h), 10,000 6, 8000 6, 8000 4 (h), 10,000 6, 15,000 4 (h), 10,000 4 (h), 10,000 6, 8000 4 (h), 10,000 4 (h), 10,000 6, 8000 4 (h), 10,000 4 (h), 10,000 Disk Size (mm), Quantity (µg) MIA >7680 MIA >7680 18 0 0 0 6.5 0 MIA >7680 6 25 14 0 0 MIA 7680 MIA 7680 0 10 0 0 MIA 7680 0 0 MIA 2880 9.5 0 Inhibition Zone (mm) Cantore et al. (2004) Cantore et al. (2004) Ertürk et al. (2006) continued Yousef and Tawil (1980) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Cantore et al. (2004) Ertürk et al. (2006) Schelz et al. (2006) Yousef and Tawil (1980) Deans and Ritchie (1987) Cantore et al. (2004) Cantore et al. (2004) Deans and Ritchie (1987) Ertürk et al. (2006) Deans and Ritchie (1987) Deans and Ritchie (1987) Cantore et al. (2004) Deans and Ritchie (1987) Deans and Ritchie (1987) Cantore et al. (2004) Deans and Ritchie (1987) Deans and Ritchie (1987) Ref. In Vitro Antimicrobial Activities of Essential Oils 365 BacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBac+ Bac+ Bac+ Bac+ Bac+ Bac+ Pseudomonas syringae pv. apata Pseudomonas syringae pv. apii Pseudomonas syringae pv. atrofaciens Pseudomonas syringae pv. Glycinea Pseudomonas syringae pv. lachrymans Pseudomonas syringae pv. maculicola Pseudomonas syringae pv. phaseolicola Pseudomonas syringae pv. pisi Pseudomonas syringae pv. syringae Pseudomonas syringae pv. tomato Pseudomonas tolaasii Pseudomonas viridiflava Salmonella pullorum Salmonella typhimurium Serratia marcescens Xanthomonas campestris pv. campestris Xanthomonas campestris pv. phaesoli var. fuscans Xanthomonas campestris pv. phaesoli var. phaesoli Xanthomonas campestris pv. Vesicatoria Yersinia enterocolitica Bacillus megaterium Bacillus subtilis Bacillus subtilis Brevibacterium linens Brochotrix thermosphacta Clavibacter michiganensis subsp. Michiganensis MO Class Pseudomonas reactans (continued) Pseudomonas corrugate Microorganism TABLE 12.4 WA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 18 h, 37°C ISA, 48 h, 25°C WA, 48 h, 25°C ISA, 48 h, 25°C WA, 48 h, 25°C WA, 48 h, 25°C WA, 48 h, 25°C WA, 48 h, 25°C ISA, 48 h, 25°C MHA, 48 h, 27°C ISA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C Test Parameters 6, 8000 4 (h), 10,000 4 (h), 10,000 6 (h), pure 4 (h), 10,000 6, 8000 4 (h), 10,000 6, 8000 6, 8000 6, 8000 6, 8000 4 (h), 10,000 6, 15,000 4 (h), 10,000 6, 8000 6, 8000 6, 8000 6, 8000 6, 8000 6, 8000 6, 8000 6, 8000 6, 8000 6, 8000 6, 8000 6, 8000 6, 8000 6, 8000 Disk Size (mm), Quantity (µg) MIA 7680 0 7.5 18 MIA >7680 0 0 MIA 1440 MIA 480 MIA 720 MIA 5760 0 8 MIA >7680 0 MIA >7680 MIA 7680 MIA >7680 MIA >7680 MIA >7680 MIA >7680 MIA >7680 MIA 960 MIA >7680 MIA 3840 MIA >7680 MIA >7680 MIA >7680 Inhibition Zone (mm) Cantore et al. (2004) Deans and Ritchie (1987) Deans and Ritchie (1987) Yousef and Tawil (1980) Deans and Ritchie (1987) Cantore et al. (2004) Deans and Ritchie (1987) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Deans and Ritchie (1987) Ertürk et al. (2006) Deans and Ritchie (1987) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Ref. 366 Handbook of Essential Oils Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Clostridium sporogenes Curtobacterium flaccunfaciens pv. betae Curtobacterium flaccunfaciens pv. flaccunfaciens Lactobacillus plantarum Leuconostoc cremoris Micrococcus luteus Mycobacterium phlei Rhodococcus fascians Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus epidermidis Staphylococcus epidermidis Streptococcus faecalis Streptomyces venezuelae Absidia cormybifera Alternaria solani Alternaria sp. Aspergillus candidus Aspergillus flavus Aspergillus fumigatus Aspergillus fumigatus Aspergillus nidulans Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus niger Cladosporium herbarum Cunninghamella echinulata Fusarium oxysporum Helminthosporium sacchari Bac+ Clavibacter michiganensis subsp. sepedonicus EYA, 48 h, 45°C SMA, 2–7 d, 20°C PDA, 18 h, 37°C PDA, 18 h, 37°C PDA, 18 h, 37°C PDA, 18 h, 37°C SMA, 2–7 d, 20°C PDA, 18 h, 37°C SMA, 2–7 d, 20°C PDA, 18 h, 37°C MHA, 48 h, 27°C SDA, 8 d, 30°C PDA, 18 h, 37°C PDA, 18 h, 37°C PDA, 18 h, 37°C PDA, 18 h, 37°C SMA, 2–7 d, 20°C ISA, 48 h, 25°C NA, 18 h, 37°C MHA, 48 h, 27°C NA, 18 h, 37°C MHA, 48 h, 27°C ISA, 48 h, 25°C WA, 48 h, 25°C NA, 18 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C WA, 48 h, 25°C WA, 48 h, 25°C ISA, 48 h, 25°C WA, 48 h, 25°C 5, sd 6.35, sd 6, sd 6, sd 6, sd 6, sd 6.35, sd 6, sd 6.35, sd 6, sd 6, 15,000 6 (h), pure 6, sd 6, sd 6, sd 6, sd 6.35, sd 5 (h), -30,000 4 (h), 10,000 6, 15,000 6 (h), pure 6, 15,000 4 (h), 10,000 6, 8000 6 (h), pure 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 6, 8000 6, 8000 4 (h), 10,000 6, 8000 0 6 12 0 7 0 12 9 0 12 12 31 12.5 21 0 16 10 0 15 12 17 16 7.5 MIA 1920 20 0 9.5 MIA >7680 0 MIA >7680 0 MIA 960 continued Nigam and Rao (1979) Maruzzella and Liguori (1958) Sharma and Singh (1979) Sharma and Singh (1979) Sharma and Singh (1979) Sharma and Singh (1979) Maruzzella and Liguori (1958) Sharma and Singh (1979) Maruzzella and Liguori (1958) Sharma and Singh (1979) Ertürk et al. (2006) Yousef and Tawil (1980) Sharma and Singh (1979) Sharma and Singh (1979) Sharma and Singh (1979) Sharma and Singh (1979) Maruzzella and Liguori (1958) Deans and Ritchie (1987) Schelz et al. (2006) Ertürk et al. (2006) Yousef and Tawil (1980) Ertürk et al. (2006) Deans and Ritchie (1987) Cantore et al. (2004) Yousef and Tawil (1980) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Cantore et al. (2004) Cantore et al. (2004) Deans and Ritchie (1987) Cantore et al. (2004) In Vitro Antimicrobial Activities of Essential Oils 367 (continued) Helminthosporium sativum Humicola grisea var. thermoidea Microsporum gypseum Mucor mucedo Mucor mucedo Mucor sp. Nigrospora panici Penicillium aculeatum Penicillium chrysogenum Penicillium chrysogenum Penicillium digitatum Penicillium digitatum Penicillium javanicum Penicillium jensenii Penicillium lividum Penicillium notatum Penicillium obscurum Penicillium sp. I Penicillium sp. II Penicillium sp. III Rhizopus nigricans Rhizopus nigricans Rhizopus sp. Sporotrichum thermophile Thermoascus aurantiacis Microorganism TABLE 12.4 Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi MO Class SMA, 2–7 d, 20°C EYA, 48 h, 45°C PDA, 18 h, 37°C SMA, 2–7 d, 20°C PDA, 18 h, 37°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C CA, 48 h, 27°C CA, 48 h, 27°C SDA, 8 d, 30°C PDA, 18 h, 37°C SMA, 2–7 d, 20°C CA, 48 h, 27°C CA, 48 h, 27°C CA, 48 h, 27°C CA, 48 h, 27°C CA, 48 h, 27°C CA, 48 h, 27°C CA, 48 h, 27°C CA, 48 h, 27°C SMA, 2–7 d, 20°C PDA, 18 h, 37°C SDA, 8 d, 30°C EYA, 48 h, 45°C EYA, 48 h, 45°C Test Parameters 6.35, sd 5, sd 6, sd 6.35, sd 6, sd 6 (h), pure 6.35, sd 5, sd 5, sd 6 (h), pure 6, sd 6.35, sd 5, sd 5, sd 5, sd 5, sd 5, sd 5, sd 5, sd 5, sd 6.35, sd 6, sd 6 (h), pure 5, sd 5, sd Disk Size (mm), Quantity (µg) 6 0 14.5 0 12 20 7 0 15 60 7 7 25 10 0 0 15 15 10 0 3 7 20 25 10 Inhibition Zone (mm) Maruzzella and Liguori (1958) Nigam and Rao (1979) Sharma and Singh (1979) Maruzzella and Liguori (1958) Sharma and Singh (1979) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Nigam and Rao (1979) Nigam and Rao (1979) Yousef and Tawil (1980) Sharma and Singh (1979) Maruzzella and Liguori (1958) Nigam and Rao (1979) Nigam and Rao (1979) Nigam and Rao (1979) Nigam and Rao (1979) Nigam and Rao (1979) Nigam and Rao (1979) Nigam and Rao (1979) Nigam and Rao (1979) Maruzzella and Liguori (1958) Sharma and Singh (1979) Yousef and Tawil (1980) Nigam and Rao (1979) Nigam and Rao (1979) Ref. 368 Handbook of Essential Oils Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Thermomyces lanuginosa Thielava minor Trichophyton rubrum Trichothecium roseum Brettanomyces anomalus Candida albicans Candida albicans Candida albicans Candida krusei Candida lipolytica Candida tropicalis Cryptococcus neoformans Cryptococcus rhodobenhani Debaryomyces hansenii Geotrichum candidum Hansenula anomala Kloeckera apiculata Kluyveromyces fragilis Lodderomyces elongisporus Metchnikowia pulcherrima Pichia membranaefaciens Rhodotorula rubra Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Torula glabrata Torula thermophila MYA, 4 d, 30°C EYA, 48 h, 45°C EYA, 48 h, 45°C EYA, 48 h, 45°C PDA, 18 h, 37°C PDA, 18 h, 37°C MYA, 4 d, 30°C SMA, 2–7 d, 20°C MHA, 48 h, 27°C SDA, 18 h, 30°C SMA, 2–7 d, 20°C MYA, 4 d, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C SMA, 2–7 d, 20°C NA, 24 h, 20°C 5 (h), -30,000 5, 10% sol. sd 5, sd 5, sd 5, sd 6, sd 6, sd 5, 10% sol. sd 6.35, sd 6, 15,000 6 (h), pure 6.35, sd 5, 10% sol. sd 6.35, sd 6.35, sd 6.35, sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 6.35, sd 7 20 15 0 8 9 0 0 12 24 0 0 0 15 20 0 0 0 10 0 0 0 0 7 0 3 11 Conner and Beuchat (1984) Nigam and Rao (1979) Nigam and Rao (1979) Nigam and Rao (1979) Sharma and Singh (1979) Sharma and Singh (1979) Conner and Beuchat (1984) Maruzzella and Liguori (1958) Ertürk et al. (2006) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Maruzzella and Liguori (1958) Schelz et al. (2006) In Vitro Antimicrobial Activities of Essential Oils 369 370 Handbook of Essential Oils TABLE 12.5 Inhibitory Data of Bitter Fennel Oil Obtained in the Dilution Test Microorganism MO Class Test Parameters 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) Escherichia coli Bac- NB, Tween 20, 18 h, 37°C 12,500 Yousef and Tawil (1980) 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) Bacillus subtilis Bac+ NB, Tween 20, 18 h, 37°C 800 Yousef and Tawil (1980) Mycobacterium phlei Bac+ NB, Tween 20, 18 h, 37°C 400 Yousef and Tawil (1980) Staphylococcus aureus Bac+ MHB, 24 h, 37°C 4880 Ertürk et al. (2006) Staphylococcus aureus Bac+ NB, Tween 20, 18 h, 37°C 12,500 Yousef and Tawil (1980) Staphylococcus epidermidis Bac+ TYB, 18 h, 37°C 3000 Schelz et al. (2006) Staphylococcus epidermidis Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast MHB, 24 h, 37°C 9760 Ertürk et al. (2006) MEB, 72 h, 28°C MEB, 72 h, 28°C MEB, 72 h, 28°C NB, Tween 20, 8 d, 30°C MHB, 24 h, 37°C MEB, 72 h, 28°C Cited MEB, 72 h, 28°C MEB, 72 h, 28°C MEB, 72 h, 28°C MEB, 72 h, 28°C MEB, 72 h, 28°C MEB, 72 h, 28°C Cited NB, Tween 20, 8 d, 30°C NB, Tween 20, 8 d, 30°C Cited MEB, 72 h, 28°C MEB, 72 h, 28°C MEB, 72 h, 28°C NB, Tween 20, 8 d, 30°C MEB, 72 h, 28°C MEB, 72 h, 28°C NB, Tween 20, 18 h, 37°C MHB, 24 h, 37°C YPB, 24 h, 20°C 1500 1800–2700 1700–2200 200 9760 1800–2000 250 1800–2200 2000–2200 1200–1500 1200–1300 800–1500 1000–1200 500 3200 400 500 2800–3000 2500–2800 800–1300 12,500 2700–3200 1000–1200 800 4880 800 Mimica-Dukic et al. (2003) Mimica-Dukic et al. (2003) Mimica-Dukic et al. (2003) Yousef and Tawil (1980) Ertürk et al. (2006) Mimica-Dukic et al. (2003) Okazaki and Oshima (1953) Mimica-Dukic et al. (2003) Mimica-Dukic et al. (2003) Mimica-Dukic et al. (2003) Mimica-Dukic et al. (2003) Mimica-Dukic et al. (2003) Mimica-Dukic et al. (2003) Okazaki and Oshima (1953) Yousef and Tawil (1980) Yousef and Tawil (1980) Okazaki and Oshima (1953) Mimica-Dukic et al. (2003) Mimica-Dukic et al. (2003) Mimica-Dukic et al. (2003) Yousef and Tawil (1980) Mimica-Dukic et al. (2003) Mimica-Dukic et al. (2003) Yousef and Tawil (1980) Ertürk et al. (2006) Schelz et al. (2006) Alternaria alternata Aspergillus flavus Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus ochraceus Aspergillus oryzae Aspergillus terreus Aspergillus versicolor Cladosporium cladosporoides Epidermophyton floccosum Fusarium tricinctum Microsporum canis Mucor racemosus Mucor sp. Penicillium chrysogenum Penicillium chrysogenum Penicillium funiculosum Penicillium ochroyloron Phomopsis helianthi Rhizopus sp. Trichoderma viride Trichophyton mentagrophytes Candida albicans Candida albicans Saccharomyces cerevisiae Bac- Bac- Bac- Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Yeast Escherichia coli Neisseria sp. Salmonella typhi Salmonella typhi Bacillus megaterium Bacillus subtilis var. aterrimus Corynebacterium diphtheriae Mycobacterium avium Staphylococcus aureus Streptococcus faecalis Streptococcus faecalis Streptococcus pyogenes Candida albicans MO Class Microorganism NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Test Parameters TABLE 12.6 Inhibitory Data of Bitter Fennel Oil Obtained in the Vapor Phase Test ~20,000 ~20,000 ~20,000 6.35, sd ~20,000 ~20,000 6.35, sd ~20,000 6.35, sd ~20,000 6.35, sd ~20,000 ~20,000 Quantity (µg) NG +++ NG + + +++ + +++ + +++ + ++ NG Activity Kellner and Kober (1954) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Ref. In Vitro Antimicrobial Activities of Essential Oils 371 NA, 18 h, 37°C 4 (h), 10,000 ISA, 48 h, 25°C Bac- Bac- Bac- Bac- Bac- Bac- Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas fluorescens Salmonella pullorum Salmonella sp. Bac- Bac- Proteus vulgaris Pseudomonas aeruginosa —, sd NA, 24 h, 37°C Bac- Bac- Neisseria perflava NA, 24 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C Cited, 18 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C NA, 18 h, 37°C NA, 24 h, 37°C 4 (h), 10,000 4, — 4 (h), 10,000 4, — 6, 2500 —, sd 4 (h), 10,000 6 (h), pure —, sd 4 (h), 10,000 Proteus vulgaris ISA, 48 h, 25°C Bac- ISA, 48 h, 25°C 6 (h), pure 4 (h), 10,000 Bac- ISA, 48 h, 25°C 4 (h), 10,000 6, 2500 4, — 9.5, 2000 —, sd 4 (h), 10,000 5000 on agar 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 —, sd 4 (h), 10,000 Disk Size (mm), Quantity (µg) Klebsiella pneumoniae Bac- Flavobacterium suaveolens ISA, 48 h, 25°C Cited, 18 h, 37°C NA, 24 h, 37°C TYA, 18–24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C Test Parameters Moraxella sp. Bac- Bac- Escherichia coli Escherichia coli Escherichia coli Bac- Bac- Escherichia coli Bac- Bac- Escherichia coli Bac- Erwinia carotovora Escherichia coli Bac- Bac- Enterobacter aerogenes Enterococcus faecalis Bac- Bac- Beneckea natriegens Citrobacter freundii Bac- Bac- Aeromonas hydrophila Alcaligenes faecalis Bac- Bac- Acinetobacter calcoaceticus MO Class Aerobacter aerogenes Microorganism TABLE 12.7 Inhibitory Data of Caraway Oil Obtained in the Agar Diffusion Test 10 6.5 4 7.3 2 0 0 9.5 0 0 14.5 5.5 0 18 9.5 10.3 4 0 0 7 0 0 10.5 7 5 8.5 0 30 Inhibition Zone (mm) El-Gengaihi and Zaki (1982) Deans and Ritchie (1987) El-Gengaihi and Zaki (1982) Janssen et al. (1986) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Yousef and Tawil (1980) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Yousef and Tawil (1980) Deans and Ritchie (1987) Janssen et al. (1986) El-Gengaihi and Zaki (1982) Morris et al. (1979) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Di Pasqua et al. (2005) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Ref. 372 Handbook of Essential Oils NA, 24 h, 37°C —, sd 4 (h), 10,000 ISA, 48 h, 25°C Bac+ Bac+ Bac+ Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Bac+ Mycobacterium phlei Bac+ Bac+ Mycobacterium phlei Sarcina lutea Bac+ Micrococcus sp. Sarcina lutea Bac+ Bac+ Micrococcus luteus Bac+ Bac+ Leuconostoc cremoris Listeria monocytogenes 5000 on agar NA, 24 h, 37°C Bac+ NA, 24 h, 37°C TYA, 18–24 h, 37°C NA, 24 h, 37°C NA, 48 h, 37°C NA, 24 h, 37°C NA, 18 h, 37°C NA, 24 h, 37°C NA, 48 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C 4 (h), 10,000 4, — 9.5, 2000 —, sd 4, — —, sd 6 (h), pure 4, — 4, — 4 (h), 10,000 5000 on agar 5000 on agar Lactococcus lactis subsp. lactis NA, 24 h, 37°C ISA, 48 h, 25°C 5000 on agar Bac+ NA, 24 h, 37°C 5000 on agar Bac+ Bac+ Lactobacillus plantarum NA, 24 h, 37°C 9.5, 2000 4 (h), 10,000 6, 2500 Lactobacillus plantarum Bac+ Lactobacillus delbrueckii TYA, 18–24 h, 37°C ISA, 48 h, 25°C Cited, 18 h, 37°C —, sd Lactococcus garvieae Bac+ Corynebacterium sp. 4, — NA, 24 h, 37°C Bac+ Bac+ Clostridium butyricum 4 (h), 10,000 4 (h), 10,000 ISA, 48 h, 25°C ISA, 48 h, 25°C Bac+ Bac+ Brevibacterium linens Brochotrix thermosphacta Clostridium sporogenes 6 (h), pure NA, 18 h, 37°C Bac+ Bac+ Bacillus subtilis Bacillus subtilis NA, 24 h, 37°C 4 (h), 10,000 Bac+ Bacillus subtilis ISA, 48 h, 25°C Bac+ Bac+ 4, — —, sd 4, — 4 (h), 10,000 4 (h), 10,000 Bacillus subtilis NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C Bacillus subtilis Bac+ Bac+ Bacillus mesentericus Bac- Yersinia enterocolitica Actinomyces sp. Bac- Bac- Serratia marcescens Serratia marcescens 2 4 0 0 4 0 28 10 4 8.5 0 0 0 0 5 0 0 0 0 0 0 0 20 8.3 9 6 4 2 10 8.5 13 Maruzzella and Lichtenstein (1956) continued El-Gengaihi and Zaki (1982) Morris et al. (1979) Maruzzella and Lichtenstein (1956) El-Gengaihi and Zaki (1982) Maruzzella and Lichtenstein (1956) Yousef and Tawil (1980) El-Gengaihi and Zaki (1982) El-Gengaihi and Zaki (1982) Deans and Ritchie (1987) Di Pasqua et al. (2005) Deans and Ritchie (1987) Di Pasqua et al. (2005) Di Pasqua et al. (2005) Deans and Ritchie (1987) Di Pasqua et al. (2005) Di Pasqua et al. (2005) Morris et al. (1979) Deans and Ritchie (1987) El-Gengaihi and Zaki (1982) Deans and Ritchie (1987) Deans and Ritchie (1987) Yousef and Tawil (1980) Janssen et al. (1986) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) El-Gengaihi and Zaki (1982) Maruzzella and Lichtenstein (1956) El-Gengaihi and Zaki (1982) Deans and Ritchie (1987) Deans and Ritchie (1987) In Vitro Antimicrobial Activities of Essential Oils 373 6.35, sd 5, sd 5, 5000 6.35, sd 6, sd 6, sd 6, sd 6.35, sd 6, sd 6, sd 6, sd 5, 5000 6.35, sd 6 (h), pure 6, sd 6, sd 6, sd 5, 5000 6, sd 6.35, sd 5, sd 6, sd 6.35, sd 6, sd 6 (h), pure 6.35, sd SMA, 2–7 d, 20°C EYA, 48 h, 45°C PDA, 72 h, 28°C SMA, 2–7 d, 20°C PDA, 18 h, 37°C PDA, 18 h, 37°C PDA, 18 h, 37°C SMA, 2–7 d, 20°C PDA, 18 h, 37°C PDA, 18 h, 37°C PDA, 18 h, 37°C PDA, 48 h, 28°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C PDA, 18 h, 37°C PDA, 18 h, 37°C PDA, 18 h, 37°C PDA, 72 h, 28°C PDA, 18 h, 37°C SMA, 2–7 d, 20°C EYA, 48 h, 45°C PDA, 18 h, 37°C SMA, 2–7 d, 20°C PDA, 18 h, 37°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Streptomyces venezuelae Absidia corMYBifera Alternaria porri Alternaria solani Alternaria sp. Aspergillus candidus Aspergillus flavus Aspergillus fumigatus Aspergillus fumigatus Aspergillus nidulans Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus niger Cladosporium herbarum Cunninghamella echinulata Fusarium oxysporum Fusarium oxysporum f.sp. cicer Helminthosporium sacchari Helminthosporium sativum Humicola grisea var. thermoidea Microsporum gypseum Mucor mucedo Mucor mucedo Mucor sp. Nigrospora panici 6 (h), pure 4 (h), 10,000 NA, 18 h, 37°C ISA, 48 h, 25°C Bac+ Bac+ 6, 2500 4 (h), 10,000 Disk Size (mm), Quantity (µg) Staphylococcus aureus Cited, 18 h, 37°C ISA, 48 h, 25°C Test Parameters Streptococcus faecalis Bac+ Bac+ MO Class Staphylococcus aureus (continued) Staphylococcus aureus Microorganism TABLE 12.7 0 7.4 15 0 13.5 0 10 12 8 0 7 13 42 0 21 6 11 6.5 12 0 11 9 15 28 15 7 0 17 7.7 7 Inhibition Zone (mm) Nigam and Rao (1979) Pawar and Thaker (2007) Maruzzella and Liguori (1958) Sharma and Singh (1979) Sharma and Singh (1979) Sharma and Singh (1979) Maruzzella and Liguori (1958) Sharma and Singh (1979) Sharma and Singh (1979) Sharma and Singh (1979) Pawar and Thaker (2006) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Sharma and Singh (1979) Sharma and Singh (1979) Sharma and Singh (1979) Pawar and Thaker (2007) Sharma and Singh (1979) Maruzzella and Liguori (1958) Nigam and Rao (1979) Sharma and Singh (1979) Maruzzella and Liguori (1958) Sharma and Singh (1979) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Deans and Ritchie (1987) Yousef and Tawil (1980) Janssen et al. (1986) Deans and Ritchie (1987) Ref. 374 Handbook of Essential Oils Penicillium aculeatum Penicillium chrysogenum Penicillium chrysogenum Penicillium digitatum Penicillium digitatum Penicillium javanicum Penicillium jensenii Penicillium lividum Penicillium notatum Penicillium obscurum Penicillium sp. I Penicillium sp. II Penicillium sp. III Rhizopus nigricans Rhizopus nigricans Rhizopus sp. Sporotrichum thermophile Thermoascus aurantiacis Thermomyces lanuginosa Thielava minor Trichophyton rubrum Trichothecium roseum Brettanomyces anomalus Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Candida krusei Candida lipolytica Candida tropicalis Candida utilis Cryptococcus neoformans Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast CA, 48 h, 27°C CA, 48 h, 27°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C PDA, 18 h, 37°C CA, 48 h, 27°C CA, 48 h, 27°C CA, 48 h, 27°C CA, 48 h, 27°C CA, 48 h, 27°C CA, 48 h, 27°C CA, 48 h, 27°C CA, 48 h, 27°C PDA, 18 h, 37°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C EYA, 48 h, 45°C EYA, 48 h, 45°C EYA, 48 h, 45°C EYA, 48 h, 45°C PDA, 18 h, 37°C PDA, 18 h, 37°C MYA, 4 d, 30°C SMA, 2–7 d, 20°C TYA, 18–24 h, 37°C NA, 24 h, 37°C Cited, 18 h, 37°C SDA, 18 h, 30°C SMA, 2–7 d, 20°C MYA, 4 d, 30°C SMA, 2–7 d, 20°C NA, 24 h, 37°C SMA, 2–7 d, 20°C 5, sd 5, sd 6 (h), pure 6.35, sd 6, sd 5, sd 5, sd 5, sd 5, sd 5, sd 5, sd 5, sd 5, sd 6, sd 6.35, sd 6 (h), pure 5, sd 5, sd 5, sd 5, sd 6, sd 6, sd 5, 10% sol. sd 6.35, sd 9.5, 2000 4, — 6, 2500 6 (h), pure 6.35, sd 5, 10% sol. sd 6.35, sd 4, — 6.35, sd 0 0 60 7 78 10 15 0 15 20 10 15 0 0 6 0 10 10 10 0 7 20 0 0 0 0 9.7 30 0 0 0 0 14 continued Nigam and Rao (1979) Nigam and Rao (1979) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Sharma and Singh (1979) Nigam and Rao (1979) Nigam and Rao (1979) Nigam and Rao (1979) Nigam and Rao (1979) Nigam and Rao (1979) Nigam and Rao (1979) Nigam and Rao (1979) Nigam and Rao (1979) Sharma and Singh (1979) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Nigam and Rao (1979) Nigam and Rao (1979) Nigam and Rao (1979) Nigam and Rao (1979) Sharma and Singh (1979) Sharma and Singh (1979) Conner and Beuchat (1984) Maruzzella and Liguori (1958) Morris et al. (1979) El-Gengaihi and Zaki (1982) Janssen et al. (1986) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Maruzzella and Liguori (1958) El-Gengaihi and Zaki (1982) Maruzzella and Liguori (1958) In Vitro Antimicrobial Activities of Essential Oils 375 (continued) Cryptococcus rhodobenhani Debaryomyces hansenii Geotrichum candidum Hansenula anomala Kloeckera apiculata Kluyveromyces fragilis Lodderomyces elongisporus Metchnikowia pulcherrima Pichia membranaefaciens Rhodotorula rubra Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Torula glabrata Torula thermophila Microorganism TABLE 12.7 Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast MO Class SMA, 2–7 d, 20°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C SMA, 2–7 d, 20°C NA, 24 h, 37°C MYA, 4 d, 30°C MYA, 4 d, 30°C EYA, 48 h, 45°C Test Parameters 6.35, sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 6.35, sd 4, — 5, 10% sol. sd 5, 10% sol. sd 5, sd Disk Size (mm), Quantity (µg) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10 Inhibition Zone (mm) Ref. Maruzzella and Liguori (1958) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Maruzzella and Liguori (1958) El-Gengaihi and Zaki (1982) Conner and Beuchat (1984) Conner and Beuchat (1984) Nigam and Rao (1979) 376 Handbook of Essential Oils Bac Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Bacteria Escherichia coli Escherichia coli Escherichia coli Escherichia coli Helicobacter pylori Pseudomonas aeruginosa Pseudomonas fluorescens Pseudomonas sp. Salmonella typhimurium Serratia marcescens Bacillus subtilis Bacillus subtilis Brochotrix thermosphacta Corynebacterium sp. Micrococcus sp. Mycobacterium phlei Mycobacterium phlei Sarcina sp. Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Aspergillus flavus Aspergillus flavus Aspergillus flavus Aspergillus flavus Aspergillus niger Alternaria citri MO Class Microorganism PDA, 7–14 d, 28°C PDA, 6–8 h 20°C, spore germ. inh. CA, 7 d, 28°C NB, Tween 20, 8 d, 30°C PDA, 5 d, 27°C PDA, 8 d, 22°C NB, Tween 20, 18 h, 37°C TYB, 18–24 h, 37°C NA, 1–3 d, 30°C TSB, 24 h, 37°C NA, 1–3 d, 30°C NB, Tween 20, 18 h, 37°C NA, 1–3 d, 30°C NA, 1–3 d, 30°C TYB, 18–24 h, 37°C M17, 24 h, 20°C NB, Tween 20, 18 h, 37°C NA, 1–3 d, 30°C NA, 1–3 d, 30°C TSB, 24 h, 37°C TSB, 24 h, 37°C NA, 1–3 d, 30°C NB, Tween 20, 18 h, 37°C Cited, 20 h, 37°C NB, Tween 20, 18 h, 37°C NA, 1–3 d, 30°C TSB, 24 h, 37°C 5% Glucose, 9 d, 37°C TYB, 18–24 h, 37°C Test Parameters TABLE 12.8 Inhibitory Data of Caraway Oil Obtained in the Dilution Test >1000 2000 50–100 84–96% inh. 500 3200 >1000 6400 500 1250 10,000 1250 200 750 1000 500 10,000 3200 1000 >10,000 2500 6000 >50,000 2000 273.1 6400 >10,000 2000 >1000 2000 MIC (µg/ml) continued Soliman and Badeaa (2002) Thompson (1986) Kumar et al. (2007) Yousef and Tawil (1980) Thompson and Cannon (1986) Arras and Usai (2001) Yousef and Tawil (1980) Morris et al. (1979) Farag et al. (1989) Di Pasqua et al. (2005) Farag et al. (1989) Yousef and Tawil (1980) Farag et al. (1989) Farag et al. (1989) Morris et al. (1979) Di Pasqua et al. (2005) Yousef and Tawil (1980) Farag et al. (1989) Farag et al. (1989) Di Pasqua et al. (2005) Di Pasqua et al. (2005) Farag et al. (1989) Yousef and Tawil (1980) Weseler et al. (2005) Yousef and Tawil (1980) Farag et al. (1989) Di Pasqua et al. (2005) Buchholtz (1875) Morris et al. (1979) Ref. In Vitro Antimicrobial Activities of Essential Oils 377 Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Rhizopus chinensis Rhizopus circinans Rhizopus japonicus Rhizopus kazanensis Rhizopus oryzae Rhizopus pymacus Rhizopus sp. Rhizopus stolonifer Yeast Yeast Yeast Fungi Rhizopus arrhizus Candida albicans Candida albicans Saccharomyces cerevisiae Fungi Rhizopus 66-81-2 Fungi Fungi Penicillium italicum Trichophyton rubrum Fungi Fungi Fungi Mucor sp. Penicillium chrysogenum Penicillium digitatum Fungi Fungi Mucor racemosus f. racemosus Fungi Fungi Mucor mucedo Trichophyton mentagrophytes Fungi Fungi Fungi Epidermophyton floccosum Fusarium moniliforme Mucor hiemalis Rhizopus tritici Fungi Fungi Fungi Aspergillus parasiticus Aspergillus parasiticus Botrytis cinera MO Class Fungi Fungi (continued) Aspergillus ochraceus Aspergillus parasiticus Microorganism TABLE 12.8 NB, Tween 20, 18 h, 37°C TYB, 18–24 h, 37°C NA, 1–3 d, 30°C SA, Tween 80, 21 d, 20°C SA, Tween 80, 21 d, 20°C PDA, 5 d, 27°C NB, Tween 20, 8 d, 30°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 8 d, 22°C NB, Tween 20, 8 d, 30°C NB, Tween 20, 8 d, 30°C PDA, 8 d, 22°C PDA, 5 d, 27°C PDA, 5 d, 27°C SA, Tween 80, 21 d, 20°C PDA, 7–14 d, 28°C PDA, 5 d, 27°C PDA, 7–14 d, 28°C PDA, 6–8 h 20°C, spore germ. inh. PDA, 8 d, 22°C PDA, 7–14 d, 28°C PDA, 5 d, 27°C Test Parameters <300 1600 500 1000 <300 >1000 >1000 >1000 3200 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 800 1600 >1000 >1000 >1000 300–625 3000 >1000 2000 50–100 3000 MIC (µg/ml) Yousef and Tawil (1980) Morris et al. (1979) Farag et al. (1989) Janssen et al. (1988) Janssen et al. (1988) Thompson and Cannon (1986) Yousef and Tawil (1980) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Arras and Usai (2001) Yousef and Tawil (1980) Yousef and Tawil (1980) Arras and Usai (2001) Thompson and Cannon (1986) Thompson and Cannon (1986) Janssen et al. (1988) Soliman and Badeaa (2002) Thompson and Cannon (1986) Soliman and Badeaa (2002) Thompson (1986) Arras and Usai (2001) Soliman and Badeaa (2002) Thompson and Cannon (1986) Ref. 378 Handbook of Essential Oils BacBacBacBacBac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Yeast Escherichia coli Neisseria sp. Salmonella typhi Salmonella typhi Bacillus megaterium Bacillus subtilis var. aterrimus Corynebacterium diphtheriae Mycobacterium avium Staphylococcus aureus Streptococcus faecalis Streptococcus faecalis Streptococcus pyogenes Candida albicans MO Class Microorganism NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Test Parameters TABLE 12.9 Inhibitory Data of Caraway Oil Obtained in the Vapor Phase Test ~20,000 ~20,000 ~20,000 6.35, sd ~20,000 ~20,000 6.35, sd ~20,000 6.35, sd ~20,000 6.35, sd ~20,000 ~20,000 Quantity (µg) NG +++ NG NG +++ NG +++ NG +++ NG NG NG NG Activity Kellner and Kober (1954) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Ref. In Vitro Antimicrobial Activities of Essential Oils 379 Bac- Bac- Bac- Bac- Bac- Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Escherichia coli Proteus vulgaris Pseudomonas aeruginosa Pseudomonas aeruginosa Salmonella pullorum Bacillus subtilis Corynebacterium pyogenes Listeria monocytogenes Pasteurella multocida Staphylococcus aureus Staphylococcus aureus Alternaria porri Aspergillus niger Fusarium oxysporum f.sp. cicer Bac- Escherichia coli MO Class Enterobacter aerogenes Microorganism PDA, 72 h, 28°C PDA, 48 h, 28°C PDA, 72 h, 28°C MHA, 24 h, 37°C BA, 24–48 h, 37°C BlA, 24–48 h, 37°C ISA, 48 h, 25°C BA, 24–48 h, 37°C BA, 24–48 h, 37°C BA, 24–48 h, 37°C MHA, 24 h, 30°C BA, 24–48 h, 37°C BA, 24–48 h, 37°C MHA, 24 h, 30°C BA, 24–48 h, 37°C MHA, 24 h, 30°C Test Parameters TABLE 12.10 Inhibitory Data of Cassia Oil Obtained in the Agar Diffusion Test 5, 5000 5, 5000 5, 5000 6, 15,000 12.7, sd 12.7, sd 4 (h), 10,000 12.7, sd 12.7, sd 12.7, sd 6, 15,000 12.7, sd 12.7, sd 6, 15,000 12.7, sd 6, 15,000 Disk Size (mm), Quantity (µg) 45 40 37 30 0 19 20 13.5 13.5 13.5 19 18 17.5 21 14 18 Inhibition Zone (mm) Pawar and Thaker (2007) Pawar and Thaker (2006) Pawar and Thaker (2007) Rossi et al. (2007) Goutham and Purohit (1974) Goutham and Purohit (1974) Lis-Balchin et al. (1998) Goutham and Purohit (1974) Goutham and Purohit (1974) Goutham and Purohit (1974) Rossi et al. (2007) Goutham and Purohit (1974) Goutham and Purohit (1974) Rossi et al. (2007) Goutham and Purohit (1974) Rossi et al. (2007) Ref. 380 Handbook of Essential Oils Alternaria alternata Aspergillus niger Aspergillus ochraceus Aspergillus oryzae Aspergillus sp. Fusarium culmorum Fusarium sp. Microsporum gypseum. Mucor racemosus Penicillium chrysogenum Trichophyton mentagrophytes Trichophyton rubrum Candida albicans Candida glabrata Candida krusei Candida tropicalis Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Staphylococcus aureus Staphylococcus aureus 300 MHA, Tween 20, 24 h, 37°C BacBac+ Yersinia enterocolitica 75–600 75–600 NA, cited NA, cited BacBac- Vibrio cholerae Vibrio parahaemolyticus Listeria monocytogenes 250 BHI, 48 h, 35°C BacBac- Salmonella typhimurium PDA, 7 d, 28°C YES broth, 10 d YES broth, 10 d Cited NA, cited YES broth, 10 d NA, cited NA, cited Cited Cited NA, cited NA, cited NA, cited NA, cited NA, cited NA, cited BHI, 48 h, 35°C NA, cited BHI, 48 h, 35°C NA, cited 100% inh. 300 87% inh. 10,000 89% inh. 10,000 500 75–150 54% inh. 10,000 75–150 18.8–37.5 500 500 18.8–37.5 18.8–37.5 100–450 100–450 100–450 100–450 250 75–600 300 75–600 75–600 Salmonella typhimurium NA, cited BacBac- 500 75–600 MIC (µg/mL) Pseudomonas aeruginosa BHI, 48 h, 35°C NA, cited Test Parameters Escherichia coli O157:H7 Bac- MO Class Escherichia coli Microorganism TABLE 12.11 Inhibitory Data of Cassia Oil Obtained in the Dilution Test Feng and Zheng (2007) Lis-Balchin et al. (1998) Lis-Balchin et al. (1998) Okazaki and Oshima (1953) Ooi et al. (2006) Lis-Balchin et al. (1998) Ooi et al. (2006) Ooi et al. (2006) Okazaki and Oshima (1953) Okazaki and Oshima (1953) Ooi et al. (2006) Ooi et al. (2006) Ooi et al. (2006) Ooi et al. (2006) Ooi et al. (2006) Ooi et al. (2006) Oussalah et al. (2006) Ooi et al. (2006) Oussalah et al. (2006) Rossi et al. (2007) Ooi et al. (2006) Ooi et al. (2006) Oussalah et al. (2006) Ooi et al. (2006) Ooi et al. (2006) Oussalah et al. (2006) Ooi et al. (2006) Ref. In Vitro Antimicrobial Activities of Essential Oils 381 Bac+ Bac+ Yeast Streptococcus faecalis Streptococcus pyogenes Candida albicans Bac+ Bac+ Streptococcus faecalis Bac+ Staphylococcus aureus Streptococcus faecalis Bac+ Bac+ Mycobacterium avium Mycobacterium avium Bac+ Bac+ Corynebacterium diphtheriae Staphylococcus aureus Bac+ Bacillus subtilis var. aterrimus Staphylococcus aureus Bac+ Bac+ Bacillus subtilis var. aterrimus Bac- Bac+ Bac- Bacillus megaterium Bac- Salmonella typhi Salmonella typhi Salmonella typhi Bac- Bac- Escherichia coli Neisseria sp. MO Class Microorganism NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Test Parameters TABLE 12.12 Inhibitory Data of Cassia Oil Obtained in the Vapor Phase Test ~20,000 ~20,000 6.35, sd ~20,000 6.35, sd 6.35, sd ~20,000 6.35, sd 6.35, sd ~20,000 6.35, sd 6.35, sd ~20,000 6.35, sd 6.35, sd ~20,000 6.35, sd ~20,000 ~20,000 Quantity (µg) NG + NG + ++ NG ++ + NG NG ++ NG ++ ++ NG ++ NG NG NG Activity Kellner and Kober (1954) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Maruzzella and Liguori (1958) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Maruzzella and Liguori (1958) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Maruzzella and Liguori (1958) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Maruzzella and Liguori (1958) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Maruzzella and Liguori (1958) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Ref. 382 Handbook of Essential Oils Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Aerobacter aerogenes Aeromonas hydrophila Alcaligenes faecalis Beneckea natriegens Campylobacter jejuni Citrobacter freundii Enterobacter aerogenes Erwinia carotovora Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Flavobacterium suaveolens Klebsiella pneumoniae Klebsiella sp. Moraxella sp. Neisseria perflava Proteus sp. Proteus vulgaris Proteus vulgaris Pseudomonas aeruginosa Pseudomonas aeruginosa MO Class Acinetobacter calcoaceticus Microorganism NA, 24 h, 37°C Cited ISA, 48 h, 25°C NA, 24 h, 37°C Cited NA, 24 h, 37°C ISA, 48 h, 25°C Cited ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 30°C NA, 24 h, 37°C Cited, 18 h, 37°C NA, 18 h, 37°C ISA, 48 h, 25°C Cited TSA, 24 h, 35°C NA, 24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C TSA, 24 h, 42°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C Test Parameters —, sd 15, 2500 4 (h), 10,000 —, sd 15, 2500 —, sd 4 (h), 10,000 15, 2500 4 (h), 10,000 4 (h), 10,000 Drop, 5000 10 (h), 2000 6, 2500 6 (h), pure 4 (h), 10,000 15, 2500 4 (h), 25,000 —, sd 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 4 (h), 25,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 —, sd 4 (h), 10,000 Disk Size (mm), Quantity (µg) TABLE 12.13 Inhibitory Data of Ceylon Cinnamon Bark Oil Obtained in the Agar Diffusion Test 4 2 15.5 12 17 0 0 11 18 22 45 35 14.7 15.5 13 13 10.1 5 16 13 12 8.9 27 18 15 0 16.5 Inhibition Zone (mm) continued Maruzzella and Lichtenstein (1956) Pizsolitto et al. (1975) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Pizsolitto et al. (1975) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Pizsolitto et al. (1975) Deans and Ritchie (1987) Deans and Ritchie (1987) Hili et al. (1997) Singh et al. (2007) Janssen et al. (1986) Yousef and Tawil (1980) Deans and Ritchie (1987) Pizsolitto et al. (1975) Smith-Palmer et al. (1998) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Ref. In Vitro Antimicrobial Activities of Essential Oils 383 Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Yersinia enterocolitica Bacillus cereus Bacillus mesentericus Bacillus sp. Bacillus subtilis Bacillus subtilis Bacillus subtilis Bacillus subtilis Bacillus subtilis Brevibacterium linens Bac+ Bac- Shigella sp. Micrococcus luteus Bac- Serratia sp. Bac+ Bac- Serratia marcescens Listeria monocytogenes Bac- Serratia marcescens Bac+ Bac- Salmonella typhi Bac+ Bac- Salmonella sp. Leuconostoc cremoris Bac- Salmonella pullorum Lactobacillus plantarum Bac- Salmonella enteritidis Bac+ Bac- Pseudomonas aeruginosa Bac+ Bac- Pseudomonas aeruginosa Clostridium sporogenes Bac- Pseudomonas aeruginosa Brochotrix thermosphacta Bac- Pseudomonas aeruginosa MO Class Bac- (continued) Pseudomonas aeruginosa Microorganism TABLE 12.13 ISA, 48 h, 25°C TSA, 24 h, 35°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C NA, 18 h, 37°C NA, 24 h, 37°C Cited, 18 h, 37°C Cited NA, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C Cited Cited ISA, 48 h, 25°C NA, 24 h, 37°C NA, 24 h, 37°C Cited ISA, 48 h, 25°C TSA, 24 h, 35°C NA, 24 h, 37°C NA, 24 h, 30°C NA, 18 h, 37°C Cited, 18 h, 37°C ISA, 48 h, 25°C Test Parameters 4 (h), 10,000 4 (h), 25,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 10 (h), 2000 4 (h), 10,000 6 (h), pure —, sd 6, 2500 15, 2500 —, sd 10 (h), 2000 4 (h), 10,000 15, 2500 15, 2500 4 (h), 10,000 —, sd 10 (h), 2000 15, 2500 4 (h), 10,000 4 (h), 25,000 10 (h), 2000 Drop, 5000 6 (h), pure 6, 2500 4 (h), 10,000 Disk Size (mm), Quantity (µg) 18 6.8 8 19 0 5 35 56 23.5 25 21 16 25 16 27 20 13 12 12 0 41 12 21.2 10.9 60 25 17.5 7.7 13 Inhibition Zone (mm) Ref. Deans and Ritchie (1987) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Singh et al. (2007) Deans and Ritchie (1987) Yousef and Tawil (1980) Maruzzella and Lichtenstein (1956) Janssen et al. (1986) Pizsolitto et al. (1975) Maruzzella and Lichtenstein (1956) Singh et al. (2007) Deans and Ritchie (1987) Pizsolitto et al. (1975) Pizsolitto et al. (1975) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Singh et al. (2007) Pizsolitto et al. (1975) Deans and Ritchie (1987) Smith-Palmer et al. (1998) Singh et al. (2007) Hili et al. (1997) Yousef and Tawil (1980) Janssen et al. (1986) Deans and Ritchie (1987) 384 Handbook of Essential Oils Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Sarcina lutea Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus epidermidis Streptococcus faecalis Streptococcus viridans Streptomyces venezuelae Alternaria porri Alternaria solani Aspergillus fumigatus Aspergillus fumigatus Aspergillus niger Aspergillus niger Aspergillus niger Fusarium oxysporum f.sp. cicer Helminthosporium sativum Keratinomyces afelloi Keratinophyton terreum Microsporum gypseum Mucor mucedo Mucor sp. Nigrospora panici Penicillium chrysogenum Penicillium digitatum Rhizopus nigricans Rhizopus sp. Bac+ Mycobacterium phlei PDA, 72 h, 28°C SMA, 2–7 d, 20°C SDA, 3 d, 28°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C PDA, 48 h, 28°C SDA, 8 d, 30°C PDA, 72 h, 28°C SMA, 2–7 d, 20°C SDA, 3 d, 28°C SDA, 3 d, 28°C SDA, 3 d, 28°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C Cited ISA, 48 h, 25°C Cited NA, 24 h, 37°C NA, 24 h, 30°C NA, 18 h, 37°C Cited TSA, 24 h, 35°C ISA, 48 h, 25°C Cited, 18 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 18 h, 37°C 5, 5000 6.35, sd 6, sd 6.35, sd 6.35, sd 5, 5000 6 (h), pure 5, 5000 6.35, sd 6, sd 6, sd 6, sd 6.35, sd 6 (h), pure 6.35, sd 6 (h), pure 6.35, sd 6.35, sd 6 (h), pure 6.35, sd 15, 2500 4 (h), 10,000 15, 2500 10 (h), 2000 Drop, 5000 6 (h), pure 15, 2500 4 (h), 25,000 4 (h), 10,000 6, 2500 —, sd —, sd 6 (h), pure 50 12 18 19 16 43 60 40 15 18 12 21 12 40 15 60 17 7 19 15 8 9 20 57 45 27.3 25 7.5 10 10 0 0 40 continued Pawar and Thaker (2007) Maruzzella and Liguori (1958) Saksena and Saksena (1984) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Pawar and Thaker (2006) Yousef and Tawil (1980) Pawar and Thaker (2007) Maruzzella and Liguori (1958) Saksena and Saksena (1984) Saksena and Saksena (1984) Saksena and Saksena (1984) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Pizsolitto et al. (1975) Deans and Ritchie (1987) Pizsolitto et al. (1975) Singh et al. (2007) Hili et al. (1997) Yousef and Tawil (1980) Pizsolitto et al. (1975) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Janssen et al. (1986) Maruzzella and Lichtenstein (1956) Maruzzella and Lichtenstein (1956) Yousef and Tawil (1980) In Vitro Antimicrobial Activities of Essential Oils 385 (continued) Trichophyton equinum Trichophyton rubrum Brettanomyces anomalus Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Candida krusei Candida lipolytica Candida tropicalis Candida tropicalis Cryptococcus neoformans Cryptococcus rhodobenhani Debaryomyces hansenii Geotrichum candidum Hansenula anomala Kloeckera apiculata Kluyveromyces fragilis Lodderomyces elongisporus Metchnikowia pulcherrima Pichia membranaefaciens Rhodotorula rubra Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Schizosaccharomyces pombe Torula glabrata Torula utilis Microorganism TABLE 12.13 Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast MO Class SDA, 3 d, 28°C SDA, 3 d, 28°C MYA, 4 d, 30°C SDA, 3 d, 28°C SMA, 2–7 d, 20°C Cited, 18 h, 37°C NA, 24 h, 30°C SDA, 18 h, 30°C SMA, 2–7 d, 20°C MYA, 4 d, 30°C SMA, 2–7 d, 20°C SDA, 3 d, 28°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C MYA, 4 d, 30°C SMA, 2–7 d, 20°C MYA, 4 d, 30°C NA, 24 h, 30°C NA, 24 h, 30°C MYA, 4 d, 30°C NA, 24 h, 30°C Test Parameters 6, sd 6, sd 5, 10% sol. sd 6, sd 6.35, sd 6, 2500 Drop, 5000 6 (h), pure 6.35, sd 5, 10% sol. sd 6.35, sd 6, sd 6.35, sd 6.35, sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 6.35, sd 5, 10% sol. sd Drop, 5000 Drop, 5000 5, 10% sol. sd Drop, 5000 Disk Size (mm), Quantity (µg) 28 28 18 14 15 27 39 48 4 21 8 21 18 13 29 18 16 15 8 17 28 11 17 6 22 53 43 20 42 Inhibition Zone (mm) Ref. Saksena and Saksena (1984) Saksena and Saksena (1984) Conner and Beuchat (1984) Saksena and Saksena (1984) Maruzzella and Liguori (1958) Janssen et al. (1986) Hili et al. (1997) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Maruzzella and Liguori (1958) Saksena and Saksena (1984) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Hili et al. (1997) Hili et al. (1997) Conner and Beuchat (1984) Hili et al. (1997) 