Bioactive Natural Products For Pharmaceutical Applications 2021

Advanced Structured Materials
Dilipkumar Pal
Amit Kumar Nayak Editors
Bioactive
Natural Products
for Pharmaceutical
Applications
Volume 140
Series Editors
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Applied Sciences, Esslingen, Germany
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University Magdeburg, Magdeburg, Sachsen-Anhalt, Germany
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Dilipkumar Pal Amit Kumar Nayak
•
Editors
Bioactive Natural Products

for Pharmaceutical
Applications
123
Editors
Dilipkumar Pal Amit Kumar Nayak
Institute of Pharmaceutical Sciences Department of Pharmaceutics
Guru Ghasidas Vishwavidyalaya Seemanta Institute of Pharmaceutical
(A Central University) Science
Bilaspur, Chhattisgarh, India Mayurbhanj, Odisha, India
ISSN 1869-8433 ISSN 1869-8441 (electronic)

ISBN 978-3-030-54026-5 ISBN 978-3-030-54027-2 (eBook)
https://doi.org/10.1007/978-3-030-54027-2
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Foreword
Bioactive products derived from natural resources possess several biological and
pharmacological significances. Due to numerous health-promoting potential, these
bioactive natural products are widely used by human as a source of medication
since ancient times. For example, many secondary metabolites have already found
huge significance in nutrition and therapeutics. In this perspective, the exploitation
of natural materials possessing different pharmacological actions (such as antioxi-
dant, antitumor, immunomodulatory, anti-inflammatory, antidiabetic, cardioactiv-
ity, diuretic, osteogenic, wound healing, etc.) presents a wide range of therapeutic
activities and hence they are being used in many pharmaceutical applications. In
addition, the assessment of naturally derived bioactive molecules for their inclusive
therapeutic potential has led to the discovery of numerous drug leads in recent
times. Even, these products are also being used as functional additives in many
pharmaceutical preparations. Therefore, the discovery of such bioactive natural
products has inspired many scientists and researchers to explore their potential
pharmaceutical applications. In this regard, many natural resources are being
explored to produce and accomplish the demand for bioactive natural products for
various ranges of pharmaceutical applications.
A thorough understanding of different bioactive natural products is very much
required in order to promote the drug discovery research and their utility in phar-
maceutical fields. I believe this book Bioactive Natural Products for
Pharmaceutical Applications edited by Dr. Dilipkumar Pal and Dr. Amit Kumar
Nayak surely provides updated information on the various aspects of bioactive
natural products for pharmaceutical and pharmacological uses to graduate and
undergraduate students, academicians, industry persons, researchers, pharmaceuti-
cal formulators, and healthcare professionals involved in natural product and
pharmaceutical fields. This new book is a reflection of the rapid development in this
area, contains 25 important chapters presenting the latest research updates on the
recent innovations relating to various bioactive natural products and their uses in
v
vi Foreword
various pharmaceutical fields. I congratulate the editors: Dr. Dilipkumar Pal and
Dr. Amit Kumar Nayak and all contributing authors for bringing the collection
of their noble piece of works.
I shall be happy if this book receives wide attention.
Prof. (Dr.) Upal Kanti Mazumder

Ex-Professor
Department of Pharmaceutical Technology
Jadavpur University
Kolkata, India
Preface
In the present day, there are tough arguments and huge significance in terms of the
safety concerns of naturally derived materials for their potential uses in various
pharmaceutical applications. In this perspective, the exploitation of various bioac-
tive natural products possessing a variety of biological and pharmacological
activities has been discussed and these bioactive natural products are also consid-
ered to have beneficial effects in nutrition and health. Thus, bioactive natural
products are a rich source of novel therapeutics. Natural materials are also currently
utilized in different pharmaceutical preparations mainly as functional additives.
Therefore, the exploration of bioactive molecules from the natural resources con-
tinues to play a significant role in fashioning new pharmaceutical uses. In this
context, bioactive natural products and their utility in pharmaceutical fields need to
be thoroughly understood.
The book entitled Bioactive Natural Products for Pharmaceutical Applications
contains 25 important chapters, which present the latest research updates on the
recent innovations relating to various bioactive natural products (such as alkaloids,
glycosides, flavonoids, anthraquinones, steroids, polysaccharides, tannins and
polyphenolic compounds, volatile oils, fixed oils, fats and waxes, proteins and
peptides, vitamins, marine products, camptothecin, piperines, carvacrol, gedunin,
GABA, ginsenosides, etc.) and their applications in various pharmaceutical fields.
Chapter 1 gives a brief account of secondary metabolites isolated from plant
sources. This is followed by a discussion of bioactive natural products and their
general applications in Chap. 2. Chapter 3 presents various pharmaceutical appli-
cations of polysaccharides derived from plant resources. The role of phytochemi-
cals in cancer prevention and cure has been discussed in Chap. 4. Further, the role
of stress and defense in plant secondary metabolites production is also important,
which has been discussed in Chap. 5. Chapter 6 presents a brief discussion of
various natural compounds extracted from medicinal plants and their
immunomodulatory activities. Biological activities of marine products and nutri-
tional importance are accounted in Chap. 7. Chapter 8 presents a comprehensive
review on the capillary electrophoresis as a new evolutionary platform of plant
secondary metabolites. The occurrence, chemistry, and mode of action of
vii
viii Preface
camptothecin as anticancer drug have been presented in Chap. 9. Chapter 10

accounts a brief discussion on elicitor signal transduction leading to the production
of plant secondary metabolites. Sources, properties, applications, and biotechno-
logical production of piperine as an important bioactive molecule have been dis-
cussed in Chap. 11. Chapter 12 presents the discussion of the antimicrobial
applications of phytoconstituents from turmeric and garlic. In Chap. 13, therapeutic
properties and molecular mechanisms of carvacrol (Origanum vulgare) have been
discussed. Current findings and future directions of pharmaceutical application of
bioactives from Alstonia genus have been presented in Chap. 14. The role of natural
bioactive compounds as antidiabetic agents has been discussed in Chap. 15. In
Chap. 16, bioactivities of gedunin have been comprehensively reviewed.
Antibacterial and antifungal plant metabolites isolated from the tropical medicinal
plants have been accounted in Chap. 17. The role of natural biomaterial in cardiac
tissue engineering has been discussed in Chap. 18. Chapter 19 presents a discussion
on the importance of natural products in cosmetics. In Chap. 20, the encapsulation
of bioactive compounds and their therapeutic potential have been reviewed.
Advances and perspectives of gamma-aminobutyric acid as a bioactive compound
in food have been accounted in Chap. 21. Chapter 22 gives a brief account of
pharmaceutical and therapeutic applications of fenugreek gum. Protein and
enzymes isolated from plant sources and their utilization in pharmaceutical field
have been reviewed in Chap. 23. Chapter 24 presents a comprehensive discussion
on tannins and polyphenols extracted from natural plants and their versatile
application. In the last chapter, the medicinal attribution of ginsenoside as a huge
source of plant bioactive compound has been discussed.
We would like to convey our sincere thanks to all the authors of the chapters for
providing timely and valuable contributions. We specially thank the publisher
Springer International Publishing and Dr. Mayra Castro for their invaluable
support in organization of the editing process. We specially thank Mr. Ashok
Arumairaj, Project Coordinator, Books Production, Springer Nature for his
priceless support right through the beginning to finishing point of this book. We
gratefully acknowledge the permissions to reproduce copyright materials from
various sources. Finally, we would like to thank our family members, respected
teachers, friends, colleagues, and dear students for their continuous encourage-
ments, inspirations, and moral supports during the preparation of the current book.
Together with our contributing authors and the publishers, we will be extremely
pleased if our efforts fulfill the needs of graduate and postgraduate students, aca-
demicians, industry persons, researchers, pharmaceutical formulators, and health-
care professionals involved in natural products and pharmaceutical fields.
Bilaspur, India Dr. Dilipkumar Pal

Mayurbhanj, India Dr. Amit Kumar Nayak
About This Book
A thorough review of the natural bioactive compounds collected from plants,

microbes, and algae has been presented in this book. It contributes detailed and
specialized information on different types of phytoconstituents involving alkaloids,
glycosides, tannins, phenolic compounds, polysaccharides, gum, proteins, enzymes,
etc. and their versatile applications in the field of pharmaceutical and medical
sciences, cosmetics, nutrition, tissue engineering, food, immunology, and biome-
dicine. The role of different phytocompounds in cancer preventions and cure;
diabetic control; and antimicrobial applications including antibacterial and anti-
fungal properties, cardiac tissue engineering, and cosmetics has been discussed in
this book. Some special topics related to secondary metabolites production, stress
and defense mechanism, and elicitor signal transduction in plant secondary
metabolites, camptothecin, carvacrol, GABA, ginsenoside, etc. are presented in this
book in detail. In a summary, the present book possesses a valuable resource with
respect to its exclusivity on various bioactive natural products and their applications
in pharmaceutical fields. This book is a valuable resource for research scholars,
academics, students, industrialists, and subject experts working in the multidisci-
plinary fields like medicinal chemistry, biochemistry, pharmacology, natural pro-
duct chemistry, and other areas related to drug discovery and research.
ix
Contents
1 Elicitor Signal Transduction Leading to the Production of Plant

Secondary Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1
Supriyo Saha and Dilipkumar Pal
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 2
1.2 Types of Plant Metabolites . . . . . . . . . . . . . . . . . . . . . . . . .. 3
1.3 Elicitor and Its Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 5
1.4 Application of Elicitors on the Production of Secondary
Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 6
1.4.1 Production of Secondary Metabolites Using
Abiotic Elicitors . . . . . . . . . . . . . . . . . . . . . . . . . .. 6
1.4.2 Production of Secondary Metabolites Using Biotic
Elicitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 16
Abiotic-Biotic Dual Elicitors . . . . . . . . . . . . . . . . .. 24
1.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 31
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 36
2 An Introduction to Bioactive Natural Products and General
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Tijjani Ahmadu and Khairulmazmi Ahmad
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
2.2 Bioactive Natural Compounds: Distribution
and Geographical Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.3 Phytochemistry of Bioactive Natural Compounds . . . . . . . . . . 64
2.4 General Applications of Bioactive Natural Products . . . . . . . . 71
2.4.1 Traditional Medicine . . . . . . . . . . . . . . . . . . . . . . . . 71
2.4.2 Plant Based Pesticides and Agrochemical Industry . . . 72
2.4.3 Pharmacological Applications in Drugs
Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
2.4.4 Cosmetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
xi
xii Contents
2.5 Delivery Technology for Bioactive Natural Products . . . . . . . . 76

2.5.1 Components of Nanoemulsion . . . . . . . . . . . . . . . . . 77
2.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
3 Plant Polysaccharides in Pharmaceutical Applications . . . . . ..... 93
Amit Kumar Nayak, Md Saquib Hasnain, Amal Kumar Dhara,
and Dilipkumar Pal
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
3.2 Classifications and Sources of Plant Polysaccharides . . . . . . . . 95
3.2.1 Plant Gums . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
3.2.2 Plant Mucilages . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
3.2.3 Plant Starches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
3.3 Applications of Plant Polysaccharides in Pharmaceutical
Dosage Forms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
3.3.1 Emulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
3.3.2 Suspensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
3.3.3 Tablets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
3.3.4 Capsules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
3.3.5 Beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
3.3.6 Microparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
3.3.7 Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
3.3.8 Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
3.3.9 Transdermal Formulations . . . . . . . . . . . . . . . . . . . . 112
3.3.10 Buccal Formulations . . . . . . . . . . . . . . . . . . . . . . . . 113
3.3.11 Nasal Formulations . . . . . . . . . . . . . . . . . . . . . . . . . 115
3.3.12 Ophthalmic Formulations . . . . . . . . . . . . . . . . . . . . 116
3.3.13 Colon-Targeting Formulations . . . . . . . . . . . . . . . . . 116
3.3.14 Dental Formulations . . . . . . . . . . . . . . . . . . . . . . . . 117
3.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
4 The Role of Phytochemicals in Cancer Prevention and Cure . . . . . 127
Braganza Cilwyn, Soundararajan Vijayarathna, Shanmugapriya,
Rameshwar Naidu Jegathambigai, Subramaniam Sreeramanan,
Yeng Chen, and Sreenivasan Sasidharan
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
4.2 Role of Phytochemicals in Cancer Prevention
via Antioxidant Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Via Pro-Oxidant Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
4.4 Role of Phytochemicals in Cancer Cure Via Apoptosis
Induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
4.5 Role of Phytochemicals in Cancer Cure Via Necrosis
Induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Contents xiii
4.6 Role of Phytochemicals in Cancer Cure Via Autophagy

Induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
4.7 Role of Phytochemicals in Cancer Cure Via Regulation
of miRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
4.8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
5 Role of Stress and Defense in Plant Secondary Metabolites
Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Humberto Aguirre-Becerra, Ma Cristina Vazquez-Hernandez,
Diana Saenz de la O, Aurora Alvarado-Mariana,
Ramon G. Guevara-Gonzalez, Juan Fernando Garcia-Trejo,
and Ana Angélica Feregrino-Perez
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
5.2 Abiotic Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
5.2.1 Electromagnetic Sources . . . . . . . . . . . . . . . . . . . . . 154
5.2.2 Acoustic Emissions . . . . . . . . . . . . . . . . . . . . . . . . . 160
5.2.3 Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
5.2.4 Metals and Salt Metals . . . . . . . . . . . . . . . . . . . . . . 172
5.2.5 Volatile Organic Compounds . . . . . . . . . . . . . . . . . . 173
5.2.6 Nutrient Deficiency . . . . . . . . . . . . . . . . . . . . . . . . . 174
5.3 Biotic Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
5.3.1 Bacteria and Viruses . . . . . . . . . . . . . . . . . . . . . . . . 176
5.3.2 Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
5.3.3 Phytohormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
5.3.4 miRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
5.4 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
6 Natural Compounds Extracted from Medicinal Plants
and Their Immunomodulatory Activities . . . . . . . . . . . . . . . . . . . . 197
Vinod Kumar Gurjar and Dilipkumar Pal
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
6.2 Immunomodulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
6.2.1 Plant-Derived Bioactive as Immunomodulators . . . . . 214
6.2.2 Classification of Immunomodulators . . . . . . . . . . . . 220
6.2.3 Low Molecular Weight Immunomodulators . . . . . . . 222
6.2.4 High Molecular Weight Immunomodulators . . . . . . . 223
6.2.5 High Throughput Screening (HTS) for Plants
and Bioactive Compounds . . . . . . . . . . . . . . . . . . . . 226
6.3 Immunomodulatory Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
6.3.1 Acacia Catechu/Senegalia Catechu (Family:
(Fabaceae)) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
6.3.2 Acorus Calamus (Family: Acoraceae) . . . . . . . . . . . 236
xiv Contents
6.3.3 Allium Sativum (Family: Amaryllidaceae) . . . . . . . . . 237

6.3.4 Andrographis Paniculate (Family: Acanthaceae) . . . . 238
6.3.5 Azadirachta Indica (Family: Meliaceae) . . . . . . . . . . 239
6.3.6 Boerhavia Diffusa (Family: Nyctaginaceae) . . . . . . . 240
6.3.7 Clerodendrum Splendens (Family: Lamiaceae) . . . . . 241
6.3.8 Curcuma Longa (Family: Zingiberaceae) . . . . . . . . . 241
6.3.9 Cynodon Dactylon (Family: Poaceae) . . . . . . . . . . . . 242
6.3.10 Ficus Benghalensis (Family: Moraceae) . . . . . . . . . . 242
6.3.11 Glycyrrhiza Uralensis Fisch (Family:
Fabaceae) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
6.3.12 Murraya Koenigii (Family: Rutaceae) . . . . . . . . . . . 243
6.3.13 Ocimum Sanctum (Family: Lamiaceae) . . . . . . . . . . 243
6.3.14 Panax Ginseng (Family: Araliaceae) . . . . . . . . . . . . 244
6.3.15 Picrorhiza Scrophulariiflora (Family:
Scrophulariaceae) . . . . . . . . . . . . . . . . . . . . . . . . . . 245
6.3.16 Syzygium Aromaticum (Family: Myrtaceae) . . . . . . . 246
6.3.17 Terminalia Arjuna (Family: Combretaceae) . . . . . . . 246
6.3.18 Tinospora Cordifolia (Family: Menispermaceae) . . . 247
6.4 Conclusion and Future Perspective . . . . . . . . . . . . . . . . . . . . . 247
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
7 Antibacterial and Antifungal Plant Metabolites from the Tropical
Medicinal Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
Luiz Everson da Silva, Camila Confortin,
and Mallappa Kumara Swamy
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
7.2 Target for Antimicrobial Agents and Resistance
Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
7.3 Tropical Plants with Relevant Antibacterial Activities . . . . . . . 269
7.4 Synergisms of Phytochemicals and Conventional
Antimicrobial Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
7.5 Antibiotic Resistance—Combination Therapy
(Antibiotic + Phytochemical/Plant Extract) . . . . . . . . . . . . . . . 273
7.6 Essential Oils with Antimicrobial Activity
from Tropical Medicinal Plants . . . . . . . . . . . . . . . . . . . . . . . 275
7.7 Plant-Derived Natural Products with Antifungal Activity . . . . . 276
7.8 Fungal Resistance and Modulatory Potential of Extracts,
Fractions and Essential Oil from Tropical Medicinal
Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
7.9 Conclusions and Future Prospects . . . . . . . . . . . . . . . . . . . . . 280
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
Contents xv
8 Capillary Electrophoresis: A New Evolutionary Platform of Plant

Secondary Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
Dilipkumar Pal and Souvik Mukherjee
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
8.1.1 Principle of the Technique . . . . . . . . . . . . . . . . . . . 289
8.1.2 Nature of Spt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
8.2 Factors Effecting Spt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
8.2.1 I.P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
8.2.2 Esp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
8.3 Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
8.4 Difference Between C.e and Others Spt Technique . . . . . . . . . 293
8.5 Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
8.5.1 Quaternary Alkaloidal Compounds . . . . . . . . . . . . . 295
8.5.2 Flavonoid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
8.5.3 Terpenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
8.5.4 Coumarin Derivatives . . . . . . . . . . . . . . . . . . . . . . . 299
8.5.5 Quinones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
8.5.6 Polyamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
8.6 Capillary Electrophoresis Non Aquas (C.eN) for Bioactive
Compound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
8.7 Online C.e Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
8.8 C.e Versus C.e MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
8.9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
9 Camptothecin: Occurrence, Chemistry and Mode of Action . . . . . . 311
Mallappa Kumara Swamy, Boregowda Purushotham,
and Uma Rani Sinniah
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
9.2 Camptothecin Discovery and Its Chemistry . . . . . . . . . . . . . . . 313
9.3 Natural Sources of Camptothecin . . . . . . . . . . . . . . . . . . . . . . 316
9.3.1 Camptothecin from Plants . . . . . . . . . . . . . . . . . . . . 316
9.3.2 Camptothecin from Endophytes . . . . . . . . . . . . . . . . 317
9.4 Mode of Action of Camptothecin . . . . . . . . . . . . . . . . . . . . . . 320
9.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
10 Secondary Metabolites from Plant Sources . . . . . . . . . . . . . . . . . . . 329
Chandi Charan Kandar
10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
10.1.1 Comparison of Secondary Metabolites
with Primary Metabolites . . . . . . . . . . . . . . . . . . . . 332
10.2 Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
10.3 Saponins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336
xvi Contents
10.4 Phenolic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340

10.4.1 Simple Phenolics . . . . . . . . . . . . . . . . . . . . . . . . . . 342
10.4.2 Coumarins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
10.4.3 Flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
10.4.4 Chromones and Xanthones . . . . . . . . . . . . . . . . . . . 347
10.4.5 Tannins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
10.4.6 Stilbenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
10.4.7 Lignans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
10.5 Terpenes and Terpenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
10.5.1 Hemiterpenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
10.5.2 Monoterpenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
10.5.3 Sesquiterpenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
10.5.4 Diterpenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
10.5.5 Sesterterpenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
10.5.6 Triterpenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
10.5.7 Sesquarterpenes . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
10.5.8 Tetraterpenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
10.5.9 Polyterpenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
10.6 Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
10.7 Lipids (Fixed Oil, Fats, Waxes and Phospholipids) . . . . . . . . . 366
10.7.1 Fixed Oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
10.7.2 Waxes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368
10.7.3 Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
10.7.4 Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
10.8 Glycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
10.9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374
11 Pharmaceutical and Therapeutic Applications
of Fenugreek Gum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
Purusottam Mishra, Amit Kumar Srivastava, Tara Chand Yadav,
Vikas Pruthi, and Ramasare Prasad
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
11.1.1 Natural Gums . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
11.1.2 Fenugreek Gum . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
11.1.3 Chemical Composition of Fenugreek Seed . . . . . . . . 382
11.2 Galactomannan: The Chief Constituent of Fenugreek
Gum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
11.2.1 Structural Properties . . . . . . . . . . . . . . . . . . . . . . . . 383
11.2.2 Physicochemical Properties . . . . . . . . . . . . . . . . . . . 383
11.2.3 Biosafety and Toxicological Studies . . . . . . . . . . . . . 386
11.3 Drug Delivery Applications of Fenugreek Gum . . . . . . . . . . . 387
11.3.1 Ophthalmic Drug Delivery . . . . . . . . . . . . . . . . . . . 387
11.3.2 Gastroretentive Drug Delivery . . . . . . . . . . . . . . . . . 388
Contents xvii
11.3.3 Colon Drug Delivery . . . . . . . . . . . . . . . . . . . . . . . 389

11.3.4 Vaginal Drug Delivery . . . . . . . . . . . . . . . . . . . . . . 389
11.3.5 Aerogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
11.4 Fenugreek Gum as a Pharmaceutical Excipient . . . . . . . . . . . . 391
11.4.1 Retarding Agent . . . . . . . . . . . . . . . . . . . . . . . . . . . 391
11.4.2 Super-disintegrating Agent . . . . . . . . . . . . . . . . . . . 391
11.4.3 Mucoadhesive and Bioadhesive Agent . . . . . . . . . . . 392
11.4.4 Matrix Forming Agent . . . . . . . . . . . . . . . . . . . . . . 393
11.4.5 Bioavailability Enhancer . . . . . . . . . . . . . . . . . . . . . 394
11.4.6 Microencapsulation of Probiotic . . . . . . . . . . . . . . . . 394
11.5 Therapeutic Applications of Fenugreek Gum . . . . . . . . . . . . . 395
11.5.1 Antidiabetic Property . . . . . . . . . . . . . . . . . . . . . . . 395
11.5.2 Hypolipidemic Potential and Role in Fat
Accumulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
11.5.3 Anti-inflammatory Potential . . . . . . . . . . . . . . . . . . . 400
11.5.4 Anticancer Potential . . . . . . . . . . . . . . . . . . . . . . . . 400
11.5.5 Hepatoprotective Potential . . . . . . . . . . . . . . . . . . . . 401
11.6 Conclusion and Future Perspectives . . . . . . . . . . . . . . . . . . . . 401
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402
12 Antimicrobial Application Potential of Phytoconstituents
from Turmeric and Garlic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
Shiv Kumar Prajapati, Gaurav Mishra, Akanksha Malaiya, Ankit Jain,
Nishi Mody, and Ashok M. Raichur
12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410
12.2 Turmeric . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
12.2.1 Phytochemistry of Turmeric . . . . . . . . . . . . . . . . . . 412
12.2.2 Antimicrobial Mechanism of Curcuminoids . . . . . . . 412
12.3 Garlic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414
12.3.1 Phytochemistry of Garlic . . . . . . . . . . . . . . . . . . . . . 415
12.3.2 Antimicrobial Mechanism of Phytochemicals
of Garlic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416
12.4 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418
12.4.1 Antimicrobial Applications of Phytoconstituents
from Turmeric . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418
of Garlic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419
12.4.3 Nanoformulation Based Applications . . . . . . . . . . . . 421
12.4.4 Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 422
12.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 428
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
xviii Contents
13 Carvacrol (Origanum vulgare): Therapeutic Properties

and Molecular Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437
Arijit Mondal, Sankhadip Bose, Kamalika Mazumder,
and Ritu Khanra
13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438
13.2 Literature Search Methodology . . . . . . . . . . . . . . . . . . . . . . . 439
13.3 Extraction and Isolation of Carvacrol . . . . . . . . . . . . . . . . . . . 439
13.4 Biosynthesis of Carvacrol . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
13.5 Physical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441
13.6 Metabolism and Excretion of Carvacrol . . . . . . . . . . . . . . . . . 441
13.7 Acute Toxicity of Carvacrol . . . . . . . . . . . . . . . . . . . . . . . . . 443
13.8 Antioxidant Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444
13.9 Antimicrobial Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445
13.10 Anticancer Effect of Carvacrol and the Related Mechanism
of Actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447
13.11 Carvacrol Derivatives with Pharmacological
Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
13.12 Conclusion and Future Perspectives . . . . . . . . . . . . . . . . . . . . 455
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456
14 Pharmaceutical Application of Bio-actives from Alstonia Genus:
Current Findings and Future Directions . . . . . . . . . . . . . . . . . . . . . 463
Atish T. Paul, Ginson George, Nisha Yadav, Arjun Jeswani,
and Prashant S. Auti
14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464
14.2 Botanical Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466
14.3 Phytochemistry and Pharmacological Activities . . . . . . . . . . . . 466
14.3.1 Phytochemistry and Pharmacological Activities
of A. Scholaris . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466
of A. Macrophylla . . . . . . . . . . . . . . . . . . . . . . . . . 489
of A. Angustifolia . . . . . . . . . . . . . . . . . . . . . . . . . . 498
of A. Boonei . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501
of A. Venenata . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
of A. Yunnanensis . . . . . . . . . . . . . . . . . . . . . . . . . . 508
of A. Spatulata . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
of A. Rupestris . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513
of A. Rostrata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515
Contents xix

of A. Pneumatophora . . . . . . . . . . . . . . . . . . . . . . . 516
of A. Penangiana . . . . . . . . . . . . . . . . . . . . . . . . . . 518
of A. Mairei . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519
of A. Congensis . . . . . . . . . . . . . . . . . . . . . . . . . . . 519
of A. Angustiloba . . . . . . . . . . . . . . . . . . . . . . . . . . 519
of A. Actinophylla . . . . . . . . . . . . . . . . . . . . . . . . . . 522
14.4 Pharmacokinetics and Metabolite Identification . . . . . . . . . . . . 522
14.5 Intellectual Property Rights (IPR) Values of Alstonia
Genus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 524
14.6 Conclusion and Future Perspective . . . . . . . . . . . . . . . . . . . . . 528
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529
15 Role of Natural Bio-active Compounds as Antidiabetic Agents . . . . 535
Sandra N. Jimenez-Garcia, Lina Garcia-Mier,
Moises A. Vazquez-Cruz, Xochitl S. Ramirez-Gomez,
15.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 536
15.2 Mechanisms of Action of Antidiabetic Substances . . . . . . . . . 537
15.3 a-Amilase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 538
15.4 a-Glucosidase Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . 541
15.5 Activation of Glucose Transporters . . . . . . . . . . . . . . . . . . . . 545
15.6 Activation of Insulin Secretion . . . . . . . . . . . . . . . . . . . . . . . . 547
15.7 Future Perspective and Conclusion . . . . . . . . . . . . . . . . . . . . . 553
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553
16 An Overview of the Bioactivities of Gedunin . . . . . . . . . . . . . . . . . 563
Yong Sze Ong, Kooi Yeong Khaw, Loh Teng-Hern Tan,
Peng-Nian Yew, Kai-Boon Tan, Wei Hsum Yap, Siah Ying Tang,
Liang Ee Low, Learn-Han Lee, and Bey-Hing Goh
16.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564
16.2 Bioactivities of Gedunin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565
16.2.1 Anti-Cancer Properties of Gedunin . . . . . . . . . . . . . 565
16.2.2 Anti-neurological Disorders and Cryoprotective
Effects of Gedunin . . . . . . . . . . . . . . . . . . . . . . . . . 577
16.2.3 Anti-inflammatory Effects of Gedunin . . . . . . . . . . . 578
16.2.4 Anti-parasitic Effects of Gedunin and
Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
xx Contents
16.2.5 Antimicrobial Effects of Gedunin and Derivatives . . . 580

16.2.6 Insect Growth Inhibition Effects of Gedunin
and Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580
16.3 Commercial Potential of Gedunin . . . . . . . . . . . . . . . . . . . . . . 581
16.4 Conclusion and Gaps of Knowledge . . . . . . . . . . . . . . . . . . . . 581
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582
17 Biological Activities of Marine Products and Nutritional
Importance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587
Dilipkumar Pal and Khushboo Raj
17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588
17.2 Marine Organisms: Source of Nutrition . . . . . . . . . . . . . . . . . 588
17.2.1 Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588
17.2.2 Lipids and Fatty Acids . . . . . . . . . . . . . . . . . . . . . . 589
17.2.3 Sterols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
17.2.4 Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594
17.2.5 Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596
17.2.6 Vitamins and Minerals . . . . . . . . . . . . . . . . . . . . . . 597
17.3 Marine Sources for Pharmacological Effect . . . . . . . . . . . . . . . 598
17.3.1 Anti-cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598
17.3.2 Anti-Cardiovascular Effect . . . . . . . . . . . . . . . . . . . . 603
17.3.3 Anti-coagulant Activity . . . . . . . . . . . . . . . . . . . . . . 604
17.3.4 Anti-obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604
17.3.5 Bone Growth and Healing . . . . . . . . . . . . . . . . . . . . 606
17.3.6 Anti-inflammatory Activities . . . . . . . . . . . . . . . . . . 607
17.3.7 Neuroprotective Agents . . . . . . . . . . . . . . . . . . . . . . 608
17.4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609
17.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
18 Cardiac Tissue Engineering: A Role for Natural Biomaterials . . . . 617
Pallavi Pushp and Mukesh Kumar Gupta
18.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 618
18.2 Approaches for CTE Using Natural Biomaterials,
Cell-Sheet and Decellularized Tissues . . . . . . . . . . . . . . . . . . 619
18.3 Natural Biomaterials Used in ‘Classical’ CTE . . . . . . . . . . . . . 620
18.3.1 Collagen I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 621
18.3.2 Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623
18.3.3 Fibrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624
18.3.4 Alginate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624
18.3.5 Chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625
18.3.6 Fibroin/Silk Fibroin . . . . . . . . . . . . . . . . . . . . . . . . 625
18.3.7 Decellularized ECM . . . . . . . . . . . . . . . . . . . . . . . . 627
18.3.8 Other Natural Biomaterials . . . . . . . . . . . . . . . . . . . 627
Contents xxi
18.4 Enhancing the Elasticity and Electrical Conductivity

of Natural Biomaterials for CTE . . . . . . . . . . . . . . . . . . . . . . 629
18.5 Enhancing the Mechanical Properties of Natural Polymers
for CTE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 630
18.6 Natural Biomaterials as Coating Materials . . . . . . . . . . . . . . . 630
18.7 Types of Scaffolds for CTE from Natural
Biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 631
18.7.1 Fibrous Scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . 631
18.7.2 Porous Scaffolds/sponges . . . . . . . . . . . . . . . . . . . . 632
18.7.3 Hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 632
18.7.4 Bio-Fabricated/Micro-Fabricated Scaffolds . . . . . . . . 633
18.8 Conclusion and Future Direction . . . . . . . . . . . . . . . . . . . . . . 634
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 634
19 The Importance of Natural Products in Cosmetics . . . . . . . . . . . . . 643
Nagarjuna Reddy Desam and Abdul Jabbar Al-Rajab
19.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 644
19.2 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645
19.3 From Past to Future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 647
19.4 Source of Natural Products . . . . . . . . . . . . . . . . . . . . . . . . . . 647
19.5 Extraction and Isolation of Natural Products
or Essential Oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 648
19.6 Plants-Derived Cosmetics and Cosmeceuticals . . . . . . . . . . . . 648
19.6.1 Manufacture of Plant-Derived Cosmetics
and Cosmeceuticals . . . . . . . . . . . . . . . . . . . . . . . . . 649
19.6.2 Substances of Plant-Based Cosmetics
and Cosmeceuticals . . . . . . . . . . . . . . . . . . . . . . . . . 651
19.7 Applications of Natural Products in Cosmetics . . . . . . . . . . . . 662
19.7.1 Natural Products as Skin Care Agents . . . . . . . . . . . 662
19.7.2 Natural Products as Hair Care Agents . . . . . . . . . . . 667
19.7.3 Essential Oils Used as Cosmetics . . . . . . . . . . . . . . . 669
19.8 New Trends in Cosmetics (Plant Origin of By-Products) . . . . . 672
19.8.1 By-Products from Citrus Fruits . . . . . . . . . . . . . . . . 673
19.8.2 By-Products from Tomato and Olive . . . . . . . . . . . . 674
19.8.3 By-Products Processing from Coffee . . . . . . . . . . . . 674
19.9 Future Prospects and Conclusions . . . . . . . . . . . . . . . . . . . . . 675
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 676
20 Encapsulation of Bioactive Compound and Its Therapeutic
Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 687
Lalduhsanga Pachuau, Laldinchhana, Probin Kumar Roy,
James H. Zothantluanga, Supratim Ray, and Sanjib Das
20.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 688
20.2 Rationale for Encapsulation of Bioactive Compounds . . . . . . . 690
xxii Contents
20.3 Encapsulation of Bioactive Compounds . . . . . . . . . . . . . . . . . 691

20.3.1 Microencapsulation . . . . . . . . . . . . . . . . . . . . . . . . . 691
20.3.2 Nano-based Encapsulation Platforms for Bioactive
Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 694
20.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 705
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 705
21 Tannins and Polyphenols Extracted from Natural Plants
and Their Versatile Application . . . . . . . . . . . . . . . . . . . . . . . . . . . 715
Suvadeep Mal and Dilipkumar Pal
21.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 716
21.2 Tannin Occurrence: Plants Containing Tannins . . . . . . . . . . . . 717
21.3 Classification of Tannins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 723
21.3.1 Gallotannins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 723
21.3.2 Ellagitannins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 724
21.3.3 Condensed Tannins . . . . . . . . . . . . . . . . . . . . . . . . . 724
21.3.4 Complex Tannins . . . . . . . . . . . . . . . . . . . . . . . . . . 725
21.4 Biosynthetic Pathways of Tannins . . . . . . . . . . . . . . . . . . . . . 726
21.4.1 Biosynthesis of Hydrolysable Tannins . . . . . . . . . . . 727
21.4.2 Biosynthesis of Condensed and Complex
Tannins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 728
21.5 Extraction Process of Tannins . . . . . . . . . . . . . . . . . . . . . . . . 731
21.6 Physical and Chemical Properties of Tannins . . . . . . . . . . . . . 732
21.7 Chemical Tests of Tannins . . . . . . . . . . . . . . . . . . . . . . . . . . 733
21.7.1 Qualitative Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . 733
21.7.2 Quantitative Tests . . . . . . . . . . . . . . . . . . . . . . . . . . 735
21.8 Activities of Tannins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 736
21.8.1 Anti-oxidant Properties . . . . . . . . . . . . . . . . . . . . . . 736
21.8.2 Anti-microbial Properties . . . . . . . . . . . . . . . . . . . . . 738
21.9 Anti-viral Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 738
21.9.1 Cardioprotective Activity . . . . . . . . . . . . . . . . . . . . . 739
21.9.2 Anti-histaminic Property . . . . . . . . . . . . . . . . . . . . . 740
21.9.3 Cytotoxic and Anticancer Activity . . . . . . . . . . . . . . 740
21.9.4 Anti-diabetic Property . . . . . . . . . . . . . . . . . . . . . . . 743
21.9.5 Anti-obesity Action . . . . . . . . . . . . . . . . . . . . . . . . . 744
21.9.6 Anti-inflammatory Action . . . . . . . . . . . . . . . . . . . . 744
21.9.7 Anti-aging Properties . . . . . . . . . . . . . . . . . . . . . . . 745
21.9.8 Other Therapeutic Activities of Tannins . . . . . . . . . . 745
21.10 Tannins in Industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 746
21.11 Tannins in Cosmeceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . 746
21.12 Tannins in Neutraceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . 747
21.13 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748
Contents xxiii
22 Piperine: Sources, Properties, Applications, and Biotechnological

Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 759
Neetu Sachan, Dilipkumar Pal, and Phool Chandra
22.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760
22.2 Biosynthesis of Piperine . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760
22.3 Extraction Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760
22.4 Effect on Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760
22.5 Effect on Pentobarbitone Sleeping Time . . . . . . . . . . . . . . . . . 762
22.6 Bioavailability of Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763
22.7 Effect on Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763
22.8 Effects on Antioxidant Pathways in Tissues
from Diabetic Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 764
22.9 Effect on the CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765
22.10 Effect on Acute Kidney Injury . . . . . . . . . . . . . . . . . . . . . . . . 766
22.11 Anticonvulsant Mechanisms of Piperine . . . . . . . . . . . . . . . . . 767
22.12 Immunomodulatory and Antitumor Activity . . . . . . . . . . . . . . 768
22.13 Larvicidal Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 769
22.14 Inhibits B Lymphocyte Activation and Effector Functions . . . . 769
22.15 The Anti-tumor Effectiveness and Mechanisms Accompanied
with the Combination of Docetaxel-Piperine . . . . . . . . . . . . . . 769
22.16 Allergic Encephalomyelitis . . . . . . . . . . . . . . . . . . . . . . . . . . 770
22.17 Memory Enhancer and Restoration of Myelin Damage . . . . . . 770
22.18 Effect on Carbamazepine Metabolism . . . . . . . . . . . . . . . . . . . 770
22.19 Bioenhancer Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 773
22.20 Ayurvedic Formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . 773
22.21 Death of Cerebellar Granule Neurons Induced by Piperine
is Distinct from that Induced by Low Potassium Medium . . . . 775
22.22 KV Channel as Therapeutic Target for Prostate Cancer
Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 776
22.23 Piperine Impairs the Migration and T Cell-Activating
Function of Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . 776
22.24 Piperine-Laden Nanoparticles with Increased Dissolving
and Improved Bioavailability for Controlling Epilepsy . . . . . . 780
22.25 In Vitro Cytotoxic and In Silico Activity . . . . . . . . . . . . . . . . 782
22.26 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784
23 Protein and Enzymes Isolated from Plant Sources
and Their Utilization in Pharmaceutical Field . . . . . . . . . . . . . . . . 793
Om Prakash Panda, Sitansu Sekhar Nanda, Dong Kee Yi,
Dilipkumar Pal, and Souvik Mukherjee
23.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794
23.1.1 Classification of pRt . . . . . . . . . . . . . . . . . . . . . . . . 794
23.1.2 Elemental Composition of Proteins . . . . . . . . . . . . . 796
xxiv Contents
23.1.3 Classification Ama . . . . . . . . . . . . . . . . . . . . . . . . . . 798

23.1.4 Properties of pRt . . . . . . . . . . . . . . . . . . . . . . . . . . . 799
23.2 pRt Sources: Animals and PlT . . . . . . . . . . . . . . . . . . . . . . . . . 802
23.3 Some Important pRt, Their Characteristics and Uses . . . . . . . . 804
23.3.1 Soybean . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 804
23.3.2 Wheat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 805
23.3.3 Corn Zein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 805
23.3.4 Pea pRt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 806
23.3.5 Rice pRt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 806
23.3.6 Sunflower pRt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 806
23.4 Isolation of pRt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 807
23.4.1 Selective Precipitation Methods . . . . . . . . . . . . . . . . 807
23.4.2 Isoionic Precipitation . . . . . . . . . . . . . . . . . . . . . . . . 807
23.4.3 Reversed-Phase High-Performance Liquid
Chromatography (RP-HPLC) . . . . . . . . . . . . . . . . . . 808
23.4.4 Mass Spectrometry of pRt . . . . . . . . . . . . . . . . . . . . 808
23.5 Application of pRt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 809
23.6 Introduction of Ezm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 809
23.6.1 Nomenclature and Classification of Ezm . . . . . . . . . . 809
23.6.2 Chemical Nature and Properties of Ezm . . . . . . . . . . 810
23.6.3 Mechanism of Ezm Action . . . . . . . . . . . . . . . . . . . . 811
23.6.4 Important Industrial Ezm and Their Sources . . . . . . . 811
23.6.5 Ezm Derived from PlT Sources . . . . . . . . . . . . . . . . . 813
23.7 Pharmaceutical Applications . . . . . . . . . . . . . . . . . . . . . . . . . 815
23.8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 816
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 816
24 Advances and Perspectives of Gamma-Aminobutyric Acid
as a Bioactive Compound in Food . . . . . . . . . . . . . . . . . . . . . . . . . 819
Priti Jain and Mangesh S. Ghodke
24.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 819
24.1.1 Why is GABA Important? . . . . . . . . . . . . . . . . . . . . 821
24.1.2 Alternative Synthetic Methods of GABA . . . . . . . . . 821
24.2 Pharmaceutical Properties of GABA . . . . . . . . . . . . . . . . . . . . 823
24.2.1 Anti-Hypertensive Effect of GABA . . . . . . . . . . . . . 823
24.2.2 GABA as Neuroprotective Compound
and for Neurological Disorders . . . . . . . . . . . . . . . . 824
24.2.3 GABA as Anti-obesity Agent . . . . . . . . . . . . . . . . . 825
24.2.4 Antimutagenic and Antimicrobial Activities
of c-Aminobutyric Acid . . . . . . . . . . . . . . . . . . . . . 825
24.2.5 GABA as Anti-stress Compound . . . . . . . . . . . . . . . 826
24.2.6 Gamma-Aminobutyric Acid in Thyroid
Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 827
24.2.7 GABA as Renoprotective . . . . . . . . . . . . . . . . . . . . 827
Contents xxv
24.3 GABA as Bioactive Compound in Food . . . . . . . . . . . . . . . . . 828

24.3.1 Microorganisms as Sources of GABA/GAD . . . . . . . 829
24.3.2 Plants as a Source of GABA and GABA
Enriched Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . 829
24.3.3 Dairy Products and Beverages as GABA
Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 830
24.3.4 Marine Sources of GABA . . . . . . . . . . . . . . . . . . . . 831
24.4 Techniques for GABA Enrichment and Advances
in GABA Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 832
24.4.1 GABA Production by LAB . . . . . . . . . . . . . . . . . . . 832
24.4.2 GABA Production by Other Microorganisms . . . . . . 833
24.4.3 Factors Affecting GABA Levels . . . . . . . . . . . . . . . 833
24.4.4 Advances in GABA Production Techniques . . . . . . . 837
24.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839
25 Medicinal Attribution of Ginsenoside: A Huge Source of Plant
Bioactive Compound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 845
Dilipkumar Pal, Souvik Mukherjee, Satish Balasaheb Nimse,
and K. K. Chandra
25.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 846
25.2 Biosynthesis of GND . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 847
25.3 Biotransformation of GND . . . . . . . . . . . . . . . . . . . . . . . . . . . 849
25.4 Medicinal and Nutraceutical Applications . . . . . . . . . . . . . . . . 852
25.4.1 Anti-carcinogenic Effects . . . . . . . . . . . . . . . . . . . . . 852
25.4.2 Cytotoxic and Anti TUm Activity . . . . . . . . . . . . . . . 852
25.4.3 Inhibition of TUm CeL Invasion and MaTs . . . . . . . . . 854
25.4.4 Inhibition of TUm Angiogenesis . . . . . . . . . . . . . . . . 854
25.4.5 Immunomodulatory Effects . . . . . . . . . . . . . . . . . . . 855
25.4.6 Anti-inflammatory Activity . . . . . . . . . . . . . . . . . . . 856
25.4.7 Antistress Activity . . . . . . . . . . . . . . . . . . . . . . . . . 858
25.4.8 Memory, Learning, and NEur Protection . . . . . . . . . . 858
25.4.9 Anti-diabetic Activity . . . . . . . . . . . . . . . . . . . . . . . 859
25.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 859
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 860
About the Editors
Dr. Dilipkumar Pal, (born in West Bengal, India)

Ph.D., M.Pharm., Chartered Chemist, PostDoc
(Australia), is an Associate Professor in the Department
of Pharmaceutical Sciences, Guru Ghasidash
Vishwavidyalaya (A Central University), Bilaspur,
Chhattisgarh, India. He received his Master’s and
Ph.D. degrees from Jadavpur University, Kolkata and
performed postdoctoral research as “Endeavor
Post-Doctoral Research Fellow” in University of
Sydney, Australia. His areas of research interest include
“Isolation, structure Elucidation and biological evalua-
tion of indigenous plants” and “natural biopolymers.” He
has published 172 full research papers in peer-reviewed
reputed national and international scientific journals,
having good impact factor and contributed 114 abstracts
in different national and international conferences. He
has written 1 book, 52 book chapters, and edited 6 books
published by reputed international publishers. His
research publications have acquired a highly remarkable
cited record in Scopus and Google Scholar (H-Index: 40;
i–10–index: 97; total citations 5286 till date). He, in his
21 years of research-oriented teaching profession,
received 13 prestigious national and international pro-
fessionals awards also. He has received Endeavour
Post-Doctoral Research Fellowship Award (Australia)
2010; ISTE Rajalaxmi Award, 2009; APTI young
Pharmacy Teacher Award, 2010; SPER Innovative
Researcher Award-2016. He has guided 8 Ph.D. and 41
Master’s students for their dissertation thesis. He is the
xxvii
xxviii About the Editors
reviewer and editorial board member of 30 and 29

scientific journals, respectively. He is the member and
life member of 15 professional organizations.
Dr. Amit Kumar Nayak (M.Pharm., Ph.D.) is cur-

rently working as Associate Professor at Seemanta
Institute of Pharmaceutical Sciences, Odisha, India. He
has earned his Ph.D. from IFTM University,
Moradabad, Uttar Pradesh, India. He worked as
Senior Research Associate at IIT, Kanpur in a CSIR
sponsored project. He has over 12 years of research
experiences in the field of pharmaceutics, especially in
the development and characterization of novel biopoly-
meric and nanostructured drug delivery systems made
of natural biopolymers. Till date, he has authored over
120 research and review publications in various
high-impact peer-reviewed journals and 76 book chap-
ters and 7 books to his credit. Overall, he has earned
highly impressive publishing and cited record in
Scopus (h-index: 35) and in Google Scholar (h-index:
39, i10-Index: 104). He has presented his research work
at several conferences. He has received University
Foundation Day Research Award-2019 by Biju Patnaik
University of Technology, Odisha. He is life member of
Association of Pharmaceutical Teachers of India
(APTI) and a Registered Pharmacist.
Chapter 1
Elicitor Signal Transduction Leading
to the Production of Plant Secondary
Metabolites
Supriyo Saha and Dilipkumar Pal
Abstract Plant metabolites are highly effective as medicine with a higher efficacy
and lower adverse effect. Two basic metabolites are obtained from nature, namely
primary metabolites and secondary metabolites. Alkaloids, glycosides, terpenoids,
flavonoids are the principal secondary metabolites, and also the primary source for
the drug discovery and development. Elicitors are the substances which under stress
conditions induce the biosynthesis of secondary metabolites of plants. Both biotic
and abiotic elicitors are used in the process. Most common secondary metabolites
Ferulic acid, cinnamic acid, vanillin, coumaric acid, silymarin, affinin, hypocrellin
A, steroiside, menthone, piperitone, glycyrrhizic acid, colchicine, thiocolchicoside,
phenolic acid, gymnemic acid, flavonoids are utilized the elicitation technique.
Elicitors are two types such as: abiotic and biotic. Abiotic elicitors such as salicylic
acid, methyl jasmonate, hydrogen peroxide, lanthanum, different hormones, light,
gamma rays and controlled temperature are used to generate secondary metabolites
of wheat grass, Thymus vulgaris, Silybum marianum, Shiraia bambusicola, Ajuga
bracteosa, broccoli plant, etc. Biotic elicitors like chitosan, rhizobacteria,
Rhizobium leguminosum, Aspergillus tenius, Agrobacterium tumefacians, car-
rageenan, Streptomyces, Rhizopus, dextran, yeast are used to develop or improvise
secondary metabolites of Khus, Mentha pulegium, Tavernia cuneifolia, chickpea, -
Vitis vinifera, Rumex gmelini Turcz, Cupressus lusitanica, etc. Some secondary
metabolites of Coleus aromaticus Benth, Rhododendron tomentosum, Fagonia
indica, Rauwolfia serpentine, Solanum khasianum, Ocimum tenuiflorum, Stevia
rebaudiana etc. are used both abiotic and biotic elicitors.