386 Handbook of Essential Oils BacBacBacBacBacBacBacBacBacBacBac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Escherichia coli Escherichia coli Escherichia coli Escherichia coli O157:H7 Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Salmonella enteritidis Salmonella typhimurium Bacillus subtilis Listeria monocytogenes Listeria monocytogenes Listeria monocytogenes Mycobacterium phlei Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Streptococcus faecalis Aspergillus flavus Aspergillus flavus Aspergillus flavus Aspergillus niger Aspergillus ochraceus Aspergillus parasiticus Aspergillus parasiticus Bac- Escherichia coli MO Class Campylobacter jejuni Microorganism PDA, 6–8 h 20°C, spore germ. inh. PDA, 5 d, 27°C PDA, 7–14 d, 28°C NB, Tween 20, 8 d, 30°C PDA, 7–14 d, 28°C PDA, 6–8 h 20°C, spore germ. inh. PDA, 5 d, 27°C NB, 16 h, 37°C MYB, DMSO, 40 h, 30°C TSB, 24 h, 35°C NB, Tween 20, 18 h, 37°C MHB, Tween 80, 24 h, 37°C NB, 16 h, 37°C BHI, 48 h, 35°C NB, Tween 20, 18 h, 37°C BHI, 48 h, 35°C TSB, 10 d, 4°C TSB, 24 h, 35°C NB, Tween 20, 18 h, 37°C BHI, 48 h, 35°C TSB, 24 h, 35°C MYB, DMSO, 40 h, 30°C NB, Tween 20, 18 h, 37°C NB, 16 h, 37°C BHI, 48 h, 35°C MYB, DMSO, 40 h, 30°C TSB, 24 h, 35°C NB, Tween 20, 18 h, 37°C NB, 16 h, 37°C TSB, 24 h, 42°C Test Parameters TABLE 12.14 Inhibitory Data of Ceylon Cinnamon Bark Oil Obtained in the Dilution Test MIC (µg/mL) 50–100 1000 1000 100 1000 50–100 1000 200 70% inh. 500 400 400 390 350 250 320 500 300 300 200 500 500 85% inh. 500 400 300 250 67% inh. 500 500 400 200 500 Ref. continued Thompson (1986) Thompson and Cannon (1986) Soliman and Badeaa (2002) Yousef and Tawil (1980) Soliman and Badeaa (2002) Thompson (1986) Thompson and Cannon (1986) Lens-Lisbonne et al. (1987) Hili et al. (1997) Smith-Palmer et al. (1998) Yousef and Tawil (1980) Bastide et al. (1987) Lens-Lisbonne et al. (1987) Oussalah et al. (2006) Yousef and Tawil (1980) Oussalah et al. (2006) Smith-Palmer et al. (1998) Smith-Palmer et al. (1998) Yousef and Tawil (1980) Oussalah et al. (2006) Smith-Palmer et al. (1998) Hili et al. (1997) Yousef and Tawil (1980) Lens-Lisbonne et al. (1987) Oussalah et al. (2006) Hili et al. (1997) Smith-Palmer et al. (1998) Yousef and Tawil (1980) Lens-Lisbonne et al. (1987) Smith-Palmer et al. (1998) In Vitro Antimicrobial Activities of Essential Oils 387 Fungi Yeast Yeast Yeast Yeast Yeast Trichophyton rubrum Candida albicans Candida albicans Saccharomyces cerevisiae Schizosaccharomyces pombe Torula utilis MO Class Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi (continued) Aspergillus parasiticus Botrytis cinera Colletotrichum musae Epidermophyton floccosum Fusarium moniliforme Fusarium proliferatum Geotrichum citri-aurantii Lasiodiplodia theobromae Mucor hiemalis Mucor mucedo Mucor racemosus f. racemosus Mucor sp. Penicillium chrysogenum Penicillium digitatum Phytophthora citrophthora Rhizopus 66-81-2 Rhizopus arrhizus Rhizopus chinensis Rhizopus circinans Rhizopus japonicus Rhizopus kazanensis Rhizopus oryzae Rhizopus pymacus Rhizopus sp. Rhizopus stolonifer Rhizopus tritici Trichophyton mentagrophytes Microorganism TABLE 12.14 Test Parameters NB, Tween 20, 18 h, 37°C MYB, DMSO, 40 h, 30°C MYB, DMSO, 40 h, 30°C MYB, DMSO, 40 h, 30°C MYB, DMSO, 40 h, 30°C SA, Tween 80, 21 d, 20°C PDA, 7–14 d, 28°C PDA, Tween 20, 7 d, 24°C SMKY, EtOH, 7 d, 28°C SA, Tween 80, 21 d, 20°C PDA, 7–14 d, 28°C SMKY, EtOH, 7 d, 28°C PDA, Tween 20, 7 d, 24°C SMKY, EtOH, 7 d, 28°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C NB, Tween 20, 8 d, 30°C NB, Tween 20, 8 d, 30°C PDA, Tween 20, 7 d, 24°C PDA, Tween 20, 7 d, 24°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C NB, Tween 20, 8 d, 30°C PDA, 5 d, 27°C PDA, 5 d, 27°C SA, Tween 80, 21 d, 20°C MIC (µg/mL) <300 80 72% inh. 500 59% inh. 500 96% inh. 500 100% inh. 500 <300 1000 25% inh. 1000 300 300–625 1000 500 30% inh. 1000 350 500 1000 1000 100 100 32% inh. 1000 38% inh. 1000 1000 500 500 500 1000 500 1000 500 400 500 1000 Ref. Yousef and Tawil (1980) Hili et al. (1997) Hili et al. (1997) Hili et al. (1997) Hili et al. (1997) Janssen et al. (1988) Soliman and Badeaa (2002) Bouchra et al. (2003) Ranasinghe et al. (2002) Janssen et al. (1988) Soliman and Badeaa (2002) Ranasinghe et al. (2002) Bouchra et al. (2003) Ranasinghe et al. (2002) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Yousef and Tawil (1980) Yousef and Tawil (1980) Bouchra et al. (2003) Bouchra et al. (2003) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Yousef and Tawil (1980) Thompson and Cannon (1986) Thompson and Cannon (1986) Janssen et al. (1988) 388 Handbook of Essential Oils Bac- Bac- Bac- Bac- Bac- Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Escherichia coli Escherichia coli Haemophilus influenzae Neisseria sp. Salmonella typhi Salmonella typhi Bacillus megaterium Bacillus subtilis var. aterrimus Corynebacterium diphtheriae Mycobacterium avium Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Streptococcus faecalis Streptococcus faecalis Streptococcus pneumoniae Streptococcus pyogenes Streptococcus pyogenes Aspergillus niger Aspergillus ochraceus Aspergillus terreus Fusarium graminearum Fusarium moniliforme Penicillium citrinum Penicillium viridicatum Candida albicans Aspergillus flavus MO Class Microorganism CDA, 6 d CDA, 6 d CDA, 6 d CDA, 6 d CDA, 6 d NA, 24 h, 37°C CDA, 6 d CDA, 6 d CDA, 6 d MHA, 18 h, 37°C NA, 24 h, 37°C MHA, 18 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C MHA, 18 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C MHA, 18 h, 37°C BlA, 18 h, 37°C NA, 24 h, 37°C Test Parameters ~20,000 12, 6000 12, 6000 12, 6000 12, 6000 12, 6000 12, 6000 12, 6000 12, 6000 ~20,000 MICair MICair ~20,000 6.35, sd MICair ~20,000 6.35, sd ~20,000 6.35, sd ~20,000 6.35, sd ~20,000 6.35, sd ~20,000 MICair ~20,000 MICair Quantity (µg) TABLE 12.15 Inhibitory Data of Ceylon Cinnamon Bark Oil Obtained in the Vapor Phase Test Activity ++ NG NG NG NG NG NG + +++ 6.25 NG + 1.56–3.13 NG ++ 6.25 + NG ++ NG ++ NG NG NG 3.13 12.5 NG Ref. Singh et al. (2007) Singh et al. (2007) Singh et al. (2007) Singh et al. (2007) Singh et al. (2007) Kellner and Kober (1954) Singh et al. (2007) Singh et al. (2007) Singh et al. (2007) Inouye et al. (2001) Kellner and Kober (1954) Inouye et al. (2001) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Inouye et al. (2001) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Kellner and Kober (1954) Inouye et al. (2001) Inouye et al. (2001) Kellner and Kober (1954) In Vitro Antimicrobial Activities of Essential Oils 389 Alternaria porri Aspergillus niger Fusarium oxysporum f.sp. cicer Candida albicans Bac+ Bac+ Fungi Fungi Fungi Yeast Staphylococcus aureus Staphylococcus aureus 9.5, 2000 TYA, 18–24 h, 37°C Bac+ Bac+ Corynebacterium sp. 10 (h), 2000 10 (h), 2000 NA, 24 h, 37°C NA, 24 h, 37°C Bac+ Bac+ Bacillus cereus Bacillus subtilis Listeria monocytogenes 10 (h), 2000 NA, 24 h, 37°C Bac- Bac- Pseudomonas aeruginosa PDA, 72 h, 28°C PDA, 48 h, 28°C PDA, 72 h, 28°C TYA, 18–24 h, 37°C NA, 24 h, 37°C TYA, 18–24 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C 5, 5000 5, 5000 5, 5000 9.5, 2000 10 (h), 2000 9.5, 2000 4 (h), 10,000 10 (h), 2000 10 (h), 2000 Salmonella typhi NA, 24 h, 37°C Bac- Bac- 9.5, 2000 Disk Size (mm), Quantity (µg) Escherichia coli TYA, 18–24 h, 37°C Test Parameters Escherichia coli MO Class Microorganism TABLE 12.16 Inhibitory Data of Ceylon Cinnamon Leaf Oil Obtained in the Agar Diffusion Test 50 30 35 14 48 18 20 12 90 32 17 90 25 17 Inhibition Zone (mm) Ref. Pawar and Thaker (2007) Pawar and Thaker (2006) Pawar and Thaker (2007) Morris et al. (1979) Singh et al. (2007) Morris et al. (1979) Lis-Balchin et al. (1998) Morris et al. (1979) Singh et al. (2007) Singh et al. (2007) Singh et al. (2007) Singh et al. (2007) Singh et al. (2007) Morris et al. (1979) 390 Handbook of Essential Oils Bac- Bac- Bac- Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Escherichia coli Escherichia coli O157:H7 Salmonella typhimurium Corynebacterium sp. Listeria monocytogenes Staphylococcus aureus Staphylococcus aureus Aspergillus flavus Aspergillus flavus Aspergillus niger Aspergillus ochraceus Aspergillus parasiticus Aspergillus parasiticus Colletotrichum musae Fusarium culmorum Fusarium proliferatum Lasiodiplodia theobromae Mucor hiemalis Mucor mucedo Mucor racemosus f. racemosus Rhizopus 66-81-2 Rhizopus arrhizus Rhizopus chinensis Rhizopus circinans Rhizopus japonicus Rhizopus kazanensis Rhizopus oryzae Rhizopus pymacus Rhizopus stolonifer Rhizopus tritici Candida albicans MO Class Microorganism PDA, 6–8 h 20°C, spore germ. inh. PDA, 5 d, 27°C YES broth, 10 d YES broth, 10 d PDA, 6–8 h 20°C, spore germ. inh. PDA, 5 d, 27°C SMKY, EtOH, 7 d, 28°C YES broth, 10 d SMKY, EtOH, 7 d, 28°C SMKY, EtOH, 7 d, 28°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C TYB, 18–24 h, 37°C BHI, 48 h, 35°C TYB, 18–24 h, 37°C BHI, 48 h, 35°C TYB, 18–24 h, 37°C BHI, 48 h, 35°C BHI, 48 h, 35°C TYB, 18–24 h, 37°C Test Parameters TABLE 12.17 Inhibitory Data of Ceylon Cinnamon Leaf Oil Obtained in the Dilution Test 50–100 500 95% inh. 10,000 94% inh. 10,000 50–100 500 500 73% inh. 10,000 500 600 500 500 500 500 500 100 500 500 500 500 500 500 500 500 500 500 2000 500 1000 1000 1000 MIC (µg/mL) Thompson (1986) Thompson and Cannon (1986) Lis-Balchin et al. (1998) Lis-Balchin et al. (1998) Thompson (1986) Thompson and Cannon (1986) Ranasinghe et al. (2002) Lis-Balchin et al. (1998) Ranasinghe et al. (2002) Ranasinghe et al. (2002) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Morris et al. (1979) Oussalah et al. (2006) Morris et al. (1979) Oussalah et al. (2006) Morris et al. (1979) Oussalah et al. (2006) Oussalah et al. (2006) Morris et al. (1979) Ref. In Vitro Antimicrobial Activities of Essential Oils 391 Bac- Bac- Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Escherichia coli Neisseria sp. Salmonella typhi Bacillus megaterium Corynebacterium diphtheriae Staphylococcus aureus Streptococcus faecalis Streptococcus pyogenes Aspergillus flavus Aspergillus niger Aspergillus niger Aspergillus ochraceus Aspergillus terreus Endomyces filbuliger Eurotium amstelodami Eurotium herbarum Eurotium repens Eurotium repens Eurotium rubrum Fusarium graminearum Fusarium moniliforme Penicillium citrinum Penicillium corylophilum Penicillium corylophilum Penicillium roqueforti Penicillium viridicatum Candida albicans Aspergillus flavus Aspergillus flavus MO Class Microorganism CDA, 6 d NA, 24 h, 37°C Bread, 14 d, 25°C WFA, 42 d, 25°C CDA, 6 d CDA, 6 d CDA, 6 d WFA, 42 d, 25°C Bread, 14 d, 25°C WFA, 42 d, 25°C WFA, 42 d, 25°C WFA, 42 d, 25°C Bread, 14 d, 25°C Bread, 14 d, 25°C CDA, 6 d CDA, 6 d WFA, 42 d, 25°C CDA, 6 d CDA, 6 d WFA, 42 d, 25°C Bread, 14 d, 25°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Test Parameters ~20,000 12, 6000 30,000 Disk, 50,000 12, 6000 12, 6000 12, 6000 Disk, 50,000 30,000 Disk, 50,000 Disk, 50,000 Disk, 50,000 30,000 30,000 12, 6000 12, 6000 Disk, 50,000 12, 6000 12, 6000 ~20,000 Disk, 50,000 30,000 ~20,000 ~20,000 ~20,000 ~20,000 ~20,000 ~20,000 ~20,000 Quantity (µg) TABLE 12.18 Inhibitory Data of Ceylon Cinnamon Leaf Oil Obtained in the Vapor Phase Test +++ NG NG +++ +++ NG NG NG NG NG +++ NG NG NG + + +++ NG NG NG NG NG NG NG NG NG NG NG NG Activity Singh et al. (2007) Kellner and Kober (1954) Suhr and Nielsen (2003) Guynot et al. (2003) Singh et al. (2007) Singh et al. (2007) Singh et al. (2007) Guynot et al. (2003) Suhr and Nielsen (2003) Guynot et al. (2003) Guynot et al. (2003) Guynot et al. (2003) Suhr and Nielsen (2003) Suhr and Nielsen (2003) Singh et al. (2007) Singh et al. (2007) Guynot et al. (2003) Singh et al. (2007) Singh et al. (2007) Guynot et al. (2003) Suhr and Nielsen (2003) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Ref. 392 Handbook of Essential Oils Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Aerobacter aerogenes Brucella abortus Campylobacter jejuni Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Klebsiella pneumonia Klebsiella pneumonia subsp. oceanae Klebsiella pneumoniae Neisseria perflava Proteus OX19 Proteus vulgaris Proteus vulgaris Proteus vulgaris Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas fluorescens Pseudomonas mangiferae indicae Salmonella enteritidis Salmonella enteritidis MO Class Aerobacter aerogenes Microorganism NA, 24 h, 37°C Cited NA, 36–48 h, 37°C NA, 24 h, 37°C Agar, 24 h, 37°C NA, 18 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Agar, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Agar, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Agar, 24 h, 37°C TGA, 18–24 h, 37°C NA, 18 h, 37°C Cited Cited NA, 24 h, 37°C NA, 24 h, 37°C Cited NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Conditions TABLE 12.19 Inhibitory Data of Citronella Oil Obtained in the Agar Diffusion Test —, sd 5 × 20, 1000 6, 15,000 6, sd 5 × 20, 1000 6, 6000 6 (h), pure 5 × 20, 1000 —, sd 6, 6000 5 × 20, 1000 —, sd 5 × 20, 1000 —, sd 6, 6000 5 × 20, 1000 5 × 20, 1000 6, 6000 9.5, 2000 6 (h), pure 20,000 6, 15,000 5 × 20, 1000 —, sd 6, 15,000 5 × 20, 1000 5 × 20, 1000 Maruzzella and Lichtenstein (1956) a Wannissorn et al. (2005) Maruzzella and Lichtenstein (1956) Möse et al. (1957) Wannissorn et al. (2005) 40a 0a 0–1a 10.5a Garg and Garg (1980) Wannissorn et al. (2005) Möse et al. (1957) 11 6–10a 0–1a 12.8a Jirovetz et al. (2006) Möse et al. (1957) 0 Yousef and Tawil (1980) continued Möse et al. (1957) 0a 0 Jirovetz et al. (2006) Maruzzella and Lichtenstein (1956) Möse et al. (1957) 0a 0a Maruzzella and Lichtenstein (1956) 0a 10 Möse et al. (1957) 2–5a Jirovetz et al. (2006) Maruzzella and Lichtenstein (1956) 0a Möse et al. (1957) 2–5a 0 Jirovetz et al. (2006) Möse et al. (1957) 2–5a Morris et al. (1979) Yousef and Tawil (1980) 7 0 0 Lemos et al. (1992) Möse et al. (1957) 2–5a 0 Möse et al. (1957) 0a 0 Ref. Inhibition Zone (mm) In Vitro Antimicrobial Activities of Essential Oils 393 Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Salmonella sp. Salmonella typhi Salmonella typhi Salmonella typhimurium Serratia marcescens Serratia marcescens Vibrio albicans Vibrio cholera Vibrio cholera Bacillus anthracis Bacillus cereus Bacillus mesentericus Bacillus mycoides Bacillus pumilus Bacillus subtilis Bacillus subtilis Bacillus subtilis Bacillus subtilis Clostridium perfringens Corynebacterium diphtheria Enterococcus faecalis Listeria monocytogenes Mycobacterium phlei Sarcina alba Sarcina beige Sarcina citrea Sarcina lutea MO Class Salmonella paratyphi B (continued) Salmonella paratyphi Microorganism TABLE 12.19 NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 18 h, 37°C NA, 24 h, 37°C Agar, 24 h, 37°C NA, 24 h, 37°C Cited NA, 18 h, 37°C NA, 36–48 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 36–48 h, 37°C NA, 36–48 h, 37°C NA, 24 h, 37°C Cited NA, 24 h, 37°C NA, 36–48 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Cited NA, 36–48 h, 37°C NA, 24 h, 37°C Agar, 24 h, 37°C NA, 24 h, 37°C NA, 36–48 h, 37°C Conditions —, sd 5 × 20, 1000 5 × 20, 1000 5 × 20, 1000 6 (h), pure 5 × 20, 1000 6, 6000 5 × 20, 1000 6, 15,000 6 (h), pure 6, sd —, sd 5 × 20, 1000 6, sd 6, sd —, sd 20,000 5 × 20, 1000 6, sd 5 × 20, 1000 5 × 20, 1000 —, sd 5 × 20, 1000 6, 15,000 6, sd 5 × 20, 1000 6, 6000 5 × 20, 1000 6, sd Möse et al. (1957) a 8a Möse et al. (1957) Möse et al. (1957) Möse et al. (1957) Maruzzella and Lichtenstein (1956) 16–23a 0a 0a Yousef and Tawil (1980) 6–10a 16 Jirovetz et al. (2006) Möse et al. (1957) Möse et al. (1957) 6–10a 1a Wannissorn et al. (2005) 39.5a 10 Yousef and Tawil (1980) 15.5 Garg and Garg (1980) Maruzzella and Lichtenstein (1956) 1a 12 Garg and Garg (1980) Möse et al. (1957) 12 Garg and Garg (1980) Maruzzella and Lichtenstein (1956) 12 Lemos et al. (1992) 3a 1a 20 Garg and Garg (1980) Möse et al. (1957) 0 6–10 Möse et al. (1957) 6–10a Maruzzella and Lichtenstein (1956) Möse et al. (1957) 0a 0 Garg and Garg (1980) Wannissorn et al. (2005) 18 21a Jirovetz et al. (2006) Möse et al. (1957) 0 2–5a Garg and Garg (1980) Möse et al. (1957) 1a Ref. 14 Inhibition Zone (mm) 394 Handbook of Essential Oils Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Staphylococcus albus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus epidermidis Streptococcus haemolyticus Streptococcus viridians Streptomyces venezuelae Alternaria solani Aspergillus fumigatus Aspergillus niger Aspergillus niger Aspergillus niger Helminthosporium sativum Microsporon gypseum Mucor mucedo Mucor sp. Nigrospora panici Penicillium chrysogenum Penicillium digitatum Rhizopus nigricans Rhizopus sp. Sporothrix schenkii Candida albicans Candida albicans Bac+ Bac+ Sporococcus sarc. Bac+ Sarcina rosa Sarcina lutea SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SDA, 7–10 d, 28°C SMA, 2–7 d, 20°C SDA, 7–10 d, 28°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SDA, 7–10 d, 28°C TGA, 18–24 h, 37°C Cited SMA, 2–7 d, 20°C NA, 24 h, 37°C NA, 24 h, 37°C Cited NA, 18 h, 37°C NA, 36–48 h, 37°C TGA, 18–24 h, 37°C Agar, 24 h, 37°C NA, 24 h, 37°C Cited NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 36–48 h, 37°C sd sd 6 (h), pure sd 5, 5000 sd 5, 5000 sd 6 (h), pure sd 6 (h), pure sd sd 6 (h), pure 5, 5000 9.5, 2000 20,000 sd 5 × 20, 1000 5 × 20, 1000 20,000 6 (h), pure 6, sd 9.5, 2000 6, 6000 5 × 20, 1000 20,000 —, sd 5 × 20, 1000 5 × 20, 1000 5 × 20, 1000 6, sd Möse et al. (1957) Möse et al. (1957) Möse et al. (1957) 6–10a 1a 6 6 15 7 8 2 34 6 18 6 40 5 1 12 12 1 17 continued Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Saikia et al. (2001) Maruzzella and Liguori (1958) Saikia et al. (2001) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Saikia et al. (2001) Morris et al. (1979) Lemos et al. (1992) Maruzzella and Liguori (1958) Lemos et al. (1992) 12a 4 Yousef and Tawil (1980) Garg and Garg (1980) Morris et al. (1979) 12.6 12 11 10 Jirovetz et al. (2006) Lemos et al. (1992) 2–5a 0 a Maruzzella and Lichtenstein (1956) Möse et al. (1957) 6–10a 0a Möse et al. (1957) 26–33a Garg and Garg (1980) Möse et al. (1957) 0 11–15a In Vitro Antimicrobial Activities of Essential Oils 395 (continued) a Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast MO Class Citronella oil obtained from Cymbopogon nardus (Ceylon-type). Candida albicans Candida albicans Candida albicans Candida albicans Candida krusei Candida tropicalis Candida tropicalis Cryptococcus neoformans Cryptococcus rhodobenhani Saccharomyces cerevisiae Microorganism TABLE 12.19 SDA, 18 h, 30°C Agar, 24 h, 37°C SMA, 2–7 d, 20°C SDA, 7–10 d, 28°C SMA, 2–7 d, 20°C Cited SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C Conditions 6 (h), pure 6, 6000 sd 5, 5000 sd 20,000 sd sd sd sd 17 28 6 7 0 0 3 2 7 2 Inhibition Zone (mm) Yousef and Tawil (1980) Jirovetz et al. (2006) Maruzzella and Liguori (1958) Saikia et al. (2001) Maruzzella and Liguori (1958) Lemos et al. (1992) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Ref. 396 Handbook of Essential Oils BacBacBacBacBacBacBacBac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Escherichia coli Escherichia coli Escherichia coli O157:H7 Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas cepacia Salmonella typhimurium Bacillus subtilis Listeria monocytogenes Mycobacterium phlei Staphylococcus aureus Staphylococcus aureus Staphylococcus intermedius Aspergillus flavus Aspergillus flavus Aspergillus fumigatus Aspergillus niger Aspergillus niger Aspergillus oryzae Aspergillus versicolor Eurotium amstelodami Eurotium chevalieri Aspergillus candidus MO Class Microorganism Conditions Cited Cited PDA, 28 d, 21°C NA, Tween 80, 24 h, 37°C SDB, 7–10 d, 28°C NB, Tween 20, 8 d, 30°C Cited Cited Cited Cited NA, Tween 80, 24 h, 37°C NB, Tween 20, 18 h, 37°C BHI, 48 h, 35°C NB, Tween 20, 18 h, 37°C BHI, 48 h, 35°C NB, Tween 20, 18 h, 37°C BHI, 48 h, 35°C NA, Tween 80, 24 h, 37°C NB, Tween 20, 18 h, 37°C NA, Tween 80, 24 h, 37°C BHI, 48 h, 35°C NB, Tween 20, 18 h, 37°C MHB, 24 h, 36°C TABLE 12.20 Inhibitory Data of Citronella Oil Obtained in the Dilution Test Yousef and Tawil (1980) >250a >250a >250a continued Nakahara et al. (2003) Nakahara et al. (2003) Thanaboripat et al. (2004) Koba et al. (2004) Saikia et al. (2001) Yousef and Tawil (1980) Okazaki and Oshima (1953) Nakahara et al. (2003) Nakahara et al. (2003) a >250 4000–10,000 200a 2500 6400 250 Koba et al. (2004) Nakahara et al. (2003) a Oussalah et al. (2006) Yousef and Tawil (1980) Oussalah et al. (2006) Yousef and Tawil (1980) Oussalah et al. (2006) Koba et al. (2004) Yousef and Tawil (1980) Koba et al. (2004) Oussalah et al. (2006) Yousef and Tawil (1980) Duarte et al. (2006) Ref. >250a >500 6400 500 1250 4000 400 >500a 4000 >500a 50,000 >8000 50,000 2000–8000 MIC In Vitro Antimicrobial Activities of Essential Oils 397 Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Penicillium islansicum Rhizopus sp. Sporothrix schenkii Trichophyton mentagrophytes Candida albicans Candida albicans Candida albicans Cryptococcus neoformans Malassezia pachydermis Citronella oil obtained from Cymbopogon nardus (Ceylon-type). Fungi Penicillium griseofulvum a Fungi Fungi Fungi Penicillium chrysogenum Penicillium chrysogenum Penicillium citrinum MO Class Fungi Fungi Fungi Fungi Fungi Fungi (continued) Microsporon canis Microsporon gypseum Microsporon gypseum Mucor racemosus Mucor sp. Penicillium adametzii Microorganism TABLE 12.20 Conditions SDB, 7–10 d, 28°C NB, Tween 20, 18 h, 37°C NA, Tween 80, 24 h, 37°C NA, Tween 80, 24 h, 37°C NB, Tween 20, 8 d, 30°C SDB, 7–10 d, 28°C NA, Tween 80, 24 h, 37°C NA, Tween 80, 24 h, 37°C Cited Cited NB, Tween 20, 8 d, 30°C Cited Cited NA, Tween 80, 24 h, 37°C NA, Tween 80, 24 h, 37°C SDB, 7–10 d, 28°C Cited NB, Tween 20, 8 d, 30°C Cited MIC >500a 1250 6400 500a 150a >250 12,500 1250 150a a >250a >250a >250a 12,500 250 200a 500a 625 500 6400 Ref. Saikia et al. (2001) Yousef and Tawil (1980) Koba et al. (2004) Koba et al. (2004) Yousef and Tawil (1980) Saikia et al. (2001) Koba et al. (2004) Koba et al. (2004) Nakahara et al. (2003) Nakahara et al. (2003) Yousef and Tawil (1980) Okazaki and Oshima (1953) Nakahara et al. (2003) Koba et al. (2004) Koba et al. (2004) Saikia et al. (2001) Okazaki and Oshima (1953) Yousef and Tawil (1980) Nakahara et al. (2003) 398 Handbook of Essential Oils Bac- Bac- Bac- Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Neisseria sp. Salmonella typhi Salmonella typhi Bacillus megaterium Bacillus subtilis var. aterrimus Corynebacterium diphtheriae Mycobacterium avium Staphylococcus aureus Staphylococcus aureus Streptococcus faecalis Streptococcus faecalis Streptococcus pyogenes Aspergillus fumigatus Aspergillus sulphureus Aspergillus versicolor Eurotium amstelodami Eurotium chevalieri Mucor fragilis Penicillium adametzii Penicillium citrinum Penicillium griseofulvum Penicillium islansicum Rhizopus stolonifer Candida albicans b a Formosan citronella oil. Citronella oil obtained from Cymbopogon nardus (Ceylon-type). Aspergillus candidus Aspergillus flavus Aspergillus flavus MO Class Microorganism Escherichia coli NA, 24 h, 37°C PDA, 10 d, 25°C PDA, 10 d, 25°C PDA, 3–5 d, 27°C PDA, 3–5 d, 27°C PDA, 3–5 d, 27°C PDA, 10 d, 25°C PDA, 3–5 d, 27°C PDA, 3–5 d, 27°C PDA, 3–5 d, 27°C PDA, 3–5 d, 27°C PDA, 10 d, 25°C PDA, 3–5 d, 27°C PDA, 3–5 d, 27°C PDA, 10 d, 25°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Conditions TABLE 12.21 Inhibitory Data of Citronella Oil Obtained in the Vapor Phase Test ~20,000 50 50 250 mg/L air 250 mg/L air 250 mg/L air 50 250 mg/L air 250 mg/L air 250 mg/L air 250 mg/L air 50 ~20,000 250 mg/L air 250 mg/L air 50 ~20,000 ~20,000 sd sd ~20,000 sd ~20,000 sd ~20,000 ~20,000 sd ~20,000 Ref. ++ NG Kellner and Kober (1954) Sarbhoy et al. (1978) Sarbhoy et al. (1978) Nakahara et al. (2003) Nakahara et al. (2003) Nakahara et al. (2003) Sarbhoy et al. (1978) Nakahara et al. (2003) Nakahara et al. (2003) Nakahara et al. (2003) Nakahara et al. (2003) Sarbhoy et al. (1978) Nakahara et al. (2003) Nakahara et al. (2003) Sarbhoy et al. (1978) ++ NG NG NGb NGb NGb NG NGb NGb NGb NGb Kellner and Kober (1954) NGb NGb Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) NG +++a NG Maruzzella and Sicurella (1960) Maruzzella and Sicurella (1960) +++a NG Kellner and Kober (1954) NGa Maruzzella and Sicurella (1960) Kellner and Kober (1954) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Kellner and Kober (1954) +a NG + +++ NG a + NG Activity In Vitro Antimicrobial Activities of Essential Oils 399 Bac- Bac- Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Yeast Yeast Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Bacillus pumilus Bacillus subtilis Enterococcus faecalis Listeria monocytogenes Staphylococcus aureus Staphylococcus epidermidis Alternaria porri Aspergillus niger Aspergillus niger Fusarium oxysporum f.sp. cicer Candida albicans Saccharomyces cerevisiae MO Class Microorganism PDA, 72 h, 28°C MHA, 48 h, 30°C PDA, 48 h, 28°C PDA, 72 h, 28°C MHA, 48 h, 30°C MHA, 48 h, 30°C MHA, 24 h, 37°C MHA, 24 h, 37°C ISA, 48 h, 25°C MHA, 24 h, 37°C MHA, 24 h, 37°C MHA, 24 h, 37°C MHA, 24 h, 37°C MHA, 24 h, 37°C MHA, 24 h, 37°C Conditions TABLE 12.22 Inhibitory Data of Clary Sage Oil Obtained in the Agar Diffusion Test 5, 5000 6, 10,000 5, 5000 5, 5000 6, 10,000 6, 10,000 6, 10,000 6, 10,000 4 (h), 10,000 6, 10,000 6, 10,000 6, 10,000 6, 10,000 6, 10,000 6, 10,000 0 0 0 9 0 0 15 15 9–15 9 17 16 0 10 14 Inhibition Zone (mm) Pawar and Thaker (2007) Yousefzadi et al. (2007) Pawar and Thaker (2006) Pawar and Thaker (2007) Yousefzadi et al. (2007) Yousefzadi et al. (2007) Yousefzadi et al. (2007) Yousefzadi et al. (2007) Lis-Balchin et al. (1998) Yousefzadi et al. (2007) Yousefzadi et al. (2007) Yousefzadi et al. (2007) Yousefzadi et al. (2007) Yousefzadi et al. (2007) Yousefzadi et al. (2007) Ref. 400 Handbook of Essential Oils BacBacBacBacBacBacBacBacBacBacBacBacBacBacBac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Aeromonas sobria Erwinia carotovora Escherichia coli Escherichia coli Escherichia coli Escherichia coli Klebsiella pneumoniae Klebsiella pneumoniae Proteus vulgaris Pseudomonas aeruginosa Salmonella typhi Salmonella typhimurium Serratia marcescens Bacillus cereus Bacillus circulans Bacillus megaterium Bacillus pumilus Bacillus subtilis Bacillus subtilis var. aterrimus Enterococcus faecalis Enterococcus faecalis Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus MO Class Acinetobacter baumannii Microorganism Conditions Cited MHA, Tween 20, 48 h, 35°C NA, 48 h, 37°C MHA, Tween 20, 48 h, 35°C MHB, Tween 80, 24 h, 37°C NA, 48 h, 37°C MHB, Tween 80, 24 h, 37°C MHB, Tween 80, 24 h, 37°C NA, 48 h, 37°C NA, 48 h, 37°C NA, 48 h, 37°C MHA, Tween 20, 48 h, 35°C MHA, Tween 20, 48 h, 35°C NA, 48 h, 37°C MHA, Tween 20, 48 h, 35°C NA, 48 h, 37°C MHA, Tween 20, 48 h, 35°C MHB, Tween 80, 24 h, 37°C Cited MHB, Tween 80, 24 h, 37°C MHA, Tween 20, 48 h, 35°C NA, 48 h, 37°C NA, 48 h, 22°C MHA, Tween 20, 48 h, 35°C MHA, Tween 20, 48 h, 35°C TABLE 12.23 Inhibitory Data of Clary Sage Oil Obtained in the Dilution Test >20,000 1500–2000 >2000 >20,000 >15,000 100 7500 7500 250 100 >20,000 250 >20,000 >2000 >20,000 >2000 >20,000 >15,000 1500–2000 >20,000 15,000 >2000 >2000 >20,000 >20,000 MIC continued Peana et al. (1999) Hammer et al. (1999) Maruzzella (1963) Hammer et al. (1999) Yousefzadi et al. (2007) Maruzzella (1963) Yousefzadi et al. (2007) Yousefzadi et al. (2007) Maruzzella (1963) Maruzzella (1963) Maruzzella (1963) Hammer et al. (1999) Hammer et al. (1999) Maruzzella (1963) Hammer et al. (1999) Maruzzella (1963) Hammer et al. (1999) Yousefzadi et al. (2007) Peana et al. (1999) Yousefzadi et al. (2007) Hammer et al. (1999) Maruzzella (1963) Maruzzella (1963) Hammer et al. (1999) Hammer et al. (1999) Ref. In Vitro Antimicrobial Activities of Essential Oils 401 MO Class Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Aspergillus ochraceus Fusarium culmorum Fusarium oxysporum f.sp. dianthi Geotrichum candidum Gibberella fugikuroi Helminthosporium truicicum Microsporum gypseum Microsporum gypseum Penicillium italicum Phoma betae Pityrosporum ovale Sclerotina cepivorum Sclerotina sclerotiorum Trichophyton equinum Trichophyton mentagrophytes Trichophyton rubrum Trichophyton rubrum Candida albicans Candida albicans Candida albicans Alternaria alternata Aspergillus flavus Aspergillus niger Bac+ Bac+ Fungi Fungi Fungi Staphylococcus epidermidis Staphylococcus epidermidis Bac+ (continued) Staphylococcus aureus Microorganism TABLE 12.23 Conditions Cited MHA, Tween 20, 48 h, 35°C PDA, Tween 20, 4 d, 23°C PDA, Tween 20, 4 d, 23°C SDA, 7 d, 30°C SDA, 6–8 h, 20°C SDA, 7 d, 30°C SDA, 6–8 h, 20°C NA, 48 h, 37°C NA, 5 d, 22°C SDA, 7 d, 30°C SDA, 6–8 h, 20°C SDA, 6–8 h, 20°C NA, 5 d, 22°C NA, 5 d, 22°C NA, 5 d, 22°C PDA, Tween 20, 4 d, 23°C NA, 5 d, 22°C YES broth, 10 d YES broth, 10 d SDA, 6–8 h, 20°C SDA, 6–8 h, 20°C YES broth, 10 d MHB, Tween 80, 24 h, 37°C Cited MHB, Tween 80, 24 h, 37°C MIC >20,000 1500–2000 >2000 >2000 94% inh. 1000 1000 400, 30% inh. 500, 40% inh. 400, 43% inh. 500, 43% inh. >2000 >2000 400, 56% inh. 500, 56% inh. 500, 59% inh. >2000 >2000 - 69% inh. 10,000 72% inh. 2000 - >10,000 - 92% inh. 10,000 500, 62% inh. 500, 52% inh. 7500 1500–2000 7500 Ref. Peana et al. (1999) Hammer et al. (1999) Pitarokili et al. (2002) Pitarokili et al. (2002) Dikshit and Husain (1984) Dikshit et al. (1986) Dikshit and Husain (1984) Dikshit et al. (1986) Maruzzella (1963) Maruzzella (1963) Dikshit and Husain (1984) Dikshit et al. (1986) Dikshit et al. (1986) Maruzzella (1963) Maruzzella (1963) Maruzzella (1963) Pitarokili et al. (2002) Maruzzella (1963) Lis-Balchin et al. (1998) Lis-Balchin et al. (1998) Dikshit et al. (1986) Dikshit et al. (1986) Lis-Balchin et al. (1998) Yousefzadi et al. (2007) Peana et al. (1999) Yousefzadi et al. (2007) 402 Handbook of Essential Oils Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Streptococcus faecalis Colletotrichum gleosporoides Fusarium oxysporum Geotrichum candidum Gibberella fugikuroi Helminthosporium truicicum Phoma betae Pityrosporum ovale Pythium ultimum Rhizoctonia solani Candida albicans Annotation: Clary sage absolute tested (Maruzzella, 1963). Botrytis cinera Bac+ Bac+ Staphylococcus aureus Mycobacterium avium Staphylococcus aureus Bac+ Bac+ Bacillus subtilis var. aterrimus NA, 48 h, 37°C PDA, 3 d, 25°C PDA, 3 d, 25°C NA, 5 d, 22°C NA, 5 d, 22°C NA, 5 d, 22°C NA, 5 d, 22°C NA, 5 d, 22°C PDA, 3 d, 25°C PDA, 3 d, 25°C PDA, 3 d, 25°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 48 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C 500 μL in cover 1000 1000 500 μL in cover 500 μL in cover 500 μL in cover 500 μL in cover 500 μL in cover 1000 1000 1000 sd sd 500 μL in cover sd sd 500 μL in cover +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ NG +++ NG + +++ sd NA, 48 h, 37°C NA, 24 h, 37°C Bac- Bac+ Salmonella typhi Bacillus subtilis var. aterrimus +++ NG 500 μL in cover NA, 48 h, 37°C NA, 48 h, 37°C Bac- Bac- Escherichia coli Proteus vulgaris 500 μL in cover Activity Conditions MO Class Microorganism TABLE 12.24 Inhibitory Data of Clary Sage Oil Obtained in the Vapor Phase Test Maruzzella (1963) Lee et al. (2007) Lee et al. (2007) Maruzzella (1963) Maruzzella (1963) Maruzzella (1963) Maruzzella (1963) Maruzzella (1963) Lee et al. (2007) Lee et al. (2007) Lee et al. (2007) Maruzzella and Sicurella (1960) Maruzzella and Sicurella (1960) Maruzzella (1963) Maruzzella and Sicurella (1960) Maruzzella and Sicurella (1960) Maruzzella (1963) Maruzzella and Sicurella (1960) Maruzzella (1963) Maruzzella (1963) Ref. In Vitro Antimicrobial Activities of Essential Oils 403 Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Aerobacter aerogenes Aeromonas hydrophila Alcaligenes faecalis Beneckea natriegens Campylobacter jejuni Campylobacter jejuni Citrobacter freundii Citrobacter sp. Enterobacter aerogenes Enterobacter sp. Erwinia carotovora Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Flavobacterium suaveolens Klebsiella pneumoniae Klebsiella sp. Moraxella sp. Neisseria perflava Proteus sp. MO Class Acinetobacter calcoaceticus Microorganism Cited NA, 24 h, 37°C ISA, 48 h, 25°C Cited ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 30°C Cited Cited, 18 h, 37°C TGA, 18–24 h, 37°C TSA, 24 h, 35°C NA, 18 h, 37°C ISA, 48 h, 25°C Cited NA, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C Cited TSA, 24 h, 42°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C Conditions TABLE 12.25 Inhibitory Data of Clove Oil Obtained in the Agar Diffusion Test 15, 2500 —, sd 4 (h), 10,000 15, 2500 4 (h), 10,000 4 (h), 10,000 Drop, 5000 6, 15,000 6, 2500 9.5, 2000 4 (h), 25,000 6 (h), pure 4 (h), 10,000 15, 2500 11, sd —, sd 4 (h), 10,000 11, sd 4 (h), 10,000 11, sd 4 (h), 10,000 6, 15,000 4 (h), 25,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 —, sd 4 (h), 10,000 4 0 0 3 7 15 38 16.5 13.7 19 9.7 16 8.5 6 0 0 14.5 16 8.5 0 9 22.5 9 13 1 16.5 0 16 Inhibition Zone (mm) Pizsolitto et al. (1975) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Pizsolitto et al. (1975) Deans and Ritchie (1987) Deans and Ritchie (1987) Hili et al. (1997) Wannissorn et al. (2005) Janssen et al. (1986) Morris et al. (1979) Smith-Palmer et al. (1998) Yousef and Tawil (1980) Deans and Ritchie (1987) Pizsolitto et al. (1975) Prasad et al. (1986) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Prasad et al. (1986) Deans and Ritchie (1987) Prasad et al. (1986) Deans and Ritchie (1987) Wannissorn et al. (2005) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Ref. 404 Handbook of Essential Oils Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Proteus vulgaris Proteus vulgaris Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas sp. Salmonella enteritidis Salmonella enteritidis Salmonella pullorum Salmonella saintpaul Salmonella sp. Salmonella sp. B Salmonella typhimurium Salmonella weltevreden Serratia marcescens Serratia marcescens Serratia sp. Shigella sp. Yersinia enterocolitica Bacillus anthracis Bacillus mesentericus Bacillus saccharolyticus Bacillus sp. Bacillus stearothermophilus Bacillus subtilis NA, 24 h, 37°C NA, 24 h, 37°C Cited NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C Cited Cited ISA, 48 h, 25°C NA, 24 h, 37°C NA, 24 h, 37°C Cited NA, 24 h, 37°C Cited NA, 24 h, 37°C ISA, 48 h, 25°C Cited TSA, 24 h, 35°C NA, 24 h, 37°C NA, 24 h, 30°C ISA, 48 h, 25°C Cited, 18 h, 37°C Cited NA, 24 h, 37°C NA, 18 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C —, sd 11, sd 15, 2500 11, sd —, sd 11, sd 4 (h), 10,000 15, 2500 15, 2500 4 (h), 10,000 —, sd 11, sd 6, 15,000 11, sd 15, 2500 11, sd 4 (h), 10,000 6, 15,000 4 (h), 25,000 11, sd Drop, 5000 4 (h), 10,000 6, 2500 15, 2500 —, sd 6 (h), pure 4 (h), 10,000 —, sd 7 20 9 20 4 25 7.5 6 2 25 0 22 19.5 26 7 22 16 14.3 11.1 0 23 11 9 1 0 0 9.5 2 continued Maruzzella and Lichtenstein (1956) Prasad et al. (1986) Pizsolitto et al. (1975) Prasad et al. (1986) Maruzzella and Lichtenstein (1956) Prasad et al. (1986) Deans and Ritchie (1987) Pizsolitto et al. (1975) Pizsolitto et al. (1975) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Prasad et al. (1986) Wannissorn et al. (2005) Prasad et al. (1986) Pizsolitto et al. (1975) Prasad et al. (1986) Deans and Ritchie (1987) Wannissorn et al. (2005) Smith-Palmer et al. (1998) Prasad et al. (1986) Hili et al. (1997) Deans and Ritchie (1987) Janssen et al. (1986) Pizsolitto et al. (1975) Maruzzella and Lichtenstein (1956) Yousef and Tawil (1980) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) In Vitro Antimicrobial Activities of Essential Oils 405 Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Clostridium perfringens Clostridium sporogenes Corynebacterium sp. Lactobacillus casei Lactobacillus plantarum Lactobacillus plantarum Leuconostoc cremoris Listeria monocytogenes Listeria monocytogenes Micrococcus glutamicus Micrococcus luteus Mycobacterium phlei Sarcina lutea Sarcina lutea Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Bac+ Bac+ Brochotrix thermosphacta Staphylococcus aureus Bac+ Brevibacterium linens Bac+ Bac+ Bacillus thurengiensis Staphylococcus aureus Bac+ Bacillus subtilis Bac+ Bac+ Bacillus subtilis Bac+ Bac+ Bacillus subtilis Staphylococcus aureus Bac+ Bacillus subtilis Staphylococcus aureus MO Class (continued) Microorganism TABLE 12.25 NA, 18 h, 37°C TGA, 18–24 h, 37°C Cited, 18 h, 37°C TSA, 24 h, 35°C ISA, 48 h, 25°C Cited NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 18 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C TSA, 24 h, 35°C ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C TGA, 18–24 h, 37°C ISA, 48 h, 25°C Cited ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C NA, 18 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C Cited, 18 h, 37°C Conditions 6 (h), pure 9.5, 2000 6, 2500 4 (h), 25,000 4 (h), 10,000 15, 2500 —, sd 11, sd —, sd 6 (h), pure 4 (h), 10,000 11, sd 4 (h), 10,000 4 (h), 25,000 4 (h), 10,000 11, sd 4 (h), 10,000 11, sd 9.5, 2000 4 (h), 10,000 6, 15,000 4 (h), 10,000 4 (h), 10,000 11, sd 6 (h), pure 11, sd 4 (h), 10,000 6, 2500 14.3 14 11 8 7 5 0 32 4 20 9 44 20 8.4 13 50 11 22 15 0 20.5 5.5 14 19 22.5 22 12.5 16 Inhibition Zone (mm) Ref. Yousef and Tawil (1980) Morris et al. (1979) Janssen et al. (1986) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Pizsolitto et al. (1975) Maruzzella and Lichtenstein (1956) Prasad et al. (1986) Maruzzella and Lichtenstein (1956) Yousef and Tawil (1980) Deans and Ritchie (1987) Prasad et al. (1986) Lis-Balchin et al. (1998) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Prasad et al. (1986) Deans and Ritchie (1987) Prasad et al. (1986) Morris et al. (1979) Deans and Ritchie (1987) Wannissorn et al. (2005) Deans and Ritchie (1987) Deans and Ritchie (1987) Prasad et al. (1986) Yousef and Tawil (1980) Prasad et al. (1986) Deans and Ritchie (1987) Janssen et al. (1986) 406 Handbook of Essential Oils Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Staphylococcus aureus Staphylococcus epidermidis Staphylococcus sp. Streptococcus faecalis Streptococcus viridans Streptomyces venezuelae Alternaria porri Alternaria solani Aspergillus fumigatus Aspergillus niger Aspergillus niger Aspergillus niger Fusarium oxysporum f.sp. cicer Helminthosporium sativum Mucor mucedo Mucor sp. Nigrospora panici Penicillium chrysogenum Penicillium digitatum Rhizopus nigricans Rhizopus sp. Brettanomyces anomalus Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Candida krusei Candida lipolytica Candida tropicalis Cryptococcus neoformans Bac+ Staphylococcus aureus PDA, 72 h, 28°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C PDA, 48 h, 28°C SDA, 8 d, 30°C PDA, 72 h, 28°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C MPA, 4 d, 30°C SMA, 2–7 d, 20°C TGA, 18–24 h, 37°C SDA, 18 h, 30°C Cited, 18 h, 37°C NA, 24 h, 30°C SMA, 2–7 d, 20°C MPA, 4 d, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C Cited ISA, 48 h, 25°C NA, 24 h, 37°C Cited NA, 24 h, 37°C NA, 24 h, 30°C 5, 5000 sd sd sd 5, 5000 6 (h), pure 5, 5000 sd sd 6 (h), pure sd 6 (h), pure sd sd 6 (h), pure 5, 10% sol. sd sd 9.5, 2000 6 (h), pure 6, 2500 Drop, 5000 sd 5, 10% sol. sd sd sd sd 15, 2500 4 (h), 10,000 11, sd 15, 2500 11, sd Drop, 5000 40.5 9 10 10 28 34 12 7 7 20 8 47 9 7 20 18 3 19 20 28.3 40 5 27 3 8 9 1 8.5 26 10 30 21 continued Pawar and Thaker (2007) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Pawar and Thaker (2006) Yousef and Tawil (1980) Pawar and Thaker (2007) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Conner and Beuchat (1984) Maruzzella and Liguori (1958) Morris et al. (1979) Yousef and Tawil (1980) Janssen et al. (1986) Hili et al. (1997) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Pizsolitto et al. (1975) Deans and Ritchie (1987) Prasad et al. (1986) Pizsolitto et al. (1975) Prasad et al. (1986) Hili et al. (1997) In Vitro Antimicrobial Activities of Essential Oils 407 (continued) Cryptococcus rhodobenhani Debaryomyces hansenii Geotrichum candidum Hansenula anomala Kloeckera apiculata Kluyveromyces fragilis Lodderomyces elongisporus Metchnikowia pulcherrima Pichia membranaefaciens Rhodotorula rubra Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Schizosaccharomyces pombe Torula glabrata Torula utilis Microorganism TABLE 12.25 Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast MO Class SMA, 2–7 d, 20°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C SMA, 2–7 d, 20°C MPA, 4 d, 30°C NA, 24 h, 30°C NA, 24 h, 30°C MPA, 4 d, 30°C NA, 24 h, 30°C Conditions sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd sd 5, 10% sol. sd Drop, 5000 Drop, 5000 5, 10% sol. sd Drop, 5000 13 26 19 19 21 14 15 29 13 18 5 19 50 34 15 39 Inhibition Zone (mm) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Hili et al. (1997) Hili et al. (1997) Conner and Beuchat (1984) Hili et al. (1997) Ref. 408 Handbook of Essential Oils BacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBac- Brucella abortus Brucella melitensis Brucella suis Campylobacter jejuni Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli O157:H7 Klebsiella pneumoniae Klebsiella pneumoniae Proteus vulgaris Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas fluorescens Salmonella enteritidis Salmonella haddar Salmonella paratyphi A Salmonella paratyphi B Salmonella typhimurium Salmonella typhimurium Serratia marcescens MO Class Acinetobacter baumannii Microorganism Conditions NA, 1–3 d, 30°C MHA, Tween 20, 48 h, 35°C BHI, 48 h, 35°C Cited Cited LA, 18 h, 37°C TSB, 24 h, 35°C NA, 1–3 d, 30°C NB, Tween 20, 18 h, 37°C MHA, Tween 20, 48 h, 35°C MPB, DMSO, 40 h, 30°C Cited Cited MHA, Tween 20, 48 h, 35°C Cited BHI, 48 h, 35°C NB, Tween 20, 18 h, 37°C MHA, Tween 20, 48 h, 35°C NA, 1–3 d, 30°C TGB, 18–24 h, 37°C MPB, DMSO, 40 h, 30°C Cited TSB, 24 h, 35°C LA, 18 h, 37°C TSB, 24 h, 42°C Cited Cited Cited MHA, Tween 20, 48 h, 35°C TABLE 12.26 Inhibitory Data of Clove Oil Obtained in the Dilution Test >20,000 1500 >500 1000 500 500 400 1500 >20,000 50,000 >500 75% inh. 500 250 >500 2500 1000 50,000 2500 1250 1000 74% inh. 500 500 400 500–1000 500 15 500 250 2500 MIC Farag et al. (1989) Hammer et al. (1999) continued Oussalah et al. (2006) Okazaki and Oshima (1952) Okazaki and Oshima (1952) Remmal et al. (1993) Smith-Palmer et al. (1998) Farag et al. (1989) Yousef and Tawil (1980) Hammer et al. (1999) Hili et al. (1997) Okazaki and Oshima (1952) Okazaki and Oshima (1952) Hammer et al. (1999) Okazaki and Oshima (1952) Oussalah et al. (2006) Yousef and Tawil (1980) Hammer et al. (1999) Farag et al. (1989) Morris et al. (1979) Hili et al. (1997) Okazaki and Oshima (1952) Smith-Palmer et al. (1998) Remmal et al. (1993) Smith-Palmer et al. (1998) Okazaki and Oshima (1952) Okazaki and Oshima (1952) Okazaki and Oshima (1952) Hammer et al. (1999) Ref. In Vitro Antimicrobial Activities of Essential Oils 409 BacBac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Shigella dysenteriae II Bacillus megaterium Bacillus subtilis Bacillus subtilis Corynebacterium sp. Enterococcus faecalis Listeria monocytogenes Listeria monocytogenes Listeria monocytogenes Micrococcus sp. Mycobacterium phlei Mycobacterium phlei Mycobacterium tuberculosis Sarcina sp. Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus citreus Achorion gypseum Alternaria alternata Alternaria citri Aspergillus flavus BacBac- MO Class Shigella dysenteriae I (continued) Serratia marcescens Microorganism TABLE 12.26 Cited, 15 d RPMI, 1.5% EtOH, 7 d, 30°C PDA, 8 d, 22°C PDA, 8 h, 20°C, spore germ. inh. Cited NB, Tween 20, 18 h, 37°C MHA, Tween 20, 48 h, 35°C LA, 18 h, 37°C NA, 1–3 d, 30°C MPB, DMSO, 40 h, 30°C BHI, 48 h, 35°C TGB, 18–24 h, 37°C Cited TSB, 24 h, 35°C NA, 1–3 d, 30°C Cited NB, Tween 20, 18 h, 37°C NA, 1–3 d, 30°C NA, 1–3 d, 30°C BHI, 48 h, 35°C TSB, 24 h, 35°C TSB, 10 d, 4°C MHA, Tween 20, 48 h, 35°C TGB, 18–24 h, 37°C NB, Tween 20, 18 h, 37°C NA, 1–3 d, 30°C LA, 18 h, 37°C Cited Cited MHA, Tween 20, 48 h, 35°C Conditions 125 156–312 500 50–100 500 6400 2500 1000 750 83% inh. 500 500 500 500 400 500 125 1600 500 250 2000 300 200 5000 500 50,000 500 500–1000 500 500 2500 MIC Okazaki and Oshima (1952) Tullio et al. (2006) Arras and Usai (2001) Thompson (1986) Okazaki and Oshima (1952) Yousef and Tawil (1980) Hammer et al. (1999) Remmal et al. (1993) Farag et al. (1989) Hili et al. (1997) Oussalah et al. (2006) Morris et al. (1979) Okazaki and Oshima (1952) Smith-Palmer et al. (1998) Farag et al. (1989) Okazaki and Oshima (1952) Yousef and Tawil (1980) Farag et al. (1989) Farag et al. (1989) Oussalah et al. (2006) Smith-Palmer et al. (1998) Smith-Palmer et al. (1998) Hammer et al. (1999) Morris et al. (1979) Yousef and Tawil (1980) Farag et al. (1989) Remmal et al. (1993) Okazaki and Oshima (1952) Okazaki and Oshima (1952) Hammer et al. (1999) Ref. 410 Handbook of Essential Oils Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Aspergillus flavus Aspergillus flavus Aspergillus flavus var. columnaris Aspergillus fumigatus Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus ochraceus Aspergillus oryzae Aspergillus parasiticus Aspergillus parasiticus Botrytis cinera Cladosporium cladosporoides Colletotrichum musae Epidermophyton floccosum Epidermophyton floccosum Epidermophyton inguinale Fusarium culmorum Fusarium oxysporum Fusarium proliferatum Lasiodiplodia theobromae Microsporum canis Microsporum gypseum Mucor hiemalis Mucor mucedo Mucor racemosus Mucor racemosus f. racemosus Mucor sp. Mucor sp. Penicillium chrysogenum Penicillium chrysogenum Penicillium digitatum Penicillium frequentans Penicillium italicum Cited NB, Tween 20, 8 d, 30°C PDA, 8 d, 22°C RPMI, 1.5% EtOH, 7 d, 30°C PDA, 8 d, 22°C Cited, 15 d YES broth, 10 d RPMI, 1.5% EtOH, 7 d, 30°C SMKY, EtOH, 7 d, 28°C SMKY, EtOH, 7 d, 28°C RPMI, 1.5% EtOH, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C PDA, 5 d, 27°C PDA, 5 d, 27°C Cited PDA, 5 d, 27°C NB, Tween 20, 8 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C PDA, 5 d, 27°C RPMI, 1.5% EtOH, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C NB, Tween 20, 8 d, 30°C YES broth, 10 d YES broth, 10 d Cited PDA, 8 h, 20°C, spore germ. inh. PDA, 5 d, 27°C PDA, 8 d, 22°C RPMI, 1.5% EtOH, 7 d, 30°C SMKY, EtOH, 7 d, 28°C RPMI, 1.5% EtOH, 7 d, 30°C SA, Tween 80, 21 d, 20°C >10,000 250 1600 500 1250 500 <300 125 73% inh. 10,000 625 500 450 125–500 125–250 100 100 2 100 1600 100 2500 1250 5000 2500 3200 95% inh. 10,000 94% inh. 10,000 250 50–100 100 500 78–156 400 125 continued Okazaki and Oshima (1953) Yousef and Tawil (1980) Arras and Usai (2001) Tullio et al. (2006) Arras and Usai (2001) Okazaki and Oshima (1952) Lis-Balchin et al. (1998) Tullio et al. (2006) Ranasinghe et al. (2002) Ranasinghe et al. (2002) Tullio et al. (2006) Tullio et al. (2006) Thompson and Cannon (1986) Thompson and Cannon (1986) Okazaki and Oshima (1953) Thompson and Cannon (1986) Yousef and Tawil (1980) Tullio et al. (2006) Thompson and Cannon (1986) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Yousef and Tawil (1980) Lis-Balchin et al. (1998) Lis-Balchin et al. (1998) Okazaki and Oshima (1953) Thompson (1986) Thompson and Cannon (1986) Arras and Usai (2001) Tullio et al. (2006) Ranasinghe et al. (2002) Tullio et al. (2006) Janssen et al. (1988) In Vitro Antimicrobial Activities of Essential Oils 411 Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Rhizopus sp. Rhizopus stolonifer Rhizopus tritici Scopulariopsis brevicaulis Trichophyton asteroides Trichophyton interdigitale Trichophyton mentagrophytes Trichophyton mentagrophytes Trichophyton purpureum Trichophyton rubrum Trichophyton schoenleinii Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Saccharomyces cerevisiae Saccharomyces cerevisiae Schizosaccharomyces pombe Torula utilis MO Class Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi (continued) Penicillium lanosum Rhizopus 66-81-2 Rhizopus arrhizus Rhizopus chinensis Rhizopus circinans Rhizopus japonicus Rhizopus kazanensis Rhizopus oryzae Rhizopus pymacus Rhizopus sp. Microorganism TABLE 12.26 Conditions Cited, 15 d TGB, 18–24 h, 37°C MPB, DMSO, 40 h, 30°C MHA, Tween 20, 48 h, 35°C MHB, Tween 80, 48 h, 35 NB, Tween 20, 18 h, 37°C MPB, DMSO, 40 h, 30°C NA, 1–3 d, 30°C MPB, DMSO, 40 h, 30°C MPB, DMSO, 40 h, 30°C Cited, 15 d SA, Tween 80, 21 d, 20°C NB, Tween 20, 8 d, 30°C PDA, 5 d, 27°C PDA, 5 d, 27°C RPMI, 1.5% EtOH, 7 d, 30°C Cited, 15 d Cited, 15 d RPMI, 1.5% EtOH, 7 d, 30°C SA, Tween 80, 21 d, 20°C RPMI, 1.5% EtOH, 7 d, 30°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C RPMI, 1.5% EtOH, 7 d, 30°C MIC <300 125 500 61% inh. 500 1200 1200 50,000 99% inh. 500 750 94% inh. 500 95% inh. 500 <300 125 >10,000 50,000 100 100 10,000 125 125 250–500 5000 100 100 100 100 100 100 100 100 Ref. Okazaki and Oshima (1952) Morris et al. (1979) Hili et al. (1997) Hammer et al. (1999) Hammer et al. (1998) Yousef and Tawil (1980) Hili et al. (1997) Farag et al. (1989) Hili et al. (1997) Hili et al. (1997) Okazaki and Oshima (1952) Janssen et al. (1988) Yousef and Tawil (1980) Thompson and Cannon (1986) Thompson and Cannon (1986) Tullio et al. (2006) Okazaki and Oshima (1952) Okazaki and Oshima (1952) Tullio et al. (2006) Janssen et al. (1988) Tullio et al. (2006) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Tullio et al. (2006) 412 Handbook of Essential Oils 413 In Vitro Antimicrobial Activities of Essential Oils TABLE 12.27 Inhibitory Data of Clove Oil Obtained in the Vapor Phase Test Microorganism MO Class Escherichia coli Bac- NA, 24 h, 37°C ~20,000 NG 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 NG Kellner and Kober (1954) Salmonella typhi Bac- NA, 24 h, 37°C ~20,000 sd ++ Bacillus megaterium Bac+ NA, 24 h, 37°C NG Bacillus subtilis var. aterrimus Corynebacterium diphtheriae Mycobacterium avium Bac+ NA, 24 h, 37°C ~20,000 sd Maruzzella and Sicurella (1960) Kellner and Kober (1954) Bac+ NA, 24 h, 37°C ~20,000 NG Bac+ NA, 24 h, 37°C sd + Staphylococcus aureus Bac+ NA, 24 h, 37°C Bac+ NA, 24 h, 37°C ~20,000 sd NG Staphylococcus aureus Streptococcus faecalis ~20,000 sd NG Conditions Activity Bac+ NA, 24 h, 37°C Streptococcus faecalis Bac+ NA, 24 h, 37°C Streptococcus pyogenes Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi RPMI, 7 d, 30°C WFA, 42 d, 25°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C WFA, 42 d, 25°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C ~20,000 MICair Disk, 50,000 MICair MICair Disk, 50,000 MICair MICair Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi WFA, 42 d, 25°C WFA, 42 d, 25°C WFA, 42 d, 25°C WFA, 42 d, 25°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C WFA, 42 d, 25°C RPMI, 7 d, 30°C Penicillium lanosum Fungi Rhizopus sp. Scopulariopsis brevicaulis Trichophyton mentagrophytes Candida albicans Alternaria alternata Aspergillus flavus Aspergillus flavus Aspergillus fumigatus Aspergillus niger Aspergillus niger Cladosporium cladosporoides Eurotium amstelodami Eurotium herbarum Eurotium repens Eurotium rubrum Fusarium oxysporum Microsporum canis Microsporum gypseum Mucor sp. Penicillium corylophilum Penicillium frequentans +++ +++ Ref. Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) NG Maruzzella and Sicurella (1960) Kellner and Kober (1954) 312–625 NG 625–1250 625–1250 NG 625–1250 78–156 Tullio et al. (2006) Guynot et al. (2003) Tullio et al. (2006) Tullio et al. (2006) Guynot et al. (2003) Tullio et al. (2006) Tullio et al. (2006) Disk, 50,000 Disk, 50,000 Disk, 50,000 Disk, 50,000 MICair MICair MICair MICair Disk, 50,000 MICair NG NG NG NG 312 312–1250 156–312 625 NG Guynot et al. (2003) Guynot et al. (2003) Guynot et al. (2003) Guynot et al. (2003) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Guynot et al. (2003) Tullio et al. (2006) RPMI, 7 d, 30°C MICair RPMI, 7 d, 30°C RPMI, 7 d, 30°C MICair MICair >10,000 125 312–1250 Tullio et al. (2006) Fungi Fungi Fungi RPMI, 7 d, 30°C MICair 78–156 Tullio et al. (2006) Yeast NA, 24 h, 37°C ~20,000 NG Kellner and Kober (1954) NA, 24 h, 37°C +++ >10,000 Tullio et al. (2006) Tullio et al. (2006) BacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBac- Aerobacter aerogenes Aeromonas hydrophila Agrobacterium tumefaciens Alcaligenes faecalis Beneckea natriegens Burkholderia gladioli pv. agaricicola Citrobacter freundii Enterobacter aerogenes Enterobacter aerogenes Erwinia carotovora Erwinia carotovora subsp. atroseptica Erwinia carotovora subsp. carotovora Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Flavobacterium suaveolens Klebsiella pneumoniae Moraxella sp. Neisseria perflava Proteus vulgaris Proteus vulgaris Pseudomonas aeruginosa MO Class Acinetobacter calcoaceticus Microorganism NA, 18 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C WA, 48 h, 25°C MHA, 48 h, 27°C NA, 18 h, 37°C NA, 24 h, 30°C ISA, 48 h, 25°C Cited, 18 h, 37°C TGA, 18–24 h, 37°C NA, 24 h, 37°C WA, 48 h, 25°C WA, 48 h, 25°C ISA, 48 h, 25°C MHA, 48 h, 27°C ISA, 48 h, 25°C ISA, 48 h, 25°C WA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C WA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C Conditions TABLE 12.28 Inhibitory Data of Coriander Oil Obtained in the Agar Diffusion Test 6 (h), pure 4 (h), 10,000 —, sd —, sd 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 6, 8000 6, 15,000 6 (h), pure Drop, 5000 4 (h), 10,000 6, 2500 9.5, 2000 —, sd 6, 8000 6, 8000 4 (h), 10,000 6, 15,000 4 (h), 10,000 4 (h), 10,000 6, 8000 4 (h), 10,000 4 (h), 10,000 6, 8000 4 (h), 10,000 —, sd 4 (h), 10,000 0 6 0 0 0 8 13 MIA 870 20 10.5 13 7.6 10 0 0 MIA 435 MIA 435 8 12 7.5 8.5 MIA 3480 0 6 MIA 435 8.5 0 0 Inhibition Zone (mm) Yousef and Tawil (1980) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Cantore et al. (2004) Ertürk et al. (2006) Yousef and Tawil (1980) Hili et al. (1997) Deans and Ritchie (1987) Janssen et al. (1986) Morris et al. (1979) Maruzzella and Lichtenstein (1956) Cantore et al. (2004) Cantore et al. (2004) Deans and Ritchie (1987) Ertürk et al. (2006) Deans and Ritchie (1987) Deans and Ritchie (1987) Cantore et al. (2004) Deans and Ritchie (1987) Deans and Ritchie (1987) Cantore et al. (2004) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Ref. 414 Handbook of Essential Oils 6, 8000 6, 8000 6, 8000 WA, 48 h, 25°C WA, 48 h, 25°C WA, 48 h, 25°C BacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBac- Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas agarici Pseudomonas chichorii Pseudomonas corrugate Pseudomonas reactans Pseudomonas syringae pv. apata Pseudomonas syringae pv. atrofaciens Pseudomonas syringae pv. glycinea Pseudomonas syringae pv. lachrymans Pseudomonas syringae pv. maculicola Pseudomonas syringae pv. phaseolicola Pseudomonas syringae pv. pisi Pseudomonas syringae pv. syringae Pseudomonas syringae pv. tomato Pseudomonas tolaasii Pseudomonas viridiflava Salmonella pullorum Salmonella typhimurium Serratia marcescens Serratia marcescens Xanthomonas campestris pv. campestris Xanthomonas campestris pv. phaesoli var. fuscans Xanthomonas campestris pv. phaesoli var. phaesoli Xanthomonas campestris pv. vesicatoria Yersinia enterocolitica ISA, 48 h, 25°C WA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C MHA, 48 h, 27°C ISA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C KBA, 48 h, 25°C MHA, 48 h, 27°C Cited, 18 h, 37°C NA, 24 h, 30°C 4 (h), 10,000 6, 8000 4 (h), 10,000 —, sd 6, 15,000 4 (h), 10,000 6, 8000 6, 8000 6, 8000 6, 8000 6, 8000 6, 8000 6, 8000 6, 8000 6, 8000 6, 8000 6, 8000 6, 8000 6, 8000 6, 8000 6, 8000 6, 15,000 6, 2500 Drop, 5000 4 (h), 10,000 —, sd Pseudomonas aeruginosa ISA, 48 h, 25°C Bac- Pseudomonas aeruginosa NA, 24 h, 37°C Bac- Pseudomonas aeruginosa 7 MIA 217 MIA 217 MIA 217 MIA 217 7.5 3 7 MIA >6960 11 MIA >6960 MIA 3480 MIA 3480 MIA 2610 MIA 2610 MIA >6960 MIA 870 MIA 870 MIA 6960 MIA >6960 MIA 3480 MIA 3480 MIA 6960 MIA 3480 12 9 6 0 0 Deans and Ritchie (1987) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Deans and Ritchie (1987) continued Maruzzella and Lichtenstein (1956) Ertürk et al. (2006) Deans and Ritchie (1987) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Cantore et al. (2004) Ertürk et al. (2006) Janssen et al. (1986) Hili et al. (1997) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) In Vitro Antimicrobial Activities of Essential Oils 415 6, 8000 6, 8000 4 (h), 10,000 6, 8000 4 (h), 10,000 WA, 48 h, 25°C WA, 48 h, 25°C ISA, 48 h, 25°C WA, 48 h, 25°C ISA, 48 h, 25°C Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bacillus subtilis Bacillus subtilis Brevibacterium linens Brochotrix thermosphacta Clavibacter michiganensis subsp. michiganensis Clavibacter michiganensis subsp. sepedonicus Clostridium sporogenes Corynebacterium sp. Curtobacterium flaccunfaciens pv. betae Curtobacterium flaccunfaciens pv. flaccunfaciens Lactobacillus plantarum Leuconostoc cremoris Micrococcus luteus Mycobacterium phlei Rhodococcus fascians Sarcina lutea Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Cited, 18 h, 37°C NA, 18 h, 37°C NA, 24 h, 30°C ISA, 48 h, 25°C TGA, 18–24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C WA, 48 h, 25°C NA, 18 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C WA, 48 h, 25°C TGA, 18–24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 18 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C 6, 2500 6 (h), pure Drop, 5000 4 (h), 10,000 9.5, 2000 —, sd —, sd 6, 8000 6 (h), pure 4 (h), 10,000 4 (h), 10,000 6, 8000 9.5, 2000 4 (h), 10,000 4 (h), 10,000 6 (h), pure 4 (h), 10,000 —, sd 6, 2500 —, sd Bacillus subtilis Cited, 18 h, 37°C NA, 24 h, 37°C Bac+ 6, 8000 Bacillus subtilis WA, 48 h, 25°C Conditions Bacillus mesentericus MO Class Bac+ (continued) Bacillus megaterium Microorganism TABLE 12.28 11.3 14 12 5 0 0 0 MIA 435 19 7 7.5 9 MIA 435 MIA 632 0 0 MIA 435 MIA 374 14 9 27 7.5 11 7.3 4 MIA 435 Inhibition Zone (mm) Ref. Janssen et al. (1986) Yousef and Tawil (1980) Hili et al. (1997) Deans and Ritchie (1987) Morris et al. (1979) Maruzzella and Lichtenstein (1956) Maruzzella and Lichtenstein (1956) Cantore et al. (2004) Yousef and Tawil (1980) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Cantore et al. (2004) Cantore et al. (2004) Morris et al. (1979) Deans and Ritchie (1987) Cantore et al. (2004) Cantore et al. (2004) Deans and Ritchie (1987) Deans and Ritchie (1987) Yousef and Tawil (1980) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Janssen et al. (1986) Maruzzella and Lichtenstein (1956) Cantore et al. (2004) 416 Handbook of Essential Oils Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Staphylococcus epidermidis Streptococcus faecalis Streptomyces venezuelae Alternaria porri Alternaria solani Alternaria sp. Aspergillus candidus Aspergillus flavus Aspergillus fumigatus Aspergillus fumigatus Aspergillus nidulans Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus niger Cladosporium herbarum Cunninghamella echinulata Fusarium oxysporum Fusarium oxysporum f.sp. cicer Helminthosporium sacchari Helminthosporium sativum Microsporum gypseum Mucor mucedo Mucor mucedo Mucor sp. Nigrospora panici Penicillium chrysogenum Penicillium digitatum Penicillium digitatum Rhizopus nigricans Rhizopus nigricans Bac+ Staphylococcus aureus PDA, 72 h, 28°C SMA, 2–7 d, 20°C PDA, 18 h, 37°C PDA, 18 h, 37°C PDA, 18 h, 37°C PDA, 18 h, 37°C SMA, 2–7 d, 20°C PDA, 18 h, 37°C PDA, 48 h, 28°C MHA, 48 h, 27°C PDA, 18 h, 37°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C PDA, 18 h, 37°C PDA, 18 h, 37°C PDA, 18 h, 37°C PDA, 72 h, 28°C PDA, 18 h, 37°C SMA, 2–7 d, 20°C PDA, 18 h, 37°C PDA, 18 h, 37°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SDA, 8 d, 30°°C PDA, 18 h, 37°C SMA, 2–7 d, 20°C PDA, 18 h, 37°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C ISA, 48 h, 25°C MHA, 48 h, 27°C MHA, 48 h, 27°C 5, 5000 sd 6, sd 6, sd 6, sd 6, sd sd 6, sd 5, 5000 6, 15,000 6, sd sd 6 (h), pure 6, sd 6, sd 6, sd 5, 5000 6, sd sd 6, sd 6, sd sd 6 (h), pure sd 6 (h), pure 6, sd sd 6, sd sd sd 4 (h), 10,000 6, 15,000 6, 15,000 12 18 9.5 0 0 12 21 0 0 0 16 21 60 21.5 20 0 11.5 13 11 8 12 25 21 28 60 12 14 11 13 11 0 10 18 continued Pawar and Thaker (2007) Maruzzella and Liguori (1958) Sharma and Singh (1979) Sharma and Singh (1979) Sharma and Singh (1979) Sharma and Singh (1979) Maruzzella and Liguori (1958) Sharma and Singh (1979) Pawar and Thaker (2006) Ertürk et al. (2006) Sharma and Singh (1979) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Sharma and Singh (1979) Sharma and Singh (1979) Sharma and Singh (1979) Pawar and Thaker (2007) Sharma and Singh (1979) Maruzzella and Liguori (1958) Sharma and Singh (1979) Sharma and Singh (1979) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Sharma and Singh (1979) Maruzzella and Liguori (1958) Sharma and Singh (1979) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Deans and Ritchie (1987) Ertürk et al. (2006) Ertürk et al. (2006) In Vitro Antimicrobial Activities of Essential Oils 417 (continued) Rhizopus sp. Trichophyton rubrum Trichothecium roseum Brettanomyces anomalus Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Candida krusei Candida lipolytica Candida tropicalis Cryptococcus neoformans Cryptococcus rhodobenhani Debaryomyces hansenii Geotrichum candidum Hansenula anomala Kloeckera apiculata Kluyveromyces fragilis Lodderomyces elongisporus Metchnikowia pulcherrima Pichia membranaefaciens Rhodotorula rubra Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Schizosaccharomyces pombe Torula glabrata Torula utilis Microorganism TABLE 12.28 Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast MO Class SDA, 8 d, 30°C PDA, 18 h, 37°C PDA, 18 h, 37°C MPA, 4 d, 30°C TGA, 18–24 h, 37°C SMA, 2–7 d, 20°C MHA, 48 h, 27°C Cited, 18 h, 37°C SDA, 18 h, 30°C NA, 24 h, 30°C SMA, 2–7 d, 20°C MPA, 4 d, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C SMA, 2–7 d, 20°C NA, 24 h, 30°C NA, 24 h, 30°C MPA, 4 d, 30°C NA, 24 h, 30°C Conditions 6 (h), pure 6, sd 6, sd 5, 10% sol. sd 9.5, 2000 sd 6, 15,000 6, 2500 6 (h), pure Drop, 5000 sd 5, 10% sol. sd sd sd sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd sd Drop, 5000 Drop, 5000 5, 10% sol. sd Drop, 5000 19 10 8 0 0 5 10 11 28 29 0 0 9 5 20 0 0 12 8 0 0 0 0 0 0 7 32 33 7 37 Inhibition Zone (mm) Yousef and Tawil (1980) Sharma and Singh (1979) Sharma and Singh (1979) Conner and Beuchat (1984) Morris et al. (1979) Maruzzella and Liguori (1958) Ertürk et al. (2006) Janssen et al. (1986) Yousef and Tawil (1980) Hili et al. (1997) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Maruzzella and Liguori (1958) Hili et al. (1997) Hili et al. (1997) Conner and Beuchat (1984) Hili et al. (1997) Ref. 418 Handbook of Essential Oils 419 In Vitro Antimicrobial Activities of Essential Oils 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 Bac- MHB, 24 h, 37°C >1000 2150 Morris et al. (1979) Escherichia coli Escherichia coli Bac- NB, Tween 20, 18 h, 37°C 3200 Yousef and Tawil (1980) Escherichia coli O157:H7 Ertürk et al. (2006) Bac- BHI, 48 h, 35°C 2000 Oussalah et al. (2006) Pseudomonas aeruginosa Bac- MPB, DMSO, 40 h, 30°C 74% inh. 500 Hili et al. (1997) Pseudomonas aeruginosa Bac- MHB, 24 h, 37°C 4350 Ertürk et al. (2006) Bac- NB, Tween 20, 18 h, 37°C 12,500 Yousef and Tawil (1980) Pseudomonas aeruginosa Salmonella typhimurium Bac- BHI, 48 h, 35°C 2000 Oussalah et al. (2006) Salmonella typhimurium Bac- MHB, 24 h, 37°C 17,260 Ertürk et al. (2006) Bacillus subtilis Bac+ NB, Tween 20, 18 h, 37°C 800 Yousef and Tawil (1980) Corynebacterium sp. Bac+ TGB, 18–24 h, 37°C 500 Morris et al. (1979) Listeria monocytogenes Bac+ BHI, 48 h, 35°C Bac+ NB, Tween 20, 18 h, 37°C >8000 200 Oussalah et al. (2006) Mycobacterium phlei Staphylococcus aureus Bac+ MPB, DMSO, 40 h, 30°C 44% inh. 500 Hili et al. (1997) Yousef and Tawil (1980) Staphylococcus aureus Bac+ TGB, 18–24 h, 37°C 1000 Morris et al. (1979) Staphylococcus aureus Bac+ MHB, 24 h, 37°C 1070 Ertürk et al. (2006) Staphylococcus aureus Bac+ BHI, 48 h, 35°C 2000 Oussalah et al. (2006) Staphylococcus aureus Bac+ MHB, Tween 80, 24 h, 37°C 3120 Bastide et al. (1987) Staphylococcus aureus Bac+ NB, Tween 20, 18 h, 37°C 3200 Yousef and Tawil (1980) Staphylococcus epidermidis Aspergillus flavus Bac+ MHB, 24 h, 37°C 8630 Ertürk et al. (2006) Fungi 50–100 Thompson (1986) Aspergillus flavus Fungi PDA, 8 h, 20°C, spore germ. inh. PDA, 5 d, 27°C Fungi Fungi Fungi CDA, cited NB, Tween 20, 8 d, 30°C MHB, 24 h, 37°C >1000 3000 3200 Thompson and Cannon (1986) Aspergillus flavus Aspergillus niger Aspergillus niger Aspergillus parasiticus Fungi Aspergillus parasiticus Fungi PDA, 8 h, 20°C, spore germ. inh. PDA, 5 d, 27°C >1000 Thompson and Cannon (1986) Epidermophyton floccosum Mucor hiemalis Fungi SA, Tween 80, 21 d, 20°C <300 Janssen et al. (1988) 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 f. racemosus Mucor sp. Penicillium chrysogenum Rhizopus 66-81-2 Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon (1986) Fungi Fungi Fungi NB, Tween 20, 8 d, 30°C NB, Tween 20, 8 d, 30°C PDA, 5 d, 27°C 800 800 >1000 Yousef and Tawil (1980) Yousef and Tawil (1980) 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) >20,000 50–100 Dubey et al. (1990) Yousef and Tawil (1980) Ertürk et al. (2006) Thompson (1986) continued 420 Handbook of Essential Oils TABLE 12.29 (continued) Inhibitory Data of Coriander Oil Obtained in the Dilution Test Microorganism MO Class 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 Thompson and Cannon (1986) Rhizopus sp. Rhizopus stolonifer Fungi Fungi NB, Tween 20, 8 d, 30°C PDA, 5 d, 27°C >1000 1600 Rhizopus tritici Fungi PDA, 5 d, 27°C Trichophyton mentagrophytes Trichophyton rubrum Fungi SA, Tween 80, 21 d, 20°C Fungi SA, Tween 80, 21 d, 20°C Candida albicans Candida albicans Candida albicans Yeast Yeast Yeast MPB, DMSO, 40 h, 30°C NB, Tween 20, 18 h, 37°C TGB, 18–24 h, 37°C Candida albicans Candida albicans Saccharomyces cerevisiae Schizosaccharomyces pombe Torula utilis Yeast Yeast Yeast Yeast MHB, Tween 80, 48 h, 35°C MHB, 24 h, 37°C MPB, DMSO, 40 h, 30°C MPB, DMSO, 40 h, 30°C >1000 2500 4310 68% inh. 500 18% inh. 500 Yeast MPB, DMSO, 40 h, 30°C 87% inh. 500 Conditions MIC Ref. Yousef and Tawil (1980) Thompson and Cannon (1986) >1000 >1000 300,625 Thompson and Cannon (1986) <300 75% inh. 500 800 Janssen et al. (1988) Janssen et al. (1988) Hili et al. (1997) Yousef and Tawil (1980) Morris et al. (1979) Hammer et al. (1998) Ertürk et al. (2006) Hili et al. (1997) Hili et al. (1997) Hili et al. (1997) TABLE 12.30 Inhibitory Data of Coriander Oil Obtained in the Vapor Phase Test Microorganism MO Class Escherichia coli Bac- NA, 24 h, 37°C Conditions Escherichia coli Bac- Haemophilus influenzae Bac- Neisseria sp. Bac- Salmonella typhi Activity NG Kellner and Kober (1954) BLA, 18 h, 37°C 250 Inouye et al. (2001) MHA, 18 h, 37°C MICair 12.5 Inouye et al. (2001) NA, 24 h, 37°C ~20,000 NG Kellner and Kober (1954) ~20,000 sd NG Kellner and Kober (1954) ++ NG Maruzzella and Sicurella (1960) Bac- NA, 24 h, 37°C Salmonella typhi Bac- NA, 24 h, 37°C Bacillus megaterium Bac+ NA, 24 h, 37°C Bacillus subtilis var. aterrimus Bac+ NA, 24 h, 37°C Corynebacterium diphtheriae Bac+ NA, 24 h, 37°C Mycobacterium avium Bac+ NA, 24 h, 37°C ~20,000 sd ~20,000 sd Staphylococcus aureus Bac+ NA, 24 h, 37°C Staphylococcus aureus Bac+ NA, 24 h, 37°C ~20,000 sd Staphylococcus aureus Bac+ MHA, 18 h, 37°C MICair Streptococcus faecalis Bac+ NA, 24 h, 37°C Streptococcus faecalis Bac+ NA, 24 h, 37°C ~20,000 sd Streptococcus pneumoniae Bac+ MHA, 18 h, 37°C MICair Streptococcus pyogenes Bac+ NA, 24 h, 37°C Streptococcus pyogenes Bac+ Yeast Candida albicans Ref. ~20,000 MICair Kellner and Kober (1954) Maruzzella and Sicurella (1960) +++ NG Kellner and Kober (1954) NG Maruzzella and Sicurella (1960) NG Kellner and Kober (1954) +++ 50 Maruzzella and Sicurella (1960) NG Kellner and Kober (1954) +++ 25 Maruzzella and Sicurella (1960) NG Kellner and Kober (1954) MHA, 18 h, 37°C ~20,000 MICair 25 Inouye et al. (2001) NA, 24 h, 37°C ~20,000 NG Kellner and Kober (1954) Inouye et al. (2001) Inouye et al. (2001) 421 In Vitro Antimicrobial Activities of Essential Oils TABLE 12.31 Inhibitory Data of Dwarf Pine Oil Obtained in the Agar Diffusion Test Inhibition Zone (mm) Ref. —, sd 2 Maruzzella and Lichtenstein (1956) —, sd 0 Maruzzella and Lichtenstein (1956) 6, 2500 11 NA, 24 h, 37°C —, sd 0 Maruzzella and Lichtenstein (1956) Bac- NA, 24 h, 37°C —, sd 5 Maruzzella and Lichtenstein (1956) Bac- NA, 24 h, 37°C —, sd 0 Maruzzella and Lichtenstein (1956) Pseudomonas aeruginosa Bac- Cited, 18 h, 37°C Serratia marcescens Bac- NA, 24 h, 37°C Bacillus mesentericus Bac+ Bacillus subtilis Bac+ Bacillus subtilis Bac+ Cited, 18 h, 37°C Sarcina lutea Bac+ NA, 24 h, 37°C Staphylococcus aureus Microorganism MO Class Conditions Aerobacter aerogenes Bac- NA, 24 h, 37°C Escherichia coli Bac- NA, 24 h, 37°C Escherichia coli Bac- Cited, 18 h, 37°C Neisseria perflava Bac- Proteus vulgaris Pseudomonas aeruginosa 6, 2500 Janssen et al. (1986) 8.3 Janssen et al. (1986) —, sd 0 Maruzzella and Lichtenstein (1956) NA, 24 h, 37°C —, sd 0 Maruzzella and Lichtenstein (1956) NA, 24 h, 37°C —, sd 5 Maruzzella and Lichtenstein (1956) 6, 2500 16.7 —, sd 0 Maruzzella and Lichtenstein (1956) —, sd 3 Maruzzella and Lichtenstein (1956) 8.7 Janssen et al. (1986) Janssen et al. (1986) Bac+ NA, 24 h, 37°C Staphylococcus aureus Bac+ Cited, 18 h, 37°C 6, 2500 Streptomyces venezuelae Bac+ Fungi Fungi Fungi Fungi SMA, 2–7 d, 20°C sd 2 Maruzzella and Liguori (1958) SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C sd sd sd sd 0 0 0 0 Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C Cited, 18 h, 37°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C sd sd sd sd sd 6, 2500 sd sd sd 0 0 0 0 0 32.3 0 0 0 Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Janssen et al. (1986) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Yeast SMA, 2–7 d, 20°C sd 0 Maruzzella and Liguori (1958) Yeast SMA, 2–7 d, 20°C sd 0 Maruzzella and Liguori (1958) Alternaria solani Aspergillus fumigatus Aspergillus niger Helminthosporium sativum Mucor mucedo Nigrospora panici Penicillium digitatum Rhizopus nigricans Candida albicans Candida albicans Candida krusei Candida tropicalis Cryptococcus neoformans Cryptococcus rhodobenhani Saccharomyces cerevisiae TABLE 12.32 Inhibitory Data of Dwarf Pine Oil Obtained in the Dilution Test Microorganism MO Class Conditions MIC Ref. Epidermophyton floccosum 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) BacBacBacBac+ Bac+ Bac+ Bac+ Bac+ Yeast Escherichia coli Neisseria sp. Salmonella typhi Bacillus megaterium Corynebacterium diphtheriae Staphylococcus aureus Streptococcus faecalis Streptococcus pyogenes Candida albicans MO Class Microorganism NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Conditions ~20,000 ~20,000 ~20,000 ~20,000 ~20,000 ~20,000 ~20,000 ~20,000 ~20,000 TABLE 12.33 Inhibitory Data of Dwarf Pine Oil Obtained in the Vapor Phase Test ++ NG + NG NG +++ NG +++ NG Activity Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Ref. 422 Handbook of Essential Oils 4 (h), 10,000 ISA, 48 h, 25°C 5 (h), -30,000 4 (h), 10,000 NA, 18 h, 37°C Bac- Bac- Pseudomonas aeruginosa Bac- Proteus sp. Bac- Bac- Neisseria perflava Proteus vulgaris Bac- Moraxella sp. Proteus vulgaris Bac- Bac- Klebsiella pneumoniae Klebsiella sp. Bac- Bac- Escherichia coli Flavobacterium suaveolens 6 (h), pure NA, 18 h, 37°C Bac- Bac- Escherichia coli Escherichia coli Cited NA, 24 h, 37°C ISA, 48 h, 25°C Cited NA, 24 h, 37°C ISA, 48 h, 25°C Cited ISA, 48 h, 25°C ISA, 48 h, 25°C Cited TSA, 24 h, 35°C 15, 2500 —, sd 4 (h), 10,000 15, 2500 —, sd 4 (h), 10,000 15, 2500 4 (h), 10,000 15, 2500 4 (h), 25,000 6, 2500 Bac- Escherichia coli Cited, 18 h, 37°C Bac- Bac- 6, 15,000 —, sd 4 (h), 10,000 9.5, 2000 4 (h), 10,000 6, 15,000 4 (h), 10,000 4 (h), 10,000 Escherichia coli MHA, 24 h, 30°C NA, 24 h, 37°C ISA, 48 h, 25°C TGA, 18–24 h, 37°C ISA, 48 h, 25°C MHA, 24 h, 30°C ISA, 48 h, 25°C ISA, 48 h, 25°C Escherichia coli Bac- Bac- Escherichia coli Bac- Escherichia coli Escherichia coli Bac- Bac- Enterobacter aerogenes Erwinia carotovora Bac- Bac- Citrobacter freundii 4 (h), 10,000 4 (h), 25,000 ISA, 48 h, 25°C TSA, 24 h, 42°C Bac- Bac- Beneckea natriegens Campylobacter jejuni Enterobacter aerogenes 4 (h), 10,000 ISA, 48 h, 25°C Bac- Bac- Aeromonas hydrophila 4 (h), 10,000 —, sd Alcaligenes faecalis ISA, 48 h, 25°C NA, 24 h, 37°C Bac- Bac- Conditions Acinetobacter calcoaceticus MO Class Aerobacter aerogenes Microorganism TABLE 12.34 Inhibitory Data of Eucalyptus Oil Obtained in the Agar Diffusion Test 0 17 0 10 4 0 1 0 10 22 20 16 10.3 8 6 3 0 0 6 7 0 6 8.3 0 0 7.5 0 7 Inhibition Zone (mm) Pizsolitto et al. (1975) continued Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Pizsolitto et al. (1975) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Pizsolitto et al. (1975) Deans and Ritchie (1987) Deans and Ritchie (1987) Schelz et al. (2006) Yousef and Tawil (1980) Pizsolitto et al. (1975) Smith-Palmer et al. (1998) Janssen et al. (1986) Rossi et al. (2007) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Morris et al. (1979) Deans and Ritchie (1987) Rossi et al. (2007) Deans and Ritchie (1987) Deans and Ritchie (1987) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Ref. In Vitro Antimicrobial Activities of Essential Oils 423 6, 15,000 4 (h), 10,000 15, 2500 ISA, 48 h, 25°C Cited Bac- Bac- Serratia marcescens Bac+ Sarcina lutea 4 (h), 10,000 ISA, 48 h, 25°C Bac+ Bac+ Bac+ Listeria monocytogenes Listeria monocytogenes Bac+ Bac+ Leuconostoc cremoris Micrococcus luteus 4 (h), 25,000 TSA, 24 h, 35°C Bac+ Mycobacterium phlei 9.5, 2000 TGA, 18–24 h, 37°C Bac+ Corynebacterium sp. Lactobacillus plantarum NA, 24 h, 37°C NA, 18 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C —, sd 6 (h), pure 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 Bac+ Clostridium sporogenes ISA, 48 h, 25°C ISA, 48 h, 25°C Bac+ Bac+ 4 (h), 10,000 —, sd 6 (h), pure 6, 2500 4 (h), 10,000 4 (h), 10,000 Brochotrix thermosphacta ISA, 48 h, 25°C NA, 24 h, 37°C NA, 18 h, 37°C Cited, 18 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C Brevibacterium linens Bac+ Bac+ Bacillus subtilis Bac+ Bacillus subtilis Bacillus subtilis 15, 2500 Cited Bac+ Bac+ Bacillus sp. Bacillus subtilis —, sd NA, 24 h, 37°C Bac- Bac+ Yersinia enterocolitica 15, 2500 Bacillus mesentericus Cited Bac- Bac- Serratia sp. —, sd Shigella sp. NA, 24 h, 37°C 4 (h), 10,000 15, 2500 Serratia marcescens Cited Bac- 4 (h), 25,000 6, 2500 6 (h), pure Bac- ISA, 48 h, 25°C 4 (h), 10,000 —, sd Salmonella pullorum TSA, 24 h, 35°C Cited, 18 h, 37°C NA, 18 h, 37°C MHA, 24 h, 30°C NA, 24 h, 37°C ISA, 48 h, 25°C Conditions Salmonella sp. Bac- Bac- Pseudomonas aeruginosa Salmonella enteritidis Bac- Bac- Pseudomonas aeruginosa Pseudomonas aeruginosa Bac- Bac- MO Class Pseudomonas aeruginosa (continued) Pseudomonas aeruginosa Microorganism TABLE 12.34 4 29 0 5.4 6–20 9.5 0 0 0 0 4.5 34 23.5 8.3 7 4 10 6 13 2 5.5 7 10 6 7.5 9.7 13 6 4 0 Inhibition Zone (mm) Maruzzella and Lichtenstein (1956) Yousef and Tawil (1980) Deans and Ritchie (1987) Smith-Palmer et al. (1998) Lis-Balchin et al. (1998) Deans and Ritchie (1987) Deans and Ritchie (1987) Morris et al. (1979) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Yousef and Tawil (1980) Janssen et al. (1986) Deans and Ritchie (1987) Pizsolitto et al. (1975) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Pizsolitto et al. (1975) Pizsolitto et al. (1975) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Pizsolitto et al. (1975) Deans and Ritchie (1987) Smith-Palmer et al. (1998) Janssen et al. (1986) Yousef and Tawil (1980) Rossi et al. (2007) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Ref. 424 Handbook of Essential Oils NA, 24 h, 37°C NA, 18 h, 37°C ISA, 48 h, 25°C Bac+ Bac+ Staphylococcus epidermidis Staphylococcus epidermidis MHA, 24 h, 37°C —, sd sd Yeast Saccharomyces cerevisiae SMA, 2–7 d, 20°C 5 (h), -30,000 sd sd sd sd 6 (h), pure sd sd 6 (h), pure sd 6 (h), pure sd sd 6 (h), pure 9.5, 2000 sd 6, 2500 6 (h), pure sd sd sd sd SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C TGA, 18–24 h, 37°C SMA, 2–7 d, 20°C Cited, 18 h, 37°C SDA, 18 h, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C NA, 24 h, 20°C Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Alternaria solani Aspergillus fumigatus Aspergillus niger Aspergillus niger Helminthosporium sativum Mucor mucedo Mucor sp. Nigrospora panici Penicillium chrysogenum Penicillium digitatum Rhizopus nigricans Rhizopus sp. Candida albicans Candida albicans Candida albicans Candida albicans Candida krusei Candida tropicalis Cryptococcus neoformans Cryptococcus rhodobenhani Saccharomyces cerevisiae Streptomyces venezuelae 15, 2500 Cited Bac+ Bac+ Streptococcus faecalis 5 (h), -30,000 4 (h), 10,000 15, 2500 6 (h), pure 6, 15,000 4 (h), 25,000 6, 2500 15, 2500 4 (h), 10,000 9.5, 2000 Streptococcus viridans Cited NA, 18 h, 37°C Bac+ Bac+ Staphylococcus aureus TSA, 24 h, 35°C Cited, 18 h, 37°C Cited ISA, 48 h, 25°C TGA, 18–24 h, 37°C Staphylococcus aureus Bac+ Bac+ Staphylococcus aureus Staphylococcus aureus Bac+ Bac+ Staphylococcus aureus Staphylococcus aureus Bac+ Bac+ Staphylococcus aureus Staphylococcus aureus 0 8 10 13 7 10 9 9 10 10 13 4 3 0 0 5 11.7 31 5 3 8 7 16–21 13 0 0 15 15 30 16 8.5 8 5 0 0 Maruzzella and Lichtenstein (1956) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Morris et al. (1979) Maruzzella and Liguori (1958) Janssen et al. (1986) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Schelz et al. (2006) Maruzzella and Liguori (1958) Pizsolitto et al. (1975) Deans and Ritchie (1987) Schelz et al. (2006) Pizsolitto et al. (1975) Yousef and Tawil (1980) Rossi et al. (2007) Smith-Palmer et al. (1998) Janssen et al. (1986) Pizsolitto et al. (1975) Deans and Ritchie (1987) Morris et al. (1979) In Vitro Antimicrobial Activities of Essential Oils 425 10,000 3200 ISB, Tween 80, 20–24 h, 37°C NB, Tween 20, 18 h, 37°C BacBac+ Shigella flexneri Bacillus subtilis 20,000 ISB, Tween 80, 20–24 h, 37°C Bac+ Bac+ Bac+ Listeria monocytogenes Mycobacterium phlei Staphylococcus aureus Bac+ Bac+ Bac+ Enterococcus faecalis Enterococcus faecium Bac+ Listeria monocytogenes 20,000 ISB, Tween 80, 20–24 h, 37°C Bac+ Enterococcus durans TGB, 18–24 h, 37°C NB, Tween 20, 18 h, 37°C TSB, 24 h, 35°C ISB, Tween 80, 20–24 h, 37°C ISB, Tween 80, 20–24 h, 37°C TGB, 18–24 h, 37°C >1000 3200 750 2500 >1000 20,000 20,000 Corynebacterium sp. ISB, Tween 80, 20–24 h, 37°C Bac+ Bac+ Bacillus subtilis Corynebacterium pseudodiphtheriae ISB, Tween 80, 20–24 h, 37°C >10,000 2500 Bac- 20,000 >40,000 12,500 20,000 5000 6400 >40,000 2800 >10,000 >1000 20,000 Bac- TSB, 24 h, 35°C MIC >10,000 10,000 Salmonella enteritidis ISB, Tween 80, 20–24 h, 37°C NB, Tween 20, 18 h, 37°C ISB, Tween 80, 20–24 h, 37°C ISB, Tween 80, 20–24 h, 37°C ISB, Tween 80, 20–24 h, 37°C NB, Tween 20, 18 h, 37°C TGB, 18 h, 37°C ISB, Tween 80, 20–24 h, 37°C TSB, 24 h, 35°C TGB, 18–24 h, 37°C ISB, Tween 80, 20–24 h, 37°C ISB, Tween 80, 20–24 h, 37°C TSB, 24 h, 42°C Conditions Salmonella choleraesuis Bac- Bac- Escherichia coli Bac- Bac- Escherichia coli Pseudomonas aeruginosa Bac- Escherichia coli Pseudomonas aeruginosa Bac- Escherichia coli Bac- Bac- Escherichia coli Bac- Bac- Enterobacter aerogenes Klebsiella pneumoniae Bac- Proteus mirabilis Bac- Citrobacter freundii MO Class Campylobacter jejuni Microorganism TABLE 12.35 Inhibitory Data of Eucalyptus Oil Obtained in the Dilution Test Morris et al. (1979) Yousef and Tawil (1980) Smith-Palmer et al. (1998) Harkenthal et al. (1999) Harkenthal et al. (1999) Harkenthal et al. (1999) Harkenthal et al. (1999) Morris et al. (1979) Harkenthal et al. (1999) Yousef and Tawil (1980) Harkenthal et al. (1999) Harkenthal et al. (1999) Smith-Palmer et al. (1998) Harkenthal et al. (1999) Yousef and Tawil (1980) Harkenthal et al. (1999) Harkenthal et al. (1999) Harkenthal et al. (1999) Yousef and Tawil (1980) Schelz et al. (2006) Harkenthal et al. (1999) Smith-Palmer et al. (1998) Morris et al. (1979) Harkenthal et al. (1999) Harkenthal et al. (1999) Smith-Palmer et al. (1998) Ref. 426 Handbook of Essential Oils Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Staphylococcus aureus Staphylococcus aureus Staphylococcus epidermidis Staphylococcus epidermidis Staphylococcus saprophyticus Staphylococcus xylosus Aspergillus ochraceus Aspergillus oryzae Botrytis cinera Epidermophyton floccosum Fusarium culmorum Geotrichum citri-aurantii Mucor racemosus Mucor sp. Penicillium chrysogenum Penicillium chrysogenum Penicillium digitatum Phytophthora citrophthora Rhizopus sp. Trichophyton mentagrophytes Trichophyton rubrum Candida albicans Candida albicans Saccharomyces cerevisiae Alternaria alternata Aspergillus niger Aspergillus niger Bac+ Staphylococcus aureus SA, Tween 80, 21 d, 20°C TGB, 18–24 h, 37°C NB, Tween 20, 18 h, 37°C YPB, 24 h, 20°C NB, Tween 20, 8 d, 30°C NB, Tween 20, 8 d, 30°C Cited PDA, Tween 20, 7 d, 24°C PDA, Tween 20, 7 d, 24°C NB, Tween 20, 8 d, 30°C SA, Tween 80, 21 d, 20°C PDA, Tween 20, 7 d, 24°C Cited Cited PDA, Tween 20, 7 d, 24°C SA, Tween 80, 21 d, 20°C YES broth, 10 d YES broth, 10 d PDA, 7 d, 28°C NB, Tween 20, 8 d, 30°C YES broth, 10 d ISB, Tween 80, 20–24 h, 37°C ISB, Tween 80, 20–24 h, 37°C ISB, Tween 80, 20–24 h, 37°C TGB, 18 h, 37°C NB, Tween 20, 18 h, 37°C ISB, Tween 80, 20–24 h, 37°C TSB, 24 h, 35°C 1000 >1250 625–1250 1000 1600 700 >500 12,500 25,000 500 2% inh. 1000 38% inh. 1000 6400 - 78% inh. 10,000 0% inh. 1000 - 61% inh. 10,000 500 2% inh. 1000 625–1250 - 87% inh. 10,000 0% inh. 500 50,000 5000 5000 5000 2800 6400 20,000 Janssen et al. (1988) Morris et al. (1979) Yousef and Tawil (1980) Schelz et al. (2006) Yousef and Tawil (1980) Yousef and Tawil (1980) Okazaki and Oshima (1953) Bouchra et al. (2003) Bouchra et al. (2003) Yousef and Tawil (1980) Janssen et al. (1988) Bouchra et al. (2003) Okazaki and Oshima (1953) Okazaki and Oshima (1953) Bouchra et al. (2003) Janssen et al. (1988) Lis-Balchin et al. (1998) Lis-Balchin et al. (1998) Feng and Zheng (2007) Yousef and Tawil (1980) Lis-Balchin et al. (1998) Harkenthal et al. (1999) Harkenthal et al. (1999) Harkenthal et al. (1999) Schelz et al. (2006) Yousef and Tawil (1980) Harkenthal et al. (1999) Smith-Palmer et al. (1998) In Vitro Antimicrobial Activities of Essential Oils 427 PDA, 3 d, 25°C PDA, 3 d, 25°C Bac- Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Salmonella typhi Salmonella typhi Bacillus megaterium Bacillus subtilis var. aterrimus Corynebacterium diphtheriae Mycobacterium avium Staphylococcus aureus Staphylococcus aureus Streptococcus faecalis Streptococcus faecalis Streptococcus pyogenes Colletotrichum gleosporoides Fusarium oxysporum Fungi Fungi Yeast Pythium ultimum Rhizoctonia solani Candida albicans Botrytis cinera Bac- Neisseria sp. NA, 24 h, 37°C PDA, 3 d, 25°C PDA, 3 d, 25°C PDA, 3 d, 25°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Bac- Escherichia coli Conditions MO Class Microorganism TABLE 12.36 Inhibitory Data of Eucalyptus Oil Obtained in the Vapor Phase Test ~20,000 1000 1000 1000 1000 ~20,000 1000 ~20,000 sd ~20,000 sd ~20,000 sd ~20,000 sd ~20,000 sd ~20,000 ~20,000 +++ NG +++ +++ +++ +++ ++ NG +++ NG NG NG +++ NG +++ NG NG NG NG Activity Kellner and Kober (1954) Lee et al. (2007) Lee et al. (2007) Lee et al. (2007) Lee et al. (2007) Lee et al. (2007) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Ref. 428 Handbook of Essential Oils Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Escherichia coli Escherichia coli Escherichia coli Escherichia coli Neisseria perflava Proteus vulgaris Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Serratia marcescens Bacillus mesentericus Bacillus subtilis Bacillus subtilis Sarcina lutea Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus epidermidis Streptomyces venezuelae Alternaria porri Alternaria solani Aspergillus flavus Aspergillus fumigatus Aspergillus niger Bac- Enterobacter aerogenes MO Class Aerobacter aerogenes Microorganism PDA, 72 h, 28°C SMA, 2–7 d, 20°C SDA, 72 h, 26°C SMA, 2–7 d, 20°C SDA, 72 h, 26°C SMA, 2–7 d, 20°C NA, 18 h, 37°C MHA, 24 h, 37°C Cited, 18 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Cited, 18 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Cited, 18 h, 37°C MHA, 24 h, 30°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C MHA, 24 h, 30°C NA, 24 h, 37°C NA, 18 h, 37°C Cited, 18 h, 37°C MHA, 24 h, 30°C NA, 24 h, 37°C Conditions TABLE 12.37 Inhibitory Data of Juniper Oil Obtained in the Agar Diffusion Test 5, 5000 sd 8, 25,000 sd 8, 25,000 5 (h), -30,000 sd 6, 15,000 6, 2500 —, sd —, sd 6, 2500 —, sd —, sd —, sd 6, 2500 6, 15,000 —, sd —, sd —, sd 6, 15,000 5 (h), -30,000 —, sd 6, 2500 6, 15,000 —, sd 11 2 0 13 0 0 0 17 9 3 5 7.7 3 3 4 8 6 0 10 0 11 1 0 0 8 0 Inhibition Zone (mm) continued Pawar and Thaker (2007) Maruzzella and Liguori (1958) Shin (2003) Maruzzella and Liguori (1958) Shin (2003) Maruzzella and Liguori (1958) Schelz et al. (2006) Rossi et al. (2007) Janssen et al. (1986) Maruzzella and Lichtenstein (1956) Maruzzella and Lichtenstein (1956) Janssen et al. (1986) Maruzzella and Lichtenstein (1956) Maruzzella and Lichtenstein (1956) Maruzzella and Lichtenstein (1956) Janssen et al. (1986) Rossi et al. (2007) Maruzzella and Lichtenstein (1956) Maruzzella and Lichtenstein (1956) Maruzzella and Lichtenstein (1956) Rossi et al. (2007) Maruzzella and Lichtenstein (1956) Schelz et al. (2006) Janssen et al. (1986) Rossi et al. (2007) Maruzzella and Lichtenstein (1956) Ref. In Vitro Antimicrobial Activities of Essential Oils 429 Yeast Saccharomyces cerevisiae MO Class Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Yeast (continued) Aspergillus niger Aspergillus niger Botrytis cinera Cercospora beticola Fusarium graminearum Fusarium oxysporum f.sp. cicer Fusarium oxysporum lycopersici Helminthosporium oryzae Helminthosporium sativum Mucor mucedo Nigrospora panici Penicillium digitatum Phytophthora capsici Pyricularia oryzae Pythium ultimum Rhizoctonia solani Rhizopus nigricans Sclerotium rolfsii Septoria tritici Candida albicans Candida albicans Candida krusei Candida tropicalis Cryptococcus neoformans Cryptococcus rhodobenhani Saccharomyces cerevisiae Microorganism TABLE 12.37 SMA, 2–7 d, 20°C PDA, 48 h, 28°C SMA, 2–7 d, 20°C PDA, few days, 24°C PDA, few days, 24°C PDA, few days, 24°C PDA, 72 h, 28°C PDA, few days, 24°C PDA, few days, 24°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C PDA, few days, 24°C PDA, few days, 24°C PDA, few days, 24°C PDA, few days, 24°C SMA, 2–7 d, 20°C PDA, few days, 24°C PDA, few days, 24°C SMA, 2–7 d, 20°C Cited, 18 h, 37°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C NA, 24 h, 20°C Conditions 5 (h), -30,000 sd 5, 5000 sd 12.7, sd 12.7, sd 12.7, sd 5, 5000 12.7, sd 12.7, sd sd sd sd sd 12.7, sd 12.7, sd 12.7, sd 12.7, sd sd 12.7, sd 12.7, sd sd 6, 2500 sd sd sd sd 0 5 6 0 0 0 7.5 0 0 0 0 0 0 0 0 0 0 6 0 0 0 15 0 0 11 6 15–17 Inhibition Zone (mm) Ref. Maruzzella and Liguori (1958) Pawar and Thaker (2006) Maruzzella and Liguori (1958) Angioni et al. (2003) Angioni et al. (2003) Angioni et al. (2003) Pawar and Thaker (2007) Angioni et al. (2003) Angioni et al. (2003) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Angioni et al. (2003) Angioni et al. (2003) Angioni et al. (2003) Angioni et al. (2003) Maruzzella and Liguori (1958) Angioni et al. (2003) Angioni et al. (2003) Maruzzella and Liguori (1958) Janssen et al. (1986) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Schelz et al. (2006) 430 Handbook of Essential Oils BacBacBacBacBacBacBacBacBacBac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Escherichia coli Escherichia coli Escherichia coli O157:H7 Pseudomonas aeruginosa Pseudomonas aeruginosa Salmonella typhimurium Yersinia enterocolitica Yersinia enterocolitica Bacillus cereus Enterococcus faecalis Enterococcus faecium VRE Listeria monocytogenes Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus MRSA Staphylococcus epidermidis Aspergillus flavus Aspergillus flavus Aspergillus flavus Aspergillus fumigatus Alternaria alternate Aspergillus flavus MO Class Microorganism Escherichia coli RPMI, DMSO, 72 h, 37°C SDA, 6–8 h, 20°C RPMI, DMSO, 72 h, 37°C RPMI, 24 h, 37°C SDA, 6–8 h, 20°C MYB, 72 h, 26°C TGB, 18 h, 37°C HIB, Tween 80, 18 h, 37°C NB, 24 h, 37°C NB, DMSO, 24 h, 37°C NB, 24 h, 37°C HIB, Tween 80, 18 h, 37°C NB, 24 h, 37°C NB, 24 h, 37°C MHA, Tween 20, 24 h, 37°C NB, 24 h, 37°C NB, 24 h, 37°C NB, 24 h, 37°C NB, DMSO, 24 h, 37°C NB, 24 h, 37°C TGB, 18 h, 37°C NB, 24 h, 37°C NB, DMSO, 24 h, 37°C Conditions TABLE 12.38 Inhibitory Data of Juniper Oil Obtained in the Dilution Test >900 20,000 500, 11% inh. 10,000 >25,000 >11,300 500, 13% inh. >20,000 >900 >900 >900 >20,000 >900 >900 >900 2500 >900 >900 >900 >900 >900 5400 >900 MIC Ref. continued Cavaleiro et al. (2006)a Dikshit et al. (1986) Cavaleiro et al. (2006)a Cosentino et al. (2003) Dikshit et al. (1986) Shin (2003) Schelz et al. (2006) Nelson (1997) Cosentino et al. (2003) Angioni et al. (2003) Cosentino et al. (2003) Nelson (1997) Cosentino et al. (2003) Cosentino et al. (2003) Rossi et al. (2007)a Cosentino et al. (2003) Cosentino et al. (2003) Cosentino et al. (2003) Angioni et al. (2003) Cosentino et al. (2003) Schelz et al. (2006) Cosentino et al. (2003) Angioni et al. (2003) In Vitro Antimicrobial Activities of Essential Oils 431 Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Aspergillus niger Epidermophyton floccosum Epidermophyton floccosum Microsporum canis Microsporum gypseum Microsporum gypseum Penicillium italicum Trichophyton mentagrophytes Trichophyton mentagrophytes Trichophyton mentagrophytes Trichophyton rubrum Trichophyton rubrum Trichophyton rubrum Candida albicans Candida albicans Candida glabrata Candida krusei Candida parapsilosis Candida tropicalis Saccharomyces cerevisiae Saccharomyces cerevisiae Juniper communis ssp. alpine. Fungi Aspergillus niger a MO Class (continued) Microorganism TABLE 12.38 YPB, 24 h, 20°C RPMI, DMSO, 24 h, 37°C RPMI, DMSO, 24 h, 37°C RPMI, DMSO, 24 h, 37°C RPMI, DMSO, 24 h, 37°C RPMI, DMSO, 24 h, 37°C SB, 24 h, 37°C RPMI, DMSO, 72 h, 37°C SDA, 6–8 h, 20°C NB, DMSO, 24 h, 30°C RPMI, DMSO, 72 h, 37°C SDA, 6–8 h, 20°C SA, Tween 80, 21 d, 20°C RPMI, DMSO, 72 h, 37°C RPMI, DMSO, 72 h, 37°C RPMI, DMSO, 72 h, 37°C SDA, 6–8 h, 20°C SDA, 6–8 h, 20°C SA, Tween 80, 21 d, 20°C RPMI, DMSO, 72 h, 37°C SA, Tween 80, 21 d, 20°C MYB, 72 h, 26°C Conditions >900 2700 >900 1250–10,000 5000 5000 5000 20,000 >1250 1250 500, 53% inh. >1250 1250 500, 48% inh. >1250 1250 1250 2500 500, 48% inh. 500, 20% inh. >25,000 10,000–20,000 MIC Schelz et al. (2006) Cavaleiro et al. (2006)a Cavaleiro et al. (2006)a Cavaleiro et al. (2006)a Cavaleiro et al. (2006)a Cavaleiro et al. (2006)a Cosentino et al. (2003) Cavaleiro et al. (2006)a Dikshit et al. (1986) Angioni et al. (2003) Cavaleiro et al. (2006)a Dikshit et al. (1986) Janssen et al. (1988) Cavaleiro et al. (2006)a Cavaleiro et al. (2006)a Cavaleiro et al. (2006)a Dikshit et al. (1986) Dikshit et al. (1986) Janssen et al. (1988) Cavaleiro et al. (2006)a Janssen et al. (1988) Shin (2003) Ref. 432 Handbook of Essential Oils BacBacBacBac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Yeast Escherichia coli Neisseria sp. Salmonella typhi Bacillus megaterium Corynebacterium diphtheriae Staphylococcus aureus Streptococcus faecalis Streptococcus pyogenes Colletotrichum gleosporoides Fusarium oxysporum Pythium ultimum Rhizoctonia solani Candida albicans Botrytis cinera MO Class Microorganism NA, 24 h, 37°C PDA, 3 d, 25°C PDA, 3 d, 25°C PDA, 3 d, 25°C PDA, 3 d, 25°C PDA, 3 d, 25°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Conditions TABLE 12.39 Inhibitory Data of Juniper Oil Obtained in the Vapor Phase Test ~20,000 1000 1000 1000 1000 ~20,000 1000 ~20,000 ~20,000 ~20,000 ~20,000 ~20,000 ~20,000 ~20,000 +++ NG +++ +++ +++ +++ + NG + NG + NG ++ NG Activity Kellner and Kober (1954) Lee et al. (2007) Lee et al. (2007) Lee et al. (2007) Lee et al. (2007) Lee et al. (2007) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Ref. In Vitro Antimicrobial Activities of Essential Oils 433 4 (h), 10,000 6, 2500 Cited, 18 h, 37°C TGA, 18–24 h, 37°C 4 (h), 10,000 ISA, 48 h, 25°C Bac- Serratia marcescens 4 (h), 10,000 ISA, 48 h, 25°C Bac- Bac- Bac- Salmonella enteritidis Salmonella pullorum Bac- Pseudomonas sp. Salmonella typhimurium — Cited, 24 h, 37°C Bac- NA, 24 h, 37°C Cited, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C —, sd — 5000 on agar 4 (h), 10,000 6, 2500 —, sd Pseudomonas aeruginosa NA, 24 h, 37°C Cited, 18 h, 37°C Bac- Bac- 6 (h), pure —, sd Pseudomonas aeruginosa NA, 18 h, 37°C NA, 24 h, 37°C Pseudomonas aeruginosa Bac- Bac- Proteus vulgaris Pseudomonas aeruginosa —, sd NA, 24 h, 37°C Bac- Bac- Neisseria perflava 4 (h), 10,000 4 (h), 10,000 Proteus vulgaris ISA, 48 h, 25°C Bac- 4 (h), 10,000 Bac- ISA, 48 h, 25°C 9.5, 2000 4 (h), 10,000 Klebsiella pneumoniae ISA, 48 h, 25°C ISA, 48 h, 25°C Moraxella sp. Bac- Bac- Escherichia coli Bac- Bac- Escherichia coli Flavobacterium suaveolens — Cited, 24 h, 37°C Bac- Escherichia coli —, sd Escherichia coli NA, 24 h, 37°C 6 (h), pure 4 (h), 10,000 5000 on agar 4 (h), 10,000 4 (h), 10,000 Bac- NA, 18 h, 37°C 4 (h), 10,000 4 (h), 10,000 Bac- ISA, 48 h, 25°C 4 (h), 10,000 —, sd Escherichia coli Bac- Erwinia carotovora NA, 24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C Conditions Escherichia coli Bac- Bac- Enterobacter aerogenes Enterococcus faecalis Bac- Bac- Beneckea natriegens Citrobacter freundii Bac- Bac- Aeromonas hydrophila Alcaligenes faecalis Bac- Bac- Acinetobacter calcoaceticus MO Class Aerobacter aerogenes Microorganism TABLE 12.40 Inhibitory Data of Lavender Oil Obtained in the Agar Diffusion Test <12 2 <12 7 0 0 0 0 0 9 0 2 9 0 0 <12 7.7 7.5 0 0 0 6 0 7.5 8.5 0 0 8 2 10 Inhibition Zone (mm) Maruzzella and Lichtenstein (1956) Rota et al. (2004) Deans and Ritchie (1987) Rota et al. (2004) Di Pasqua et al. (2005) Deans and Ritchie (1987) Janssen et al. (1986) Maruzzella and Lichtenstein (1956) Yousef and Tawil (1980) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Janssen et al. (1986) Rota et al. (2004) Deans and Ritchie (1987) Morris et al. (1979) Maruzzella and Lichtenstein (1956) Yousef and Tawil (1980) Deans and Ritchie (1987) Di Pasqua et al. (2005) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Ref. 434 Handbook of Essential Oils ISA, 48 h, 25°C 4 (h), 10,000 4 (h), 10,000 9, 20,000 ISA, 48 h, 25°C MRS, cited Bac+ Bac+ Bac+ Lactobacillus plantarum Lactobacillus plantarum —, sd NA, 24 h, 37°C Bac+ Bac+ Bac+ Bac+ Bac+ Listeria monocytogenes Micrococcus luteus Micrococcus luteus Micrococcus ureae Mycobacterium phlei Bac+ Bac+ Bac+ Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Bac+ Bac+ Listeria monocytogenes Bac+ Bac+ Listeria monocytogenes Sarcina lutea Bac+ Leuconostoc cremoris Sarcina ureae 6 (h), pure NA, 18 h, 37°C Bac+ NA, 24 h, 37°C NA, 24 h, 37°C TGA, 18–24 h, 37°C MHA, cited MHA, cited ISA, 48 h, 25°C MHA, cited Cited, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C —, sd 5000 on agar 9.5, 2000 9, 20,000 9, 20,000 4 (h), 10,000 9, 20,000 — 5000 on agar 4 (h), 10,000 4 (h), 10,000 5000 on agar 5000 on agar Lactococcus lactis subsp. lactis NA, 24 h, 37°C Bac+ Bac+ Lactobacillus sp. 5000 on agar 5000 on agar Lactococcus garvieae NA, 24 h, 37°C NA, 24 h, 37°C 9.5, 2000 Lactobacillus delbrueckii TGA, 18–24 h, 37°C Bac+ 4 (h), 10,000 Bac+ ISA, 48 h, 25°C 5000 on agar 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 Corynebacterium sp. Bac+ NA, 24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C Clostridium sporogenes Bac+ Brochotrix thermosphacta Brochotrix thermosphacta 6 (h), pure NA, 18 h, 37°C Bac+ Bac+ Bacillus subtilis Brevibacterium linens 6, 2500 Cited, 18 h, 37°C Bac+ Bac+ Bacillus subtilis —, sd —, sd Bacillus subtilis NA, 24 h, 37°C Bac+ — Bac+ NA, 24 h, 37°C 4 (h), 10,000 — Bacillus mesentericus Cited, 24 h, 37°C ISA, 48 h, 25°C Cited, 24 h, 37°C Bacillus subtilis Bac- Bac- Yersinia enterocolitica Yersinia enterocolitica Bac- Bac- Serratia marcescens Shigella flexneri 7.5 3 0 >90 0 0 22 22 9.5 <12 0–22 0 0–18 0 0 20– >90 0 7.5 0 0 0 0 0 5.5 8.5 43 13.7 12.5 17 <12 4 <12 9 Deans and Ritchie (1987) continued Maruzzella and Lichtenstein (1956) Di Pasqua et al. (2005) Morris et al. (1979) Pellecuer et al. (1980) Maruzzella and Lichtenstein (1956) Yousef and Tawil (1980) Pellecuer et al. (1980) Deans and Ritchie (1987) Pellecuer et al. (1980) Rota et al. (2004) Di Pasqua et al. (2005) Lis-Balchin et al. (1998) Deans and Ritchie (1987) Di Pasqua et al. (2005) Di Pasqua et al. (2005) Pellecuer et al. (1980) Deans and Ritchie (1987) Di Pasqua et al. (2005) Di Pasqua et al. (2005) Morris et al. (1979) Deans and Ritchie (1987) Di Pasqua et al. (2005) Deans and Ritchie (1987) Deans and Ritchie (1987) Yousef and Tawil (1980) Janssen et al. (1986) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Maruzzella and Lichtenstein (1956) Rota et al. (2004) Deans and Ritchie (1987) Rota et al. (2004) In Vitro Antimicrobial Activities of Essential Oils 435 Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Staphylococcus aureus Staphylococcus epidermidis Streptococcus D Streptococcus faecalis Streptococcus micros Streptomyces venezuelae Alternaria porri Alternaria solani Aspergillus flavus Aspergillus fumigatus Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus niger Fusarium oxysporum f.sp. cicer Helminthosporium sativum Mucor mucedo Mucor sp. Nigrospora panici Penicillium chrysogenum Penicillium digitatum Rhizopus nigricans Rhizopus sp. Candida albicans Candida albicans Candida albicans Candida albicans Candida krusei Candida tropicalis Cryptococcus neoformans Cryptococcus rhodobenhani Saccharomyces cerevisiae Bac+ Staphylococcus aureus MO Class Bac+ (continued) Staphylococcus aureus Microorganism TABLE 12.40 PDA, 72 h, 28°C SMA, 2–7 d, 20°C SDA, 72 h, 26°C SMA, 2–7 d, 20°C SDA, 72 h, 26°C SMA, 2–7 d, 20°C PDA, 48 h, 28°C SDA, 8 d, 30°C PDA, 72 h, 28°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C TGA, 18–24 h, 37°C SMA, 2–7 d, 20°C SDA, 18 h, 30°C Cited, 18 h, 37°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C MHA, cited ISA, 48 h, 25°C MHA, cited MHA, cited NA, 18 h, 37°C Cited, 18 h, 37°C ISA, 48 h, 25°C Conditions 5, 5000 sd 8, 25,000 sd 8, 25,000 sd 5, 5000 6 (h), pure 5, 5000 sd sd 6 (h), pure sd 6 (h), pure sd sd 6 (h), pure 9.5, 2000 sd 6 (h), pure 6, 2500 sd sd sd sd sd sd 9, 20,000 4 (h), 10,000 9, 20,000 9, 20,000 6 (h), pure 6, 2500 4 (h), 10,000 13.3 0 4 7 2 8 10 32 9.8 7 10 21 5 16 3 8 14 0 7 11.5 14.3 2 6 0 7 3 15 26 14 0–29 14 14.3 12.3 8 Inhibition Zone (mm) Ref. Pawar and Thaker (2007) Maruzzella and Liguori (1958) Shin (2003) Maruzzella and Liguori (1958) Shin (2003) Maruzzella and Liguori (1958) Pawar and Thaker (2006) Yousef and Tawil (1980) Pawar and Thaker (2007) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Morris et al. (1979) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Janssen et al. (1986) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Pellecuer et al. (1980) Deans and Ritchie (1987) Pellecuer et al. (1980) Pellecuer et al. (1980) Yousef and Tawil (1980) Janssen et al. (1986) Deans and Ritchie (1987) 436 Handbook of Essential Oils BacBacBacBacBacBacBacBacBacBacBacBacBac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Escherichia coli Escherichia coli Escherichia coli Escherichia coli Haemophilus influenza Klebsiella pneumoniae Moraxella glucidolytica Neisseria catarrhalis Neisseria flava Pseudomonas aeruginosa Salmonella typhimurium Bacillus subtilis Bacillus subtilis Corynebacterium pseudodiphtheriae Corynebacterium sp. Enterococcus faecium VRE Lactobacillus sp. Micrococcus flavus Micrococcus luteus Micrococcus ureae Mycobacterium phlei Sarcina lutea Sarcina ureae Staphylococcus aureus Staphylococcus aureus MO Class Bordetella bronchiseptica Microorganism Conditions Cited TGB, 18–24 h, 37°C MHB, cited Cited NB, Tween 20, 18 h, 37°C MHB, cited MHB, cited Cited MRS, cited HIB, Tween 80, 18 h, 37°C TGB, 18–24 h, 37°C Cited Cited NB, Tween 20, 18 h, 37°C TSB, 24 h, 37°C NB, Tween 20, 18 h, 37°C Cited Cited Cited Cited Cited NB, Tween 20, 18 h, 37°C TSB, 24 h, 37°C TGB, 18–24 h, 37°C Cited Cited TABLE 12.41 Inhibitory Data of Lavender Oil Obtained in the Dilution Test >1000 2000 0.31–0.62 2000 100 2.5 1.25–2.5 1000 5 5000–10,000 1000 1000 1000 400 >50,000 10,000 500 250 2000 1000 2000 >10,000 50,000 >1000 500 2000 MIC continued Pellecuer et al. (1976) Morris et al. (1979) Pellecuer et al. (1980) Pellecuer et al. (1976) Yousef and Tawil (1980) Pellecuer et al. (1980) Pellecuer et al. (1980) Pellecuer et al. (1976) Pellecuer et al. (1980) Nelson (1997) Morris et al. (1979) Pellecuer et al. (1976) Pellecuer et al. (1976) Yousef and Tawil (1980) Di Pasqua et al. (2005) Yousef and Tawil (1980) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Yousef and Tawil (1980) Di Pasqua et al. (2005) Morris et al. (1979) Pellecuer et al. (1976) Pellecuer et al. (1976) Ref. In Vitro Antimicrobial Activities of Essential Oils 437 Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus MRSA Staphylococcus epidermidis Staphylococcus epidermidis Streptococcus D Streptococcus micros Streptococcus pyogenes Alternaria alternata Aspergillus flavus Aspergillus flavus Aspergillus flavus Aspergillus flavus var. columnaris Aspergillus fumigatus Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus ochraceus Cladosporium cladosporoides Epidermophyton floccosum Epidermophyton floccosum Epidermophyton floccosum Fusarium culmorum MO Class Bac+ (continued) Staphylococcus aureus Microorganism TABLE 12.41 Conditions Cited RPMI, 1.5% EtOH, 7 d, 30°C YES broth, 10 d RPMI, 1.5% EtOH, 7 d, 30°C SA, Tween 80, 21 d, 20°C RPMI, 1.5% EtOH, 7 d, 30°C NB, Tween 20, 8 d, 30°C YES broth, 10 d RPMI, 1.5% EtOH, 7 d, 30°C Cited MYB, 72 h, 26°C YES broth, 10 d RPMI, 1.5% EtOH, 7 d, 30°C CA, 7 d, 28 MYB, 72 h, 26°C RPMI, 1.5% EtOH, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C Cited MHB, cited MHB, cited Cited MHB, cited HIB, Tween 80, 18 h, 37°C MHB, Tween 80, 24 h, 37°C NB, Tween 20, 18 h, 37°C TSB, 3% EtOH, 24 h, 37°C MIC - 89% inh. 10,000 <300 1000 5000 - 90% inh. 10,000 2500–10,000 - 93% inh. 10,000 10,000 25,000 >10,000 10,000 1000 3120 5000 5–8% inh. 500 3120 10,000 >5 2000 0.62–5 2000 5 5000 12,500 12,500 10,000 Ref. Pellecuer et al. (1976) Tullio et al. (2006) Lis-Balchin et al. (1998) Tullio et al. (2006) Janssen et al. (1988) Tullio et al. (2006) Yousef and Tawil (1980) Lis-Balchin et al. (1998) Tullio et al. (2006) Pellecuer et al. (1976) Shin (2003) Lis-Balchin et al. (1998) Tullio et al. (2006) Kumar et al. (2007) Shin (2003) Tullio et al. (2006) Tullio et al. (2006) Pellecuer et al. (1976) Pellecuer et al. (1980) Pellecuer et al. (1980) Pellecuer et al. (1976) Pellecuer et al. (1980) Nelson (1997) Bastide et al. (1987) Yousef and Tawil (1980) Rota et al. (2004) 438 Handbook of Essential Oils Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Fusarium oxysporum Microsporum canis Microsporum canis Microsporum gypseum Microsporum gypseum Microsporum gypseum Mucor sp. Mucor sp. Penicillium chrysogenum Penicillium frequentans Penicillium lanosum Rhizopus sp. Rhizopus sp. Scopulariopsis brevicaulis Trichophyton equinum Trichophyton interdigitale Trichophyton mentagrophytes Trichophyton mentagrophytes Trichophyton rubrum Trichophyton rubrum Candida albicans Candida albicans Candida albicans Candida albicans Candida mycoderma Candida paraspilosis Candida pelliculosa Candida tropicalis Geotrichum asteroides Hansenula sp. Saccharomyces carlsbergensis SDA, 7 d, 30°C TGB, 18–24 h, 37°C Cited MHB, Tween 80, 48 h, 35 NB, Tween 20, 18 h, 37°C Cited Cited Cited Cited Cited Cited Cited NB, Tween 20, 8 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C SDA, 7 d, 30°C Cited RPMI, 1.5% EtOH, 7 d, 30°C SA, Tween 80, 21 d, 20°C SA, Tween 80, 21 d, 20°C NB, Tween 20, 8 d, 30°C NB, Tween 20, 8 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C SDA, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C MBA, Tween 80, 10 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C MBA, Tween 80, 10 d, 30°C <300 400, 21% inh. 1000 2000 5000 6400 2000 4000 1000 4000 2000 2000 2000 >10,000 12,500 10,000 400, 10% inh. 1000 5000–10,000 300–625 >10,000 50,000 12,500 5000 10,000 50– >300 400, 24% inh. 75– >300 5000–10,000 156 2500–5000 Dikshit and Husain (1984) Morris et al. (1979) Pellecuer et al. (1976) Hammer et al. (1998) Yousef and Tawil (1980) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Yousef and Tawil (1980) Tullio et al. (2006) Dikshit and Husain (1984) Pellecuer et al. (1976) Tullio et al. (2006) Janssen et al. (1988) Janssen et al. (1988) Yousef and Tawil (1980) Yousef and Tawil (1980) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Dikshit and Husain (1984) Tullio et al. (2006) Tullio et al. (2006) Perrucci et al. (1994) Tullio et al. (2006) Tullio et al. (2006) Perrucci et al. (1994) In Vitro Antimicrobial Activities of Essential Oils 439 Bac- Bac- Bac- Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Salmonella typhi Salmonella typhi Bacillus megaterium Bacillus subtilis var. aterrimus Corynebacterium diphtheriae Lactobacillus sp. Micrococcus luteus Micrococcus ureae Mycobacterium avium Sarcina ureae Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus epidermidis Streptococcus D Bac- Escherichia coli Haemophilus influenzae Bac- Escherichia coli Neisseria sp. MO Class Microorganism MHB, cited MHB, cited MHA, 18 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C MHB, cited NA, 24 h, 37°C MHB, cited MHB, cited MRS, cited NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C MHA, 18 h, 37°C BLA, 18 h, 37°C NA, 24 h, 37°C Conditions TABLE 12.42 Inhibitory Data of Lavender Oil Obtained in the Vapor Phase Test Disk, 20,000? Disk, 20,000? MICair ~20,000 sd Disk, 20,000? sd Disk, 20,000? Disk, 20,000? ~20,000 Disk, 20,000? ~20,000 sd ~20,000 sd ~20,000 MICair ~20,000 MICair +++ ++ +++ 100 + NG ++ NG +++ +++ +++ NG + +++ NG NG >1600 25 + Activity Pellecuer et al. (1980) Pellecuer et al. (1980) Inouye et al. (2001) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Pellecuer et al. (1980) Maruzzella and Sicurella (1960) Pellecuer et al. (1980) Pellecuer et al. (1980) Pellecuer et al. (1980) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Kellner and Kober (1954) Inouye et al. (2001) Inouye et al. (2001) Kellner and Kober (1954) Ref. 440 Handbook of Essential Oils Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Streptococcus faecalis Streptococcus micros Streptococcus pneumoniae Streptococcus pyogenes Streptococcus pyogenes Alternaria alternata Aspergillus flavus Aspergillus fumigatus Aspergillus niger Cladosporium cladosporoides Fusarium oxysporum Microsporum canis Microsporum gypseum Mucor sp. Penicillium frequentans Penicillium lanosum Rhizopus sp. Scopulariopsis brevicaulis Trichophyton mentagrophytes Candida albicans Bac+ Streptococcus faecalis RPMI, 7 d, 30°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C NA, 24 h, 37°C MHA, 18 h, 37°C NA, 24 h, 37°C MHA, 18 h, 37°C MHB, cited NA, 24 h, 37°C NA, 24 h, 37°C ~20,000 MICair MICair MICair MICair MICair MICair MICair MICair MICair MICair MICair MICair MICair MICair ~20,000 MICair MICair Disk, 20,000? ~20,000 sd NG 625–2500 2500 1250–2500 1250 156–312 5000 312–1250 312 1250 625 625 2500 125 312–625 NG 50 NG +++ 50 +++ Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Kellner and Kober (1954) Inouye et al. (2001) Kellner and Kober (1954) Inouye et al. (2001) Pellecuer et al. (1980) Maruzzella and Sicurella (1960) Kellner and Kober (1954) In Vitro Antimicrobial Activities of Essential Oils 441 CAB, 24 h, 42°C 4 (h), 10,000 10 (h), 100,000 5 × 20, 1000 NA, 24 h, 37°C NA, 24 h, 37°C Bac- Bac- Bac- Bac- Bac- Bac- Flavobacterium suaveolens Klebsiella pneumonia Klebsiella pneumonia subsp. oceanae Klebsiella pneumoniae Klebsiella sp. Cited ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C Disk, 10,000 15, 2500 4 (h), 10,000 5 × 20, 1000 4 (h), 10,000 6 (h), pure Klebsiella aerogenes NA, 18 h, 37°C Bac- 4 (h), 25,000 4 (h), 10,000 9.5, 2000 —, sd 10 (h), 100,000 15, 2500 5 × 20, 1000 4 (h), 10,000 Bac- NA, 24 h, 37°C Disk, 10,000 4 (h), 10,000 Escherichia coli TSA, 24 h, 35°C ISA, 48 h, 25°C TGA, 18–24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Cited NA, 24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C Escherichia coli Bac- Escherichia coli Bac- Bac- Escherichia coli Escherichia coli Bac- Escherichia coli Escherichia coli Bac- Bac- Escherichia coli Bac- Bac- Escherichia coli Bac- Enterobacter aerogenes Erwinia carotovora Bac- Bac- Campylobacter jejuni Citrobacter freundii 4 (h), 25,000 5 × 20, 1000 TSA, 24 h, 42°C NA, 24 h, 37°C Bac- Bac- Brucella abortus 4 (h), 10,000 4 (h), 10,000 ISA, 48 h, 25°C ISA, 48 h, 25°C Bac- Bac- Alcaligenes faecalis Beneckea natriegens Campylobacter jejuni —, sd 4 (h), 10,000 NA, 24 h, 37°C ISA, 48 h, 25°C Bac- Bac- Aerobacter aerogenes Aeromonas hydrophila 4 (h), 10,000 5 × 20, 1000 ISA, 48 h, 25°C NA, 24 h, 37°C Bac- Bac- Conditions Acinetobacter calcoaceticus MO Class Aerobacter aerogenes Microorganism TABLE 12.43 Inhibitory Data of Lemon Oil Obtained in the Agar Diffusion Test 0 0 2–5 6–10 0 13.5 18.5 21 4 0 0 0 0 0 0–10 6 0 0 41 4.6 2–5 0 0 6.5 0 1–10 0 Inhibition Zone (mm) Pizsolitto et al. (1975) Deans and Ritchie (1987) Möse et al. (1957) Möse et al. (1957) Narasimha Rao and Nigam (1970) Deans and Ritchie (1987) Yousef and Tawil (1980) Fisher and Phillips (2006) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Morris et al. (1979) Maruzzella and Lichtenstein (1956) Narasimha Rao and Nigam (1970) Pizsolitto et al. (1975) Möse et al. (1957) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Fisher and Phillips (2006) Smith-Palmer et al. (1998) Möse et al. (1957) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Möse et al. (1957) Deans and Ritchie (1987) Ref. 442 Handbook of Essential Oils 15, 2500 Cited Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Proteus vulgaris Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas fluorescens Pseudomonas mangiferae indicae Salmonella enteritidis Salmonella enteritidis Salmonella paratyphi Salmonella paratyphi B Salmonella pullorum Salmonella sp. Salmonella typhi Salmonella typhi Salmonella typhi Serratia marcescens Serratia marcescens Serratia marcescens Bac- Bac- Bac- Vibrio albicans Vibrio cholera Vibrio cholerae Bac- Bac- Proteus vulgaris Bac- Bac- Proteus vulgaris Serratia sp. Bac- Proteus sp. Shigella sp. 5 × 20, 1000 NA, 24 h, 37°C Bac- Proteus OX19 NA, 24 h, 37°C NA, 36–48 h, 37°C NA, 24 h, 37°C Cited ISA, 48 h, 25°C NA, 24 h, 37°C NA, 36–48 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Cited ISA, 48 h, 25°C NA, 24 h, 37°C NA, 36–48 h, 37°C TSA, 24 h, 35°C NA, 24 h, 37°C NA, 36–48 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C NA, 18 h, 37°C Cited ISA, 48 h, 25°C NA, 24 h, 37°C NA, 24 h, 37°C Cited NA, 24 h, 37°C 5 × 20, 1000 6, sd 5 × 20, 1000 15, 2500 4 (h), 10,000 —, sd 6, sd 10 (h), 10,0000 5 × 20, 1000 15, 2500 4 (h), 10,000 5 × 20, 1000 6, sd 4 (h), 25,000 5 × 20, 1000 6, sd 5 × 20, 1000 5 × 20, 1000 4 (h), 10,000 —, sd 6 (h), pure 15, 2500 4 (h), 10,000 —, sd 5 × 20, 1000 15, 2500 5 × 20, 1000 —, sd 4 (h), 10,000 Bac- NA, 24 h, 37°C Neisseria perflava ISA, 48 h, 25°C Bac- Moraxella sp. 6–10 12 11–13 0 0 1 0 0 28 0 2–5 0 7 6–10 16 4 6–10 14 1 1 0 0 0 0 0 0 2–5 0 2–5 0 0 Möse et al. (1957) Garg and Garg (1980) Möse et al. (1957) Pizsolitto et al. (1975) Pizsolitto et al. (1975) Möse et al. (1957) Deans and Ritchie (1987) continued Maruzzella and Lichtenstein (1956) Garg and Garg (1980) Narasimha Rao and Nigam (1970) Möse et al. (1957) Pizsolitto et al. (1975) Deans and Ritchie (1987) Möse et al. (1957) Garg and Garg (1980) Smith-Palmer et al. (1998) Möse et al. (1957) Garg and Garg (1980) Möse et al. (1957) Möse et al. (1957) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Yousef and Tawil (1980) Pizsolitto et al. (1975) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Möse et al. (1957) Pizsolitto et al. (1975) Möse et al. (1957) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) In Vitro Antimicrobial Activities of Essential Oils 443 5 × 20, 1000 Disk, 10,000 NA, 24 h, 37°C BHA, 24 h, 30°C 15, 2500 Cited 4 (h), 10,000 ISA, 48 h, 25°C Bac+ Bac+ Bac+ Sarcina beige Bac+ Micrococcus luteus Mycobacterium phlei Bac+ Listeria monocytogenes Sarcina alba Bac+ Bac+ Listeria monocytogenes Listeria monocytogenes Bac+ Bac+ Leuconostoc cremoris 9.5, 2000 4 (h), 10,000 TGA, 18–24 h, 37°C ISA, 48 h, 25°C Bac+ Bac+ Corynebacterium sp. Lactobacillus plantarum Listeria monocytogenes 10 (h), 10,0000 NA, 24 h, 37°C Bac+ Bac+ Corynebacterium diphtheria NA, 24 h, 37°C NA, 24 h, 37°C NA, 18 h, 37°C ISA, 48 h, 25°C LSA, 24 h, 37°C TSA, 24 h, 35°C ISA, 48 h, 25°C NA, 24 h, 37°C NA, 24 h, 37°C 4 (h), 10,000 5 × 20, 1000 5 × 20, 1000 6 (h), pure 4 (h), 10,000 Disk, 10,000 4 (h), 25,000 4 (h), 10,000 5 × 20, 1000 5 × 20, 1000 4 (h), 10,000 Corynebacterium diphtheria ISA, 48 h, 25°C ISA, 48 h, 25°C Bac+ Bac+ 4 (h), 10,000 6 (h), pure 6, sd 4 (h), 10,000 —, sd 5 × 20, 1000 6, sd Brochotrix thermosphacta ISA, 48 h, 25°C NA, 18 h, 37°C NA, 36–48 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 36–48 h, 37°C Clostridium sporogenes Bac+ Bac+ Brevibacterium linens Bac+ Bacillus subtilis Bacillus subtilis Bac+ Bac+ Bacillus subtilis Bacillus subtilis Bac+ Bac+ Bacillus sp. Bacillus subtilis 6, sd NA, 36–48 h, 37°C Bac+ Bac+ Bacillus mycoides —, sd Bacillus pumilus NA, 24 h, 37°C Bac+ Bac+ Bacillus cereus Bacillus mesentericus 4 (h), 10,000 Bac+ 10 (h), 10,0000 Bacillus anthracis NA, 24 h, 37°C ISA, 48 h, 25°C Bac- Conditions Bac- MO Class Vibrio cholerae (continued) Yersinia enterocolitica Microorganism TABLE 12.43 6–10 2–5 24 0 41 5.3 3 2–5 7 0 0 0 1 0 8 6 19.5 16 6 5 1 0 16 12 4 19 6–10 0 22 Inhibition Zone (mm) Möse et al. (1957) Möse et al. (1957) Yousef and Tawil (1980) Deans and Ritchie (1987) Fisher and Phillips (2006) Smith-Palmer et al. (1998) Lis-Balchin et al. (1998) Möse et al. (1957) Deans and Ritchie (1987) Deans and Ritchie (1987) Morris et al. (1979) Narasimha Rao and Nigam (1970) Möse et al. (1957) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Yousef and Tawil (1980) Garg and Garg (1980) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Möse et al. (1957) Pizsolitto et al. (1975) Garg and Garg (1980) Garg and Garg (1980) Maruzzella and Lichtenstein (1956) Fisher and Phillips (2006) Möse et al. (1957) Deans and Ritchie (1987) Narasimha Rao and Nigam (1970) Ref. 444 Handbook of Essential Oils Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Sarcina lutea Sarcina lutea Sarcina rosa Sporococcus sarc. Staphylococcus albus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus epidermidis Staphylococcus epidermidis Streptococcus faecalis Streptococcus haemolyticus Streptococcus sp. Streptococcus viridans Streptococcus viridians Streptomyces venezuelae Alternaria porri Alternaria solani Aspergillus fumigatus Aspergillus niger Aspergillus niger Aspergillus niger Fusarium oxysporum f.sp. cicer Helminthosporium sativum Bac+ Sarcina citrea PDA, 72 h, 28°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C PDA, 48 h, 28°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C PDA, 72 h, 28°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C NA, 24 h, 37°C Cited NA, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C Cited NA, 18 h, 37°C BHA, 24 h, 37°C NA, 36–48 h, 37°C TSA, 24 h, 35°C ISA, 48 h, 25°C TGA, 18–24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Cited NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 36–48 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C 5, 5000 sd sd 5, 5000 sd 6 (h), pure 5, 5000 sd sd 5 × 20, 1000 15, 2500 10 (h), 100,000 5 × 20, 1000 4 (h), 10,000 10 (h), 100,000 15, 2500 6 (h), pure Disk, 10,000 6, sd 4 (h), 25,000 4 (h), 10,000 9.5, 2000 —, sd 10 (h), 100,000 15, 2500 5 × 20, 1000 5 × 20, 1000 5 × 20, 1000 5 × 20, 1000 6, sd —, sd 5 × 20, 1000 7.3 6 9 7 9 18 13.2 16 11 1 0 0 6–10 7 18 0 22 14 14 6 0 0 0 0 0 11–15 6–10 6–10 6–10 17 0 2–5 continued Pawar and Thaker (2007) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Pawar and Thaker (2006) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Pawar and Thaker (2007) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Möse et al. (1957) Pizsolitto et al. (1975) Narasimha Rao and Nigam (1970) Möse et al. (1957) Deans and Ritchie (1987) Narasimha Rao and Nigam (1970) Pizsolitto et al. (1975) Yousef and Tawil (1980) Fisher and Phillips (2006) Garg and Garg (1980) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Morris et al. (1979) Maruzzella and Lichtenstein (1956) Narasimha Rao and Nigam (1970) Pizsolitto et al. (1975) Möse et al. (1957) Möse et al. (1957) Möse et al. (1957) Möse et al. (1957) Garg and Garg (1980) Maruzzella and Lichtenstein (1956) Möse et al. (1957) In Vitro Antimicrobial Activities of Essential Oils 445 (continued) Mucor mucedo Mucor sp. Nigrospora panici Penicillium chrysogenum Penicillium digitatum Rhizopus nigricans Rhizopus sp. Brettanomyces anomalus Candida albicans Candida albicans Candida albicans Candida krusei Candida lipolytica Candida tropicalis Cryptococcus neoformans Cryptococcus rhodobenhani Debaryomyces hansenii Geotrichum candidum Hansenula anomala Kloeckera apiculata Kluyveromyces fragilis Lodderomyces elongisporus Metchnikowia pulcherrima Pichia membranaefaciens Rhodotorula rubra Saccharomyces cerevisiae Saccharomyces cerevisiae Torula glabrata Microorganism TABLE 12.43 Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast MO Class SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C MPA, 4 d, 30°C TGA, 18–24 h, 37°C SMA, 2–7 d, 20°C SDA, 18 h, 30°C SMA, 2–7 d, 20°C MPA, 4 d, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C SMA, 2–7 d, 20°C MPA, 4 d, 30°C Conditions sd 6 (h), pure sd 6 (h), pure sd sd 6 (h), pure 5, 10% sol. sd 9.5, 2000 sd 6 (h), pure sd 5, 10% sol. sd sd sd sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd sd 5, 10% sol. sd 11 14 10 17 11 0 0 0 0 2 33.5 3 0 1 5 4 0 0 0 0 0 0 0 0 0 0 7 0 Inhibition Zone (mm) Ref. Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Conner and Beuchat (1984) Morris et al. (1979) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Maruzzella and Liguori (1958) Conner and Beuchat (1984) 446 Handbook of Essential Oils Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Campylobacter jejuni Escherichia coli Escherichia coli Escherichia coli Escherichia coli Pseudomonas aeruginosa Pseudomonas aeruginosa Salmonella schottmuelleri Salmonella senftenberg Serratia marcescens Bacillus cereus Bacillus subtilis Bacillus subtilis Corynebacterium sp. Lactobacillus plantarum Listeria monocytogenes Micrococcus sp. Mycobacterium phlei Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus MO Class Aerobacter aerogenes Microorganism Conditions NB, Tween 20, 18 h, 37°C TGB, 18–24 h, 37°C NB, 24–72 h, 37°C BHA, 24 h, 37°C NB, Tween 20, 18 h, 37°C NA, pH 7 LSA, 24 h, 37°C NA, pH 7 TGB, 18–24 h, 37°C NB, Tween 20, 18 h, 37°C NA, pH 7 BHA, 24 h, 30°C NA, pH 7 NB, 24–72 h, 37°C NA, pH 7 NB, 24–72 h, 37°C NB, Tween 20, 18 h, 37°C NB, 24–72 h, 37°C NB, Tween 20, 18 h, 37°C NA, 24 h, 37°C TGB, 18–24 h, 37°C CAB, 24 h, 42°C NA, pH 7 TABLE 12.44 Inhibitory Data of Lemon Oil Obtained in the Dilution Test 6400 500 >40,000 10,000 800 2000 2500 >1000 1000 3200 2000 >2000 10,000 >2000 98% inh. 10,000 90% inh. 10,000 50,000 98% inh. 10,000 25,000 >1000 10,000 >40,000 >2000 MIC Ref. continued Yousef and Tawil (1980) Morris et al. (1979) Dabbah et al. (1970) Fisher and Phillips (2006) Yousef and Tawil (1980) Subba et al. (1967) Fisher and Phillips (2006) Subba et al. (1967) Morris et al. (1979) Yousef and Tawil (1980) Subba et al. (1967) Fisher and Phillips (2006) Subba et al. (1967) Dabbah et al. (1970) Subba et al. (1967) Dabbah et al. (1970) Yousef and Tawil (1980) Dabbah et al. (1970) Yousef and Tawil (1980) Fisher and Phillips (2006) Morris et al. (1979) Fisher and Phillips (2006) Subba et al. (1967) In Vitro Antimicrobial Activities of Essential Oils 447 Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Aspergillus flavus Aspergillus flavus Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus ochraceus Aspergillus oryzae Aspergillus parasiticus Aspergillus parasiticus Botrytis cinera Fusarium culmorum Geotrichum citri-aurantii Mucor hiemalis Mucor mucedo Mucor racemosus Mucor racemosus f. racemosus Mucor sp. Penicillium chrysogenum Penicillium chrysogenum Aspergillus awamorii Aspergillus flavus MO Class Bac+ Fungi (continued) Streptococcus faecalis Microorganism TABLE 12.44 Conditions NB, Tween 20, 8 d, 30°C NB, Tween 20, 8 d, 30°C Cited Cited PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 8 h, 20°C, spore germ. inh. PDA, Tween 20, 7 d, 24°C YES broth, 10 d PDA, Tween 20, 7 d, 24°C PDA, 5 d, 27°C PDA, 5 d, 27°C YES broth, 10 d NB, Tween 20, 8 d, 30°C YES broth, 10 d Cited PDA, 8 h, 20°C, spore germ. inh. PDA, pH 4.5 PDA, pH 4.5 PDA, 5 d, 27°C PDA, pH 4.5 NA, pH 7 MIC >1000 25,000 1600 500 >1000 500 >1000 >1000 50–100 4% inh. 1000 0% inh. 10,000 0% inh. 1000 >500 >2000 4% inh. 10,000 800 22% inh. 10,000 >2000 50–100 >1000 >2000 1000 Ref. Yousef and Tawil (1980) Yousef and Tawil (1980) Okazaki and Oshima (1953) Okazaki and Oshima (1953) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson (1986) Bouchra et al. (2003) Lis-Balchin et al. (1998) Bouchra et al. (2003) Thompson and Cannon (1986) Thompson and Cannon (1986) Lis-Balchin et al. (1998) Yousef and Tawil (1980) Lis-Balchin et al. (1998) Okazaki and Oshima (1953) Thompson (1986) Subba et al. (1967) Subba et al. (1967) Thompson and Cannon (1986) Subba et al. (1967) Subba et al. (1967) 448 Handbook of Essential Oils Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Penicillium digitatum Penicillium digitatum Penicillium italicum Phytophthora citrophthora Rhizopus 66-81-2 Rhizopus arrhizus Rhizopus chinensis Rhizopus circinans Rhizopus japonicus Rhizopus kazanensis Rhizopus oryzae Rhizopus pymacus Rhizopus sp. Rhizopus stolonifer Rhizopus tritici Candida albicans Candida albicans Candida albicans Saccharomyces cerevisiae Torula utilis Zygosaccharomyces mellis MHB, Tween 80, 48 h, 35°C TGB, 18–24 h, 37°C NB, Tween 20, 18 h, 37°C PDA, pH 4.5 PDA, pH 4.5 PDA, pH 4.5 PDA, 5 d, 27°C NB, Tween 20, 8 d, 30°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C SDB, 5 d, 20°C, MIC = ED50 PDA, Tween 20, 7 d, 24°C PDA, 5 d, 27°C SDB, 5 d, 20°C, MIC = ED50 PDA, Tween 20, 7 d, 24°C >2000 >1000 20,000 500 6400 500 1000 >1000 >1000 12,500 >1000 >1000 >1000 >1000 >1000 >1000 >1000 20% inh. 1000 1000–2500 0% inh. 1000 500–1000 Hammer et al. (1998) Morris et al. (1979) Yousef and Tawil (1980) Subba et al. (1967) Subba et al. (1967) Subba et al. (1967) Thompson and Cannon (1986) Yousef and Tawil (1980) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Bouchra et al. (2003) Thompson and Cannon (1986) Caccioni et al. (1998) Bouchra et al. (2003) Caccioni et al. (1998) In Vitro Antimicrobial Activities of Essential Oils 449 Bac+ Bac+ Bac+ Bac+ Fungi Staphylococcus aureus Streptococcus faecalis Streptococcus pneumoniae Streptococcus pyogenes Streptococcus pyogenes Fungi Fungi Fungi Yeast Penicillium corylophilum Pythium ultimum Rhizoctonia solani Candida albicans WFA, 42 d, 25°C Fungi Fungi Eurotium rubrum Fusarium oxysporum WFA, 42 d, 25°C Fungi Fungi Eurotium herbarum Eurotium repens PDA, 3 d, 25°C NA, 24 h, 37°C PDA, 3 d, 25°C PDA, 3 d, 25°C WFA, 42 d, 25°C PDA, 3 d, 25°C WFA, 42 d, 25°C WFA, 42 d, 25°C Fungi Fungi Colletotrichum gleosporoides PDA, 3 d, 25°C WFA, 42 d, 25°C WFA, 42 d, 25°C MHA, 18 h, 37°C NA, 24 h, 37°C MHA, 18 h, 37°C NA, 24 h, 37°C MHA, 18 h, 37°C BHA, 24 h, 37°C NA, 24 h, 37°C Eurotium amstelodami Fungi Fungi Aspergillus niger Botrytis cinera Aspergillus flavus Bac+ Bac+ Staphylococcus aureus Bac+ Bac+ Staphylococcus aureus LSA, 24 h, 37°C ~20,000 1000 1000 Disk, 50,000 1000 Disk, 50,000 Disk, 50,000 Disk, 50,000 Disk, 50,000 1000 1000 Disk, 50,000 Disk, 50,000 ~20,000 MICair ~20,000 MICair MICair ~20,000 Disk, 10,000 ~20,000 Disk, 10,000 Bac+ Listeria monocytogenes ~20,000 NA, 24 h, 37°C NA, 24 h, 37°C Bacillus cereus NA, 24 h, 37°C ~20,000 Bac+ Bac- Salmonella typhi NA, 24 h, 37°C MICair MICair Bacillus megaterium Bac- Neisseria sp. MHA, 18 h, 37°C BLA, 18 h, 37°C Corynebacterium diphtheriae Bac- Haemophilus influenzae BHA, 24 h, 30°C Bac- Escherichia coli NA, 24 h, 37°C Disk, 10,000 ~20,000 Disk, 10,000 Bac+ Bac- Escherichia coli NA, 24 h, 37°C CAB, 24 h, 42°C Conditions ~20,000 Disk, 10,000 BacBac- Campylobacter jejuni MO Class Escherichia coli Microorganism TABLE 12.45 Inhibitory Data of Lemon Oil Obtained in the Vapor Phase Test +++ NG +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ 200 NG 400 NG +++ 800 +++ NG + NG +++ NG NG >1600 200 +++ ++ +++ Activity Kellner and Kober (1954) Lee et al. (2007) Lee et al. (2007) Guynot et al. (2003) Lee et al. (2007) Guynot et al. (2003) Guynot et al. (2003) Guynot et al. (2003) Guynot et al. (2003) Lee et al. (2007) Lee et al. (2007) Guynot et al. (2003) Guynot et al. (2003) Inouye et al. (2001) Kellner and Kober (1954) Inouye et al. (2001) Kellner and Kober (1954) Inouye et al. (2001) Fisher and Phillips (2006) Kellner and Kober (1954) Fisher and Phillips (2006) Kellner and Kober (1954) Kellner and Kober (1954) Fisher and Phillips (2006) Kellner and Kober (1954) Kellner and Kober (1954) Inouye et al. (2001) Inouye et al. (2001) Fisher and Phillips (2006) Kellner and Kober (1954) Fisher and Phillips (2006) Ref. 450 Handbook of Essential Oils Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Aerobacter aerogenes Aeromonas hydrophila Alcaligenes faecalis Beneckea natriegens Campylobacter jejuni Campylobacter jejuni Citrobacter freundii Enterobacter aerogenes Enterobacter aerogenes Erwinia carotovora Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Flavobacterium suaveolens Klebsiella pneumoniae Moraxella sp. Neisseria perflava Proteus vulgaris MO Class Acinetobacter calcoaceticus Microorganism ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C MHA, 24 h, 30°C TSA, 24 h, 35°C Cited ISA, 48 h, 25°C TGA, 18–24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C MHA, 24 h, 30°C ISA, 48 h, 25°C ISA, 48 h, 25°C Cited TSA, 24 h, 42°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C Conditions TABLE 12.46 Inhibitory Data of Mandarin Oil Obtained in the Agar Diffusion Test 4 (h), 10,000 —, sd 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 6, 15,000 4 (h), 25,000 6, 15,000 4 (h), 10,000 9.5, 2000 —, sd 4 (h), 10,000 6, 15,000 4 (h), 10,000 4 (h), 10,000 6, 15,000 4 (h), 25,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 —, sd 4 (h), 10,000 0 0 0 0 0 8 4 0 0 0 0 0 7 0 0 11 4 0 0 6 1 0 Inhibition Zone (mm) Deans and Ritchie (1987) continued Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Rossi et al. (2007) Smith-Palmer et al. (1998) Wannissorn et al. (2005) Deans and Ritchie (1987) Morris et al. (1979) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Rossi et al. (2007) Deans and Ritchie (1987) Deans and Ritchie (1987) Wannissorn et al. (2005) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Ref. In Vitro Antimicrobial Activities of Essential Oils 451 MO Class Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Proteus vulgaris Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Salmonella enteritidis Salmonella enteritidis Salmonella pullorum Salmonella typhimurium Serratia marcescens Serratia marcescens Yersinia enterocolitica Bacillus mesentericus Bacillus subtilis Bacillus subtilis Brevibacterium linens Brochotrix thermosphacta Clostridium perfringens Clostridium sporogenes Corynebacterium sp. Lactobacillus plantarum Leuconostoc cremoris (continued) Microorganism TABLE 12.46 ISA, 48 h, 25°C ISA, 48 h, 25°C TGA, 18–24 h, 37°C ISA, 48 h, 25°C Cited ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C Cited ISA, 48 h, 25°C TSA, 24 h, 35°C Cited MHA, 24 h, 30°C ISA, 48 h, 25°C NA, 24 h, 37°C NA, 24 h, 37°C Conditions 4 (h), 10,000 4 (h), 10,000 9.5, 2000 4 (h), 10,000 6, 15,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 —, sd —, sd 4 (h), 10,000 4 (h), 10,000 —, sd 6, 15,000 4 (h), 10,000 4 (h), 25,000 6, 15,000 6, 15,000 4 (h), 10,000 —, sd —, sd 7 0 0 0 35 7 0 5 3 3 0 0 0 9 0 4 0 6 0 0 9 Inhibition Zone (mm) Ref. Deans and Ritchie (1987) Deans and Ritchie (1987) Morris et al. (1979) Deans and Ritchie (1987) Wannissorn et al. (2005) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Wannissorn et al. (2005) Deans and Ritchie (1987) Smith-Palmer et al. (1998) Wannissorn et al. (2005) Rossi et al. (2007) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Maruzzella and Lichtenstein (1956) 452 Handbook of Essential Oils Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Yeast Staphylococcus aureus Staphylococcus aureus Streptococcus faecalis Streptomyces venezuelae Alternaria solani Aspergillus fumigatus Aspergillus niger Helminthosporium sativum Mucor mucedo Nigrospora panici Penicillium digitatum Rhizopus nigricans Candida albicans Candida albicans Candida krusei Candida tropicalis Cryptococcus neoformans Cryptococcus rhodobenhani Saccharomyces cerevisiae Bac+ Bac+ Staphylococcus aureus Bac+ Bac+ Sarcina lutea Staphylococcus aureus Bac+ Micrococcus luteus Staphylococcus aureus Bac+ Listeria monocytogenes SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C TGA, 18–24 h, 37°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C ISA, 48 h, 25°C MHA, 24 h, 37°C TSA, 24 h, 35°C ISA, 48 h, 25°C TGA, 18–24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C TSA, 24 h, 35°C sd sd sd sd sd sd sd sd sd 9.5, 2000 sd sd sd sd sd sd 4 (h), 10,000 6, 15,000 4 (h), 25,000 4 (h), 10,000 9.5, 2000 —, sd —, sd 4 (h), 10,000 4 (h), 25,000 7 11 12 22 3 6 7 2 0 0 0 2 10 7 5 7 0 17 4.1 0 0 0 15 0 4.2 Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Morris et al. (1979) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Deans and Ritchie (1987) Rossi et al. (2007) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Morris et al. (1979) Maruzzella and Lichtenstein (1956) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Smith-Palmer et al. (1998) In Vitro Antimicrobial Activities of Essential Oils 453 454 Handbook of Essential Oils TABLE 12.47 Inhibitory Data of Mandarin Oil Obtained in the Dilution Test Microorganism MO Class Escherichia coli Conditions MIC Ref. Bac- TGB, 18–24 h, 37°C Escherichia coli Bac- NB, 24–72 h, 37°C >1000 98% inh. 10,000 Dabbah et al. (1970) Morris et al. (1979) Pseudomonas aeruginosa Bac- NB, 24–72 h, 37°C 87% inh. 10,000 Dabbah et al. (1970) Salmonella senftenberg Bac- NB, 24–72 h, 37°C Dabbah et al. (1970) Yersinia enterocolitica Bac- MHA, Tween 20, 24 h, 37°C >10,000 2500 Corynebacterium sp. Bac+ TGB, 18–24 h, 37°C >1000 Morris et al. (1979) Staphylococcus aureus Bac+ TGB, 18–24 h, 37°C Morris et al. (1979) Staphylococcus aureus Bac+ Yeast NB, 24–72 h, 37°C >1000 10,000 TGB, 18–24 h, 37°C 1000 Morris et al. (1979) Candida albicans Rossi et al. (2007) 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 Microorganism MO Class Conditions Activity Ref. Aspergillus flavus Fungi WFA, 42 d, 25°C Aspergillus niger Fungi WFA, 42 d, 25°C Disk, 50,000 +++ Guynot et al. (2003) Disk, 50,000 +++ Eurotium amstelodami Fungi Guynot et al. (2003) WFA, 42 d, 25°C Disk, 50,000 +++ Eurotium herbarum Guynot et al. (2003) 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) BacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBac- Aerobacter aerogenes Aeromonas hydrophila Alcaligenes faecalis Beneckea natriegens Brucella abortus Citrobacter freundii Enterobacter aerogenes Erwinia carotovora Escherichia coli Escherichia coli Escherichia coli Escherichia coli Flavobacterium suaveolens Klebsiella pneumonia Klebsiella pneumonia subsp. Oceanae Klebsiella pneumoniae Moraxella sp. Proteus OX19 Proteus vulgaris Proteus vulgaris Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas fluorescens MO Class Acinetobacter calcoaceticus Microorganism NA, 24 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C NA, 18 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C TGA, 18–24 h, 37°C NA, 18 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C Conditions TABLE 12.49 Inhibitory Data of Matricaria Oil Obtained in the Agar Diffusion Test 5 × 20, 1000 4 (h), 10,000 5 × 20, 1000 6 (h), pure 4 (h), 10,000 5 × 20, 1000 5 × 20, 1000 4 (h), 10,000 4 (h), 10,000 5 × 20, 1000 5 × 20, 1000 4 (h), 10,000 4 (h), 10,000 5 × 20, 1000 9.5, 2000 6 (h), pure 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 5 × 20, 1000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 5 × 20, 1000 4 (h), 10,000 0 0 0 0 0 0 0 0 0 2–5 2–5 0 0 0 0 0 0 0 0 0 0 0 0 0 7 Inhibition Zone (mm) continued Möse et al. (1957) Deans and Ritchie (1987) Möse et al. (1957) Yousef and Tawil (1980) Deans and Ritchie (1987) Möse et al. (1957) Möse et al. (1957) Deans and Ritchie (1987) Deans and Ritchie (1987) Möse et al. (1957) Möse et al. (1957) Deans and Ritchie (1987) Deans and Ritchie (1987) Möse et al. (1957) Morris et al. (1979) Yousef and Tawil (1980) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Möse et al. (1957) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Möse et al. (1957) Deans and Ritchie (1987) Ref. In Vitro Antimicrobial Activities of Essential Oils 455 BacBacBacBacBacBacBacBacBacBac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Salmonella pullorum Salmonella typhi Serratia marcescens Serratia marcescens Vibrio albicans Vibrio cholerae Yersinia enterocolitica Bacillus anthracis Bacillus subtilis Bacillus subtilis Bacillus subtilis Brevibacterium linens Brochotrix thermosphacta Clostridium sporogenes Corynebacterium diphtheria Corynebacterium sp. Lactobacillus plantarum Leuconostoc cremoris Listeria monocytogenes Listeria monocytogenes Micrococcus luteus MO Class Salmonella paratyphi B (continued) Salmonella enteritidis Microorganism TABLE 12.49 ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C TGA, 18–24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 18 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C NA, 24 h, 37°C Conditions 4 (h), 10,000 5 × 20, 1000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 9.5, 2000 5 × 20, 1000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 6 (h), pure 4 (h), 10,000 5 × 20, 1000 5 × 20, 1000 4 (h), 10,000 5 × 20, 1000 5 × 20, 1000 4 (h), 10,000 5 × 20, 1000 5 × 20, 1000 4 (h), 10,000 5 × 20, 1000 5 × 20, 1000 0 2–5 0–11 0 0 0 1 0 0 0 13.5 0 0 2–5 0 2–5 1 0 0 2–5 0 0 2–5 Inhibition Zone (mm) Deans and Ritchie (1987) Möse et al. (1957) Lis-Balchin et al. (1998) Deans and Ritchie (1987) Deans and Ritchie (1987) Morris et al. (1979) Möse et al. (1957) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Yousef and Tawil (1980) Deans and Ritchie (1987) Möse et al. (1957) Möse et al. (1957) Deans and Ritchie (1987) Möse et al. (1957) Möse et al. (1957) Deans and Ritchie (1987) Möse et al. (1957) Möse et al. (1957) Deans and Ritchie (1987) Möse et al. (1957) Möse et al. (1957) Ref. 456 Handbook of Essential Oils Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Streptococcus haemolyticus Streptococcus viridians Alternaria porri Aspergillus niger Aspergillus niger Fusarium oxysporum f.sp. cicer Mucor sp. Penicillium chrysogenum Rhizopus sp. Candida albicans Candida albicans Bac+ Bac+ Streptococcus faecalis Bac+ Bac+ Staphylococcus aureus Bac+ Staphylococcus aureus Staphylococcus aureus ISA, 48 h, 25°C PDA, 72 h, 28°C SDA, 8 d, 30°C PDA, 48 h, 28°C PDA, 72 h, 28°C SDA, 8 d, 30°C SDA, 8 d, 30°C SDA, 8 d, 30°C TGA, 18–24 h, 37°C SDA, 18 h, 30°C NA, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C NA, 18 h, 37°C NA, 24 h, 37°C 5, 5000 6 (h), pure 5, 5000 5, 5000 6 (h), pure 6 (h), pure 6 (h), pure 9.5, 2000 6 (h), pure 5 × 20, 1000 5 × 20, 1000 4 (h), 10,000 6 (h), pure 5 × 20, 1000 4 (h), 10,000 9.5, 2000 5 × 20, 1000 TGA, 18–24 h, 37°C NA, 24 h, 37°C Bac+ Bac+ Staphylococcus albus 5 × 20, 1000 5 × 20, 1000 NA, 24 h, 37°C NA, 24 h, 37°C Bac+ Bac+ Sarcina rosa Sporococcus sarc. Staphylococcus aureus 5 × 20, 1000 5 × 20, 1000 NA, 24 h, 37°C NA, 24 h, 37°C Bac+ Bac+ Sarcina beige Sarcina citrea 6 (h), pure 5 × 20, 1000 NA, 18 h, 37°C NA, 24 h, 37°C Bac+ Bac+ Mycobacterium phlei Sarcina alba 15.5 9.7 10 0 9 10 11 0 0 9.5 2–5 2–5 0 11.5 2–5 0 0 2–5 6–10 6–10 0 6–10 0 Yousef and Tawil (1980) Pawar and Thaker (2007) Yousef and Tawil (1980) Pawar and Thaker (2006) Pawar and Thaker (2007) Yousef and Tawil (1980) Yousef and Tawil (1980) Yousef and Tawil (1980) Morris et al. (1979) Yousef and Tawil (1980) Möse et al. (1957) Möse et al. (1957) Deans and Ritchie (1987) Yousef and Tawil (1980) Möse et al. (1957) Deans and Ritchie (1987) Morris et al. (1979) Möse et al. (1957) Möse et al. (1957) Möse et al. (1957) Möse et al. (1957) Möse et al. (1957) Möse et al. (1957) In Vitro Antimicrobial Activities of Essential Oils 457 MO Class BacBacBacBacBacBacBacBacBac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Microorganism Escherichia coli Escherichia coli Escherichia coli Escherichia coli Helicobacter pylori Klebsiella pneumoniae Pseudomonas aeruginosa Pseudomonas aeruginosa Bacillus subtilis Bacillus subtilis Corynebacterium sp. Mycobacterium phlei Pseudomonas aeruginosa Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Streptococcus faecalis Agar, cited NB, Tween 20, 18 h, 37°C NB, Tween 80, 24 h, 37°C Agar, cited TGB, 18–24 h, 37°C NB, Tween 80, 24 h, 37°C NB, Tween 20, 18 h, 37°C TGB, 18–24 h, 37°C NB, Tween 80, 24 h, 37°C NB, Tween 20, 18 h, 37°C NB, Tween 20, 18 h, 37°C Agar, cited Agar, cited Cited, 20 h, 37°C NB, Tween 20, 18 h, 37°C Agar, cited NB, Tween 80, 24 h, 37°C TGB, 18–24 h, 37°C Conditions TABLE 12.50 Inhibitory Data of Matricaria Oil Obtained in the Dilution Test 2500 25,000 7000 2500 >8000 1000 >1000 1600 6000 >50,000 400 7500 10,000 >50,000 35.7–70.4 >8000 10,000 >1000 MIC Kedzia et al. (1991) Yousef and Tawil (1980) Aggag and Yousef (1972) Kedzia et al. (1991) Morris et al. (1979) Aggag and Yousef (1972) Yousef and Tawil (1980) Morris et al. (1979) Aggag and Yousef (1972) Yousef and Tawil (1980) Yousef and Tawil (1980) Kedzia et al. (1991) Kedzia et al. (1991) Weseler et al. (2005) Yousef and Tawil (1980) Kedzia et al. (1991) Aggag and Yousef (1972) Morris et al. (1979) Ref. 458 Handbook of Essential Oils Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Aspergillus flavus Aspergillus niger Aspergillus niger Aspergillus ochraceus Aspergillus ochraceus Aspergillus parasiticus Botrytis cinera Fusarium culmorum Fusarium moniliforme Microsporum gypseum Mucor sp. Penicillium chrysogenum Phytophthora citrophthora Rhizopus sp. Trichophyton mentagrophytes Trichophyton mentagrophytes Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Agar, cited NB, Tween 20, 8 d, 30°C NB, Tween 20, 8 d, 30°C PDA, Tween 20, 7 d, 24°C NB, Tween 20, 8 d, 30°C SDA, 21 d, 20°C SDA, 21 d, 20°C TGB, 18–24 h, 37°C SDA, 7 d, 37°C Agar, cited NB, Tween 80, 24 h, 37°C NB, Tween 20, 18 h, 37°C PDA, 7–14 d, 28°C PDA, Tween 20, 7 d, 24°C YES broth, 10 d PDA, 7–14 d, 28°C YES broth, 10 d NB, Tween 20, 8 d, 30°C PDA, 7–14 d, 28°C YES broth, 10 d PDA, 7–14 d, 28°C >3000 1000 50,000 50,000 2% inh. 1000 50,000 1000 1000 500 1000 5000 7000 50,000 -75% inh. 10,000 >3000 0% inh. 1000 -56% inh. 10,000 >3000 -63% inh. 10,000 50,000 >3000 Kedzia et al. (1991) Yousef and Tawil (1980) Yousef and Tawil (1980) Bouchra et al. (2003) Yousef and Tawil (1980) Szalontai et al. (1977) Szalontai et al. (1977) Morris et al. (1979) Szalontai et al. (1977) Kedzia et al. (1991) Aggag and Yousef (1972) Yousef and Tawil (1980) Soliman and Badeaa (2002) Bouchra et al. (2003) Lis-Balchin et al. (1998) Soliman and Badeaa (2002) Lis-Balchin et al. (1998) Yousef and Tawil (1980) Soliman and Badeaa (2002) Lis-Balchin et al. (1998) Soliman and Badeaa (2002) In Vitro Antimicrobial Activities of Essential Oils 459 BacBacBacBac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Yeast Escherichia coli Neisseria sp. Salmonella typhi Bacillus megaterium Corynebacterium diphtheriae Staphylococcus aureus Streptococcus faecalis Streptococcus pyogenes Colletotrichum gleosporoides Fusarium oxysporum Pythium ultimum Rhizoctonia solani Candida albicans Botrytis cinera MO Class Microorganism NA, 24 h, 37°C PDA, 3 d, 25°C PDA, 3 d, 25°C PDA, 3 d, 25°C PDA, 3 d, 25°C PDA, 3 d, 25°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Conditions TABLE 12.51 Inhibitory Data of Matricaria Oil Obtained in the Vapor Phase Test ~20,000 1000 1000 1000 1000 ~20,000 1000 ~20,000 ~20,000 ~20,000 ~20,000 ~20,000 ~20,000 ~20,000 +++ +++ +++ +++ +++ +++ + ++ + + ++ +++ +++ +++ Activity Kellner and Kober (1954) Lee et al. (2007) Lee et al. (2007) Lee et al. (2007) Lee et al. (2007) Lee et al. (2007) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Ref. 460 Handbook of Essential Oils Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Aeromonas hydrophila Alcaligenes faecalis Beneckea natriegens Campylobacter jejuni Citrobacter freundii Enterobacter aerogenes Erwinia carotovora Escherichia coli Escherichia coli Escherichia coli Escherichia coli O157 Flavobacterium suaveolens Klebsiella pneumoniae Klebsiella sp. Moraxella sp. Proteus sp. Proteus vulgaris Pseudomonas aeruginosa Pseudomonas aeruginosa Salmonella agona Salmonella braenderup Salmonella derby Salmonella enteritidis Salmonella gallinarum MO Class Acinetobacter calcoaceticus Microorganism Cited Cited Cited Cited Cited ISA, 48 h, 25°C Cited ISA, 48 h, 25°C Cited ISA, 48 h, 25°C Cited ISA, 48 h, 25°C ISA, 48 h, 25°C Cited Cited Cited ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C Cited ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C Conditions TABLE 12.52 Inhibitory Data of Mint Oil Obtained in the Agar Diffusion Test 6, 15,000 6, 15,000 6, 15,000 6, 15,000 6, 15,000 4 (h), 10,000 15, 2500 4 (h), 10,000 15, 2500 4 (h), 10,000 15, 2500 4 (h), 10,000 4 (h), 10,000 6, 15,000 6, 15,000 15, 2500 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 6, 15,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 52.5 13.5 10 13 12 0 0 7 2 8 2 6 9 13.5 14.5 1 0 0 6.5 8.5 90 9.5 5 8.5 9 Inhibition Zone (mm) Ref. continued Wannissorn et al. (2005) Wannissorn et al. (2005) Wannissorn et al. (2005) Wannissorn et al. (2005) Wannissorn et al. (2005) Deans and Ritchie (1987) Pizsolitto et al. (1975) Deans and Ritchie (1987) Pizsolitto et al. (1975) Deans and Ritchie (1987) Pizsolitto et al. (1975) Deans and Ritchie (1987) Deans and Ritchie (1987) Wannissorn et al. (2005) Wannissorn et al. (2005) Pizsolitto et al. (1975) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Wannissorn et al. (2005) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) In Vitro Antimicrobial Activities of Essential Oils 461 MO Class Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Salmonella hadar Salmonella mbandaka Salmonella montevideo Salmonella pullorum Salmonella saintpaul Salmonella schwarzergrund Salmonella senftenberg Salmonella sp. Salmonella typhimurium Serratia marcescens Serratia sp. Shigella sp. Yersinia enterocolitica Bacillus sp. Bacillus subtilis Brevibacterium linens Brochotrix thermosphacta Clostridium perfringens Clostridium sporogenes Lactobacillus plantarum (continued) Microorganism TABLE 12.52 ISA, 48 h, 25°C ISA, 48 h, 25°C Cited ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C Cited ISA, 48 h, 25°C Cited Cited ISA, 48 h, 25°C Cited Cited Cited Cited Cited ISA, 48 h, 25°C Cited Cited Cited Conditions 4 (h), 10,000 4 (h), 10,000 6, 15,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 15, 2500 4 (h), 10,000 15, 2500 15, 2500 4 (h), 10,000 6, 15,000 15, 2500 6, 15,000 6, 15,000 6, 15,000 4 (h), 10,000 6, 15,000 6, 15,000 6, 15,000 9 0 90 6 0 14.5 10 6.5 2 6 4.5 47.5 4 12 11.5 12 6 12 11.5 13.5 Inhibition Zone (mm) Ref. Deans and Ritchie (1987) Deans and Ritchie (1987) Wannissorn et al. (2005) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Pizsolitto et al. (1975) Deans and Ritchie (1987) Pizsolitto et al. (1975) Pizsolitto et al. (1975) Deans and Ritchie (1987) Wannissorn et al. (2005) Pizsolitto et al. (1975) Wannissorn et al. (2005) Wannissorn et al. (2005) Wannissorn et al. (2005) Deans and Ritchie (1987) Wannissorn et al. (2005) Wannissorn et al. (2005) Wannissorn et al. (2005) 462 Handbook of Essential Oils Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Micrococcus luteus Staphylococcus aureus Staphylococcus aureus Staphylococcus epidermidis Streptococcus faecalis Streptococcus viridans Absidia corMYBifera Aspergillus flavus Aspergillus fumigatus Aspergillus sulphureus Humicola grisea var. thermoidea Mucor fragilis Rhizopus stolonifer Sporotrichum thermophile Thermoascus aurantiacis Thermomyces lanuginosa Thielava minor Torula thermophila Bac+ Leuconostoc cremoris EYA, 48 h, 45°C PDA, 10 d, 25°C PDA, 10 d, 25°C PDA, 10 d, 25°C EYA, 48 h, 45°C PDA, 10 d, 25°C PDA, 10 d, 25°C EYA, 48 h, 45°C EYA, 48 h, 45°C EYA, 48 h, 45°C EYA, 48 h, 45°C EYA, 48 h, 45°C Cited ISA, 48 h, 25°C Cited ISA, 48 h, 25°C Cited ISA, 48 h, 25°C ISA, 48 h, 25°C 5, sd 50 50 50 5, sd 50 50 5, sd 5, sd 5, sd 5, sd 5, sd 15, 2500 4 (h), 10,000 15, 2500 4 (h), 10,000 15, 2500 4 (h), 10,000 4 (h), 10,000 10 NG NG NG 15 NG NG 20 10 0 0 10 1 14 2 12 2 8.5 0 Nigam and Rao (1979) Sarbhoy et al. (1978) Sarbhoy et al. (1978) Sarbhoy et al. (1978) Nigam and Rao (1979) Sarbhoy et al. (1978) Sarbhoy et al. (1978) Nigam and Rao (1979) Nigam and Rao (1979) Nigam and Rao (1979) Nigam and Rao (1979) Nigam and Rao (1979) Pizsolitto et al. (1975) Deans and Ritchie (1987) Pizsolitto et al. (1975) Deans and Ritchie (1987) Pizsolitto et al. (1975) Deans and Ritchie (1987) Deans and Ritchie (1987) In Vitro Antimicrobial Activities of Essential Oils 463 Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Fusarium oxysporum Helminthosporium oryzae Helminthosporium oryzae Helminthosporium oryzae Helminthosporium oryzae Macrophomina phaesoli Microsporum gypseum Penicillium italicum Sclerotium rolfsii Trichophyton mentagrophytes Trichophyton rubrum Candida albicans b a Mentha arvensis var. piperascens. Dementholized oil. Alternaria alternate Aspergillus flavus Aspergillus flavus Aspergillus flavus Aspergillus niger Botryodiplodia theobromae Cladosporium cladosporoides Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Staphylococcus aureus MRSA Staphylococcus aureus MSSA Bac+ Bac+ Staphylococcus aureus MRSA 800 CA, 7 d, 28°C CA, 7 d, 28°C MA, citeda MA, pH 5.0, 6 d, 28°Ca,b MA, pH 4.0, 6 d, 28°Ca,b CA, 7 d, 28°C SDA, 6–8 h, 20°C SDA, 6–8 h, 20°C CA, 7 d, 28°C SDA, 6–8 h, 20°C SDA, 6–8 h, 20°C Cited, 48 h, 36°Ca SDA, 6–8 h, 20°C CA, 7 d, 28°C Cited SDA, 6–8 h, 20°C CA, 7 d, 28°C CA, 7 d, 28°C CA, 7 d, 28°C Cited, 24 h HIB, Tween 80, 18 h, 37°C Cited, 24 h MIC >500 83% inh. 500 500 2000 2000 500 94% inh. 500 64% inh. 500 65% inh. 500 500 69% inh. 500 36% inh. 500 1100 63% inh. 500 500 1000 65% inh. 500 81% inh. 500 87% inh. 500 400 5000 400 5000–10,000 Cited, 24 h HIB, Tween 80, 18 h, 37°C Bac- Enterococcus faecium VRE Salmonella enteritidis 400 100 Cited, 24 h Cited, 48 h BacBac- Conditions Escherichia coli MO Class Helicobacter pylori Microorganism TABLE 12.53 Inhibitory Data of Mint Oil Obtained in the Dilution Test Kumar et al. (2007) Kumar et al. (2007) Dikshit et al. (1979) Dikshit et al. (1982) Dikshit et al. (1982) Kumar et al. (2007) Dikshit et al. (1986) Dikshit et al. (1986) Kumar et al. (2007) Dikshit et al. (1986) Dikshit et al. (1986) Duarte et al. (2005) Dikshit et al. (1986) Kumar et al. (2007) Kumar et al. (2007) Dikshit et al. (1986) Kumar et al. (2007) Kumar et al. (2007) Kumar et al. (2007) Imai et al. (2001) Nelson (1997) Imai et al. (2001) Nelson (1997) Imai et al. (2001) Imai et al. (2001) Imai et al. (2001) Ref. 464 Handbook of Essential Oils Bac+ Listeria monocytogenes 6 (h), pure NA, 18 h, 37°C Bac+ Bac+ 15, 2500 —, sd Cited NA, 24 h, 37°C Bac+ Bac+ Bacillus sp. Bacillus subtilis Bacillus subtilis 10 (h), 100,000 —, sd NA, 24 h, 37°C NA, 24 h, 37°C Bac- Bac+ Vibrio cholerae Bacillus mesentericus Corynebacterium diphtheriae 15, 2500 15, 2500 Cited Cited Bac- Bac- Serratia sp. Shigella sp. ISA, 48 h, 25°C NA, 24 h, 37°C 4 (h), 10,000 10 (h), 100,000 10 (h), 100,000 —, sd Bac- Serratia marcescens NA, 24 h, 37°C NA, 24 h, 37°C Bac- Bac- 15, 2500 6 (h), pure —, sd 15, 2500 —, sd —, sd Salmonella typhi Cited NA, 18 h, 37°C NA, 24 h, 37°C Cited NA, 24 h, 37°C NA, 24 h, 37°C Salmonella sp. Bac- Bac- Pseudomonas aeruginosa Bac- Pseudomonas aeruginosa Pseudomonas aeruginosa 15, 2500 Cited Bac- Bac- 15, 2500 —, sd Cited NA, 24 h, 37°C Bac- Bac- Klebsiella sp. Neisseria perflava Proteus sp. 10 (h), 100,000 10 (h), 100,000 NA, 24 h, 37°C NA, 24 h, 37°C Bac- Bac- Escherichia coli Klebsiella aerogenes Proteus vulgaris 6 (h), pure NA, 18 h, 37°C Bac- Bac- Escherichia coli —, sd 15, 2500 Escherichia coli NA, 24 h, 37°C Cited Bac- Bac- Conditions Aerobacter aerogenes MO Class Escherichia coli Microorganism TABLE 12.54 Inhibitory Data of Neroli Oil Obtained in the Agar Diffusion Test 11–19 18 22 19 1 7 0 1 0 0 19 0 12 0 0 2 0 2 0 0 22 14 2 0 0 Inhibition Zone (mm) continued Dikshit et al. (1986) Dikshit et al. (1986) Kumar et al. (2007) Dikshit et al. (1986) Dikshit et al. (1986) Kumar et al. (2007) Dikshit et al. (1982) Dikshit et al. (1982) Dikshit et al. (1979) Kumar et al. (2007) Kumar et al. (2007) Kumar et al. (2007) Kumar et al. (2007) Kumar et al. (2007) Dikshit et al. (1986) Kumar et al. (2007) Kumar et al. (2007) Dikshit et al. (1986) Imai et al. (2001) Nelson (1997) Imai et al. (2001) Nelson (1997) Imai et al. (2001) Imai et al. (2001) Imai et al. (2001) Ref. In Vitro Antimicrobial Activities of Essential Oils 465 10 (h), 100,000 10 (h), 100,000 10 (h), 100,000 NA, 24 h, 37°C NA, 24 h, 37°C Bac+ Bac+ Staphylococcus epidermidis sd sd sd sd 6 (h), pure sd sd 6 (h), pure sd 6 (h), pure sd sd 6 (h), pure sd 6 (h), pure sd sd sd sd sd SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SDA, 18 h, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Yeast Alternaria solani Aspergillus fumigatus Aspergillus niger Aspergillus niger Helminthosporium sativum Mucor mucedo Mucor sp. Nigrospora panici Penicillium chrysogenum Penicillium digitatum Rhizopus nigricans Rhizopus sp. Candida albicans Candida albicans Candida krusei Candida tropicalis Cryptococcus neoformans Cryptococcus rhodobenhani Saccharomyces cerevisiae Streptomyces venezuelae 15, 2500 Cited Bac+ Bac+ Streptococcus sp. 15, 2500 Streptococcus viridans Cited 15, 2500 6 (h), pure Staphylococcus epidermidis NA, 18 h, 37°C Bac+ —, sd Bac+ Cited 6 (h), pure —, sd Staphylococcus aureus NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 18 h, 37°C Conditions Staphylococcus aureus Bac+ Bac+ Staphylococcus aureus Staphylococcus aureus Bac+ Bac+ MO Class Mycobacterium phlei (continued) Sarcina lutea Microorganism TABLE 12.54 9 5 9 26 9 9 22 5 56 9 6 21 8 30 0 0 6 6 4 9 0 0 22 1 13.6 2 0 0 0 20 Inhibition Zone (mm) Dikshit et al. (1986) Kumar et al. (2007) Kumar et al. (2007) Kumar et al. (2007) Kumar et al. (2007) Kumar et al. (2007) Dikshit et al. (1979) Dikshit et al. (1982) Dikshit et al. (1982) Kumar et al. (2007) Dikshit et al. (1986) Dikshit et al. (1986) Kumar et al. (2007) Dikshit et al. (1986) Dikshit et al. (1986) Duarte et al. (2005) Imai et al. (2001) Imai et al. (2001) Imai et al. (2001) Kumar et al. (2007) Kumar et al. (2007) Dikshit et al. (1986) Imai et al. (2001) Nelson (1997) Imai et al. (2001) Nelson (1997) Imai et al. (2001) Imai et al. (2001) Imai et al. (2001) Duarte et al. (2005) Ref. 466 Handbook of Essential Oils 50,000 NB, Tween 20, 18 h, 37°C NB, Tween 20, 8 d, 30°C YES broth, 10 d YES broth, 10 d Bac+ Bac+ Fungi Fungi Mycobacterium phlei Staphylococcus aureus Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Aspergillus ochraceus Aspergillus oryzae Cephalosporium sacchari Ceratocystis paradoxa Curvularia lunata Fusarium culmorum Fusarium moniliforme var. subglutinans Helminthosporium sacchari Mucor racemosus Mucor sp. Penicillium chrysogenum Penicillium chrysogenum Physalospora tucumanensis Rhizopus sp. Sclerotium rolfsii Candida albicans Aspergillus niger Aspergillus niger 3200 NB, Tween 20, 18 h, 37°C BacBac+ Bacillus subtilis 25,000 OA, EtOH, 3 d, 20°C OA, EtOH, 3 d, 20°C Cited NB, Tween 20, 8 d, 30°C Cited NB, Tween 20, 8 d, 30°C OA, EtOH, 3 d, 20°C NB, Tween 20, 8 d, 30°C OA, EtOH, 6 d, 20°C NB, Tween 20, 18 h, 37°C Cited OA, EtOH, 3 d, 20°C OA, EtOH, 3 d, 20°C OA, EtOH, 3 d, 20°C YES broth, 10 d NB, Tween 20, 18 h, 37°C MIC -71% inh. 10,000 2000 2000 500 3200 250 6400 20,000 1600 20,000 3200 -90% inh. 10,000 250 20,000 20,000 4000 -86% inh. 10,000 3200 6400 25,000 NB, Tween 20, 18 h, 37°C Bac- Escherichia coli Pseudomonas aeruginosa NB, Tween 20, 18 h, 37°C Conditions MO Class Microorganism TABLE 12.55 Inhibitory Data of Neroli Oil Obtained in the Dilution Test Narasimba Rao et al. (1971) Narasimba Rao et al. (1971) Okazaki and Oshima (1953) Yousef and Tawil (1980) Okazaki and Oshima (1953) Yousef and Tawil (1980) Narasimba Rao et al. (1971) Yousef and Tawil (1980) Narasimba Rao et al. (1971) Yousef and Tawil (1980) Okazaki and Oshima (1953) Narasimba Rao et al. (1971) Narasimba Rao et al. (1971) Narasimba Rao et al. (1971) Lis-Balchin et al. (1998) Lis-Balchin et al. (1998) Yousef and Tawil (1980) Lis-Balchin et al. (1998) Yousef and Tawil (1980) Yousef and Tawil (1980) Yousef and Tawil (1980) Yousef and Tawil (1980) Yousef and Tawil (1980) Ref. In Vitro Antimicrobial Activities of Essential Oils 467 BacBacBacBacBac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Yeast Escherichia coli Neisseria sp. Salmonella typhi Salmonella typhi Bacillus megaterium Bacillus subtilis var. aterrimus Corynebacterium diphtheriae Mycobacterium avium Staphylococcus aureus Staphylococcus aureus Streptococcus faecalis Streptococcus faecalis Streptococcus pyogenes Candida albicans MO Class Microorganism NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Conditions TABLE 12.56 Inhibitory Data of Neroli Oil Obtained in the Vapor Phase Test ~20,000 ~20,000 ~20,000 sd ~20,000 sd ~20,000 sd ~20,000 sd ~20,000 sd ~20,000 ~20,000 NG +++ NG +++ NG NG NG +++ NG +++ NG +++ +++ NG Activity Kellner and Kober (1954) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Kellner and Kober (1954) Kellner and Kober (1954) Ref. 468 Handbook of Essential Oils TSA, 24 h, 42°C 4 (h), 10,000 6 (h), pure NA, 18 h, 37°C 4 (h), 10,000 ISA, 48 h, 25°C Bac- Bac- Bac- Pseudomonas aeruginosa Salmonella enteritidis Salmonella pullorum Bac- Bac- Pseudomonas aeruginosa Pseudomonas aeruginosa Bac- Bac- Proteus vulgaris Pseudomonas aeruginosa 15, 2500 Cited Bac- Bac- Moraxella sp. ISA, 48 h, 25°C TSA, 24 h, 35°C ISA, 48 h, 25°C Cited, 18 h, 37°C NA, 18 h, 37°C Cited ISA, 48 h, 25°C 4 (h), 10,000 4 (h), 10,000 4 (h), 25,000 4 (h), 10,000 6, 2500 6 (h), pure 15, 2500 4 (h), 10,000 15, 2500 Proteus sp. ISA, 48 h, 25°C Cited Bac- Bac- 4 (h), 10,000 4 (h), 10,000 Klebsiella pneumoniae ISA, 48 h, 25°C ISA, 48 h, 25°C Klebsiella sp. Bac- Bac- Escherichia coli Flavobacterium suaveolens 4 (h), 25,000 TSA, 24 h, 35°C Bac- Bac- Escherichia coli 15, 2500 6, 2500 Escherichia coli Cited, 18 h, 37°C Bac- 9.5, 2000 4 (h), 10,000 Bac- Cited 4 (h), 25,000 4 (h), 10,000 Escherichia coli TGA, 18–24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C 4 (h), 10,000 Escherichia coli Bac- Bac- Escherichia coli Bac- Enterobacter aerogenes Erwinia carotovora Bac- Bac- Campylobacter jejuni Citrobacter freundii ISA, 48 h, 25°C 4 (h), 10,000 Bac- Bac- Alcaligenes faecalis Beneckea natriegens ISA, 48 h, 25°C 4 (h), 10,000 4 (h), 10,000 ISA, 48 h, 25°C ISA, 48 h, 25°C Bac- Bac- Conditions Acinetobacter calcoaceticus MO Class Aeromonas hydrophila Microorganism TABLE 12.57 Inhibitory Data of Nutmeg Oil Obtained in the Agar Diffusion Test 17 7.6 0 0 0 0 6 5 0 2 9 13 18 6.9 10.5 8 3 0 9 10.5 13.5 5.5 7 8 11.5 10.5 Inhibition Zone (mm) continued Deans and Ritchie (1987) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Janssen et al. (1986) Yousef and Tawil (1980) Pizsolitto et al. (1975) Deans and Ritchie (1987) Pizsolitto et al. (1975) Deans and Ritchie (1987) Pizsolitto et al. (1975) Deans and Ritchie (1987) Deans and Ritchie (1987) Yousef and Tawil (1980) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Janssen et al. (1986) Pizsolitto et al. (1975) Morris et al. (1979) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Ref. In Vitro Antimicrobial Activities of Essential Oils 469 6 (h), pure NA, 18 h, 37°C Bac+ Bac+ Bac+ Bac+ Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus epidermidis Bac+ Bac+ Staphylococcus aureus Staphylococcus aureus Bac+ Bac+ Mycobacterium phlei Staphylococcus aureus 4 (h), 10,000 ISA, 48 h, 25°C Bac+ Bac+ Listeria monocytogenes Micrococcus luteus Cited NA, 18 h, 37°C Cited, 18 h, 37°C TSA, 24 h, 35°C Cited ISA, 48 h, 25°C TGA, 18–24 h, 37°C TSA, 24 h, 35°C ISA, 48 h, 25°C 15, 2500 6 (h), pure 6, 2500 4 (h), 25,000 15, 2500 4 (h), 10,000 9.5, 2000 4 (h), 25,000 4 (h), 10,000 4 (h), 10,000 Bac+ Listeria monocytogenes ISA, 48 h, 25°C Bac+ Bac+ 4 (h), 10,000 9.5, 2000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 Leuconostoc cremoris ISA, 48 h, 25°C TGA, 18–24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C Lactobacillus plantarum Bac+ Bac+ Corynebacterium sp. Bac+ Clostridium sporogenes Brochotrix thermosphacta 6 (h), pure NA, 18 h, 37°C Bac+ Bac+ Bacillus subtilis Brevibacterium linens 6, 2500 Cited, 18 h, 37°C Bac+ Bac+ Bacillus subtilis 4 (h), 10,000 15, 2500 Bacillus subtilis ISA, 48 h, 25°C Cited Bac- Bac+ Yersinia enterocolitica 15, 2500 Bacillus sp. Cited 15, 2500 Bac- Bac- Serratia sp. Shigella sp. Cited 15, 2500 4 (h), 10,000 Cited ISA, 48 h, 25°C Bac- Conditions Bac- MO Class Salmonella sp. (continued) Serratia marcescens Microorganism TABLE 12.57 1 15.5 8.7 6.5 3 0 0 27 0 7.7 0–12 9 7 0 0 5.5 7.5 34 8.7 8 5 10.5 2 3 18 2 Inhibition Zone (mm) Pizsolitto et al. (1975) Yousef and Tawil (1980) Janssen et al. (1986) Smith-Palmer et al. (1998) Pizsolitto et al. (1975) Deans and Ritchie (1987) Morris et al. (1979) Yousef and Tawil (1980) Deans and Ritchie (1987) Smith-Palmer et al. (1998) Lis-Balchin et al. (1998) Deans and Ritchie (1987) Deans and Ritchie (1987) Morris et al. (1979) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Yousef and Tawil (1980) Janssen et al. (1986) Deans and Ritchie (1987) Pizsolitto et al. (1975) Deans and Ritchie (1987) Pizsolitto et al. (1975) Pizsolitto et al. (1975) Deans and Ritchie (1987) Pizsolitto et al. (1975) Ref. 470 Handbook of Essential Oils Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Streptococcus viridans Alternaria porri Aspergillus niger Aspergillus niger Fusarium oxysporum f.sp. cicer Mucor sp. Penicillium chrysogenum Rhizopus sp. Brettanomyces anomalus Candida albicans Candida albicans Candida albicans Candida lipolytica Debaryomyces hansenii Geotrichum candidum Hansenula anomala Kloeckera apiculata Kluyveromyces fragilis Lodderomyces elongisporus Metchnikowia pulcherrima Pichia membranaefaciens Rhodotorula rubra Saccharomyces cerevisiae Torula glabrata Bac+ Streptococcus faecalis PDA, 72 h, 28°C PDA, 48 h, 28°C SDA, 8 d, 30°C PDA, 72 h, 28°C SDA, 8 d, 30°C SDA, 8 d, 30°C SDA, 8 d, 30°C MPA, 4 d, 30°C TGA, 18–24 h, 37°C Cited, 18 h, 37°C SDA, 18 h, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C Cited ISA, 48 h, 25°C 5, 5000 5, 5000 6 (h), pure 5, 5000 6 (h), pure 6 (h), pure 6 (h), pure 5, 10% sol. sd 9.5, 2000 6, 2500 6 (h), pure 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 15, 2500 4 (h), 10,000 8.3 5 40 0 28 60 19 0 0 13 24 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Pawar and Thaker (2007) Pawar and Thaker (2006) Yousef and Tawil (1980) Pawar and Thaker (2007) Yousef and Tawil (1980) Yousef and Tawil (1980) Yousef and Tawil (1980) Conner and Beuchat (1984) Morris et al. (1979) Janssen et al. (1986) Yousef and Tawil (1980) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Pizsolitto et al. (1975) Deans and Ritchie (1987) In Vitro Antimicrobial Activities of Essential Oils 471 472 Handbook of Essential Oils TABLE 12.58 Inhibitory Data of Nutmeg Oil Obtained in the Dilution Test Microorganism MO Class Conditions MIC Campylobacter jejuni Bac- TSB, 24 h, 42°C >10,000 Escherichia coli Bac- 800 Ref. Smith-Palmer et al. (1998) Yousef and Tawil (1980) Escherichia coli Bac- NB, Tween 20, 18 h, 37°C TGB, 18–24 h, 37°C >1000 Morris et al. (1979) Escherichia coli Bac- TSB, 24 h, 35°C >10,000 Pseudomonas aeruginosa Bac- >50,000 Salmonella enteritidis Bac- NB, Tween 20, 18 h, 37°C TSB, 24 h, 35°C Smith-Palmer et al. (1998) Yousef and Tawil (1980) Bacillus subtilis Bac+ >10,000 6400 Smith-Palmer et al. (1998) Yousef and Tawil (1980) Corynebacterium sp. Bac+ NB, Tween 20, 18 h, 37°C TGB, 18–24 h, 37°C 1000 Morris et al. (1979) Listeria monocytogenes Bac+ TSB, 24 h, 35°C <100 Listeria monocytogenes Bac+ TSB, 10 d, 4°C 500 Mycobacterium phlei Bac+ 3200 Smith-Palmer et al. (1998) Smith-Palmer et al. (1998) Yousef and Tawil (1980) Staphylococcus aureus Bac+ NB, Tween 20, 18 h, 37°C TGB, 18–24 h, 37°C 500 Morris et al. (1979) Staphylococcus aureus Bac+ TSB, 24 h, 35°C >10,000 Staphylococcus aureus Bac+ 25,000 Alternaria alternata Aspergillus flavus Fungi Fungi 0% inh. 500 50–100 Feng and Zheng (2007) Thompson (1986) Aspergillus flavus Fungi NB, Tween 20, 18 h, 37°C PDA, 7 d, 28°C PDA, 8 h, 20°C, spore germ. inh. PDA, 5 d, 27°C Smith-Palmer et al. (1998) Yousef and Tawil (1980) >1000 Aspergillus niger Aspergillus niger Fungi Fungi NB, Tween 20, 8 d, 30°C YES broth, 10 d Thompson and Cannon (1986) Yousef and Tawil (1980) Lis-Balchin et al. (1998) Aspergillus ochraceus Fungi YES broth, 10 d Aspergillus parasiticus Fungi Aspergillus parasiticus Fungi PDA, 8 h, 20°C, spore germ. inh. PDA, 5 d, 27°C Epidermophyton floccosum Fusarium culmorum Fungi Mucor hiemalis Mucor mucedo 1600 -88% inh. 10,000 -86% inh. 10,000 50–100 >1000 300–625 Lis-Balchin et al. (1998) Thompson (1986) Thompson and Cannon (1986) Janssen et al. (1988) Fungi SA, Tween 80, 21 d, 20°C YES broth, 10 d ->10,000 Lis-Balchin et al. (1998) Fungi PDA, 5 d, 27°C >1000 Fungi PDA, 5 d, 27°C >1000 Thompson and Cannon (1986) Thompson and Cannon (1986) continued 473 In Vitro Antimicrobial Activities of Essential Oils TABLE 12.58 (continued) Inhibitory Data of Nutmeg Oil Obtained in the Dilution Test Microorganism MO Class Conditions MIC Ref. Mucor racemosus f. racemosus Mucor sp. Penicillium chrysogenum Rhizopus 66-81-2 Fungi PDA, 5 d, 27°C >1000 Fungi Fungi Fungi NB, Tween 20, 8 d, 30°C NB, Tween 20, 8 d, 30°C PDA, 5 d, 27°C 3200 400 >1000 Rhizopus arrhizus Fungi PDA, 5 d, 27°C >1000 Rhizopus chinensis Fungi PDA, 5 d, 27°C >1000 Rhizopus circinans Fungi PDA, 5 d, 27°C >1000 Rhizopus japonicus Fungi PDA, 5 d, 27°C >1000 Rhizopus kazanensis Fungi PDA, 5 d, 27°C >1000 Rhizopus oryzae Fungi PDA, 5 d, 27°C >1000 Rhizopus pymacus Fungi PDA, 5 d, 27°C >1000 Rhizopus sp. Rhizopus stolonifer Fungi Fungi NB, Tween 20, 8 d, 30°C PDA, 5 d, 27°C >1000 Rhizopus tritici Fungi PDA, 5 d, 27°C >1000 Trichophyton mentagrophytes Trichophyton rubrum Fungi 625–1250 300–625 Janssen et al. (1988) Candida albicans Candida albicans Yeast Yeast SA, Tween 80, 21 d, 20°C SA, Tween 80, 21 d, 20°C TGB, 18–24 h, 37°C NB, Tween 20, 18 h, 37°C Thompson and Cannon (1986) Yousef and Tawil (1980) Yousef and Tawil (1980) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Yousef and Tawil (1980) Thompson and Cannon (1986) Thompson and Cannon (1986) Janssen et al. (1988) 500 3200 Morris et al. (1979) Yousef and Tawil (1980) Fungi 50,000 TABLE 12.59 Inhibitory Data of Nutmeg Oil Obtained in the Vapor Phase Test Microorganism MO Class Conditions Activity Ref. Salmonella typhi Bac- NA, 24 h, 37°C Bacillus subtilis var. aterrimus Bac+ NA, 24 h, 37°C sd +++ Maruzzella and Sicurella (1960) sd +++ Mycobacterium avium Bac+ Maruzzella and Sicurella (1960) NA, 24 h, 37°C sd ++ Staphylococcus aureus Maruzzella and Sicurella (1960) Bac+ NA, 24 h, 37°C sd +++ Streptococcus faecalis Maruzzella and Sicurella (1960) Bac+ NA, 24 h, 37°C sd +++ Maruzzella and Sicurella (1960) Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Escherichia coli Flavobacterium suaveolens Klebsiella pneumoniae Klebsiella sp. Moraxella sp. Neisseria perflava Proteus sp. Proteus vulgaris Proteus