Keywords Secondary metabolites Abiotic elicitor Biotic elicitor Phenolic

compounds Silymarin Affinin Hypocrellin A
S. Saha (&)
School of Pharmaceutical Sciences & Technology, Sardar Bhagwan Singh University,
Dehradun, Uttarakhand 248161, India
e-mail: supriyo9@gmail.com
D. Pal
Department of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya (A Central
University), Bilaspur, Chhattisgarh 495009, India
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 1

D. Pal and A. K. Nayak (eds.), Bioactive Natural Products
for Pharmaceutical Applications, Advanced Structured Materials 140,
https://doi.org/10.1007/978-3-030-54027-2_1
2 S. Saha and D. Pal
1.1 Introduction
Nowadays, plants are the primary sources of food and medicine for mankind. High
efficacy and lesser side effects are the main advantage of plant metabolites (Sato and
Matsui 2012). Not only that, synthetic molecules are carry various side and adverse
effects to our body system (Hesketh et al. 2002). Natural sources contain diversified
medicinal component such as alkaloids, glycosides, terpenoids, flavonoids; those
are essential for maintaining body immune system. Plants are capable to cultivate a
diversified source of natural metabolites; those are important to link up the behavior
of other organism. Also, in present time exposure to air pollution, water pollution,
ultraviolet ray exposure and deforestation are the most common fact to our nature
and our life style. Natural sources are also helps to protect us from these abiotic
changes (Hiroaki et al. 2012). In this situation ancient folkloric knowledge of plants
and their metabolites is the primary weapon to conquer against various viruses,
pathogens, bacteria and their mutant strains. Also in this era different complex
structure of secondary metabolites is developed using recombinant DNA technol-
ogy. As per the definition, metabolites are compounds produced by plants for
essential functions (Buchanan et al. 2015); as growth and development (primary
metabolites), as well as specific functions as pollinator attraction or defense
mechanism. Also metabolites are organic compounds procured from
enzyme-dependent chemical reactions of organism known as metabolic pathways
(Lena 2012). Nowadays there is a tremendous demand of various plant derived
product as health supplement, natural flavors, natural colors, but due to natural
calamity and lesser growth of the secondary metabolites the producer does not able
to fulfil the demand of the market. Plant tissue culture is one of the way to cope up
with the market demand of secondary metabolites (such as alkaloids, glycosides,
terpenoids, flavonoids etc.) and helps to over produce the secondary metabolites
(Pichersky and Gang 2000). Actually plant tissue culture comprised of clonal
propagation, callus formation and formation of germplasm using a perfect combi-
nation of plant hormones, temperature, humidity and elicitors. Tissue culture is not
only helps to generate secondary metabolites but also participate in seed germi-
nation, improvement of crop quality, immobilization, biomass accumulation,
micropropagation and existence of some rare plants (those are lost during evolu-
tion) (Jensen et al. 2014). Tissue culture mainly focused on shortening of biosyn-
thetic process of plant cell, higher cell division and metabolism; these all factors are
cumulatively increase the growth of secondary metabolites. This chapter mainly
focus on the role of elicitors on the production of secondary metabolites.
1 Elicitor Signal Transduction Leading to the Production of Plant … 3
1.2 Types of Plant Metabolites
Plant metabolites were two types such as primary metabolite and secondary
metabolite.
1. Primary metabolites were the basic requirement for growth and development
and it was present in all plants, organisms or cells. Low molecular weight
primary metabolites were important components of crop plants for both con-
sumers and producers. Primary metabolites such as ethanol, lactic acid, and
certain amino acids were the most primary one. Primary metabolism was
referred as trophophase, which was characterized by balanced growth of
microorganisms. It occurred when all the nutrients needed by the organisms
were provided in the medium. Primary metabolism was essential for plant
existence and reproduction of cells. In the trophophase, the cells possessed with
optimal concentrations of all the macromolecules such as proteins, DNA, RNA
etc. In during trophophase, growth of microorganisms was followed the expo-
nential nature. During trophophase, the collective metabolic products were
known as primary metabolites (Croteau et al. 2000).
Basically primary metabolites were subdivided into two groups, such as:
a. Primary essential metabolites: These were the compounds produced in
adequate quantizes to sustain cell growth e.g. vitamins, amino acids,
nucleosides. Primary microorganisms usually do not overproduce essential
primary metabolites because it was wasted. In case of industrial overpro-
duction, the regulatory mechanisms were taken.
b. Primary metabolic end products: These were the normal and traditional end
products of fermentation process of primary metabolism. The end products
had many industrial applications such as in the form of ethanol, acetone and
lactic acid. Carbon dioxide was a metabolic end product of Saccharomyces
cerevisiae, which was essential for leavening of dough in baking industry.
Growth limitation: Due to insufficient supply of any nutrient (substrate or even
Oxygen), the growth of microorganisms was slows down. However, the
metabolism does not stop, continues until cell lives with the differentiate product
formation (Pal et al. 2019a).
2. Secondary Metabolites: These are sometimes colored, fragrant and flavorful
compounds, which typically conjugate with plant metabolite and organisms.
Interactions were included pollination through animal such as butterfly, bees,
moths, flies as well as by fungi, bacteria, nematodes, and viruses and by foliage,
gastropods and caterpillar. After the ending of exponential growth of microor-
ganisms, idiophase was initiated. Idiophase was characterized by secondary
metabolism. Secondary metabolites (produced in abundance) were not required
by the microorganisms, but it had some industrially very important mainly in the
biotechnology to produce antibiotics, steroids, alkaloids, glycosides, plant
hormones and toxins (Pal et al. 2017).
Characteristics of secondary metabolites:

a. Secondary metabolites were specifically produced by selected microorganisms.
b. Secondary metabolites were less essential for development and reproduction
of organisms.
c. Environmental factors were influenced the production of secondary metabolites
(Pal et al. 2019b).
d. Certain microorganisms can produce structurally related secondary
metabolites as a group of compounds not a single one such as anthracyclines,
produced by Streptomyces.
e. The biosynthetic pathways of were not clearly established for most secondary
metabolites.
f. The regulation of the formation of secondary metabolites was more complex
than primary metabolites (Pal et al. 2018).
Functions of secondary metabolites: There were two possible hypotheses for the
justification of secondary metabolite function as:
a. Secondary metabolites were beneficial for the cells to survive.
b. The secondary metabolites were important for the cell development.
The metabolic regulation was equally complex to achieve overproduction of
secondary metabolites. Some regulatory mechanisms were as follows:
i. Induction: Methionine addition induced certain enzymes, which enhanced
the production of cephalosporin. Also tryptophan regulated ergot alkaloid
biosynthesis.
ii. ii. End product regulation: Secondary metabolites inhibited their own
biosynthesis by negative feed regulation such as penicillin, streptomycin,
puromycin and chloramphenicol (Pal et al. 2019c).
iii. Catabolite regulation: In this process, a key enzyme was participated in the
catabolic pathway was inactivated, inhibited or repressed the process.
Catabolic repression was obtained by carbon or nitrogen sources. Where
the most common source of carbon was glucose, which was participated in
the inhibition process of several antibiotics, such as: penicillin, strepto-
mycin, bacitracin, chloramphenicol, puromycin; whereas ammonia was the
primary source of nitrogen which was catabolite regulators for the over-
production of certain antibiotics (Pal et al. 2019d).
iv. Phosphate regulation: Inorganic phosphate was essential for the growth and
multiplication of prokaryotes and eukaryotes. Concentration of inorganic
phosphate (up to 1 mM) was directly correlated with the concentration of
secondary metabolites e.g. streptomycin, tetracycline, alkaloids, gibberellins.
v. Auto regulation: Certain microorganisms such as: actinomycetes were
associated with the self-regulation for the production of secondary
metabolites. A compound (factor A) was a derivative of a hormone was
suggested to be closely involved in auto regulation for the production
streptomycin by Streptomyces griseus.
vi. Bioconversions: Biotransformation process using microorganisms was

very important for the production of several compounds e.g. vinegar,
sorbose, steroid hormones and certain amino acids. In this process,
microorganisms were responsible for the conversion of a compound to
another structurally related product in one or a few enzymatic reactions.
The bioconversions can be performed with resting cells, spores or even
killed cells. Non-growing cells were preferred for bioconversions due to its
high substrate concentration (Pal et al. 2019e).
1.3 Elicitor and Its Type
Elicitors are the substances which under stress conditions induce the biosynthesis of
secondary metabolites of plants. So elicitation is the process to create stress on the
plant, which relates with the chemical composition and growth of the plant. The
process of elicitation mainly increase the growth of the plant and its metabolites.
Elicitors are two types such as abiotic and biotic elicitor. Abiotic elicitors are
obtained from non-living source. Abiotic elicitors are mainly three types such as:
physical, chemical and hormonal abiotic elicitors. Biotic elicitors are obtained from
biological source and it has four types such as: saccharides, yeast, fungal and
bacterial biotic elicitors (Fig. 1.1).
Fig. 1.1 Elicitor and its types

1.4 Application of Elicitors on the Production

of Secondary Metabolites
1.4.1 Production of Secondary Metabolites Using Abiotic

Elicitors
1.4.1.1 Effect of Arachidonic and Jasmonic Acid Elicitors

on Secondary Metabolites of Wheatgrass
Zlotek et al. was scientifically tested the effect of abiotic elicitors as arachidonic
acid and jasmonic acid on the phenolic and flavonoid content of wheatgrass
(Triticum aestivum L.). The experiment started with slightly grown wheatgrass
plant treated with different concentration of arachidonic acid and jasmonic acid
(arachidonic acid: 0.01, 1.0 and 100 µM; jasmonic acid: 0.01, 1 and 100 µM) at a
relative humidity of 70%, 18 °C of temperature and 200 µmol m−2 s−1 of photon
flux density. Thereafter the growth of the plants were prominent, then make it dry
followed by centrifuged at 9000 g force for 30 min. The total phenolic content,
flavonoid content, Antioxidant and Anti-inflammatory activities were evaluated.
The outcomes revealed that amount of principle flavonoids (luteolin and apigenin)
and phenolic compounds (ferulic acid and syringic acid) were remain unchanged
after elicitation, some polyphenolic compounds observed with increased amount;
but the anti-inflammatory activity was markedly improved. The outcomes con-
cluded that 0.01 lM of arachidonic acid showed better elicitation behavior than
jasmonic acid (Złotek et al. 2019).
1.4.1.2 Effect Abiotic Elicitors on Secondary Metabolites of Thyme,

Greater Celandine and Parsley
Kleinwachter et al. was experimentally proved the effects of slight drought and
abiotic elicitors on the production of secondary metabolites of thyme (Thymus
vulgaris), greater celandine (Chelidonium majus) and parsley (Petroselinum cris-
pum). The experimental procedure was started with the minimization of water
supply to the plant by 50% and continue until the evaporation done by plants were
80% reduced than usual. These conditions leads to the abrupt ratio of soil and water
by (6–10)% as compare to the control plant (17–20)%. Then the samplings of
thyme and greater celandine were treated with methyl jasmonate (MJ) (0.2 mM)
and parsley sampling was treated with (2 mM) concentration, followed by addition
of salicylic acid solution. Then the samplings were quantified the production of
monoterpenes in thyme, benzylisoquinoline alkaloids in greater celandine, flavones
and essential oil in in parsley. The outcomes revealed that the quantity of ben-
zylisoquinoline in greater celandine was increased by 46% and flavones were
increased by 70% in parsley; but the minimized water supply reduced the overall
content of the plants due to its lesser growth. The presence of salicylic acid does not
create any remarkable effect and also the different ratios of MJ regulated the plant
secondary metabolites growth depends upon their species characteristics
(Kleinwachter et al. 2015).
1.4.1.3 Effect of Abiotic Elicitor on Triterpenoid Accumulation

in Centella asiatica
Buraphaka et al. was scientifically tested the effects of abiotic elicitor (MJ and
salicylic acid) on the accumulation of triterpenoid in Centella asiatica plant. The
experiment was started with the reaction between different concentrations of MJ
and salicylic acid (1, 2 and 4 mM) with the leaves of the plant under a shaker for
20 min, 40, 60 and 120 min. Then the treated leaves were divided into two groups:
first group for anti-inflammatory was dried at 50 °C whereas another group was
stored at (−) 80 °C for messenger RNA level expression analysis. The outcomes
revealed that after the treatment with salicylic acid (2 mM), the amount of triter-
penoid was doubled; whereas treatment with MJ the amount of increased content
was 1.4 times than normal. Also the elicited leaves were remarkably inhibited the
nitric oxide production in lipopolysaccharide induced RAW 264.7 macrophage
cells with increased activity of phenylalanine ammonia lyase, peroxidase and
catalase enzymes. So these data correlated with the positive effect of abiotic elicitor
on the aforementioned plant (Buraphaka and Putalun 2020).
1.4.1.4 Effect of Abiotic Elicitor on Affinin Contents in Heliopsis

longipes (Chilcuague)
Parola-Contreras et al. was experimentally proved the effects of salicylic acid and
hydrogen peroxide as aboiotic elicitor on the amount of affinin content in Heliopsis
longipes (chilcuague). The experiment was started with the treatment of the sam-
plings of chilcuague and salicylic acid (5 and 10 mM) at 150 days after trans-
planting as well as hydrogen peroxide (200 and 400 mM) at 157 days after
transplanting. Then the elicited plants were evaluated with the enzymatic activity
for superoxide dismutase, catalase, phenylalanine ammonia lyase, valine decar-
boxylase and also quantified the amount of affinin present in the plant. The out-
comes revealed that after 150 days maximum superoxide dismutase, catalase,
phenylalanine ammonia lyase and valine decarboxylase activities were observed
with 10 mM of salicylic acid, 200 mM of hydrogen peroxide, both salicylic acid
and hydrogen peroxide, 200 mM of hydrogen peroxide as abiotic elicitor. After
157 days, maximum superoxide dismutase, catalase, phenylalanine ammonia lyase
were observed with 10 mM of salicylic acid whereas maximum valine decar-
boxylase activity was observed with 200 mM of hydrogen peroxide elicitor
Fig. 1.2 Typical morphological aspect at day 164 post-transplanting of H. longipes treated with
hydrogen peroxide and salicylic acid. Copyright permission obtained from Parola-Contreras et al.
@ 2020 Elsevier B.V
(Fig. 1.2). After 150 and 157 days, maximum affinin content was observed with
10 mM of salicylic acid and 200 mM of hydrogen peroxide. These data confirmed
the positive effects of abiotic elicitors on chilcaugaue (Parola-Contreras et al. 2020).
1.4.1.5 Effects of Abiotic Elicitor on Hypocrellin a Content in Shiraia

bambusicola
Lu et al. was scientifically proved the effect of abiotic elicitor (lanthum La3+) on the
content of hypocrellin A (anticancer agent mainly against lung adenocarcinoma)
present in Shiraia bambusicola (parasitic fungi present in bamboo). The experiment
was started with mycelium of S. bambusicola and lanthum (La3+) with a concen-
tration range from (0.0–1.4 g/L). The elicited fungus was evaluated by membrane
permeabilization assay with fluorescent dye SYTOX green, generation of reactive
oxygen species using 7-dichloro dihydro fluoresceindiacetate dye followed by
antioxidative activity using NADPH oxidase produce superoxide, superoxide dis-
mutase and catalase enzymes. The outcomes showed that after lanthanum treatment
for 4 h, the hyphae became more strong and intense. The bioactivities showed that
after lanthum treatment the amount of hydrogen peroxide was higher with many
fold increased in antioxidative enzymatic activity. Also vitamin C suppressed the up
regulation of major facilitator superfamily transporter and O-methyltransferase,
polyketide synthase, ATP-binding cassette transporter, O-methyltransferase/
FAD-dependent monooxygenase and FAD/FMN-dependent oxidoreductase
genes. These data correlated with the greater production of hypocrellin A in lan-
thanum elicitor induced fungi (Lu et al. 2019a).
1.4.1.6 Effects of Abiotic Elicitor on Steviosides Production in Stevia

rebaudiana Bertoni Calli
Mejia-Espejel et al. was experimentally proved the effects of abiotic elicitor (sali-
cylic acid, MJ, citric acid, ascorbic acid) with different light (red, blue and white)
and temperature environment on the production of steviosides (diterpene glycoside)
present in Stevia rebaudiana. The experiment was started with the reaction between
3 gm of calii (grown in Murashige and Skoog (MS) medium containing
2,4-dichlorophenoxyacetic acid, BA, citric acid and ascorbic acid) and different
elicitors such as salicylic acid (10 and 100 mM), MJ (10 and 100 mM), antioxi-
dants, growth regulator, different light (white light, red light, blue light and a
combination of red light and blue light) and temperature (25 °C and 28 °C) as
physical abiotic elicitor. The outcomes revealed that maximum stevioside was
obtained at a combination of white light, 28 °C, 100 mM of salicylic acid and
10 mM of MJ. These data strictly adhere with the importance of abiotic elicitor on
the production of steviosides (Mejia-Espejel et al. 2018).
1.4.1.7 Effect of Light as Abiotic Elicitor on the Secondary Metabolite

Growth in Stevia rebaudiana
Ahmad et al. was experimentally proved the effect of light of the production of
secondary metabolites (total phenolic and flavonoid content) and antioxidative
properties of the Stevia rebaudiana plant. The experiment was started with the
development of callus from the leaves of the plant in MS medium contained with
2,4-dichlorophenoxyacetic acid, BA; then the callus was exposed to different light
medium such as green light, yellow light, blue light, red light and white fluorescent
light environments at a 25 °C temperature flowed by antioxidative property
assessment using DPPH antioxidant method. The outcomes revealed that maximum
fresh callus weight was observed in white light with greater growth kinetic. Total
phenolic and flavonoid contents were observed in blue light (Fig. 1.3). Also blue
light elicitor increased the antioxidative property of the callus. These data con-
firmed the importance of blue light on the growth of secondary metabolites of
Stevia rebaudiana (Ahmad et al. 2016).
Fig. 1.3 Effect of different spectral lights on callus morphological features in S. rebaudiana. a red
light induced callus b blue light c yellow light d green light and e control white light. Copyright
permission obtained from Ahmad et al. @ 2016 Elsevier B.V
1.4.1.8 Effect of Gamma Radiation on Secondary Metabolites

of Hypericum triquetrifolium Turra
Azeez et al. was scientifically proved the effect of abiotic elicitor (gamma radiation)
on the production of biomass and secondary metabolites (phenolic compounds and
naphtodiantrones) in Hypericum triquetrifolium Turra plant. The experiment was
started with induction of explants (obtained from leaf, stem and root) with indole
acetic acid (IAA) and thidiazuron (synthetic cytokinin) in MS medium followed by
irradiated with gamma rays of 10, 20, 30 and 40 Gy unit; then finally quantify its
average growth index based on the values of callus biomass at initial and final point.
The outcomes revealed that at 10 Gy scale maximum growth index was observed as
well as maximum accumulation of 4-hydroxybenzoic acid, chlorogenic acid and
epicatechin. At 10 Gy of gamma irradiation the accumulation of naphtodiantrones
(hypericina and pseudohypericin) were observed (Fig. 1.4). So it was quite
resemble that 10 Gy scale of gamma rays directly helps to accumulate the principle
secondary metabolites present in the plant (Azeez et al 2017).
1.4.1.9 Effects of Abiotic Elicitors on Secondary Metabolites of Vitis

vinifera Suspension Culture
Cai et al. was experimentally proved the importance of abiotic elicitor (strepto-
mycin, activated charcoal, etephon) and pressure on the growth of secondary
metabolites of Vitis vinifera plant. The experiment was started with the reaction
between plant cell cultures with the above mentioned abiotic elicitors along with a
diverse pressure treatment from (40–50) Mega Pascal unit for a seven days regime.
Here streptomycin was used to reduce the load of contamination, activated charcoal
Fig. 1.4 The morphological characteristics of Hypericum triquetrifolium Turra callus mass
accumulated from leaf explant and irradiated with 10 Gy dose after the third successive subculture
(a). Hypericum triquetrifolium T. callus mass from leaf irradiated with 30 Gy dose and harvested
after the third regular subculture (b). Copyright permission obtained from Azeez et al. @ 2017
Elsevier B.V
was utilized as source of carbon and etephon was a plant growth regulator. The
outcomes revealed that after 5th day of treatment maximum fresh and dry weight of
the callus was observed with activated charcoal and etephon treatments. Also the
amount of anthocyanins were increased with etephon and activated charcoal
treatments. But the amount of extracellular 3-O-glucosyl resveratrol and phenolic
compound were increased with the combination treatment of etephon and high
pressure. These data confirmed the importance of abiotic elicitors on the growth and
accumulation of secondary metabolites present in Vitis vinifera plant (Cai et al.
2011).
1.4.1.10 Effect of Salicylic Acid on Alkaloid Biosynthesis in Marine

Microalgae Arthrospira platensis
Hadizadeh et al. experimentally proved the importance of salicylic acid as abiotic

elicitor on the biosynthesis of pharmaceutical alkaloid present in marine microalgae
Arthrospira platensis. The experiment as started with addition of salicylic acid (0,
5, 20 and 100) µM concentration to A. platensis suspension culture after 3, 7, 10
and 14 days of culture; followed by estimation of dry weight of biomass and total
alkaloid content. The outcomes revealed that maximum growth was observed
between 8 to 10 days of culture and at 15 days maximum dry weight of biomass
was observed with salicylic acid (5 and 20) µM as well as at 15th day of treatment
maximum content of alkaloid was obtained with 5 µM of salicylic acid. These data
confirmed the importance of salicylic acid on the production of alkaloid in the
marine microalgae (Hadizadeh et al. 2019).
1.4.1.11 Effect of Abiotic Elicitor on Growth of Secondary Metabolites

in Broccoli Plant
Hassini et al. scientifically proved the importance of abiotic elicitors (salicylic acid,
MJ and methionine) on the growth of secondary metabolites present in broccoli
plant. The experiment was started with treating the seeds with potassium chloride,
potassium sulfate and sodium chloride followed by elicited the roots and shoots
with methionine (10 mM), salicylic acid (200 µM) and MJ (100 µM) concentra-
tions. The capacity of roots to conduct water between roots to xylem was measured
by root hydraulic conductivity using root fresh weight and pressure, followed by
assessment of plant defense mechanism using myrosinase activity and total phe-
nolic content present in the plant was also evaluated. The outcomes showed that
maximum dry weight of plant was obtained from roots using potassium sulfate and
from shoots using methionine as elicitor. The root hydraulic conductivity showed
that methionine positively culminated the plant. The myrosinase activity showed
that methionine increase the defense mechanism while sodium chloride, salicylic
acid were suppressed the defense of the plant against insect and herbivores. Two
phenolic acid component as chlorogenic acid and sinapic acid were increased in
presence of potassium chloride and potassium sulfate whereas flavonol was max-
imum obtained through methionine elicitation. These data confirmed the impor-
tance of abiotic elicitors on the growth of secondary metabolites present in broccoli
plant (Hassini et al. 2019).
1.4.1.12 Effect of Abiotic Elicitors on Steviol and Adventitious Root

Growth in Stevia rebaudiana Plant
Kazmi et al. scientifically proved the importance of abiotic elicitors (MJ, phenyl
acetic acid and melatonin) on the growth of steviol glycoside and adventitious roots
in Stevia rebaudiana plant. The experiment was started with culturing the small
pieces of root, stem and leaf portions of the plant in MS medium with BA, IAA and
naphthalene acetic acid (NAA) as growth regulators, then the explants were elicited
using different concentrations of melatonin, phenyl acetic acid and MJ for a period
of 15, 30 and 45 min. The elicited dry mass of the explants were estimated with
total phenolic and flavonoid content as well as evaluated by antioxidative assay
methods. The outcomes revealed that in case of root maximum morphogenic
response was given by combination of 2.0 mg/L of benzylaminopurine (BA) and
1.0 mg/L of NAA; in case of leaf maximum morphogenic response was obtained
from 0.5 mg/L of NAA and from stem maximum morphogenic response was
obtained using 2.0 mg/L of BA plant regulators. Maximum adventitious roots were
obtained from 0.5 mg/L of MJ after 30 min dipping time. Total phenolic and
flavonoid contents were highly obtained from in vitro plants but among the elicitors
MJ-adventitious root combination wins the race and MJ-adventitous root combi-
nation was also observed with higher antioxidative effect (Fig. 1.5). So these data
confirmed the importance of the abiotic elicitors on Stevia rebaudiana plant (Kazmi
et al. 2019).
1.4.1.13 Effect of Ultrasound on Secondary Metabolite Accumulation

in Tomato Plant
Lu et al. experimentally proved the importance of high intensity ultrasound on

secondary metabolite accumulation and antioxidative efficiency of Solanum
lycopersicum (tomato) plant. The experiment was started with ultrasound treatment
of tomatoes with ultrasound (25 kHz) for (1–4) min at room temperature with
26 W/L of acoustic power density. After 0 h, 24 h and 48 h of treatment, the
elicited tomatoes were checked for firmness, total phenolic content, lycopene
content, total carotenoid content and ascorbic acid followed by antioxidative effi-
ciency evaluation. The outcomes showed that firmness of tomatoes were not
remarkably changed so it can stored for longer time; total phenolic content was
maximum after 48 h of storage with 2 min of ultrasound treatment; maximum
lycopene and carotenoid contents were obtained after 48 h of storage with (1–4)
min of treatment as well as ascorbic acid content was higher with 3 min of
Fig. 1.5 In vitro morphogenesis and adventitious root (AR) formation a In vitro germinated
plants, b and c response of root and stem explants to 6-benzyladenine (BA), d–f different
morphological responses by leaf explants, d callus formation, e AR induction from callus, f direct
AR formation, g–j AR formation in leaf explants pretreated with elicitors for defined time periods,
g Melatonin (Mel) induced AR, h: Phenyl acetic acid (PAA) induced AR, i and j Methyl
jasmonate (Me-J) induced AR. Copyright permission obtained from Kazmi et al. @ 2019
Elsevier B.V
ultrasound treatment after 48 h. Maximum phenylalanine ammonia-lyase activity

was also observed after 48 h of storage with (1–4) min of ultrasound treatment.
These data confirmed the importance of abiotic elicitors on secondary metabolite
accumulation in tomato plant (Lu et al. 2020).
1.4.1.14 Effect of Abiotic Elicitors on Metabolite Accumulation

of Trifolium resupinatum
Twaij et al. experimentally proved the importance of abiotic elicitors and precursors
on the accumulation of secondary metabolites in Trifolium resupinatum. The
experiment was started with culture propagation of T. resupinatum shoot in MS
medium containing BA (2.0 mg/L), IAA (0.5 mg/L), sucrose and phytage at pH 7.0
until calli was full grown. Then the calli further transferred into the MS medium
with sucrose, NAA and kinetin as growth regulators. Then the grown explants were
divided into four categories such as shade grown plant, somatic embryos, light
induced and dark induced followed by treated with MJ, salicylic acid and glu-
tathione with concentration range (0, 10, 20, 40, 80 and 160) µM for a period of (0–
4) weeks. Then total phenolic content, total flavonoid and antioxidative efficiency
using DPPH radical scavenging and ferric reducing antioxidant potential assay
activity were checked and evaluated for/by elicited explant. The outcomes revealed
that for shade grown plant, somatic embryos and light induced and dark induced
conditions antioxidative efficiencies against DPPH and ferric reducing antioxidative
processes were observed with greater efficiency in MJ, salicylic acid and glu-
tathione elicitors. Maximum effects as antioxidative and enzymatic activities
against glutathione peroxidase, glutathione reductase and glutathione-S-transferase
were observed with salicylic acid as elicitor. These data correlated abiotic elicitor
and secondary metabolites accumulation in Trifolium resupinatum plant (Twaij
et al. 2019).
1.4.1.15 Effect of MJ Abiotic Elicitor on Anthraquinone Production

in Rubia tinctorum
Perassolo et al. experimentally proved the effects of combined culture medium and
MJ as abiotic elicitor on the production of anthraquinone in Rubia tinctorum plant.
The process was started with culturing of young small clones of leaf margin or
aerial stem of the plant with Agrobaterium rhizogenes in Gamborg B5 medium with
sucrose and ampicillin as source of carbon and strengthening of natural defense
system. Then after two to four weeks roots were developed for cloning in Gamborg
B5 and Llyod-McCown woody plant medium with thiamine, pyridoxine, nicotinic
acid and myoinositol as growth regulators. After fourteen days, inoculum cultures
were elicited in presence of MJ (100 µM) concentration. After a time interval of 0,
2, 4 and 7 days, the amount of anthraquinone and growth of biomass were eval-
uated. The outcomes revealed that after six weeks of treatment biomass in woody
plant medium was higher than Gamborg medium and for anthraquinone production
Gamborg medium was perfectly suited. But after seven days treatment with MJ,
anthraquinone content was remarkably good as compare to dimethylsulfoxide.
These information stated the importance of MJ and growth regulators for the
production of anthraquinone (Perassolo et al. 2017).
1.4.1.16 Effect of Polyunsaturated Fatty Acids on Gymnemic Acid

Production in Gymnema Sylvestre
Praveen et al. scientifically proved the importance of polyunsaturated fatty acids

(oleic acid and linoleic acid) on the production of gymnemic acid present in
Gymnema sylvestre plant. The experiment was started with formation of hairy root
culture of the plant with the plantation in MS medium with sucrose as carbon
source and these were sub-cultured for fortnight period. After the incubation period,
cultures were elicited with oleic acid and linoleic acid with concentration range (0,
1, 5, 10 and 50) µM. The dry biomass, amount of gymnemic acid, total phenolic
content, total flavonoid content were checked as well as antioxidative property was
also evaluated. The outcomes revealed that the growth ratio between fresh and dry
biomass was higher with linoleic acid (1.0 µM) elicitation. Highest amount of
gymnemic acid and greater antioxidative property were observed with linoleic acid
(5.0 µM) elicitation. These data confirmed the importance of linoleic acid for the
production of gymnemic acid (Praveen et al. 2014).
1.4.1.17 Effect of Magnesium Oxide Nanoparticle on Secondary

Metabolite in Atropa belladonna
Tian et al. experimentally proved the importance of magnesium oxide nanoparticle

on the growth and accumulation of antioxidative metabolites present in Atropa
belladonna plant. The experiment was started with germination of the plant in MS
medium containing sodium hypochlorite, then the clones of root and shoot were
elicited with magnesium oxide nanoparticle of (25, 50, 100 and 200) mg/L con-
centration without any presence of cytokinin and auxin. Then the shoot/root
number, length and fresh weights were evaluated; evaluation of relative water
content, chlorophyll content, malondialdehyde and membrane stability index,
enzymatic activities against superoxide dismutase, ascorbate peroxidase were also
evaluated. Also the total phenolic flavonoid content, total alkaloid and antioxidative
efficiencies were measured. The outcomes revealed that shoot/root number, length
and fresh weight, relative water, chlorophyll and membrane stability index were
high with 25 mg/L of magnesium oxide nanoparticle presence. Total phenolic and
flavonoid contents were observed with (100 mg/L) magnesium oxide nanoparticle;
maximum alkaloid content was observed with (25 mg/L) of nanoparticle was well
as maximum antioxidative effect of the elicited plant was observed with (200 mg/L)
magnesium oxide nanoparticle (Tian et al. 2018).
1.4.1.18 Effect of Temperature on Secondary Metabolite

Accumulation in Cold Environment Soil Fungi
Ulaganathan et al. experimentally suggested the effect of temperature on secondary

metabolite accumulation in forty soil isolated fungal strains. The experiment was
started with cultivation of isolated fungi into potato dextrose agar plate for the
mycelia formation followed by antimicrobial assessment against gram positive
(Bacillus subtilis, Enterococcus facaellis and Bacillus cereus) and gram negative
(Pseudomonas aeruginosa and Escherichia coli) strains. As per the first screening
test, the pass over fungal strains [HND 10 (Atradidymella sp), AK 102
(Pseudogymnoascus sp.) and HND 11 (Penicillium flavigenum)] were evaluated
against bacterial strains (E. coli, B. subtilis, S. aureus, P. aeruginosa and Candida
albicans) at different temperature modules as 4, 10, 15 and 28 °C. The outcomes
showed that the growth of E. coli, B. subtilis, S. aureus and C. albicans were highly
inhibited by AK 102 at 4 and 15˚C temperature; P. aeruginosa was not observed
with any susceptibility against strains. So it was quite justified the importance of
temperature on microbial growth inhibition (Ulaganathan et al. 2017).
1.4.1.19 Effect of Polyunsaturated Fatty Acids on Secondary

Metabolite Production in Panax ginseng
Wu et al. experimentally proved the effect of polyunsaturated fatty acids (linoleic

and alpha linolenic acid) on secondary metabolite accumulation and biomass pro-
duction of Panax ginseng plant. The experiment was started with culturing of
adventitious root of ginseng into MS medium containing indole butyric acid and
sucrose. When the roots were 5 fresh weight per litre, these were inoculated into the
same medium with addition of linoleic acid and alpha-linolenic acid with con-
centrations of (1.0, 2.5, 5.0, 10.0 and 20.0) lmol per litre. Total ginsenosides, diol
ginsenoside, triol ginsenoside, total phenolic and total flavonoid contents were
checked for polyunsaturated acid elicited ginseng plant adventitious roots. The
outcomes showed that maximum amount of total ginsenosides, diol ginsenoside,
triol ginsenoside and total flavonoid contents were observed with 5.0 lmol/l of fatty
acid elicitation, whereas total phenolic content was maximum with 5.0 lmol/l of
linoleic acid and 10 lmol/l of alpha linolenic acid. The enzymatic activity data
expressed that maximum superoxide dismutase activity was observed with
20 lmol/l of linoleic acid and 5.0 lmol/l of alpha linolenic acid, maximum catalase
activity was obtained with 2.5 lmol/l of linoleic acid and 5.0 lmol/l of alpha
linolenic acid, maximum ascorbate peroxidase activity was obtained with 20 lmol/l
of linoleic acid and 5.0 lmol/l of alpha linolenic acid as well as glutathione per-
oxidase was higher in case of 2.5 lmol/l of linoleic acid and alpha linolenic acid.
These data confirmed the importance of fatty acids on the secondary metabolite
production and antioxidative properties of ginseng plant (Wu et al. 2009).
1.4.2 Production of Secondary Metabolites Using Biotic

Elicitors
1.4.2.1 Plant Growth Regulating Rhizobacteria Stimulated Secondary

Metabolite Growth in Pennyroyal
Asghari et al. experimentally proved the effect of plant growth regulating rhi-
zobacteria on the biosynthesis of secondary metabolites in pennyroyal (Mentha
pulegium) plant under droght situation. The experiment was started with culturing
the seeds of pennyroyal plant with rhizobacteria in four different categories such as
control group without rhizobacteria, treated with Azotobacter chroococcum, treated
with Azospirillum brasilense and final group treated with a combination of A.
chroococcum and A. brasilense followed by treat in the environment with enough
water, moderate water and less water condition. Relative water content, chlorophyll
fluorescence, total phenolic content, total flavonoid content, antioxidative activities
using glutathione peroxidase, catalase and superoxide dismutase and radical scav-
enging activity against DPPH were checked and evaluated by the elicited plant. The
outcomes showed that relative water and chlorophyll contents were highest in
enough water condition and lowest with less water condition; the glutathione
peroxidase, catalase and superoxide dismutase activities were maximum less water
condition with A. chroococcum elicitated condition followed by A. brasilense and
its combination. Abscisic acid content, total phenolic content, total flavonoid
content, radical scavenging activities as well as presence of essential oils such as
1,8-cineloe, menthone and pulegone were maximum in case of less water condition
with A. chroococcum and A. brasilense combination; whereas piperitone was
maximum obtained with moderate water condition with A. chroococcum and A.
brasilense combination. So these data directly stated the importance of rhizobac-
teria on pennyroyal plant (Asghari et al. 2020).
1.4.2.2 Effect of Carrageenan on Secondary Metabolite Growth

in Chickpea and Maize Plant
Bi et al. scientifically proved the importance of carrageenan (polysaccharide) on the

growth of secondary metabolite present in chick pea and maize plants. The process
was started with formation of two types of elicitors as liquid carrageenan elicitor of
100 µg glucose concentration and solid carrageenan elicitor (mixer of hot aqueous
extract of carrageenan with soil). The treatment module had three types: (i) 50 ml of
liquid elicitor (ii) 5 g of solid elicitor (iii) 5 ml of liquid elicitor. These modules
were applied to both chickpea and maize plants. Plant height, number of pods/
branches/leaves per plant, and plant height, number of cobs per plant, stem diam-
eter, number of leaves per plant, days to flowering, secondary metabolite produc-
tion were evaluated for maize plant. Maximum results were obtained from treatment
with 50 ml of liquid elicitor. So these data confirmed the importance of carrageenan
in liquid form for the growth and production of secondary metabolite present in
chickpea and maize plants (Bi et al. 2011).
1.4.2.3 Effect of Fungal Elicitor on Phenylalanine Ammonia Lyase

Activity in French Bean Cells
Bolwell et al. scientifically proved the importance of fungal pathogen obtained from
Colletotrichum lindemuthianum fungal pathogen on phenylalanine ammonia lyase
activity of cultured french bean cells. The experiment was started with treatment of
cultured french bean cells with calcium ionophore, calmodulin inhibitor (triflur-
operazine), calcium channel blocker (verapamil), pertussis toxin, cholera toxin,
polyether antibiotic (monensin), labdane diterpene (forskolin), local anesthetic
(procaine), caffeine and fungal elicitor. The outcomes revealed that on the activity
of elicitor, caffeine and procaine had no direct effect whereas most effect on elic-
itation was observed with ionophore and most negative effect on elicitation was
observed with monensin (Bolwell et al. 1991).
1.4.2.4 Effect of Endophytic Fungi on Secondary Metabolite

Accumulation in Rumex gmelini Turcz
Ding et al. scientifically proved the importance endophytic fungi (Aspergillus sp.,
Fusarium sp., and Ramularia sp.) on the secondary metabolite accumulation of R.
gmelini Turcz plant. The experiment was started with the development of explant
using rhizomes of the plant in MS medium. After fifteen days, roots were
co-cultured with endophytic fungi with three concentration range (1000, 10,000 and
100,000 mL−1) followed by measured the amount of secondary metabolites
(polydatin, resveratrol, chrysophaein, musizin, emodin, chrysophanol and phys-
cion) accumulation. The outcomes revealed that Aspergillus sp. was used to
increase the production of resveratrol, chrysophaein, musizin, emodin, chryso-
phanol and physcion whereas Fusarium sp. was used for polydatin. In case of
combination treatment, R. gmelini seedling cultured with three endophytic fungi
(Aspergillus sp. = 1000 mL−1; Fusarium sp. = 10,000 mL−1 and Ramularia
sp. = 100,000 mL−1) for a period of 20 days created positive effects on polydatin,
resveratrol, chrysophaein, musizin, chrysophanol and physcion whereas produc-
tivity of emodin was increased many folds with combination of R. gmelini seedling
cultured with two endophytic fungi Aspergillus sp. = 1000 mL−1; Fusarium
sp. = 10,000 mL−1 after 15 days of treatment. These data cumulatively stated the
importance of endophytic fungi on the production of secondary metabolites present
in R. gmelini (Ding et al. 2018).
1.4.2.5 Effect of Chitosan on Flavonoid Productivity in Isatis tinctoria

L. Hairy Root Cultures
Jiao et al. experimentally proved the importance of chitosan on the increased

production of flavonoid and antioxidative efficiency of Isatis tinctoria L. The
experiment was started with formation of hairy root of I. tinctoria in MS medium
with sucrose as principle constituent. After 24 days, chitosan in acetic acid (con-
centration = 50, 100, 150, 200 and 400 mg/l) were added to hairy roots of the plant
followed by incubated for a period of 0, 6, 12, 18, 24, 30, 36, 48, 60, 72, and 96 h.
Total flavonoid content especially (rutin, neohesperidin, buddleoside, liquiritigenin,
quercetin, isorhamnetin, kaempferol, and isoliquiritigenin) were checked after the
chitosan treatment. The outcomes revealed that total flavonoid content was
increased with increasing concentration of chitosan (upto 200 mg/l); incase of
400 mg/l chitosan, the productivity of total flavonoid was decreased. Among other
flavonoids, rutin, followed quercetin and isorhamnetin were highly expressed
(Fig. 1.6). Antioxidative efficiencies of elicited hairy root cultures of I. tinctoria
(150 mg/l) were good as compare to ascorbic acid (as per percent radical scav-
enging activity) and butylated hydroxytoluene (as per percent bleaching inhibition
activity). These data confirmed the effects of chitosan on I. tinctoria secondary
metabolite production (Jiao et al. 2018).
Fig. 1.6 Phenotype comparison of hairy root tissues from control and ITHRCs elicited by
150 mg/L chitosan for 36 h. Copyright permission obtained from Jiao et al. @ 2018 Elsevier B.V
1.4.2.6 Effect of Biotic Elicitor on Glyceollin in Soybean (Glycine max)
Kalli et al. scientifically proved the cumulative effects of reactive oxygen species
and a mixture of fungal strains (Rhizopus oligosporus and Rhizopus oryzae) on the
production of natural antimicrobial prenylated pterocarpan glyceollin and iso-
flavonoid present in soybean plant. The experiment was started with the formation
of seedling using two different cultivar (I and II) in three phase (soaking, germi-
nation and priming). Soaking was done for one day followed by germination for
two days. Priming was done by two phase such as early priming with reactive
oxygen species and stress priming with slicing and sonication of seed for two days
as well as late priming by mixture of fungal strains for five days. The outcomes
revealed that the amount of glyceollin, glycinol (source of glyceollin) and iso-
flavonoid were maximum with primed and elicited (reactive oxygen species with
fungal mixture) and sonication with fungal mixture dual effect. These data con-
firmed the importance of fungal mixture, wounding stress and reactive oxygen
species on the production of glyceollin and isoflavonoid present in soybean plant
(Kalli et al. 2020).
1.4.2.7 Effect of Fungal Biotic Elicitor in Sign-Al Transduction

in Potato Tubers
Kawakita et al. experimentally confirmed the positive role of fungal elicitor

(Phytophthora infestans) in the involvement of guanosine triphosphate and gua-
nosine triphosphatase activity in the tubers of potato. The experiment was started
with reaction between potato tuber and hyphae wall component of P. infestans in
presence of sorbitol, potassium metametabisulfate, and salicylhydroxamic acid and
phenylmethylsulfonyl fluoride followed by centrifugation at 14,000 rotations per
minute for fifteen minutes and again recentrifuged at one hour. The outcomes
revealed that relative gamma guanosine triphosphatase activity was increased with
1 micromolar concentration of adenosine triphosphate upto 5 h with P. infestans
elicitor. So these outcomes confirmed the importance of fungal strains in signal
transduction in potato tuber (Kawakita and Doke 1994).
1.4.2.8 Effect of Carbohydrate on Secondary Metabolite

Accumulation in Fagonia indica
Khan et al. scientifically proved the importance of carbohydrates (sucrose, glucose,

fructose and maltose) on the accumulation of secondary metabolites (phenolic
compounds and chlorophyll) in Fagonia indica plant. The experiment was started
with the formation of aforementioned plant callus culture by treating the explant of
plant stem portion with different concentrations (1, 3 and 5%) of sucrose, glucose,
fructose and maltose in MS medium followed by estimation of callus dimension,
maximum production of biomass, total phenolic compound production, chlorophyll
and estimation of free radical scavenging activity. The outcomes revealed the
maximum width and height of callus were obtained with 5% and 3% sucrose
concentration, respectively; maximum biomass production from fresh weight and
dry weight were obtained from 3 and 5% sucrose concentration, respectively;
maximum phenolic compounds were gained from 5% of maltose; phloroglucinol
was the most accumulated phenolic compound obtained from all the carbohydrate
callus as well as maximum radical scavenging activity was observed with 3%
glucose elicitation (Fig. 1.7). So these data confirmed the importance of different
carbohydrates on secondary metabolite accumulation in Fagonia indica (Khan et al.
2018).
Fig. 1.7 Callus cultures as affected by various concentrations of Sucrose (S), Glucose (G),
Fructose (F) and Maltose (M) 42 days after culture initiation. Copyright permission obtained from
Khan et al. @ 2018 Elsevier B.V
1.4.2.9 Effect of Dextran on Secondary Metabolite Accumulation

and Improve Defense in Tomato Fruit
Lu et al. experimentally proved the importance of dextran on the accumulation of

phenylpropanoid and flavonoids along with improved defense mechanism against
grey mold infection in tomato (Solanum lycopersicum) fruit. The experiment was
started with treatment between sterilized wounded tomato fruit (sodium
hypochlorite treated) and dextran (0.1%, 0.5% and 1.0%) followed by ruthenium
red treatment on the site of wound to view the effect of calcium channel blockers on
site of wound as well as activity against grey mold infection Botrytis cinerea
inoculation with dextran elicitor. The checked activities enlisted with percent dis-
ease incidence and development of lesion with dextran elicitor alone or along with
ruthenium red after one and three days of inoculation on original and synthetic
wounds after two days; total phenolic and flavonoid accumulation; phenylalanine
lyase activity along with percent grey mold infection germination rate. The out-
comes revealed that development of disease incidence and induction lesion were
decreased with gradual increase in dextran concentration but the incidence was
slightly higher with dextran and ruthenium red combination; accumulation of total
phenolic and flavonoid compounds were higher with dextran elicitation after one
day and two days of inoculation, respectively; the phenylalanine lyase activity was
higher with dextran treatment after twelve hour of inoculation but it hits the lowest
point after two of inoculation also the rate of spore germination in presence of
B. cinerea was gradually decreased with dextran treatment. So these data confirm
the importance of dextran as biotic elicitor to strengthening the plant against grey
mold and greater accumulation of phenolic compound as well as flavonoid content
in tomato fruit (Lu et al. 2019b).
1.4.2.10 Effect of Yeast on Vincristine and Vinblastine Production

in Catharanthus roseus Plant
Maqsood et al. scientifically proved the importance yeast as biotic elicitor on the
production of vincristine and vinblastine obtained from protoplast sourced tissues
and plant of Catharanthus roseus (commonly known as periwinkle). The experi-
ment was commenced from protoplast culture involving incubation between sus-
pended cells and enzymes treatment (cellulose, pectinase, macroenzyme and
driselase) in MS medium followed by treatments with NAA, dichlorophenoxyacetic
acid, BA and gibberellin within the same medium to culture the callus and embryo
formation followed by elicitation using different concentrations of yeast (0.5, 1.0,
1.5 and 2.0 g/L). The proliferation, maturation and germination of vincristine and
vinblastine after different yeast elicitation. The outcomes showed that maximum
observations were observed with 1.5 and 2.0 g/L yeast concentrations, so these
elicitations were selected for enzymatic activities (catalase, superoxide dismutase,
ascorbate peroxidase and glutathione reductase) in the medium with different plant
regulators. The results showed that maximum catalase and ascorbate peroxidase
activities were observed with leaf harvested tissues with 2.0 g/L yeast in the
medium contained BA and NAA; the superoxide dismutase and glutathione per-
oxidase activities were observed with 2.0 g/L yeast in the medium contained BA
and NAA in embryo germination stage (Fig. 1.8). So these outcomes confirmed the
importance of yeast on production of vincristine and vinblastine in Catharanthus
roseus plant (Maqsood and Mujib 2017).
Fig. 1.8 a Isolated protoplasts; b development of embryogenic callus from protoplasts;

c individual embryo developed from PDEC treated with T3 treatment of YE and d regenerated
plantlet from protoplast derived embryo, grown in MS medium containing yeast. Copyright
permission obtained from Maqsood et al. @ 2017 Sociedade Brasileira de Farmacognosia
1.4.2.11 Effect of Chitosan on Curcumin Production and Improved

Defense in Curcuma longa Plant
Sathiyabama et al. experimentally proved the importance of chitosan polysaccha-

ride on the curcumin production, increased biomass production from leaf and
rhizome followed by estimation of protein and enzymatic activities against beta

glucanase, peroxidase and polyphenol oxidase enzymes. The experiment was
started with the reaction between new root/leaf of turmeric obtained from sodium
hypochlorite treated turmeric rhizomes and chitosan (0.1% weight/volume) fol-
lowed by elicited for a time period of six month. During the period, proper dosing
of chitosan elicitation was provided after seven month leaves and roots were
evaluated for the activities. The outcomes revealed that rhizome biomass was
higher with fresh weight culture; chitosan elicitation was increased the curcumin
production followed by elicitated leaves produced higher amount of protein as well
as greater enzymatic activities against beta glucanase, peroxidase and polyphenol
oxidase enzymes than elicitated rhizome. So these data confirmed the importance of
chitosan on the accumulation of curcumin and enzymatic activity of turmeric plant
(Sathiyabamaa et al. 2016).

Abiotic-Biotic Dual Elicitors
1.4.3.1 Effects of Microbial Elicitors on Glycyrrhizic Acid Production

in Taverniera cuneifolia Culture
Awad et al. experimentally proved the importance of microbial elicitors on the

production of glycyrrhizic acid from root cultures of Taverniera cuneifolia (Indian
liquorice) plant. The experiment was started with the development of root from
synthetic roots of the plant followed by treatment with fungal (Aspergillus niger,
Aspergillus tenius, Penicillium fellutanum, Fusarium moniliforme, Mucor hiemalis)
and bacterial (Bacillus aminovorans, A. rhizogenes, A. tumefacians, B. cereus,
Rhizobium leguminosarum) cultures. Another elicitation process include treatment
of root cultures (after 6th week) with MJ (1, 2.5, 5, 10, 100 and 1000) lM con-
centration. After three days treatment both elicited root cultures were stored and
measured for glycyrrhizic acid production. The outcomes revealed that maximum
glycyrrhizic acid was produced from Fusarium moniliforme and R. leguminosarum
microbial elicitors. Another outcomes showed that glycyrrhizic acid was maximum
obtained with MJ from plant biomass with 100 lM concentration (Fig. 1.9) These
data confirmed the importance of microbial elicitors and MJ on the production of
glycyrrhizic acid present in T. cuneifolia plant (Awad et al. 2014).
1.4.3.2 Effects of Yeast and MJ on b-thujaplicin in Cupressus

lusitanica Culture
Zhao et al. scientifically proved the importance of yeast as biotic elicitor on the
production of natural antimicrobial agent generated from geranyl pyrophosphate
Fig. 1.9 Root cultures of T. cuneifolia. a Mother culture used for root culture initiation and b six
weeks old root culture used for elicitation. Copyright permission obtained from Awad et al. @
2014 Elsevier B.V
present in Cupressus lusitanica cell culture. The process was stared with the for-
mation of fresh suspension culture of the plant from fresh cells (4 g) followed by
addition yeast (1 mg/ml) on to the five days old culture. Then in a single treatment
all plant growth regulators such as calcium ionophore, cholera toxin, mastopaoran
and actinomycin were added alone or along with twenty minutes prior to elicitation.
The outcomes revealed that b-thujaplicin was greater in production with yeast
elicitor within 2–4 days of incubation. The enzymatic activities against isopentenyl
pyrophosphate isomerase, geranyl pyrophosphate synthase and monoterpene syn-
thase showed that maximum activity within 1–2 days incubation with yeast elicitor.
In all the cases MJ was just one step down than yeast elicitation but greater than
dimethylsulfoxide control. These data confirmed the importance of yeast on the
natural antimicrobial b-thujaplicin production from C. lusitanica callus culture
(Zhao et al. 2006).
1.4.3.3 Effects of Yeast, Chitosan, MJ and Heat on Secondary

Metabolite Accumulation in Khus Root Extracts
Moon et al. scientifically proved the importance of yeast, chitosan, MJ and heat as
dual abiotic-biotic elicitor on the production of para hydroxyl benzoic acid, vanillin,
para coumaric acid, ferulic acid, total phenolic content followed by antioxidative
effects using DPPH (Diphenyl picrylhydrazyl), ABTS (Azino bisethyl benzothia-
zoline sulfonic acid) and FRAP (Ferric reducing ability of plasma) methods as well
as acetycholinesterase activity. The experiment was started with reaction between
(5–6) long root of khus plant and chitosan biotic elicitor with 100, 200 and 300 mg/
l or MJ with 25, 50 and 75 micromolar concentration or yeast elicitor with 5, 10 and
15 mg/ml concentration with or without treatment with 100 °C for twenty minute
period. The outcomes revealed that maximum para hydroxyl benzoic acid, vanillin,
para coumaric acid, ferulic acid, total phenolic content were obtained from heat
treated chitosan (200 mg/l) concentration elicited khus dry weight. Antioxidative
efficiency and acetycholinesterase data depicted that best activity observed with
heat treated chitosan (200 mg/l) concentration elicited khus dry weight. So these
data confirmed the importance chitosan on the production of secondary metabolite
from khus plant (Moon et al. 2020).
1.4.3.4 Effects of Yeast and MJ on Silymarin Production in Silybum

marianum Culture
Sanchez-Sampedro et al. experimentally proved the importance of yeast and methyl

as dual abiotic-biotic elicitor on the production and accumulation of silymarin
obtained from Silybum marianum cell culture. The process was started with
development of cell suspension culture from hypocotyl callus of the plant cultured
in MS medium containing sucrose, dichlorophenoxyacetic acid, benzyladenine,
then the suspension culture was elicited with 1 mg/ml concentration of yeast and
100 micromolar concentration of MJ for a period of 3 days. Also the silymarin
production was monitored in presence of calcium antagonist, calcium effectors and
inhibitors of protein kinase and protein phosphatase enzymes. The outcomes
showed that MJ increased silymarin production many folds than yeast. It was also
observed that silymarin production was exponentially increased with ruthenium red,
neomycin and diphenylene iodonium treated both MJ and yeast elicitors. These data
confirmed the importance of MJ and yeast elicitors on silymarin production present
in S. marianum suspension culture (Sanchez-Sampedro et al. 2008).
1.4.3.5 Effects of MJ, Phenylacetic Acid and Light Elicitors on Ajuga

bracteosa
Ali et al. scientifically proved the importance of MJ, phenylacetic acid and light as
dual abiotic and biotic elicitors on the production of volatile oils (a-phelendrene,
Sabinene, a-terpinene, limonene, 1,8-cineole, c-terpinene, b-pinene, b-myrcene,
D-limonene, b-phelendrene, b-ocimene, Thujyl alcohol, cis-sabinol, b-linalool,
1-terpinene-4-ol, Cis-geraniol, a-terpineol, myrtenol, myrtenal, nerol, caryophyl-
lene, b-farnesene, carvone, citronellyl acetate, p-cymen-7-ol, bornyl acetate) from
cell cultures of Ajuga bracteosa. The process was started with the reaction between
callus culture and plant growth regulators (kinetin, BA, dichlorophenoxyacetic acid,
indole butyric acid) and elicitors (MJ and phenyl acetic acid) with 0.5, 1.0, 1.5 g/l
of concentrations in three different luminous condition such as dark for one day,
16 h light with 8 h dark and complete light for one day. The amount of dry
biomass, total phenolic content, total flavonoid content, free radical scavenging
activity as well as volatile oils productions were evaluated. The outcomes showed
that amount of dry biomass was maximum (11.3 g/l) with BA (1.0 g/l) in complete
dark condition; total phenolic content was maximum (7.0 mg of gallic acid
equivalent/g) with MJ (0.5 g/l) in complete light condition; total flavonoid content
was maximum (3.8 mg quercetin equivalent/g) with MJ (0.5 g/l) in complete light
condition; percent free radical scavenging activity was greater (86%) with MJ
(0.5 g/l) in complete light condition; higher amount of volatile oils was observed
with MJ (0.5 g/l) and BA (1.0 g/l) in dark period. So these data confirmed the
importance of MJ, phenylacetic acid, other plant regulators and light treatment on
the production and accumulation of secondary metabolites obtained from A.
bracteosa cell culture (Ali et al. 2018).
1.4.3.6 Effects of Salicylic Acid, Yeast and Casein Hydrolysate

on Colchicine and Thiocolchicoside Production from Gloriosa
superba Plant
Mahendran et al. scientifically proved the importance of salicylic acid, yeast, casein
hydrolysate and silver nitrate on the accumulation of colchicine and thio-
colchicoside in Gloriosa superba (Calihari) plant. The process was started with
reaction between cell suspension culture (generated from plant rhizomes cultured in
MS medium contained sucrose, dichlorophenoxyacetic acid, NAA) and salicylic
acid (13.812, 27.624, 41.436, 55.248 and 69.060 mg/l), yeast, casein hydrolysate
and silver nitrate with (100, 200, 300, 400 and 500 mg/l) followed by estimation of
colchicine and thiocolchicoside after fifteen and thirty days of elicitation. The
outcomes showed that a proper callus induction was happen with a combination of
2.0 mg/l of dichlorophenoxyacetic acid and 0.5 mg/l of NAA as well as maximum
colchicine was obtained from casein hydrolysate (300 mg) and (27.624 mg) of
salicylic acid after fifteen and thirty days of elicitation respectively whereas max-
imum thiocolchicoside was obtained from silver nitrate (200 mg) and (300 mg) of
silver nitrate after fifteen and thirty days of elicitation respectively. So these data
confirmed the importance of mixed abiotic and biotic elicitors on the accumulation
of colchicine and thiocolchicoside in G. superba plant (Mahendran et al. 2018).
1.4.3.7 Effects of Yeast and MJ on Polyphenolic Compound in Aster

scaber Plant
Ghimire et al. scientifically proved the importance of biotic elicitor yeast and abiotic
elicitor MJ on the production of polyphenolic compounds (flavonols, hydroxy
cinnamic acid derivative, hydroxybenzoic acid derivative, vanillin, resveratrol and
homogentisic), biomass production, total phenolic and flavonoid content as well as
effects on antioxidative properties of the hairy root culture of the plant using
diphenylpicraylhydrazyl and ferric reducing potential processes. The experiment
was started with the formation of hairy root culture of A. scaber plant (Chwinamul)
by the germination of seeds in MS medium contained with sucrose, cefotaxime
antibiotic and excised with A. rhizogenes followed by elicited with yeast with
50 mg/l, 100 mg/l, 200 mg/l concentration and MJ with 50, 100, 200 micromolar
concentration. The outcomes revealed that maximum biomass was obtained with
MS medium, Sucrose (3%) with twenty seven days of interval. Maximum flavonol,
hydroxy cinnamic acid derivative, hydroxybenzoic acid derivative, vanillin,
resveratrol and homogentisic were obtained with MJ elicitation (Fig. 1.10). Total
phenolic and flavonoid contents were higher with increased dose of both elicitors as
well as MJ (100 micromolar concentration) was showed maximum antioxidative
property. So these data confirmed the importance of MJ and yeast as elicitors on the
accumulation of polyphenols in A. scaber plant (Ghimire et al. 2019).
Fig. 1.10 Agrobacterium rhizogenes-mediated hairy root cultures in Aster scaber. a Hairy roots
induction, b Hairy root cultures in hormone-freeMS liquid medium, c PCR analysis of the rolC
gene in the transgenic root lines. DNA ladder marker Lane M, pRiKCTC2703 DNA C(+),
transgenic root lines induced by A. rhizogenes L1–L5, roots from a non-transgenic plant C(−).
Copyright permission obtained from Ghimire et al. @ 2019 Elsevier B.V
1.4.3.8 Effects of Phenylalanine, Salicylic Acid and Chitosan

on Secondary Metabolites of Coleus aromaticus Benth
Govindaraju et al. experimentally confirmed the importance of phenylalanine,

salicylic acid and chitosan as dual abiotic-biotic elicitor on the accumulation of
alkaloid, flavonoid, saponin, terpenoids, total phenolic content followed by
expression of phenylalanine messenger ribonucleic acid in the root/shoot culture
and regenerated Coleus aromaticus Benth plant. The experiment was started from
the induction of shoot followed by development of explants in the MS medium with
activated charcoal and ascorbic acid as carbon and antioxidant source. The effect of
BA and kinetin were also tested. The outcomes revealed that healthy explants were
maximized with (7.5 g/l) activated charcoal and (1.0 mg/l) ascorbic acid. The
percent induction of explants and number of shoots were maximum with a com-
bination of (1 mg/l) of BA and kinetin. Different concentration of phenylalanine
(0.5, 1.0, 1.5, 2.0, and 2.5) mg/l, salicylic acid (0.2, 0.4, 0.6, 0.8, and 1.0) mg/l and
chitosan (20, 40, 60, and 80) mg/l were used as elicitors with BA (1 mg/l) con-
centration. The outcomes showed that maximum number of shoots were observed
with 40 mg/l of chitosan cultured with BA as well as maximum development of
roots was observed with (0.5 mg/l) of a combination NAA and IAA (Fig. 1.11).
The outcomes also showed that regenerated explants observed with greater amounts
of alkaloid, flavonoid, saponin, terpenoids, total phenolic content as well as higher
Fig. 1.11 In vitro propagation of C. aromaticus, a shoot bud induction, b multiple shoot bud
development from in vitro derived nodal explants, c multiple shoots (1.0 mg BAP + 40 mg/l Ch),
d root initiation and e, f rooted plant growing in the plastic cup with soil and sand in the ratio of
1:1. Copyright permission obtained from Govindaraju et al. @ 2016 Elsevier B.V
expression of phenylalanine messenger ribonucleic acid. These data confirmed the

effects of phenylalanine, salicylic acid and chitosan on secondary metabolites of C.
aromaticus Benth (Govindaraju and Arulselvi 2018).
1.4.3.9 Effects of MJ, Chitosan and Microbial Lysates on Essential Oil

Accumulation in Rhododendron tomentosum
Jesionek et al. experimentally proved the importance of abiotic elicitors (copper,

nickel, methyl jasmoante in dimethyl sulfoxide and ethanol, dimethylsulfoxide,
ethanol) and biotic elicitors (chitosan hydrochloride, ergosterol and aphid extract in
ethanol, lysates of C. albicans, E. coli, Enterobacter sakazaki, Pectobacterium
Carotovorum, Dickeya dadantii) on the quantity of essential oils (monoterpene
hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated
sesquiterpenes, aromadendrane-type sesquiterpenes) in R. tomentosum plant. The
experiment was started with formation of in vitro culture of the plant on
Schenk-Hildebrandt medium contained ammonium nitrate and isopentenyladenine
within a period of one month. The formation of biomass was done using micro-
shoots of the plant within sterilized bioreactor with proper illumination followed by
elicitation using the aforementioned abiotic and biotic elicitors. The fresh weight
and dry weight of biomass with growth index reflected that nickel, MJ in ethanol,
ergosterol in ethanol and lysate of Enterobacter sakazaki showed greater produc-
tion. The productions of monoterpene hydrocarbons, oxygenated monoterpenes,
sesquiterpene hydrocarbons, oxygenated sesquiterpene and aromadendrane-type
sesquiterpenes were maximized with copper and moderate with Escherichia coli
lysates, nickel and ethanolic ergosterol and ethanolic MJ, respectively. The out-
comes stated the importance of both abiotic and biotic elicitors on the production of
volatile oils in R. tomentosum plant (Jesionek et al. 2018).
1.4.3.10 Effects of Yeast, MJ and Salicylic Acid on Ursolic Acid

and Eugenol Production in Ocimum tenuiflorum L.
Sharan et al. experimentally proved the importance of abiotic elicitors [(MJ: 0, 3, 60

and 120 mg/l) and (salicylic acid: 0, 30, 60 and 120 mg/l)] and biotic elicitor (yeast:
0, 25, 50 and 100 mg/l) on the production of ursolic acid and eugenol in Ocimum
tenuiflorum L (holy basil) plant. The experiment was started with formation of hairy
root culture from explants cultured in MS medium with cefotaxime and sucrose as
antibiotic and sugar source, respectively followed by elicitation using aforemen-
tioned elicitors and evaluated the effects of A. rhizogenes bacterial strains (LBA
9402 and A4) on the transformation into leaf and stems as well as evaluated the
production of biomass, ursolic acid and eugenol contents for a time period of thirty
five days and finally quantified the effects of elicitors on seventeen and twenty one
old hairy roots tendency to accumulate ursolic acid and eugenol within four to
twelve days period. The outcomes revealed that LBA 9402 strain of A. rhizogenes
facilitated the transformation of explants into leaf and stem; highest amounts of
biomass, ursolic acid and eugenol accumulations were observed within twenty to
twenty five days of incubation as well as maximum ursolic acid was accumulated
with (50 mg/l) yeast, (60 mg/l) of methyl jasmoante and (60 mg/l) of salicylic acid
from seventeen day old hairy roots and (50 mg/l) yeast, (0 mg/l) of methyl jas-
moante and (0 mg/l) of salicylic acid from twenty one day old hairy roots; whereas
maximum eugenol was accumulated with (50 mg/l) yeast, (60 mg/l) of methyl
jasmoante and (0 mg/l) of salicylic acid from seventeen day old hairy roots and
(0 mg/l) yeast, (0 mg/l) of methyl jasmoante and (0 mg/l) of salicylic acid from
twenty one day old hairy roots. These data confirmed the importance of yeast, MJ
and salicylic acid contents present in O. tenuiflorum L plant (Sharan et al. 2019).
1.5 Conclusion
This chapter provides a detailed information about What is Elicitation? Types of

Elicitors along with applications of elicitors to increase productivity of secondary
metabolites. This chapter also includes the information about secondary metabolites
and its types such as alkaloid, glycoside, flavonoid and terpenoids etc. Elicitors are
the physical/chemical/biological/microbial substances which under stress condi-
tions induce the biosynthesis of secondary metabolites of plants. Both biotic and
abiotic elicitors are used in the process. Most common secondary metabolites
Ferulic acid, cinnamic acid, vanillin, coumaric acid, silymarin, affinin, hypocrellin
A, steroiside, menthone, piperitone, glycyrrhizic acid, colchicine, thiocolchicoside,
phenolic acid, gymnemic acid, flavonoids are utilized the elicitation technique.
Elicitors are two types such as: abiotic and biotic. Abiotic elicitors such as salicylic
acid, methyl jasmonate, hydrogen peroxide, lanthanum, different hormones, light,
gamma rays and controlled temperature are used to generate secondary metabolites
of wheat grass, Thymus vulgaris, Silybum marianum, Shiraia bambusicola, Ajuga
bracteosa, broccoli plant, etc. Biotic elicitors like chitosan, rhizobacteria,
Rhizobium leguminosum, A. tenius, A. tumefacians, carrageenan, Streptomyces,
Rhizopus, dextran, yeast are used to develop or improvise secondary metabolites of
Khus, M. pulegium, T. cuneifolia, chickpea, V. vinifera, R. gmelini Turcz, C.
lusitanica, etc. Some secondary metabolites of C. aromaticus Benth, R. tomento-
sum, F. indica, R. serpentine, S. khasianum, O. tenuiflorum, S. rebaudiana etc. are
used both abiotic and biotic elicitors. In the Table 1.1 detailed tabulated information
of elicitors used to accumulate and increased productivity of secondary metabolites.
Elicitors are not only increase secondary metabolite production as well as increase
the defense mechanism, antioxidative, antimicrobial and enzymatic activities. This
data collectively express the detailed knowledge about the elicitors and its signal
transduction behavior. So if scientific minds hunts for secondary metabolites with
greater activity but with very lesser abundance, then elicitation is the future.
Table 1.1 Detailed effects of elicitors on different plants and secondary metabolites
SN Types of Description of elicitors Accumulated Reference
elicitation secondary metabolites
1 Abiotic Arachidonic acid, Flavonoid and Hassini et al.
Jasmonic acid polyphenolic (2019)
compounds in Triticum
aestivum L.)
2 Methyl jasmonate, Benzylisoquinoline Hesketh et al.
Salicylic acid flavonoids in Thymus (2002)
vulgaris, Chelidonium
majus and
Petroselinum crispum
3 Methyl jasmonate, Triterpenoid in Hiroaki et al.
Salicylic acid Centella asiatica (2012)
4 Salicylic acid, Affinin contents in Jensen et al. (2014)
Hydrogen peroxide Heliopsis longipes
5 Lanthum (La3+) Hypocrellin A content Jesionek et al.
in Shiraia bambusicola (2018)
6 Salicylic acid, Methyl Steviosides in Stevia Jiao et al. (2018)
jasmonate, Citric acid, rebaudiana Bertoni
Ascorbic acid calli
7 Light Total phenolic and Kalli et al. (2020)
flavonoid contents in
Stevia rebaudiana
8 Gamma rays Phenolic compounds Kawakita and Doke
and naphtodiantrones (1994)
in Hypericum
triquetrifolium Turra
9 Streptomycin, 3-O-glucosyl Kazmi et al .(2019)
Activated charcoal and resveratrol and
Etephon phenolic compound in
Vitis vinifera
10 Salicylic acid Alkaloid in marine Khan et al. (2018)
microalgae Arthrospira
platensis
11 Salicylic acid, Methyl Chlorogenic acid and Kleinwachter et al.
jasmonate and Sinapic acid in (2015)
Methionine Broccoli plant
12 Methyl jasmonate, Steviol glycoside in Lena (2012)
Phenyl acetic acid and Stevia rebaudiana
Melatonin
13 High intensity Total phenolic content, Lu et al. (2020)
ultrasound lycopene and
carotenoid contents in
Solanum lycopersicum
(continued)
Table 1.1 (continued)

14 Methyl jasmonate, Total phenolic content Lu et al. 2019.a
Salicylic acid and and total flavonoid in
Glutathione Trifolium resupinatum
plant
15 Methyl jasmonate Anthraquinone in Lu et al. (2019b)
Rubia tinctorum plant
16 Abiotic Oleic acid and Linoleic Gymnemic acid in Mahendran et al.
acid Gymnema sylvestre (2018)
17 Magnesium oxide Total phenolic and Maqsood and
nanoparticle flavonoid contents in Mujib 2017
Atropa belladonna
18 Temperature E. coli, B. subtilis, S. Mejia-Espejel et al.
aureus C. albicans and (2018)
P. aeruginosa in soil
19 Linoleic and Alpha Total ginsenosides, diol Moon et al. (2020)
linolenic acid ginsenoside, triol
ginsenoside and total
flavonoid contents in
Panax ginseng
20 Biotic Azotobacter Relative water content, Pal et al. (2017)
chroococcum, Chlorophyll
Azospirillum fluorescence, Total
brasilense, Azotobacter phenolic content, Total
chroococcum and flavonoid contents in
Azospirillum brasilense Mentha pulegium
21 Carrageenan Plant height, number of Pal et al. (2019a)
pods/branches/leaves
per plant, and plant
height, number of cobs
per plant, stem
diameter, number of
leaves per plant, days
to flowering, secondary
metabolite productions
in Chickpea and maize
plant
22 Colletotrichum Phenylalanine Pal et al. (2018)
lindemuthianum fungal ammonia lyase activity
23 Aspergillus sp., Resveratrol, Pal et al. (2019b)
Fusarium sp., and Chrysophaein,
Ramularia sp. Musizin, Emodin,
Chrysophanol and
Physcion in Rumex
gmelini
(continued)

24 Chitosan Rutin, Neohesperidin, Pa and Saha
Buddleoside, (2019a)
Liquiritigenin,
Quercetin,
Isorhamnetin,
Kaempferol, and
Isoliquiritigenin in
Isatis tinctoria L
25 Rhizopus oligosporus Prenylated pterocarpan Pa and Saha
and Rhizopus oryzae glyceollin and (2019b)
fungal strains Isoflavonoid present in
soybean plant
26 Biotic Fungal elicitor Guanosine Pal et al. (2019c)
(Phytophthora triphosphatase activity
infestans)
27 Sucrose, Glucose, Phenolic compounds Parola-Contreras
Fructose and Maltose and chlorophyll in et al. (2020)
Fagonia indica plant
28 Dextran Phenylpropanoid and Perassolo et al.
flavonoids with (2017)
improved defense
mechanism against
grey mold infection in
tomato (Solanum
lycopersicum) fruit
29 Yeast Vincristine and Pichersky and Gang
Vinblastine in (2000)
Catharanthus roseus
30 Chitosan Curcumin in Curcuma Praveen et al.
longa plant (2014)
31 Abiotic-Biotic Fungal (Aspergillus Glycyrrhizic acid from Sanchez-Sampedro
niger, Aspergillus root cultures of et al. (2008)
tenius, Penicillium Taverniera cuneifolia
fellutanum, Fusarium
moniliforme, Mucor
hiemalis) and bacterial
(Bacillus aminovorans,
Agrobacterium
rhizogenes,
Agrobacterium
tumefacians, Bacillus
cereus, Rhizobium
leguminosarum) and
Methyl jasmonate
32 Yeast and Methyl b-thujaplicin in Sathiyabamaa et al.
jasmonate Cupressus lusitanica (2016)
(continued)

33 Yeast, Chitosan, Para hydroxyl benzoic Sato and Matsui
Methyl jasmonate and acid, Vanillin, Para (2012)
Heat coumaric acid, Ferulic
acid, Total phenolic
contents in Khus plant
34 Yeast and Methyl Silymarin production in Sharan et al. (2019)
jasmonate Silybum marianum
culture
35 Methyl jasmonate, Total phenolic and total Tian et al. (2018)
Phenylacetic acid and flavonoid contents in
Light Ajuga bracteosa
36 Salicylic acid, Yeast Colchicine and Twaij et al. (2019)
and Casein hydrolysate Thiocolchicoside in
Gloriosa superba plant
37 Yeast and Methyl Polyphenolic Ulaganathan et al.
jasmonate compounds in Aster (2017)
scaber plant
38 Abiotic-Biotic Phenylalanine, Alkaloid, Flavonoid, Wu et al. (2009)
Salicylic acid and Saponin, Terpenoids,
Chitosan Total phenolic contents
in Coleus aromaticus
Benth
39 Abiotic elicitors Essential oils Zhao et al. (2006)
(copper, nickel, methyl (monoterpene
jasmoante in dimethyl hydrocarbons,
sulfoxide and ethanol, oxygenated
dimethylsulfoxide, monoterpenes,
ethanol) and biotic sesquiterpene
elicitors (chitosan hydrocarbons,
hydrochloride, oxygenated
ergosterol and aphid sesquiterpenes,
extract in ethanol, aromadendrane-type
lysates of Candida sesquiterpenes) in
albicans, Escherichia Rhododendron
coli, Enterobacter tomentosum plant
sakazaki,
Pectobacterium
Carotovorum, Dickeya
dadantii
40 Yeast, Methyl Ursolic acid and Złotek et al. (2019)
jasmonate and Salicylic Eugenol production in
acid Ocimum tenuiflorum L
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Chapter 2
An Introduction to Bioactive Natural
Products and General Applications
Tijjani Ahmadu and Khairulmazmi Ahmad
Abstract Nature has been a powerful source of potential medicinal plants with
natural bioactive products for a long time period. The plants are known to contain
quantum of various active principles of therapeutic value and possess biological
activity against a number of diseases. These plants synthesized phytochemicals,
serving as their natural defence system and also used in medicine, dye colours,
fragrant, pharmaceutical, agrochemicals and flavouring. They also possess
antimicrobial properties that are correlated with their ability to manufacture several
secondary metabolites with antimicrobial properties like phenols, phenolic acids,
flavonoids, alkanoids, tannins, quinones, coumarins, saponins, terpenoids, triter-
penoids, glycosides and organic acids. Today, an increasing research interest in the
herbal medicine field has been gaining popularity in both developed and developing
countries. The bioactive compounds are provided by chemical diversity and natural
plant products as purified compounds or as plant crude extracts. The extracts
(crude) existed as a combination of different bioactive phytocompounds combined
with various polarities and their partition still remains a challenge in the process of
identification and characterization. Since bioactive natural products have diverse
range of uses, the present book chapter constitutes a review on their distribution and
geographical sources, phytochemistry, delivery technology, medicinal properties
and their general potential applications in agricultural plant protection and phar-
maceutical field.
T. Ahmadu (&) K. Ahmad

Department of Plant Protection, Faculty of Agriculture, University Putra Malaysia, 43400
UPM Serdang, Selangor Darul Ehsan, Malaysia
e-mail: tijjaniahmadu72@gmail.com
K. Ahmad
e-mail: tijjaniahmadu72@gmail.com
T. Ahmadu
Department of Crop Production, Faculty of Agriculture and Agricultural Technology,
Abubakar Tafawa Balewa University, Bauchi, Bauchi State P. M. B 0248, Nigeria
K. Ahmad
Institute of Plantation Studies, University Putra Malaysia, 43400 UPM Serdang, Selangor
Darul Ehsan, Malaysia

https://doi.org/10.1007/978-3-030-54027-2_2
42 T. Ahmadu and K. Ahmad

Keywords Antimicrobial properties Bioactive compounds Biological activity

Medicinal plants Phenolic acids
2.1 Introduction
Since times immemorial many countries across the planet have accumulated a rich
body of empirical knowledge tracing the occurrence of bioactive natural com-
pounds with medicinal properties from medicinal plants for the treatment of dif-
ferent illnesses throughout their long history. Natural bioactive compounds are
metabolites manufactured by plants and their uses in different fields like medicine in
form of pharmaceutical drugs and plant based bio-pesticides in agricultural plant
protection are well known and documented. Approximately, 85% of preparation of
traditional medicine involves the use of bioactive natural compounds extracted from
plant (Meenupriya et al. 2014). Medicinal plants play a key role in world health and
global economy (Meenupriya et al. 2014). Plants generate numerous categories of
bioactive natural compounds, thus, making them rich source of various types of
drugs and biopesticides (Meenupriya et al. 2014). All parts of these plants
(roots, flowers, stem, bark, leaves, essential oils and seeds have microbial qualities
and are therefore used for many purposes incuding traditional medicine (Dwivedi
and Enespa 2012; Anwar et al. 2007). The choice and use of these potential plants
and their general applications in pharmaceuticals and agriculture is due to: (1) their
safety for human consumption, (2) non environmental pollution. (3) more accept-
able to the pharmaceuticals and local farmers because they are indigenous (4) their
biological activity and dispersion in harvested tissue (5) our ability to develop
formulations that allow the delivery of non-toxic concentrations but at the same
time interfere with antimicrobial development (Sogvar et al. 2016; Bhattacharjee
and Dey 2014).
Today, research interest on medicinal plants is focus in recent time in such areas
such as pharmacognosy, phytochemistry and horticulture. Bioactive compounds
have been validated and detailed of their structural analysis have been present in the
field or area phytochemistry. Ríos et al. (2015) and Jacob and Narendhirakannan
(2019) have reported the use of such plants as fenugreek, caper, aloe, soybean,
banaba, green tea, bitter melon, turmeric, cinnamon, walnut, coffee, guava, cocoa,
garlic, sage, gymnema, nettle and yerba mate for treating illness like diabetes and its
alike and the mechanisms of natural bioactive products as antidiabetic agents, with
attention to compounds of high interest. The compounds are gurmarin, phlorizin,
berberine, gymnemic acids, palmatine, honokiol, amorfrutins, trigonelline and
fukugetin. The review described by (Jacob and Narendhirakannan 2019) has cat-
egorized 81 plants from literature that are native to Asian countries with
anti-lipidemic, antidiabetic, insulin-mimetic, hypoglycemic, and antihyperglycemic
properties. The presence of some bioactive compounds in the leaves, stem bark and
flowers of Eugenia jambolana Lam. such as n-dotricontanol, anthocyanins, acylated
flavonol glycosides delphinidin, friedelan-3-a-ol, friedelin, corilagin, petunidin,
2 An Introduction to Bioactive Natural Products … 43
ellagic acid, garlic acid, jambosine, b-sitoterol, n-hentriacontane, gallotannin,

myricitrin, quercetin, myricetin, n-nonacosane, ellagicacids, n-hepatcosane, n-tria-
contanol, isoquercetin, malvidin-diglucosides, myricetin, betulinic acid, pino-
carveol, cineole, oleanolic acid, b-sitosterol-D-glucoside, muurolol kaempferol,
myrtenol, eucarvone, noctacosanol, a-myrtenal, geranyl acetone, a-cadinol,
3-galloylglucose, pinocarvone, 3,6-hexahydroxy diphenoylglucose,
1-galloylglucose, 4,6-hexahydroxydiphenoylglucose, crategolic acid and flavonol
glycosides, myricetin 3-O-(4′′-acetyl)-a-L-rhamnopyranosides, a-terpeneol, were
also reported (Baliga et al. 2011). Bio-assays and mechanisms of action of
medicinal plants has been the interest in the area of pharmacognosy. The interest in
horticultural research on medicinal plants has been on optimal growth and yield in
cultivation. In this regards, bioactive compounds have been found to be good in
improving postharvest quality, crop immunity and growth parameters (Freire et al.
2015; Mustafa et al. 2014). Advancement in search for alternatives to synthetic
drugs and pesticides led to the wild harvesting of these plants yet the growth
conditions have not been optimized. The consequences could be loss of biodiver-
sity, improper identification of plant and potential variation in medicinal plant
quality. Additionally, the feature of medicinal plants is seemingly being endangered
and by complacency regarding their preservation. Stocks and herbs of these plants
are in menace of extinction and diminishing in developing countries due to the
demands for the growing market for cheaper alternative healthcare products and
new plant-based therapeutic markets in preference to more expensive target-specific
drugs and biopharmaceuticals.
Extracts of plant origin are found to be the most pressing sources of natural
bioactive compounds and can be screened from local traditional plants (Mari et al.
2016; Freire et al. 2015). These plant bioactive compounds are extracted using
solvents of different polarities and are shown to acquire antidiabetic, antifungal,
antimicrobial, antioxidant and antibacterial properties etc. (Bhattacharjee and Dey
2014). Bioactive compounds obtainable from these plants have been shown to be
the best alternative for pharmaceutical drugs and chemical fungicides (Bahare et al.
2019; Mari et al. 2016; Sogvar et al. 2016). Extraction of these bioactive natural
compounds from species of medicinal plants is done by using various solvents and
methods of extraction. Bioactive natural compounds are mostly aromatic or organic
compounds that are saturated in nature. As a result, they are collected frequently
through initial extraction with ethanol or methanol. Ethyl aceted, hexane,
dicholro-methane, chloroform, acetone, butanol among others, and/or their appro-
priate ratio combination have been suggested by researchers (Gurjar et al. 2012) to
get the best solvent systems for their extraction. Filtration is a common method for
the extraction of bioactive natural compounds from plants which involves dis-
solving fresh ground sample (wet or dried) into certain volume of solvent, followed
by vigorous shaking and filtration after sometimes usually after 24 h. Serial
exhaustive is another extraction method for the bioactive compounds suggested by
Green and Beestman (2007) which includes successive extraction with solvents of
different polarity beginning from non-polar to higher polar solvents in order to
make necessary extraction of crucial quantum compounds with enough and
excellent polarity range. Gurjar et al. (2012) reported the extraction of phyto-
chemical compounds from dried plant material with soxhlet method using organic
solvent. High Pressure Liquid Chromatography is an analytical method that have
been commonly used with other chromatography methods such as gas chro-
matography mass spectrometry (GC–MS) and liquid chromatography mass
spectrometry/mass spectrometry (LC–MS/MS) for the separation and identification
of bioactive natural compounds (Snyder et al. 2012; Villas-Boas et al. 2005). GC–
MS had high ability in the partitioning and identification of compounds from
multiple biological mixtures (Villas-Boas et al. 2005). More so, GC–MS technique
can identify various bioactive natural compounds with lower molecular weight,
volatile in nature and have different functions (Huang et al. 2012; Wang et al.
2012a, b). LC–MS is another chromatography technique apart from GC–MS where
liquid chromatography is attached to spectrometry together with electrospray ion-
ization and atmospheric pressure chemical ionization (Villas-Boas et al. 2005). LC–
MS can analyse and identify huge number of non-volatile compounds even in
smaller quantities. Nuclear magnetic resonance (1H NMR) and thin layer chro-
matography (TLC) are other methods utilized in the validation, characterization and
separation of active compounds (Sharma and Paliwal 2013).
2.2 Bioactive Natural Compounds: Distribution

and Geographical Sources
Table 2.1 has 251 plants species belonging to 98 genera and 56 families distributed in
virtually 96 different continents and countries across the planet which includes
Africa, Algeria, America, Asia, Bangladesh, Brazil, Cameroon, central America,
China, Chinese medicines, Cuba, east Asia, Egypt, Ethiopia, Ghana, India, India
(Ayurveda), India (Ayurveda, Unani), India (Ayurveda, Unani, Siddha and home-
opathy), Japan, Ivory Coast, Iraq, Iran, Indonesia, Jordan, Kenya, Korea, Laos, Latin
America, Marshall Islands, Mali, Mauritius, Mexico, Malaysia, New Guinea,
Morocco, Nigeria, Pakistan, Papua, Paraguay, Philippines, Puerto Rico, Sierra
Leone, Saudi Arabia, South Africa, Senegal, South East Asia, Nepal, Sri lanka,
Sudan, South East Asian Countries,Taiwan, Tanzania, Thailand, Tobago, Togo,
Trinidad and Tobago, Trinidad, Turkey, Uganda, Vietnam, West Indies. Of the 98
genera given, some have more than 8 species giving different biological activity.
Eighteen (18) species were observed in Ficus, 14 in Artemisia, 11 in Terminalia,
while Cinnamomum, Phyllanthus and Ziziphus with 9 each and Acacia and Zizigium
with 8 each in that order. Of the 18 species in the Ficus genus, the most prominent and
important species are, Ficus elastic, Ficus benghalensis and F. hispida. Indian
Banyan tree also known as F. benghalensis is a frequent used plant to avert most
illness diabetes inclusive (Jaya Kumari et al. 2016) and is used in folk medicines,
Ayurveda, Unani, Siddha (Gopukumar and Praseetha 2015), and homeopathy (Deepa
et al. 2018). F. benghalensis, F. glomerata, F. carica, F. glumosa, F. religiosa and
Table 2.1 Distribution and Geographical Sources of Bioactive Natural Compounds
Species Bioactive compounds Geographic zone References
Allium cepa Allyl propyl disulphide, S-methyl cysteine sulphoxide Mauritius, Algeria Mootoosamy and Fawzi
Mahomoodally Ý(2014)
Allium porrum S-methyl cysteine sulphoxide, Allyl propyl disulphide Turkey Bahare et al. (2019)
Allium sativum Allyl propyl disulphide China, India (Ayurveda), Iran, Indonesia, García Mesa (2014),
Cuba, Mauritius, Togo Sukandar et al. (2015)
Allium Allyl propyl disulphide Iran Bahare et al. (2019)
stipitatum
Amaranthus Allyl propyl disulphide Mauritius Mondal et al. (2015)
hybridus
Amaranthus Allyl propyl disulphide Taiwan Bahare et al. (2019)
spinosus
Mangifera Mangiferin, Flavonoid, Phenolics, Vietnam Nguyen et al. (2016)
mekongensis
Panax Saponin China Bahare et al. (2019)
2 An Introduction to Bioactive Natural Products …
notoginseng
Panax Saponin China, Korea Bahare et al. (2019)
quinquefolius
Aloe marlothii Lophenol, Luteolin South Africa Sudha et al. (2011)
Aloe ferox 24-methyl-lophenol, Luteolin, 24ethyllophenol India (Ayurveda) Kamel et al. (2017)
Aloe vera 24-methyl-lophenol, Luteolin, 24ethyllophenol, Saudi Arabia, Ghana, Philippines, Uganda Mina and Mina (2017),
Lophenol, cycloartanol, 24-methylene-cycloartanol Tanzania India, Pakistan, Iran, Ghana, Asase and Yohonu (2016),
Mauritius, Chinese medicines Ssenyange et al. (2015)
Artemisia Polysaccharide Africa Islam et al. (2014)
absinthium
Artemisia Polysaccharide Africa Islam et al. (2014)
capillaris
(continued)
45
46

Artemisia Polysaccharide Morocco Dib et al. (2017)
campestris
Artemisia Polysaccharide Morocco Ota and Ulrih (2017)
dracunculus
Artemisia Polysaccharide Jordan Abu-Darwish et al. (2016)
judaica
Artemisia Polysaccharide Mexico Anaya-Eugenio et al. (2014)
ludoviciana
Artemisia Polysaccharide Algeria, Iraq, Jordan Nedjimi and Beladel (2015)
herba-alba Abu-Darwish et al. (2016)
Artemisia Polysaccharide India Stanifer et al. (2015)
parviflora
Artemisia Polysaccharide Asia Bahare et al. (2019)
pallens
Artemisia Polysaccharide Asia Bahare et al. (2019)
princeps
Artemisia Polysaccharide China Shah et al. (2016a; b)
roxburghiana
Artemisia Polysaccharide China Yuan et al. (2010)
sacrorum
Artemisia Polysaccharide Jordan Shah et al. (2016a, b)
sphaerocephala
Eugenia Pandanus odorus India (Ayurveda) Kumar et al. (2013)
jambolana
Eugenia Tarpen Paraguay Bahare et al. (2019)
uniflora
(continued)
T. Ahmadu and K. Ahmad
Eugenia Tarpen Indonesia, India Jerang et al. (2015)
polyantha
Brassica rapa Isorhamnetin diglucoside India Jokar et al. (2016)
Brassica Isorhamnetin diglucoside India Jokar et al. (2016)
oleracea
Terminalia Phenolics Vietnam Nguyen et al. (2016)
alata
Terminalia Phenolics Bangladesh, Sri lanka, South East Asia, Nguyen et al. (2016), Tanaka
bellirica Vietnam, India et al. (2016)
Terminalia Phenolics Bangladesh, India (Ayurveda), Nguyen et al. (2016)
arjuna
Terminalia Phenolics Bangladesh, Iran, India (Ayurveda), Thailand Sudha et al. (2011)
chebula Kadir et al. (2012)
Terminalia Phenolics Bangladesh Biswas et al. (2014)
citrina
Terminalia Phenolics Vietnam Nguyen et al. (2016)
corticosa
Terminalia Phenolics Africa Pham et al. (2014)
macroptera
Terminalia Phenolics Cameroon Biswas et al. (2014)
glaucescens
Terminalia Phenolics India Padmashree and Pandey
superba (2010)
Terminalia Phenolics Thailand Nkobole et al. (2011)
sericea
(continued)
47
48

Tremella Phenolics India (Ayurveda) Singh et al. (2014),
mesenterica Mamun-or-Rashid et al.
(2014)
Calendula isorhamnetin India Sudha et al. (2011)
officinalis
Coccinia indica B-amyrin, Cucurbitacin B, Lupeol India (Ayurveda) Singh et al. (2014),
Mamun-or-Rashid et al.
(2014)
Coccinia B-amyrin, Cucurbitacin B, Lupeol India(Ayurveda), Sri Lank Pulbutr et al. (2017)
grandis
Cucumis sativus B-carotne, Fatty acid Malaysia Panwar et al. (2014)
Cucumis Fatty acid, B-carotne India Jamal et al. (2011)
metuliferus
Cucumis B-carotne India Jamal et al. (2011)
callosus
Momordica Luteolin, Saponin, Cucurbitacin, Alkaloid, Carotene, South Africa Attanayake et al. (2015)
balsamina Fixed oil, Vitamin C, Resin acid, Momordicin
Momordica Cucurbitacin, Glycoside, Steroidal glycoside or Nigeria, South Africa Balkhande and Surwase
cymbalaria phenolics, Luteolin (2013)
Momordica Charantin, Cucurbitacin glycoside lanosterol, Luteolin, Bangledash, Nigeria, India (Ayurveda), Akharaiyi et al. (2017),
charantia Momordicin, Galactosebinding lectin Non-bitter, Taiwan, Vietnam, Philippines, Mauritius, Giovannini et al. (2016)
Cholesterol, Diosgenin, b-sitosterol, central America, Trinidad and Tobago
Momordica Phenolics or Steroidal glycoside, Luteolin China Di et al. (2011)
grosvenori
Momordica Cucurbitacin, Saponin, Alkaloid Luteolin South Africa Mahomoodally and
foetida Ramalingum (2015)
(continued)
Ibervillea Fatty acid, Monoglyceride (MG) India Mamun-or-Rashid et al.
sonorae (2014)
Diospyros Lupeol, Betulin, Betulinic acid, Gallic acid, India Kiran Kumar et al. (2012)
peregrina Hexacosane, Hexacosanol
Diospyros Phenolics Sri Lanka, India Dewanjee et al. (2011)
melanoxylon
Diospyros Betulinic acid Cameroon Kuete and Efferth (2011)
canaliculata
Diospyros Phenolics, Hexacosane, Hexacosanol Cameroon Kuete and Efferth (2011)
crassiflora
Diospyros lotus Phenolics, Sitosterol India Singh et al. (2014),
(2014)
Vaccinium Anthocyanoside, Phenolic India Sánchez-Villavicencio et al.
angustifolium (2017)
Vaccinium Anthocyanoside, Phenolic China Qian et al. (2017)
bracteatum
Vaccinium Anthocyanoside, Phenolic Iran Nickavar and Amin (2010
arctostaphylos
Vaccinium vitis Anthocyanoside, Phenolic Beaulieu et al. (2010)
Vaccinium Anthocyanoside, Phenolic China Kellogg et al. (2010)
myrtillus
Vaccinium Anthocyanoside, Phenolic China, India Mishra et al. (2013)
ovalifolium
Emblica Tannoid India, Vietnam Mamun-or-Rashid et al.
officinalis (2014), Singh et al. (2014)
(continued)
49
50