vulgaris Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Salmonella enteritidis Bac- Escherichia coli Bac- Bac- Escherichia coli Bac- Bac- Erwinia carotovora Escherichia coli Bac- Enterobacter aerogenes Escherichia coli Bac- Citrobacter freundii Bac- Bac- Campylobacter jejuni Escherichia coli Bac- Beneckea natriegens Bac- Bac- Alcaligenes faecalis Bac- Bac- Aeromonas hydrophila Escherichia coli Bac- Escherichia coli Bac- Aerobacter aerogenes MO Class Acinetobacter calcoaceticus Microorganism TSA, 24 h, 35°C ISA, 48 h, 25°C Cited, 18 h, 37°C NA, 24 h, 37°C NA, 18 h, 37°C Cited ISA, 48 h, 25°C NA, 24 h, 37°C Cited NA, 24 h, 37°C ISA, 48 h, 25°C Cited ISA, 48 h, 25°C ISA, 48 h, 25°C TSA, 24 h, 35°C Cited, 18 h, 37°C NA, 18 h, 37°C ISA, 48 h, 25°C Cited NA, 24 h, 37°C TGA, 18–24 h, 37°C NA, 18 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C TSA, 24 h, 42°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C Conditions 4 (h), 25,000 4 (h), 10,000 6, 2500 —, sd 6 (h), pure 15, 2500 4 (h), 10,000 —, sd 15, 2500 —, sd 4 (h), 10,000 15, 2500 4 (h), 10,000 4 (h), 10,000 4 (h), 25,000 5 (h), -30,000 6, 2500 4 (h), 10,000 15, 2500 —, sd 9.5, 2000 6 (h), pure 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 4 (h), 25,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 —, sd 4 (h), 10,000 TABLE 12.60 Inhibitory Data of Peppermint Oil Obtained in the Agar Diffusion Test 6.3 9 0 0 0 0 8 0 10 2 0 0 11 12 6.8 9.7 23 9 5 3 0 0 11 9 8 7.1 16.5 8.5 13 0 14.5 Inhibition Zone (mm) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Janssen et al. (1986) Maruzzella and Lichtenstein (1956) Yousef and Tawil (1980) Pizsolitto et al. (1975) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Pizsolitto et al. (1975) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Pizsolitto et al. (1975) Deans and Ritchie (1987) Deans and Ritchie (1987) Smith-Palmer et al. (1998) Janssen et al. (1986) Schelz et al. (2006) Deans and Ritchie (1987) Pizsolitto et al. (1975) Maruzzella and Lichtenstein (1956) Morris et al. (1979) Yousef and Tawil (1980) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Ref. 474 Handbook of Essential Oils Bac- Bac- Bac- Bac- Bac- Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Serratia sp. Shigella sp. Vibrio cholera Yersinia enterocolitica Bacillus anthracis Bacillus mesentericus Bacillus pumilus Bacillus sp. Bacillus subtilis Bacillus subtilis Bacillus subtilis Bacillus subtilis Brevibacterium linens Brochotrix thermosphacta Clostridium sporogenes Corynebacterium sp. Lactobacillus plantarum Leuconostoc cremoris Listeria monocytogenes Listeria monocytogenes Micrococcus luteus Mycobacterium phlei Sarcina lutea Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Bac- Salmonella sp. Serratia marcescens Bac- Salmonella pullorum Serratia marcescens Bac- Salmonella paratyphi NA, 18 h, 37°C ISA, 48 h, 25°C Cited NA, 24 h, 37°C TGA, 18–24 h, 37°C NA, 24 h, 37°C NA, 18 h, 37°C ISA, 48 h, 25°C TSA, 24 h, 35°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C TGA, 18–24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C Cited, 18 h, 37°C NA, 18 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C Cited NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C Cited Cited ISA, 48 h, 25°C NA, 24 h, 37°C Cited ISA, 48 h, 25°C NA, 24 h, 37°C 6 (h), pure 4 (h), 10,000 15, 2500 —, sd 9.5, 2000 —, sd 6 (h), pure 4 (h), 10,000 4 (h), 25,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 9.5, 2000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 6, 2500 6 (h), pure 4 (h), 10,000 —, sd 15, 2500 6, sd —, sd 6, sd 4 (h), 10,000 6, sd 15, 2500 15, 2500 4 (h), 10,000 —, sd 15, 2500 4 (h), 10,000 6, sd 12 8 8 3 0 5 16 9 5.3 13–20 10 11 0 0 7 12 14.7 21.5 10 8 10 13 3 17 13 16 3 3 20 2 1 15 18 Yousef and Tawil (1980) Deans and Ritchie (1987) Pizsolitto et al. (1975) continued Maruzzella and Lichtenstein (1956) Morris et al. (1979) Maruzzella and Lichtenstein (1956) Yousef and Tawil (1980) Deans and Ritchie (1987) Smith-Palmer et al. (1998) Lis-Balchin et al. (1998) Deans and Ritchie (1987) Deans and Ritchie (1987) Morris et al. (1979) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Janssen et al. (1986) Yousef and Tawil (1980) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Pizsolitto et al. (1975) Dube and Rao (1984) Maruzzella and Lichtenstein (1956) Dube and Rao (1984) Deans and Ritchie (1987) Dube and Rao (1984) Pizsolitto et al. (1975) Pizsolitto et al. (1975) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Pizsolitto et al. (1975) Deans and Ritchie (1987) Dube and Rao (1984) In Vitro Antimicrobial Activities of Essential Oils 475 Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Staphylococcus aureus Staphylococcus epidermidis Staphylococcus epidermidis Streptococcus faecalis Streptococcus viridans Streptomyces venezuelae Absidia cormybifera Alternaria porri Alternaria solani Aspergillus flavus Aspergillus fumigatus Aspergillus fumigatus Aspergillus fumigatus Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus sulphureus Fusarium oxysporum f.sp. cicer Helminthosporium sativum Humicola grisea var. thermoidea Keratinomyces afelloi Keratinophyton terreum Microsporum gypseum Mucor fragilis Mucor mucedo Mucor sp. Nigrospora panici Penicillium chrysogenum Penicillium digitatum Bac+ MO Class Staphylococcus aureus (continued) Staphylococcus aureus Microorganism TABLE 12.60 EYA, 48 h, 45°C PDA, 72 h, 28°C SMA, 2–7 d, 20°C PDA, 10 d, 25°C PDA, 10 d, 25°C SMA, 2–7 d, 20°C SDA, 3 d, 28°C PDA, 48 h, 28°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C PDA, 10 d, 25°C PDA, 72 h, 28°C SMA, 2–7 d, 20°C EYA, 48 h, 45°C SDA, 3 d, 28°C SDA, 3 d, 28°C SDA, 3 d, 28°C PDA, 10 d, 25°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C Cited ISA, 48 h, 25°C NA, 18 h, 37°C Cited TSA, 24 h, 35°C Cited, 18 h, 37°C NA, 24 h, 37°C Conditions 5, sd 5, 5000 sd 10 10 sd 6, sd 5, 5000 sd 6 (h), pure 10 5, 5000 sd 5, sd 6, sd 6, sd 6, sd 10 sd 6 (h), pure sd 6 (h), pure sd sd 15, 2500 5 (h), -30,000 4 (h), 10,000 15, 2500 4 (h), 25,000 6, 2500 6, sd 0 10.6 0 – – 4 10 0 6 60 – 10 7 30 18 0 10 – 8 23 8 60 8 3 0 0 15 10 6.4 19 13 Inhibition Zone (mm) Nigam and Rao (1979) Pawar and Thaker (2007) Maruzzella and Liguori (1958) Sarbhoy et al. (1978) Sarbhoy et al. (1978) Maruzzella and Liguori (1958) Saksena and Saksena (1984) Pawar and Thaker (2006) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Sarbhoy et al. (1978) Pawar and Thaker (2007) Maruzzella and Liguori (1958) Nigam and Rao (1979) Saksena and Saksena (1984) Saksena and Saksena (1984) Saksena and Saksena (1984) Sarbhoy et al. (1978) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Pizsolitto et al. (1975) Deans and Ritchie (1987) Schelz et al. (2006) Pizsolitto et al. (1975) Smith-Palmer et al. (1998) Janssen et al. (1986) Dube and Rao (1984) Ref. 476 Handbook of Essential Oils Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Rhizopus nigricans Rhizopus sp. Rhizopus stolonifer Sporotrichum thermophile Thermoascus aurantiacis Thermomyces lanuginosa Thielava minor Trichophyton equinum Trichophyton rubrum Brettanomyces anomalus Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Candida krusei Candida lipolytica Candida tropicalis Candida tropicalis Cryptococcus neoformans Cryptococcus rhodobenhani Debaryomyces hansenii Geotrichum candidum Hansenula anomala Kloeckera apiculata Kluyveromyces fragilis Lodderomyces elongisporus Metchnikowia pulcherrima Pichia membranaefaciens Rhodotorula rubra Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Torula glabrata Torula thermophila MPA, 4 d, 30°C EYA, 48 h, 45°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C PDA, 10 d, 25°C EYA, 48 h, 45°C EYA, 48 h, 45°C EYA, 48 h, 45°C EYA, 48 h, 45°C SDA, 3 d, 28°C SDA, 3 d, 28°C MPA, 4 d, 30°C TGA, 18–24 h, 37°C SMA, 2–7 d, 20°C Cited, 18 h, 37°C SDA, 18 h, 30°C SDA, 3 d, 28°C SMA, 2–7 d, 20°C MPA, 4 d, 30°C SMA, 2–7 d, 20°C SDA, 3 d, 28°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C SMA, 2–7 d, 20°C NA, 24 h, 20°C 5 (h), -30,000 5, 10% sol. sd 5, sd sd 6 (h), pure 10 5, sd 5, sd 5, sd 5, sd 6, sd 6, sd 5, 10% sol. sd 9.5, 2000 sd 6, 2500 6 (h), pure 6, sd sd 5, 10% sol. sd sd 6, sd sd sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd sd 8 0 – 0 0 0 15 14 16 0 0 4 10 16 18 1 0 3 23 3 2 0 7 0 0 0 0 9 0 7 0 2 12–15 7 22 Conner and Beuchat (1984) Nigam and Rao (1979) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Sarbhoy et al. (1978) Nigam and Rao (1979) Nigam and Rao (1979) Nigam and Rao (1979) Nigam and Rao (1979) Saksena and Saksena (1984) Saksena and Saksena (1984) Conner and Beuchat (1984) Morris et al. (1979) Maruzzella and Liguori (1958) Janssen et al. (1986) Yousef and Tawil (1980) Saksena and Saksena (1984) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Maruzzella and Liguori (1958) Saksena and Saksena (1984) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Maruzzella and Liguori (1958) Schelz et al. (2006) In Vitro Antimicrobial Activities of Essential Oils 477 Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Capnocytophaga sp. Eikenella corrodens Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Fusobacterium nucleatum Helicobacter pylori Helicobacter pylori Porphyromonas gingivalis Prevotella buccae Prevotella intermedia Prevotella nigrescens Pseudomonas aeruginosa Salmonella enteritidis Salmonella enteritidis Selenomonas artemidis Actinomyces viscosus Bacillus subtilis Corynebacterium sp. Listeria monocytogenes Mycobacterium phlei NB, Tween 20, 18 h, 37°C TSB, 24 h, 35°C TGB, 18–24 h, 37°C NB, Tween 20, 18 h, 37°C HS, 16–24, 37°C HS, 72 h, 37°C TSB, 24 h, 35°C Cited, 24 h NB, Tween 20, 18 h, 37°C HS, 72 h, 37°C HS, 72 h, 37°C HS, 72 h, 37°C HS, 72 h, 37°C Cited, 20 h, 37°C Cited, 48 h HS, 72 h, 37°C TSB, 24 h, 35°C MHB, 24 h, 36°C TGB, 18 h, 37°C NB, Tween 20, 18 h, 37°C TGB, 18–24 h, 37°C Cited, 24 h HS, 72 h, 37°C MIC 400 >1000 300 1600 5000 >10,000 1000 >50,000 400 2000 3000 2000 2000 135.6 100 >10,000 2000 >10,000 5700 >1000 1600 800 2000 3000 1000 TSB, 24 h, 42°C Bac- HS, 72 h, 37°C 1000 2000 3000 Conditions HS, 72 h, 37°C HS, 72 h, 37°C HS, 72 h, 37°C Bac Bac MO Class Treponema denticola Treponema vincentii Actinobacillus actinomycetemcomitans Campylobacter jejuni Microorganism TABLE 12.61 Inhibitory Data of Peppermint Oil Obtained in the Dilution Test Yousef and Tawil (1980) Smith-Palmer et al. (1998) Morris et al. (1979) Yousef and Tawil (1980) Shapiro et al. (1994) Shapiro et al. (1994) Smith-Palmer et al. (1998) Imai et al. (2001) Yousef and Tawil (1980) Shapiro et al. (1994) Shapiro et al. (1994) Shapiro et al. (1994) Shapiro et al. (1994) Weseler et al. (2005) Imai et al. (2001) Shapiro et al. (1994) Smith-Palmer et al. (1998) Duarte et al. (2006) Schelz et al. (2006) Yousef and Tawil (1980) Morris et al. (1979) Imai et al. (2001) Shapiro et al. (1994) Shapiro et al. (1994) Smith-Palmer et al. (1998) Shapiro et al. (1994) Shapiro et al. (1994) Shapiro et al. (1994) Ref. 478 Handbook of Essential Oils Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus MRSA Staphylococcus aureus MSSA Staphylococcus epidermidis Streptococcus sanguinis Streptococcus sobrinus Aspergillus niger Aspergillus niger Aspergillus ochraceus Aspergillus oryzae Aspergillus parasiticus Aspergillus parasiticus Botrytis cinera Epidermophyton floccosum Fusarium culmorum Geotrichum citri-aurantii Microsporum gypseum Mucor hiemalis Mucor mucedo Mucor racemosus Mucor racemosus f. racemosus Mucor sp. Penicillium chrysogenum Penicillium chrysogenum Penicillium digitatum Phytophthora citrophthora Rhizopus 66-81-2 Aspergillus flavus Aspergillus flavus Bac+ Peptostreptococcus anaerobius NB, Tween 20, 8 d, 30°C Cited NB, Tween 20, 8 d, 30°C PDA, Tween 20, 7 d, 24°C PDA, Tween 20, 7 d, 24°C PDA, 5 d, 27°C Cited PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, Tween 20, 7 d, 24°C SDA, 7 d, 30°C PDA, 5 d, 27°C PDA, Tween 20, 7 d, 24°C SA, Tween 80, 21 d, 20°C YES broth, 10 d Cited PDA, 8 h, 20°C, spore germ. inh. PDA, 5 d, 27°C YES broth, 10 d NB, Tween 20, 8 d, 30°C YES broth, 10 d PDA, 8 h, 20°C, spore germ. inh. PDA, 5 d, 27°C HS, 16–24, 37°C HS, 16–24, 37°C TGB, 18 h, 37°C Cited, 24 h Cited, 24 h NB, Tween 20, 18 h, 37°C TGB, 18–24 h, 37°C TSB, 24 h, 35°C HS, 72 h, 37°C >1000 >1000 1600 500 3200 0% inh. 1000 14% inh. 1000 >1000 500 >1000 - >10,000 0% inh. 1000 400, 37% inh. >1000 0% inh. 1000 300–625 - 93% inh. 10,000 500 50–100 - 98% inh. 10,000 >1000 800 50–100 3000 6000 5700 200 200 6400 1000 400 2000 continued Yousef and Tawil (1980) Okazaki and Oshima (1953) Yousef and Tawil (1980) Bouchra et al. (2003) Bouchra et al. (2003) Thompson and Cannon (1986) Okazaki and Oshima (1953) Thompson and Cannon (1986) Thompson and Cannon (1986) Bouchra et al. (2003) Dikshit and Husain (1984) Thompson and Cannon (1986) Bouchra et al. (2003) Janssen et al. (1988) Lis-Balchin et al. (1998) Okazaki and Oshima (1953) Thompson (1986) Thompson and Cannon (1986) Lis-Balchin et al. (1998) Yousef and Tawil (1980) Lis-Balchin et al. (1998) Thompson (1986) Thompson and Cannon (1986) Shapiro et al. (1994) Shapiro et al. (1994) Schelz et al. (2006) Imai et al. (2001) Imai et al. (2001) Yousef and Tawil (1980) Morris et al. (1979) Smith-Palmer et al. (1998) Shapiro et al. (1994) In Vitro Antimicrobial Activities of Essential Oils 479 MO Class Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Rhizopus arrhizus Rhizopus chinensis Rhizopus circinans Rhizopus japonicus Rhizopus kazanensis Rhizopus oryzae Rhizopus pymacus Rhizopus sp. Rhizopus stolonifer Rhizopus tritici Trichophyton equinum Trichophyton mentagrophytes Trichophyton rubrum Trichophyton rubrum Candida albicans Candida albicans Candida albicans Saccharomyces cerevisiae (continued) Microorganism TABLE 12.61 MHB, Tween 80, 48 h, 35°C YPB, 24 h, 20°C SDA, 7 d, 30°C SA, Tween 80, 21 d, 20°C SDA, 7 d, 30°C SA, Tween 80, 21 d, 20°C NB, Tween 20, 18 h, 37°C TGB, 18–24 h, 37°C PDA, 5 d, 27°C NB, Tween 20, 8 d, 30°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C Conditions >1000 5000 400 >1000 400, 51% inh. 625–1250 400, 61% inh. 300–625 800 >1000 >1000 6400 >1000 >1000 >1000 >1000 >1000 >1000 MIC Hammer et al. (1998) Schelz et al. (2006) Dikshit and Husain (1984) Janssen et al. (1988) Dikshit and Husain (1984) Janssen et al. (1988) Yousef and Tawil (1980) Morris et al. (1979) Thompson and Cannon (1986) Yousef and Tawil (1980) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Ref. 480 Handbook of Essential Oils Bac+ Bac+ Bac+ Fungi Streptococcus pneumoniae Streptococcus pyogenes Streptococcus pyogenes WFA, 42 d, 25°C WFA, 42 d, 25°C WFA, 42 d, 25°C Fungi Eurotium herbarum PDA, 3 d, 25°C Yeast Candida albicans PDA, 3 d, 25°C Fungi Fungi Pythium ultimum Penicillium corylophilum Rhizoctonia solani WFA, 42 d, 25°C Fungi Fungi Fusarium oxysporum NA, 24 h, 37°C PDA, 3 d, 25°C PDA, 3 d, 25°C WFA, 42 d, 25°C Fungi Fungi Eurotium repens Eurotium rubrum WFA, 42 d, 25°C Fungi Fungi PDA, 3 d, 25°C Colletotrichum gleosporoides Botrytis cinera WFA, 42 d, 25°C MHA, 18 h, 37°C NA, 24 h, 37°C MHA, 18 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C MHA, 18 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Eurotium amstelodami Fungi Fungi Aspergillus niger Aspergillus flavus Bac+ Bac+ Streptococcus faecalis Bac+ Staphylococcus aureus Streptococcus faecalis Bac+ Staphylococcus aureus NA, 24 h, 37°C Bac+ Bac+ Corynebacterium diphtheriae Mycobacterium avium Bac+ Bac+ Bacillus subtilis var. aterrimus Staphylococcus aureus NA, 24 h, 37°C Bac+ Bacillus megaterium NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C MHA, 18 h, 37°C Bac- Neisseria sp. Bac- Bac- Haemophilus influenzae NA, 24 h, 37°C BLA, 18 h, 37°C Salmonella typhi Bac- Escherichia coli Conditions Salmonella typhi BacBac- Escherichia coli MO Class Microorganism TABLE 12.62 Inhibitory Data of Peppermint Oil Obtained in the Vapor Phase Test ~20,000 1000 1000 Disk, 50,000 1000 Disk, 50,000 Disk, 50,000 Disk, 50,000 Disk, 50,000 1000 1000 Disk, 50,000 Disk, 50,000 ~20,000 MICair MICair ~20,000 sd MICair ~20,000 sd ~20,000 sd ~20,000 sd ~20,000 sd ~20,000 MICair ~20,000 MICair + +++ +++ + +++ +++ ++ +++ ++ +++ +++ +++ +++ 25 NG +++ 25 + ++ 25 + + NG ++ + +++ + NG >1600 12.5 ++ Activity Kellner and Kober (1954) Lee et al. (2007) Lee et al. (2007) Guynot et al. (2003) Lee et al. (2007) Guynot et al. (2003) Guynot et al. (2003) Guynot et al. (2003) Guynot et al. (2003) Lee et al. (2007) Lee et al. (2007) Guynot et al. (2003) Guynot et al. (2003) Inouye et al. (2001) Kellner and Kober (1954) Inouye et al. (2001) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Inouye et al. (2001) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Kellner and Kober (1954) Inouye et al. (2001) Inouye et al. (2001) Kellner and Kober (1954) Ref. In Vitro Antimicrobial Activities of Essential Oils 481 Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Bordetella bronchiseptica Citrobacter freundii Escherichia coli Escherichia coli Escherichia coli 1 Escherichia coli 2 Klebsiella pneumoniae Neisseria perflava Proteus mirabilis Proteus vulgaris Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Serratia marcescens Bacillus mesentericus Bacillus sp. Bacillus subtilis Bacillus subtilis Clostridium perfringens MO Class Aerobacter aerogenes Microorganism MHA, 18 h, 37°C Cited, 18 h, 37°C NA, 24 h, 37°C MHA, 18 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C Cited, 18 h, 37°C MHA, 18 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C MHA, 18 h, 37°C NA, 24 h, 37°C MHA, 18 h, 37°C MHA, 18 h, 37°C MHA, 18 h, 37°C Cited, 18 h, 37°C NA, 24 h, 37°C MHA, 18 h, 37°C MHA, 18 h, 37°C NA, 24 h, 37°C Conditions 6, 17,500 6, 2500 —, sd 6, 17,500 —, sd —, sd 6, 2500 6, 17,500 —, sd —, sd 6, 17,500 —, sd 6, 17,500 6, 17,500 6, 17,500 6, 2500 —, sd 6, 17,500 6, 17,500 —, sd TABLE 12.63 Inhibitory Data of Pinus sylvestris Oil Obtained in the Agar Diffusion Test 23 8.7 3 11 2 0 0 0 0 4 0 3 0 0 8 11.7 1 0 0 0 Inhibition Zone (mm) Schales et al. (1993) Janssen et al. (1986) Maruzzella and Lichtenstein (1956) Schales et al. (1993) Maruzzella and Lichtenstein (1956) Maruzzella and Lichtenstein (1956) Janssen et al. (1986) Schales et al. (1993) Maruzzella and Lichtenstein (1956) Maruzzella and Lichtenstein (1956) Schales et al. (1993) Maruzzella and Lichtenstein (1956) Schales et al. (1993) Schales et al. (1993) Schales et al. (1993) Janssen et al. (1986) Maruzzella and Lichtenstein (1956) Schales et al. (1993) Schales et al. (1993) Maruzzella and Lichtenstein (1956) Ref. 482 Handbook of Essential Oils Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Yeast Sarcina lutea Staphylococcus aureus Staphylococcus aureus Staphylococcus sp. Streptomyces venezuelae Alternaria solani Aspergillus fumigatus Aspergillus niger Helminthosporium sativum Mucor mucedo Nigrospora panici Penicillium digitatum Rhizopus nigricans Candida albicans Candida albicans Candida krusei Candida tropicalis Cryptococcus neoformans Cryptococcus rhodobenhani Saccharomyces cerevisiae Bac+ Enterococcus sp. SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C Cited, 18 h, 37°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C MHA, 18 h, 37°C Cited, 18 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C MHA, 18 h, 37°C sd sd sd sd sd sd sd sd sd 6, 2500 sd sd sd sd sd sd 6, 17,500 6, 2500 —, sd —, sd 6, 17,500 8 9 12 12 15 12 10 5 5 16.3 5 10 6 14 18 7 6 7.7 2 0 15 Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Janssen et al. (1986) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Schales et al. (1993) Janssen et al. (1986) Maruzzella and Lichtenstein (1956) Maruzzella and Lichtenstein (1956) Schales et al. (1993) In Vitro Antimicrobial Activities of Essential Oils 483 Alternaria alternata Aspergillus flavus Aspergillus flavus var. columnaris Aspergillus fumigatus Aspergillus niger Aspergillus niger Aspergillus versicolor Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Staphylococcus aureus Staphylococcus aureus CA, 7 d, 25°C Bac+ Bac+ Rhodococcus sp. CA, 7 d, 25°C MHA, Tween 20, 48 h, 35°C Bac+ Bac+ Bacillus sp. Enterococcus faecalis Staphylococcus aureus MHA, Tween 20, 48 h, 35°C BacBac- Salmonella typhimurium Serratia marcescens MHA, Tween 20, 48 h, 35°C RPMI, 1.5% EtOH, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C CA, 7 d, 25°C CA, 7 d, 25°C MHB, Tween 80, 24 h, 37°C MHB, Tween 80, 24 h, 37°C MHA, Tween 20, 48 h, 35°C MHA, Tween 20, 48 h, 35°C MHB, Tween 80, 24 h, 37°C BacBac- Pseudomonas aeruginosa MHB, Tween 80, 24 h, 37°C MHA, Tween 20, 48 h, 35°C MHB, Tween 80, 24 h, 37°C MHB, Tween 80, 24 h, 37°C MHA, Tween 20, 48 h, 35°C MHB, Tween 80, 24 h, 37°C MHA, Tween 20, 48 h, 35°C MHA, Tween 20, 48 h, 35°C Conditions Pseudomonas aeruginosa BacBac- Klebsiella pneumoniae Bac- Klebsiella pneumonia Proteus mirabilis BacBac- Escherichia coli Escherichia coli Escherichia coli BacBac- Aeromonas sobria Bac- MO Class Acinetobacter baumannii Microorganism TABLE 12.64 Inhibitory Data of Pinus sylvestris Oil Obtained in the Dilution Test 10,000 5000 1250 5000–10,000 5000 7500–15,000 7500–15,000 4000 >20,000 3500 >20,000 5000 >20,000 5000 >20,000 >29,000 >20,000 >29,000 >20,000 3500 64,300 >29,000 20,000 20,000 20,000 MIC Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Motiejunaite and Peciulyte (2004) Motiejunaite and Peciulyte (2004) Bastide et al. (1987) Chalchat et al. (1989) Hammer et al. (1999) Motiejunaite and Peciulyte (2004) Hammer et al. (1999) Motiejunaite and Peciulyte (2004) Hammer et al. (1999) Hammer et al. (1999) Chalchat et al. (1989) Hammer et al. (1999) Chalchat et al. (1989) Hammer et al. (1999) Chalchat et al. (1989) Bastide et al. (1987) Hammer et al. (1999) Chalchat et al. (1989) Hammer et al. (1999) Hammer et al. (1999) Ref. 484 Handbook of Essential Oils Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Aureobasidum pullulans Chaetomium globosum Cladosporium cladosporoides Cladosporium cladosporoides Epidermophyton floccosum Fusarium oxysporum Microsporum canis Microsporum gypseum Mucor sp. Paecilomyces variotii Penicillium chrysogenum Penicillium frequentans Penicillium lanosum Phoma glomerata Phoma sp. Rhizopus sp. Rhizopus stolonifer Scopulariopsis brevicaulis Stachybotrys chartarum Trichoderma viride Trichophyton mentagrophytes Candida albicans Candida albicans Candida lipolytica Geotrichum candida CA, 7 d, 25°C RPMI, 1.5% EtOH, 7 d, 30°C CA, 7 d, 25°C CA, 7 d, 25°C RPMI, 1.5% EtOH, 7 d, 30°C MHB, Tween 80, 24 h, 37°C MHA, Tween 20, 48 h, 35°C CA, 7 d, 25°C CA, 7 d, 25°C CA, 7 d, 25°C RPMI, 1.5% EtOH, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C CA, 7 d, 25°C CA, 7 d, 25°C RPMI, 1.5% EtOH, 7 d, 30°C CA, 7 d, 25°C CA, 7 d, 25°C RPMI, 1.5% EtOH, 7 d, 30°C CA, 7 d, 25°C RPMI, 1.5% EtOH, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C CA, 7 d, 25°C >10,000 5000–7500 10,000 10,000–15,000 5500 2500–5000 14,000 20,000 5000 3500–5000 10,000–25,000 1250 10,000 10,000–25,000 7500 >10,000 5000–7500 5000 1250–2500 5000 1250 312 1250–5000 2500–5000 Motiejunaite and Peciulyte (2004) Tullio et al. (2006) Motiejunaite and Peciulyte (2004) Motiejunaite and Peciulyte (2004) Tullio et al. (2006) Chalchat et al. (1989) Hammer et al. (1999) Motiejunaite and Peciulyte (2004) Motiejunaite and Peciulyte (2004) Motiejunaite and Peciulyte (2004) Motiejunaite and Peciulyte (2004) Tullio et al. (2006) Motiejunaite and Peciulyte (2004) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Thanaboripat et al. (2004) Motiejunaite and Peciulyte (2004) Motiejunaite and Peciulyte (2004) Tullio et al. (2006) Tullio et al. (2006) Motiejunaite and Peciulyte (2004) Motiejunaite and Peciulyte (2004) Tullio et al. (2006) In Vitro Antimicrobial Activities of Essential Oils 485 MO Class Fungi Fungi Fungi Fungi Fungi Microorganism Botrytis cinera Colletotrichum gleosporoides Fusarium oxysporum Pythium ultimum Rhizoctonia solani PDA, 3 d, 25°C PDA, 3 d, 25°C PDA, 3 d, 25°C PDA, 3 d, 25°C PDA, 3 d, 25°C Conditions Table 12.65 Inhibitory Data of Pinus sylvestris Oil Obtained in the Vapor Phase Test 1000 1000 1000 1000 1000 +++ +++ +++ +++ +++ Activity Ref. Lee et al. (2007) Lee et al. (2007) Lee et al. (2007) Lee et al. (2007) Lee et al. (2007) 486 Handbook of Essential Oils 4 (h), 10,000 ISA, 48 h, 25°C Bac- Bac- Bac- Bac- Escherichia coli Escherichia coli Escherichia coli Escherichia coli Bac- Bac- Escherichia coli Escherichia coli Bac- Bac- Escherichia coli Escherichia coli Bac- Bac- Escherichia coli Escherichia coli Bac- Bac- Escherichia coli Escherichia coli Bac- Bac- Enterobacter aerogenes Erwinia carotovora Bac- Bac- Citrobacter freundii Enterobacter aerogenes 4 (h), 25,000 TSA, 24 h, 42°C Bac- Bac- Campylobacter jejuni NA, 24 h, 37°C NA, 18 h, 37°C NA, 18 h, 37°C TSA, 24 h, 35°C Cited, 18 h, 37°C MHA, 24 h, 30°C Cited, 24 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C NA, 24 h, 30°C Cited Cited ISA, 48 h, 25°C ISA, 48 h, 25°C MHA, 24 h, 30°C ISA, 48 h, 25°C 10 (h), 10,0000 6 (h), pure 5 (h), -30,000 4 (h), 25,000 6, 2500 6, 15,000 — 4 (h), 10,000 —, sd Drop, 5000 15, 2500 (h) 20,000 4 (h), 10,000 4 (h), 10,000 6, 15,000 4 (h), 10,000 4 (h), 10,000 Beneckea natriegens ISA, 48 h, 25°C Bac- (h) 20,000 4 (h), 10,000 Bac- Cited ISA, 48 h, 25°C 4 (h), 10,000 —, sd Alcaligenes faecalis Bac- Aerobacter tumefaciens NA, 24 h, 37°C ISA, 48 h, 25°C Conditions Aeromonas hydrophila Bac- Bac- Acinetobacter calcoaceticus MO Class Aerobacter aerogenes Microorganism TABLE 12.66 Inhibitory Data of Rosemary Oil Obtained in the Agar Diffusion Test 18 13.5 15 8.7 8.7 <12 12 12 3 1 - to +++ 1 7.5 8.5 7 8 9.3 0 0 7 — 3 15 Inhibition Zone (mm) continued Narasimha Rao and Nigam (1970) Yousef and Tawil (1980) Schelz et al. (2006) Smith-Palmer et al. (1998) Janssen et al. (1986) Rossi et al. (2007) Rota et al. (2004) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Hili et al. (1997) Pizsolitto et al. (1975) Hethenyi et al. (1989) Deans and Ritchie (1987) Deans and Ritchie (1987) Rossi et al. (2007) Deans and Ritchie (1987) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Hethenyi et al. (1989) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Ref. In Vitro Antimicrobial Activities of Essential Oils 487 MO Class Conditions Bac- Salmonella enteritidis (h) 20,000 Cited Bac- Bac- Pseudomonas pisi Pseudomonas tabaci 6, 2500 Cited, 18 h, 37°C Bac- Bac- Pseudomonas aeruginosa TSA, 24 h, 35°C Cited NA, 18 h, 37°C —, sd 4 (h), 25,000 (h) 20,000 6 (h), pure 6, 15,000 Pseudomonas aeruginosa NA, 24 h, 37°C MHA, 24 h, 30°C Bac- Bac- Drop, 5000 4 (h), 10,000 15, 2500 (h) 20,000 4 (h), 10,000 —, sd w 20,000 15, 2500 (h) 20,000 —, sd 4 (h), 10,000 Pseudomonas aeruginosa NA, 24 h, 30°C ISA, 48 h, 25°C Cited Cited ISA, 48 h, 25°C NA, 24 h, 37°C Cited Cited Cited NA, 24 h, 37°C ISA, 48 h, 25°C Pseudomonas aeruginosa Bac- Bac- Pseudomonas aeruginosa Pseudomonas aeruginosa Bac- Bac- Pseudomonas aeruginosa Pseudomonas aeruginosa Bac- Bac- Proteus vulgaris Proteus vulgaris Bac- Bac- Proteus vulgaris Bac- Proteus sp. Peptobacterium carotovorum 4 (h), 10,000 ISA, 48 h, 25°C Bac- Bac- Moraxella sp. Neisseria perflava 15, 2500 Cited Bac- Bac- Klebsiella pneumoniae (h) 20,000 10 (h), 10,0000 Klebsiella sp. Cited NA, 24 h, 37°C Bac- Bac- 4 (h), 10,000 Haemophilus influenza ISA, 48 h, 25°C Klebsiella aerogenes Bac- (continued) Flavobacterium suaveolens Microorganism TABLE 12.66 +++ 9.3 ++ 11 11 6 2 0 0 +++ 0 8.5 +++ 0 +++ 0 3 9.5 0 6 20 — 8.5 Inhibition Zone (mm) Ref. Smith-Palmer et al. (1998) Hethenyi et al. (1989) Hethenyi et al. (1989) Janssen et al. (1986) Yousef and Tawil (1980) Rossi et al. (2007) Maruzzella and Lichtenstein (1956) Hili et al. (1997) Deans and Ritchie (1987) Pizsolitto et al. (1975) Hethenyi et al. (1989) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Hethenyi et al. (1989) Pizsolitto et al. (1975) Hethenyi et al. (1989) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Pizsolitto et al. (1975) Deans and Ritchie (1987) Narasimha Rao and Nigam (1970) Hethenyi et al. (1989) Deans and Ritchie (1987) 488 Handbook of Essential Oils 15, 2500 Cited Bac- Bac- Bac+ Yersinia enterocolitica Bacillus mesentericus Bac+ Bac+ Bac+ Bac+ Bac+ Clostridium sporogenes Corynebacterium diphtheriae Corynebacterium fascians Lactobacillus plantarum 4 (h), 10,000 ISA, 48 h, 25°C Bac+ Brevibacterium linens Brochotrix thermosphacta 6 (h), pure NA, 18 h, 37°C Bac+ Bac+ Bacillus subtilis ISA, 48 h, 25°C Cited NA, 24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C —, sd 4 (h), 10,000 (h) 20,000 10 (h), 100,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 6, 2500 Bacillus subtilis NA, 24 h, 37°C Cited, 18 h, 37°C Bac+ Bac+ (h) 20,000 4 (h), 10,000 (h) 20,000 10 (h), 100,000 —, sd Bacillus subtilis Cited ISA, 48 h, 25°C Cited NA, 24 h, 37°C NA, 24 h, 37°C Bacillus subtilis Bac+ —, sd NA, 24 h, 37°C Bac- Xanthomonas versicolor Bac+ 15, 2500 Cited Bac- Shigella sp. Vibrio cholerae Bacillus sp. 15, 2500 (h) 20,000 Cited Cited Bac- Bac- Serratia sp. Shigella sonnei Bacillus subtilis 4 (h), 10,000 ISA, 48 h, 25°C Bac- Bac- Serratia marcescens 10 (h), 100,000 Serratia marcescens 15, 2500 Cited NA, 24 h, 37°C Bac- Bac- Salmonella sp. Salmonella typhi — 4 (h), 10,000 Cited, 24 h, 37°C ISA, 48 h, 25°C Bac- Bac- Salmonella enteritidis Salmonella pullorum ++ 8 30 13.5 6 5.5 22.5 20 8 8 0 3 +++ 0 22 ++ 3 0 7.5 4 18 0 <12 5 Rota et al. (2004) Deans and Ritchie (1987) Hethenyi et al. (1989) continued Narasimha Rao and Nigam (1970) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Yousef and Tawil (1980) Deans and Ritchie (1987) Janssen et al. (1986) Maruzzella and Lichtenstein (1956) Hethenyi et al. (1989) Pizsolitto et al. (1975) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Hethenyi et al. (1989) Narasimha Rao and Nigam (1970) Pizsolitto et al. (1975) Hethenyi et al. (1989) Pizsolitto et al. (1975) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Narasimha Rao and Nigam (1970) Pizsolitto et al. (1975) Deans and Ritchie (1987) In Vitro Antimicrobial Activities of Essential Oils 489 4 (h), 10,000 9, 20,000 ISA, 48 h, 25°C MHA, cited Bac+ Bac+ Micrococcus luteus Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Staphylococcus epidermidis Staphylococcus epidermidis Staphylococcus epidermidis Staphylococcus epidermidis Streptococcus aegui Streptococcus D Staphylococcus aureus Staphylococcus aureus Bac+ Staphylococcus aureus Staphylococcus aureus Bac+ Bac+ Staphylococcus aureus Bac+ Bac+ Staphylococcus aureus Bac+ Staphylococcus aureus Bac+ Staphylococcus aureus Staphylococcus aureus 9, 20,000 MHA, cited Bac+ Bac+ Sarcina ureae Staphylococcus aureus —, sd NA, 24 h, 37°C Bac+ Bac+ Pneumococcus sp. MHA, cited Cited NA, 24 h, 37°C MHA, cited NA, 18 h, 37°C Cited NA, 18 h, 37°C Cited, 18 h, 37°C MHA, 24 h, 37°C TSA, 24 h, 35°C ISA, 48 h, 25°C Cited NA, 24 h, 37°C NA, 24 h, 30°C NA, 24 h, 37°C Cited Cited 9, 20,000 (h) 20,000 10 (h), 100,000 5 (h), -30,000 9, 20,000 15, 2500 6 (h), pure 6, 2500 6, 15,000 4 (h), 25,000 4 (h), 10,000 15, 2500 —, sd Drop, 5000 10 (h), 10,0000 (h) 20,000 (h) 20,000 6 (h), pure Sarcina lutea NA, 18 h, 37°C Bac+ Bac+ Micrococcus ureae 9, 20,000 Mycobacterium phlei MHA, cited 4 (h), 25,000 4 (h), 10,000 Micrococcus luteus ISA, 48 h, 25°C Bac+ Bac+ TSA, 24 h, 35°C 9, 20,000 4 (h), 10,000 Listeria monocytogenes ISA, 48 h, 25°C MRS, cited Conditions Listeria monocytogenes Bac+ Bac+ MO Class Lactobacillus sp. (continued) Leuconostoc cremoris Microorganism TABLE 12.66 + 0–30 22 12 0 0 17.9 10.7 16 5.9 12 10 2 0 0 >90 — ++ 0 23 21 6 18–26 16 7.1 16.5 – >90 8.5 Inhibition Zone (mm) Pellecuer et al. (1980) Hethenyi et al. (1989) Narasimha Rao and Nigam (1970) Pellecuer et al. (1980) Schelz et al. (2006) Pizsolitto et al. (1975) Yousef and Tawil (1980) Janssen et al. (1986) Rossi et al. (2007) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Pizsolitto et al. (1975) Maruzzella and Lichtenstein (1956) Hili et al. (1997) Narasimha Rao and Nigam (1970) Hethenyi et al. (1989) Pellecuer et al. (1980) Maruzzella and Lichtenstein (1956) Hethenyi et al. (1989) Yousef and Tawil (1980) Pellecuer et al. (1980) Deans and Ritchie (1987) Pellecuer et al. (1980) Lis-Balchin et al. (1998) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Pellecuer et al. (1980) Ref. 490 Handbook of Essential Oils Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Streptococcus viridans Streptomyces venezuelae Alternaria porri Alternaria solani Aspergillus flavus Aspergillus fumigatus Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus niger Fusarium moniliforme Fusarium oxysporum f.sp. cicer Fusarium solani Helminthosporium sativum Mucor mucedo Mucor sp. Nigrospora panici Ophiobolus graminis Penicillium chrysogenum Penicillium digitatum Rhizopus nigricans Rhizopus sp. Brettanomyces anomalus Bac+ Bac+ Streptococcus sp. Streptococcus sp. Bac+ Bac+ Streptococcus haemolyticus Streptococcus micros Bac+ Bac+ Streptococcus faecalis Streptococcus faecalis Cited PDA, 72 h, 28°C SMA, 2–7 d, 20°C SDA, 72 h, 26°C SMA, 2–7 d, 20°C SDA, 72 h, 26°C SMA, 2–7 d, 20°C PDA, 48 h, 28°C SDA, 8 d, 30°C Cited PDA, 72 h, 28°C Cited SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C Cited SDA, 8 d, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C MPA, 4 d, 30°C SMA, 2–7 d, 20°C Cited NA, 24 h, 37°C Cited MHA, cited Cited ISA, 48 h, 25°C (h) 20,000 5, 5000 sd 8, 25,000 sd 8, 25,000 sd 5, 5000 6 (h), pure (h) 20,000 5, 5000 (h) 20,000 sd sd 6 (h), pure sd (h) 20,000 6 (h), pure sd sd 6 (h), pure 5, 10% sol. sd sd 15, 2500 10 (h), 100,000 (h) 20,000 9, 20,000 (h) 20,000 4 (h), 10,000 21 7 2 6 0 3 7 10 — 11 — 9 5 18 5 — 20 6 6 0 0 10 0 + 30 + 18 ++ 15 Hethenyi et al. (1989) continued Pawar and Thaker (2007) Maruzzella and Liguori (1958) Shin (2003) Maruzzella and Liguori (1958) Shin (2003) Maruzzella and Liguori (1958) Pawar and Thaker (2006) Yousef and Tawil (1980) Hethenyi et al. (1989) Pawar and Thaker (2007) Hethenyi et al. (1989) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Hethenyi et al. (1989) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Conner and Beuchat (1984) Maruzzella and Liguori (1958) Pizsolitto et al. (1975) Narasimha Rao and Nigam (1970) Hethenyi et al. (1989) Pellecuer et al. (1980) Hethenyi et al. (1989) Deans and Ritchie (1987) In Vitro Antimicrobial Activities of Essential Oils 491 Yeast Yeast Yeast Yeast Saccharomyces cerevisiae Schizosaccharomyces pombe Torula glabrata Torula utilis MO Class Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast (continued) Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Candida krusei Candida lipolytica Candida tropicalis Cryptococcus neoformans Cryptococcus rhodobenhani Debaryomyces hansenii Geotrichum candidum Hansenula anomala Kloeckera apiculata Kluyveromyces fragilis Lodderomyces elongisporus Metchnikowia pulcherrima Pichia membranaefaciens Rhodotorula rubra Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Microorganism TABLE 12.66 NA, 24 h, 30°C NA, 24 h, 30°C MPA, 4 d, 30°C NA, 24 h, 30°C Cited SMA, 2–7 d, 20°C NA, 24 h, 30°C Cited, 18 h, 37°C SDA, 18 h, 30°C SMA, 2–7 d, 20°C MPA, 4 d, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C Cited MPA, 4 d, 30°C SMA, 2–7 d, 20°C NA, 24 h, 20°C Conditions 5 (h), -30,000 Drop, 5000 Drop, 5000 5, 10% sol. sd Drop, 5000 (h) 20,000 sd Drop, 5000 6, 2500 6 (h), pure sd 5, 10% sol. sd sd sd sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd (h) 20,000 5, 10% sol. sd sd 12 16 0 10 — 2 7 13 24 1 0 2 6 6 0 0 0 0 0 0 0 0 8 — 0 7 10 Inhibition Zone (mm) Hili et al. (1997) Hili et al. (1997) Conner and Beuchat (1984) Hili et al. (1997) Hethenyi et al. (1989) Maruzzella and Liguori (1958) Hili et al. (1997) Janssen et al. (1986) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Hethenyi et al. (1989) Conner and Beuchat (1984) Maruzzella and Liguori (1958) Schelz et al. (2006) Ref. 492 Handbook of Essential Oils BacBacBac- Klebsiella pneumoniae Klebsiella pneumoniae Moraxella glucidolytica BacBacBacBacBacBacBac- Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas fluorescens Salmonella enteritidis Bac- Bac- Helicobacter pylori Bac- Bac- Haemophilus influenza Neisseria catarrhalis Bac- Escherichia coli Neisseria flava BacBac- Escherichia coli Bac- Escherichia coli Escherichia coli BacBac- Escherichia coli Bac- Escherichia coli Escherichia coli BacBac- Bac- Bordetella bronchiseptica Campylobacter jejuni Bac- Escherichia coli Bac- Aeromonas sobria MO Class Acinetobacter baumannii Microorganism Conditions TSB, 24 h, 35°C NA, 1–3 d, 30°C NB, Tween 20, 18 h, 37°C MHA, Tween 20, 48 h, 35°C NB, DMSO, 24 h, 37°C MPB, DMSO, 40 h, 30°C TSB, Tween 80, 48 h, 35°C Cited Cited Cited MHA, Tween 20, 48 h, 35°C Cited Cited, 20 h, 37°C Cited NB, Tween 20, 18 h, 37°C MHA, Tween 20, 48 h, 35°C TSB, 24 h, 35°C NA, 1–3 d, 30°C Cited NB, DMSO, 24 h, 37°C MPB, DMSO, 40 h, 30°C TSB, Tween 80, 48 h, 35°C TSB, 24 h, 42°C Cited MHA, Tween 20, 48 h, 35°C MHA, Tween 20, 48 h, 35°C TABLE 12.67 Inhibitory Data of Rosemary Oil Obtained in the Dilution Test >10,000 >20,000 50,000 >900 >40 54% inh. 500 1250 1250 2500 20,000 2500 137 2500 25,000 >10,000 10,000 3500 >900 2500 25% inh. 500 40 500 2500 5000 10,000 MIC continued Thanaboripat et al. (2004) Farag et al. (1989) Smith-Palmer et al. (1998) Yousef and Tawil (1980) Hammer et al. (1999) Angioni et al. (2004) Hili et al. (1997) Panizzi et al. (1993) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Hammer et al. (1999) Pellecuer et al. (1976) Weseler et al. (2005) Pellecuer et al. (1976) Yousef and Tawil (1980) Hammer et al. (1999) Smith-Palmer et al. (1998) Farag et al. (1989) Pellecuer et al. (1976) Angioni et al. (2004) Hili et al. (1997) Panizzi et al. (1993) Smith-Palmer et al. (1998) Pellecuer et al. (1976) Hammer et al. (1999) Hammer et al. (1999) Ref. In Vitro Antimicrobial Activities of Essential Oils 493 Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Sarcina ureae Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Mycobacterium phlei Sarcina lutea Bac+ Mycobacterium phlei Sarcina sp. Bac+ Bac+ Micrococcus ureae Bac+ Micrococcus sp. 1250 Cited Bac+ Bac+ Bac+ Listeria monocytogenes Micrococcus flavus Bac+ Micrococcus luteus 200 TSB, 24 h, 35°C Bac+ Listeria monocytogenes MPB, DMSO, 40 h, 30°C TSB, 24 h, 35°C TSB, Tween 80, 48 h, 35°C TSB, 3% EtOH, 24 h, 37°C MHB, cited NA, 1–3 d, 30°C Cited NA, 1–3 d, 30°C NB, Tween 20, 18 h, 37°C MHB, cited NA, 1–3 d, 30°C MHB, cited TSB, 3% EtOH, 24 h, 37°C MRS, cited 1250 1% inh. 500 400 20 30,000–50,000 2.5 2000 1250 1250 400 5 1500 2.5–5 7000–10,000 >20,000 5 Lactobacillus sp. Cited MHA, Tween 20, 48 h, 35°C Bac+ 1250 800 750 >6000 10 1250 10,000–15,000 >20,000 20,000 >20,000 11,000 Bac+ Cited MIC 10,000–13,000 Corynebacterium pseudodiphtheriae Bac+ Bacillus subtilis NB, Tween 20, 18 h, 37°C NA, 1–3 d, 30°C TSB, Tween 80, 48 h, 35°C HS, 16–24, 37°C MHA, Tween 20, 24 h, 37°C TSB, 3% EtOH, 24 h, 29°C TSB, 3% EtOH, 24 h, 37°C MHA, Tween 20, 48 h, 35°C NA, 1–3 d, 30°C MHA, Tween 20, 48 h, 35°C TSB, 3% EtOH, 24 h, 37°C Conditions Enterococcus faecalis Bac+ Bac+ Bacillus subtilis Bacillus subtilis Bac+ Bac- Yersinia enterocolitica Bac+ Bac- Yersinia enterocolitica Bacillus subtilis Bac- Shigella flexneri Actinomyces viscosus BacBac- Serratia marcescens Bac- Serratia marcescens Bac- MO Class Salmonella typhimurium (continued) Salmonella typhimurium Microorganism TABLE 12.67 Hili et al. (1997) Smith-Palmer et al. (1998) Panizzi et al. (1993) Rota et al. (2004) Pellecuer et al. (1980) Farag et al. (1989) Pellecuer et al. (1976) Farag et al. (1989) Yousef and Tawil (1980) Pellecuer et al. (1980) Farag et al. (1989) Pellecuer et al. (1980) Pellecuer et al. (1976) Smith-Palmer et al. (1998) Rota et al. (2004) Pellecuer et al. (1980) Hammer et al. (1999) Pellecuer et al. (1976) Pellecuer et al. (1976) Yousef and Tawil (1980) Farag et al. (1989) Panizzi et al. (1993) Shapiro et al. (1994) Rossi et al. (2007) Rota et al. (2004) Rota et al. (2004) Hammer et al. (1999) Farag et al. (1989) Hammer et al. (1999) Rota et al. (2004) Ref. 494 Handbook of Essential Oils Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Aspergillus flavus Aspergillus flavus Aspergillus giganteus Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus ochraceus Aspergillus oryzae Aspergillus oryzae Aspergillus parasiticus Aspergillus parasiticus Aspergillus repens Cephalosporium sacchari Absidia glauca Aspergillus chevalieri Aspergillus clavatus Aspergillus flavus Aspergillus flavus Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Streptococcus sanguinis Bac+ Streptococcus pyogenes Streptococcus sobrinus Bac+ Bac+ Staphylococcus epidermidis Streptococcus micros Bac+ Staphylococcus epidermidis Bac+ Bac+ Staphylococcus aureus Bac+ Bac+ Staphylococcus epidermidis Bac+ Staphylococcus aureus Staphylococcus aureus Streptococcus D Bac+ Bac+ Staphylococcus aureus Staphylococcus aureus NB, DMSO, 24 h, 37°C Cited OA, EtOH, 3 d, 20°C Cited MYB, 72 h, 26°C Cited Cited YES broth, 10 d MYB, 72 h, 26°C NB, Tween 20, 8 d, 30°C YES broth, 10 d Cited Cited PDA, 8 h, 20°C, spore germ. inh. PDA, 5 d, 27°C Cited Cited Cited PDA, 8 h, 20°C, spore germ. inh. PDA, 5 d, 27°C HS, 16–24, 37°C HS, 16–24, 37°C Cited