Jatropha curcas Diterpene Nigeria, India, China Mamun-or-Rashid et al.
(2014),
Singh et al. (2014)
Phyllanthus Tannin India Mamun-or-Rashid et al.
acidus (2014)
Phyllanthus Tannin Thailand, Southeast Asia, India (Ayurveda) Mamun-or-Rashid et al.
emblica (2014), Singh et al. (2014)
Phyllanthus Tannin Malaysia, Nigeria, Vietnam, India(Ayurveda, Sarin et al. (2014), Adedapo
amarus Unani, Siddha and homeopathy) and Ofuegbe (2014)
Phyllanthus Tannin Tanzania Bahare et al. (2019)
engleri
Phyllanthus Tannin India Muthulakshmi et al. (2014)
gardnerianus
Phyllanthus Tannin Vietnam Bharati et al. (2016)
urinaria
Phyllanthus Tannin Asia, Vietnam Ramasamy et al. (2013)
watsonii
Phyllanthus Tannin Asia, Vietnam Bharati et al. (2016)
niruri
phyllanthus Tannin Asia, Vietnam Hashim et al. (2013)
virgatus
Acacia modesta Polyphenol, Tannin Pakistan, India Rao et al. (2015)
Acacia senegal Polyphenol, Tannin Sudan Hilmi et al. (2014)
Acacia Polyphenol, Tannin Sudan Deb and Dash (2013)
ferruginea
Acacia tortilis Polyphenol, Tannin Sudan Vadivel and Biesalski (2012)
(continued)
Acacia Polyphenol, Tannin Bangladesh Kingsley et al. (2013)
farnesiana
Acacia catechu Polyphenol, Tannin India, Nepal Rao et al. (2015)
Acacia nilotica Polyphenol, Tannin India Rao et al. (2015)
Butea frondosa Palasonin, Butein, Genistein India Rao et al. (2015)
Butea Stigmasterol-3, Genistein, Butein, b-D- India Kumar et al. (2012)
monosperma glucopyranoside
Bauhinia Quercetin-3-O-rutinoside India Bahare et al. (2019)
monandra
Cassia Triterpenoid, Sterol, Tannin, Flavonoid Tanzania, India Mamun-or-Rashid et al.
auriculata (2014)
Cassia Triterpenoid, Sterol, Tannin, Flavonoid Nigeria Salihu Shinkafi et al. (2015)
sieberiana
Cassia Triterpenoid, Sterol, Tannin, Flavonoid China He et al. (2014)

obtusifolia
Cassia fistula Triterpenoid, Sterol, Tannin, Flavonoid India Thakur et al. (2016)
Cassia Triterpenoid, Sterol, Tannin, Flavonoid India Mamun-or-Rashid et al.
spectabilis (2014)
Clitoria kaempferol-3-neohesperidoside Asia Singh et al. (2014)
ternatea
Glycine max 3-O-methyl-D-chiro-inositol Africa, Asia, Latin America Singh et al. (2014)
Tamarindus Polysaccharide, Flavonoid Africa, Asia Mamun-or-Rashid et al.
indica (2014)
Xanthocercis Castanospermine, Fagomine, 4-O-beta-D- Africa, Asia Mamun-or-Rashid et al.
zambesiaca glucopyranosylfagomine (2014), Singh et al. (2014)
Pongamia Pongaflavonol India Bahare et al. (2019)
pinnata
51
(continued)
52

Tephrosia Purpurin India Bahare et al. (2019)
purpurea
Cinnamomum Cinnamaldehyde Orhan et al. (2013)
burmannii
Cinnamomum Cinnamaldehyde Malaysia Mustaffa et al. (2014)
iners
Cinnamomum Cinnamaldehyde Korea Seo et al. (2013)
japonicum
Cinnamomum Cinnamaldehyde India (Ayurveda) Bahare et al. (2019)
tamala
Cinnamomum Cinnamaldehyde India (Ayurveda) Sudha et al. (2011)
verum
Cinnamomum Cinnamaldehyde Bangladesh Grover et al. (2012)
obtusifolium
Cinnamomum Cinnamaldehyde India Zaidi et al. (2015)
impressinervium
Cinnamomum Cinnamaldehyde India (Ayurveda, Unani), China, Japan, South Zaidi et al. (2015)
cassia Africa
Persea Mineral, fat, Protein, Vitamin America Mamun-or-Rashid et al.
americana (2014)
Mentha arvensis Flavonoid, Terpen, Essential oil, Zinc, Vanadium, India Rao et al. (2015)
Chromium, Iron, Copper, Sodium, Potassium, Nickel,
Luteolin-7-O-glycoside
Mentha Flavonoid, Terpen, Essential oil, Zinc, Vanadium, India Rao et al. (2015)
longifolia Chromium, Iron, Copper, Sodium, Potassium, Nickel,
(continued)
Mentha piperita Flavonoid, Terpen, Essential oil, Zinc, Vanadium, India Bahare et al. (2019)
Chromium, Iron, Copper, Sodium, Potassium, Nickel,
Ocimum canum Saponin, Phenolics, Tannin, Eugenol Ghana Berhow and Affum (2012)
(1-hydroxy-2-methoxy-4-allylbenzene),
Ocimum Saponin, Tannin, Eugenol Trinidad and Tobago Bahare et al. (2019)
campechianum (1-hydroxy-2-methoxy-4-allylbenzene), Phenolics
Ocimum Saponin, Phenolics, Tannin, Eugenol Bangladesh, China, India (Ayurveda) Upadhyay (2017)
sanctum (1-hydroxy-2-methoxy-4- allylbenzene),
Ocimum Saponin, Tannin, Eugenol Nigeria, Bangladesh Akharaiyi et al. (2017)
gratissimum (1-hydroxy-2-methoxy-4-allylbenzene), Phenolics
Ocimum Saponin, Tannin, Phenolics India (Ayurveda) Mousavi et al. (2016)
tenuiflorum
Salvia officinalis Flavonoid India Mamun-or-Rashid et al.
(2014)
Glyccheriza Mimosoidea India Sudha et al. (2011)
glabra
Thespesia Populnetin, Quercetin, Populneol, Herbacetin India Singh et al. (2014),
populnea Mamun-or-Rashid et al.
(2014)
Melia Liminoid India Ranganathan et al. (2012)
azadirachta
Melia orientalis Liminoid India (Ayurveda) Marimuthu et al. (2013)
Melia dubia Liminoid Mexico Manosroi et al. (2011)
Moringa Phenolics, Tannin, Saponin, Flavonoid, Steroid, West Indies Ullah et al. (2015)
peregrina Glycoside, Quercetin, Stearic acid, Malic acid
(continued)
53
54

Moringa Phenolics, Tannin, Saponin, Flavonoid, Steroid, Ethiopia Geleta et al. (2016)
stenopetala Glycoside, Quercetin, Stearic acid, Malic acid
Artocarpus Sapogenin Nigeria Bahare et al. (2019)
communis
Artocarpus Sapogenin Indonesia, Mauritius Trinidad and Tobago Mahomoodally and
altilis Ramalingum (2015),
Wahyudin et al. (2017)
Artocarpus Sapogenin Marshall Islands Englberger et al. (2014)
mariannensis
Ficus Leucopelargonidin Southeast Asia, India (Ayurveda, Siddha, Gopukumar and Praseetha
bengalensis Unani, homoeopathy) (2015)
Ficus religiosa Leucopelargonidin India (Ayurveda) Singh et al. (2011)
Ficus virens Leucopelargonidin India (Ayurveda) Babu et al. (2010)
Ficus racemosa Leucopelargonidin Bangladesh, Southeast Asia, India Shah et al. (2016a, b)
Ficus cunia Leucopelargonidin India Sheikh et al. (2015)
Ficus carica Leucopelargonidin India (Ayurveda, Siddha, Unani, Badgujar et al. (2014)
homoeopathy)
Ficus thonningii Leucopelargonidin Africa Ahmed et al. (2012)
Ficus Leucopelargonidin Africa Awolola et al. (2014)
sansibarica
Ficus lutea Leucopelargonidin Africa Olaokun et al. (2014)
Ficus palmata Leucopelargonidin Africa Shahreen et al. (2012)
Ficus Leucopelargonidin south Asia Akhtar et al. (2016), Chan
microcarpa et al. (2017)
Ficus hispida Leucopelargonidin Bangladesh Singh et al. (2014)
(continued)
Ficus glumosa Leucopelargonidin Cameroon, Nigeria Zayyanu et al. (2015)
Ficus elastica Leucopelargonidin Philippines Kamel et al. (2017)
Ficus deltoidea Leucopelargonidin Southeast Asia, Malaysia Fidele et al. (2015)
Ficus Leucopelargonidin Nigeria, Ivory Coast, Cameroon, Sierra Leone Salihu Shinkafi et al. (2015)
exasperata
Ficus glomerata Leucopelargonidin India (Ayurveda, Siddha, Unani, Vaishnav et al. (2015)
homoeopathy)
Ficus Leucopelargonidin India (Ayurveda, Siddha, Unani) Arunachalam and
amplissima Parimelazhagan (2013)
Musa sapientum Flavonoid, Glycoside, Steroid, Pectin Africa, India Jayamurthy et al. (2013)
Musa Flavonoid, Glycoside, Steroid, Pectin Africa, India Venkatesh et al. (2013)
acuminata
Eucalyptus Calytoside Iran Asgharpour et al. (2012)
globules
Psidium Polysaccharide, Flavonoid, Terpen, Pedunculagin, China, Togo, New Guinea, Sri Lanka, Japan, Deguchi and Miyazaki
guajava Isostrictinin, Strictinin Mauritius, Central America, Papua, (2010)
Psidium Polysaccharide, Flavonoid, Terpen, Pedunculagin, East Asia Im et al. (2012)
cattleianum Isostrictinin, Strictinin
Syzygium Gallic acid, Malic acid, Citric acid, Anthocyanin India Adefegha et al. (2014)
aromaticum
Syzygium Gallic acid, Malic acid, Citric acid, Anthocyanin India Muthusamy and
densiflorum Krishnasamy (2016)
Syzygium Gallic acid, Malic acid, Citric acid, Anthocyanin Puerto Rico Gavillán-Suárez and
jambosa Aguilar-Perez (2012)
Syzygium Gallic acid, Malic acid, Citric acid, Anthocyanin Puerto Rico Kasetti et al. (2012)
alternifolium
(continued)
55
56

Syzygium Gallic acid, Malic acid, Citric acid, Anthocyanin Bangladesh Shahreen et al. (2012)
samarangense
Syzygium Gallic acid, Malic acid, Citric acid, Anthocyanin Bangladesh, Brazil, India (Ayurveda), Bansode and Salalkar
cumini (2015), Sharma et al. (2017)
Syzygium Gallic acid, Malic acid, Citric acid, Anthocyanin India (Ayurveda) Baliga et al. (2013)
jambolanum
Syzygium Gallic acid, Malic acid, Citric acid, Anthocyanin India Mamun-or-Rashid et al.
cordatum (2014)
Biophytum Phenol India Mamun-or-Rashid et al.
sensitivum (2014), Singh et al. (2014)
Butea Flavonoid India Rao et al. (2015)
monosperma
Piper guineense Saponin, Tannin, Terpen, Terpenoid, Flavonoid Nigeria van de Venter et al. (2008)
Piper betle Saponin, Tannin, Terpen, Terpenoid, Flavonoid Asia Srividya et al. (2015)
Piper Saponin, Tannin, Terpen, Terpenoid, Flavonoid South East Asia Sh Ahmed et al. (2017)
sarmentosum
Piper cubeba Saponin, Tannin, Terpen, Terpenoid, Flavonoid India Sh Ahmed et al. (2017)
Piper crocatum Saponin, Tannin, Terpen, Terpenoid, Flavonoid India Sh Ahmed et al. (2017)
Piper nigrum Saponin, Tannin, Terpen, Terpenoid, Flavonoid India Mishra et al. (2011)
Triticum Albumin India, Nigeria Singh et al. (2014)
vulgare Mamun-or-Rashid et al.
(2014)
Panax Tannin India Bahare et al. (2019)
quinquefolius
Panax ginseng Tannin Korea Lee et al. (2016)
(continued)
Ziziphus Fatty acid, Christinin-A, Alkaloid China, India (Ayurveda), Pakistan Modi et al. (2014)
xylopyrus
Ziziphus Fatty acid, Christinin-A, Flavonoid, Alkaloid, Tannin, Pakistan Ahmad et al. (2017)
oxyphylla Saponin
Ziziphus Fatty acid, Christinin-A, Flavonoid, Alkaloid, Tannin, Nigeria Ibrahim and Islam (2017)
mucronata Saponin
Ziziphus Fatty acid, Christinin-A, Flavonoid, Alkaloid, Tannin, India Goyal et al. (2011)
nummularia Saponin
Ziziphus lotus Fatty acid, Flavonoid, Alkaloid, Tannin, Saponin Algeria Benammar et al. (2010)
Ziziphus Christinin-A, Tannin, Flavonoid, Alkaloid, Saponin Mali, Southeast Asia Marwat et al. (2014)
mauritiana
Ziziphus jujuba Flavonoid, Alkaloid, Tannin, Saponin Turkey Sadegh-Nejadi et al. (2018)
Ziziphus amole Fatty acid, Christinin-A, Flavonoid, Alkaloid, Tannin, Mali Romero-Castillo et al.
Saponin (2013)
Mucuna Alkaloid, Carbazole India (Ayurveda) Bahare et al. (2019)

pruriens
Limonia Polysaccharide India Singh et al. (2014),
acidissima Mamun-or-Rashid et al.
(2014)
Aegle marmelos Alkaloid, Coumarin, Aegeline 2, Flavonoid India Singh et al. (2014),
(2014)
Citrus Sterol, Essential oil China Choi et al. (2012)
aurantium
Citrus sinensis Sterol, Essential oil India Shakthi Deve et al. (2014)
(continued)
57
58

Citrus reticulata Sterol, Essential oil China Choi et al. (2012)
Citrus grandis Sterol, Essential oil China Tzeng et al. (2011)
Citrus paradisi Sterol, Essential oil Nigeria, Trinidad and Tobago, Cuba García Mesa (2014),
Adeneye (2008)
Citrus medica Hesperidin China Sudha et al. (2011)
Feronia Bergapten,Triterpenoid, Bioflavonoid, Stigma sterol India Singh et al. (2014),
elephantum Mamun-or-Rashid et al.
(2014)
Bacopa Tannin, Luteolin India Sudha et al. (2011)
moneirra
Physalis Polysaccharide India Ramasamy et al. (2013)
alkekengi
Physalis Polysaccharide India Maobe et al. (2013)
peruviana
Physalis mnima Polysaccharide India Ayyanar and Ignacimuthu
(2011)
Physalis Polysaccharide India Ramasamy et al. (2013)
angulata
Withania Alkaloid, Withanolide India, Ayurveda Rehman et al. (2015),
somnifera Mishra et al. (2013), Maurya
et al. (2008)
Withania Alkaloid, Fatty oil, Milk-coagulating enzyme, Pakistan, India, Ayurveda Jonathan et al. (2015)
coagulans Esterase, Essential oil,
Lycium Polysaccharide China Potterat (2010)
barbarum
(continued)
Lycium Polysaccharide China Dhar et al. (2011)
ruthenicum
Lycium Polysaccharide China Potterat (2010), Wang and
chinense Ye (2016), Chan et al.
(2009)
Helicteres Terpenoid, Steroid, Alkaloid, Phenolics, Carbohydrate, Southeast Asia Varghese et al. (2012)
hirsuta
Urtica dioica Lectin, Flavonoid, Coumarin Turkey, Iran, Kenya Rezaei Aref et al. (2012),
Nickavar and Yousefian
(2011)
Urtica Coumarin Africa Zhang et al. (2012)
angustifolia
Curcuma Curcuminoid Bangladesh, Indonesia, Laos Peltzer et al. (2016)
xanthorrhiza
Curcuma longa Curcuminoid China, Laos Indonesia, Bangladesh, India Yadav and Chaudhury
(Ayurveda) (2016), Peltzer et al. (2016),
Mishra et al. (2011)
Curcuma Curcuminoid India Sushma et al. (2015)
domestica
Zingiber Ethanol, Gingerol China Chen et al. (2016)
striolatum
Zingiber Ethanol, Gingerol Africa, Latin America India (Ayurveda) Sudha et al. (2011), Ranilla
officinale et al. (2010), Morakinyo
et al. (2011)
Andrographis 5-hydroxy-7,8-dimethoxyflavone India, Nigeria Sudha et al. (2011)
paniculata
(continued)
59
60

Allium S-allyl cysteine, Ajoene, Allyl propyl disulfide,, S-allyl Iran Rahimi-Madiseh et al.
ampeloprasum mercaptocysteine, Diallyl disulphide oxide (2017)
Amaranthus Allyl propyl disulphide Kenya Mahomoodally and
cruentus Ramalingum (2015)
Mangifera Mangiferin, Flavonoid, Phenolics, Nigeria, India Sudha et al. (2011)
indica
Cuminum Aldehyde India Mnif and Aifa (2015)
cyminum
Catharanthus Alkaloid, Vinculin South Africa, China, Malaysia, Trinidad, Marwat et al. (2014),
roseus South East Asian countries, India Rasineni et al. (2010)
Panax ginseng Saponin Korea Xia et al. (2014)
Aloe Lophenol, 24-methyl-lophenol, 24ethyllophenol, India (Ayurveda) Bhaludra et al. (2013)
barbadensis Luteolin
Artemisia afra Polysaccharide Africa Bahare et al. (2019)
Betula pendula Quercetrin Indonesia, India Sudha et al. (2011)
Oroxylum Chrysin India Bahare et al. (2019)
indicum
Brassica juncea Isorhamnetin diglucoside India (Ayurveda) Bahare et al. (2019)
Opuntia dillenii Polysaccharide India Mamun-or-Rashid et al.
(2014), Singh et al. (2014)
Viburnum Tanin Africa, India Singh et al. (2014)
opulus
Carica papaya Alkaloid, Flavonod, Saponin, Tannin, Glycoside, Africa, Asia Mamun-or-Rashid et al.
Reducing sugar, Anthraquinone (2014)
Beta vulgaris Polydextrose, Sugar beet pectin India Mamun-or-Rashid et al.
(2014)
(continued)
Terminalia Phenolics India Venkatalakshmi et al. (2014)
catappa
Cannabis sativa Quercetin Latin America, India Sudha et al. (2011)
Coccinia B-amyrin, Cucurbitacin B, Lupeol India Singh et al. (2014)
cordifolia
Vaccinium Anthocyanoside, Phenolic Iran Singh et al. (2014)
uliginosum
Acalypha indica Kaempferol glycoside India Sudha et al. (2011)
Acacia arabica Polyphenol, Tannin India Yasir et al. (2010)
Ganoderma Polysaccharide Fraction India Singh et al. (2014)
lucidum
Leonotis Terpen India Mamun-or-Rashid et al.
leonurus (2014)
Cinnamomum Cinnamaldehyde Orhan et al. (2013)

zeylanicum
Cajanus cajan 7R*,9as*)-7-phenyloctahydroquinolizin-2-one Africa, Asia Mamun-or-Rashid et al.
(2014)
Lyophyllum Polysaccharide India Mamun-or-Rashid et al.
decastes (2014)
Brysoni (+)-catechin India Geleta et al. (2016)
macrassa
Abelmoschus Carbohydrate, Flavonoid, Gum, Phenolics, Mucilage, India Mamun-or-Rashid et al.
esculentus Tannin Protein, Phytosterol, Volatile oil (2014)
Azadirachta Nimbidin, Quercetin India, Africa Mamun-or-Rashid et al.
indica (2014)
(continued)
61
62

Grifola Disaccharide India Mamun-or-Rashid et al.
frondosa (2014)
Artocarpus Sapogenin Mauritius, India (Ayurveda) Bahare et al. (2019)
heterophyllus
Moringa Phenolics, Tannin, Saponin, Flavonoid, Steroid, India, Nigeria, Kenya, South Africa, Senegal, Geleta et al. (2016)
oleifera Glycoside, Quercetin, Stearic acid, Malic acid Mexico, Mauritius
Musa Pectin, Dietary fibre Africa, India Venkatesh et al. (2013)
paradisiaca
Eucalyptus Calytoside Nigeria Guillen et al. (2015)
torreliana
Nelumbo Tolbutamide India Mamun-or-Rashid et al.
nucifera (2014)
Averrhoa Tannin, Terpen China Singh et al. (2014),
bilimbi Mamun-or-Rashid et al.
(2014)
Lodoicea Flavonoid, Carbohydrate India Mamun-or-Rashid et al.
sechellarum (2014), Singh et al. (2014)
Butea frondosa Flavonoid India Kumar and Malik (2012)
Passiflora Vitexin India Sankaranarayanan et al.
incarnate (2010)
Piper Saponin, Tannin, Terpen, Terpenoid, Flavonoid Latin America Ranilla et al. (2010)
angustifolium
Hordeum Beta-glucan, Carbohydrate, Flavonoids Asia, Africa Mamun-or-Rashid et al.
vulgare (2014)
Panax Tannin China Xia et al. (2014), Yang et al.
notoginseng (2010)
(continued)
Nigella sativa Thymoquinone India Mamun-or-Rashid et al.
(2014)
Ziziphus Fatty acid, Christinin-A, Egypt Singh et al. (2014)
spina-christi
Morinda Flavonoid, Saponin, steroid, Triterpene India Singh et al. (2014),
citrifolia Mamun-or-Rashid et al.
(2014)
Murraya Alkaloid, Carbazole Nigeria Mamun-or-Rashid et al.
panicutata (2014)
Scoparia dulcis Tannin India Singh et al. (2014)
Capsicum Capsaicin Africa, Asia Singh et al. (2014)
frutescens
Helicteres isora Terpenoid, Steroid, Alkaloid, Phenolics, Carbohydrate, India (Ayurveda) Varghese et al. (2012)
Tilia cordata Hyperoside India Bahare et al. (2019)
Turnera diffusa Terpen, Flavonoid Africa Mamun-or-Rashid et al.

(2014)
Urtica urens Lectin, Flavonoid, Coumarin China, India Nencu et al. (2015)
Clerodendrum Pectolinarigenin Sushma et al. (2015)
phlomidis
Curcuma Curcuminoid India Sushma et al. (2015)
angustifolia
63
F. racemosa have shown excellent biological activity with different modes of action
and are reported as candidates for controlling diabetes disease (Deepa et al. 2018).
2.3 Phytochemistry of Bioactive Natural Compounds
The leaves, roots, stem bark, flowers, seeds, pulp, rhizomes and the essential oils of
medicinal plants are known to possess various bioactive natural compounds
(Bahare et al. 2019; Baliga et al. 2011). Essentially, these phytochemicals could be
produced by these plants as flavonoids, saponins, phenolic acids, alkaloids, tannin,
glycosides, terpenoids, polysaccharides and stilbenes which are investigated
intensively for their chemical constituents. Flavonoids are a group of natural
bioactive compounds with more than 4000 varieties that have been identified
(Shashank and Abhay 2013). Flavonoids are chemically a structure with a skeleton
of fifteen-carbon that exist as 2 rings of benzene (Fig. 2.1a, b) connected through a
pyrane heterocyclic ring (Fig. 2.1c). Therefore on the basis of chemical structure,
flavonoids can be categorized into different number of classes that includes flavo-
nols (i.e., quercetin, myricetin, kaempferol and fisetin), flavones (i.e., apigenin,
Chrysin, flavone and luteolin), flavanones (i.e., hesperetin, flavanone and narin-
genin), isoflavone (i.e., daidzin and Genistin), Anthocyanidin (i.e., Apigenidin and
cyanidin), flavanonol (i.e., taxifolin) and others. The different classes of flavonoids
differ from one another in the level of oxidation and pattern of substitution of the C
ring, while individual compounds within a class differ in the pattern of substitution
of the A and B rings.
Normally aglycones, glycosides, and methylated derivatives are the most com-
mon forms that flavonoids exist. Aglycone is the structural base for flavonoid
(Fig. 2.1). A six-member ring usually condensed or compressed with the benzene
ring which is either a a-pyrone or its dihydroderivative. The site of the benzenoid
substitution categorizes flavonoid into flavonoids and isoflavonoids (i.e., 2-position
and 3-position). At the 3 position and a C2–C3 double bond there is a hydroxyl
group that differentiates flavonols from flavanones structurally (Narayana et al.
2001). Naturally, acetylesters and methylethers of the alcohol group are known to
Fig. 2.1 Basic chemical

structure of flavonoids
occur. Flavonoids are often hydroxylated in positions 3, 5, 7, 2, 3′, 4′, and 5′. When
glycosides are formed, the glycosidic linkage is normally placed in positions 3 or 7
and the carbohydrate can be glucorhamnose, galactose, L-rhamnose, D-glucose, or
arabinose (Narayana et al. 2001).
The presence of bioactive natural compounds (phenolic compounds) in plant
products like vegetables, fruits and legumes offer protection afforded by their
consumption. Phenolic compounds are well-known phytochemicals found in all
plants. Phenolic compounds are manufactured by plants as a result of reaction to
physiological or ecological pressures such as pests and diseases attack, wounding
and UV radiation (Ali et al. 2013; Kennedy and Wightman 2011) The aromatic ring
having one or more hydroxyl groups forms the basic in the structures of phenolic
compounds (Fig. 2.2) (Ali et al. 2013). The location and number of hydroxyl
groups on the aromatic ring gave rise to the variation in phenolic acids (Ali et al.
2013). Phenolic compounds in plants are divided into polyphenols or simple
phenols depending phenol units present in the molecule. This therefore shows that
phenolics in plant exist as benzoic and cinnamic acid, simple phenols, coumarins,
lignans, lignins, condensed and hydrolysable tannins, phenolic acids and flavonoids
(Ali et al. 2013; Soto-Vaca et al. 2012) Phenolic acids as one class of phenols have
hydroxycinnamic and hydroxybenzoic acid as their parent structures. The deriva-
tives of hydroxybenzoic acid exit as protocatechuic acids, syringic, gallic and
vanillic. The derivatives of hydroxycinnamic acid derivatives include p-coumaric,
sinapic acids, ferulic and caffeic. Cell wall phenolics is a phenolic compound group
that are found to be insoluble and although capable of making complexes with other
components of the cell. Vanholme et al. (2010) indicated that lignins and
hydroxycinnamic acids are the main cell wall phenolics. At the time plant growth,
the compounds play a key role in the cell wall against such stresses like infection
and wounding (Ali et al. 2013).
The structure of saponins consists of water soluble glucidic chain and liposol-
uble structure in that they are glycosylated compounds (Chaieb 2010; Eskandar and
Somayeh 2015). The structure of saponin is given in Fig. 2.3 (Chaieb 2010). The
aglycone and glycone are the non-sugar and sugar components respectively. The
steroid backbone or triterpenoid are the main component of aglycone portion (Abid
Ali Khan et al. 2012). Some of the main components of saponin include among
others L-arabinose, D-galactose, D-glucose, D-xylose, D-glucuronic acid, D-fructose,
L-rhamnose (Chaieb 2010). The aglycone has glycosylation sites where the sugar
moiety is linked to it via one or two ether glycosidic linkage. It is important to note
Fig. 2.2 a hydroxybenzoic acid, b hydroxycinnamic acid, c flavonoids

Fig. 2.3 Chemical structure of saponin
that the aglycone may consist in it one or more unsaturated carbon to carbon (C–C)
bonds. If the chain (oligosaccharide) is connected to the site of C3, then the
molecule is known as monodesmosidic, whereas if additional sugar moiety were
present at the site of C26 or C28 of the saponin, such saponins are called
bidesmosidic. The saponins structure is affected by the amount and types of sugars
present, as well as the composition of steroid ring. Abid Ali Khan et al. (2012)
observed high saponin composition in plants at tender age than plants at their peak
age, even though many factors like environmental factors and physiological state
affect the saponin contents. Classification of saponins depends on the nature of their
aglycone and as result two classes were identified as steroidic aglycone saponosides
and triterpenic aglycone saponosides. The triterpenic aglycones come from the
cyclization of the (3S)-2,3-epoxy-2,3 dihydrosqualene. The skeleton of steroidic
aglycones was observed to have 27 carbon atoms. The molecules were from
cetalisation (intramolecular) that intervenes after oxidation in C16, C22 and C26 of a
cholestanic precursor taking into consideration spironature of C22; which is usually
shown by the spirostane term. It is known as furostane, if the structure is penta-
cyclic. This process of cyclization gives rise to pentacyclic compounds such as
ursanes, dammaranes, hopanes and oleananes. The majority of triterpenic sapo-
genins belong to these four basic skeletons.
It is well known that bioactive natural compounds are many and we are giving
an introduction, therefore, in this section, some bioactive natural compounds, their
chemical structures and sources will be presented as are available in the literature
(Table 2.2).
Table 2.2 Some bioactive natural compounds, their chemical structures and sources
Compound Structure Source Reference
Fisetin Acacia berlandieri Prasath et al.
Acacia greggii (2014)
Butea fronds
Gleditschia
triacanthow
Quebracho colorado
Gleditsia triacanthos
Rhus cotinus
Cotinus coggygria
Rhus vemiciflua
Gambogic acid Garcinia hanburyi Cui et al.
Garcinia cambogia (2018)
Garcinia indica
Chrysin Passiflora caerulea Amjid et al.
Oroxylum indicum (2014), Oza and
Passiflora incarnata Kulkarni (2016)
Berberine Argemone mexicana Cui et al.
Berberis vulgaris (2018)
Berberis aristata
Berberis aquifolium
Eschscholzia
californica
Coptis chinensi,
Hydrastis canadensis
Xanthorhiza
simplicissima
Tinospora cordifolia
Phellodendron
amurense
Butein Toxicodendron Benzler et al.
vernicifluum (2015)
Dalbergia odorifera,
Cyclopia subternata,
Semecarpus
anacardium Creopsis
tungtoria
Boldine Peumus boldus Oza and
Kulkarni (2016)
Baicalein Scutellaria Oza and

baicalensis Kulkarni
Oroxylum indicum, (2016), Ahad
et al (2014)
Embelin Embelia ribes, Ahad et al. et al.
Lysimachia punctata, (2014)
Lysimachia
erythrorhiza
(continued)

Curcumin Curcuma longa Kunwar and
Zingiberaceae plants Priyadarsini
(2016)
Withanolides Withania somnifera Ahad et al.
(2014)
Piperine Piper nigrum Atal et al.

Piper longum (2016)
Embelin Embelia ribes Durg et al.
Lysimachia (2017)
erythrorhiza Cui et al.
Lysimachia punctata (2018)
Erianin Dendrobium Yu et al. (2016)
chrysotoxum
Luteolin Leonotis leonurus Wang et al.
Mentha arvensis (2012a, b)
Mentha longifolia
Mentha piperita
Ocimum canum
Ocimum
campechianum
Ocimum sanctum
Ocimum gratissimum
Ocimum tenuiflorum
Salvia officinalis
Silymarin Silybum marianum Oza and
Kulkarni (2016)
Ursolic acid Calluna vulgaris Lee et al.

Crataegus laevigata (2014), Castro
Eriobotrya japonica et al. (2015)
Eugenia jambolana
Melissa officinalis
Mentha piperita
Ocimum sanctum
Rosmarinus
officinalis Thymus
vulgaris
Dracocephalum
heterrophyllum
Hyssopus
seravshanicus
Triptolide Tripterygium Lee et al.
wilfordii (2014)
Sanguinarine Sanguinaria Lee et al.

canadensis (2014)
(continued)

Resveratrol Vitis vinifera Do et al. (2012)
Vaccinium
corymbosum
Pistacia vera
Quercetin in many grains, fruits, Li et al. (2013)
leaves, vegetables
Piceatannol Passiflora edulis Uchida-Maruki
et al. (2015)
Neferine Nelumbo nucifera Oza and

Kulkarni (2016)
Oxymatrine Sophora flavescens Oza and
Kulkarni (2016)
Naringenin Citrus paradisi Tsai et al.

(2012)
Catechins Theobroma cacao Oza and
Camellia sinensis Kulkarni (2016)
Garcinol Garcinia indica Mali et al.
(2017)
Honokiol Magnolia officinalis Sun et al.

(2015)
Lupanine Lupinus perennis Madhuri and
Naik (2017)
Morin Morus alba Pandey et al.
Psidium guajava (2019)
Maclura pomifera
Prunus dulcis
Maclura
Chlorophora
tinctoria tinctoria
Castanea sativ
Inulin Helianthus tuberosus Ma et al. (2011)
Boswellic acids Boswellia carteri Razavi et al.

Boswellia serrata (2018)
Kaempferol Acalypha indica Alkhalidy et al.

(2015)
Genistein Butea monospermea Shashank and
Butea frondosa Abhay (2013)
Mangiferin Anemarrhena Razavi et al.
asphodeloides (2018)
(continued)

Oleanoic acid Aralia cortex Taguchi et al.
Phellodendron cortex (2016)
Oleanoic acid-28-O- Aralia cortex Taguchi et al.

beta-D-glucopyranoside Phellodendron cortex (2016)
Kaempferitrin Bauhinia forficata Taguchi et al.

(2016)
Apigenin Buddleja officinalis Alkhalidy et al.
(2015)
Bassic acid Bumelia sartorum Alkhalidy et al.
(2015)
Ellagic acid Caesalpinia ferrea Uchida-Maruki

et al. (2015)
Alpha-homonojirimycin Commelina Pandey et al.
communis (2019)
Ursolicacid Cornus officinalis Uchida-Maruki

et al. (2015)
Cryptolepine Cryptolepis Pandey et al.
sanguinolenta (2019)
Dioscoretine Dioscorea Alkhalidy et al.
dumetorum (2015)
Leucopelargonidin Ficus bengalensis Alkhalidy et al.
(2015)
Achyrophuran Achyrocline Taguchi et al.
satureioides (2016)
Maturine Psacalium Baileyand Day
decompositum (2004)
Psacalium peltatum
Acourtia thurberi
Metformin Galega officinalis Bailey and Day
(2004)
Isoliquiritigenin Glycyrrhizae radix Taguchi et al.
(2016)
Coutaraegenin Hintonia latiflora Mali et al.
(2017)
Hydnocarpin Hydnocarpus Reddy et al.
wightiana (2005)
Hedearagenin Kalopanax pictus Taguchi et al.
(2016)
Masopropol Larrea tridentata Taguchi et al.
(2016)
(continued)

Maesanin Maesa lancelata Taguchi et al.
(2016)
Flaviolin Maesa lancelata Taguchi et al.
(2016)
Caffeic acid Origanum vulgare Kim et al.
(2005)
Rosmarinicacid Origanum vulgare Mali et al.
(2017)
Bakuchiol Otholobium Mali et al.
pubescens (2017)
Maurine Psacalium Mali et al.
decompositum (2017)
Psoralidin Psoralea corylifolia Kim et al.
(2005)
Marsupin Pterocarpus Pandey et al.
marsupium (2019)
Trigonella
foenumgraecum
Swerchirin Swertia chirayita Pandey et al.
(2019)
Fagomine Xanthocercis Pandey et al.
zambesiaca (2019)
2.4 General Applications of Bioactive Natural Products
2.4.1 Traditional Medicine
The practise of traditional medicine is worldwide in spread, China, Japan, India,

Pakistan, Thailand and Sri Lanka inclusive. Folklore and traditional systems of
medicine bequeathed from generation to generation is rich in domestic recipes and
communal practice. Medicinal plants have the great potential used in various tra-
ditional systems of medicine like in the Ayurveda, Siddha, Unani, in the Tibetan, in
the Srilankan, and in the Homeopathy systems of alternative and complementary
medicine (Meenupriya et al. 2014). The best known examples of traditional med-
icine, differing in concept and protocol, are well-developed systems such as ayur-
vedic and acupuncture medicine that have been widely used to conserve human
health in India, China and native Indians from Brazil in the Amazonian region
(Meenupriya et al. 2014). In Chinese medicinal folk practice, the search for new
medicines to treat emergent and old diseases such as AIDS and malaria, attention is
now being geared towards discovering the active ingredients encountered in the
treasury of over 5,000 Chinese plants, herbs and roots that have been used routinely
and traditionally. Chaihu and Quinghaosu are two such examples. Whereas the
former, called artemisinin and obtained from Artemisia annua is expected to yield,
in the coming millennium, a potent new class of antimalarials, the latter, obtained
from Bupleurum chinense and used as a popular remedy for hepatitis is the focus of
intense research by the Japanese pharmaceutical industry (Baliga et al. 2011).
The practise of traditional medicine using medicinal plants such as Panax
quinquefolium, Podophyllum peltatum and Eupatorium perfoliatum has long been
associated with the American Indians in the United States of America. These
medicinal plants have also been recognised and appreciated for their aesthetic and
ornamental value. In Central America medicinal plants have been widely used by
the Maya Indians in Mexico, the Pipiles in El Salvador, the Miskitos and Sumus in
Nicaragua and Honduras, the Pech, Xicaques and Lencas in Honduras,
the Guaymis and Kunas in Panama and the Talamancas in Costa Rica. In Europe,
more than 1,500 species of medicinal plants are widely used in Albania, Croatia,
Bulgaria, France, Germany, Poland, Hungary, Turkey, Spain, and the United
Kingdom. The islands of Maltese constitute a good example where medicinal plants
are widely used in everyday life as part of folk medicinal remedies.
Africa is another rich source of medicinal plants rich in bioactive natural com-
pounds. Perhaps, the best known species include that of Moringa oleifera reported
by different researchers in the literatures to have important biological potentials
include antimicrobial activities (Arora and Onsare 2014; Arora et al. 2013), anti-
fungal (Kadhim and AL-Shammaa 2014), antioxidant (Satish et al. 2014),
antibacterial and antiulcer (Belay and Sisay 2014), anti-inflamatory, diuretic,
antispasmodic (Krishnamurthy et al. 2015; Araujo et al. 2013), antimutagenic and
antioxidant (Satish et al. 2014), antistress (Luqman et al. 2012), anticancer
(Krishnamurthy et al. 2015; Pinto et al. 2015), cytotoxic activities (Araujo et al.
2013; Asare et al. 2012), antitumour, antipyretic, antiepileptic, antinociceptive and
antidiabetic (Vinoth et al. 2012). Other notable examples are Catharanthus roseus,
which yields anti-tumour agents such as vinblastine and vincristine; and Ricinus
communis, which yields the laxative castor oil. In Botswana, Namibia, Lesotho and
South Africa, Harpagophytum procumbens is produced as a crude drug for export.
Similarly, Hibiscus sabdariffa is exported from Egypt and Sudan. Other exports
are Pausinystalia yohimbe from Cameroon, Nigeria and Rwanda, which yields
yohimbine; and Rauwolfia vomitoria, from Madagascar, Mozambique and Zaire,
which is exploited to yield ajmaline and reserpine.
2.4.2 Plant Based Pesticides and Agrochemical Industry
The plant world is rich store house of natural chemicals that could be exploited for
use as biopesticides (Dwivedi and Enespa 2012). Medicinal plants are now
emerging as safer and more compatible approach to chemical control of pests and
diseases. All parts of these plants including roots, flowers, bark, stem, leaves, seeds
and essential oils possess antimicrobial properties and are therefore used for
medicinal and other purposes (Dwivedi and Enespa 2012). The use of these
potential plants as biopesticides has emerged as a result of the drawbacks associated

with the use of synthetic chemical methods notably among them are their high cost,
their carcinogenicity, teratogenicity, high and acute residual toxicity, long degra-
dation period, environmental pollution and possible side-effects on human health
through the food chain (Ai-ying et al. 2011). These drawbacks coupled with public
concern have increased interest in developing further alternative control methods,
particularly those that are eco-friendly, biodegradable, feasible to the farmers,
non-toxic to human and animals, specific in their action and have a broad spectrum
of antimicrobial activity (Marino and Bersani 2001; Abhishek et al. 2013).
Scientific investigations on different morphological parts of medicinal plants
indicated the presence of biologically active compounds which possesses an array
of antimicrobial properties acting on different types of microorganisms. These
bioactive compounds were evaluated in vitro and further subjected to in vivo testing
to validate their efficacy in controlling the incidence and severity of diseases in
crops and insect infestations. Recently, Batista et al. (2014) reported the antifungal
inhibitory activity of Moringa-Chitin Binding Protein (Mo-CBP3) purified from the
seeds of M. oleifera Lam. against mycelial growth and spore germination of
Fusarium solani at 0.05 mg mL−1. Similarly, in another study, Gifoni et al. (2012)
also showed the antifungal efficacy of Mo-CBP3 chitin-binding protein purified also
from M. oleifera seed against phyopathogenic fungi Fusarium oxysporium, F.
solani, Colletotrichum gloesporiodes and Pythium oligandrum at 0.05 mg mL−1
and 0.1 mg mL−1 respectively. Dwivedi and Enespa (2012) reported that M. olei-
fera extracts (leaves, bark and seeds) at 75% (v/v) give significant inhibitory effect
on the mycelial growth of F. solani and Fusarium oxysporum f. sp. Lycopersici.
The fungicidal effect of Moringa extracts on some soil-borne fungi such as
Rhizoctonia, Pythium and Fusarium causing tomato rots was also reported (Moyo
et al. 2012). Again, El-Mohamedy and Abdalla (2014) reported the fungicidal effect
of M. oleifera against F. oxysporm, F. solani, Alternaria alternate, A. solani.
Rhizoctonia solani, Sclerotium rolfsii and Macrophomina phaseolina causing rots
on fruit and other perishables. Ukeh and Chiejina (2012) reported that phytopes-
ticides from two plants including Afromomium meleguata and Zingiber officinale
inhibited Penicillium digitatum, Mucor piriformis, Aspergillus niger and
Heminsthoporium solani causal agents involved with soft rot of tomato. Ijato et al.
(2011) reported the antimicrobial activity of Vernonia amygdalina and Tridax
procumbens each with two varying formulations and concentrations (aqueous
extracts: 80 and 60% and ethanol extracts: 30 and 20%) against A. niger, F.
oxysporum, G. candidum and Rhizopus stolonifer. Again, the aqueous and organic
solvents (water and ethanol) extracts from leaves of Chromolaena odorantum and
Azachirachta indica were reported to have antifungal activity against fungal
pathogens that cause tomato rots (A. niger, F. oxysporum, R. stolonifer and G.
candidium) by poisoned food method (Ijato et al. 2011).They showed that among
the various extracts with varying concentrations, ethanol extracts of 30% A. indica
had the best inhibitory effect (83.30%) against A. niger followed by 30% ethanol
extract of C. odorata (80.00%) against G. candidium which proved the potentiality
of the plant extracts for the control of postharvest and transit fungal rot of tomato
fruit. Ijato et al. (2011) reported that these plants extracts in addition to their ability
to retard mycelial growth of the fungi, they also inhibit their spore germination.
Other reports on the antifungal activity of plant-based pesticides include that of
Prapassom et al. (2012) who reported the efficacy of 14 crude leaf extracts including
Piper sarmentosum, Cymbopogon citratus, Citrus hystrix, Murraya paniculata,
Ocimum basilicum, Ocimum canum, Annona squamosal, M. oleifera, Psidium
guajava, Ocimum sanctum, Eucalyptus camaldulensis, Artocarpus heterophyllus,
Cassia siamea, Mentha cordifolia, using ethanol, methanol, and chloroform (80%)
as solvents against C. gloesporioides (Penz.) and found that crude methanol extract
of P. sarmentosum leaves effectively inhibited the growth of fungal mycelium
(100%), followed by crude chloroform extract(81.85%). Similarly, extracts from
the bark, roots, and leaves of A. indica at 400 and 500 mg/ml concentrations tested
against C. gloesporioides, a causal agent of field soft rot of fruit completely inhibit
the growth of the fungus (Ijato et al. 2011).
The challenge in agrochemical industry today is not only to formulate pesticides
with high efficiency, but also need to developed better brands with improved safety
to the users and have less impact to the environment (Polychniatou and Tzia 2014).
Not only that, such formulations should be an adequate delivery system that will
resolve the problem of inconsistency associated with bipesticides that reduce their
rate of competition with long standing synthetic pesticides in the market (Su et al.
2014). Since bipesticides are formulated with active ingredients and inert ingredi-
ents (Jiang et al. 2011), the primary aim of formulation process is to make the active
ingredient easy to handle, use, ensure that that it is stable during storage and
transport, and achieved an adequate shelf life in the formulation. The active
ingredient should possess antimicrobial or chemical properties that can control the
target pest. Specifically, the active ingredient should function as antifungal,
antibacterial, antiviral, antioxidant, cytotoxic, repellent, destroyer, killer, or mitigate
pest, or as plant regulator, desiccant or defoliant. Bioactive compounds (active
ingredients) of biopesticides come from various sources that include those extracted
from botanicals (plants) such as rotenone, MoCBP3, nicotine, pyrethrum, saponins
for the preparation of plant-based biopesticides. Typical example of plant-based
biopesticide is that of MILSANA and REGALIA formulated from anthraquinone
containing extracts of giant knotweed (Reynoutria sachalinensis). Both biopesti-
cides are commercially developed and marketed by Marrone Bio Innovatives Inc
sold as MILSANA® and REGALIA®. Both have antifungal and antibacterial
properties against various fungal and bacterial pathogens. In addition, they also
serve as plant defence inducers and act in the accumulation of fungistatic phenolic
compounds in the plants (Huang and Campbell 2016; Su et al. 2014).
2.4.3 Pharmacological Applications in Drugs Development
Another application of bioactive natural compounds is in the field of drugs devel-

opment as a result of their efficacy and safety concerns of the hundreds of millions of
individuals which are currently seeking better management of diseases