MHB, cited MHB, cited Cited NB, DMSO, 24 h, 37°C MHB, cited NB, Tween 20, 18 h, 37°C MHA, Tween 20, 48 h, 35°C Cited NA, 1–3 d, 30°C >1000 2000 20,000 >1000 2000 12,500 2000 2000 12% inh. 10,000 12,500 50,000 14% inh. 10,000 250 2000 50–100 >6000 2000 2000 2000 50–100 >6000 625 5 2.5–5 >900 1250 5 12,500 10,000 1250 >900 1000 Angioni et al. (2004) continued Pellecuer et al. (1976) Narasimba Rao et al. (1971) Pellecuer et al. (1976) Shin (2003) Pellecuer et al. (1976) Pellecuer et al. (1976) Lis-Balchin et al. (1998) Shin (2003) Yousef and Tawil (1980) Lis-Balchin et al. (1998) Okazaki and Oshima (1953) Pellecuer et al. (1976) Thompson (1986) Thompson and Cannon (1986) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Thompson (1986) Thompson and Cannon (1986) Shapiro et al. (1994) Shapiro et al. (1994) Pellecuer et al. (1976) Pellecuer et al. (1980) Pellecuer et al. (1980) Pellecuer et al. (1976) Angioni et al. (2004) Pellecuer et al. (1980) Yousef and Tawil (1980) Hammer et al. (1999) Pellecuer et al. (1976) Farag et al. (1989) In Vitro Antimicrobial Activities of Essential Oils 495 Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fusarium culmorum Fusarium moniliforme var. subglutinans Helminthosporium sacchari Microsporum canis Microsporum gypseum Mucor hiemalis Mucor mucedo Mucor mucedo Mucor racemosus Mucor racemosus f. racemosus Mucor sp. Penicillium chrysogenum Penicillium chrysogenum Penicillium chrysogenum Penicillium liliacinum Penicillium rubrum Physalospora tucumanensis Rhizopus 66-81-2 Rhizopus arrhizus Rhizopus chinensis Rhizopus circinans Rhizopus japonicus Rhizopus kazanensis Rhizopus nigricans Rhizopus oryzae MO Class Fungi Fungi Fungi Fungi Fungi (continued) Ceratocystis paradoxa Cladosporium herbarum Curvularia lunata Epidermophyton floccosum Epidermophyton floccosum Microorganism TABLE 12.67 Cited PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C NB, Tween 20, 8 d, 30°C Cited Cited NB, Tween 20, 8 d, 30°C Cited Cited OA, EtOH, 3 d, 20°C PDA, 5 d, 27°C Cited PDA, 5 d, 27°C Cited PDA, 5 d, 27°C PDA, 5 d, 27°C MBA, Tween 80, 10 d, 30°C YES broth, 10 d OA, EtOH, 3 d, 20°C OA, EtOH, 3 d, 20°C MBA, Tween 80, 10 d, 30°C OA, EtOH, 3 d, 20°C Cited OA, EtOH, 3 d, 20°C SA, Tween 80, 21 d, 20°C Cited Conditions >1000 >1000 2000 >1000 >1000 >1000 >1000 >1000 >1000 6400 500 2000 12,500 2000 2000 20,000 >1000 500 >1000 1000 >300 300– >300 >4000 0% inh. 10,000 20,000 20,000 4000 2000 20,000 625–1250 MIC Pellecuer et al. (1976) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Yousef and Tawil (1980) Okazaki and Oshima (1953) Pellecuer et al. (1976) Yousef and Tawil (1980) Pellecuer et al. (1976) Pellecuer et al. (1976) Narasimba Rao et al. (1971) Thompson and Cannon (1986) Okazaki and Oshima (1953) Thompson and Cannon (1986) Pellecuer et al. (1976) Thompson and Cannon (1986) Thompson and Cannon (1986) Perrucci et al. (1994) Lis-Balchin et al. (1998) Narasimba Rao et al. (1971) Narasimba Rao et al. (1971) Perrucci et al. (1994) Narasimba Rao et al. (1971) Pellecuer et al. (1976) Narasimba Rao et al. (1971) Janssen et al. (1988) Pellecuer et al. (1976) Ref. 496 Handbook of Essential Oils Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Candida albicans Candida albicans Candida albicans Candida mycoderma Candida paraspilosis Candida pelliculosa Candida tropicalis Geotrichum asteroides Geotrichum candidum Hansenula sp. Saccharomyces carlsbergensis Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Schizosaccharomyces pombe Torula utilis Escherichia coli Saccharomyces cerevisiae Bac+ Yeast Bac- Yeast Yeast Yeast Candida albicans Candida albicans Candida albicans Staphylococcus epidermidis Fungi Fungi Trichophyton mentagrophytes Trichophyton rubrum YPB, 24 h, 20°C TGB, 18 h, 37°C Cited NB, Tween 20, 18 h, 37°C MHA, Tween 20, 48 h, 35°C Cited Cited Cited Cited Cited Cited Cited Cited SDB, Tween 80, 48 h, 35°C MPB, DMSO, 40 h, 30°C NA, 1–3 d, 30°C MPB, DMSO, 40 h, 30°C MPB, DMSO, 40 h, 30°C TGB, 18 h, 37°C SDB, Tween 80, 48 h, 35 MPB, DMSO, 40 h, 30°C NB, DMSO, 24 h, 30°C SA, Tween 80, 21 d, 20°C SA, Tween 80, 21 d, 20°C OA, EtOH, 6 d, 20°C Cited Cited Cited PDA, 5 d, 27°C Fungi Fungi Fungi Fungi Fungi Rhizopus tritici Sclerotium rolfsii Scopulariopsis brevicaulis Syncephalastrum racemosum Trichophyton interdigitale PDA, 5 d, 27°C NB, Tween 20, 8 d, 30°C PDA, 5 d, 27°C Fungi Fungi Fungi Rhizopus pymacus Rhizopus sp. Rhizopus stolonifer 2800 11,300 >900 1000 3200 10,000 2000 1000 2000 2000 4000 2000 2000 4000 5 88% inh. 500 2000 86% inh. 500 2% inh. 500 11,300 <300 10 8% inh. 500 >4000 300–625 >1000 4000 2000 2000 >1000 >1000 25,000 Schelz et al. (2006) Schelz et al. (2006) Pellecuer et al. (1976) Yousef and Tawil (1980) Hammer et al. (1999) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Panizzi et al. (1993) Hili et al. (1997) Farag et al. (1989) Hili et al. (1997) Hili et al. (1997) Schelz et al. (2006) Panizzi et al. (1993) Hili et al. (1997) Angioni et al. (2004) Janssen et al. (1988) Janssen et al. (1988) Narasimba Rao et al. (1971) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Thompson and Cannon (1986) Yousef and Tawil (1980) Thompson and Cannon (1986) Thompson and Cannon (1986) In Vitro Antimicrobial Activities of Essential Oils 497 Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Aerobacter tumefaciens Escherichia coli Escherichia coli Haemophilus influenza Neisseria sp. Peptobacterium carotovorum Proteus vulgaris Pseudomonas aeruginosa Pseudomonas pisi Pseudomonas tabaci Salmonella typhi Salmonella typhi Shigella sonnei Xanthomonas versicolor Bacillus megaterium Bacillus subtilis Bacillus subtilis var. aterrimus Corynebacterium diphtheriae Corynebacterium fascians Lactobacillus sp. Micrococcus luteus Micrococcus ureae Mycobacterium avium Pneumococcus sp. Sarcina ureae Staphylococcus aureus Staphylococcus aureus Staphylococcus epidermidis MO Class Microorganism MHB, cited Cited NA, 24 h, 37°C MHB, cited Cited NA, 24 h, 37°C MHB, cited MHB, cited MRS, cited Cited NA, 24 h, 37°C NA, 24 h, 37°C Cited NA, 24 h, 37°C Cited Cited NA, 24 h, 37°C NA, 24 h, 37°C Cited Cited Cited Cited Cited NA, 24 h, 37°C Cited Cited NA, 24 h, 37°C Cited Conditions TABLE 12.68 Inhibitory Data of Rosemary Oil Obtained in the Vapor Phase Test Disk, 20,000 Disk, 20,000? ~20,000 Disk, 20,000 Disk, 20,000? Disk, 20,000 sd Disk, 20,000? Disk, 20,000? Disk, 20,000? ~20,000 Disk, 20,000 sd ~20,000 Disk, 20,000 Disk, 20,000 Disk, 20,000 ~20,000 sd Disk, 20,000 Disk, 20,000 Disk, 20,000 Disk, 20,000 ~20,000 Disk, 20,000 Disk, 20,000 ~20,000 Disk, 20,000 +++ NG ++ NG ++ NG +++ ++ ++ +++ + +++ + NG +++ +++ +++ + +++ +++ +++ +++ +++ NG NG to +++ NG ++ NG Activity Pellecuer et al. (1980) Hethenyi et al. (1989) Kellner and Kober (1954) Pellecuer et al. (1980) Hethenyi et al. (1989) Maruzzella and Sicurella (1960) Pellecuer et al. (1980) Pellecuer et al. (1980) Pellecuer et al. (1980) Hethenyi et al. (1989) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Hethenyi et al. (1989) Kellner and Kober (1954) Hethenyi et al. (1989) Hethenyi et al. (1989) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Hethenyi et al. (1989) Hethenyi et al. (1989) Hethenyi et al. (1989) Hethenyi et al. (1989) Hethenyi et al. (1989) Kellner and Kober (1954) Hethenyi et al. (1989) Hethenyi et al. (1989) Kellner and Kober (1954) Hethenyi et al. (1989) Ref. 498 Handbook of Essential Oils Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Streptococcus pyogenes Streptococcus sp. Aspergillus flavus Aspergillus flavus Aspergillus niger Botrytis cinera Colletotrichum gleosporoides Endomyces filbuliger Eurotium amstelodami Eurotium herbarum Eurotium repens Eurotium repens Eurotium rubrum Fusarium moniliforme Fusarium oxysporum Fusarium solani Ophiobolus graminis Penicillium corylophilum Penicillium corylophilum Penicillium roqueforti Pythium ultimum Rhizoctonia solani Candida albicans Candida albicans Saccharomyces cerevisiae Bac+ Streptococcus faecalis Streptococcus haemolyticus Bac+ Bac+ Streptococcus faecalis Streptococcus D Streptococcus faecalis Bac+ Bac+ Streptococcus aegui Cited Cited Cited NA, 24 h, 37°C PDA, 3 d, 25°C PDA, 3 d, 25°C Bread, 14 d, 25°C Bread, 14 d, 25°C Cited Cited WFA, 42 d, 25°C Cited PDA, 3 d, 25°C WFA, 42 d, 25°C Bread, 14 d, 25°C WFA, 42 d, 25°C WFA, 42 d, 25°C WFA, 42 d, 25°C Bread, 14 d, 25°C PDA, 3 d, 25°C PDA, 3 d, 25°C WFA, 42 d, 25°C Bread, 14 d, 25°C WFA, 42 d, 25°C Cited NA, 24 h, 37°C Cited NA, 24 h, 37°C Cited NA, 24 h, 37°C MHB, cited Disk, 20,000 ~20,000 Disk, 20,000 Disk, 20,000 1000 1000 30,000 30,000 Disk, 20,000 Disk, 20,000 Disk, 50,000 Disk, 20,000 1000 Disk, 50,000 30,000 Disk, 50,000 Disk, 50,000 Disk, 50,000 30,000 1000 1000 Disk, 50,000 30,000 Disk, 50,000 ~20,000 Disk, 20,000 Disk, 20,000 sd ~20,000 Disk, 20,000 Disk, 20,000? NG NG +++ NG +++ +++ +++ ++ +++ NG NG +++ NG +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ NG NG +++ NG + NG ++ NG Hethenyi et al. (1989) Hethenyi et al. (1989) Hethenyi et al. (1989) Kellner and Kober (1954) Lee et al. (2007) Lee et al. (2007) Suhr and Nielsen (2003) Suhr and Nielsen (2003) Hethenyi et al. (1989) Hethenyi et al. (1989) Guynot et al. (2003) Hethenyi et al. (1989) Lee et al. (2007) Guynot et al. (2003) Suhr and Nielsen (2003) Guynot et al. (2003) Guynot et al. (2003) Guynot et al. (2003) Suhr and Nielsen (2003) Lee et al. (2007) Lee et al. (2007) Guynot et al. (2003) Suhr and Nielsen (2003) Guynot et al. (2003) Hethenyi et al. (1989) Kellner and Kober (1954) Hethenyi et al. (1989) Maruzzella and Sicurella (1960) Hethenyi et al. (1989) Kellner and Kober (1954) Pellecuer et al. (1980) In Vitro Antimicrobial Activities of Essential Oils 499 Bac+ Bacillus cereus 4 (h), 10,000 ISA, 48 h, 25°C Bac- Bac- Serratia marcescens Bac- Bac- Salmonella pullorum Salmonella typhi Yersinia enterocolitica 10 (h), 2000 NA, 24 h, 37°C Bac- Pseudomonas aeruginosa NA, 24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C Cited, 18 h, 37°C 10 (h), 2000 4 (h), 10,000 4 (h), 10,000 10 (h), 2000 6, 2500 4 (h), 10,000 Bac- Pseudomonas aeruginosa ISA, 48 h, 25°C Bac- Bac- 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 10 (h), 2000 6, 2500 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 Pseudomonas aeruginosa ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C Cited, 18 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C 4 (h), 10,000 Proteus vulgaris Bac- Bac- Bac- Flavobacterium suaveolens Moraxella sp. Bac- Escherichia coli Klebsiella pneumoniae Bac- Bac- Escherichia coli Escherichia coli Bac- Bac- Enterobacter aerogenes Erwinia carotovora Bac- Citrobacter freundii ISA, 48 h, 25°C 4 (h), 10,000 Bac- Bac- Alcaligenes faecalis Beneckea natriegens ISA, 48 h, 25°C 4 (h), 10,000 4 (h), 10,000 ISA, 48 h, 25°C ISA, 48 h, 25°C Bac- Bac- Conditions Acinetobacter calcoaceticus MO Class Aeromonas hydrophila Microorganism TABLE 12.69 Inhibitory Data of Star Anise Oil Obtained in the Agar Diffusion Test 0 9 5.5 30.1 6 20.3 6.7 0 0 0 0 9 18.5 7 0 0 0 0 5 5 6 0 Inhibition Zone (mm) Singh et al. (2006) Deans and Ritchie (1987) Deans and Ritchie (1987) Singh et al. (2006) Deans and Ritchie (1987) Singh et al. (2006) Janssen et al. (1986) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Singh et al. (2006) Janssen et al. (1986) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Ref. 500 Handbook of Essential Oils Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Bacillus subtilis Bacillus subtilis Brevibacterium linens Brochotrix thermosphacta Clostridium sporogenes Lactobacillus plantarum Leuconostoc cremoris Micrococcus luteus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Streptococcus faecalis Brettanomyces anomalus Candida albicans Candida lipolytica Debaryomyces hansenii Geotrichum candidum Hansenula anomala Kloeckera apiculata Kluyveromyces fragilis Lodderomyces elongisporus Metchnikowia pulcherrima Pichia membranaefaciens Rhodotorula rubra Saccharomyces cerevisiae Torula glabrata Bac+ Bacillus subtilis MPA, 4 d, 30°C Cited, 18 h, 37°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C ISA, 48 h, 25°C Cited, 18 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C Cited, 18 h, 37°C ISA, 48 h, 25°C 5, 10% sol. sd 6, 2500 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 4 (h), 10,000 6, 2500 4 (h), 10,000 10 (h), 2000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 10 (h), 2000 6, 2500 4 (h), 10,000 0 8.7 7 0 0 0 8 0 0 0 0 0 0 0 0 9 0 0 6 0 0 0 0 0 26.2 6.7 0 Conner and Beuchat (1984) Janssen et al. (1986) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Deans and Ritchie (1987) Janssen et al. (1986) Deans and Ritchie (1987) Singh et al. (2006) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Singh et al. (2006) Janssen et al. (1986) Deans and Ritchie (1987) In Vitro Antimicrobial Activities of Essential Oils 501 502 Handbook of Essential Oils TABLE 12.70 Inhibitory Data of Star Anise Oil Obtained in the Dilution Test Microorganism Epidermophyton floccosum Trichophyton mentagrophytes Trichophyton rubrum MO Class Conditions MIC Ref. Fungi Fungi Fungi SA, Tween 80, 21 d, 20°C SA, Tween 80, 21 d, 20°C SA, Tween 80, 21 d, 20°C 300–625 300–625 300–625 Janssen et al. (1988) Janssen et al. (1988) Janssen et al. (1988) TABLE 12.71 Inhibitory Data of Star Anise Oil Obtained in the Vapor Phase Test Microorganism MO Class Conditions Escherichia coli Bac- NA, 24 h, 37°C ~20,000 Bac- NA, 24 h, 37°C Neisseria sp. Salmonella typhi Activity Ref. Kellner and Kober (1954) ~20,000 + NG Kellner and Kober (1954) Kellner and Kober (1954) Bac- NA, 24 h, 37°C ~20,000 NG Bacillus megaterium Bac+ NA, 24 h, 37°C ~20,000 Kellner and Kober (1954) Corynebacterium diphtheriae Bac+ NA, 24 h, 37°C ~20,000 + NG 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 Kellner and Kober (1954) Streptococcus pyogenes Bac+ Fungi NA, 24 h, 37°C ~20,000 12, 6000 + NG Aspergillus flavus CDA, 6 d + Kellner and Kober (1954) Kellner and Kober (1954) 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) Fusarium moniliforme Penicillium citrinum Fungi Fungi CDA, 6 d CDA, 6 d 12, 6000 12, 6000 +++ NG Penicillium viridicatum Fungi CDA, 6 d 12, 6000 Candida albicans Yeast NA, 24 h, 37°C ~20,000 + ++ NG Singh et al. (2006) Singh et al. (2006) Singh et al. (2006) Kellner and Kober (1954) Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Aerobacter aerogenes Aeromonas hydrophila Alcaligenes faecalis Beneckea natriegens Campylobacter jejuni Campylobacter jejuni Citrobacter freundii Enterobacter aerogenes Enterobacter aerogenes Erwinia carotovora Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Flavobacterium suaveolens Klebsiella pneumoniae Klebsiella sp. Moraxella sp. Neisseria perflava Proteus sp. MO Class Acinetobacter calcoaceticus Microorganism Cited NA, 24 h, 37°C ISA, 48 h, 25°C Cited ISA, 48 h, 25°C ISA, 48 h, 25°C TGA, 18–24 h, 37°C NA, 18 h, 37°C NA, 24 h, 37°C Cited MHA, 24 h, 30°C Cited, 18 h, 37°C TSA, 24 h, 35°C NA, 18 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C MHA, 24 h, 30°C ISA, 48 h, 25°C ISA, 48 h, 25°C TSA, 24 h, 42°C CAB, 24 h, 42°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C ISA, 48 h, 25°C Conditions 15, 2500 —, sd 4 (h), 10,000 15, 2500 4 (h), 10,000 4 (h), 10,000 9.5, 2000 6 (h), pure Disk, 10,000 15, 2500 6, 15,000 6, 2500 5 (h), -30,000 4 (h), 25,000 4 (h), 10,000 —, sd 4 (h), 10,000 6, 15,000 4 (h), 10,000 4 (h), 10,000 4 (h), 25,000 Disk, 10,000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 —, sd 4 (h), 10,000 TABLE 12.72 Inhibitory Data of Sweet Orange Oil Obtained in the Agar Diffusion Test 3 0 0 2 0 10 20 19.5 18 16 8 7 4 0 0 0 0 6 0 0 4 0 0 0 0 0 0 Inhibition Zone in mm Pizsolitto et al. (1975) continued Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Pizsolitto et al. (1975) Deans and Ritchie (1987) Deans and Ritchie (1987) Morris et al. (1979) Yousef and Tawil (1980) Fisher and Phillips (2006) Pizsolitto et al. (1975) Rossi et al. (2007) Janssen et al. (1986) Smith-Palmer et al. (1998) Schelz et al. (2006) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Rossi et al. (2007) Deans and Ritchie (1987) Deans and Ritchie (1987) Smith-Palmer et al. (1998) Fisher and Phillips (2006) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Ref. In Vitro Antimicrobial Activities of Essential Oils 503 MO Class Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac- Bac+ Bac+ Bac+ Bac+ Bac+ Proteus vulgaris Proteus vulgaris Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas mangiferae indicae Pseudomonas mangiferae indicae Salmonella enteritidis Salmonella paratyphi Salmonella paratyphi Salmonella pullorum Salmonella sp. Salmonella typhi Serratia marcescens Serratia marcescens Serratia sp. Shigella sp. Vibrio cholera Vibrio cholera Xanthomonas campestris Yersinia enterocolitica Bacillus anthracis Bacillus cereus Bacillus mesentericus Bacillus mycoides Bacillus mycoides (continued) Microorganism TABLE 12.72 NA, 24 h, 28°C NA, 36–48 h, 37°C NA, 24 h, 37°C BHA, 24 h, 30°C NA, 24 h, 28°C ISA, 48 h, 25°C NA, 24 h, 28°C NA, 24 h, 28°C NA, 36–48 h, 37°C Cited Cited ISA, 48 h, 25°C NA, 24 h, 37°C NA, 36–48 h, 37°C Cited ISA, 48 h, 25°C NA, 24 h, 28°C NA, 36–48 h, 37°C TSA, 24 h, 35°C NA, 24 h, 28°C NA, 36–48 h, 37°C MHA, 24 h, 30°C NA, 24 h, 37°C Cited ISA, 48 h, 25°C Cited, 18 h, 37°C NA, 18 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C Conditions 6, sd 6, sd —, sd Disk, 10,000 6, sd 4 (h), 10,000 6, sd 6, sd 6, sd 15, 2500 15, 2500 4 (h), 10,000 —, sd 6, sd 15, 2500 4 (h), 10,000 6, sd 6, sd 4 (h), 25,000 6, sd 6, sd 6, 15,000 —, sd 15, 2500 4 (h), 10,000 6, 2500 6 (h), pure 4 (h), 10,000 —, sd 17 0 2 36 23 0 20.5 13 13 13 2 0 0 24 15 0 19 14 4 9 0 6 2 2 0 0 0 0 0 Inhibition Zone in mm Kindra and Satyanarayana (1978) Garg and Garg (1980) Maruzzella and Lichtenstein (1956) Fisher and Phillips (2006) Kindra and Satyanarayana (1978) Deans and Ritchie (1987) Kindra and Satyanarayana (1978) Kindra and Satyanarayana (1978) Garg and Garg (1980) Pizsolitto et al. (1975) Pizsolitto et al. (1975) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Garg and Garg (1980) Pizsolitto et al. (1975) Deans and Ritchie (1987) Kindra and Satyanarayana (1978) Garg and Garg (1980) Smith-Palmer et al. (1998) Kindra and Satyanarayana (1978) Garg and Garg (1980) Rossi et al. (2007) Maruzzella and Lichtenstein (1956) Pizsolitto et al. (1975) Deans and Ritchie (1987) Janssen et al. (1986) Yousef and Tawil (1980) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Ref. 504 Handbook of Essential Oils Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Leuconostoc cremoris Listeria monocytogenes Listeria monocytogenes Listeria monocytogenes Micrococcus luteus Mycobacterium phlei Sarcina lutea Sarcina lutea Staphylococcus aureus Staphylococcus aureus Bac+ Bac+ Lactobacillus plantarum Staphylococcus aureus Bac+ Corynebacterium sp. Bac+ Bac+ Clostridium sporogenes Staphylococcus aureus Bac+ Brochotrix thermosphacta Bac+ Bac+ Brevibacterium linens Staphylococcus aureus Bac+ Bacillus subtilis Bac+ Bac+ Bacillus subtilis Staphylococcus aureus Bac+ Bacillus subtilis Bac+ Bac+ Bacillus subtilis Staphylococcus aureus Bac+ Bacillus subtilis Bac+ Bac+ Bacillus subtilis Bac+ Bac+ Bacillus sp. Staphylococcus aureus Bac+ Bacillus pumilus Staphylococcus aureus Bac+ Bacillus pumilus NA, 18 h, 37°C MHA, 24 h, 37°C Cited, 18 h, 37°C Cited TSA, 24 h, 35°C ISA, 48 h, 25°C NA, 36–48 h, 37°C TGA, 18–24 h, 37°C NA, 24 h, 37°C NA, 36–48 h, 37°C NA, 24 h, 37°C NA, 18 h, 37°C ISA, 48 h, 25°C LSA, 24 h, 37°C ISA, 48 h, 25°C TSA, 24 h, 35°C ISA, 48 h, 25°C ISA, 48 h, 25°C TGA, 18–24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 28°C NA, 18 h, 37°C NA, 36–48 h, 37°C Cited, 18 h, 37°C ISA, 48 h, 25°C NA, 24 h, 37°C Cited NA, 24 h, 28°C NA, 36–48 h, 37°C 6, sd 6 (h), pure 6, 15,000 6, 2500 15, 2500 4 (h), 25,000 4 (h), 10,000 6, sd 9.5, 2000 —, sd 6, sd —, sd 6 (h), pure 4 (h), 10,000 Disk, 10,000 4 (h), 10,000 4 (h), 25,000 4 (h), 10,000 4 (h), 10,000 9.5, 2000 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 6, sd 6 (h), pure 6, sd 6, 2500 4 (h), 10,000 —, sd 15, 2500 6, sd 0 20.6 17 7 6 4 0 0 0 0 0 0 23.5 >90 0 10 4 7.5 0 22 0 8 0 21.5 19 14 10.3 10 4 7 16 Yousef and Tawil (1980) Rossi et al. (2007) Janssen et al. (1986) Pizsolitto et al. (1975) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Garg and Garg (1980) Morris et al. (1979) continued Maruzzella and Lichtenstein (1956) Garg and Garg (1980) Maruzzella and Lichtenstein (1956) Yousef and Tawil (1980) Deans and Ritchie (1987) Fisher and Phillips (2006) Lis-Balchin et al. (1998) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Deans and Ritchie (1987) Morris et al. (1979) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Kindra and Satyanarayana (1978) Yousef and Tawil (1980) Garg and Garg (1980) Janssen et al. (1986) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Pizsolitto et al. (1975) Kindra and Satyanarayana (1978) Garg and Garg (1980) In Vitro Antimicrobial Activities of Essential Oils 505 Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Staphylococcus epidermidis Streptococcus faecalis Streptococcus viridans Streptomyces venezuelae Alternaria porri Alternaria solani Aspergillus fumigatus Aspergillus niger Aspergillus niger Aspergillus niger Fusarium oxysporum f.sp. cicer Helminthosporium sativum Mucor mucedo Mucor sp. Nigrospora panici Penicillium chrysogenum Penicillium digitatum Rhizopus nigricans Rhizopus sp. Bac+ Staphylococcus epidermidis MO Class Bac+ (continued) Staphylococcus aureus Microorganism TABLE 12.72 Conditions PDA, 72 h, 28°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C PDA, 48 h, 28°C SDA, 8 d, 30°C PDA, 72 h, 28°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SDA, 8 d, 30°C SMA, 2–7 d, 20°C Cited ISA, 48 h, 25°C Cited NA, 18 h, 37°C BHA, 24 h, 37°C 5, 5000 sd sd sd 5, 5000 6 (h), pure 5, 5000 sd sd 6 (h), pure sd 6 (h), pure sd sd 6 (h), pure sd 15, 2500 4 (h), 10,000 15, 2500 5 (h), –30,000 Disk, 10,000 10 13 8 4 6 28 10 7 4 18 6 25 7 0 0 5 0 0 12 0 46 Inhibition Zone in mm Ref. Pawar and Thaker (2007) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Pawar and Thaker (2006) Yousef and Tawil (1980) Pawar and Thaker (2007) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Pizsolitto et al. (1975) Deans and Ritchie (1987) Pizsolitto et al. (1975) Schelz et al. (2006) Fisher and Phillips (2006) 506 Handbook of Essential Oils Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Brettanomyces anomalus Candida albicans Candida albicans Candida albicans Candida albicans Candida krusei Candida lipolytica Candida tropicalis Cryptococcus neoformans Cryptococcus rhodobenhani Debaryomyces hansenii Geotrichum candidum Hansenula anomala Kloeckera apiculata Kluyveromyces fragilis Lodderomyces elongisporus Metchnikowia pulcherrima Pichia membranaefaciens Rhodotorula rubra Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Torula glabrata MPA, 4 d, 30°C SMA, 2–7 d, 20°C MPA, 4 d, 30°C MPA, 4 d, 30°C TGA, 18–24 h, 37°C SMA, 2–7 d, 20°C Cited, 18 h, 37°C SDA, 18 h, 30°C SMA, 2–7 d, 20°C MPA, 4 d, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C NA, 24 h, 20°C 5 (h), -30,000 5, 10% sol. sd sd 5, 10% sol. sd 5, 10% sol. sd 9.5, 2000 sd 6, 2500 6 (h), pure sd 5, 10% sol. sd sd sd sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 0 18 10 0 0 5 13 22.5 5 7 4 6 4 0 0 0 0 0 0 0 0 0 7–8 Conner and Beuchat (1984) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Conner and Beuchat (1984) Morris et al. (1979) Maruzzella and Liguori (1958) Janssen et al. (1986) Yousef and Tawil (1980) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Schelz et al. (2006) In Vitro Antimicrobial Activities of Essential Oils 507 BacBac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bacillus cereus Bacillus subtilis Bacillus subtilis Corynebacterium sp. Lactobacillus plantarum Listeria monocytogenes Micrococcus sp. Mycobacterium phlei Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus BacBac- Salmonella oranienburg Salmonella schottmuelleri Bac- BacBac- Salmonella heidelberg Salmonella montevideo Yersinia enterocolitica Bac- Pseudomonas aeruginosa Serratia marcescens 93% inh. 10,000 NB, 24–72 h, 37°C Bac- Pseudomonas aeruginosa Bac- NB, 1–2 d, 35–37°C NA, pH 7 Bac- Helicobacter pylori Bac- 90% inh., 1000 1000 NB, 1–2 d, 35–37°C NB, 1–2 d, 35–37°C Bac- Escherichia coli Salmonella senftenberg 90% inh., 1000 90% inh., 1000 NB, 24–72 h, 37°C Bac- Escherichia coli Salmonella typhimurium >50,000 87% inh. 10,000 NB, Tween 20, 18 h, 37°C Bac- NB, 24–72 h, 37°C TGB, 18–24 h, 37°C NB, Tween 20, 18 h, 37°C NB, Tween 20, 18 h, 37°C NA, pH 7 LSA, 24 h, 37°C NA, pH 7 TGB, 18–24 h, 37°C NB, Tween 20, 18 h, 37°C NA, pH 7 BHA, 24 h, 30°C MHA, Tween 20, 24 h, 37°C NA, pH 7 NB, 1–2 d, 35–37°C Cited, 20 h, 37°C TGB, 18 h, 37°C NB, 24–72 h, 37°C NA, 24 h, 37°C TGB, 18–24 h, 37°C 10,000 500 12,500 800 1000 2500 1000 500 3200 >40,000 2000 >2000 1250 90% inh., 1000 >11,300 65.1 93% inh. 10,000 >1000 10,000 3200 Escherichia coli 2000 Bac- NB, Tween 20, 18 h, 37°C Escherichia coli NA, pH 7 Bac- MIC Bac- Conditions Escherichia coli MO Class Aerobacter aerogenes Microorganism TABLE 12.73 Inhibitory Data of Sweet Orange Oil Obtained in the Dilution Test Dabbah et al. (1970) Morris et al. (1979) Yousef and Tawil (1980) Yousef and Tawil (1980) Subba et al. (1967) Fisher and Phillips (2006) Subba et al. (1967) Morris et al. (1979) Yousef and Tawil (1980) Subba et al. (1967) Fisher and Phillips (2006) Rossi et al. (2007) Subba et al. (1967) Dabbah et al. (1970) Dabbah et al. (1970) Subba et al. (1967) Dabbah et al. (1970) Dabbah et al. (1970) Dabbah et al. (1970) Dabbah et al. (1970) Yousef and Tawil (1980) Weseler et al. (2005) Schelz et al. (2006) Dabbah et al. (1970) Fisher and Phillips (2006) Morris et al. (1979) Yousef and Tawil (1980) Subba et al. (1967) Ref. 508 Handbook of Essential Oils Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Streptococcus faecalis Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus ochraceus Aspergillus oryzae Aspergillus parasiticus Aspergillus parasiticus Botrytis cinera Cephalosporium sacchari Ceratocystis paradoxa Curvularia lunata Epidermophyton floccosum Fusarium culmorum Fusarium moniliforme var. subglutinans Geotrichum citri-aurantii Helminthosporium sacchari Mucor hiemalis Mucor mucedo Mucor racemosus Mucor racemosus f. racemosus Mucor sp. Penicillium chrysogenum Penicillium chrysogenum Penicillium digitatum Penicillium digitatum Penicillium italicum Aspergillus awamorii Aspergillus flavus Aspergillus flavus Aspergillus flavus Bac+ Staphylococcus epidermidis SDB, 5 d, 20°C, MIC = ED50 SDB, 5 d, 20°C, MIC = ED50 PDA, Tween 20, 7 d, 24°C NB, Tween 20, 8 d, 30°C Cited NB, Tween 20, 8 d, 30°C Cited PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, Tween 20, 7 d, 24°C OA, EtOH, 3 d, 20°C PDA, 5 d, 27°C YES broth, 10 d OA, EtOH, 3 d, 20°C OA, EtOH, 3 d, 20°C OA, EtOH, 3 d, 20°C SA, Tween 80, 21 d, 20°C PDA, Tween 20, 7 d, 24°C OA, EtOH, 3 d, 20°C PDA, pH 4.5 NB, Tween 20, 8 d, 30°C YES broth, 10 d YES broth, 10 d Cited PDA, 8 h, 20°C, spore germ. inh. PDA, 5 d, 27°C PDA, pH 4.5 PDA, pH 4.5 PDA, 8 h, 20°C, spore germ. inh. PDA, 5 d, 27°C NA, pH 7 TGB, 18 h, 37°C 32% inh. 1000 3000–5500 >1000 12,500 500 25,000 1000–2400 >1000 500 >1000 >20,000 7% inh. 1000 20,000 >1250 84% inh. 10,000 >20,000 20,000 20,000 >1000 4% inh. 1000 >1000 2000 50,000 0% inh. 10,000 34% inh. 10,000 500 50–100 2000 2000 50–100 >11,300 1000 continued Bouchra et al. (2003) Caccioni et al. (1998) Yousef and Tawil (1980) Okazaki and Oshima (1953) Yousef and Tawil (1980) Caccioni et al. (1998) Okazaki and Oshima (1953) Thompson and Cannon (1986) Thompson and Cannon (1986) Bouchra et al. (2003) Narasimba Rao et al. (1971) Thompson and Cannon (1986) Lis-Balchin et al. (1998) Narasimba Rao et al. (1971) Narasimba Rao et al. (1971) Narasimba Rao et al. (1971) Janssen et al. (1988) Bouchra et al. (2003) Narasimba Rao et al. (1971) Subba et al. (1967) Yousef and Tawil (1980) Lis-Balchin et al. (1998) Lis-Balchin et al. (1998) Okazaki and Oshima (1953) Thompson (1986) Thompson and Cannon (1986) Subba et al. (1967) Subba et al. (1967) Thompson (1986) Thompson and Cannon (1986) Subba et al. (1967) Schelz et al. (2006) In Vitro Antimicrobial Activities of Essential Oils 509 Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Rhizopus arrhizus Rhizopus chinensis Rhizopus circinans Rhizopus japonicus Rhizopus kazanensis Rhizopus oryzae Rhizopus pymacus Rhizopus sp. Rhizopus stolonifer Rhizopus tritici Sclerotium rolfsii Trichophyton mentagrophytes Trichophyton rubrum Candida albicans Candida albicans Saccharomyces cerevisiae Saccharomyces cerevisiae Torula utilis Zygosaccharomyces mellis MO Class Fungi Fungi Fungi (continued) Physalospora tucumanensis Phytophthora citrophthora Rhizopus 66-81-2 Microorganism TABLE 12.73 NB, Tween 20, 18 h, 37°C TGB, 18–24 h, 37°C PDA, pH 4.5 YPB, 24 h, 20°C PDA, pH 4.5 PDA, pH 4.5 SA, Tween 80, 21 d, 20°C OA, EtOH, 6 d, 20°C SA, Tween 80, 21 d, 20°C PDA, 5 d, 27°C NB, Tween 20, 8 d, 30°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C OA, EtOH, 3 d, 20°C PDA, Tween 20, 7 d, 24°C PDA, 5 d, 27°C Conditions >1250 1600 500 1000 2800 1000 1000 >1250 >1000 20,000 >1000 >1000 3200 >1000 >1000 >1000 >1000 >1000 >1000 >1000 4000 13% inh. 1000 MIC Yousef and Tawil (1980) Morris et al. (1979) Subba et al. (1967) Schelz et al. (2006) Subba et al. (1967) Subba et al. (1967) Janssen et al. (1988) Narasimba Rao et al. (1971) Janssen et al. (1988) Thompson and Cannon (1986) Yousef and Tawil (1980) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Narasimba Rao et al. (1971) Bouchra et al. (2003) Thompson and Cannon (1986) Ref. 510 Handbook of Essential Oils BacBacBacBacBac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Escherichia coli Neisseria sp. Salmonella typhi Bacillus cereus Bacillus megaterium Corynebacterium diphtheriae Listeria monocytogenes Staphylococcus aureus Staphylococcus aureus Streptococcus faecalis Streptococcus pyogenes Aspergillus niger Botrytis cinera Colletotrichum gleosporoides Endomyces filbuliger Eurotium amstelodami Eurotium herbarum Eurotium repens Eurotium repens Eurotium rubrum Fusarium oxysporum Penicillium corylophilum Penicillium corylophilum Penicillium roqueforti Pythium ultimum Rhizoctonia solani Candida albicans Aspergillus flavus Aspergillus flavus Bac- Escherichia coli MO Class Campylobacter jejuni Microorganism NA, 24 h, 37°C PDA, 3 d, 25°C PDA, 3 d, 25°C Bread, 14 d, 25°C Bread, 14 d, 25°C WFA, 42 d, 25°C PDA, 3 d, 25°C WFA, 42 d, 25°C WFA, 42 d, 25°C Bread, 14 d, 25°C WFA, 42 d, 25°C WFA, 42 d, 25°C Bread, 14 d, 25°C PDA, 3 d, 25°C PDA, 3 d, 25°C WFA, 42 d, 25°C Bread, 14 d, 25°C WFA, 42 d, 25°C NA, 24 h, 37°C NA, 24 h, 37°C BHA, 24 h, 37°C NA, 24 h, 37°C LSA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C BHA, 24 h, 30°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C CAB, 24 h, 42°C Conditions ~20,000 1000 1000 30,000 30,000 Disk, 50,000 1000 Disk, 50,000 Disk, 50,000 30,000 Disk, 50,000 Disk, 50,000 30,000 1000 1000 Disk, 50,000 ~20,000 30,000 Disk, 50,000 ~20,000 ~20,000 Disk, 10,000 ~20,000 Disk, 10,000 ~20,000 ~20,000 Disk, 10,000 ~20,000 ~20,000 Disk, 10,000 Disk, 10,000 TABLE 12.74 Inhibitory Data of Sweet Orange Oil Obtained in the Vapor Phase Test +++ NG +++ +++ +++ + +++ +++ +++ + +++ +++ +++ +++ +++ +++ +++ NG NG +++ NG +++ NG NG +++ NG NG +++ NG + +++ Activity Kellner and Kober (1954) Lee et al. (2007) Lee et al. (2007) Suhr and Nielsen (2003) Suhr and Nielsen (2003) Guynot et al. (2003) Lee et al. (2007) Guynot et al. (2003) Guynot et al. (2003) Suhr and Nielsen (2003) Guynot et al. (2003) Guynot et al. (2003) Suhr and Nielsen (2003) Lee et al. (2007) Lee et al. (2007) Guynot et al. (2003) Suhr and Nielsen (2003) Guynot et al. (2003) Kellner and Kober (1954) Kellner and Kober (1954) Fisher and Phillips (2006) Kellner and Kober (1954) Fisher and Phillips (2006) Kellner and Kober (1954) Kellner and Kober (1954) Fisher and Phillips (2006) Kellner and Kober (1954) Kellner and Kober (1954) Fisher and Phillips (2006) Kellner and Kober (1954) Fisher and Phillips (2006) Ref. In Vitro Antimicrobial Activities of Essential Oils 511 4 (h), 10,000 Drop, 5000 ISA, 48 h, 25°C NA, 24 h, 30°C Bac- Bac+ Pseudomonas aeruginosa Drop, 5000 Yeast Yeast Yeast Saccharomyces cerevisiae Schizosaccharomyces pombe Torula utilis NA, 24 h, 30°C NA, 24 h, 30°C NA, 24 h, 30°C 5 (h), -30,000 Drop, 5000 Drop, 5000 Drop, 5000 5 (h), -30,000 5, 5000 8, 25,000 5, 5000 8, 25,000 5, 5000 Drop, 5000 NA, 18 h, 37°C PDA, 72 h, 28°C SDA, 72 h, 26°C PDA, 48 h, 28°C SDA, 72 h, 26°C PDA, 72 h, 28°C NA, 24 h, 30°C NA, 24 h, 20°C Bac+ Fungi Fungi Fungi Fungi Fungi Yeast Yeast Alternaria porri Aspergillus flavus Aspergillus niger Aspergillus niger Fusarium oxysporum f.sp. cicer Candida albicans Saccharomyces cerevisiae Staphylococcus epidermidis 12.7, 30,000 MHA, 24 h, 37°C Bac+ Bac+ Staphylococcus aureus Staphylococcus aureus MRSA NA, 24 h, 30°C 5 (h), -30,000 Drop, 5000 Listeria monocytogenes NA, 24 h, 30°C NA, 18 h, 37°C Bac- Bac- Escherichia coli Conditions Escherichia coli MO Class Microorganism TABLE 12.75 Inhibitory Data of Tea Tree Oil Obtained in the Agar Diffusion Test 13 20 47 15.3 9 7 8 10.5 11 19–21 14 21–33 7 20 5 17 14 Inhibition Zone (mm) Hili et al. (1997) Hili et al. (1997) Hili et al. (1997) Pawar and Thaker (2007) Shin (2003) Pawar and Thaker (2006) Shin (2003) Pawar and Thaker (2007) Hili et al. (1997) Schelz et al. (2006) Schelz et al. (2006) Carson et al. (1995) Hili et al. (1997) Lis-Balchin et al. (1998) Hili et al. (1997) Schelz et al. (2006) Hili et al. (1997) Ref. 512 Handbook of Essential Oils BacBacBac- Porphyromonas gingivalis Prevotella sp. Bac- Klebsiella pneumoniae Bac- Bac- Klebsiella pneumoniae Klebsiella pneumoniae Bac- Gardnerella vaginalis Klebsiella pneumoniae BacBac- Fusobacterium sp. Bac- Escherichia coli Fusobacterium nucleatum BacBac- Escherichia coli Bac- Escherichia coli Bac- Escherichia coli Bac- Enterobacter aerogenes Escherichia coli Bac- Coliform bacilli Bac- Bac- Coliform bacilli Bac- Bac- Citrobacter freundii Enterococcus faecalis Bac- Bacteroides sp. Escherichia coli BacBac- Aeromonas sobria Bac- Actinobacillus actinomycetemcomitans Bac Bac Bac MO Class Mycoplasma fermentans Mycoplasma hominis Mycoplasma pneumoniae Acinetobacter baumannii Microorganism Conditions VC, Tween 80 HS, 72 h, 37°C MHA, Tween 20, 48 h, 35°C ISB, Tween 20, 24 h, 37°C ISB, Tween 80, 20–24 h, 37°C HIB, Tween 80, 24 h, 35°C VC, Tween 80 VC, Tween 80 HS, 72 h, 37°C TGB, 18 h, 37°C MPB, DMSO, 40 h, 30°C ISB, Tween 80, 20–24 h, 37°C ISB, Tween 80, 16–20 h, 37°C MHA, Tween 20, 48 h, 35°C ISB, Tween 20, 24 h, 37°C ISB, Tween 20, 24 h, 37°C ISB, Tween 80, 20–24 h, 37°C BA, 24 h, 37°C MHB, Tween 80, 24 h, 37°C ISB, Tween 80, 20–24 h, 37°C VC, Tween 80 MHA, Tween 20, 48 h, 35°C HS, 72 h, 37°C Cited Cited Cited HIB, Tween 80, 24 h, 35°C TABLE 12.76 Inhibitory Data of Tea Tree Oil Obtained in the Dilution Test 300–2500 1100 5000 3000 2500 1200–50,000 600 >6000 600–2500 5600 28% inh. 500 2500 2500 2500 2000 5000–7500 2500 5000 10,000–20,000 5000 300–5000 2500 1100 100–600 600–1200 100 600–10,000 MIC continued Hammer et al. (1999) Shapiro et al. (1994) Hammer et al. (1999) Griffin et al. (2000) Harkenthal et al. (1999) Hammer et al. (1996) Hammer et al. (1999) Hammer et al. (1999) Shapiro et al. (1994) Schelz et al. (2006) Hili et al. (1997) Harkenthal et al. (1999) Gustafson et al. (1998) Hammer et al. (1999) Griffin et al. (2000) Griffin et al. (2000) Harkenthal et al. (1999) Banes-Marshall et al. (2001) Banes-Marshall et al. (2001) Harkenthal et al. (1999) Hammer et al. (1999) Hammer et al. (1999) Shapiro et al. (1994) Furneri et al. (2006) Furneri et al. (2006) Furneri et al. (2006) Hammer et al. (1996) Ref. In Vitro Antimicrobial Activities of Essential Oils 513 MO Class BacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBacBac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Proteus mirabilis Proteus vulgaris Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas fluorescens Pseudomonas putida Pseudomonas putida Salmonella choleraesuis Salmonella typhimurium Serratia marcescens Serratia marcescens Serratia marcescens Shigella flexneri Actinomyces viscosus Anaerobic cocci Bacillus cereus Bacillus subtilis Bacillus subtilis Corynebacterium pseudodiphtheriae Corynebacterium sp. Corynebacterium sp. Enterococcus durans Enterococcus faecalis (continued) Microorganism TABLE 12.76 ISB, Tween 80, 20–24 h, 37°C ISB, Tween 80, 20–24 h, 37°C HIB, Tween 80, 24 h, 35°C ISB, Tween 20, 24 h, 37°C ISB, Tween 80, 20–24 h, 37°C ISB, Tween 20, 24 h, 37°C ISB, Tween 80, 20–24 h, 37°C ISB, Tween 20, 24 h, 37°C VC, Tween 80 HS, 16–24, 37°C ISB, Tween 80, 20–24 h, 37°C MHA, Tween 20, 48 h, 35°C HIB, Tween 80, 24 h, 35°C ISB, Tween 20, 24 h, 37°C MHA, Tween 20, 48 h, 35°C ISB, Tween 80, 20–24 h, 37°C MHB, 18–24 h, 37°C ISB, Tween 20, 24 h, 37°C MHB, 18–24 h, 37°C MPB, DMSO, 40 h, 30°C MHA, Tween 20, 48 h, 35°C MHB, 18–24 h, 37°C HIB, Tween 80, 24 h, 35°C MHB, Tween 80, 24 h, 37°C ISB, Tween 20, 24 h, 37°C ISB, Tween 80, 20–24 h, 37°C MCA, 24 h, 37 ISB, Tween 20, 24 h, 37°C ISB, Tween 80, 20–24 h, 37°C Conditions 1000 1000 600–20,000 2000–3000 5000 3000 2500 3000 600–2500 6000 2500 5000 2500–50,000 1000–3000 5000 2500 >20,000 10,000 40,000 75% inh. 500 5000 40,000 20,000–50,000 10,000– >20,000 10,000–80,000 >40,000 >20,000 3000 2500 MIC Harkenthal et al. (1999) Harkenthal et al. (1999) Hammer et al. (1996) Griffin et al. (2000) Harkenthal et al. (1999) Griffin et al. (2000) Harkenthal et al. (1999) Griffin et al. (2000) Hammer et al. (1999) Shapiro et al. (1994) Harkenthal et al. (1999) Hammer et al. (1999) Hammer et al. (1996) Griffin et al. (2000) Hammer et al. (1999) Harkenthal et al. (1999) Papadopoulos et al. (2006) Griffin et al. (2000) Papadopoulos et al. (2006) Hili et al. (1997) Hammer et al. (1999) Papadopoulos et al. (2006) Hammer et al. (1996) Banes-Marshall et al. (2001) Griffin et al. (2000) Harkenthal et al. (1999) Banes-Marshall et al. (2001) Griffin et al. (2000) Harkenthal et al. (1999) Ref. 514 Handbook of Essential Oils Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Staphylococcus aureus Bac+ Bac+ Staphylococcus aureus Staphylococcus capitis Bac+ Staphylococcus aureus Bac+ Bac+ Propionibacterium acnes Staphylococcus capitis Bac+ Propionibacterium acnes Bac+ Bac+ Peptostreptococcus anaerobius Staphylococcus aureus MRSA Bac+ Peptostreptococcus anaerobius Bac+ Bac+ Mobiluncus sp. Staphylococcus aureus MRSA Bac+ Micrococcus varians Bac+ Bac+ Micrococcus sp. Bac+ Bac+ Micrococcus luteus Staphylococcus aureus MRSA Bac+ Micrococcus luteus Staphylococcus aureus MRSA Bac+ Listeria monocytogenes Bac+ Bac+ Enterococcus faecium VRE Bac+ Bac+ Enterococcus faecium Staphylococcus aureus MRSA Bac+ Enterococcus faecalis Staphylococcus aureus MRSA Bac+ Enterococcus faecalis ISB, Tween 80, 20–24 h, 37°C HIB, Tween 80, 24 h, 35°C HIB, Tween 80, 24 h, 37°c Cited HIB, Tween 80, 18 h, 37°C ISB, Tween 20, 24 h, 37°C MHB, Tween 80, 24 h, 37°C BA, 24 h, 37°C Agar, 24 h, 37°C MHB, Tween 80, 24 h, 37°C MHA, Tween 20, 48 h, 35°C MPB, DMSO, 40 h, 30°C ISB, Tween 80, 20–24 h, 37°C MHB, Tween 80, 24 h, 37°C ISB, Tween 20, 24 h, 37°C HIB, Tween 80, 24 h, 35°C BA, 24 h, 37°C ISB, Tween 20, 24 h, 37°C Agar, 24 h, 37°C VC, Tween 80 HS, 72 h, 37°C VC, Tween 80 HIB, Tween 80, 24 h, 35°C HIB, Tween 80, 24 h, 35°C HIB, Tween 80, 24 h, 35°C ISB, Tween 20, 24 h, 37°C ISB, Tween 80, 20–24 h, 37°C HIB, Tween 80, 18 h, 37°C ISB, Tween 80, 20–24 h, 37°C MHB, Tween 80, 24 h, 37°C MHA, Tween 20, 48 h, 35°C 1200–2500 10,000–10,0000 2500–5000 2500 2500 2000–3000 20,000–40,000 10,000 6300–12,500 5000 5000 43% inh. 500 2500 20,000 2000 1200–5000 10,000 5000 3100–6300 600–2500 2000 300–600 5000–10,000 600–5000 600–5000 2000–3000 2500 5000–10,000 1000 80,000 10,000 continued Harkenthal et al. (1999) Hammer et al. (1996) Carson et al. (1995) Carson and Messager (2005) Nelson (1997) Griffin et al. (2000) Banes-Marshall et al. (2001) Banes-Marshall et al. (2001) Raman et al. (1995) Banes-Marshall et al. (2001) Hammer et al. (1999) Hili et al. (1997) Harkenthal et al. (1999) Banes-Marshall et al. (2001) Griffin et al. (2000) Hammer et al. (1996) Banes-Marshall et al. (2001) Griffin et al. (2000) Raman et al. (1995) Hammer et al. (1999) Shapiro et al. (1994) Hammer et al. (1999) Hammer et al. (1996) Hammer et al. (1996) Hammer et al. (1996) Griffin et al. (2000) Harkenthal et al. (1999) Nelson (1997) Harkenthal et al. (1999) Banes-Marshall et al. (2001) Hammer et al. (1999) In Vitro Antimicrobial Activities of Essential Oils 515 Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Bac+ Fungi Fungi Fungi Fungi Staphylococcus epidermidis Staphylococcus epidermidis Staphylococcus epidermidis Staphylococcus haemolyticus Staphylococcus haemolyticus Staphylococcus hominis Staphylococcus hominis Staphylococcus saprophyticus Staphylococcus saprophyticus Staphylococcus warneri Staphylococcus xylosus Staphylococcus xylosus Streptococci beta-haemolytic