(Mancha-Ramirez and Slaga 2016). Medicinal plants produce a wide array of
phytoconstituents which include alkaloids, flavonoids, phenolic acids, saponins,
glycosides, polysaccharides and tannin, which are intensively investigated for their
bioactive effects. They are today, the main quarry for discovering promising lead
candidates and play an imperative role in the upcoming drug development programs
(Sharifi-Rad et al. 2018a, b, c; Salehi et al. 2018). They have low cost, ease of
availability and least side effects which make plant-based preparations the main key
player of all available therapies, especially in rural areas (Arya et al. 2011).
Moreover, many plants provide a rich source of bioactive phytochemicals, which are
free from undesirable side effects and possess powerful pharmacological actions
(Abdolshahi et al 2018; Mishra et al. 2018a, b; Sharifi-Rad et al. 2018a, b, c; Singab
et al. 2014;). These plants also have been an exemplary source of drugs with many of
the currently available drugs being obtained directly or indirectly from them
(Abdolshahi et al. 2018; Mishra et al. 2018a, b; Arumugam et al. 2013). Despite the
presence of drugs in the pharmaceutical market, the treatment of most diseases with
medicinal plants is often successful. Herbal medicines and plant components with
insignificant toxicity and no side effects are notable therapeutic options for the
treatment of diseases around the world (Bahare et al. 2019). Furthermore, reliance on
the use of medicinal plants that is increasing in the industrialised societies has been
traced to the extraction of bioactive natural compound from medicinal plants and
development of several drugs and chemotherapeutics from these plants as well as
from traditionally used rural herbal remedies (Baliga et al. 2011). Validated phar-
macological properties of the bioactive natural compounds are antiviral, antifungal,
antibacterial, free radical scavenging and antitoxic effect, Gastroprotective effects,
anti-inflamatory, Hepatoprotective effects, antidiabetic activities, Cardioprotective
effects, Radioprotective effects, Chemopreventive effects, Anticlastogenic effects,
Antineoplastic effects, Neuropsychopharmacological effects, Antipyretic effects,
Anti-diarrheal effects, Antifertility activity, Anti-allergic effects, Hypolipidemic
effects (Baliga et al. 2011; Jabeen and Javaid 2010).
2.4.4 Cosmetics
Bioactive natural compounds used in the production of herbal products have today
gained increase acceptance and popularity, expanding which are sold as herbal
cosmetics (Ribeiro et al. 2015; Lee et al. 2013). Phenolic components such as
quercetin, kaempherol and carbohydrates such as glucose, galacturonic acid, ara-
binose and rhamnose are some of the substances present in the chemical compo-
sition of many medicinal plants that the cosmetic industry has interest in them as
anti-aging and moisturizers products (Zhang et al. 2011; Ribeiro et al. 2015). Dry
skin is a common problem among the human populace. Its main characteristic is a
phenomenon called xerosis, which is a rough peeling and appearance. To accom-
plish the main function of the human skin as a barrier provider between the internal
contents external environment, moisturizers made from bioactive natural products

are needed. Moisturizers are cosmetic products that prevent the skin against xerosis
and delaying of premature ageing and for their use to help dermatological therapies
in a wide variety of skin disorders (Capitani et al. 2012). The mode of action of
moisturizers should not be overemphasized as 1. They may act by an occlusive
mode thereby impairing the evaporation of skin moisture by forming an epicuta-
neous lipidic film that prevents water loss, when oils and lipids are used as mois-
turizers; or 2. As humectants that act by attracting water from the other layers of the
epidermis to the stratum corneum i.e., glycerine and sodium pyrrolidone carboxylic
acid; are used as moisturizers. Other moisturizing modes of action include active
hydration by rearranging the stratum corneum and aquaporin formation. The
aquaporins are transmembrane proteins which form water channels and facilitate
water flux through the cell plasma membrane thus being important to maintain
aconstant water content in viable epidermis (Capitani et al. 2012).
2.5 Delivery Technology for Bioactive Natural Products
Nanotechnology has been considered as a promising delivery system with respect to

transdermal drugs delivery and eco-friendly pesticide formulation, including those
with natural plant products incorporated as active ingridients (Angajala et al. 2014;
Da Costa et al. 2014; Rodríguez-Rojo et al. 2012). Nanotechnology is a useful
system for delivering chemical compounds across the cuticle and it has the potential
of application in the food industry, disease treatment delivery system, food pack-
aging and bioactive compound delivery to target sites. The widespread commercial
application of nanoemulsion technology especially in pharmaceutical and agro-
chemical industries is due to its small droplet size (10–100 nm) as suggested by
some researchers (Rai et al. 2015; Polychniatou and Tzia 2014), that causes a
reduction in the Brownian motion and gravitational strength (Mishra et al. 2014;
Tadros et al. 2004). Similarly, the small size of droplets spread uniformly on the
surface and helps to enhanced wetting, spreading and penetration as a result of the
low surface tension. Additionally, it can prevent flocculation and coalescence of the
droplets and enables the system to remain dispersed without separation.
Furthermore, the surfactant film thickness prevents disruption and thinning of liquid
film between the droplets. Nanoemulsion can encapsulate active ingredients within
their droplets and this help in reducing chemical degradation (McClements and
Decker 2000) and hence expand cell wall penetration of the fungus, due to their
smaller size (Zahid et al. 2012)). Other benefits include, less amount of energy
requirement, increase rate of absorption and eliminates variation on absorption, and
increases bioavailability (Mishra et al. 2014).
2.5.1 Components of Nanoemulsion
Basically, a nanoemulsion consists of an active ingredient, an oil phase, surfactant

and aqueous phase. The oil phase is usually the carrier(s) which are mineral oils
and/or vegetable oils (Xu et al. 2011). Today, vegetable oils and their esterified
derivatives are gaining interest compared to the mineral oils because of their
biodegradability and renewability (Xu et al. 2011). Castor oil, copaiba oil, olive oil,
soy oil, Agnique® AMD 10, Edenol SP 100 is few examples of vegetable oils used
in nanoemulsion formulations. The oil phase in nanoemulsion formulations
enhance the spread and penetration of droplets on the surface of the skin as well as
plants; split open the cuticle to increase both the fluidity of cuticular components
and pesticide diffusion rates (Xu et al. 2011).
2.5.1.1 Surfactants
Surfactants, also called surface-active agents are organic compounds that are
amphiphilic in nature (having both hydrophobic groups and hydrophilic groups)
that reduce surface and interfacial tensions by accumulating at the interface of
immiscible fluids and increase solubility, bioavailability, mobility and subsequent
biodegradation of hydrophobic or insoluble organic compounds (Singh et al. 2011).
They can be found and used as emulsifiers, de-emulsifiers, wetting agents, forming
agents, detergents in petroleum, petrochemicals, functional food ingredients,
environmental management, foods and beverages, agrochemicals, cosmetic and
pharmaceuticals, and in the mining and metallurgical industries (Singh et al. 2011).
Surfactant emulsifiers are added to nanoemusion formulations to ensure sponta-
neous emulsification with good stability qualities in the spray tank. Surfactants can
be classified as ionic and non-ionic. Non-ionic surfactants are preferred in pesticide
formulation system (Mehmood 2015) than the ionic ones due to their less toxicity,
enhanced solubility, spreading, adsorption, translocation and penetration of active
ingredients into the target. Myers (2005) define non-ionic surfactant as a surfactant
that carries no electric charge, as its water solubility is derived from the occurrence
of polar functionalities capable of significant hydrogen bonding relations with water
(e.g. polyglycidols and polyoxyethylenes) (Shafiq et al. 2007). Although stable
nanoemulsions are best formulated with surfactants or a combination of surfactants
having hydrophile-lipophile balance (HLB) values close to that required for the oil
phase, it is important to know that there are no specified rules to resolve the ratio of
surfactants in the blend surfactants. However, guidance can be obtained from the
(HLB) system.
2.6 Conclusions
The current book chapter tries to review the global efforts by scientists regarding
their investigation on natural bioactive compounds from medicinal plants that will
serve as a novel chemotherapeutants and their general applications in various fields.
Alongside, this provides a prospect for further research in order to find novel
antimicrbial agents, their potentials, modes of action and synergistic effects for the
eventual formulation of herbal mixtures and their combination with synthetic
medicines. Such efforts will be more lucrative if their toxicological effects are
confirmed by necessary and careful studies. It will be more scientific to standardize
techniques of extraction, in vitro and in vivo antimicrobial efficacy testing so that
the search for new biologically active compounds could be more systematic and
results interpretation would be facilitated. The phytochemicals responsible for the
potential properties of the bioactive natural compounds are mainly phenolic acids,
alkaloids, flavonoids, saponins, glycosides, polysaccharides, tannins and stilbenes.
Notwithstaanding, the phytochemical composition in plant depend highly on sev-
eral factors, including plant specie, genetic traits, plant organs used, and the
growing, drying, and storing condition. Moreover, new techniques must be
employed to get excellent plant products in high amounts and to determine their
potential applications more accurately in food industry, traditional medicine, cos-
metics, drugs development and plant based pesticides and agrochemical industry.
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Chapter 3
Plant Polysaccharides in Pharmaceutical
Applications
Amit Kumar Nayak, Md Saquib Hasnain, Amal Kumar Dhara,

and Dilipkumar Pal
Abstract Plant polysaccharides are the by-products of photosynthesis within the

plants and are being extracted from different parts of the plants, such as leaves,
pods, fruits, seeds, cereals, stems, roots, rhizomes, corms, exudates, etc. The
important advantages for the uses of plant polysaccharides include easy availability
from the nature as plant resources are abundant, sustainable and low cost produc-
tion, biodegradability, biocompatibility, water solubility, swelling ability, etc. Since
long, numerous plant polysaccharides have already been explored and exploited as
excipients in a variety of common pharmaceutical dosage forms, such as suspen-
sions, emulsions, gels, tablets, capsules, beads, microparticles, nanoparticles,
liposomes, transdermal formulations, buccal formulations, nasal formulations,
ophthalmic formulations, etc. The current chapter presents a brief review on the
pharmaceutical applications of various plant polysaccharides.
Keywords Plant polysaccharides Biopolymers Excipient Pharmaceutical

applications Drug delivery
A. K. Nayak
Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Mayurbhanj,
Odisha 757086, India
Md. S. Hasnain
Department of Pharmacy, Shri Venkateshwara University, Gajraula, Amroha, UP, India
A. K. Dhara
Department of Pharmacy, Contai Polytechnic, Darua, Contai, Purba Medinipur,
West Bengal 721406, India
D. Pal (&)
Department of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya
(A Central University), Koni, Bilaspur, C.G 495009, India
e-mail: drdilip2003@yahoo.co.in

https://doi.org/10.1007/978-3-030-54027-2_3
94 A. K. Nayak et al.
3.1 Introduction
An inspiring array of plant-derived materials and products have already been

revealed for the uses in many applications including pharmaceuticals, cosmeceu-
ticals, biomedical, foods, textiles, paints, paper-making, etc. (Ali et al. 2019;
George and Suchithra 2019; Hasnain et al. 2019a; Nayak et al. 2020; Pal et al.
2019a; Sinha Mahapatra et al. 2011; Xie et al. 2016). Amongst the different
plant-derived materials, plant polysaccharides have already been demonstrated as a
useful group of polymeric biomacromolecules possessing some outstanding merits
over the synthetic polymers and these merits include easy availability from the
nature as plant resources are abundant, sustainable and low cost production,
biodegradability, biocompatibility, water solubility, swelling ability, etc. (Nayak
and Hasnain 2019a; Nayak et al. 2018a). The most important reason for the rising
interest for the uses of various plant polysaccharides is the advantages of easy
cultivation and harvesting to offer a constant supply of raw plant materials for the
polysaccharide extractions. Plant polysaccharides are the by-products of photo-
synthesis within the plants and are being extracted from different parts of the plants,
such as leaves, pods, fruits, seeds, cereals, stems, roots, rhizomes, corms, exudates,
etc. (Prajapati et al. 2013). These are high molecular weight biopolymers possessing
many monosaccharidic units as building blocks, which are linked each other by
O-glycosidic linkages in different patterns (Nayak and Pal 2016). Similar or dif-
ferent monosaccharidic units are arranged as extremely complex molecular struc-
tures with the variations in sequences, linkages, branching patterns and distributions
of side chains. In addition, the molecular structural features of plant polysaccharides
possess the presence of many functional groups, which can be modified or tailored
to produce polysaccharides of desirable quality (Nayak and Pal 2018; Nayak et al.
2018b). During past few decades, an extensive volume of research efforts have been
directed to use various plant polysaccharides in many biomedical and medical
applications including their pharmaceutical uses as the dosage formulation excip-
ients and dosage performance enhancers in terms of desired pattern of drug
releasing, improved drug stability, enhanced bioavailability, desired target speci-
ficity, etc. (Nayak and Hasnain 2019a; b; Pal and Nayak 2017).
Various useful plant polysaccharides have already been explored and exploited
as biopolymeric agents in many healthcare area including pharmaceutical industry
and researches. Some of the widely used plant polysaccharides as efficient excip-
ients in various kinds of pharmaceutical dosage forms are gum Arabic (Nayak and
Hasnain 2019c), gum tragacanth (Dhupal et al. 2019), pectin (Nayak and Pal 2016),
guar gum (Jana et al. 2019), locust bean gum (Hasnain et al. 2019b; Nayak and
Hasnain 2019d), sterculia gum (Bera et al. 2019; Nayak and Hasnain 2019e),
tamarind gum (Dey et al. 2019; Nayak, 2016; Nayak and Hasnain 2019f), cashew
gum (Nayak et al. 2019), okra gum (Nayak and Hasnain 2019g; Nayak et al.
2018b), gum odina (Samanta et al. 2019), fenugreek seed mucilage (Nayak and
Hasnain 2019h; Pal et al. 2019b), linseed polysaccharides (Nayak and Hasnain
2019i), ispaghula mucilages (Guru et al. 2018), plant starches (Nayak and Pal
3 Plant Polysaccharides in Pharmaceutical Applications 95
2017a), etc. Important plant starches used as pharmaceutical excipients are rice
starch, potato starch, maize starch, jackfruit seed starch, sago starch, etc. (Nayak
and Hasnain 2019i; Nayak and Pal 2017a). The current chapter presents a brief
review on the pharmaceutical applications of various plant polysaccharides.
3.2 Classifications and Sources of Plant Polysaccharides
Plant polysaccharides possess a wider ranging of structurally diversified

biomacromolecules with different useful physicochemical properties (Nayak et al.
2015). These useful biomacromolecules are occurred abundantly in the plant-
resources in forms of gums, mucilages and starches (Avachat et al. 2011; Prajapati
et al. 2013).
3.2.1 Plant Gums
Plant gums are commonly known as useful class of polysaccharides comprising

multiple numbers of sugar units, which are linked together (Nayak and Pal 2016).
Upon the hydrolysis process, these yield different sugar units, such as glucose,
galactose, arabinose, xylose, mannose or uronic acids, etc., which are occurred in
the respective plant gums (Prajapati et al. 2013; Nayak and Pal 2016; Pal and Nayak
2017). This type of plant polysaccharides are commonly occurred in various parts
of higher plants, such as leaves, pods, fruits, seeds, cereals, stems, roots, rhizomes,
corms, exudates, etc., on account of their own defense mechanisms following
natural injury or harvesting via the break-down of cell walls (Prajapati et al. 2013).
This gum generation process in plants is commonly known as gummosis (Rana
et al. 2011). Plant gums are mainly pathologically generated products containing
complex substances, commonly known as polyuronides. These gums are also
capable of producing 3-dimensional gel networks like other gums derived from
animal as well as microbiological resources (Rana et al. 2011).
The sources of some important plant gums, which have already been studied for
pharmaceutical applications, are listed in Table 3.1.
3.2.2 Plant Mucilages
Mucilages originate in the plants either as a part of contents of the cell or as a part of
the wall thereof (Malviya et al. 2011). These serve as food reserve and membrane
thickener and aid in water storage and seed germination (Pal and Nayak 2017).
Chemically, the molecular structural feature of mucilages possess complex structure
of polysaccharides containing uronic acid and sugar residues. Principally, plant
Table 3.1 Sources of some important plant gums for pharmaceutical applications
Plant gums Common sources
Scientific name of the plant Family
Gum Arabica/gum acacia Acacia Arabica, Leguminoseae
Acacia Senegal
Gum tragacanth Astragalus gummifer Leguminosae
Locust bean gum Ceratonia siliqua Fabaceae
Guar gum Cyamompsis tetraganolobus Leguminoseae
Okra gum Hibiscus esculantus Malvaceae
Tamarind gum Tamarindus indica Leguminoseae
Sterculia (karaya) gum Sterculia urens Sterculiaceae
Gum kondagogu Cochlospermum gossypium Colchospermaceae
Gum odina Lannea woodier Anacardiaceae
Gum cordia Cordia obliqua Boraginaceae
Moringa gum Moringa oleifera Moringaceae
Albizia gum Albizia procera Leguminoseae
Khaya gum Khaya grandifoliola Meliaceae
Terminalia gum Terminalia randii Combretaceae
Gum ghatti Anogeissus latifolia Combretaceae
Honey locust gum Gleditsia triacanthos Leguminosea
Abelmoschus gum Abelmoschus esculantus Malavaceae
Gum copal Bursera bipinnata Burseraceae
Gum dammar Shorea Wiesneri Dipterocarpaceae
Tara gum Caesalpinia spinosa Leguminosae
Moi gum Lannea coromandelica Anacardiaceae
Bahera gum Terminalia bellerica roxb Combretaceae
Hakea gum Hakea gibbosa Proteaceae
Leucaena gum Leucaena leucocephata Fabaceae
Grewia gum Grewia mollis Malvaceae
Balangu gum Lallemantia royleana Labiatae
Dillenia fruit gum Dillenia indica L Dilleniaceae
Cashew tree gum Anacardium occidentale Anacardiaceae
mucilages are the sulphuric acid esters (Malviya et al. 2011). Due to the high
concentration of hydroxyl groups in their structures, these have high water binding
capacity and this has led to studies of their role in plant water relations (Nayak and
Pal 2016).
The sources of some important plant mucilages, which have already been studied
for pharmaceutical applications, are listed in Table 3.2.
Table 3.2 Sources of some important plant mucilages for pharmaceutical applications
Plant mucilages Common sources
Scientific name of the Family
plant
Ispaghula mucilage Plantago ovata Plantaginaceae
Fenugreek seed mucilage Trigonella Fabaceae
foenum-graecum
Aloe mucilage Aloe barbadensis Liliaceae
Flaxseed mucilage Linum usitatissimum Linaceae
Mimosa pudica seed mucilage Mimosa pudica Mimosaceae
Spinacia oleraceae leaves mucilage Spinacia oleraceae Amaranthaceae
Basella alba leaves and stem mucilages Basella alba Basellaceae
Prosopis juliflora seed mucilage Prosopis juliflora Fabaceae
Hibiscus rosasinensis L. leaves mucilages Hibiscus rosasinensis Malvaceae
Basil seed mucilage Ocimum basilicum Lamiaceae
Clove basil seed mucilage Ocimum gratissimum Lamiaceae
Amaranthus viridis L. (Leotia) leaves Amaranthus viridis Amaranthaceae
mucilage
Yellow mustard seed mucilage Sinapis alba Brassicaceae,
Lepidium perfoliatum seed. mucilage Lepidium perfoliatum Brassicaceae
3.2.3 Plant Starches
Starches are the widely studied storage carbohydrates. The molecular structure of
starches comprises of 2 co-polymers: amylose (20–30%) and amylopectin (70–
80%) (Nayak and Pal 2012). Amylose is essentially linear with the glucose units
bound together through a-(1, 4)-linkages and very few a-(1, 6)-bonds. Amylose
chains have a degree of polymerization up to 6,000 dependent on source and a
molecular mass of 105–106 g/mol (Nayak and Pal 2017a). Amylopectin has a
molecular mass of 107–109 g/mol. It is highly branched with a-(1, 6)-bonds in the
branching points and has an average degree of polymerization of 2 million, making
it one of the largest molecules in nature (Malakar et al. 2013a; Nayak and Pal
2017a). Many starches are also being extracted from different plant resources as
these are occurred in various parts of these plant resources, such as seeds, cereals,
rhizomes, roots, tubers, corms, etc. (Builders and Arhewoh 2016). Starches are
exists as microscopic granules with characteristically origin-specific shapes and
sizes (Nayak and Pal 2017a).
The sources of some important plant starches, which have already been studied
for pharmaceutical applications, are listed in Table 3.3.
Table 3.3 Sources of some important plant starches for pharmaceutical applications
Plant starches Common sources
Scientific name of the plant Family
Rice starch Oryza sativa Poaceae
Potato starch Solanum tuberosum Solanaceae
Sweet potato starch Ipomoea batatas Convolvulaceae
Maize starch Zea mays Poaceae
Tapioca starch Manihot esculenta Euphorbiaceae
Sago starch Metroxylon sagu Palmae
Cocoyam starch or Xanthosoma sagittifolium Araceae
Malanga starch
Jackfruit seed starch Artocarpus heterophyllus Moraceae
Sorghum starch Sorghum bicolor Poaceae
Dioscorea starch Dioscorea dumetorum, Dioscorea Dioscoreaceae
oppositifolia, Dioscorea alata,
Dioscorea rotundata, etc
Arrowroot starch Maranta arundinacea Marantaceae
Ginger starch Zingiber officinale Zingiberaceae
Indian palo rhizome Curcuma angustifolia Roxb Zingiberaceae
starch
Tiger nut starch Cyperus esculentus Cyperaceae
3.3 Applications of Plant Polysaccharides

in Pharmaceutical Dosage Forms
Various plant polysaccharides have extensively been utilized as thickeners, sus-

pending agents, emulsifiers, stabilizers, gel forming agents, binders, disintegrating
agents, matrix formers, release retardants, film formers, coating materials, mucoad-
hesive agents, etc., in a variety of common pharmaceutical dosage forms, such as
suspensions, emulsions, gels, tablets, capsules, beads, microparticles, nanoparticles,
liposomes, transdermal formulations, buccal formulations, nasal formulations, oph-
thalmic formulations, etc. (Avachat et al. 2011; Nayak and Hasnain 2019a; Nayak and
Pal 2017a, b; Nayak et al. 2018c; Prajapati et al. 2013; Rana et al. 2011).
3.3.1 Emulsions
Plant polysaccharides are used as emulsifiers in various emulsions. These materials

can efficiently stabilize the emulsion via the interfacial absorption and the mini-
mizations of droplet-coalescence. Porto and Cristianini (2014) investigated the
emulsifying property of cashew (Anacardium occidentale L.) gum and compared its
emulsifying property with that of the gum Arabic. Emulsions prepared using gum
Arabic were found more uniform than the emulsions prepared using cashew gum.
The smaller oil droplets were formed in the emulsions prepared using gum Arabic.
Figure 3.1a presents the microscopic observations of emulsions prepared using both
emulsifiers (gum Arabic and cashew gum). Figure 3.1b presents a confocal
microscopy image of emulsions prepared using both emulsifiers (gum Arabic and
cashew gum). From the results, a prominent flocculation was noticed in the
emulsions prepared using gum Arabic as emulsifier in comparison with emulsions
prepared using cashew gum. This flocculation behavior of emulsions was found to
be directly influenced the emulsion instability. In a research, gum odina has been
studied as an emulsifier in the preparation of primary emulsion, where gum odina at
low concentration produced more stable primary emulsion than that of gum acacia
(Samanta et al. 2010). In another research, the same research group studied the
efficacy of gum odina as a stabilizer for the formulation of W/O/W multiple
emulsions of lamivudine (Jena et al. 2018). The in vitro lamivudine releasing from
these W/O/W multiple emulsions stabilized by gum odina produced more sustained
lamivudine release over 6 h, which was comparable that of the W/O/W multiple
emulsions stabilized by Tween 80. Verma and Razdan (2003) studied the use of
leucaena gum (extracted from the seeds of Leucaena leucocephala) as emulsifier
via the preparation of 30% liquid paraffin emulsions (o/w) employing 1–4% w/v
leucaena seed gum. The emulsions prepared using leucaena seed gum as emulsifier
were compared with the emulsions prepared using another plant-derived gum, gum
acacia. The results demonstrated enhanced emulsifying property of leucaena seed
gum as emulsifier in comparison with that of gum acacia in liquid paraffin emul-
sions. Lago et al. (2019) prepared o/w nanoemulsions using mucilage extracted
from Pereskia aculeata Miller leaves via the ultra-sound assisted technique. These
Fig. 3.1 a Optical microscopy images of emulsions prepared with both gum Arabic and cashew
gum after 24 h of quiescent storage. Horizontal bar displayed on far right-bottom of figure
corresponds to 1 mm extent. b Confocal scanning laser microscopy images of o/w emulsions
prepared with gum Arabic and cashew gum. Red: emulsifier; green: D-limonene; black: water.
Horizontal bar displayed on far right-bottom of figure corresponds to 20 mm extent. (Porto and
Cristianini 2014; Copyright @ 2014, with permission from Elsevier Ltd.)
o/w nanoemulsions exhibited good stability when 1–1.5% Pereskia aculeata Miller
leave mucilage was used as emulsifier. In a research, Gemede et al. (2018) studied
the emulsifying property of Ethiopian okra (Abelmoschus esculentus) pod mucilage
and the prepared emulsions showed good stability. Avlani et al. (2019) explored the
emulsifying property of sweet basil (Ocimum basilicum L.) seed mucilage to for-
mulate surfactant-free stable sunflower oil emulsions. The sunflower oil emulsions
stabilized by 0.3–0.5% w/v sweet basil seed mucilage exhibited good stability.
Khunkitti et al. (2006) evaluated the emulsifying property of jackfruit seed starch
(1–5% w/v). Although the jackfruit seed starch was found to thicken the external
phase of the emulsions prepared, it demonstrated poor emulsifying property. Zhao
et al. (2017) formulated and characterized o/w soybean oil emulsions using and
gelatinized kudzu starch as emulsifier. 10% (w/w) soybean oil prepared using 3%
(w/w) gelatinized kudzu starch showed comparatively good stability.
3.3.2 Suspensions
As suspending agents, numerous plant polysaccharides are being investigated and

successfully used in various pharmaceutical suspensions. In suspensions, plant
polysaccharides augment the tensile strength of the hydration layer produced
around the suspended particles, via hydrogen bonding as well as molecular inter-
actions. These materials are generally hydrophilic colloids and capable of forming
aqueous dispersions to enhance the viscosity of continuous phase, in order that the
solid particles remain suspended in it over a longer period. In a research, Ogaji and
Hoang (2011) prepared ibuprofen pediatric oral suspensions using 0.5% w/v grewia
gum as suspending agent. The activity of grewia gum as suspending agent was
compared to semi-synthetic conventional suspending agents, namely hydroxyl-
propyl methylcellulose and sodium carboxymethylcellulose. The grewia gum
showed better redispersion property with minimal alterations in viscosity on storage
in comparison with that of hydroxylpropyl methylcellulose and sodium car-
boxymethylcellulose as suspending agent. Thus, the results demonstrated that
grewia gum may serve as a good suspending agent. In a research, Ogaji (2011)
found the excellent performance of okra gums extracted by different procedures,
when used as suspending agents in the formulations of acetaminophen pediatric
suspensions. Rao et al. (2005) investigated the use of moringa gum (extracted from
the exudate material of Moringa oleifera plant) as a suspending agent in sul-
phamethoxazole suspension and they found the prospective results. Avlani et al.
(2019) explored the use of sweet basil (Ocimum basilicum L.) seed mucilage to
formulate adult and pediatric paracetamol suspensions. The formulated suspensions
prepared using 1% w/v sweet basil seed mucilage showed flocculated property with
enhanced stability because of the high sedimentation volume as well as good
redispersibility. In a research, Pal et al. (2010) evaluated the suitability of Basella
alba L. leaves mucilage as suspending agent in zinc oxide (20% w/v) suspensions.
The Basella alba L. leaves mucilage as suspending agent showed better results than
that of both gum tragacanth and bentonite. Even, zinc oxide suspensions prepared
using Basella alba L. leaves mucilage as suspending agent were found easy
redispersible. Nayak et al. (2010) assessed the suitability of spinach (Spinacia
oleracea L.) leaves as suspending agent in zinc oxide (20% w/v) suspensions. The
spinach (Spinacia oleracea L.) leaves mucilage as suspending agent showed better
degree of flocculation and redispersibilty than those of both gum tragacanth and
bentonite. The same research group also studied the suitability of fenugreek
(Trigonella foenum-graecum L.) seed mucilage as suspending agent in zinc oxide
(20% w/v) suspensions (Nayak et al. 2012). The fenugreek seed mucilage per-
formed as better suspending agent exhibiting better degree of flocculation and
redispersibilty than those of gum tragacanth, gum acacia and bentonite.
Piriyaprasarth et al. (2010) studied the uses of arrowroot (Maranta arundinacea)
starch and yam (Dioscorea sp.) starch as suspending agents in paracetamol sus-
pension. From the results of the study, it was noticed that the optimal concentrations
of arrowroot starch was 5–6% and yam starch was 7–8% as suspending agents in
paracetamol suspensions. Khunkitti et al. (2006) evaluated the use of jackfruit seed
starch as suspending agent and jackfruit seed starch (1–5% w/v) was found able to
flocculate titanium dioxide suspensions.
3.3.3 Tablets
Since long different plant polysaccharides are being extensively used in many
pharmaceutical tablets as binders, disintegrating agents, matrix formers, and release
retardants. The excellent binding property of these materials is due to their adhesive
characteristics, which impart cohesiveness to the powdered mass to prepare gran-
ules, which are used for further compression in the preparations of tablets. Plant
polysaccharides are also employed as disintegrating agents in many tablets
(Prajapati et al. 2013). The disintegrating property of plant polysaccharides is
attributable to their capability to absorb water and swelling. Plant polysaccharides
are also being employed as matrix formers, and release retardants in many sustained
drug releasing tablets, especially in matrix tablets. Because of the hydrophilic and
high swelling ability, when the matrix tablets containing plant polysaccharides
come in contact with water, these get highly hydrated and produce viscous gels onto
the surface of the tablets, which produce sustained drug releasing over a longer
period.
In a study, Menon et al. (2011) studied the application of orange peel pectin as
tablet binder in ibuprofen tablets and they found tablets prepared using 30 mg
orange peel pectin exhibited better friability and disintegrating time. Even, these
tablets (prepared using 30 mg orange peel pectin as binder) exhibited 82%
ibuprofen releasing, in vitro, and were comparable to that of the same amount of
starch. In another study, Srivastava et al. (2010) also found the excellent tablet
binding property of orange peel pectin in the formulations of paracetamol tablets.
Jena et al. (2014) studied the usefulness of gum odina as tablet binder to prepare
paracetamol tablets. The in vitro dissolution results showed that 98.55% parac-
etamol releasing within 30 min from the paracetamol tablets prepared using 0.125%
gum odina as binder. However, the in vitro paracetamol releasing was slowed from
the paracetamol tablets prepared using 0.25 and 0.375% gum odina. Gum odina
was also studied as controlled release matrix former for the preparations of tol-
terodine tartarate and pioglitazone HCl matrix tablets (Sinha et al. 2011). Both the
tolterodine tartarate and pioglitazone HCl matrix tablets exhibited a controlled drug
releasing over a prolonged period (Fig. 3.2). Pachuau and Mazumdar (2012)
assessed the efficacy of Albizia procera gum as release retardant excipients in
matrix tablets. They formulated paracetamol tablets using Albizia procera gum and
observed a controlled sustained releasing of drug from these matrix tablets over12
h. Ofori-Kwakye et al. (2016) prepared matrix tablets of diclofenac sodium and
metformin HCl by direct compression using cashew gum, xanthan gum and
hydroxypropyl methylcellulose as release retardants. These matrix tablets exhibited
extended release of drugs over a longer period. Hasnain et al. (2017a) used cashew
Fig. 3.2 a In vitro mean cumulative percentage release of pioglitazone HCl from matrices
containing various proportions of gum odina. b In vitro mean cumulative% release of tolterodine
tartarate from matrices containing various proportions of gum odina. Each point is the mean
value of three samples (n = 3) (Sinha et al. 2011; Copyright @ 2010, with permission from
Elsevier Ltd.)
gum with hydroxypropylmethylcellulose to prepare matrix tablets of hydralazine

HCl. These matrix tablets showed a prolonged sustained drug releasing along with
good floating as well as bioadhesive behaviors. Kaleemullah et al. (2017) evaluated
the use of Hibiscus rosa-sinensis leaves mucilage as matrix former and release
retardant excipients in the formulations of sustained release ketoprofen matrix
tablets. The in vitro dissolution studies of formulated ketoprofen matrix tablets
exhibited sustained ketoprofen releasing up to 24 h. Gaikar and Sandhya (2012)
evaluated the tablet binding properties of Aegle marmelos fruit mucilage at different
concentrations in the formulations of paracetamol tablets. Aegle marmelos fruit
mucilage of 3% w/w was found to produce comparable results with starch paste of
10% w/w as tablet binder. Ahuja et al. (2013a) studied tablet binding and disin-
tegrant properties of Mimosa pudica seed mucilage. The tablet binding property
was assessed via the preparation of the paracetamol tablets using Mimosa pudica
seed mucilage (6, 8 and 10% w/w) as tablet binder and compared with the standard
binders (polyvinyl pyrrolidone K25 and gum acacia). Mimosa pudica seed muci-
lage at 10% (w/w) concentration produced tablets possessing adequate hardness as
well as friability. The tablet disintegrant property was assessed via the preparation
of directly compressed hydrochlorothiazide tablets using Mimosa pudica seed
mucilage of 1–10% (w/w) as disintegrant and compared with the standard disin-
tegrants (Ac-Di-Sol and starch). The Mimosa pudica seed mucilage at 3% (w/w)
concentration showed better disintegrant property of the hydrochlorothiazide
tablets. In a research, Okunlola and Odeku (2011) evaluated tablet binding prop-
erties of starches from four Dioscorea species, namely Dioscorea alata, Dioscorea
dumetorum, Dioscorea rotundata, Dioscorea oppositifolia, etc., in chloroquine
phosphate tablet. The results of the research suggested that starches of Dioscorea
alata, and Dioscorea rotundata could be functional for faster disintegration. On the
other hand, starches of Dioscorea oppositifolia and Dioscorea dumetorum could be
useful for the minimizations the tablet defects like capping and lamination in tablet
formulations. Manek et al. (2012) employed the starch extracted from Cyperus
esculentus as binder in formulation of metronidazole tablets. Metronidazole tablets
prepared using 5, 7.5 and 10% Cyperus esculentus starch were compared with
tablets prepared using 10% potato starch. Metronidazole tablets prepared using 10%
Cyperus esculentus starch showed better tablet binding property than others. In a
research, Builders et al. (2013) studied the efficacy of tiger nut starch as direct
compression excipient in the formulations of acetylsalicylic acid tablets and
reported the potential of tiger nut starch as direct compression excipient for the
compressible tablets along with enhanced flow properties and improved disinte-
gration. Recently, Peerapattana et al. (2020) evaluated the potential of spray-dried
glutinous rice starch as direct compression excipient to prepare hydrophilic matrix
tablets of propranolol. The content of spray-dried glutinous rice starch was found to
be influenced the propranolol releasing rate from the matrix tablets, significantly.
3.3.4 Capsules
Plant polysaccharides are also investigated in the formulations of capsules, where

these are used to prepare capsule shells, diluents and capsule matrices. Misale et al.
(2008) studied sago starch in the preparation capsule shell as because of its film
forming ability. The results of the study indicated that sago starch made capsule
shells were moderately comparable to that of gelatin made capsule shells. The
in vitro drug releasing rate using sago starch capsule shells exhibited a slower and
sustained drug releasing as compared to that of gelatin capsule shells.
3.3.5 Beads
Recent years, many plant polysaccharides have been used to formulate polymeric
beads loaded with a variety of drugs and the uses of plant polysaccharides to
formulate beads is mainly due to the matrix forming and release retarding properties
(Nayak and Hasnain 2019k). Most of these polymeric beads made up of plant
polysaccharides are intended for oral administrations. Sometimes, mucoadhesive
plant polymers are being used to formulate bioadhesive beads, which have been
proved useful by facilitating better drug delivery (Nayak and Pal 2016, 2017a, b;
Pal and Nayak 2017). In a work, Bera et al. (2015a) developed interpenetrating
polymer network beads of ziprasidone HCl employing two plant polysaccharides,
namely pectin and sterculia gum via the simultaneous ionotropic crosslinking by
zinc acetate and covalent crosslinking by glutaraldehyde. These zinc pectinate-
sterculia gum beads were of spherically shaped with characteristic large wrinkles
and cracks on the bead surface (Fig. 3.3). These beads showed with controlled drug
release over 8 h (Fig. 3.4) with excellent buoyancy and excellent mucoadhesivity
onto the goat gastric mucosal membrane. Guru et al. (2018) prepared ispaghula
Fig. 3.3 Scanning electron microphotograph of zinc pectinate-sterculia gum interpenetrating

polymer network beads of ziprasidone HCl showing rough surface: a 75 and b 900 (Bera
et al. 2015a; Copyright @ 2015, with permission from Elsevier Ltd.)
Fig. 3.4 In vitro drug release from zinc pectinate-sterculia gum interpenetrating polymer network
beads of ziprasidone HCl (Bera et al. 2015a; Copyright @ 2015, with permission from Elsevier
Ltd.)
husk mucilage-zinc pectinate beads for controlled delivery of aceclofenac. These

beads showed a controlled aceclofenac releasing pattern over 10 h with favorable
pH dependent swelling. Nayak et al. (2014a; b) developed metformin HCl releasing
calcium pectinate-ispagula husk mucilage mucoadhesive beads and calcium
pectinate-tamarind seed polysaccharide mucoadhesive beads. Both the mucoadhe-
sive bead formulations showed controlled in vitro drug releasing with good ex vivo
mucoadhesion and in vivo antidiabetic activity in diabetic rats. The same group also
developed similar kinds of calcium pectinate-fenugreek seed mucilage mucoadhe-
sive beads for oral delivery of metformin HCl (Nayak et al. 2013a). These calcium
pectinate-fenugreek (Trigonella foenum-graecum L.) seed mucilage mucoadhesive
beads of metformin HCl were of spherical in shape with rough surface morphology
(Fig. 3.5) and exhibited controlled in vitro drug releasing over 10 h (Fig. 3.6). In
addition, these mucoadhesive beads showed significant antidiabetic action in
alloxan-induced diabetic rats, in vivo (Fig. 3.7). In another research, an almost
similar result of in vivo antidiabetic action in alloxan-induced diabetic rats was
noticed by the fenugreek seed mucilage-calcium alginate beads of metformin HCl
(Fig. 3.8) (Nayak et al. 2013b). The same research group developed metformin HCl
releasing ispaghula husk mucilage-gellan gum mucoadhesive beads for oral
administration (Nayak et al. 2014c). These ispaghula husk mucilage-gellan gum
mucoadhesive beads exhibited controlled in vitro release of metformin HCl with
good ex vivo bioadhesion. They also developed similar kinds of controlled
releasing mucoadhesive beads of metformin HCl using polymeric blends of
tamarind seed polysaccharide-gellan gum (Nayak et al. 2014b), fenugreek seed
mucilage-gellan gum (Nayak and Pal 2014) and jackfruit (Artocarpus heterophyllus
L.) seed starch-gellan gum (Nayak et al. 2014e). Tamarind seed
polysaccharide-calcium alginate beads of metformin HCl were formulated for the
use in oral administration (Nayak et al. 2016; Nayak and Pal 2013a). In these
biopolymeric beads, tamarind seed polysaccharide was employed as release retar-
dant and bioadhesive polymeric excipients. These beads showed a prolonged
Fig. 3.5 Scanning electron microphotograph of the optimized calcium pectinate-fenugreek

(Trigonella foenum-graecum L.) seed mucilage mucoadhesive beads of metformin HCl (Nayak
et al. 2013a; Copyright @ 2013, with permission from Elsevier Ltd.)
Fig. 3.6 In vitro drug release from various calcium pectinate-fenugreek (Trigonella
foenum-graecum L.) seed mucilage mucoadhesive beads of metformin HCl [mean ± S.D.,
n = 3] (Nayak et al. 2013a; Copyright @ 2013, with permission from Elsevier Ltd.)
metformin HCl release over 10 h, in vitro. Even these beads exhibited excellent
bioadhesion onto goat intestinal mucosa, ex vivo and significant antidiabetic action
in alloxan-induced diabetic rats, in vivo. Sinha et al. (2015a) used okra gum as
release retardant polymeric blend with alginate to prepare zinc alginate-okra gum
beads for sustained release of diclofenac sodium (over 8 h). The same research
group, in another research, formulated okra gum-calcium alginate mucoadhesive
beads for controlled releasing glibenclamide (over 8 h) and these beads showed
excellent bioadhesion onto goat intestinal mucosa, ex vivo (Sinha et al. 2015b).
Hasnain et al. (2018a) prepared mucoadhesive beads of diclofenac sodium using
Linum usitatisimum mucilage with sodium alginate. In these beads, Linum
Fig. 3.7 a Comparative in vivo blood glucose level in alloxan-induced diabetic rats after oral
administration of pure metformin HCl and optimized calcium pectinate-fenugreek (Trigonella
foenum-graecum L.) seed mucilage mucoadhesive beads of metformin HCl.The data were
analyzed for significant differences (*p < 0.05) by paired samples t-test, and b comparative in vivo
mean percentage reduction in blood glucose level in alloxan-induced diabetic rats after oral
administration of pure metformin HCl and optimized calcium pectinate-fenugreek seed mucilage
mucoadhesive beads of metformin HCl (Nayak et al. 2013a; Copyright @ 2013, with permission
from Elsevier Ltd.)
Fig. 3.8 a Comparative in vivo blood glucose level in alloxan-induced diabetic rats after oral
administration of pure metformin HCl and fenugreek seed mucilage-calcium alginate beads of
metformin HCl. The data were analyzed for significant differences (p < 0.05) by paired samples
t-test. b Comparative in vivo mean percentage reduction in blood glucose level in alloxan-induced
diabetic rats after oral administration of pure metformin HCl and fenugreek seed mucilage-calcium
alginate beads of metformin HCl (Nayak et al. 2013b; Copyright @ 2012, with permission from
Elsevier B.V.)
usitatisimum mucilage was used as matrix former, release retardant and mucoad-
hesive agents. These beads exhibited prolonged in vitro drug releasing with
excellent ex vivo biomucoadhesion. The use of jackfruit seed starch-low methoxy
pectin blends (Nayak and Pal 2013b) and jackfruit seed starch-sodium alginate
blends (Nayak and Pal 2013c) to formulate two different kinds of mucoadhesive
beads of metformin HCl by ionotropic gelation was also studied and reported.
These beads showed a prolonged metformin HCl release over 10 h, in vitro and
significant antidiabetic effects, in vivo. In another research, the applicability of
jackfruit seed starch-sodium alginate blends for the preparation of controlled drug
releasing was investigated using pioglitazone as a model drug (Nayak et al. 2013c).
Malakar et al. (2013b) studied the efficacy of potato starch used as release retardant
blends with sodium alginate to formulate potato starch-alginate beads of tolbu-
tamide and these beads showed a controlled tolbutamide releasing pattern, in vitro.
Even various plant polysaccharides have been used to formulate buoyant beads for
the uses in floating drug delivery. Bera et al. (2015b) developed alginate-sterculia
gum gel-coated oil-entrapped calcium alginate beads of resperidone for gas-
trorentive floating drug delivery. In this work, sterculia gum coat was applied for its
bioadhesive nature and these alginate-sterculia gum gel-coated buoyant bioadhesive
beads exhibited prolonged release resperidone over 8 h in gastric pH medium.
Scanning electron microphotographs exhibited rough surface morphology of the
uncoated oil-entrapped calcium alginate beads of resperidone; whereas in case of
the alginate-sterculia gum gel-coated oil-entrapped calcium alginate beads of res-
peridone, comparatively smooth surface morphology was noticed. The cross-
sectional view of the alginate-sterculia gum gel-coated oil-entrapped calcium
alginate beads of resperidone exhibited a sponge like structural morphology, in
which the oil was entrapped with the beads. In another work, the same research
group developed risperidone-loaded alginate gel-coated oil-entrapped alginate–ta-
marind gum–magnesium stearate buoyant beads for gastrorentive floating drug
delivery (Bera et al. 2015c). The use of tamarind gum in these beads imparted
sustained release and bioadhesive behavior. In a work, emulsion-gelled groundnut
oil-entrapped buoyant beads of diclofenac sodium were developed using sodium
alginate and tamarind seed polysaccharide-blends (Nayak et al. 2013d). These
groundnut oil-entrapped buoyant beads showed sustained drug releasing and
excellent floating pattern, in vitro. Guru et al. (2013) also formulated oil-entrapped
beads of aceclofenac using sterculia gum-sodium alginate blends. These beads
exhibited excellent floating behavior with prolonged sustained release of encap-
sulated aceclofenac.
3.3.6 Microparticles
Since past few years, many plant polysaccharides have already been exploited for
the formulation of microparticles to deliver numerous drugs due to the matrix
forming and release retarding properties of plant polysaccharides. Pal and Nayak
(2012) formulated gliclazide-loaded tamarind seed polysaccharide-calcium alginate
mucoadhesive microspheres for oral adminstration. In these biopolymeric
mucoadhesive microspheres, tamarind seed polysaccharide was employed as
release retardant and bioadhesive polymeric excipients. These beads showed a
prolonged metformin HCl release in vitro and significant antidiabetic activity in

alloxan induced diabetic rats, in vivo. Das et al. (2014) developed alginate-based
microbeads encapsulated with isoxsuprine HCl using carboxymethyl cashew gum.
The scanning electron micrographs of these microbeads demonstrated that these
microbeads were of spherically shaped and no agglomeration of particles was
noticed. The micrographs also exhibited a rough surface morphology (Fig. 3.9).
These microbeads of isoxsuprine HCl showed a prolonged drug releasing pattern
(Fig. 3.10). Jana et al. (2013) assessed the use of tamarind seed polysaccharide to
prepare chitosan-based interpenetrating polymeric network microparticles of ace-
clofenac. These microparticles exhibited sustained aceclofenac releasing over 8 h
and anti-inflammatory activity was noticed in the carrageenin-induced rats, in vivo,
after oral administration (Fig. 3.11) Mohanty et al. (2015) prepared microcapsules
of lornoxicam using two plant polysaccharides like Dillenia indica pectin and gum
dikamali. These formulated microcapsules exhibited sustained release of lornoxi-
cam over a longer period. Nayak et al. (2018d) developed starch-blended Ca2+-
Zn2+-alginate microparticles of aceclofenac. During in vitro release study at pH 1.2
(for initial 2 h), more than 20% aceclofenac was released from these beads and at
pH 7.4, it was found to produce sustained release of encapsulated aceclofenac over
7 h Jha and Bhattacharya (2008) investigated the usefulness of sweet potato starch
blends with sodium alginate to prepare microbeads for sustained releasing of
ibuprofen. These sweet potato starch-alginate microbeads of ibuprofen exhibited
sustained drug releasing. Sachan and Bhattyacharya (2009) studied the sustained
drug releasing matrix properties of Assam bora rice starch blends with sodium
Fig. 3.9 Scanning electron micrographs of the surface of optimized zinc alginate-carboxymethyl
cashew gum microbeads containing isoxsuprine HCl: a 75 , b 200 , c 1500 , d 2000 ,
e 4000 and f 10,000 (Das et al. 2014; Copyright @ 2014, with permission from Elsevier B.
V.)
Fig. 3.10 In vitro drug release from various zinc alginate-carboxymethyl cashew gum microbeads
containing isoxsuprine HCl [mean ± S.D., n = 3] (Das et al. 2014; Copyright @ 2014, with
permission from Elsevier B.V.)
Fig. 3.11 The percentages inhibition of paw oedema swelling in carageenan induced rat paw
oedema model for the standard and chitosan-based interpenetrating polymeric network micropar-
ticles of aceclofenac at various time intervals (Jana et al. 2013; Copyright @ 2013, with permission
from Elsevier B.V.)
alginate to prepare ionotropically-gelled microbeads of metformin HCl. These

Assam bora rice starch-alginate microbeads showed a sustained drug releasing
profile over a longer period.
3.3.7 Nanoparticles
The utilization of plant polysaccharides in the designing of nanoparticles is still in

its infancy and relatively few studies are reported where plant polysaccharides have
been investigated for the preparation of nanoparticles. Pitombeira et al. (2015)
prepared nanoparticles using acetylated cashew gum via the self-assembled tech-
nique by dialysis. These self-assembled cashew gum-based nanoparticles were
loaded with indomethacin. Indomethacin-loaded self-assembled cashew gum-based

nanoparticles were of spherically shaped as evidenced in scanning electron
microscopy and the particle size characterization demonstrated a unimodal distri-
bution of nanoparticles with an average size of 179 nm (Fig. 3.12). In vitro drug
release tests demonstrated a preliminary burst releasing of loaded indomethacin
from these cashew gum-based nanoparticles in the initial 2 h followed by a con-
trolled releasing pattern up to 72 h. Burapapadh et al. (2012) prepared pectin-based
nanoparticles for delivery of itraconazole via the nanoemulsion templates. The
nanoemulsion templates were formed by means of a high-pressure homogenization
process employing different types of pectins, such as high methoxyl pectin, low
methoxyl pectin, amidated low methoxyl pectin. The results of this investigation
indicated that nanoparticles prepared using high methoxyl pectin produced better
in vivo absorption than others. Soumya et al. (2010) synthesized the lipase func-
tionalized guar gum-based nanoparticles based via the nanoprecipitation and
cross-linking. They tested these formulated nanoparticles as the carrier for anti-
hypertensive drug. The drug release evaluation results demonstrated that the rate as
well as quantity of encapsulated drug releasing from the lipase functionalized guar
gum-based nanoparticles was elevated up to 24 h and subsequently, these were
found to be decreased. Sadrjavadi et al. (2018) prepared de-esterified gum
tragacanth-chitosan nanoparticles of methotrexate. These gum tragacanth-chitosan
nanoparticles of methotrexate were found to be endocytosed via the asialoglyco-
protein receptors with sustained release of encapsulated methotrexate for 9 days.
Tan et al. (2016) prepared gum Arabic-based nanoparticles via polyelectrolyte
complexation with chitosan for the delivery of curcumin. The curcumin loading and
curcumin encapsulation efficiency of gum Arabic-chitosan nanoparticles were 3.8
and 90%, respectively. These nanoparticles demonstrated a delayed releasing of
curcumin in the simulated gastrointestinal milieu, in vitro.
Fig. 3.12 Scanning electron microscopy and the particle size distribution of self-assembled
cashew gum-based nanoparticles without indimethacin (a) and with indomethacin (b) (Pitombeira
et al. 2015; Copyright @ 2014, with permission from Elsevier Ltd.)
3.3.8 Liposomes
Plant polysaccharides have also been used to coat liposomes for their better activity.
Haghighi et al. (2018) employed coating of pectin onto the nanoliposomes of
phloridzin to enhance their targeting property. The pectin-coated nanoliposomes of
phloridzin exhibited improved drug entrapment efficiency and storage stability. In
another research, Zhou et al. (2014) employed the coating of coated with high
methoxyl pectin or low methoxyl pectin onto vitamin C liposomes. These
pectin-coated vitamin C liposomes exhibited the enhanced the stability, especially
with the coating of high methoxyl pectin.
3.3.9 Transdermal Formulations
Plant polysaccharides have already been investigated to formulate different trans-

dermal gels and films due to gel forming and film forming properties, respectively.
Hadebe et al. (2014) formulated transdermal patches using amidated pectin for
delivery of insulin. The slow releasing insulin from these amidated pectin-based
transdermal patches improved various diabetic parameters in streptozotocin-
induced diabetic rats. Hasnain et al. ( 2020a) used dillenia (Dillenia indica L.)
fruit gum as gel-forming agent to prepare lidocaine HCl topical gels. In the ex vivo
permeation study, lidocaine HCl was permeated across the porcine ear skin
membrane from formulated 4% lidocaine HCl topical gels and the results showed a
sustained drug permeation profile over 7 h. The same research group also reported
the potential of the application of cashew gum as a potential gel forming material
with hydroxypropyl methylcellulose K4M to prepare 4% lidocaine HCl topical
gels, which exhibited good skin permeation (Hasnain et al. 2017b). In another
research, Das et al. (2013) formulated same type of 4% lidocaine HCl topical gels
using cashew gum with a synthetic polymer, Carbopol 940. The in vitro permeation
of lidocaine HCl from 4% lidocaine HCl gels through porcine skin was found to be
sustained over 7 h (Fig. 3.13). Panda et al. (2006) evaluated gel-forming ability of
moringa (Moringa oleifera) gum to prepare diclofenac gels. The gels prepared
using 8% moringa gum showed good results and found comparable to the marketed
formulation Mundhe et al. (2012) studied the gel-forming potential fenugreek seed
mucilage to prepare topical gels of diclofenac potassium. In another study, Rao
et al. (2010) assessed the Cocculas hirsutus leaf powder as gel base to prepare gel
of flurbiprofen. From the results of the research, it was found that the in vitro release
of flurbiprofen form from the formulated gels and in vivo anti-inflammatory activity
were better than that of the marketed gel compared. Nazim et al. (2011) developed
hydrotrope potato starch topical gels of rofecoxib. The in vitro rofecoxib releasing
results demonstrated that these 1% rofecoxib hydrotrope-gelled potato starch based
topical formulation containing 5% w/w potato starch and 15% w/w sodium sali-
cylate exhibited rofecoxib releasing of 16.65% within 6 h; while topical
Fig. 3.13 In vitro lidocaine HCl permeation profile through porcine skin per unit area from 4%
lidocaine HCl topical gels containing cashew gum and Carbopol 940 [mean ± S.D., n = 3] (Das
et al. 2013; Copyright @ 2013, with permission from Elsevier B.V.)
formulation containing 10% w/w potato starch and 15% w/w sodium salicylate
exhibited rofecoxib releasing of 16.39% within 6 h.
3.3.10 Buccal Formulations
Due to film forming properties and excellent bioadhesivity, various plant

polysaccharides have already been investigated to formulate different buccal drug
delivery systems including. Buccal tablets, buccal films and patches (Adhikari and
Panda 2017; Hasnain et al. 2020b). Gowthamarajan et al. (2012) prepared buccal
tablets of curcumin using cashew gum considering its mucoadhesive potential. The
buccal tablets of curcumin formulated using 20% cashew gum, 0.1% methol, 40 mg
ethylcellulose (as backing layer forming agent) with a compression force of 2 tons/
cm2 for 10 s was identified as an optimized buccoadhesive tablet formulations on
the basis of residence time and mucoadhesive strength. The buccal acceptance
evaluation of optimized buccoadhesive tablet formulations of curcumin is presented
in Fig. 3.14. The in vitro release of curcumin of these buccoadhesive tablets was
found to be dependent on the cashew gum concentration used in the formulations.
The in vitro release of curcumin from buccoadhesive tablets prepared using 20%
cashew gum and 20% hydroxypropyl methylcellulose was compared and the
in vitro curcumin release results demonstrated the faster release of curcumin from
the buccoadhesive tablets prepared using cashew gum after 8 h (Fig. 3.15).
Curcumin release results indicated that cashew gum could be used as a mucoad-
hesive polymeric excipient to formulate buccoadhesive tablets of curcumin.
Avachat et al. (2013) prepared tamarind seed glucan-based buccoadhesive buccal
Fig. 3.14 Comparative results of buccoadhesive tablet acceptance for curcumin buccal tablets
(Gowthamarajan et al. 2012; Copyright @ 2012, with permission from Elsevier Ltd.)
Fig. 3.15 The comparative in vitro release of curcumin from buccoadhesive tablets prepared
using 20% cashew gum and 20% hydroxypropyl methylcellulose (Gowthamarajan et al. 2012;
Copyright @ 2012, with permission from Elsevier Ltd.)
films for rizatriptan benzoate delivery. In these buccal films, tamarind seed
xyloglucan and Carbopol 934 P were used as mucoadhesive agents. The in vitro
rizatriptan benzoate permeation from these buccal films across the porcine buccal
mucosal membrane exhibited desirable flux over a prolonged time. Hasnain et al.
(2020b) investigated the application of dillenia fruit gum as mucoadhesive film
former excipient in the formulation of atenolol buccal patches to treat hypertension.
They formulated atenolol buccal patches composed of a mucoadhesive layer of
dillenia fruit gum-hydroxypropyl methylcellulose K4M and a backing layer
(drug-free) of 1% ethyl cellulose via solvent-casting method. The ex vivo atenolol
permeation across the porcine buccal mucosa showed the sustained atenolol
permeation over a period of 12 h along with excellent bioadhesion. Ahuja et al.
(2013b) evaluated the mucoadhesive property of gum cordia in the preparation of
buccal discs containing fluconazole. These gum cordia buccal discs showed good
ex vivo buccoadhesion onto the buccal mucosa and it was found to be significantly
affected by the compression pressure during preparation.The optimized buccal discs
was made by using gum cordia to lactose ratio of 0.66, fluconazole of 20 mg
compression pressure of 6600 kg, which showed ex vivo buccoadhesion of 22 h
and in vitro fluconazole release of 80% within 24 h. In a research, Mylangam et al.
(2016) prepared metoprolol succinate buccoadhesive tablets employing badam gum
as mucoadhesive agent by wet granulation technique. These showed good buc-
coadhesive retention profile. Adhikari and Panda (2017) assessed the mucoadhe-
sivity potential of fenugreek seed mucilage in the formulation of atenolol-releasing
buccal patches. They formulated atenolol buccal patches composed of a mucoad-
hesive layer of fenugreek seed mucilage-hydroxypropyl methylcellulose K4M and a
backing layer (drug-free) of 1% ethyl cellulose via solvent-casting method. The
sustained atenolol permeation across the porcine buccal mucosa over 12 h was
measured along with excellent boaddhesion. Nerkar and Gattani (2011) prepared
buccomucoadhesive microspheres of venlafaxine using linseed mucilage as
mucoadhesive excipient. They evaluated in vitro as well as in vivo performances of
these buccomucoadhesive microspheres. The results exhibited high encapsulation
of venlafaxine, higher swelling and good buccoadhesion.
3.3.11 Nasal Formulations
Plant polysaccharides have already been investigated to formulate different nasal

drug delivery systems due to gel-forming properties and excellent bioadhesivity.
Datta and Bandyopadhyay (2006) assessed the potential of gel-forming property
and mucoadhesive property of tamarind seed polysaccharide for the preparation of
diazepam nasal gels. The mucoadhesivity and gelling characteristics of tamarind
seed polysaccharide was found higher than that of the conventionally used synthetic
gel-forming agents, Carbopol 934 and hydroxypropyl methylcellulose. In vitro
diazepam releasing from these nasal gels was carried out by Franz-diffusion cell
using excised bovine nasal membrane, the results of which was found better than
the synthetic gel-forming agents. The same research group evaluated the potential
use of Linum usitatissimum L. seed mucilage in the formulation of midazolam
mucoadhesive nasal gels (Basu et al. 2007). In this work, Linum usitatissimum L.
seed mucilage was proved as better mucoadhesive agent than Carbopol 934 and
hydroxypropyl methylcellulose. The midazolam nasal gels prepared using Linum
usitatissimum L. seed mucilage demonstrated favorable mucoadhesive character-
istics, which facilitate to adhere onto the nasal mucosal surface over a prolonged
period. Thus, enhancement of drug absorption can be achieved when administered
via the intra-nasal route. Sahu et al. (2011) accessed the use of mucoadhesive agent
extracted from Dillenia fruits in the formulation of felodipine nasal gels. The nasal
gels exhibited controlled releasing of felodipine. In another work, Ketousetuo and
Bandyopadhyay (2007) also studied the preparation oxytocin nasal gel using
mucoadhesive agent extracted from Dillenia fruits.
3.3.12 Ophthalmic Formulations
Plant polysaccharides have already been investigated as potential excipient mate-

rials in various ocular drug delivery formulations like ophthalmic solutions, gels,
and nanoparticles (Dilbaghi et al. 2013; Gheraldi et al. 2004). The high viscosity as
well as mucoadhesive characteristics of plant polysaccharides makes these excellent
excipients in various ocular formulations for enhancing the ocular residence time.
Suzuki and Lim (1994) studied the use of locust bean gum in ocular drug delivery
system. They prepared gentamicin-loaded locust bean gum/i-carrageenan
microparticles by emulsification process, and these microparticles were further
incorporated in the poly(vinyl alcohol) gel. The ocular formulations without locust
bean gum exhibited an initial burst releasing of gentamicin within the initial 6 h,
which was found to decrease by more than 50% by the incorporation of 10% locust
bean gum in the microparticle formula. Gheraldi et al. (2000) evaluated the
mucoadhesive potential of tamarind gum for the ocular administration of gentam-
icin and ofloxacin. In this study, the concentrations of gentamicin and ofloxacin
were found significantly high in the aqueous humor and cornea in the rabbit eye,
when treated with tamarind gum-based ocular formulations containing tamarind
gum than that of the free drugs. The same research group also investigated the
potential of tamarind gum as mucoadhesive polymer for the improvement of
intra-ocular penetration of rufloxacin to treat bacterial keratitis (Gheraldi et al.
2004). Mehra et al. (2010) found the improvement of miotic activity of pilocarpine
by the treatment with a tamarind gum based in situ ocular gels, which was found to
produce sustained release of pilocarpine up to 12 h Dilbaghi et al. (2013) prepared
tamarind seed xyloglucan nanoaggregates loaded with tropicamide for ocular
delivery. In this work, they evaluated the ex vivo corneal permeation of tropicamide
across the goat cornea and the results demonstrated a significantly increased ex vivo
corneal permeation of tropicamide in comparison with that of the marketed
formulation.
3.3.13 Colon-Targeting Formulations
Some plant polysaccharides have been studied for the development of biodegrad-
able carriers for colon targeting drug releasing. Vivekanandan et al. (2015) prepared
guar gum-based matrix tablets of budenoside via the wet granulation. The devel-
oped matrix tablets demonstrated 97.12 and 76.86% of budenoside release in rat
cecal medium and in the dissolution medium without cecal content, respectively.
Dodi et al. (2016) formulated rhodamine-B loaded carboxymethyl guar gum
nanoparticles via ionic gelation for colon delivery. These rhodamine-B loaded
nanoparticles (208 nm of average diameter) demonstrated a pH-responsive
rhodamine-B releasing in simulated gastrointestinal fluids. The MTT assay
results demonstrated nontoxicity of the carboxymethyl guar gum nanoparticles
prepared by crosslinking using trisodium trimetaphosphate (up to *0.3 mg/ml). In
a study, El-Gibaly, (2002) prepared orally administrable zinc pectinate micropar-
ticles for colonic delivery of ketoprofen. These ketoprofen-loaded zinc pectinate
microparticles were mixed with mixtures of pectin-dextran to prepare matrix
tablets, which were assessed for colonic delivery. These pectinate-tablets exhibited
the sigmoidal pattern releasing of ketoprofen with a sustained manner. Odeku and
Fell (2005) studied the successful uses of Albizia gum and khaya gum as com-
pression coating material for colon drug targeting. Mishra and Khandare, (2011)
prepared tamarind seed polysaccharide-based matrix tablets of ibuprofen via the
wet granulation. The in vitro ibuprofen release from these matrix tablets demon-
strated that the utmost quantity of ibuprofen was found to be released in simulated
colonic fluid pH (6.8) containing rat caecal contents (2 and 4% w/v). On the other
hand, less amounts of ibuprofen were found to be released in both simulated gastric
fluid (pH 1.2) and simulated intestinal fluid (pH 7.4) before as well as after enzyme
induction. Newton et al. (2015) evaluated the chronotherapeutic propranolol HCl
delivery matrix tablets for colon targeting therapeutics. These matrix tablets were
prepared using tamarind gum and okra gum along with chitosan. The in vitro
propranolol HCl release from these matrix tablets demonstrated that the formula-
tions prepared using tamarind gum showed the sustained the propranolol HCl
releasing for extended period in comparison with matrix tablets prepared using
other polymer based formulations.
3.3.14 Dental Formulations
Plant-derived polysaccharides are also used in several dental formulations for

loacalized drug releasing. In a research, Hasnain et al. (2018b) used cashew
(Anacardium occidentale) gum for the preparation of dental pastes containing
aceclofenac for the use in periodontitis treatment. These dental pastes of ace-
clofenac exhibited sustained in vitro aceclofenac releasing over 6 h and excellent
bioadhesion onto the oral mucosal membrane. Recently, the same research group
investigated the use of dillenia fruit gum to prepare dental pastes containing ace-
clofenac (Hasnain et al. 2020b). The in vitro aceclofenac releasing from these pastes
was found decreased as viscosity increment of these dental pastes due to incor-
poration more amount of dillenia fruit gum within paste formulations. These dental
pastes also exhibited excellent bioadhesion onto the oral mucosal membrane.
3.4 Conclusion
Since long, plant polysaccharides have gained much more attention as pharma-
ceutical as excipients in a variety of pharmaceutical dosage forms due to some
important advantages for the uses of plant polysaccharides include easy availability
from the nature as plant resources are abundant, sustainable and low cost produc-
tion, biodegradability, biocompatibility, water solubility, swelling ability, etc. Plant
polysaccharides have extensively been utilized as thickeners, suspending agents,
emulsifiers, stabilizers, gel forming agents, binders, disintegrating agents, matrix
formers, release retardants, film formers, coating materials, mucoadhesive agents,
etc., in various common pharmaceutical dosage forms, such as suspensions,
emulsions, gels, tablets, capsules, beads, microparticles, nanoparticles, liposomes,
transdermal formulations, buccal formulations, nasal formulations, ophthalmic
formulations, etc. It is anticipated that a variety of high-quality and multipurpose
pharmaceutical dosage forms will possible be formulated in the near future as a
result of the continuous research and development in the field of pharmaceutical
formulation with the successful uses of new plant polysaccharides as advanced
pharmaceutical excipient. Therefore, the future outlook in exploration and
exploitation of new plant polysaccharides as useful and multipurpose pharmaceu-
tical excipient is extremely promising.
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Chapter 4
The Role of Phytochemicals in Cancer
Prevention and Cure
Braganza Cilwyn, Soundararajan Vijayarathna, Shanmugapriya,

Rameshwar Naidu Jegathambigai, Subramaniam Sreeramanan,
Yeng Chen, and Sreenivasan Sasidharan
Abstract The comprehensive search for therapeutic agents, predominantly the

green phytochemicals derived from plants, has recently gained high momentum.
Phytochemicals are plant’s (phyto) chemical referring to various types of com-
pounds that occur naturally in plants. Green phytochemicals from Mother Nature
especially from medicinal plants are a rich source of novel therapeutics and pre-
vention agents. Interestingly, their unique mechanism of action as compared with the
conventional drug mechanism against cancer cells made them a vital target for novel
drug discovery for cancer prevention and cure. Most plants do yield a vast array of
phytochemicals that play essential roles in cancer prevention and cure via various
biological activities and novel mechanisms of actions. Since cancer has a significant
impact on human health and contributes to mortality, it is appropriate to examine the
role of phytochemicals in cancer prevention and cure. This chapter provides a
comprehensive overview of the role of phytochemicals in cancer prevention and
cancer cures via antioxidant activity, pro-oxidant activity, apoptosis induction,
necrosis induction, autophagy induction and regulation of miRNA in cancer cells.

Keywords Phytochemical Cancer Apoptosis Necrosis Autophagy miRNA
B. Cilwyn S. Vijayarathna Shanmugapriya S. Sasidharan (&)

Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia,
11800 USM, Pulau Pinang, Malaysia
e-mail: srisasidharan@yahoo.com
R. N. Jegathambigai
Department of Biochemistry, Faculty of Medicine, Asia Metropolitan University,
No 6 Jalan Lembah, 81750 Bandar Seri Alam, Johor Bahru, Malaysia
S. Sreeramanan
School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia
Y. Chen
Dental Research & Training Unit, and Oral Cancer Research and Coordinating Centre
(OC-RCC), Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia
https://doi.org/10.1007/978-3-030-54027-2_4
128 B. Cilwyn et al.
4.1 Introduction
Cancer is one of the greatest consequential cause for the death of world human
population every year (Zaorsky et al. 2017). The extensive search for therapeutic
agents, predominantly the green phytochemicals derived from medicinal plants, has
recently gained high momentum especially for cancer prevention and cure (Zhang
et al. 2020). Various plants have shown beyond doubt their worth as a source of
phytochemicals with therapeutic potential and still represent as an important source
for the discovering of new therapeutic agents which leads as the blue print for
chemically synthetic compound (Newman and Cragg 2012). Phytochemicals are
plant’s (phyto) bioactive non-nutrient chemicals referring to various types of
compounds that occur naturally in plants (Liu 2004; Mousavi et al. 2018). The
utilization of plants has a long-established history in traditional treatment of various
diseases. The breakthrough in chemical field with the advances in chemical analysis
lead to the isolation and characterization of various purified bioactive compounds of
plants, which initiated the exploration of plant sources as chemotherapeutic can-
didates for cancer (Cragg and Newman 2005). The continuous rise in cancer cases
and the failure of synthetic conventional chemotherapies due to drug resistance and
excessive toxicity towards healthy normal non-targeted tissues have also pushed to
the utilization of naturally occurring phytochemicals of plants, which is evidently
shown to improve treatment efficiency with lesser side effects. According to
increasing scientific evidences confirming the remarkable anticancer activity
induced by phytochemicals derived from plants has prompted to explore the role of
phytochemicals in cancer cure. Green phytochemical from Mother Nature espe-
cially from medicinal plants are a rich source of novel therapeutics and prevention
agents. In reality, some of the commonly used and commercially available anti-
cancer drugs were isolated from medicinal plants, namely vinblastine, and vin-
cristine (Newman et al. 2011). Interestingly, their unique mechanism of action as
compared with the conventional drug mechanism against cancer cells made them a
vital target for novel drug discovery for cancer prevention and cure. The important
characteristic of plant-based phytochemical is that they can kill the cancer cells with
least toxicity. Moreover, phytochemical also exhibits a higher selectivity towards
cancer cells in comparison to normal non-cancerous cells. Most plants do yield a
vast array of phytochemicals that play essential roles in cancer prevention and cure
via various biological activities and novel mechanisms of actions. Since cancer has
a significant impact on human health and contributes to mortality, it is appropriate
to examine the role of phytochemicals in cancer prevention and cure (Vineis and
Fecht 2018). The objective of this chapter is to furnish the role of phytochemicals in
cancer prevention and cure that addressed various mechanisms of actions of various
phytochemicals against the cancer cells as reported in the literature. Hence, this
chapter provides a comprehensive overview of the role of phytochemicals in cancer
prevention and cancer cures via antioxidant activity, pro-oxidant activity, apoptosis
induction, necrosis induction, autophagy induction and regulation of miRNA in
cancer cells as shown in Fig. 4.1.
4 The Role of Phytochemicals in Cancer Prevention and Cure 129
Fig. 4.1 The role of phytochemicals in cancer prevention and cancer cures via various modes of
actions in cancer cells

via Antioxidant Activity
A free radical is molecules (or atom) comprising one or more unpaired electrons in
outer orbit that make them unstable and extremely reactive, which leads them to
steals electrons from other molecules to achieve stability. Subsequently, the
attacked molecule converts to a free radical itself to start the chain reaction cascade,
which eventually injuries the healthy cell (Mukherji and Singh 1984). Free radicals
consist of reactive oxygen species, and reactive nitrogen species are made in the
human body naturally via numerous endogenous systems according to diverse
physiochemical or pathological circumstances (Valko et al. 2007). Free radicals are
created in the human body either from usual important metabolic routes or from
outside sources such as exposure to irradiation, ozone, pollutants, and various toxic
chemicals (Bagchi and Puri 1998). However, the generated free radicals can be
dangerous at the excessive level and lead to the damages to the main apparatuses
and biomolecules of cells such as DNA, proteins and cell membranes. The various
injuries caused by excessive level free radicals to human cells, particularly the
damage to DNA, may lead to the development of cancer and other diseases (Dreher
and Junod 1996). Antioxidants also identified as “free radical scavengers” are
chemicals such as phytochemicals that neutralize the extremely reactive free radi-
cals by donating an electron to the free radical or via quenching chain-initiating
catalyst to avoid them from instigating harm to the healthy cell, which can lead to
the development of cancer. Hence, the plants’ phytochemicals react as a natural
antioxidant to block the harmful action of free radicals preventing the development
of cancer in humans (Rohman et al. 2006). Plants’ phytochemicals are the most
commonly known antioxidants, which include ascorbate, tocopherols, polyphenols
and terpenoids (Dimitrios 2006).
Fig. 4.2 The antioxidant activity of phytochemicals in cancer prevention
The anticancer activity of phytochemicals via antioxidant activity may depend

on the diverse inter-dependent pathways, as shown in Fig. 4.2. Firstly, the phyto-
chemical will be entering the cells to start the pro-oxidant anticancer activity.
Firstly, the accumulation of phytochemicals in the cell will reduce the excessive
amount of free radicals. Subsequently, the reduced excessive amount of free radicals
will not trigger the damages to the main apparatuses and biomolecules of cells,
especially to DNA. Eventually, the phytochemicals neutralize the extremely reac-
tive free radicals in the cell which will stop the development of cancer (Fig. 4.2).
Conclusively, the various inter-dependent processes exhibit the beneficial effects of
the antioxidant activity of phytochemicals that efficiently prevent cancer develop-
ment in humans.
4.3 Role of Phytochemicals in Cancer Prevention Via

Pro-Oxidant Activity
Pro-oxidant represents any phytochemicals that persuade oxidative stress either by

the generation of free radicals or by hindering the antioxidant systems of a cell
(Rahal et al. 2014). The redox level of a cell is determining how well a healthy cell
is functioning. Besides, the protection of antioxidant systems in the cells is essential
for the survival of the cells. Free radicals play a vital part in cancer. Excessive
exposure to free radicals might be linked with a high risk of cancer for a normal
cell. Although high levels of free radicals can be a high risk of cancer for a normal
cell, a high level of free radicals can also trigger apoptosis and cell death in various
types of cancer cells. Decisively, the actions of free radicals in cancer development,
inhibition and treatment is tremendously complex and extremely challenging to
research. Alteration of redox homeostasis in cancerous and healthy cells recom-
mends that pro-oxidant based upregulation of cellular free radicals would target
specifically against cancer cells without damaging the normal healthy cells
(Wondrak 2009). Even though the antioxidant activity of various phytochemicals is
well researched and commonly applied to stop or cure cancer, multiple phyto-
chemicals also exhibit the pro-oxidant and free radicals generating activities under
unique conditions especially in cancer cells. Accordingly, a pro-oxidant activity
reached phytochemical might attack the cancerous cells, which is already at an
extraordinary level of free radicals with high oxidative stress without affecting the
well-tolerated non-cancerous cells (González-Bártulos et al. 2015). Several phy-
tochemicals that target the cellular redox balance produce an exercise amount of
reactive oxygen species (ROS) (Kruk et al. 2019) and eventually leads to cell death.
Moreover, the transition metal-based phytochemicals could be favourable phyto-
chemicals for pro-oxidant therapies (Rahal et al. 2014). Once, the metal-based
phytochemicals accumulate metals, namely iron and copper, they induced the
cycling redox reactions in the cancer cells, which will lead to the productions of the
excessive amount of free radicals, mainly the extremely damaging hydroxyl radical
species via the Fenton reaction. The phytochemicals belong to the flavonoid group,
such as quercetin and kaempferol that have been reported to exhibit the pro-oxidant
activity when a transition metal is available (Halliwell 2008).
The anticancer activity of phytochemicals via pro-oxidant activity may depend
on diverse inter-dependent routes, as shown in Fig. 4.3. Firstly, the phytochemical
will be entering the cells to start the pro-oxidant anticancer activity. Secondly, the
accumulation of phytochemicals will trigger the cell to produce an excessive
amount of free radicals. Subsequently, the excessive amount of free radicals will
trigger DNA fragmentation and DNA damages via oxidative mechanisms. An
excessive amount of free radicals in the cells will react with the cellular DNA, thus
altering its structure, and disturbing the normal function of the DNA is one of the
main reasons for DNA damage induced by pro-oxidant activity of the phyto-
chemical (Beckman and Ames 1997). Even though the DNA molecule is an intact
molecule, free radicals can act against the DNA and can cause various types of
harm, namely alteration of DNA molecule bases, single- and double-strand DNA
molecule disruptions, loss of purines in the DNA, destruction to the deoxyribose
sugar, cross-link between DNA and protein and destruction of the naturally
occurred DNA repair systems (Srinivas et al. 2019). The DNA fragmentation will
lead to the induction of cell cycle arrest. Eventually, the cell cycle arrest will lead
the cell to apoptotic cell death (Fig. 4.3). Conclusively, the various inter-dependent
processes exhibit the beneficial effects of the pro-oxidant activity of phytochemicals
that efficiently kills the cancer cells.
Fig. 4.3 The pro-oxidant activity of phytochemicals in cancer prevention
4.4 Role of Phytochemicals in Cancer Cure Via Apoptosis

Induction
Major plant-based phytochemicals such as terpenoids, phenolic acids and alkaloids

have shown potentially promising anticancer property by fine-tuning the reactive
oxygen species (ROS) signalling pathways (Chirumbolo et al. 2018). In line with
the anti-oxidative and ROS-scavenging properties of phytochemicals, numerous
phytochemicals have been studied and scientifically demonstrated to induce
apoptosis through the ROS generation. An established plant-based anticancer agent
can exemplify this, resveratrol, which was shown to induce caspase-8/
Caspase-3-dependent apoptosis in human colon cancer cells, HCT29 and
COLO201 by significantly increasing the intracellular ROS levels (Miki et al.
2012). Another commonly known phytochemical exhibiting anticancer activity,
quercetin, was also inhibiting cancer growth by inducing apoptosis via
cyclooxygenase-2 (COX-2)-dependent ROS generation.
Furthermore, numerous phytochemicals showed potentially promising anti-
cancer activity by inducing apoptosis via upregulating the expression of caspases.
This can be exemplified by a phytochemical called hyperforin which was reported
to promote caspase-dependent apoptosis in various leukemia cell lines by upreg-
ulating caspase-9, caspase-8 and caspase-3 (Hostanska et al. 2003). One such
phytochemical is corosolic acid reported to promoting caspase activation, leading to
mitochondria-mediated signalling pathways to induce cell death in HeLa cells (Xu
et al. 2009). The purified bioactive compound, Pyranocycloartobiloxanthone A,
was also reported to play an imperative role in inducing apoptosis in breast cancer
cells by upregulating Bcl-2 expression and downregulating Bax expression, which
eventually lead to the release of cytochrome c, initiating the caspase cascade
(Mohan et al. 2012).
Interestingly, another phytochemical from the class of phenolics, known as
Scutellarin, has been proven to promote apoptosis by activating the p53 pathway
(Yang et al. 2017). Scutellarin was found to suppress the anti-apoptotic protein
Bcl-2, which eventually activates the pro-apoptotic protein, p53, leading to the
upregulation of Bax protein to induce caspase-3 dependent apoptosis in human
colon cancer (Yang et al. 2017). Another such phenolic compound, gallic acid, also
reported inducing apoptosis in cancer cells via the upregulation of the p53. This, in
turn, depolarizes the mitochondrial membrane potential, facilitates the release of
caspase-activator, cytochrome c and induces an intrinsic apoptotic pathway (Yang
et al. 2018). Role of another important phytochemical, capsaicin to induce
p53-mediated apoptosis in various cancer cells have been well elucidated in pre-
vious studies (Jin et al. 2014; Clark and Lee 2016; Garufi et al. 2016; Lee and Clark
2016).
Various other phytochemicals have been reported to play an essential role in
cancer cure by targeting nuclear factor kappa B (NF-kB), to promote cancer cell
death (Kumar et al. 2016). The fact that NF-kB is highly expressed in cancer cells is
inevitable due to its function in regulating anti-apoptotic and apoptotic genes (Tse
et al. 2007; Manu and Kuttan 2008; Oh et al. 2012; Kumar et al. 2016).
Intriguingly, various phytochemicals, including alkaloids and flavonoids, are
known to induce apoptosis in cancer cells by specifically targeting NF-kB sig-
nalling pathway. For instance, known phytochemicals including xanthohumol
(Colgate et al. 2007), Magnolol (Tse et al. 2007), Morusin (Lee et al. 2008),
urosolic acid (Manu et al. 2008), Corilagin (Gambari et al. 2012) were ostensibly
demonstrated to significantly down-regulate the expression of NF-kB in various
cancer cells. The suppression of this apoptosis-inhibitor, NF-kB, eventually leads to
tumour necrotic factor-a (TNF-a)-induced apoptosis. Recent review collectively
elucidated TNF-a induced activation of NF-kB in mitochondria to stimulate pro-
grammed cell death by the release of cytochrome c to the cytoplasm, followed by
the activation of a caspase cascade (Albensi 2019).
Besides, there are also several plant-based secondary metabolites reported to
induce the extrinsic pathway of apoptosis in cancer cells. A form of flavonol can
depict this, kaempferol, which has been previously reported to up-regulate the
expression of FasL, leading to the activation of caspase-8 in colon cancer cells (Lee
et al. 2014). Bid protein, which is cleaved by the activated caspase-8 in the means
of extrinsic apoptosis pathway, is then translocated into mitochondria, promoting
intrinsic apoptosis (Lim et al. 2014). Another phytochemical known to encourage
the extrinsic pathway is a phenolic compound called hispidin. Hispidin was sci-
entifically proven to increase the level of death receptor 3 in colon cancer cells,
leading to activation of the caspases involved in the extrinsic apoptosis pathway,
namely, caspase-1 and caspase-8, along with the cleavage of PARP to induce cell
death (Hengartner 2000).
Cumulatively, it can be suggested that these plant-derived biologically active

compounds induce apoptosis mainly through the mitochondria-dependent mecha-
nism. In short, phytochemicals promote ROS generation in cancer cells, causing
polarization of mitochondria membrane potential leading to the release of various
toxin proteins, including cytochrome c. Eventually, cytochrome c actively binds to
apoptotic protease activating factor-1 (Apaf-1), which activates caspase-9 leading to
the formation of cytochrome c/Apaf-1/caspase 9 complexes termed as the apop-
tosome which activates the executioner caspase 3 resulting in apoptosis (Hengartner
2000). On the other hand, phytochemicals were also found to be inducing the
extrinsic apoptotic pathway by upregulation of death ligands such as FasL, TNF-a
and TRAIL. These ligands are responsible for the activation of caspase-8 by
binding to death receptors such as FAS, TNFR and other death receptors. Active
caspase-8 cleaves Bid protein into tBid, which is then translocated to mitochondria
to promote BAX and BAK proteins, allowing the intrinsic pathway to take place by
the activation of the caspase cascade. Figure 4.4 shows the intrinsic and extrinsic
apoptotic pathways induced by phytochemicals.
Fig. 4.4 Intrinsic and extrinsic apoptosis mechanisms induced by phytochemicals

4.5 Role of Phytochemicals in Cancer Cure Via Necrosis

Induction
Phytochemicals have been shown to protect cells by interfering with their molecular
pathways that regulate the cell cycle, survival, angiogenesis and cell death. These
properties made phytochemicals an essential source of drug for the prevention and
treatment of cancer. Many anticancer drugs such as paclitaxel and vinblastine are
derived from phytochemicals, and still, many more are under investigation.
Notably, most of these compounds target the apoptotic mechanism mainly by
interfering with caspase-dependent pathways (Ashraf 2020). However, other
non-apoptotic cell death pathways such as necrosis also play an essential role in
checking neoplastic cells and destroying tumour cells. Inducing necrosis is a known
mechanism of some anticancer drugs such as DNA-alkylating agents in treating
human cancers (Cho and Park 2014). Therefore, understanding the role of phyto-
chemicals in signalling cascades involved in the induction of necrotic cell death will
allow us to develop a novel drug to treat cancer.
Depending on the physiological and pathological conditions, a cell would either
take the apoptotic or necrotic pathway. Unlike apoptosis, necrosis does not have a
dedicated molecular pathway, instead it overlaps with many of those
caspase-independent apoptotic pathways, which culminates in disruption of organelle
and loss of membrane integrity resulting in the spillover of cellular contents (Lee et al.
2018). In a tumour microenvironment, induction of necrotic pathway would cause
more damage as it destroys the cells around, and the contents released from these cells
create a pro-inflammatory environment (Lee et al. 2018). Phytochemicals with
enhanced necrosis may help to exert more effective tumour suppression property.
Well-controlled, a programmed form of necrosis is known as necroptosis, which
is mainly triggered by extracellular stimuli similar to the extrinsic apoptotic path-
way. Necroptosis is primarily orchestrated by serine/threonine kinase
receptor-interacting protein 1/3 (RIP1 and RIP3) to induce necrotic cell death. RIP1
and RIP3 can be activated by signalling via tumour necrotic factor receptor 1/2
(TNF-R1/2), Toll-like receptor 3/4 (TLR3/4), DNA damage-induced Poly
[ADP-ribose] polymerase 1 (PARP1) pathways, especially when caspase-8 is either
downregulated, non-responsive or inactivated by other regulators (De Giffoni De
Carvalho et al. 2019). Finally, RIP1 and RIP3 form a dimer which is one of the
ways of induction of necroptosis by the activation of mixed lineage kinase
domain-like protein (MLKL) that destroys the integrity of plasma membrane or via
activation of mitochondrial protein phosphatases PGAM5 and Drp1 (mitochondrial
fission protein) which lead to mitochondrial dysregulation (Fig. 4.5) (Mishra et al.
2018). However, progression depends on the level of caspase-8 and Fas-associated
protein with death domain (FADD) that regulates RIP1/RIP3 levels. Experiments
have demonstrated downregulation of Caspase 8 and FADD promotes
RIP3-dependent necrosis (Wattanathamsan et al. 2019). Further, reactive oxygen
species (ROS), advanced glycation end products (AGE), calcium, cyclophilin D
(CypD), NO/NOS, phospholipase A2 (PLA2), calpains, cathepsin B, ceramide,
methylglyoxal and high mobility group box 1 (HMGB1), act as important media-
tors in the necrotic pathway. In addition, the necrotic pathway can be triggered by
oncogenic metabolic stress and hypoxia by inducing transcription factors Snail and
Dlx-2 (Lee et al. 2018). Polymorphisms and defects in necroptosis regulators such
as RIP3 have been shown to have a positive correlation with tumour progression in
non-Hodgkin’s lymphoma (Mishra et al. 2018). There is also increasing evidence of
impaired necroptosis in cancer cells (Lalaoui and Brumatti, 2017). Hence, targeting
necroptosis is very promising to treat various cancers, and trials of repurposing
anticancer drugs for inducing necroptosis has been explored (Fulda 2018).
Moreover, phytochemicals have been shown to induce necrosis by targeting many
of the above mediators. Therefore, it is imperative to evaluate the effects of phy-
tochemicals on the above molecular mediators to understand their role in inducing
necrosis/necroptosis.
It is beginning to unravel mounting evidence on the molecular regulation of
phytochemicals by RIP1/RIP3 upregulation of mitochondrial disruption by ROS
generation, ATP depletion and fragmentation. Several in vitro and in vivo studies
have reported the involvement of phytochemicals in necrotic/necroptotic signalling
in cancer (Fig. 4.5). For example, Solamargine has shown to induce necrosis in
melanoma and non-melanoma skin cell lines by targeting the lysosomal mito-
chondrial death pathway in lung cancer, breast cancer, squamous cell carcinoma
and leukemia cell lines (Al Sinani et al. 2016). Similarly, Phenethyl isothiocyanate
and Shikonin induce necroptosis in lung cancer cells via ROS, and b-Lapachone
induces necroptosis in human hepatocellular carcinoma SK-Hep1 cells through the
RIP1-PARP-AIF-dependent pathway (Diederich and Cerella 2016).
Green tea polyphenol is shown to induce necroptosis in p53-deficient Hep3B
cells through mitochondrial-associated signalling via activation of Bax/Bak
translocation (Lin and Tongyi 2014). Polyphenols resveratrol and analogs from
roots of Fallopia japonica has shown to induce necrosis in MCF-7 breast cancer and
C6 glioma cell lines. Curcumin and analogs have shown to induce necrosis in
prostate (DU-145) cancer cells by generating ROS, and in bladder cancer xeno-
grafts by downregulating NF-kB, cyclin D1 with increased p21 expression.
Genistein from Genista tinctorial has shown to cause necrosis in cervical cancer
(HeLa) cell lines (Gali-Muhtasib et al. 2015). Alkaloids such as berberine and
analogs showed necrosis in melanoma (B16) Prostate (RM-1) cell lines. Colchicin
and analogs inhibit cell division and cause necrosis in Lung, colorectal, ovarian,
prostate and breast mouse model. Terpenoids parthenolide and analogs were shown
to cause necrosis in leukemia (HL60, Jurkat), breast (MDA-MB-231) cancer cell
lines by ROS generation, induce dissolution of mitochondria membrane potential
and RIP1 activation. Organosulfur sulforaphane and analogs induce necrosis in the
breast (MCF-7), Colorectal (Caco-2) cancer cells by regulating CDK1
(Gali-Muhtasib H et al. 2015). Flavonoids such as quercetin shown to promote
necroptosis in MCF-7 cells. Artemisinin (ARS) derivatives such as artemether
(ARM) has been shown to exert necrosis in PG 100 gastric cancer cell line and
artesunate (ART) induce necroptosis in RT4 schwannoma cell line (Efferth 2017).
Fig. 4.5 Mechanisms of phytochemical induced necrosis and necroptosis. Various phytochem-
icals follow different molecular pathways to induce necrosis and necroptosis. Molecular
mechanism of NF-kB induced necrosis is well understood with RIP1, RIP3, PARP1 which are
key players while CYLD is the regulator of RIP1. Phosphorylation of MLKL1 by RIP3 will induce
necrosis either by direct disintegration of plasma membrane or via PGAM5/Drp1 causing
mitochondrial damage leading to necrosis/necroptosis. HMGB1 proteins are released by necrotic
cells that stimulate immune response
Even the extracts of plants have been demonstrated to induce necrosis in liver
cancer cell line Huh7it (Ficus carica leaves and fruits) (Purnamasari et al. 2019).
Further, Hymenocallis speciosa extract in acute myeloid leukaemia cell line showed
necroptosis and Jacaranda decurrens extract induced necrosis in K562 ery-
throleukaemia cells (De Giffoni De Carvalho et al. 2019).
Overall, the role of phytochemicals in necrosis induction and molecular mech-
anisms are less explored and has the potential to contribute to cancer cure. Drugs
targeting necrotic pathways are mainly useful to treat tumour cells that are resistant
to apoptotic drugs either by upregulation of anti-apoptotic proteins (Bcl-2) or
impaired pro-apoptotic proteins such as p53. This resistance, either intrinsic such as
mutations in p53 or acquired to anticancer drugs, is a significant reason for poor
treatment outcomes. Besides, necrosis-inducing phytochemicals could be used in
combination therapy to simultaneously hit both apoptosis and necrosis pathways
and thus provide an effective cure for cancer.
4.6 Role of Phytochemicals in Cancer Cure Via

Autophagy Induction
Autophagy, a word originated from Greek meaning self-eating, is a self-digestion

approach leading to cellular degradation of the subcellular materials such as
organelles and proteins to generate energy and metabolic precursors for prolonging
cell survival (Glick et al. 2010). The cytoplasmic proteins and cellular organelles
will be enveloped in autophagosomes and degraded by fusion with lysosomes
during this process. The cellular stress response that usually serves as a quality
control mechanism leads to apoptosis-independent cell death. Various forms of
autophagy have been described, including macroautophagy, microautophagy and
chaperone-mediated autophagy (Sharma et al. 2014). It is a morphological
description, and no decisive indication of precise mechanisms underlying
autophagy-persuaded cell death can be witnessed (Tsujimoto and Shimizu 2005).
Numerous phytochemicals, namely phenols, alkaloids, flavones and organic acids
are stated to be autophagy regulators, and it was revealed that autophagy might
exhibit either cytoprotective or cytotoxic role in natural products phytochemicals
treated cancer cells (Wang and Feng 2014).
Characteristics of cancer cells, including enhanced cell proliferation, altered
apoptotic pathways, and reprogrammed cellular metabolism, all together have
shown to influence the autophagic route (Linder and Kögel 2019). Most
chemotherapeutic drugs promote autophagy, which is generally considered a
cytoprotective response in that its inhibition frequently promotes apoptotic cell
death (Sharma et al. 2014). In some circumstances, both autophagy and apoptosis
are required in parallel pathways to contribute to cell death (Mettlin 1997). Under
stress conditions, it can induce programmed cell death, called
“autophagy-dependent cell death” (ADCD). Several reports have indicated that a
variety of naturally occurring compounds play roles in the prevention or therapy of
cancer, and their bioactive compounds lead to autophagy. Recent studies have
suggested the role of phytochemicals in modulating the autophagy pathway. The
Resveratrol, found abundantly in grape skins and red wine, induced cell death and
growth inhibition in ovarian cancer cell lines, through autophagocytosis (Kim et al.
2011). Resveratrol induced molecular features of apoptosis, including the mito-
chondrial release of cytochrome c and caspase activation, resveratrol-treated cells
exhibited the morphologic and ultrastructural changes indicative of autophagocytic
death (Opipari et al. 2004). Soy-derived isoflavone, genistein, was also shown to
induce both apoptosis and autophagy (Kueck et al. 2007). Genistein induced
autophagy in ovarian cancer cells, indicating recruitment and localization of LC3-II
to autophagosomes (Gossner et al. 2007).
4.7 Role of Phytochemicals in Cancer Cure Via