Streptococci beta-haemolytic Streptococci beta-haemolytic Gp.D Streptococci, faecal Streptococci, faecal Streptococcus equi Streptococcus equisimilis Streptococcus pyogenes Streptococcus pyogenes Streptococcus sobrinus Streptococcus sp. group G Streptococcus zooepidemicus Alternaria alternata Alternaria sp. Alternaria sp. Aspergillus flavus Bac+ MO Class Staphylococcus epidermidis (continued) Staphylococcus epidermidis Microorganism TABLE 12.76 PDA, 7 d, 28°C Cited data RPMI, Tween 80, 48 h, 30°C Cited data THB, Tween 80, 24 h, 35°C THB, Tween 80, 24 h, 35°C HS, 16–24, 37°C MHB, Tween 80, 24 h, 37°C THB, Tween 80, 24 h, 35°C THB, Tween 80, 24 h, 35°C THB, Tween 80, 24 h, 35°C BA, 24 h, 37°C MHB, Tween 80, 24 h, 37°C MHB, Tween 80, 24 h, 37°C MHB, Tween 80, 24 h, 37°C BA, 24 h, 37°C ISB, Tween 80, 20–24 h, 37°C HIB, Tween 80, 24 h, 35°C HIB, Tween 80, 24 h, 35°C ISB, Tween 80, 20–24 h, 37°C HIB, Tween 80, 24 h, 35°C ISB, Tween 80, 20–24 h, 37°C HIB, Tween 80, 24 h, 35°C ISB, Tween 80, 20–24 h, 37°C HIB, Tween 80, 24 h, 35°C Agar, 24 h, 37°C TGB, 18 h, 37°C ISB, Tween 20, 24 h, 37°C ISB, Tween 80, 20–24 h, 37°C HIB, Tween 80, 24 h, 35°C Conditions 62% inh. 500 160–1200 160–1200 3100–7000 600 1200 6000 20,000 1200 1200 1200 >80,000 10,000 5000–20,000 80,000 1200–5000 2500 10,000–30,000 20,000–80,000 2500–5000 20,000–30,000 1200 10,000–40,000 2500–5000 10,000–40,000 6300–12,500 5600 5000 2500 1200–40,000 MIC Feng and Zheng (2007) Carson et al. (2006) Carson and Riley (2002) Carson et al. (2006) Carson et al. (1996) Carson et al. (1996) Shapiro et al. (1994) Banes-Marshall et al. (2001) Carson et al. (1996) Carson et al. (1996) Carson et al. (1996) Banes-Marshall et al. (2001) Banes-Marshall et al. (2001) Banes-Marshall et al. (2001) Banes-Marshall et al. (2001) Banes-Marshall et al. (2001) Harkenthal et al. (1999) Hammer et al. (1996) Hammer et al. (1996) Harkenthal et al. (1999) Hammer et al. (1996) Harkenthal et al. (1999) Hammer et al. (1996) Harkenthal et al. (1999) Hammer et al. (1996) Raman et al. (1995) Schelz et al. (2006) Griffin et al. (2000) Harkenthal et al. (1999) Hammer et al. (1996) Ref. 516 Handbook of Essential Oils Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Aspergillus flavus Aspergillus flavus Aspergillus flavus Aspergillus flavus Aspergillus fumigatus Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus ochraceus Blastoschizomyces capitatus Cladosporium sp. Cladosporium sp. Epidermophyton floccosum Epidermophyton floccosum Fusarium culmorum Fusarium sp. Fusarium sp. Malassezia sympodialis Microsporum canis Microsporum canis Microsporum gypseum Penicillium sp. Penicillium sp. Pleurotus ferulae Pleurotus nebrodensis Pleurotus nebrodensis Trichophyton interdigitale Trichophyton mentagrophytes Trichophyton mentagrophytes Trichophyton mentagrophytes Trichophyton rubrum YES broth, 10 d Cited data RPMI, Tween 80, 72 h, 30°C Cited data RPMI, Tween 80, 96 h, 30°C Cited data YES broth, 10 d Cited data RPMI, Tween 80, 48 h, 35°C Cited data Cited data RPMI, Tween 80, 96 h, 30°C RPMI, Tween 80, 96 h, 30°C Cited data RPMI, Tween 80, 48 h, 35°C SDA, 7 d, 25°C SDA, 7 d, 25°C SDA, 7 d, 25°C RPMI, Tween 80, 96 h, 30°C Cited data MA, Tween 20, 24 h, 30°C RPMI, Tween 80, 96 h, 30°C MA, Tween 20, 24 h, 30°C MYB, 72 h, 26°C MA, Tween 20, 24 h, 30°C MA, Tween 20, 24 h, 30°C RPMI, Tween 80, 48 h, 35°C RPMI, Tween 80, 48 h, 35°C Cited data MA, Tween 20, 24 h, 30°C MYB, 72 h, 26°C RPMI, Tween 80, 48 h, 35°C YES broth, 10 d >10,000 91% inh. 10,000 2500 160–1200 80–1200 80–300 80–7000 76% inh. 10,000 80–2500 80–2500 160–1200 300–5000 40–300 160–300 300–600 300–600 72–82% inh. 1000 64–88% inh. 1000 83–88% inh. 1000 80–300 1100–4400 3000–4000 80–600 10,000 3120 4000–5000 5000–7000 600–1200 600–1200 160–4000 3000–4000 3120 600–1200 continued Lis-Balchin et al. (1998) Carson et al. (2006) Carson and Riley (2002) Carson et al. (2006) Carson and Riley (2002) Carson et al. (2006) Lis-Balchin et al. (1998) Carson et al. (2006) Carson and Riley (2002) Carson et al. (2006) Carson et al. (2006) Carson and Riley (2002) Carson and Riley (2002) Carson et al. (2006) Carson and Riley (2002) Angelini et al. (2008) Angelini et al. (2008) Angelini et al. (2008) Carson and Riley (2002) Carson et al. (2006) Griffin et al. (2000) Carson and Riley (2002) Banes-Marshall et al. (2001) Shin (2003) Griffin et al. (2000) Griffin et al. (2000) Carson and Riley (2002) Carson and Riley (2002) Carson et al. (2006) Griffin et al. (2000) Shin (2003) Carson and Riley (2002) Lis-Balchin et al. (1998) In Vitro Antimicrobial Activities of Essential Oils 517 (continued) Trichophyton rubrum Trichophyton rubrum Trichophyton tonsurans Trichophyton tonsurans Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Candida capitatus Candida famata Candida glabrata Candida glabrata Candida glabrata Candida glabrata Candida glabrata Candida guilliermondii Candida incospigua Candida krusei Candida krusei Candida krusei Candida lipolytica Microorganism TABLE 12.76 Fungi Fungi Fungi Fungi Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast MO Class Cited data RPMI, Tween 80, 96 h, 30°C Cited data RPMI, Tween 80, 96 h, 30°C RPMI, Tween 80, 48 h, 30°C MEB, Tween 20, 24 h, 37 RPMI, Tween 80, 48 h, 35°C MHB, Tween 80, 48 h, 35°C MPB, DMSO, 40 h, 30°C MHA, Tween 20, 48 h, 35°C SDA, 24 h, 37°C RPMI, Tween 80, 48 h, 30°C SDA, 24 h, 37°C MHB, Tween 80, 48 h, 35°C SDA, 24 h, 37°C RPMI, Tween 80, 48 h, 30°C Cited data RPMI, Tween 80, 48 h, 35°C RPMI, Tween 80, 48 h, 30°C RPMI, Tween 80, 48 h, 30°C RPMI, Tween 80, 48 h, 30°C RPMI, Tween 80, 48 h, 35°C SDA, 24 h, 37°C RPMI, Tween 80, 48 h, 30°C Conditions 300–6000 80–300 40–160 40–160 1250 2000 2500 2500–5000 37% inh. 500 5000 5000–10,000 1250–2500 2500 1200–5000 2500–5000 300–1250 300–8000 600 125ß 300 1250 2500 5000 600–1250 MIC Carson et al. (2006) Carson and Riley (2002) Carson et al. (2006) Carson and Riley (2002) Oliva et al. (2003) Griffin et al. (2000) Mondello et al. (2006) Hammer et al. (1998) Hili et al. (1997) Hammer et al. (1999) Banes-Marshall et al. (2001) Oliva et al. (2003) Banes-Marshall et al. (2001) Hammer et al. (1998) Banes-Marshall et al. (2001) Oliva et al. (2003) Carson et al. (2006) Mondello et al. (2006) Oliva et al. (2003) Oliva et al. (2003) Oliva et al. (2003) Mondello et al. (2006) Banes-Marshall et al. (2001) Oliva et al. (2003) Ref. 518 Handbook of Essential Oils Candida lusitaniae Candida parapsilosis Candida parapsilosis Candida parapsilosis Candida parapsilosis Candida sp. Candida sp. Candida tropicalis Candida tropicalis Cryptococcus neoformans Cryptococcus neoformans Malassezia furfur Malassezia furfur Malassezia globosa Malassezia obtusa Malassezia slooffiae Malassezia sympodialis Rhodotorula rubra Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Schizosaccharomyces pombe Torula utilis Trichophyton tonsurans Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast RPMI, Tween 80, 48 h, 30°C RPMI, Tween 80, 48 h, 35°C MHB, Tween 80, 48 h, 35°C Cited data RPMI, Tween 80, 48 h, 30°C MHB, Tween 80, 48 h, 35°C MHB, Tween 80, 24 h, 37°C Cited data RPMI, Tween 80, 48 h, 35°C Cited data RPMI, Tween 80, 48 h, 35°C MMA, Tween 20, 7 d Cited data MMA, Tween 20, 7 d MMA, Tween 20, 7 d MMA, Tween 20, 7 d MMA, Tween 20, 7 d Cited data MEB, Tween 20, 24 h, 37°C Cited data YPB, 24 h, 20°C MPB, DMSO, 40 h, 30°C MPB, DMSO, 40 h, 30°C MPB, DMSO, 40 h, 30°C Cited data 1250 1250 2500–5000 300–5000 600–1250 1200–5000 5000 1200–20,000 600 150–600 300 1200–2500 300–1200 300–1200 1200 1200–2500 160–2500 600 2000 2500 2800 69% inh. 500 74% inh. 500 33% inh. 500 1200–2200 Oliva et al. (2003) Mondello et al. (2006) Hammer et al. (1998) Carson et al. (2006) Oliva et al. (2003) Hammer et al. (1998) Banes-Marshall et al. (2001) Carson et al. (2006) Mondello et al. (2006) Carson et al. (2006) Mondello et al. (2006) Hammer et al. (2000) Carson et al. (2006) Hammer et al. (2000) Hammer et al. (2000) Hammer et al. (2000) Hammer et al. (2000) Carson et al. (2006) Griffin et al. (2000) Carson et al. (2006) Schelz et al. (2006) Hili et al. (1997) Hili et al. (1997) Hili et al. (1997) Carson et al. (2006) In Vitro Antimicrobial Activities of Essential Oils 519 MICair sd MHA, 18 h, 37°C NA, 24 h, 37°C BacBac+ Bac+ Bac+ Bac+ Bacillus subtilis var. aterrimus Mycobacterium avium Staphylococcus aureus Staphylococcus aureus PDA, 3 d, 25°C PDA, 3 d, 25°C Fungi Fungi Fungi Fungi Colletotrichum gleosporoides Fusarium oxysporum Pythium ultimum Rhizoctonia solani Botrytis cinera Bac+ Fungi Streptococcus pyogenes PDA, 3 d, 25°C PDA, 3 d, 25°C PDA, 3 d, 25°C MHA, 18 h, 37°C MHA, 18 h, 37°C Bac+ Bac+ Streptococcus faecalis Streptococcus pneumoniae NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C MHA, 18 h, 37°C 1000 1000 1000 1000 1000 MICair MICair sd sd sd sd MICair Salmonella typhi MICair Bac- Haemophilus influenzae BLA, 18 h, 37°C Bac- Escherichia coli Conditions MO Class Microorganism TABLE 12.77 Inhibitory Data of Tea Tree Oil Obtained in the Vapor Phase Test +++ +++ +++ +++ +++ 50 +++ 50 +++ 50 +++ NG +++ 25 50 Activity Lee et al. (2007) Lee et al. (2007) Lee et al. (2007) Lee et al. (2007) Lee et al. (2007) Inouye et al. (2001) Inouye et al. (2001) Maruzzella and Sicurella (1960) Inouye et al. (2001) Maruzzella and Sicurella (1960) Maruzzella and Sicurella (1960) Maruzzella and Sicurella (1960) Maruzzella and Sicurella (1960) Inouye et al. (2001) Inouye et al. (2001) Ref. 520 Handbook of Essential Oils 4 (h), 10,000 Drop, 5000 ISA, 48 h, 25°C NA, 24 h, 30°C Bac- Bac- Escherichia coli 15, 2500 4 (h), 10,000 Cited ISA, 48 h, 25°C Bac- Bac- Klebsiella pneumoniae —, sd 4 (h), 10,000 NA, 24 h, 37°C ISA, 48 h, 25°C Bac- Bac- Bac- Bac- Proteus vulgaris Proteus vulgaris Pseudomonas aeruginosa Cited Cited 15, 2500 15, 2500 —, sd Proteus sp. NA, 24 h, 37°C Bac- Bac- Moraxella sp. 4 (h), 10,000 Neisseria perflava ISA, 48 h, 25°C 4 (h), 10,000 Klebsiella sp. ISA, 48 h, 25°C Bac- Bac- Escherichia coli Flavobacterium suaveolens TGA, 18–24 h, 37°C 6, 2500 5 (h), -30,000 9.5, 2000 Escherichia coli Cited, 18 h, 37°C NA, 18 h, 37°C Bac- Bac- 15, 2500 8 (h), pure 4 (h), 25,000 —, sd 4 (h), 10,000 4 (h), 10,000 4 (h), 10,000 Escherichia coli Cited NA, 24 h, 37°C TSA, 24 h, 35°C NA, 24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C ISA, 48 h, 25°C Escherichia coli Bac- Bac- Escherichia coli Escherichia coli Bac- Bac- Escherichia coli Escherichia coli Bac- Bac- 4 (h), 10,000 ISA, 48 h, 25°C Bac- Citrobacter freundii Erwinia carotovora 4 (h), 10,000 4 (h), 25,000 ISA, 48 h, 25°C TSA, 24 h, 42°C Bac- Bac- Beneckea natriegens Campylobacter jejuni Enterobacter aerogenes 4 (h), 10,000 ISA, 48 h, 25°C Bac- Bac- Aeromonas hydrophila 4 (h), 10,000 —, sd Alcaligenes faecalis ISA, 48 h, 25°C NA, 24 h, 37°C Bac- Bac- Conditions Acinetobacter calcoaceticus MO Class Aerobacter aerogenes Microorganism TABLE 12.78 Inhibitory Data of Thyme Oil Obtained in the Agar Diffusion Test 0 20 0 16 3 24 12 19 38 41 22.5 27 26 19.3 21 16 8.3 4 21.5 25.5 21.5 10.4 20 20 22.5 0 19 Inhibition Zone (mm) Pizsolitto et al. (1975) Deans and Ritchie (1987) continued Maruzzella and Lichtenstein (1956) Pizsolitto et al. (1975) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Pizsolitto et al. (1975) Deans and Ritchie (1987) Deans and Ritchie (1987) Hili et al. (1997) Deans and Ritchie (1987) Morris et al. (1979) Schelz et al. (2006) Janssen et al. (1986) Pizsolitto et al. (1975) Fawzi (1991) Smith-Palmer et al. (1998) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Ref. In Vitro Antimicrobial Activities of Essential Oils 521 Drop, 5000 15, 2500 15, 2500 4 (h), 10,000 8 (h), pure —, sd 15, 2500 Cited Cited ISA, 48 h, 25°C NA, 24 h, 37°C NA, 24 h, 37°C Cited Bac- Bac- Bac- Bac- Bac+ Bac+ Serratia marcescens Shigella sp. Yersinia enterocolitica Bacillus cereus Bacillus mesentericus 4 (h), 10,000 ISA, 48 h, 25°C Bac+ Bac+ Bac+ Listeria monocytogenes Micrococcus luteus Micrococcus luteus Bac+ Bac+ Bac+ Lactobacillus sp. Listeria monocytogenes Bac+ Lactobacillus plantarum Leuconostoc cremoris Bac+ Bac+ Clostridium sporogenes Corynebacterium sp. Bac+ Bac+ Brevibacterium linens Brochotrix thermosphacta 6, 2500 Cited, 18 h, 37°C Bac+ Bac+ Bacillus subtilis MHA, cited ISA, 48 h, 25°C TSA, 24 h, 35°C ISA, 48 h, 25°C ISA, 48 h, 25°C MRS, cited ISA, 48 h, 25°C TGA, 18–24 h, 37°C ISA, 48 h, 25°C ISA, 48 h, 25°C NA, 24 h, 37°C 9, 20,000 4 (h), 10,000 4 (h), 25,000 4 (h), 10,000 4 (h), 10,000 9, 20,000 4 (h), 10,000 9.5, 2000 4 (h), 10,000 4 (h), 10,000 —, sd 4 (h), 10,000 Bacillus subtilis ISA, 48 h, 25°C Bac+ Bac+ Bacillus sp. 4 (h), 10,000 Bacillus subtilis ISA, 48 h, 25°C —, sd Serratia sp. NA, 24 h, 37°C Bac- 15, 2500 4 (h), 10,000 4 (h), 25,000 4 (h), 10,000 6, 2500 Bac- ISA, 48 h, 25°C Cited —, sd 8 (h), pure Serratia marcescens Bac- Salmonella pullorum TSA, 24 h, 35°C ISA, 48 h, 25°C Cited, 18 h, 37°C NA, 24 h, 30°C NA, 24 h, 37°C NA, 24 h, 37°C Conditions Salmonella sp. Bac- Bac- Pseudomonas aeruginosa Salmonella enteritidis Bac- Bac- Pseudomonas aeruginosa Pseudomonas aeruginosa Bac- Bac- MO Class Pseudomonas aeruginosa (continued) Pseudomonas aeruginosa Microorganism TABLE 12.78 40 32 10 6–20 25– >90 0 18 16 0 16 24.5 33.2 25 12.5 26 0 21 23 8 5 20.5 4 10 26 11.1 22.5 8.7 14 8 0 Inhibition Zone (mm) Pellecuer et al. (1980) Deans and Ritchie (1987) Smith-Palmer et al. (1998) Lis-Balchin et al. (1998) Deans and Ritchie (1987) Pellecuer et al. (1980) Deans and Ritchie (1987) Morris et al. (1979) Deans and Ritchie (1987) Deans and Ritchie (1987) Deans and Ritchie (1987) Janssen et al. (1986) Maruzzella and Lichtenstein (1956) Deans and Ritchie (1987) Pizsolitto et al. (1975) Maruzzella and Lichtenstein (1956) Fawzi (1991) Deans and Ritchie (1987) Pizsolitto et al. (1975) Pizsolitto et al. (1975) Deans and Ritchie (1987) Maruzzella and Lichtenstein (1956) Pizsolitto et al. (1975) Deans and Ritchie (1987) Smith-Palmer et al. (1998) Deans and Ritchie (1987) Janssen et al. (1986) Hili et al. (1997) Fawzi (1991) Maruzzella and Lichtenstein (1956) Ref. 522 Handbook of Essential Oils Bac+ Bac+ Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Streptococcus viridans Streptomyces venezuelae Alternaria porri Alternaria solani Aspergillus fumigatus Aspergillus niger Aspergillus niger Aspergillus niger Fusarium oxysporum f.sp. cicer Helminthosporium sativum Mucor mucedo Nigrospora panici Penicillium digitatum Rhizopus nigricans Bac+ Bac+ Streptococcus micros 9, 20,000 MHA, cited Bac+ PDA, 72 h, 28°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C PDA, 48 h, 28°C SMA, 2–7 d, 20°C SDA, 3 d, 30 PDA, 72 h, 28°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C Cited MHA, cited ISA, 48 h, 25°C 5, 5000 sd sd 5, 5000 sd 8 (h), pure 5, 5000 sd sd sd sd sd sd 15, 2500 9, 20,000 4 (h), 10,000 15, 2500 Drop, 5000 6, 2500 4 (h), 10,000 Streptococcus D Streptococcus faecalis Cited NA, 24 h, 30°C Cited, 18 h, 37°C ISA, 48 h, 25°C 9, 20,000 Staphylococcus aureus TGA, 18–24 h, 37°C 5 (h), -30,000 9.5, 2000 MHA, cited Bac+ Staphylococcus aureus NA, 18 h, 37°C 8 (h), pure Bac+ Bac+ Staphylococcus aureus NA, 24 h, 37°C 15, 2500 4 (h), 25,000 Bac+ Bac+ Staphylococcus aureus Cited TSA, 24 h, 35°C Staphylococcus epidermidis Bac+ Staphylococcus aureus —, sd Staphylococcus epidermidis Bac+ Staphylococcus aureus NA, 24 h, 37°C 9, 20,000 Bac+ Bac+ Staphylococcus aureus MHA, cited 9, 20,000 —, sd Bac+ Bac+ Sarcina ureae MHA, cited NA, 24 h, 37°C Staphylococcus aureus Bac+ Sarcina lutea Staphylococcus aureus Bac+ Bac+ Micrococcus ureae 40 26.6 22 21 12 22 35 10.5 20 22 16 17 14 21 >90 3 17– >90 19.5 40 16 51 33.7 29 25 24 19 14 8.5 >90 0 0 continued Pawar and Thaker (2007) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Pawar and Thaker (2006) Maruzzella and Liguori (1958) Fawzi (1991) Pawar and Thaker (2007) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Pizsolitto et al. (1975) Pellecuer et al. (1980) Deans and Ritchie (1987) Pellecuer et al. (1980) Pellecuer et al. (1980) Pizsolitto et al. (1975) Hili et al. (1997) Janssen et al. (1986) Deans and Ritchie (1987) Morris et al. (1979) Schelz et al. (2006) Fawzi (1991) Pizsolitto et al. (1975) Smith-Palmer et al. (1998) Maruzzella and Lichtenstein (1956) Pellecuer et al. (1980) Maruzzella and Lichtenstein (1956) Pellecuer et al. (1980) In Vitro Antimicrobial Activities of Essential Oils 523 Yeast Yeast Yeast Yeast Yeast Yeast Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Schizosaccharomyces pombe Torula glabrata Torula utilis MO Class Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast (continued) Brettanomyces anomalus Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Candida krusei Candida lipolytica Candida tropicalis Cryptococcus neoformans Cryptococcus rhodobenhani Debaryomyces hansenii Geotrichum candidum Hansenula anomala Kloeckera apiculata Kluyveromyces fragilis Lodderomyces elongisporus Metchnikowia pulcherrima Pichia membranaefaciens Rhodotorula rubra Saccharomyces cerevisiae Microorganism TABLE 12.78 Conditions SMA, 2–7 d, 20°C MPA, 4 d, 30°C NA, 24 h, 30°C NA, 24 h, 30°C MPA, 4 d, 30°C NA, 24 h, 30°C MPA, 4 d, 30°C SMA, 2–7 d, 20°C TGA, 18–24 h, 37°C SDA, 24 h, 37°C Cited, 18 h, 37°C NA, 24 h, 30°C SMA, 2–7 d, 20°C MPA, 4 d, 30°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C SMA, 2–7 d, 20°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C MPA, 4 d, 30°C NA, 24 h, 20°C 5 (h), -30,000 sd 5, 10% sol. sd Drop, 5000 Drop, 5000 5, 10% sol. sd Drop, 5000 5, 10% sol. sd sd 9.5, 2000 8 (h), pure 6, 2500 Drop, 5000 sd 5, 10% sol. sd sd sd sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 5, 10% sol. sd 16 25 80 69 15 67 31 14 14 37 40.7 61 13 18 12 25 14 15 34 18 19 17 16 38 34 21 23–25 Inhibition Zone (mm) Ref. Maruzzella and Liguori (1958) Conner and Beuchat (1984) Hili et al. (1997) Hili et al. (1997) Conner and Beuchat (1984) Hili et al. (1997) Conner and Beuchat (1984) Maruzzella and Liguori (1958) Morris et al. (1979) Fawzi (1991) Janssen et al. (1986) Hili et al. (1997) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Maruzzella and Liguori (1958) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Conner and Beuchat (1984) Schelz et al. (2006) 524 Handbook of Essential Oils BacBac- Enterococcus faecalis Escherichia coli BacBacBacBacBac- Escherichia coli O157:H7 Escherichia coli O157:H7 Haemophilus influenza Helicobacter pylori Bac- Escherichia coli O157:H7 Escherichia coli O157:H7 BacBac- Escherichia coli Bac- Escherichia coli Escherichia coli BacBac- Escherichia coli Bac- Escherichia coli Escherichia coli BacBac- Escherichia coli Escherichia coli BacBac- Escherichia coli Escherichia coli BacBac- Escherichia coli Escherichia coli Bac- Bac- Campylobacter jejuni Bac- Bac- Bordetella bronchiseptica Escherichia coli Bac- Aeromonas sobria Escherichia coli Bac- Bac MO Class Bacteria Acinetobacter baumannii Microorganism Conditions Cited, 20 h, 37°C Cited BHI, 48 h, 35°C BHI, 48 h, 35°C BHI, 48 h, 35°C BHI, 48 h, 35°C MHB, 24 h, 36°C NA, 1–3 d, 30°C MPB, DMSO, 40 h, 30°C TSB, 24 h, 37°C MHB, DMSO, 24 h, 37°C TSB, 24 h, 35°C Cited TGB, 18–24 h, 37°C NB, 24 h, Tween 20, 37 LA, 18 h, 37°C TSB, 3% EtOH, 24 h, 37°C NB, 16 h, 37°C TSB, Tween 80, 48 h, 35°C TGB, 18 h, 37°C MHA, Tween 20, 48 h, 35°C M17, 24 h, 20°C TSB, 24 h, 42°C Cited MHA, Tween 20, 48 h, 35°C 5% Glucose, 9 d, 37 MHA, Tween 20, 48 h, 35°C TABLE 12.79 Inhibitory Data of Thyme Oil Obtained in the Dilution Test 275.2 1000 8000 500 >8000 500 >10,000 750 75% inh. 500 700 62.5 500 500 500 400 375–500 3000–8000 200 2 1500 >10,000 1200 400 250 1200 500–1000 1200 MIC Weseler et al. (2005) continued Pellecuer et al. (1976) Oussalah et al. (2006) Oussalah et al. (2006) Oussalah et al. (2006) Oussalah et al. (2006) Oussalah et al. (2006) Farag et al. (1989) Hili et al. (1997) Di Pasqua et al. (2005) Al-Bayati (2008) Smith-Palmer et al. (1998) Pellecuer et al. (1976) Morris et al. (1979) Fawzi (1991) Remmal et al. (1993) Rota et al. (2004) Lens-Lisbonne et al. (1987) Panizzi et al. (1993) Schelz et al. (2006) Hammer et al. (1999) Di Pasqua et al. (2005) Smith-Palmer et al. (1998) Pellecuer et al. (1976) Hammer et al. (1999) Buchholtz (1875) Hammer et al. (1999) Ref. In Vitro Antimicrobial Activities of Essential Oils 525 1000 1500 NA, 1–3 d, 30°C TSB, 24 h, 37°C BacBacBacBacBacBac- Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas fluorescens BacBacBac- Salmonella typhimurium Serratia marcescens Bac- Salmonella typhimurium Salmonella typhimurium BacBac- Salmonella typhimurium Bac- Salmonella typhimurium Salmonella typhimurium Bac- Salmonella typhimurium 250 MHB, DMSO, 24 h, 37°C BacBac- Salmonella typhi Salmonella typhimurium 500 LA, 18 h, 37°C BacBac- Salmonella enteritidis NA, 1–3 d, 30°C TSB, 3% EtOH, 24 h, 37°C BHI, 48 h, 35°C BHI, 48 h, 35°C TSB, 24 h, 37°C BHI, 48 h, 35°C MHB, DMSO, 24 h, 37°C BHI, 48 h, 35°C MHA, Tween 20, 48 h, 35°C TSB, 3% EtOH, 24 h, 37°C 1250 5000 500 4000 300 2000 125 >20,000 1000 4000–8000 400 Salmonella haddar TSB, 24 h, 35°C BacBac- Pseudomonas sp. 800 >50,000 77% inh. 500 >500 >40 Salmonella enteritidis NB, 16 h, 37°C MPB, DMSO, 40 h, 30°C NB, 24 h, Tween 20, 37°C MHB, DMSO, 24 h, 37°C TSB, Tween 80, 48 h, 35°C MIC >20,000 31.2 62.5 500 Bac- MHA, Tween 20, 48 h, 35°C MHB, DMSO, 24 h, 37°C MHB, DMSO, 24 h, 37°C Cited 125 Pseudomonas aeruginosa Bac- Neisseria flava Cited 1000 5000 Bac- Bac- Neisseria catarrhalis Cited Cited Bac- Bac- Moraxella glucidolytica 2500 500 Proteus vulgaris Bac- Klebsiella pneumoniae MHB, DMSO, 24 h, 37°C MHA, Tween 20, 48 h, 35°C Conditions Proteus mirabilis BacBac- MO Class Klebsiella pneumoniae (continued) Klebsiella pneumoniae Microorganism TABLE 12.79 Farag et al. (1989) Rota et al. (2004) Oussalah et al. (2006) Oussalah et al. (2006) Di Pasqua et al. (2005) Oussalah et al. (2006) Al-Bayati (2008) Oussalah et al. (2006) Hammer et al. (1999) Al-Bayati (2008) Remmal et al. (1993) Rota et al. (2004) Smith-Palmer et al. (1998) Di Pasqua et al. (2005) Farag et al. (1989) Lens-Lisbonne et al. (1987) Hili et al. (1997) Fawzi (1991) Al-Bayati (2008) Panizzi et al. (1993) Hammer et al. (1999) Al-Bayati (2008) Al-Bayati (2008) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Al-Bayati (2008) Hammer et al. (1999) Ref. 526 Handbook of Essential Oils Bac+ Bac+ Bac+ Micrococcus sp. Micrococcus ureae Bac+ Listeria monocytogenes Micrococcus luteus Bac+ Lactococcus lactis subsp. lactis Bac+ Bac+ Lactococcus garvieae Micrococcus flavus Bac+ Lactobacillus sp. Bac+ Bac+ Lactobacillus plantarum Listeria monocytogenes Bac+ Lactobacillus delbrueckii Bac+ Bac+ Enterococcus faecium VRE Listeria monocytogenes Bac+ Enterococcus faecalis Bac+ Bac+ Corynebacterium sp. Bac+ Bac+ Corynebacterium pseudodiphtheriae Listeria monocytogenes Bac+ Brochotrix thermosphacta Listeria monocytogenes Bac+ Bacillus subtilis Bac+ Bac+ Bacillus subtilis Listeria monocytogenes >8000 BHI, 48 h, 35°C Bac+ Bacillus subtilis Bac+ Bac+ Bacillus megaterium Bac+ Bac+ Bacillus cereus Listeria monocytogenes Bac+ Bacillus cereus Listeria monocytogenes <1000–5000 TSB, 3% EtOH, 24 h, 37°C Bac- MHB, cited NA, 1–3 d, 30°C MHB, cited Cited BHI, 48 h, 35°C TSB, 10 d, 4°C TSB, 24 h, 35°C BHI, 48 h, 35°C TSB, 24 h, 37°C BHI, 48 h, 35°C M17, 24 h, 20°C M17, 24 h, 20°C MRS, cited MRS, 24 h, 30°C MRS, 24 h, 37°C HIB, Tween 80, 18 h, 37°C MHA, Tween 20, 48 h, 35°C TGB, 18–24 h, 37°C Cited M17, 24 h, 20°C Cited NA, 1–3 d, 30°C TSB, Tween 80, 48 h, 35°C LA, 18 h, 37°C MHB, DMSO, 24 h, 37°C NB, 24 h, Tween 20, 37°C TSB, 3% EtOH, 24 h, 29°C 150 250 150 500 2000 200 200 1000 >8000 1000 >10,000 >10,000 >10,000 310–620 >10,000 500–10,000 5000 500 125 2500 500 250 2 375–500 >50,000 15.6 3000–5000 3000–8000 2500 Yersinia enterocolitica TSB, 3% EtOH, 24 h, 37°C Bac- Shigella flexneri MHA, Tween 20, 48 h, 35°C Bac- Serratia marcescens continued Pellecuer et al. (1980) Farag et al. (1989) Pellecuer et al. (1980) Pellecuer et al. (1976) Oussalah et al. (2006) Smith-Palmer et al. (1998) Smith-Palmer et al. (1998) Oussalah et al. (2006) Di Pasqua et al. (2005) Oussalah et al. (2006) Oussalah et al. (2006) Rota et al. (2004) Di Pasqua et al. (2005) Di Pasqua et al. (2005) Pellecuer et al. (1980) Di Pasqua et al. (2005) Di Pasqua et al. (2005) Nelson (1997) Hammer et al. (1999) Morris et al. (1979) Pellecuer et al. (1976) Di Pasqua et al. (2005) Pellecuer et al. (1976) Farag et al. (1989) Panizzi et al. (1993) Remmal et al. (1993) Al-Bayati (2008) Fawzi (1991) Rota et al. (2004) Rota et al. (2004) Hammer et al. (1999) In Vitro Antimicrobial Activities of Essential Oils 527 500 95% inh. 500 LA, 18 h, 37°C MPB, DMSO, 40 h, 30°C Bac+ Bac+ Bac+ Staphylococcus aureus Staphylococcus aureus TGB, 18–24 h, 37°C 500 Bac+ Fungi Fungi Absidia glauca Alternaria alternata Streptococcus pyogenes 78–620 MHB, cited Bac+ Bac+ Bac+ Staphylococcus epidermidis Streptococcus D Bac+ Streptococcus faecalis 620 MHB, cited Bac+ Staphylococcus epidermidis Cited RPMI, 1.5% EtOH, 7 d, 30°C Cited NB, 16 h, 37°C Cited TGB, 18 h, 37°C 500 5000 250 150 500 1500 5000 Staphylococcus epidermidis HIB, Tween 80, 18 h, 37°C Bac+ Bac+ Staphylococcus aureus 500 Staphylococcus aureus MRSA NA, 1–3 d, 30°C 4000 5 Staphylococcus aureus BHI, 48 h, 35°C TSB, Tween 80, 48 h, 35°C Bac+ Bac+ 400 31.2 2500 250 250 200 1700 150 1000 <1000–5000 1000 Staphylococcus aureus NB, 24 h, Tween 20, 37°C MHB, DMSO, 24 h, 37°C MHA, Tween 20, 48 h, 35°C BHI, 48 h, 35°C BHI, 48 h, 35°C TSB, 24 h, 35°C TSB, 24 h, 37°C NB, 16 h, 37°C BHI, 48 h, 35°C Cited TSB, 3% EtOH, 24 h, 37°C MIC Staphylococcus aureus Bac+ Bac+ Staphylococcus aureus Staphylococcus aureus Bac+ Bac+ Staphylococcus aureus Staphylococcus aureus Bac+ Bac+ Staphylococcus aureus Staphylococcus aureus Bac+ Bac+ Staphylococcus aureus Bac+ Staphylococcus aureus Staphylococcus aureus Bac+ Staphylococcus aureus 78 MHB, cited Bac+ Bac+ Sarcina ureae Staphylococcus aureus 125 NA, 1–3 d, 30°C Bac+ 125 250 Sarcina sp. NA, 1–3 d, 30°C Cited Bac+ Conditions Bac+ MO Class Mycobacterium phlei (continued) Sarcina lutea Microorganism TABLE 12.79 Pellecuer et al. (1976) Tullio et al. (2006) Pellecuer et al. (1976) Lens-Lisbonne et al. (1987) Pellecuer et al. (1980) Pellecuer et al. (1980) Pellecuer et al. (1976) Schelz et al. (2006) Nelson (1997) Hili et al. (1997) Remmal et al. (1993) Farag et al. (1989) Morris et al. (1979) Panizzi et al. (1993) Oussalah et al. (2006) Fawzi (1991) Al-Bayati (2008) Hammer et al. (1999) Oussalah et al. (2006) Oussalah et al. (2006) Smith-Palmer et al. (1998) Di Pasqua et al. (2005) Lens-Lisbonne et al. (1987) Oussalah et al. (2006) Pellecuer et al. (1976) Rota et al. (2004) Pellecuer et al. (1980) Farag et al. (1989) Pellecuer et al. (1976) Farag et al. (1989) Ref. 528 Handbook of Essential Oils Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Alternaria alternata Alternaria citri Aspergillus chevalieri Aspergillus clavatus Aspergillus flavus Aspergillus flavus Aspergillus flavus Aspergillus flavus Aspergillus flavus Aspergillus flavus Aspergillus flavus var. columnaris Aspergillus fumigatus Aspergillus giganteus Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus niger Aspergillus ochraceus Aspergillus ochraceus Aspergillus oryzae Aspergillus parasiticus Aspergillus parasiticus Aspergillus parasiticus Aspergillus repens Botrytis cinera Cladosporium cladosporoides Cladosporium herbarum Epidermophyton floccosum Epidermophyton floccosum Epidermophyton floccosum Fusarium culmorum Fusarium moniliforme Fusarium oxysporum PDA, 7–14 d, 28°C RPMI, 1.5% EtOH, 7 d, 30°C Cited RPMI, 1.5% EtOH, 7 d, 30°C YES broth, 10 d Cited PDA, 5 d, 27°C PDA, 7–14 d, 28°C PDA, 8 h, 20°C, spore germ. inh. Cited PDA, 8 d, 22°C RPMI, 1.5% EtOH, 7 d, 30°C Cited SA, Tween 80, 21 d, 20°C PDA, 7–14 d, 28°C YES broth, 10 d PDA, 7 d, 28°C PDA, 8 d, 22°C Cited Cited Cited PDA, 7–14 d, 28°C RPMI, 1.5% EtOH, 7 d, 30°C PDA, 5 d, 27°C PDA, 8 h, 20°C, spore germ. inh. CA, 7 d, 28°C RPMI, 1.5% EtOH, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C Cited Cited SB, 72 h, Tween 20, 37°C RPMI, 1.5% EtOH, 7 d, 30°C YES broth, 10 d -86% inh. 10,000 250 1250 <300 1000 625 -92% inh. 10,000 2000 500 500 50–100 250 500 1250–2500 1000 -96% inh. 10,000 500 62% inh. 500 500 250 1000 1000 250 2500 500 50–100 74–76% inh. 500 2500 2500 1000 1000 200 2500–5000 continued Soliman and Badeaa (2002) Tullio et al. (2006) Pellecuer et al. (1976) Tullio et al. (2006) Lis-Balchin et al. (1998) Pellecuer et al. (1976) Thompson and Cannon (1986) Soliman and Badeaa (2002) Thompson (1986) Pellecuer et al. (1976) Arras and Usai (2001) Tullio et al. (2006) Pellecuer et al. (1976) Janssen et al. (1988) Soliman and Badeaa (2002) Lis-Balchin et al. (1998) Feng and Zheng (2007) Arras and Usai (2001) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Soliman and Badeaa (2002) Tullio et al. (2006) Thompson and Cannon (1986) Thompson (1986) Kumar et al. (2007) Tullio et al. (2006) Tullio et al. (2006) Pellecuer et al. (1976) Pellecuer et al. (1976) Fawzi (1991) Tullio et al. (2006) Lis-Balchin et al. (1998) In Vitro Antimicrobial Activities of Essential Oils 529 Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Mucor hiemalis Mucor mucedo Mucor mucedo Mucor racemosus f. racemosus Mucor sp. Penicillium chrysogenum Penicillium digitatum Penicillium frequentans Penicillium italicum Penicillium lanosum Penicillium liliacinum Penicillium rubrum Rhizopus 66-81-2 Rhizopus arrhizus Rhizopus chinensis Rhizopus circinans Rhizopus japonicus Rhizopus kazanensis Rhizopus nigricans Rhizopus oryzae Rhizopus pymacus Rhizopus sp. Rhizopus stolonifer Rhizopus tritici Scopulariopsis brevicaulis Scopulariopsis brevicaulis Syncephalastrum racemosum MO Class Microsporum canis Microsporum gypseum Microsporum gypseum (continued) Microsporum canis Microorganism TABLE 12.79 PDA, 5 d, 27°C PDA, 5 d, 27°C Cited RPMI, 1.5% EtOH, 7 d, 30°C Cited Cited PDA, 8 d, 22°C RPMI, 1.5% EtOH, 7 d, 30°C PDA, 8 d, 22°C RPMI, 1.5% EtOH, 7 d, 30°C Cited Cited PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C PDA, 5 d, 27°C Cited PDA, 5 d, 27°C PDA, 5 d, 27°C RPMI, 1.5% EtOH, 7 d, 30°C PDA, 5 d, 27°C PDA, 5 d, 27°C Cited PDA, 5 d, 27°C RPMI, 1.5% EtOH, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C RPMI, 1.5% EtOH, 7 d, 30°C MBA, Tween 80, 10 d, 30°C MBA, Tween 80, 10 d, 30°C Conditions >10,000 500 500 1000 10,000 500 >10,000 1000 1000 2500 1000 2500 500 1000 500 500 500 500 500 1000 500 500 500 25– >300 500 100 1000 500 12.5– >300 1250–2500 1250–2500 MIC Thompson and Cannon (1986) Thompson and Cannon (1986) Pellecuer et al. (1976) Tullio et al. (2006) Pellecuer et al. (1976) Pellecuer et al. (1976) Arras and Usai (2001) Tullio et al. (2006) Arras and Usai (2001) Tullio et al. (2006) Pellecuer et al. (1976) Pellecuer et al. (1976) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Thompson and Cannon (1986) Pellecuer et al. (1976) Thompson and Cannon (1986) Thompson and Cannon (1986) Tullio et al. (2006) Thompson and Cannon (1986) Thompson and Cannon (1986) Pellecuer et al. (1976) Thompson and Cannon (1986) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Perrucci et al. (1994) Perrucci et al. (1994) Ref. 530 Handbook of Essential Oils Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Candida albicans Candida guilliermondii Candida mycoderma Candida paraspilosis Candida pelliculosa Candida tropicalis Candida tropicalis Candida utilis Geotrichum asteroides Geotrichum candidum Hansenula sp. Saccharomyces carlsbergensis Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Schizosaccharomyces pombe Torula utilis SDB, Tween 80, 48 h, 35°C MPB, DMSO, 40 h, 30°C MHA, Tween 20, 48 h, 35°C NB, 24 h, Tween 20, 37°C Cited Cited, 48 h, 36°C TGB, 18–24 h, 37°C RPMI, DMSO, 48 h, 35°C RPMI, DMSO, 48 h, 35°C Cited Cited Cited Cited RPMI, DMSO, 48 h, 35°C RPMI, DMSO, 48 h, 35°C Cited Cited Cited Cited MPB, DMSO, 40 h, 30°C SDB, Tween 80, 48 h, 35°C YPB, 24 h, 20°C NA, 1–3 d, 30°C MPB, DMSO, 40 h, 30°C MPB, DMSO, 40 h, 30°C RPMI, 1.5% EtOH, 7 d, 30°C SA, Tween 80, 21 d, 20°C Cited SA, Tween 80, 21 d, 20°C <300 1 500 1200 200 2000 2000 500 7.5–10.5 7.5 2000 4000 1000 4000 8.5 7.5 2000 2000 2000 2000 500 2 400–700 500 500 500 <300 2500 1000 Panizzi et al. (1993) Hili et al. (1997) Hammer et al. (1999) Fawzi (1991) Pellecuer et al. (1976) Duarte et al. (2005) Morris et al. (1979) 12,166 12,166 Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) 12,166 12,166 Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Pellecuer et al. (1976) Hili et al. (1997) Panizzi et al. (1993) Schelz et al. (2006) Farag et al. (1989) Hili et al. (1997) Hili et al. (1997) Tullio et al. (2006) Janssen et al. (1988) Pellecuer et al. (1976) Janssen et al. (1988) (1) Thymus vulgaris carvacroliferum, (2) Thymus vulgaris linaloliferum, (3) Thymus vulgaris thuyanoliferum, and (4) Thymus vulgaris thymoliferum. Fungi Fungi Trichophyton mentagrophytes Trichophyton rubrum Annotation: Fungi Fungi Trichophyton interdigitale Trichophyton mentagrophytes In Vitro Antimicrobial Activities of Essential Oils 531 NA, 24 h, 37°C MRS, cited Bac- Bac- Bac- Bac- Bac+ Bac+ Bac+ Haemophilus influenzae Neisseria sp. Salmonella typhi Salmonella typhi Bacillus megaterium Bacillus subtilis var. aterrimus Corynebacterium diphtheriae NA, 24 h, 37°C Fungi Alternaria alternata Aspergillus flavus Aspergillus flavus Aspergillus flavus Bac+ Bac+ Fungi Fungi Fungi Bac+ Streptococcus D Streptococcus faecalis Streptococcus pyogenes Bac+ Staphylococcus epidermidis Bac+ Bac+ Staphylococcus aureus Streptococcus pyogenes NA, 24 h, 37°C Bac+ Staphylococcus aureus Bac+ Bac+ Staphylococcus aureus Bac+ Bac+ Sarcina ureae Streptococcus faecalis Disk, 20,000 ? MHB, cited NA, 24 h, 37°C Bac+ Mycobacterium avium Streptococcus pneumoniae Disk, 20,000 ? MHB, cited Bac+ RPMI, 7 d, 30°C RPMI, 7 d, 30°C WFA, 42 d, 25°C Bread, 14 d, 25°C MHA, 18 h, 37°C NA, 24 h, 37°C MHA, 18 h, 37°C MHA, 18 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C MHB, cited NA, 24 h, 37°C MHB, cited MICair MICair Disk, 50,000 30,000 ~20,000 MICair MICair ~20,000 sd MICair ~20,000 sd Disk, 20,000 ? sd Disk, 20,000 ? Disk, 20,000 ? Micrococcus ureae MHB, cited Bac+ Bac+ Lactobacillus sp. ~20,000 Disk, 20,000 ? ~20,000 sd ~20,000 sd ~20,000 MICair ~20,000 MICair Micrococcus luteus NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C NA, 24 h, 37°C MHA, 18 h, 37°C BLA, 18 h, 37°C Bac- Bac- Escherichia coli Conditions Escherichia coli MO Class Microorganism TABLE 12.80 Inhibitory Data of Thyme Oil Obtained in the Vapor Phase Test +++ 156–312 78 NG 3.13–6.25 NG ++ 3.13–6.25 ++ NG ++ ++ 6.25–12.5 + NG + ++ ++ ++ ++ NG ++ NG NG NG 3.13 12.5 NG Activity Tullio et al. (2006) Tullio et al. (2006) Guynot et al. (2003) Suhr and Nielsen (2003) Inouye et al. (2001) Kellner and Kober (1954) Inouye et al. (2001) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Pellecuer et al. (1980) Pellecuer et al. (1980) Inouye et al. (2001) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Pellecuer et al. (1980) Maruzzella and Sicurella (1960) Pellecuer et al. (1980) Pellecuer et al. (1980) Pellecuer et al. (1980) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Maruzzella and Sicurella (1960) Kellner and Kober (1954) Kellner and Kober (1954) Inouye et al. (2001) Inouye et al. (2001) Kellner and Kober (1954) Ref. 532 Handbook of Essential Oils Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Yeast Aspergillus fumigatus Aspergillus niger Aspergillus niger Botrytis cinera Cladosporium cladosporoides Colletotrichum gleosporoides Endomyces filbuliger Eurotium amstelodami Eurotium herbarum Eurotium repens Eurotium repens Eurotium rubrum Fusarium oxysporum Fusarium oxysporum Microsporum canis Microsporum gypseum Mucor sp. Penicillium corylophilum Penicillium corylophilum Penicillium frequentans Penicillium lanosum Penicillium roqueforti Pythium ultimum Rhizoctonia solani Rhizopus sp. Scopulariopsis brevicaulis Trichophyton mentagrophytes Candida albicans RPMI, 7 d, 30°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C NA, 24 h, 37°C PDA, 3 d, 25°C PDA, 3 d, 25°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C Bread, 14 d, 25°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C RPMI, 7 d, 30°C WFA, 42 d, 25°C Bread, 14 d, 25°C WFA, 42 d, 25°C WFA, 42 d, 25°C WFA, 42 d, 25°C Bread, 14 d, 25°C WFA, 42 d, 25°C PDA, 3 d, 25°C Bread, 14 d, 25°C RPMI, 7 d, 30°C PDA, 3 d, 25°C RPMI, 7 d, 30°C WFA, 42 d, 25°C RPMI, 7 d, 30°C PDA, 3 d, 25°C ~20,000 MICair MICair MICair 1000 1000 MICair MICair 30,000 MICair MICair MICair MICair Disk, 50,000 30,000 Disk, 50,000 Disk, 50,000 Disk, 50,000 30,000 Disk, 50,000 1000 30,000 MICair 1000 MICair Disk, 50,000 MICair 1000 + 312 78 39–78 NG +++ +++ +++ 156 312 + 156 78–156 78 156 NG +++ NG NG NG NG NG + +++ 78 78–312 NG 78–156 Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Kellner and Kober (1954) Lee et al. (2007) Lee et al. (2007) Tullio et al. (2006) Tullio et al. (2006) Suhr and Nielsen (2003) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Tullio et al. (2006) Guynot et al. (2003) Suhr and Nielsen (2003) Guynot et al. (2003) Guynot et al. (2003) Guynot et al. (2003) Suhr and Nielsen (2003) Guynot et al. (2003) Lee et al. (2007) Suhr and Nielsen (2003) Tullio et al. (2006) Lee et al. (2007) Tullio et al. (2006) Guynot et al. (2003) Tullio et al. (2006) Lee et al. (2007) In Vitro Antimicrobial Activities of Essential Oils 533 534 Handbook of Essential Oils 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.). In Vitro Antimicrobial Activities of Essential Oils 535 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 officinalis 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. 536 Handbook of Essential Oils 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 officinalis 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 dissitiflora 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. In Vitro Antimicrobial Activities of Essential Oils 12.3 537 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 538 Handbook of Essential Oils 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 Bitter fennel Caraway Cassia Cinnamon bark Cinnamon leaf Citronella Clary sage Clove Coriander Dwarf pine Eucalyptus Juniper Lavender Lemon Mandarin Matricaria Mint Neroli Nutmeg Peppermint Pinus sylvestris Pinus pinaster Rosemary Star anise Sweet orange Tea tree Thyme 0–1 0–2 0–1 2 1–2 2–3 0–1 0–1 0–2 0–2 0–1 0–1 0–1 0–1 0–1 0–1 0–1 0–3 0–1 0–1 0–1 0–1 0–1 0–1 0–1 0–2 1 1–2 0–3 1–2 0–3 2–3 3 2 1–2 0–1 0–3 0–2 ND 0–1 0–2 0–3 0–1 0–2 0–3 2–3 0 0–2 0–3 0–1 0–2 0–3 ND 0–3 0–3 1–3 0–2 0–3 0–3 1–3 1–3 3 0–3 0–3 1–3 3 0–3 0–3 1–3 0–3 0–3 ND 0 ND 0–3 0 0–3 ND 1–3 0–3 2–3 0–3 0–3 2–3 0–1 0–2 0–1 2 1–3 2–3 0–1 1–2 1–3 0–2 0–1 0–1 0–1 0–3 0–1 0–1 0–1 0–1 0–1 0–1 0–1 1 0–1 0–2 0–1 0–2 1–2 0–3 0–3 1–3 1–3 3 3 2 2–3 0–3 0–3 1–3 ND 1 0 1–3 0–2 0–1 0–3 2–3 1–2 0–3 1–3 0–2 2 0–3 ND 0–2 0–3 0–3 0–2 0–2 0–3 1–3 1–3 3 0–3 0–3 0–3 0–3 2–3 0–3 2–3 0–3 0–3 ND 1–2 ND 0–3 0–1 2–3 ND 1–3 0–3 2–3 0–3 0–3 1–3 2–3 0–2 1–3 3 2–3 3 1–3 0–1 1–3 0–2 0 0–1 0–1 0–2 0–1 1 0–1 0–3 1–2 0–3 0–3 1 0–1 0–2 ND 0–2 1 1–3 0–3 2–3 0–3 1–3 2–3 1–3 1–3 0–2 0–3 0–3 ND 0–2 0–2 0–3 0–3 ND 0–2 2 1–3 0–3 0–3 0–2 2 0–2 ND 0–3 1–3 1–3 0–2 ND ND ND 0–3 0–3 3 0 3 ND ND 0 0 3 0–1 0–2 0 ND ND ND 1–3 0 ND 0–3 0–3 0–3 0 0–3 0–1 0–2 0–1 ND 2–3 2 1–2 0 1–3 0–2 0–1 1 0–1 0–1 0–1 0–1 0–1 1 0–1 0–1 0–1 1 0–1 0–1 0–1 0–1 1–3 1–3 1–2 2 2 3 2–3 2 1–3 0–2 0–2 2 0 2 2 2 2 2 0–2 2 2 2 2–3 1–2 0–3 0–2 2–3 2 1–3 2–3 0 3 3 3 3 3 3 0 3 3 1 3 3 3 3 ND 0 ND 3 ND 2 ND 2 3 3 3 ND 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 = <500 μg/mL; vapor phase test (VP): 0 = no, 1 = weak, 2 = moderate, 3 strong growth reduction. species. The characterization of essential oils with physical properties alone (e.g., density and solubility) is not sufficient for an unequivocal definition of its constituents. At best distillation, analysis, and pharmacological examination of an essential oil are done in a close-time relationship. To find out the most appropriate method for antimicrobial testing of essential oils or components thereof, literature inhibitory data of eugenol against Escherichia coli were compared with each other and it was concluded that the variation of data as it is obtained in the serial dilution test is tolerable (MIC range from 250 to 600 μg/mL) (Pauli et al., 1996). The same is true for clove oil, which was found to be active in dilution tests against Escherichia coli in the range from 400 to 1250 μg/mL in seven of nine examinations (Table 12.26). In Vitro Antimicrobial Activities of Essential Oils 539 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 540 Handbook of Essential Oils 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 In Vivoa Test Materials Anethole Lemon oil Terpineol Nutmeg oil, expressed Geraniol Eugenol Lymphnodes Viscera In Vitrob 33 49 59 61 96 89 50 55 57 73 71 84 2500 1250 625 312 625 1250 Source: Imura, K., 1935. J. Shanghai Sci. Institute/Section, 4, 1: 235–270. 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. a 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). 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