Regulation of miRNA
Cancer has become an extensive malicious disease that relies greatly on the treat-
ment of chemotherapy. The chemotherapy drugs, however, not only contrives the
intensity to cause deleterious side effects, but also triggers tumour regression after a
temporary period of improvement. Strange enough, these relapsed cancer cells
enhance their survival propensity by being chemoresistance. Hence, critical mea-
surements are indispensable to pursue an agent without or with minimal side effects.
Nature endows vegetables, fruits and herbs with chemical properties called phy-
tochemicals possessing the aptitude in treating cancer, infections and healing
proneness. These phytochemicals are found copious from a wide range of plant
products and of these, 10,000 compounds were documented and described (Russo
et al. 2010). Some of these chemicals displayed anticancer potential with almost
zero or minimal cytotoxicity to normal cell physiology (Rao et al. 2007).
Enigmatically, 47% of drugs ratified by FDA are of plant origin that can be treated
as single chemotherapeutic agent usage or by merging with other standard anti-
cancer drugs (Newman and Cragg 2007).
When phytochemicals are exerting anticancer properties, various questions
emerged in connection to microRNA’s role and regulation. The miRNAs some time
back had been linked with oncogenic and tumour suppressor action, serving a
perfect goal in cancer deterrence and therapy. The explicate mechanism that
involves anomalous expression of miRNAs discovered in many cancer types has
yet to be determined. Mounting data implicates anomalous transcription machinery,
epigenetic and miRNA biosynthesis alteration, mutations or the presence of dif-
ferent DNA number to be prompting miRNA dysregulation in human cancer (Deng
et al. 2008). Nonetheless, researchers exposed the potentiality of phytochemicals in
the modulation miRNA expression and their effect in cancer pathobiology.
There were several studies revealing the modus operandi of phytochemicals in
regulating miRNA expression with an exception to transcriptional regulation.
Epigallocatechin-3-gallate (EGCG) is a rich constituent and the most efficient
catechin in green tea where its role has been contributed to cancer treatment
(Mukherjee et al. 2015). This compound was reported to incite the binding of
hypoxia inducible factor-1a (HIF-1a) to the promoter region of miR-210, eventu-
ally deceiving the cells to stop cell proliferation and anchorage-independent growth
as noted in human non-small cell lung cancer cell lines, H1299, H460 and A549
(Wang et al. 2011). Coincidingly, Yamada et al. (2016) had remarkable discovery
on EGCG upregulating miRNA-let-7b which then activated 67 kDa laminin
receptor to inhibit cancer growth in B16 melanoma cells. Of late, ECGC was also
demonstrated to down-regulate the expression of miR-25 and escalate PARP,
pro-caspase-3 and pro-caspase-9 inducing cell apoptosis in MCF- 7 cells (Zan et al.
2019).
The resveratrol has long been insinuated in the treatment of cancer and other
diseases. In human colon cancer study, resveratrol significantly reduced the level of
miR-17, miR-21, miR-25, miR-92a-2, miR-103–1 and miR-103–2, where these

miRNAs were deliberately known to behave as oncomiRs (Tili E, J-J Michaille
2011). The biological effect of resveratrol was studied in prostate cancer (Kumar
et al. 2017) where it was found to down-regulate the expression of miR-221 while
in lung cancer, a high surge was reported in the expression of miR-200c (Bai et al.
2014). Notably, in estrogen-responsive breast cancer cells, resveratrol was reported
to dysregulated two miRNAs mainly miR-542-3p which observes a reduction while
miR-122-5p that records an increment all in together to promote apoptosis cell
death. Comparatively, the triple-negative breast cancer cells with the treatment of
resveratrol manage to only display an increase in miR-122-5p (Venkatadri et al.
2016). Osteosarcoma (OS) is considered as an aggressive cancer and when the cells
U20S and MG63 were introduced to resveratrol, impressive effects in response to
apoptosis were perceived. Expression of miR-139-5p was reduced where ante-
cedently it has the aptitude in binding to 3′UTR region of NOTCH1 and mediate the
progression of osteosarcoma (Xiao et al. 2020).
Curcumin is an all-natural occurring phytochemical dominating the root and
rhizome of Curcuma longa which has extensive established records on antioxidant,
anti-inflammatory, and anticancer properties (Gupta et al. 2013; Park et al. 2013).
Coker-Gurkan et al. demonstrated the anti-proliferation activities of curcumin in
T47D cells through the downregulation of miR-183, miR-96 and miR-182 along
with NF-jB signalling inhibition (Coker-Gurkan et al. 2019). Competently, treat-
ment with curcumin has demonstrated suppressed proliferation and increased
apoptosis of NSCLS cells by mediating the downregulating of miR-21 via PTEN
axis (Bai et al. 2014; Zhang and Bai 2014). PTEN gene implicated as the target of
miR-21 is a tumour suppressor and the suppression of the oncomiR has resulted in
upregulation of PTEN, hence resuming back to mediating the anticancer properties
as exerted by curcumin (Liu et al. 2013). In another case, an expression of elevated
miR-130a directed to target Wnt/b-catenin pathway was halted by curcumin which
may lead to an inhibition of cell proliferation in colon cancer SW480 cells (Dou
et al. 2017). Curcumin also attested to induce apoptosis in bladder cancer cell lines
(T24 and SV-HUC-1) via upregulation of miR-7641 levels, which attenuated cell
proliferation and invasion and, thereafter, inducing apoptosis by repressing p16
(Wang et al. 2018). The activity of curcumin was further investigated utilizing
SCID mice xenograft tumour model of glioblastoma multiforme. Intriguing results
exhibited a surge in the level of miR-378 through target mediator p38. This,
however, resulted in the inhibition of cellular growth thus substantiating potential
effects of curcumin (Li et al. 2017).
Similar to other polyphenols, quercetin can be detected present in apples, onions
and broccoli (Nam et al. 2016) where it is prominently studied for its anticancer
mechanism. Notably, when quercetin is applied to pancreatic cancer cells, it dis-
closed a regulated plethora of 105 miRNAs at which 25 miRNAs were
down-regulated (chiefly; miR-103a-3p, miR-125b, and miR-1202) whereas 80
miRNAs were up-regulated (namely; let-7c, miR-200a-3p, and miR-200b-3p).
Additional studies were performed on one of the most expressed let-7c with con-
nection to pancreatic cancer anti-proliferation mechanism (Nwaeburu et al. 2016).
Outstandingly, let-7c behaves in an unusual manner than the common miRNAs by

binding to 3′UTR of Numbl and evidently increasing the expression of Numbl, an
inhibitor of Notch signalling (Miele et al. 2006). As a fact, tumour progression and
apoptosis were observed in these pancreatic ductal adenocarcinoma cells. The
influence of quercetin was also studied using in vitro oral cancer cells (HSC-6 and
SCC-9). Cells treated with quercetin expressed higher miR-16 expression which
was then shown to mediate the inhibition of HOXA10 level (Zhao et al. 2019). An
earlier in vitro study revealed HOXA10 to be positively related to the outcome of
cancer cell proliferation on head and neck squamous cell carcinoma (Guo et al.
2018). Collectively, the activated expression of miR-16 acted as a tumour sup-
pressor in oral cancer by interrupting HOXA10 (Zhao et al. 2019). Likewise,
quercetin mediating expression of distinct microRNA: miR-16, miR-217 and
miR-145 were discovered through many studies conducted in lung adenocarci-
noma, osteosarcoma, and ovarian cancer cells, respectively (Sonoki et al. 2015;
Zhang et al. 2015; Zhou et al. 2015).
Sulforaphane (SFN) originates as an isothiocyanate derivative of cruciferous
plants such as cauliflower and broccoli known to be effective as anticancer agent
based on reputable in vivo studies (Zhang and Tang 2007). Importantly, SFN
treated non-small cell lung (H1299, 95C and 95D) cancer cells which disclosed a
suppression of miR-616, an oncomiR modulating GSK3b/b-catenin signalling
pathway commonly implicated in cancer invasiveness (Wang et al. 2017). In par-
ticular, the SFN targets the EGFR signalling which is associated with EMT (ep-
ithelial mesenchymal transition) of lung cancer. Studies, however, indicate EMT
largely influencing the progression of metastasis, chemoresistance and other types
of tumours (Xiao and He 2010). Another study uniformly related to EMT was
recorded in SFN-treated bladder cancer cells, (T24 and UMUC-3). Consequently,
expression of (miR-200c) was greatly enhanced where it inhibits mesenchymal
phenotype (vimentin) and upregulated epithelial phenotype (E-cadherin), thus
bringing the mechanism of EMT down to a halt (Huang et al. 2018). Combination
treatment of SFN and iberin in colorectal cancer cell lines (NCM460 and NCM356)
exhibited better potential via upregulation of miR-23b and downregulation of
oncomiR, miR-27b. The performance of these miRNAs is contributed to the
anti-invasive, anti-angiogenesis and anti-proliferation activities in cancer cells
(Slaby et al. 2013). Of the reasons why pancreatic cancer cells are resistant to
treatment is due to the loss of gap junction intercellular communication and con-
nexin 43 expression. However, PANC-1 cells treated with SFN seem to restore
these features by making cell sensitive again to anticancer drugs. This is achieved
by reducing the expression of miR-30a-3p thus enhancing the expression of Cx43, a
protein associated with gap junction (Georgikou et al. 2020). In another study of
breast cancer (MCF-7, MDA-MB-231 and SK-BR-3) cell lines, the introduction of
SFN dramatically reduced the expression of miR-23, miR-92b, miR-381 which
eventually promoted cell cycle arrest and senescence in these cells (Lewinska et al.
2017).
Genistein is a natural soy isoflavone that has featured exceeding intensity as an
anticancer agent (Spagnuolo et al. 2015; Varinska et al. 2015). In MCF-7 and
MDA-MB-435 breast cancer cells, downregulation of oncogenic miR-155 was

observed after treatment with genistein which led to the expression FOXO3, PTEN,
casein kinase, and p27. Interestingly, the inhibition of invasiveness and metastasis
were achieved as miR-155 has been demonstrated to escalate in various tumours
(De La Parra et al. 2016). When oncogenic miR-23b-3p was reduced in renal cancer
cells treated with genistein, expression of target PTEN begin to enhance followed
by reduced expression of cancer-related genes PI3K (phosphatidylinositol-3-
kinase), Akt and IL-32 (interleukin-32) (Zaman et al. 2012). Genistein increased the
levels of miR-145 in retinoblastoma cell (Y79) cell, thereby inhibiting ABCE1
target gene to demonstrate anti-proliferation and growth impediment. The gene
ABCE1 is known as a member of ATP-binding cassette (ABC) transporters which
carries the responsibility for actively transferring molecules across phospholipid
bilayers (Wei et al. 2017). Genistein exhibited apoptotic in laryngeal cancer when
the expression of miR-1469 was greatly up-regulated followed by a suppression in
myeloid cell leukemia 1, an anti-apoptotic member of Bcl-2 family (Ma et al. 2018).
In the case of pancreatic cancer cells, it was reported that following treatment with
genistein, the expression of miR-223 was significantly decreased. Further investi-
gation also indicated the up-surge of F-box/WD repeat-containing protein 7 gene, a
tumour suppressor gene which was one of the targets of miR-223 (Ma et al. 2013).
The expression of miR-29b in U266 multiple myeloma cells were revealed with the
administration of genistein. The miR-29b observes a significant twofold increase
that targets the NF-jB expression, hence promoting and restoring apoptosis in
U266 multiple myeloma cells (Xie et al. 2016).
Indole-3-carbinol (I3C) is a constituent of cruciferous vegetables that has the
prospecting value as a chemo-preventive agent (Beier 1990) according to many
studies (Chinni and Sarkar 2002; Rahman et al. 2004). It mainly works by modifying
estrogen metabolism upon consumption to aid its cancer-preventive measurements.
Concurrent researches have provided substantiate evidence indicating I3C in cell
growth arrest, cell proliferation prevention and apoptosis (Takada et al. 2005; Weng
et al. 2007; Omar et al. 2009). In human breast cancer cells (MCF-7), I3C has
demonstrated an upregulation of miR-34a (Hargraves et al. 2016). The higher
expression of miR-34a targets Bcl-2 and other anti-apoptotic proteins hinders chro-
matin silencers of p53 genes and assist the repression of CDK4, CDK6, and cyclin D1
(He et al. 2007; Hermeking, 2010). Taken together, the expression of miR-34a
modulates p53 tumour suppressor escalation and its downstream gene 21 to achieve
apoptosis (He et al. 2007). In an in vivo lung cancer study, female mice were observed
after exposing to vinyl carbamate (VC). A plethora of miRNAs were down-regulated
which includes miR-21, miR-31, miR-130a, miR-146b, and miR-377 where reversed
changes were observed in mice treated with carcinogen only. Further analysis of
miR-21 indicated PTEN, PDCD4, and reversion-inducing-cystein-rich protein with
Kazal motifs as potential targets for the oncogenic effect of miR-21 and the
chemopreventive activity of I3C (Melkamu et al. 2010). Identically, in hepatocellular
carcinoma (HCC) cell, I3C was also revealed to reduce and prevent the progression of
tumorigenicity by arresting miR-21 (Wang et al. 2015).
Ellagic acid (EA) is a phenol with anticancer property found rich in countless
fruits and vegetables (Zhang et al. 2014; González‐Sarrías et al. 2016). Human
colon cancer cells (Caco-2, HT-29, and SW480) that were subjected to EA dis-
played downregulated oncogenic miR-224 while tumour suppressor miR-215 was
up-regulated. These miRNAs induced facilitated modulation of p53 via p21
accumulation (Munagala et al. 2013). In a different case, EA treatment was con-
ducted on female ACI rats which had been previously exposed to estrogen to
develop mammary tumorigenesis. The observation of this treatment includes
upregulation of miR-182, miR-375, miR-183, miR-34c, miR-196c and miR-429
and downregulation of miR122, miR-127, miR-335, miR-205, and miR-206
expression in tumour cells. The overall inhibition of the tumour was reached with
the modulation of key proteins; ERa, cyclin D1, RASD1, FoxO3a, FoxO1, cyclin
G1, Bcl-w and Bcl-2 (Ravindranath and Chandrasekhara, 1980).
Though there are many phytochemicals which have entered clinical trials for the
treatment of cancer, lower bioavailability and poor potency of dietary
plant-compounds still pose as a great challenge to scientists (Ravindranath et al.
1980; GonzáLez-Barrio et al. 2010). Based on evidence here, phytochemicals are
shown to induce miRNAs in various cancers both in vivo and in vitro (Fig. 4.6).
Hence, the limitation of the bioavailability can be resolved by imitating a synthetic
analog of the miRNA of interest and by encapsulating their formulation into
nanoparticle component. Convincingly, phytochemicals deliver as promising agents
in regulating cancers and with the discovery of corresponding miRNA activities
together with their target gene mechanism that proposes a magnificent future
approach to combat this disease.
Fig. 4.6 Molecular targets of different phytochemicals in curbing various tumour growths/
malignancies through microRNA regulation. The red arrow (") indicates upregulation, yellow
arrow (#) represents downregulation and (*) marks the phytochemical in subject
4.8 Conclusions
Phytochemical from plants is an essential source of novel therapeutics and pre-

vention agents against cancer. This chapter discusses the various mechanism of
actions of different types of phytochemicals against the cancer cells for under-
standing the critical role played by phytochemicals in cancer prevention and
treatment. In conclusion, this chapter discussed in detail the role of phytochemicals
in cancer prevention and cures via antioxidant activity, pro-oxidant activity,
apoptosis induction, necrosis induction, autophagy induction and regulation of
miRNA in cancer cells, as reported by the worldwide researcher.
Acknowledgements This project was funded by the Fundamental Research Grant

Scheme (FRGS; Grant No.: 203/CIPPM/6711723) from the Ministry of Education Malaysia,
Government of Malaysia, Malaysia. Braganza Cilwyn is supported by Fundamental Research
Grant Scheme (FRGS; Grant No.: 203/CIPPM/6711723) from the Ministry of Education Malaysia,
Government of Malaysia, Malaysia.
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Chapter 5
Role of Stress and Defense in Plant
Secondary Metabolites Production
Humberto Aguirre-Becerra, Ma Cristina Vazquez-Hernandez,

Diana Saenz de la O, Aurora Alvarado-Mariana, Ramon
G. Guevara-Gonzalez, Juan Fernando Garcia-Trejo,
Abstract Secondary plant metabolites are natural bioactive compounds which are
an important income for the pharmaceutical, food, cosmetic, agriculture, and other
sectors due to their health-promoting properties and prevention and treatment of
some diseases. The secondary metabolites can be classified into three main groups:
phenolic compounds, terpenoids, and nitrogen compounds. The secondary meta-
bolism in plants is a mechanism of adaptation and evolution as a defense to harsh
environmental factors that induce stress. According to the hormesis curve of each
plant model, the stress can be divided into distress (bad stress that leads to damage
and ultimately plant death) or eustress (good stress that leads to activation of
secondary metabolism). The environmental factors can be divided into biotic and
abiotic which can be artificially induced to activate plant defense responses leading
to the production of secondary metabolites. Several approaches to this process
called elicitation have been proposed in the last decades with different types of
metabolism-inducing factors or elicitors. Novel elicitation using abiotic factors
includes electromagnetic waves (including several wavelengths of the light spectra,
and electric and magnetic fields), acoustic waves, nanostructures, volatile com-
pounds, nutrient deprivation, and several metals and salt soil pollutants. In the same
order, novel elicitation using biotic factors include new bacteria consortium, fungi,
phytohormones, and miRNA solutions. In general, the purpose of elicitation is to
interact with the biochemical routes in order to produce secondary metabolites in
H. Aguirre-Becerra D. Saenz de la O R. G. Guevara-Gonzalez J. F. Garcia-Trejo

A. A. Feregrino-Perez (&)
Facultad de Ingenieria, Universidad Autonoma de Queretaro,
C.P. 76010 Santiago de Querétaro, QRO, Mexico
e-mail: feregrino.angge@hotmail.com
M. C. Vazquez-Hernandez
Departamento de Ingeniería, Tecnologico Nacional de Mexico Roque, Carr.
Celaya-Juventino Rosas Km. 8, C.P. 38110 Celaya, GTO, Mexico
A. Alvarado-Mariana
Facultad de Ciencias Naturales, Horticultura Ambiental, Av. de la Ciencia s/n,
C.P. 76230 Juriquilla, QRO, Mexico
https://doi.org/10.1007/978-3-030-54027-2_5
152 H. Aguirre-Becerra et al.
high quantities, usually with negative effects in biomass production or morphology,

but increasing plant quality in terms of aroma, taste or color. The metabolic profile
and general response of elicitation vary greatly depending on the plant model, the
elicitor level or concentration, and the stimulation time. Considering these facts,
this chapter clearly and concisely discusses the most current strategies of elicitation
for the increase of secondary metabolites production in plants.
Keywords Secondary metabolites Eustress Distress Defense in plant

Elicitation
5.1 Introduction
A variety of substances or compounds called secondary metabolites are mainly

responsible for the adaptation of plants to environmental changes. Plants synthesize
a wide range of secondary metabolites such as alkaloids, flavonoids, phenolic,
steroids, anthocyanins between others, which are used as pharmaceuticals, agro-
chemicals, biopesticides, color, additives, etc. Secondary metabolites are not con-
sidered to be part of fundamental life processes of plants, however, they play a
significant role in protection from insect, pest, herbivores, phytopathogens, and
other harsh environmental variables (Thakur et al. 2019). Therefore, the synthesis
of secondary metabolites depends on internal and external factors (stressors and
stress factors, respectively) that influence plant metabolism, and can affect plant
reproduction and productivity (Kranner et al. 2010). The stress factors, that for their
nature can be biotic or abiotic, modify positively or negatively the plant metabo-
lism (Cheynier et al. 2013). Biotic stress is the result of the interaction between
plant and viral, bacterial, fungi, pheromones, phytohormones, and nucleic acids
among others. Meanwhile, abiotic stress can be physical factors (Light spectra,
temperature, water stress, acoustic waves, others) and chemical factors
(Nanostructures, gasses, nutriment, others) (Vázquez-Hernández et al. 2019).
According to data provided by Agathokleous et al. (2019), exist 109,821 pub-
lications that review the topic of “plant stress” only in the period 2000 to 2018. The
results indicated that the dose–response is not always linear, observing a biphasic
response when the doses allow it. This balance of response between the plant and
stress factors provides information on a positive (eustress) or a negative effect
(distress), a phenomenon called “Hormesis” (Agathokleous et al. 2019;
Vázquez-Hernández et al. 2019). The concept of hormesis is a term used in med-
icine for the application of toxins in low doses (Calabrese 2004). Paracelsus (1493–
1541) defined it as “All things are poison and nothing is without poison, only the
dose permits something not to be poisonous” (cited by (Vázquez-Hernández et al.
2019)). Currently, the term is applied in horticultural and agricultural practices as a
biphasic response in which doses of a toxic agent could cause inhibition (distress)
or can cause stimulation (eustress) (Vargas-Hernandez et al. 2017).
5 Role of Stress and Defense in Plant Secondary Metabolites … 153
Plants are sessile organisms susceptible to the interaction between various types
of stress, which has resulted in an evolved defense system that increases the syn-
thesis of secondary metabolites (Ghorbanpour et al. 2014). The variety of stress
factors together can affect the plant physiology, plant-plant interaction, defense
type, reproductivity, among others. For example, salinity and low/high-temperature
are conditions which restrict plant growth and productivity (Akula and Ravishankar
2011). In the signaling response to pathogens or herbivorous insects, several
response pathways are invoked, some of these are induced by infection and some
are performed regardless of the antimicrobial nature (Zaynab et al. 2018). Another
example is the interaction between plants and herbivory insects that causes the plant
to emit volatile organic compounds which influence the plant-to-plant communi-
cation, pollinators, and other insects, and increase fluidity of cell membranes for
thermo-tolerance and leaf tissue protection from atmospheric oxidants within and
around leaves (Faiola and Taipale 2020).
The foregoing indicates that plants can react in various ways in the presence of
one or more stress factors and that, in the same way, the response to the stimulus
may be the activation of a synthesis pathway of only one metabolite or a series of
secondary metabolites. For this reason, this work will focus on stress factors and the
production of secondary metabolites in plants.
5.2 Abiotic Stress
“Any unfavorable condition or substance that affects or blocks a plant's metabo-

lism, growth or development” is the definition of plant stress suggested by
Lichtenthaler (1996). Currently, this definition has been modified depending on the
stimulus origin, defining as stress factors those stimuli that are external to the plant,
biotic (fungi, insects, etc.) or abiotic (temperature, luminosity, nanoparticles, metals
and polluting salts, water, etc.) (Kranner et al. 2002; Thakur et al. 2019). Abiotic
stress origin is not biological and can be divided into chemical or physical
(Vázquez-Hernández et al. 2019). For example, the adaptation to cold environments
in some plants is the result of an increase in the synthesis of flavonoids due to
acclimatization processes at low temperatures or by the application of UV radiation
(Samanta et al. 2011; Nakabayashi et al. 2014). In Vitis vinifera, it has been
observed that stimulation with heavy metals such as Cadmium (Cd2+), Cobalt
(Co2+) and Silver (Ag+), can increase the synthesis of Resveratrol (Cai et al. 2013),
while the application of UV-C irradiation induces the synthesis of stilbene (Wang
et al. 2010; Liu et al. 2010). This indicates that the synthesis of secondary
metabolites will depend on various factors such as the type of stimulus, the con-
centration, and the form of application. These same observations are appreciated by
Feregrino-Perez et al. (2018), where the effect of nanomaterials on germination,
development of plants, and synthesis of secondary metabolites is reviewed, con-
cluding that the stress level will depend on the used nanomaterial, the dose and the
time of exposition. Low/high-temperature, relative humidity in air, drought,
microelements shortage, and CO2 reduction are classical abiotic factors for plants
elicitation. The role of novel abiotic stress factors on the production of secondary
metabolites is described below.
5.2.1 Electromagnetic Sources
Electromagnetic phenomena can be seen as an abiotic stress elicitor to affect plants.

In this context, many reports have demonstrated more advantages than disadvan-
tages when strong or weak electric fields, magnetic fields were applied to plants
(Dannehl 2018). Electromagnetic sources have been studied as another possibility
to increase plant growth and development due to the alteration in the electrostatic
balance of the plant system at the cell membrane level (Radhakrishnan 2019). An
electromagnetic field is produced by a distribution of electric current and charge.
An electric field (EF) can occur under high-voltage lines and the units in the SI are
newtons per coulomb or, equivalently volts per meter (V/m); in the same way, a
magnetic field (MF) is usually measured in terms of its magnetic flux density whose
unit is expressed as Tesla (T) (Dannehl 2018).
Pulsed electric field PEF technology consists of the application of short, high
power electrical pulses to products placed in a treatment chamber, confined between
electrodes (Soliva-Fortuny et al. 2017). A high electric field can cause cell mem-
brane disruption, whereby inner secondary metabolites are released from intracel-
lular cell compartments, resulting in a high content of bioactive compounds
(Odriozola-Serrano et al. 2009; Janositz and Knorr 2010). In the case of MF,
several studies have used small boxes with coils, iron bars, a function generator,
and a power amplifier. The application of magnetized water to plants is a novel area
of indirect application of MF that the scientific community is currently researching
(Dannehl 2018).
Light is an electromagnetic wave within the visible spectrum, however, that
definition depends upon the sense of sight involving the response of individuals
(Koshel 2004), therefore, the UV and infrared parts of the electromagnetic spec-
trum are roughly included, but will be considered in this chapter due to their
importance in the plant production of secondary metabolites. Plants sense light
through specific molecules called photoreceptors that trigger specific signals for
photomorphogenesis or other defense systems. Depending on the dose rate and
exposure time, either insufficient or excess levels, light can become a type of
eustress, producing several effects, from damage to cellular components to trig-
gering of defense systems for secondary metabolite production (Alvarado et al.
2019; Muller-Xing et al. 2014; Akula and Ravishankar 2011). The next sections of
this chapter will discuss the effect of electromagnetic sources in the production of
natural bioactive compounds.
5.2.1.1 Light
Light is one of the most important and obvious requirements for plant growth and
development, where the energy of sunlight and artificial light sources is mainly
used for photosynthesis. However, light is not only involved in the photosynthesis
process but also in the production of natural bioactive compounds, gene expression,
and synchronization of the circadian clock in the light/dark cycle (Larner et al.
2018). Changes in the light intensity, quality, direction, and duration are sensed by
specialized photoreceptors which are specially designed proteins that sense light,
triggering chain reactions that have been studied in terms of photomorphogenesis
and primary and secondary metabolites production (Alvarado et al. 2019).
Photoreceptors perceive specific light wavelengths of over a continuous spectral
range through a small cofactor or chromophore molecule (Burgie et al. 2014). Five
photosensory systems have been identified: phytochromes perceiving red (660–
700 nm) and far-red (700–750 nm), cryptochromes, phototropins, and members of
the Zeitlupe family perceiving blue (495–400 nm) and UV-A (400–315 nm), and
UV Resistance Locus 8 (UVR8) perceiving (315–280 nm) (Bantis et al. 2018;
Alvarado et al. 2019).
Recent investigation has focused on the effect of light technology in plant
growth, developmental traits, and primary and secondary metabolites by using one
or more light wavelengths, intensities, and photoperiods. It has been reported that
blue light increases phenolic compounds by promoting the production of malonyl
CoA and coumaroyl CoA, participating in the synthesis of phenolic compounds
(Qian et al. 2016). In addition, red and far-red wavelengths are perceived by the
phytochromes photoreceptors, which regulates biosynthetic pathways involved in
the synthesis of anthocyanins, molecules that belong to the phenolic compounds
known as flavonoids and have many functions in plants including pigmentation
(Alokam et al. 2002). In the same way, plants produce secondary metabolites such
as flavonoids and anthocyanins to cope with cell damage produced by UV radiation
(Jiang et al. 2017b). Serious damage to DNA, membrane, and proteins can be
caused by UV-B radiation, whereas UV-A induces DNA damage less efficiently
because of the activation of photoreactions forming reactive oxygen species
(ROS) (Hideg and Strid 2017; Häder et al. 2015).
Supplemental lighting has been accepted for improving horticultural crops.
Light-emitting diode (LED) technology has been linked to controlled environments
in horticulture for achieving crop yield, phytochemical content, nutritional value,
flowering control, transplant success, pre-harvest and postharvest product quality,
and production of regeneration material (Bantis et al. 2018; Alvarado et al. 2019).
LEDs have allowed a sustainable and highly efficient use of energy and reproduce
true spectral composition of blue, green, red, and far-red wavelengths that matches
with plant-specific photoreceptors (Singh et al. 2015). Other light technologies, as
high sodium pressure (HSP) and other high-intensity discharge (HID) lamps are still
used in greenhouse and plant experimentation, however, LED technology is
replacing these devices due to the various advantages LEDs offer. Table 5.1
summarizes some examples of the application of supplemental light on plants or
foods with a commercial interest and presents the effect on the production of natural
bioactive compounds.
5.2.1.2 Electric and Magnetic Fields
Magnetic fields (MFs) are considered an abiotic factor that can induce eustress with
significant effects on the growth and development of plants. The effect of light,
gravity, mechanical damage, and electrical signaling on plants has been studied and
documented over the past years concluding strong facts relating to phototropism,
gravitropism, and thigmotropism (Maffei 2014). The geomagnetic field (GMF) is a
natural component of our environment, however, its impact on plant growth and
development is not well-understood, moreover, the effects of artificial magnetic
fields on plants have been poorly studied (Maffei 2014). Several experiments with
lower and higher values than the GMF has been conducted with predominantly
positive effects depending on the plant, time of exposure and intensity. For
example, an increase in germination or subsequent seedling growth barley, corn,
beans, wheat, hornwort, mung bean, pea, chickpea, tomato, and okra, but it was
reduced in seeds of rice. In a similar way, the effect on roots, shoots, gravitropism,
photosynthesis, and lipid composition present a similar pattern (Maffei 2014).
Several theories and studies about the biological effect on MF have been pro-
posed. A polar structure in various chemical bonds in the organic material may be
linked to the polar water molecules and dissociated ions of mineral salts conferring
magnetic properties (Chepets et al. 1985). A MF can decrease the disease index of
plants due to the modulation of calcium signaling, and proline and polyamines
pathways (Radhakrishnan 2019). The plant cells contain about 4500 iron atoms in
the ferritin molecules involved in growth and metabolism. The magnetic rotator
moment of ultimate iron atoms creates an external MF which collectively generates
an atom re-positioning in the direction of MF that leads to an increase of the plant
temperature (Vaezzadeh et al. 2006). Photoreceptors have been also proposed to be
potential magnetoreceptors since cryptochromes and phytochromes produce radical
pairs after the exposure to their corresponding light wavelength triggers (Maffei
2014; Dhiman and Galland 2018). Cryptochrome-dependent responses such as
blue-light-dependent anthocyanin accumulation and blue-light-dependent degra-
dation of CRY2 protein were enhanced at higher magnetic intensities in
Arabidopsis mutants lacking cryptochromes (Ahmad and Jones 1979). Limited
information is available on the molecular basis and the function of the MF receptors
and their activation by physiological signals, therefore, their involvement in
directing the overall response in different plant organs is yet to be determined
(Radhakrishnan 2019).
Static magnetic field (SMF) exposition in plants has been found to be an
effective and emerging tool to control diseases and increase tolerance against the
adverse environment (Radhakrishnan 2019). However, a small number of studies
have been attempted to determine the role of MF on plant tolerance against various
Table 5.1 Effect on natural bioactive compounds of experiments where supplemental light was
the stress factor on plants or food with commercial interest
Food or cultivar/ Light condition treatment Result
Reference
Stored tomato fruit var. (T1) Darkness (control), (T2) (") Lycopene concentration.
Cappricia/Panjai et al. Darkness + UV, (T3) R, (T4) Sharply increase with T3 and
(2017) R + UV. UV of 4.98 kJ m−1 T4
per 30 min day−1 (") Concentration of
T2/T4: UV tube for 15 min in b-carotene. The highest at T3
the morning and at night after 10 days of postharvest.
T3/T4: 60% UV-B, 30% T2 had the highest value after
UV-A, 4% UV-C and 6% 15 days
visible light (") TFC. The highest with T3
R (665 nm): applied for the after 10 and 15 days of
whole storage period, postharvest
equivalent to PAR of (#) TFC. T2 showed a
113 lmol m−2 per day significant decrease at day 5
(") TPC. A sharply increase
with T3 at day 10 and peaked
on day 20
(") AC – ABTS. T4 showed
highest Hydrophilic and
Lipophilic AC 20 days after
harvesting compared to control
Stored habanero pepper Combination of B (0, 1.5, and (") TPC and TFC. All
(Capsicum chinense)/ 3 min) and UV-C (0, 0.5, and treatments with B and/or UV-C
Pérez-Ambrocio et al. 1 min) showed a significant increase
(2018) B: 48 W m−2 compared to control. The
UV-C: 11.3 W m−2 highest at 3 min of
B + 0.5 min of UV-C
(") TCC. Increase the first
10 days of storage
(") Capsaicin. Almost all
treatments (1.5 min of B and
1.5 min of B + 1 min UV-C)
presented an increase in
capsaicin
(") AC. Statically higher in all
treatments with B and UV-C
light
Green and purple basil Ten treatments: Combination (") TAC. 9–23% higher after
(Ocimum basilicum) of two PPFDs and five UV-B UV-B radiation compared to
plants/Dou et al. (2019) radiation doses. PPFDs: 160 control for green basil. Greater
and 224 lmolm−2s−1 (high under high PPFD for purple
and low) with a 16-h basil
photoperiod provided by cool (") TPC. 28–126% higher after
white fluorescent lamps with UV-B compared to control for
UV of 2.2 and green basil. Greater in high
2.5 lmol m−2 s−1, PPFD for purple and green
respectively basil. 29–63% higher under
UV-B (16.0 lmol m−2 s−1):
(continued)

Reference
(Control) No UV-B 2H2D and 2H5D for purple
(1H2D) 1 hd−1 for 2 days basil
(2H2D) 2 hd−1 for 2 days (") TFC. 80–169% higher after
(1H5D) 1 hd−1 for 5 days UV-B compared to control for
(2H5D) 2 hd−1 for 5 days green basil. 37–79% higher
under 2H2D and 2H5D for
purple basil
(") AC. Higher in green basil
under all supplemental UV-B
treatments. Only higher under
2H2D and 2H5D in purple
basil plants
Coriander (Coriandrum R (661 nm) and B (449 nm) (") AC – DPPH. 2.0, 1.6, and
sativum)/Naznin et al. ratios (R:B) of LED light 1.5 times higher, under 5:1,
(2016) Four treatments: 100% R, 5:1, 10:1, and 19:1 respectively,
10:1, and 19:1 compared to the plants under
Photoperiod of 16/8 h (day/ 100% R
night) and PPFD of
120 lmol m−2 s−1 in growth
chamber
Soybean (Glycine max Experiment 1: B (450– (") TPC and isoflavones.
L.) sprout/Azad et al. 495 nm), G (510–550 nm) Higher under B compared to G
(2018) LEDs and florescent lamps and fluorescent light.
(control). PPFD in growth Isoflavones increased at five
chamber was and six DAS
150 µmol m−2 s−1. Samples Significant increase in total
were harvested at 3rd, 4th, 5th, isoflavones with FIR of 110 °
6th, and 7th days after sowing C/120 min, nearly 2.3 times
(DAS) higher than the control. Further
Experiment 2: Far Infrared increase in the FIR temperature
(FIR) irradiation, photoperiod decreased the isoflavones
was same as experiment 1. content
Exposure time for 30, 60, and (") AC – DPPH and
120 min at 90, 110, and 130 ° FRAP. DPPH 75% higher with
C on harvested sprout B compared to 69% with G
and 58% with control. FRAP
was more than 2 times higher
in B compared to control
FIR 110 °C/120 min treatment
had the highest FRAP value
among the FIR treatments
Blueberry Leaves R (661 nm–24 µmol m−2 s−1) (") TPC. Higher under 12 h of
(Vaccinium and B (417 nm– B light compared to untreated
corymbosum L.)/ 6 µmol m−2 s−1) at 12, 24, and leaves (control)
Routray et al. (2018) 48 h (#) TPC. 48 h induced
deterioration of TPC in all
observed cases. R light led to a
decrease in TPC compared to
control
(continued)

Reference
(") Monomeric anthocyanins.
An increase with time up to
24 h with B light, after which
it decreased. Lower than the
untreated sample with R light
(") AC. The extract prepared
with leaves treated with R light
for 12 h had higher FRAP than
control and treated samples
with both R and B. For 24 and
48 h, FRAP from treatment
with B light was maintained at
a higher level as compared to
samples treated with R
Persimmon (Diospyros Two maturity stages (unripe (") TPC. Higher than control in
kaki L. cv. Vanilla) and ripe) were exposed to a unripe fruit exposed to
fruit/Denoya et al. pulsed light treatments of 20 20 kJ m−2
(2020) and 60 kJ m−2. Time of In ripe fruit, No significant
exposure: 1.2 s, Frequency: differences in TPC were found
3 Hz, 5.7 kJ m−2 per pulse, of between treatments
polychromatic light in the (") AC - ABTS, DPPH, and
wavelength range between 200 FRAP. Al treatments presented
and 1100 nm higher AC than the control
treatment in unripe fruit
Used abbreviations PAR Photosynthetic available radiation, PPFD Photosynthetic photon flux
density, LED Light-Emitting diode, R Red, B Blue, UV Ultraviolet, AC Antioxidant capacity, TCC
Total carotenoid content, TPC Total phenolic content, TFC Total flavonoid content, TAC Total
anthocyanin content, DW dry weight, FW fresh weight, (") increment of, (#) decrement of
stress conditions (Radhakrishnan 2019). The effect on secondary metabolites pro-

duction has been scarcely studied; presenting an opportunity area in this field.
Electrostatic fields (EF) has also been used in the horticultural industry. Most of
the applications of this technology are for germination and seedlings improvement
due to its importance in the supply chain. For example, drought resistance and
removal of free radicals in maize seedlings with electric field intensity 200 kV/m,
pulse width 80 ms and frequency 1 Hz were analyzed by He et al. (2017), where
the growth of root, the ability of self-organization, and the respiration metabolism
of root cells was improved. Another example with potato tubers subjected to pulsed
electric field (PEF) through a treatment voltage across the potatoes of 5 kV and a
discharge capacitance of 450 pF prior to planting had an increase in the yield of 22–
29% (Gachovska et al. 2015). In the same way, winter wheat seeds increased the
germination energy up to 32.41% and weight up to 23.8% under PEF
(Starodubtseva et al. 2018). In the experiment of Yan et al. (2017), PEF through a
high-voltage pulse power supply and arc electrode was studied on cotton seeds
vigor with different frequencies of 1, 5, 10, 20, and 50 Hz at the voltage of 16 and
20 kV, and the treatment time was 40 s, finding that when the frequency of the
electric field increased, the effects increased and reached the maximum at 10 Hz,
and after 10 Hz, as the electric field frequency increased, the effects began to
decrease. This last experiment exemplifies the hormetic curve in a vigor treatment
where there is a maximal dose at which a maximum response value is reached and
then vigor starts decreasing.
In several studies, PEF pretreatments in plants, whole fruits, or other food
sources result in an increase in the natural bioactive compounds. When biological
cells are exposed to an EF, the charge accumulates along the plasma membrane
causing electroporation, a transmembrane potential difference which causes
porosity, and thus the diffusion of intracellular components in cellular juice
increasing the extractability of natural bioactive compounds by the release of
solutes into the solvent (El Kantar et al. 2018; Vicaş et al. 2017; Barba et al. 2015;
Hendrawan et al. 2019). The time exposure and intensity of the EF are critical since
a lower EF may form smaller pores allowing the ions to pass through, but large
molecules may not get out of the cell, however, higher EF are suspected to damage
antioxidant compounds due to long exposure to high-voltage electric current
(Hendrawan et al. 2019). Table 5.2 summarizes some examples of the application
of magnetic or electric fields on plants or foods with a commercial interest and
presents the effect on the production of natural bioactive compounds.
5.2.2 Acoustic Emissions
Acoustic emissions (AE) stimulus is one of the recent physical abiotic factors
whose beneficial effects on plant growth, development, and health have been dis-
cussed. AE from ecological conditions or artificially applied can initiate diverse
signals that trigger transduction cascades, similar to other abiotic stress factors
(Alvarado et al. 2019). From a bioacoustics perspective, chewing serves as an alarm
signal to plants and has been demonstrated that applying recorded insect chewing
sounds caused an increase of phytochemical production (Appel and Cocroft 2014).
In the same way, Jeong et al. (2014), reported an improvement of natural protection
responses in rice plants caused by amplification at 100 decibels of a wide range of
frequencies between 0 and 1.5 kHz. Moreover, Hassanien et al. (2014), found a
higher disease resistance in pepper, cucumber, and tomato after AE treatments.
The biological mechanism of how sound affects plants is still under discussion.
A mechano-stimuli perception of waves has been proposed, but a reliable expla-
nation of sound-specific structure for recognition by plants has not been completely
elucidated (Alvarado et al. 2019). This mechanism consists of the second mes-
senger of calcium ion (Ca2+) signals. The channels that mediate Ca2+ flux are
possibly located in the plasmatic membrane where Ca2+ is sensed possibly through
various Ca2+ sensors and/or CDPKs (Calcium-dependent protein kinase), which
pass the message through phosphorylation/dephosphorylation to different signaling
Table 5.2 Effect on natural bioactive compounds of experiments where magnetic or electric fields
was the stress factor on plants or food with a commercial interest
Food or cultivar/Reference MF or EF treatment Result
Apples var. Golden PEF: (") AC. 24% at (a) just after
Delicious (a) 0.4 kV cm−1, 5 pulses treatment. (Not significant
(Whole fruit)/Ribas-Agustí (0.01 kJ kg−1, 20 ls total 24 h after treatment)
et al. (2019) treatment time) (#) AC. 39% at (c) just after
(b) 2.0 kV cm−1, 35 pulses treatment. At (b) 46% and
(1.8 kJ kg−1, 140 ls total (c) 62% after 24 h
treatment time) (") TPC and flavan-3-ol. 25–
(c) 3.0 kV cm−1, 65 pulses 26% in TPC and 43–35% in
(7.3 kJ kg−1, 260 ls total total flavan-3-ols just after
treatment time) and 24 h after treatment
The system supplied 4 ls (respectively) with (a)
monopolar pulses at a fixed (#) TPC by 32% at (b) and
frequency of 0.1 Hz 43% at (c) just after
treatment and 50% at (b) and
66% at (c) after 24 h
(#) Total flavan-3-ols by
19% at (b) and 51% at
(c) just after treatment and
52% at (b) and 59% at
(c) after 24 h
(#) TFC by 28% at (b) and
50% at (c) just after
treatment and 60% at (b) and
68% at (c) 24 h after
treatment
Orange, pomelo and lemon PEF of 3 kV cm−1 for whole (") by approximately 39%
fruits/El Kantar et al. (2018) fruits and 10 kV cm−1 for for orange, 66% for pomelo,
stacks of skins. Time interval and 135% for lemon in the
between pulses: 2 s. Pulse release of polyphenols from
duration: 70 ls the inner parts of the cells
Apples var. Golden PEF intensities of 0.4 – (") TPC (13%) and
delicious (Whole fruit)/ 2 kV cm−1, using 5–35 flavan-3-ol (92%). Best
Soliva-Fortuny et al. (2017) monopolar pulses of 4 ls at result with 0.008 kJ kg−1 in
a frequency of 0.1 Hz, apples stored 24 h at 22 °C
corresponding to an specific (") Flavonoids (58%). Stored
energy input of 0.008 – at 4 °C for
1.3 kJ kg−1 (") AC enhanced by 43%
after 12 h at 4 °C and by
15% after 24 h at 22 °C
Tissues of apple var. Ligol PEF intensities of 1.85, 3 (") TCC up to 11.34% in
and carrot var. Baltimore/ and 5 kV cm−1 with the carrots with 1.85 kV cm−1
Wiktor et al. (2015) combination of 10, 50 and regardless of the applied
100 exponential shaped pulse number
pulses of average 10 ls (") Maximal TPC and
width each. Interval between antioxidant activity (EC50)
pulses was set at 2 s in 10 pulses at
1.85 kV cm−1 in apple tissue
(continued)

(#) TCC up to 25.33% with
3 kV cm−1
(#) TPC and EC50 up to
35.93 and 32.95%
respectively at 5 kV cm−1
and 100 pulses
Wine of grapes Muscat Grape mash was treated with (") TPC. 1.4 times higher in
Ottonel (MO), Pinot Noir PEF and then fermented for wine of MO grapes, 2.98 in
(pn), and Merlot (MT)/Vicaş wine elaboration wine of PN grapes, and 1.72
et al. (2017) Pulses of 150 µs of in wine of MT grapes, than
7 kV cm−1 were applied the untreated one
with a frequency of 178 Hz (") TFC. The highest in PN
wine, followed by MT and
MO in which the grapes
were PEF treated before the
fermentation process
(") TAC. An increase of
11.11% in the case of PN
and only 5.22% in the case
of MT
(") AC. The highest
in PN wine treated by PEF
Blackberries (Rubus PEF of 13.3 kV cm−1 and (") TPC yield using HVED
fruticosus)/Barba et al. High Voltage Electric (333.8 mg/100 g), followed
(2015) Discharge (HVED) of by PEF (108.0 mg/100 g)
40 kV. Distance between Extracts obtained after
pulses of 2 s. 1 min pause applying PEF were more
was made after each 100 clear and stable than HVED
pulses. Damped oscillations assisted extraction
duration of 10 µs. TPC relative recovery of
Discharges PEF after supplementary
were applied with a extraction was also higher
repetition rate of 0.5 Hz, (sixfold and fourfold higher
after hot water and ethanol
extraction, respectively) than
for HVED (1.8-fold and
1.5-fold higher for hot water
and ethanol extraction,
respectively)
(") Anthocyanin Content,
similarly to TPC
Basil leaves/(Hendrawan PEF. Two factors. (") TPC. The highest
et al. (2019) Combination of 2, 3, and (115.203 ± 1.115 mg GAE
4 kV cm−1 with an exposure g−1) at 3 kV cm−1 for 2 min
time of 1, 2, and 3 min while the lowest
(23.507 ± 1.656 mg GAE
g−1) at 2 kV cm−1 for 1 min
(continued)

Medicinal herb MF and Fe3O4 magnetic (") TPC. All treatments
Dracocephlum polychaetum nanoparticles (MNP). SMF1: higher than control. The
Bornm/Taghizadeh et al. (3 days, 3 h day−1) and highest at SMF2 + MNP
(2019) SMF2: (4 days, 5 h day−1) with 5.9 mg g−1 FW (1.73
both of 30 mT fold increase compared with
control)
(") TFC. All treatments
higher than control. The
highest at treatments with
MNP with 405.65 µg g−1
FW
(") TAC. Significantly
higher by either
SMF + MNP or SMF2 as
compared to the control
samples. The highest at
SMF2 + MNP with
37.41 nmol g−1 FW
corresponding to 1.95 times
higher than control
Almonds seeds from two MF. 10 mT for 5 h per day, (") TPC compared to control
plant species (Amygdalus for 4 days group. (About 175 and
scoparia Spach 240 mg g−1 DW for A.
and A. eburnea Spach)/ scoparia and A. eburnea
Abdollahi et al. (2019) respectively)
(") TAC remarkably
compared to control
group. (About 0.015 and
0.009 mmol g−1 FW for A.
scoparia and A. eburnea
respectively)
(") AC compared to control
group. (About 45% and 40%
for A. scoparia and A.
eburnea respectively)
Note: According to the
DPPH test, SMF treated
seeds were able to convert
the stable radical DPPH into
yellow diphenyl picryl
hydrazine more readily than
those in the control group
Grapes. Two cultivars Magnetic Solutions (MS) (") TPC. All higher than
‘Rasha’ and ‘Sultana’/ Two methods for the control. The highest at T2
(Zareei et al. (2019) preparation of MS and T5, up to 53.25 and
(a) Prepared Hoagland 52.1 mg g−1 FW,
solution was passed through respectively, in Rasha. The
0.1 T (T2) and 0.2 T (T3) highest at T4 with
(continued)

(b) Distilled water was first 25.57 mg g − 1 FW in
exposed to 0.1 T (T4) and Sultana
0.2 T (T5) and then the salts (") TAC. The highest at T2
were added to the with 0.76 mg g−1 FW
magnetized water. among all treatments with
Magnetized water or nutrient Rasha. The highest at T2
solution was provided by with 0.58 mg g−1 FW
passing them through among all treatments with
magnets installed on the Sultana
pipes at the flow rate (") TFC. All higher than
of 3 L min−1 control in Rasha. The
highest at T4 and T2, 52.38
and 50.97 mg g−1 FW
respectively. No statistic
difference with Sultana
(#) AC. T4 and T5 on the
Sultana grapevines presented
73.63% and 74.83%,
respectively, values lower
than control
(") Trans-resveratrol. Based
on HPLC–DAD results, the
peak area of trans-resveratrol
increased in Rasha and
Sultana grapevines subjected
by T3 and T4, 45.03 and
44.35 ± 0.5 lg g−1 FW,
respectively,
Tea plant (Camellia sinensis MF. Eight different (") TPC. Higher than control
L.)/Azizi et al. (2019) treatments: with (b), (c), (d), and (e).
(a) 1 mT during 30 min The highest with (b), about
(b) 1 mT during 60 min 0.4 gGae g−1
(c) 2 mT during 30 min (#) TPC. Lower than control
(d) 2 mT during 60 min with (f), (g), and (h)
(e) 4 mT during 30 min (") TFC. Significantly higher
(f) 4 mT during 60 min than control with (b) and (d),
(g) 6 mT during 30 min about 225 and 140 mg g−1
(h) 6 mT during 60 min of QE respectively
All for 7 continuous days (#) TFC. Lower than control
with (h)
Note. This study exemplify a
hormetic condition, where
low doses have a beneficial
effect on the production of
secondary metabolites and
high doses have a negative
effect
(continued)

Seedlings of moringa (Two MW (Tap water after (#) TPC lower in both
species Moringa oleifera magnetization through species. M. olifera and M.
and Moringa peregrina)/ passing in a magnetron of 30 peregrine seedlings exposed
Hasan et al. (2018) mT, output 4–6 m3 h−1) to MS, SS increased TPC by
under medium (MS) and 20, 30 and 13, 29% under
severe (SS) drought stress tap water, however,
decreased by 11%, 15% and
16%, 21% with MW
(#) TFC and AC. Similar
pattern than TPC
Lens (Lens culinaris L.) MW. Tap water was treated TPC, TAC, and proline in
plants and seeds/(Azimi by the magnetic field of 110 the leaf of both control and
et al. (2018) mT MW-treated plants were
identical
(") AC. The Ferric reducing
antioxidant power in plant
and seeds leaves was
significantly increased by
MW
(") TCC. Noticeably
increased in leaf by MW
No significant difference was
observed between TFC and
TAC of seeds of both groups
( #) TPC and proline.
Significantly lower than
control in seeds
Blackberries. Wild (Rubus MF. Homogenous 50 Hz (") TAC. 9–33% in wild
sulcatus Vest) and cultivated MF with 3 mT magnetic flux blackberries and 30–129% in
(Rubus fruticosus density at 1, 2, 4, 6, and 12 h cultivated ones depending of
Thornfree)/Răcuciu and exposure times irradiation time. The highest
Oancea (2018) with 1 h, both in wild and
cultivated. Long MF
exposure (12 h) caused a
decrease in wild blackberries
by 20% compared to control
(") TPC. 5–27% in wild
blackberries and 6.5–63% in
cultivated ones depending of
irradiation time. The highest
of both was recorded after
1h
Enhanced levels of TAC and
TPC of blackberries were
obtained for samples
exposed to homogeneous
magnetic field (50 Hz, 3 mT)
for relatively short times (1–
6 h) compared to control
Used abbreviations MW Magnetized water, MF Magnetic field, PEF Pulsed electric field, AC
Antioxidant capacity, TCC Total carotenoid content, TPC Total phenolic content, TFC Total
flavonoid content, TAC Total anthocyanin content, DW dry weight, FW fresh weight, (") increment
of, (#) decrement of
proteins or to transcription factors (Mishra et al. 2016). In that way, it is strongly

suggested that AE can influence the synthesis of secondary metabolites. The most
common acoustic emission utilized for the stimulation of bioactive compounds is
ultrasound (US). Several mechanisms of how the US interacts within the cell have
been proposed. When the US is applied, cavitation bubbles creates a pressure zone
change that occurs and increases up to 400 km h−1, causing higher porosity, rupture
or removal of cell membranes, facilitating the mass transfer from the cells’ interior
when imploding (Toma et al. 2001; Vinatoru 2001). In that way, the increase of
natural bioactive compounds may be caused by better extractability due to rupture
of membranes of cell organelles, however, when a decrease is presented, it could be
explained by the creation of reactive forms of oxygen (ROS) during cavitation, and
that the collapsing bubbles release high doses of energy, raising the temperature
(>5000 K) enough to decompose polyphenols (Witrowa-Rajchert et al. 2014;
Kentish and Ashokkumar 2011). The second possible explanation is an enhance-
ment of enzymes activity when the US is applied by contact, leading to phenolic
compounds’ reduction, more significantly after longer treatment time (Wiktor et al.
2016). Ampofo and Ngadi (2020) established that elicitation of common beans with
the US, increased the accumulation of stress markers from the onset until the
process was arrested, signifying a demand for sprout protection, resulting in an
elevated stimulation of defense phenolic triggering enzymes (PAL and TAL), and
final biosynthesis of phenolic compounds. In that way, application in food or plants
of US treatment should be studied to find the optimal time, frequency, and intensity
in order to optimize the production of secondary metabolites. Table 5.3 summarizes
some examples of the application of acoustic emissions on plants or foods with a
commercial interest and presents the effect on the production of natural bioactive
compounds.
5.2.3 Nanoparticles
Nanoparticles (NPs) vary in size from 1 to 100 nm and have physicochemical

properties, due to their dimensions, which generate a high added value for the
nanotechnology industry (Yokel and MacPhail 2011). Nanoscale materials can be
found on medical imaging, drug delivery, personal care products, cosmetics,
clothing, electronics, agrochemicals, motor vehicles, among other products and
applications (Vance et al. 2015; Yokel and MacPhail 2011). The metal-based NPs
most commonly studied and found in industrial products are Cd (cadmium) in
various complexes, GaAs (gallium arsenide), Au (gold), Ni (nickel), Pt (platinum),
Ag (silver), Al2O3 (aluminum oxide or alumina), CeO2 (cerium dioxide or ceria),
SiO2 (Silicon dioxide or silica), TiO2 (titanium dioxide or titania), ZnO (zinc
oxide), CuO (copper oxide), and Fe3O4/Fe2O3 (iron oxides) (Khot et al. 2012;
Yokel and MacPhail 2011). Among the carbon-based nanomaterials often studied
are fullerene, single-walled carbon nanotubes (SWCNTs), and multiwalled carbon
nanotubes (MWCNTs) (Balbus et al. 2007).
Table 5.3 Effect on natural bioactive compounds of experiments where the magnetic or electric
field was the stress factor on plants or food with a commercial interest
Food or cultivar/ AE stimuli Result
Reference
Romaine lettuce US of 25 kHz and (") TPC. After 60 h storage,
(Lactuca sativa, var. 2 kW nominal power. The sample of 1-min had a 22.50%
longifolia)/Yu et al. acoustic energy delivered 69.4, higher TPC than control
(2016) 138.8, and 208.3 kJ for (#) TPC. After storage for
treatments at 1, 2, and 3 min, 30 h, samples treated for
respectively 1 min had significantly lower
TPC than the control
(") AC. After 60 h storage, the
DPPH inhibition for 1, 2, and
3 min was 97.84, 75.22, and
75.87%, significantly higher
than the control, respectively.
After 90 h storage, only the
AC of samples sonicated for
2 min remained significantly
higher than the control
Note: The AC with 1 and
2 min US decreased by 50.87
and 64.24% compared with
the control respectively during
the first 30 h storage, followed
by a significant increase 97.07
and 83.67% respectively
during the next 30 h
Black cumin (Nigella US pretreatment. 30, 60, and (") TPC. With enhancements
Sativa)/Moghimi et al. 90 W with the constant in US power from 30 to 90 W,
(2018) frequency equal to 25 kHz, TPC increased from 93.21 to
and irradiation time of 30, 45, 106.6 ppm. 1 h after the
and 60 min extraction process, TPC was
5% higher compared to control
(") AC. Increment in the AC
of the extracted infusions
Lavender (Lavandula US (pulse system (") AC. Essential oils obtained
Stoechas L) from Adekar 270, Italy, 26 kHz, 150 W) for by US-HD had a higher AC
and Keddara regions/ different times: 10, 20, 30, 45 than those obtained from
Lilia et al. (2018) and 60 min were applied on untreated samples. “Keddara
the plant materials as a and Adekar of treated and
pretreatment before untreated samples revealed a
hydrodistillation percent of inhibition of DPPH
of 20.22 ± 1.72% and
18.88 ± 2.08%,
23.96 ± 3.08% and
20.70 ± 4.41% respectively”
Note: Antioxidant activities of
the essential oils from
aromatic plants are mainly
attributed to the active
compounds present in them
(continued)

Reference
Commercially mature High intensity US (25 kHz (") TPC. Higher than control
tomato (Lycopersicon frequency with a nominal at all storage times. 2 h
esculentum) fruit/Lu power of 1 kW) for 1, 2, 3, storage: maximum at 2 min,
et al. (2020) and 4 min. Storage time of 2, and decrease with longer US
24, and 48 h time. 24 h storage: the highest
at 1 min US (17.05% higher
than control). 48 h storage:
2 min US presented an
increase in TPC
(") LC. 2 h storage:
significantly higher at 3 min
US. 24 h storage: the highest at
2 min US (12.21% higher than
the control fruit). 48 h storage:
the highest at 2 min US
(") TCC. 24 h storage: an
increase in all the US
treatments (significantly
higher than the control at
2 min). 48 h storage: the
highest at 1 min US (17.13%
higher compared to the
control)
(") AC. All the US treatments
had a higher DPPH than the
control in all storage times,
except the samples treated
with US for 3 min
Common bean US at power levels of 360 and (") Total Phenolic Acids. The
(Phaseolus vulgaris) 180 W (40 kHz) and time greatest (216.7 mg 100 g−1)
sprouts/Ampofo and levels of 30, 45 and 60 min with 360 W (60 min) at 96 h
Ngadi (2020) of sprouting, 11.65 folds
compared to control
(") TFC. The greatest
(203.5 mg 100 g−1) with
360 W (60 min) at 96 h of
sprouting, 6.6 folds compared
to control
(") TAC. Significantly higher
(30.35 mg 100 g−1) at 96 h of
sprouting with 360 W
(60 min), 11.54 folds
compared to control
(") AC. Significantly higher
(97.81% with DPPH and
98.34% with ABTS) at 96 h of
sprouting with 360 W
(60 min), 13.84% and 25.57%
folds compared to control,
respectively,
(continued)

Reference
Apple (Malus domestica US at 21 kHz or 40 kHz and (") TPC. 543.4 mg 100 g−1
var. Ligol) tissue/Wiktor 180 W. Sonication lasted for DW at 21 kHz - 30 min and
et al. (2016) 0, 5, 10, 20 and 30 min 1046.5 mg/100 g−1 DW at
40 kHz and 5 min, 27.4% and
145.3% higher than control,
respectively,
(#) TPC. Only at contact
sonication method for 30 min
(c_US_30), for which the
lowest amount of TPC
(298.5 mg/100 g DM) were
determined. 30% less than raw
apples
Black currant fruits US of 150 W power and (") TPC. Increased by
(Ribes nigrum L.)/ 40 kHz frequency. Three approximately 4% compared
(Oancea et al. (2014) predetermined extraction times to control at 10 min and 70%
(3, 6 and 10 min) and three amplitude in all three cases of
ultrasonic amplitudes (10, 40 sample preparation
and 70%). Sample preparation: (") TAC. Frozen: increase by
frozen, freeze-dried, and oven 4% at 3 min at 70% amplitude.
air-dried Freeze-dried: increase by 20%
at 10 min and 70% amplitude.
Oven air-dried: increase by 7%
All compared to control
(") AC – FRAP method.
Frozen: increase by 1% at
6 min and 40% amplitude.
Freeze-dried: increase by 28%
Oven air-dried: increase by
144% at 3 min and 70%
amplitude. All compared to
control
Used abbreviations US Ultrasound, LC Lycopene content, AC Antioxidant capacity, TCC Total
carotenoid content, TPC Total phenolic content, TFC Total flavonoid content, TAC Total
anthocyanin content, DW dry weight, FW fresh weight, (") increment of, (#) decrement of
In contrast to its benefits, the release of nanomaterial-containing wastes has

become a threat, since they cause pollution to air, water, and soil (Oberdörster et al.
2005). Their size, equivalent to that of cellular components, allows them to easily
permeate cells, causing adverse biological effects (plants and animals) (Shang et al.
2014; Vecchio et al. 2012). In plant cells, NPs can enter from the apoplast and cross
the plasma membrane towards cytosol or other organelles via endocytosis, specific
membrane-bound transporter proteins or through induction of new pores by using
ion carrier substances; subsequently, they can be transported between cells through
symplastic flow (Anjum et al. 2019; Marslin et al. 2017). Despite its potential of
toxicity, studies have reported positive effects on plant development and physiol-
ogy, which are dependent on the nature of the nanomaterial, dose and time of
exposure, the plant species, and growth conditions (Cox et al. 2016).
Positive physiological effects using carbon-based NPs include increased water
uptake, and enhanced assimilation of CO2 in broccoli (Martínez-Ballesta et al.
2016), promotion of seed germination and root growth in rice (Jiang et al. 2014),
enhanced germination and seedling growth in sweet corn, barley, rice soybean,
switchgrass, and tomato (Lahiani et al. 2015; Tiwari et al. 2014), increase fruit yield
in tomato and bitter melon (Khodakovskaya et al. 2013; Kole et al. 2013), among
many others. Studies using metallic nanoparticles have reported similar effects. In
wheat, CeO2 particles improved plant growth, shoot biomass, and grain yield (Rico
et al. 2014). Au nanoparticles in chinese mustard (Brassica juncea) had positive
effects on growth parameters and seed yield (Arora et al. 2012). The response of
maize exposed to ZnO nanoparticles showed enhanced germination, seedling vigor,
and zinc biofortification of grains (Subbaiah et al. 2016).
The effect of NPs on secondary plant metabolism is still largely unknown
compared to physiological and phenotypic responses. However, studies have shown
that a constant response between species is the induction of reactive oxygen species
(ROS) (Marslin et al. 2017). Studies reporting NPs-elicitation of specialized
metabolites often report a reduction in the photosynthetic rate and inhibition of
growth. These phytotoxic effects have been linked to the inhibition of
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity and decreased
photo-protective capacity of PSII (Jiang et al. 2017a; Wang et al. 2016). When NPs
permeate the cells, damage on the photosynthetic apparatus is done because of its
accumulation in chloroplasts, at the same time, when they cross the plasma
membrane they probably dissociate into ions (rather than stay as intact particles)
and bound to NADPH oxidases, causing the production of ROS at the apoplast
(Jiang et al. 2017a; Sosan et al. 2016). Besides oxidative burst, it has been reported
that NPs also induce reactive nitrogen species (*NO, nitric oxide) (Marslin et al.
2017).
Initial responses also include calcium ion (Ca2+) spikes, Ca2+ flux movements,
and upregulation/phosphorylation of mitogen-activated protein kinase (MAPK)
cascades that together with ROS production, ultimately lead to the activation of the
pathways of specialized metabolites biosynthesis (Anjum et al. 2019; Marslin et al.
2017). As expected, plants exposed to stressful concentrations of NPs have shown
to cope with the oxidative stress and lipid peroxidation through the upregulation of
enzymatic antioxidants such as superoxide dismutase (SOD), ascorbate peroxidase
(APX), glutathione-S-transferase (GST), and catalase (CAT) (Dimkpa et al. 2012;
Fu et al. 2014; Mirzajani et al. 2014; Zhao et al. 2012).
Through these findings, the concept of “nano-elicitors” have recently emerged as
a novel alternative to stimulate the production of valuable bioactive compounds that
might be used as additives in food, cosmetics, and pharmaceutical products.
The most widely used nano-elicitors with this purpose are carbon nanotubes, silver,
gold, copper, zinc oxide, and titanium dioxide (Anjum et al. 2019). The way to
supply NPs to plants can be carried out through foliar spray, directly in the soil or
through the nutrient solution applied. Table 5.4 summarizes some published studies
in recent years reporting the enhanced production of commercially important spe-
cialized metabolites using metallic-, metal oxide-, and carbon related-NPs.
Table 5.4 Summary of the effects of different types of nanoparticles used as elicitors of secondary
metabolites in different plant species
Plant species/ Treatment Result
reference
Feverfew ZnONPs (1000 ppm) sprayed (") Essential oil (0.9% V/W)
(Tanacetum during seedling stage growth in with anti-cancer compounds
parthenium L.)/ soil compared to control (0.56% V/
Shahhoseini et al. W)
(2020)
Deadly nightshade Mn2O3 (25 mg L−1) applied to (") Alkaloids (23%)
(Atropa belladonna shoot tip in MS growth media (") TPC (12%)
L.)/Tian et al. (2018) (") TFC (32%)
Aloe vera (Aloe vera TiO2 (120 mg L−1) in cell (") Aloin (118%)
L.)/Raei et al. (2014) suspension using MS media
Ag (0.625 mg L−1) in cell (") Aloin (127%)
suspension using MS media
Selfheal (Prunella Ag+ Au (1:3) supplemented to (") 1.8-Fold in TPC and TFC
vulgaris L.)/Fazal cell suspension culture in MS
et al. (2019) media with NAA
Cucumber (Cucumis Cu (20 mg L−1) in hydroponic (") TPC (2.35 mg g−1 DW)
sativus)/Zhao et al. culture at early development
(2016) stages
Salvia (Salvia Multi-walled carbon nanotubes (") Rosmarinic acid, nearly four
verticillata L.)/ (MWCNTs – 50 and 1000 mg times relative to the control
Rahmani et al. (2020) L−1) foliar sprayed to
2-month-old plants growth in
soil and greenhouse conditions
Bitter melon Fullerene (10.8 mM) during (") Anticancerous
(Momordica seed germination using B (cucurbitacin-B, 74% and
charantia)/Kole et al. potting mix under greenhouse lycopene, 82%) and antidiabetic
(2013) conditions (charantin, 20% and insulin,
90%) compounds
Used abbreviations TPC Total phenolic content, TFC Total flavonoid content, (") increment of
5.2.4 Metals and Salt Metals
Metals, at high concentrations, act as stress agents to plants, therefore, they can
induce changes in the secondary metabolism causing an elicitation effect. Exposure
of plants to metals, such as Ni, Ag, Fe, and Co, has shown increased production of
secondary metabolites in a variety of plants (Zhao et al. 2001). For instance, cad-
mium (Cd2+) and copper (Cu2+) are known for their toxicity and for not having any
value for plants (Das et al. 1997). However, Cd and Cu treatments resulted in
enhanced phenolic accumulation on the medicinal plant Gynura procumbens
(Ibrahim et al. 2017). Several factors influence the response of plants to metal
exposure, mainly depending on the chemical metal species and concentration, the
plant species, climate conditions, growth stage, among others (Lajayer et al. 2017).
The use of nonfood crops with the capacity of absorbing and accumulating
heavy metals is an alternative for remediation of contaminated environments. It has
been shown in certain medicinal and aromatic plants that this practice can lead to
the accumulation of secondary metabolites, which can be phytoextracted to obtain
high-value compounds (Lajayer et al. 2017). Metabolic changes by the action of
heavy metals can lead to inhibition of enzymes involved in the production of
photosynthetic pigments, sugars, proteins, and nonprotein thiols (Naik and
Al-Khayri 2016; Nasim and Dhir 2010). To date, many studies have shown
increases in medicinal plant performance following exposure to heavy metal stress.
For example, in a study where garden mint (Mentha crispa L., Lamiaceae) was used
for phytoaccumulation of lead (Pb), the chemical composition of the essential oil of
the plant was affected by improving the production of carvone, a major component
of essential oils (Sá et al. 2015).
Heavy metals have also shown to have a role in stress amelioration through
changes in antioxidant balance which often comes hand in hand with increased sec-
ondary metabolites. A study subjecting Camellia sinensis (L) plants to drought stress
was performed to understand the role of Zn in modulating stress conditions. Results
showed decreases in hydrogen peroxide (H2O2) and lipid peroxidation, and at the
same time increases in phenolics content and differential expression of antioxidant
enzymes, such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POX),
polyphenol peroxidase (PPO), glutathione reductase (GR), and ascorbate peroxidase
(APX) (Upadhyaya et al. 2013). Similar results were obtained in Brassica napus
exposed to cadmium (Cd) stress, exogenous application of low concentrations of
selenium (Se) increased the tolerance of plants meanwhile concentrations of ascorbic
acid and reduced glutathione were increased (Hasanuzzaman et al. 2012).
Metallic salts have also shown enhanced production of secondary metabolites
during in vitro root cultures treatments such as two tropane alkaloids, scopolamine
and hyoscyamine, by eliciting with silver nitrate (AgNO3) and cadmium chloride
(CdCl2) in Brugmansia candida (Angelova et al. 2006), increases in tanshinone
contents using AgNO3 in Perovskia abrotanoides (Zaker et al. 2015) and
sesquiterpenoid–defensive compounds using cadmium salts in Datura stramonium
(Furze et al. 1991).
5.2.5 Volatile Organic Compounds
Volatile organic compounds (VOCs) are low-molecular weight compounds that are
emitted in the atmosphere in vapors or gaseous form. VOCs are produced as
secondary metabolites by micro- (bacteria and fungi) and macro-organisms (ani-
mals and plants) and play vital roles, such as regulation of physiological processes
and inter-organismal communication (Fincheira and Quiroz 2018; Rakshit et al.
2020). Plants can emit VOCs constitutively to attract pollinators and seed dis-
persers, or in response to a stimulus as a defense against insects or predators,
plant-to-plant communication, thermo-tolerance, and environmental stress adapta-
tion (Vivaldo et al. 2017). Plant VOCs can be classified into terpenoids, fatty acid
derivatives, phenylpropanoids/benzenoids, and amino acid derivatives (Dudareva
et al. 2013). Under-ground VOC’s are emitted by plants through their roots (Rakshit
et al. 2020), where they also interact with bacteria and fungi in the rhizosphere zone
giving rise to a deep symbiotic plant-microorganisms relation (Dessaux et al. 2016).
Microorganisms benefit from root’s exudates while they produce nonvolatile
metabolites that affect the plant’s nutrient assimilation and benefits plant growth
(Dotaniya and Meena 2015).
A new plant–microbe interaction involving microbial volatile organic com-
pounds (mVOCs) was discovered by (Ryu et al. 2003). In their study, they iden-
tified that volatile compounds from Bacillus subtilis act as strong promoters of
growth in Arabidopsis thaliana. Since then, studies focusing on mVOCs as potential
compounds with practical applications on regulating characteristics of agronomic
importance have emerged. Bacterial and fungal volatile compounds may activate
defense responses against biotic and abiotic stress, induce systemic resistance,
promote growth, and enhance health processes in plants (Kanchiswamy et al. 2015;
Piechulla and Degenhardt 2014). There are approximately 1000 mVOCs produced
by bacteria and fungi reported in the literature, a few examples include 3-hydroxy-
2-butanone (acetoin), 2,3-butanediol, 2-pentylfuran, or dimethylhexadecylmine
(Fincheira and Quiroz 2018; Piechulla and Degenhardt 2014).
The idea of using VOCs to elicit secondary metabolites in plants is novel and
still very little studied. There are cases of success in the literature to this purpose,
which are summarized in Table 5.5, most of them have shown that different volatile
compounds from bacterial can increase commercially valued components of
essential oils, such as monoterpenes, pulegone, menthone, menthol, limonene,
menthyl acetate, terpineol, and eugenol (Banchio et al. 2009; Santoro et al. 2011,
2016; Zhou et al. 2016). These studies are often performed in sterile plastic boxes or
petri dishes with divided into two compartments by a physical barrier so that
microbial and plant cultures interact without physical contact.
Table 5.5 Summary of the effects of different sources of VOCs used as elicitors of secondary
metabolites in different plant species
Plant species/ Treatment Result
Reference
Arabidopsis VCs emitted by the phytopathogen (") Total chlorophyll and TCC
thaliana/ Alternaria alternata cultured in
Sánchez-López petri dishes in MS medium with
et al. (2016) 14-day-old plants
Peppermint VOCs emitted by plant (") Essential oils: monoterpenes
(Mentha piperita)/ growth-promoting rhizobacteria (twofold), pulegone (3.14-fold)
Santoro et al. (Pseudomonas fluorescens and and menthone (15.4-fold) in
(2011) Azospirillum brasilense) grown on P. fluorescens-treated plants
the same Petri dish with a center (") Menthone (13.5-fold) in A.
partition brasilense-treated plants
Peppermint VCs from three native (") Total essential oil production
(Mentha piperita)/ rhizospheric bacterial strains (") Limonene, menthol and
Santoro et al. (SJ04, SJ25, SJ48) suspended in Menthyl acetate
(2016) Hoagland solution positioned on
the same partitioned Petri dish
with plant’s young shoot in MS
solid medium
Atractylodes Nitrogenous volatiles (formamide (") Volatile oils accumulation
lancea/Zhou et al. and N,N-dimethyl-formamide) (1.8-fold)
(2016) and benzaldehyde volatile emitted
by Pseudomonas fluorescens
ALEB7B. Bacterial suspension
cultured on MS agar in petri dish
was placed beside tissue culture
plantlets in MS rooting agar with
NAA
Sweet Basil VCs emitted by soil benefic (") Essential oil components
(Bacillus subtilis)/ bacterium Bacillus subtilis GB03 content: terpineol (twofold) and
Banchio et al. by positioning plants and bacteria eugenol (tenfold)
(2009) in separate regions of a partitioned
petri dish with MS media in both
sides
Used abbreviations TCC Total carotenoid content, (") increment of
5.2.6 Nutrient Deficiency
The soil provides water and nutrients to plants. Fourteen mineral nutrients are
required for plant correct growth and development which are divided into
macronutrients (N, P, K, Ca, Mg, and S) and micronutrients (Cl, Fe, B, Mn, Zn Cu,
Ni, and Mo). Macronutrients form structural and energy compounds in plants. On
the other hand, microelements are related to enzymatic responses. For example, Zn,
Fe, Mn, and Cu are components of enzymatic antioxidants, which regulate oxi-
dation processes in the plant (Hajiboland 2012; Nath and Tuteja 2016). Due to the
indispensable role of nutrients, plant roots have developed an efficient sensing and
signaling system to maintain nutrient homeostasis. Low availability of nutrients in

the soil is detected by roots, and in response, chemical signaling and chain reactions
are produced. Plants employ signaling players as phytohormones, reactive oxygen
species (ROS), sugars, and transcription factors to maintain nutrients homeostasis
within the plant (Nath and Tuteja 2016; Isah 2019).
Nutrient deficiency can produce metabolic responses that cause an increased
accumulation of secondary metabolites. Natural bioactive compounds are sought in
bioproduction processes and improvement of nutritional quality of vegetables and
fruits, therefore a nutrient deficiency of specific macro or micronutrients may be an
alternative. However, this stress can cause a decrease in crop yields (Hawkesford
et al. 2012). To produce secondary metabolites of interest without a considerable
loss of growth and biomass, it is necessary to generate eustress in the plant
(El-Nakhel et al. 2019). Currently, nutritional eustress is a strategy used in protected
production systems, where soilless crops allow greater control of nutrient supply
through nutrient solutions.
Nitrogen is a macronutrient constituent of primary metabolites (e.g., protein,
peptides, amino acids, and nucleic acids), phytohormones and secondary metabo-
lites. Plants can uptake nitrogen as nitrate and ammonium (mineral form) (Isah
2019). Research has shown an inverse relationship between low nitrogen avail-
ability and the synthesis of phenolic compounds. According to this hypothesis, low
nitrogen availability increases synthesis of metabolites that contain C, H, and O in
their structure. Therefore, terpenes and phenolic compounds synthesis will be
favored. On the contrary, metabolites that contain N in its structure such as,
alkaloids, nonprotein amino acids, and cyanogenic compounds, will decrease its
synthesis (Nath and Tuteja 2016). For example, growing lettuce (Lactuca sativa)
increases its content of phenolic compounds and antioxidant capacity in the pres-
ence of nitrogen deficiency and drought (Galieni et al. 2015). Table 5.6 shows more
examples of the effect on phenolic compounds of experiments where nitrogen
deficiency was the stress factor on plants or food with commercial interest.
Table 5.6 Effect on phenolic compounds of experiments where nitrogen deficiency was the stress
factor on plants or food with commercial interest
Plant/Reference Treatment or culture conditions Phenolic
compounds
Matricaria chamomilla/Kováčik N deficiency and N source (NH4+ Phenolic acids
and Klejdus (2014) and NO3-) in growth chamber
Vitis vinifera ‘Cabernet Nitrogen application in field Wine flavonoids
Suavignon’/Gutiérrez-Gamboa (foliar application)
et al. (2017)
Castilleja tenuiflora/ N deficiency (1.23 mM KNO3 Phenylethanoid
Medina-Pérez et al. (2015) and 0.09 mM (NH2)2SO4) glycosides
in vitro
Lactuca sativa cv crispa and cv N deficiency (0.75 and 3 mM Flavonoids and
Satine/Becker et al. (2015) under greenhouse caffeic acid
derivatives
Phosphorus (P) is a component of molecules such as nucleic acids, lipids and

nucleotides with an energy function (ATP and ADP). Plants uptake P in the form of
inorganic orthophosphate (Pi, HPO42−, and H2PO4−) which deficiency promotes
anthocyanin synthesis (Jezek et al. 2016). Peng et al. (2019) proposed a model of
com-modulation (miR399d) and epigenetic modification as a regulatory mechanism
of anthocyanin synthesis that depends on the P availability. Sulfur is a structural
component of amino acid precursors of secondary metabolites. Therefore, its
deficiency negatively affects the biosynthesis of lycopenes and carotenoids
(Mohammed et al. 2015). Micronutrient deficiencies are shown to have a negative
impact on the synthesis of phenolic compounds and terpenes. Micronutrients such
as Cu, Fe, Mo, and Mn, act as factors for the synthesis of secondary metabolites.
5.3 Biotic Stress
Biological stressors are those considered within living organisms (plants or

pathogens) including bacteria, insect or herbivores, fungi, phytohormones, and
miRNA, among others, that results in biotic stress (Patel and Krishnamurthy 2013).
The action mechanism of this factor includes activation or inactivation of enzymes,
interaction with receptors, ion channels, stimulation of bioactive compounds, and so
forth (Joshi et al. 2019). Some biotic stress factors and their role in the synthesis of
secondary metabolites in plants are described below.
5.3.1 Bacteria and Viruses
Plants are exposed to interactions with other living things. The interaction between
microorganisms and plants can have positive effects. Microorganism colonization
(pathogens and non-pathogens) triggers the resistance mechanism of the plant,
conferring resistance against other stressors (Nejat and Mantri 2017; Choudhary
et al. 2016). Nonpathogenic microorganisms act as plant biostimulants. A plant
biostimulant is defined as any substance or microorganism applied to plants in order
to improve nutritional efficiency, tolerance to biotic and abiotic stress, and quality
(Van Oosten et al. 2017; Du Jardin 2015). Arbuscular mycorrhizal fungi,
Trichoderma, and plant growth-promoting rhizobacteria are biostimulant microor-
ganisms used in crops.
Microorganisms can confer a certain degree of tolerance against abiotic stress
conditions. Colonized plants produce a wide range of enzymes and metabolites that
allow them to generate tolerance to stress (Miliute et al. 2015). Some genera of
bacteria like Rhizobium, Bacillus, Pseudomonas, Pantoea, Paenibacillus,
Burkholderia, Achromobacter, Azospirillum, Microbacterium, Methylobacterium,
variovorax, Enterobacter, have been shown to induce tolerance to abiotic stress
(Choudhary et al. 2016; Naveed et al. 2014; Gururani et al. 2013). Tolerance
generated by pathogen attack can induce resistance to abiotic stress factors. The
biochemical response generated by the attack of the pathogen is similar to the
response generated by abiotic factors. Plants attacked by Verticillium dahliae
(pathogenic fungus) develop tolerance to drought due to the formation of xylem but
reducing the growth rate (Tani et al. 2018).
Viruses are considered symbiotes. They can behave as pathogens or mutualists
depending on the environmental conditions where the host is (Roossinck 2015).
Research suggests that the mutualistic behavior of a virus occurs when the titer
virus is low and the environmental disturbance is low (Bao and Roossinck 2013).
Plant viruses can have a positive effect like other pathogens. The presence of the
virus in the plant can increase its ability to cope with biotic and abiotic stress factors
because of the activation of the plant defense system. Metabolomic studies in
infected plants have shown a significant increase in the quantity and diversity of
secondary metabolites. This metabolic effect allows the plant to cope with the stress
caused by the infection, as well as other stressors present in the environment. For
example, Sade et al. (2015), reported a significant impact on the metabolome in
tomato plants infected with Tomato yellow leaf curl virus (TYLCV) where resistant
and susceptible cultivars showed a major expression of the phenylpropanoid
pathway which is related to the production of antioxidant compounds, among
others. In the same research, the expression in resistant cultivars was more sig-
nificant in terms of the production of flavonoids and other antioxidants. On the other
hand, rice plants infected with Brome mosaic virus (BMV) and beet plants (Beta
vulgaris) infected with Cucumber mosaic virus (CMV) increased the accumulation
of osmoprotectants and antioxidant compounds, conferring drought tolerance to
both crops (Xu et al. 2008).
5.3.2 Fungi
Plants have a strong symbiosis relationship with some fungi and bacteria present in
the substrate where they are grown. These microorganisms, endophytes or exoge-
nous, can induce eustress to the crop, increasing the production of specialized
metabolites, e.g.,Aspergillus sp. applied as an elicitor in Artemisia annua L. callus
culture, enhanced the production of artemisinin, an endoperoxide sesquiterpene
lactone and an effective antimalarial agent (Yuliani et al. 2018). Soil-borne bene-
ficial microbes have shown a protecting potential against pathogens and herbivores
via the elicitation of plant responses e.g. plant growth-promoting fungi (PGPF) and
arbuscular mycorrhizal fungi (AMF) (Pappas et al. 2018). Fungal elicitation (in-
cluding yeas extract) is one of the most used to enhance the production of sec-
ondary metabolites (Singh et al. 2018).
Fungi with a beneficial effect on plant development that associate to plant roots
are called PGPF and are considered the first prevention mechanism in the pathogen
infection. Plants need to detect PGPFs and take advantage of the presence of
microbe-associated molecular patterns (MAMPs) that can be recognized by pattern

recognition receptors (PRRs). PGPFs can stimulate the defense system of plants
involving the modification of cell walls by the accumulation of lignin, callose,
phenols, etc., preventing the growth and proliferation of pathogens. In addition,
elicitors such as chitin, chitosan, and b-glucan that are part of the fungal cell wall
have been researched (Naziya et al. 2020; Fesel and Zuccaro 2016; Li et al. 2016).
Recent studies have been performed in different plants, e.g., fungal elicitation in
Leguminosae increased the accumulation of isoflavonoids and stilbenoids
(Araya-Cloutier et al. 2017). Moola and Diana (2019) used Aspergillus niger,
Penicillium notatum, and Rhizopus oligosporus as elicitors on hairy root culture of
Beta vulgaris to enhance betalain synthesis. Trichoderma spp. is one of the most
widely used microorganisms as a pathogen biocontrol agent and elicitor. In addi-
tion, this fungus has been shown to colonize roots and can also induce systemic
resistance (ISR), which favors plant growth, increases nutrient availability and
enhances disease resistance. The mechanisms employed by Trichoderma spp. are
the modulation of plant hormonal mechanisms and the production of secondary
metabolites (Nandini et al. 2020; Guzmán-Guzmán et al. 2019; Silva et al. 2019;
Martínez‐Medina et al. 2017). PGPFs can be used as bio-fertilizers, improving the
quality and quantity of products, and reducing the contamination of the agricultural
environment by lowering the use of chemical fertilizers (Pereira et al. 2019; Zhou
et al. 2018). Table 5.7 shows some of the fungi used to induce the production of
secondary metabolites in plants.
Table 5.7 Plants elicited by fungi application to increase the production of secondary metabolites
Plant species/Reference Elicitor Secondary metabolites
Tagetes patula/Moola and Fusarium Total thiophenes
Diana (2019) conglutinans
Catharanthus roseus/Moola Penicillium Catharanthine (chemical precursor of
and Diana (2019) jasmonate vinblastine)
Hyoscyamus muticus/Moola Rhizoctonia Phytoalexins, Solavetivone, lubimin
and Diana (2019) solani
Hyoscyamus muticus/Moola Lnonotus Stimulation hyoscyamine
and Diana (2019) obliquus
Anoectochilus formosanus/ Mycena sp F-23 Kinsenoside, flavonoid content
Zhou et al. (2018)
S. miltiorrhiza/Zhou et al. Alternaria sp. Total phenolic acid, Lithospermic acid A
(2018) A-13 and Lithospermic acid B
Salvia mittiorrhiza/Halder Trichoderma Tanshinone
et al. (2019) atroviride D-16
Sinapis alba/Andini et al. Rhizopus oryzae Glucosinolates
(2019)
5.3.3 Phytohormones
Phytohormones are molecules synthesized by defined organs that regulate plant

growth and have a prominent impact on plant metabolism. Additionally, they play a
vital role in the stimulation of response mechanisms of plant defense against stress.
Auxins, gibberellins, cytokinins (CK), abscisic acid (ABA), jasmonates (jasmonic
acid (JA), methyl jasmonate (MeJA)), salicylic acid (SA), brassinosteroids,
strigolactones, cinnamic acid (CA), among others, are examples of phytohormones.
Auxins and the auxin índole-3-acetic acid (IAA) act as promoters of growth and
developmental events in plants (cell división, elongation, and differentiation). CKs
are involved in the maintaining of cellular proliferation, differentiation, and pre-
vention of senescence. ABA has an important role in the plant response to stress
and adaptation. Gibberellic acid (GA) is a plant growth regulator and has a vital role
in seed dormancy, formation of floral organs, and lateral shoot growth
(Egamberdieva et al. 2017). SA has an important role in plant stress tolerance
through modulation of antioxidative enzyme activities. JA is a lipid-derived com-
pound synthesized via the octadecanoid pathway and is important in the develop-
ment, structure, and flowering of plants (Złotek et al. 2020). CA is a bioprecursor of
podophyllotoxin and a huge number of plant substances, including tannins, flavo-
noids, etc. (Kašparová et al. 2018).
Currently, the use of phytohormones in agriculture and research has been
implemented as a strategy to induce the production of secondary metabolites
(Egamberdieva et al. 2017). The exogenous application of phytohormones by
spraying has resulted in increased production of various significant bioactive com-
pounds in plants (Akram et al. 2020, Wang et al. 2018a). For example, Garcia-Ibañez
et al. (2019), applied MeJA, SA, and SA + MeJA to Bimi® plants resulting in a
differentiated response to each elicitor, all treatments showed an increased content of
GLs in leaves and inflorescences. Table 5.8 shows the result of plant elicitation with
phytohormones in the production of bioactive compounds of recent studies.
5.3.4 miRNA
MicroRNAs (miRNAs - length of 18 to 28 nucleotides) are noncoding RNAs. The

main function of these molecules is to participate in gene expression and regulation at
the post-transcriptional level by degrading mRNA or at the translational level by
blocking protein biosynthesis at different stages (Fig. 5.1), resulting in regulation of
plant development, metabolism, and response to biotic or abiotic stress (Tripathi et al.
2019). In plants miRNAs genes are transcribed by RNA polymerase II producing
primary miRNA (Pri-miRNA) which are important for the regulation of genome
integrity, primary and secondary metabolism, development, signal transduction,
signaling pathways, homeostasis, innate immunity, and environmental stress
responses (Vargas-Hernández et al. 2019; Wang et al. 2018b; Samad et al. 2019).
Table 5.8 Secondary metabolites produced by application of phytohormones to several plant

species
Plant species/Reference Elicitor Secondary metabolite
Solanum tuberosum/Egamberdieva et al. (2017) ABA Antioxidant enzyme
peroxidase
Zea mays/Egamberdieva et al. (2017) ABA Ethylene
Calotropis gigantean/Halder et al. (2019) JA Cardenolide
Stevia rebaudiana/Vazquez-Hernandez et al. SA Steviol glycosides
(2019)
Levisticum officinale Koch cv. Elsbetha/Złotek JA Chlorophylls, vitamin C
et al. (2020) and phenolic compounds
Artemisia annua/Qi et al. (2018) JA Artemisinin
Mentha canadensis L./Qi et al. (2018) JA Mentol
Ajuga bracteosa / (Saeed et al. 2017) MeJA Phenolic content and
flavonoid conten
Bacopa monnieri/Singh and Dwivedi (2018) MeJa Bacoside A
Hyoscyamus niger Vitis vinífera/Ramirez-Estrada MeJa Scopolamine,
et al. (2016) hyoscyamine
Stilbenes and t-resveratrol
Corylus avellana, T. chinensis, T. bacccata/ SA placlitaxel
Ramirez-Estrada et al. (2016)
Brassica oleracea I. var. Italica/Villarreal-García Etilene, Glucosinolate
et al. (2016) MeJA Phenolic compounds
Rhazya stricta/Akhgari et al. (2019) MeJA Terpenoid índole alkaloids
(vindoline
Juniperus virginiana/Kašparová et al. (2018) CA Podophyllotoxin
Fig. 5.1 Clasification of

plant noncoding RNAs ncRNAs
(ncRNAs). Linear noncoding
RNAs (Linear ncRNAs),
circular noncoding RNAs
(cncRNAs), HouseKeeping Linear ncRNAs cncRNAs
noncoding RNAs (hk
ncRNAs), Regulatory
noncoding RNAs (Regulatory
ncRNAs), long noncoding Regulatory
hk ncRNAs
RNAs (lncRNAs), small ncRNAs
RNAs (sRNAs), small
interfering RNAs (siRNAs),
micro RNAs (miRNAs)
lncRNAs sRNAs
siRNAs miRNAs
The regulation mechanism of gene expression by miRNAs is via RNA inter-

ference. These small molecules are transcribed as longer precursors in the nucleus
and are then further processed into their mature forms (Wang et al. 2018b; Samad
et al. 2017). Most plant miRNA genes are located inside intergenic regions between
two adjacent genes and are transcriptionally regulated by their promoters and ter-
minators (Hossain et al. 2019). miRNAs were reported to be involved in plant
secondary metabolite regulation such as terpenoid, phenolic, fatty acid, flavonoids,
phenolic, and nitrogen-containing compound biosynthesis (Liu et al. 2017; Samad
et al. 2019).
There is a very close relationship between miRNAs and the transcription factors
(TFs), either to be switched “on” or switched “off”. Current research trends have
focused on knowing the regulation of miRNA in response to environmental stress
and how they interact with transcription factors (Fig. 5.2) (Samad et al. 2019).
The main goal of functional studies on miRNAs has been to understand the
biological processes in which the miRNAs are involved. Different technologies
Fig. 5.2 Stress mechanism and interaction between transcription factors and miRNAs
have been developed to characterize the function and action mechanisms of these
small molecules in various plant materials (Liu et al. 2017). Research on the reg-
ulation of miRNAs in Taxus callus cells has been conducted, concluding that
miRNAs are capable of direct regulation of secondary metabolism by modulating
transcriptional factors (Chen et al. 2020). The expresión level of miRNAs is mostly
governed by temperature and radiation (Tripathi et al. 2019). Studies have
demonstrated that miRNAs may act as master regulators of flavonoid biosynthesis,
e.g., miR156-SPL9 network directly influences anthocyanin production, miR163
targets S-adenosyl-Met-dependent methyltransferases that methylates secondary
metabolites and signaling molecules, and miR397 regulates lignin biosynthesis in
Arabidopsis and Populus spp (Sharma et al. 2016).
Studies carried out on different plant species indicate that the cellular levels of
miRNA have a high regulation control for optimal spatiotemporal regulation of
target genes, adding an additional layer of complexity to the signaling processes
(Gupta et al. 2017; Sharma et al. 2016). These results suggest that a strategy to
induce the production of secondary metabolites may be the use of miRNAs that
promote the expression of genes involved in plant biosynthetic pathways.
5.4 Future Perspectives
The use of AE stimuli as a new elicitor in plants has huge sustainable potential.
However, this “environmentally friendly” agricultural technology is still under
discussion and more studies should be encouraged. Currently, the optimal sound
therapy is not known since the effect could differ depending on several factors such
as plant model, amplitude, frequency or time and duration of treatment, and
application distance among others that are not explained in most studies (Alvarado
et al. 2019).
PEF and MF in food, horticulture, and biotechnology asthe postharvest pro-
cesses have increased substantially during the last few years (Xi-ran and Ting 2017;
Gilani et al. 2017; Rusakova et al. 2017). Generally, it can be noted that the
application of PEF and MF as pretreatment is a novel method to improve plant
development due to a higher production of ROS and an associated activation of
antioxidant defense systems, such as POD, SOD, and CAT. However, the effect of
electromagnetic sources is plant and treatment specific. It is difficult to have a
strong conclusion in the analyzed experiments in this chapter since the experimental
details often were incomplete, including an insufficient description of equipment
and treatment conditions. It is recommended to consider the description of
parameters, such as electric field strength (V/m), frequency (Hz), magnetic flux
density (T), electric current (A), PPFD, distance of light source to plant, light decay
among the experimental source, photoperiod, light wavelength, the duration of
application, and plant or food complete physical description (Dannehl 2018;
Alvarado et al. 2019).
Nanoparticles in agriculture have shown benefits related to physiological and

growth parameters. Moreover, the new knowledge related to its effect on secondary
metabolism has opened a field of possibilities in the production of bioactive
compounds with high commercial value using NPs. However, as with many elic-
itors, there is still a lack of knowledge about the type and size of nanoparticle and
appropriate concentrations to use depending on the species of interest. Furthermore,
a large part of the existing nanoparticles has not yet been studied and it is also
necessary to continue generating knowledge to understand the molecular mecha-
nisms of elicitation with NPs.
Metal ions have been proposed as suitable elicitors of secondary metabolism in
cell cultures (Rudrappa et al. 2004), since they can make more efficient the tissue
culture techniques during the obtention of valuable secondary metabolites. Many
studies have proposed the use of different chemical metal species to enhance
bioactive compounds in plants. This tool is very attractive in the sense that, in
addition to producing these types of compounds, it can become an environmental
remediation technique. Also, the idea of producing specialized metabolites for later
extraction eliminates the risks associated with the potential health risks to the
consumers.
Among the study of new agricultural tools in the last decade, VOCs stands out
for being considered as an eco-friendly, cheap, and effective alternative. Even
genetically modified plants with altered VOC emission and synthetic formulations
of plant VOCs are been developed as a promising technology for agriculture and
horticulture (Rakshit et al. 2020). However, there are still many unknowns, espe-
cially about the mode of action of VOCs and about the molecular and biochemical
mechanisms related to eliciting responses of interest in plants.
The application of phytohormones in different phenological stages of plants,
including in the postharvest stage, induces the production of secondary metabolites
and is an effective strategy that uses defense mechanisms to mimic stress caused by
various environmental factors. This method can be used at the agronomic level to
improve the quality of plant products by increasing the content of bioactive
molecules. Defense plant response due to different types of stress depends on the
type of crosstalk (positive or negative) between the hormone signaling pathways
rather than on the individual contributions of each hormone (Verma et al. 2016).
This suggests that for future perspectives it should be taken into account that the
results of elicitation will depend on the synergy of the used phytohormones, the
concentration, the application conditions, and the type of cultivation. For example,
there is a balance between SA and JA to regulate biotic stress in tomato (Verma
et al. 2016). In the same way, SA and gibberellins have been used as elicitor and
biostimulant to enhance the production of steviol glycosides in stevia, producing
tall plants with a greater number of leaves and a larger stem diameter
(Vazquez-Hernandez et al. 2019).
Endophytic microbes have been shown to be able to promote plant growth,
induce tolerance and production of bioactive compounds (Lata et al. 2018).
Endophytic microbes generally reside in tissues and plants without causing
symptoms. However, they activate plant defense system and induce secondary
metabolites production with potential pharmaceutical use (Jalgaonwala and

Mahajan 2014). Therefore, the inoculation of plants with this type of microor-
ganism is a strategy that can be applied to induce and/or increase the production of
natural bioactive compounds in plants of commercial interest.
Fungi elicitation is a popular practice among horticultural producers due to the
demonstrated increase in crop yield and the production of bioactive compounds.
The ability of plant roots to uptake nutrients from the substrate is enhanced by this
type of elicitation, with a positive influence on phytohormone production and gene
expression reprogramming. Fungi are used as biocontrol treatments, bioremediation
agents, and as biostimulants, which can contribute to the development of sustain-
able agriculture. In the same way, mineral nutrients play an important role in the
growth, development, and yield of crops (Nath and Tuteja 2016). Nutrient man-
agement is a common agricultural practice, especially in soilless cultivation and
controlled systems in order to guarantee high crop yield. On the other hand, con-
sumers demand natural products with high nutritional quality. Both objectives are
feasible to be induced by the nutrition of the crops. Therefore, the management of
nutrients during the production cycle allows a balance between growth and accu-
mulation of bioactive compounds.
The use of specific miRNAs to enhance the production of metabolites is a novel
technique that is still under research and discussion among the scientific commu-
nity. Research has focused on the identification of the regulatory mechanisms of
these molecules that will lead to the design of strategies for direct manipulation,
identification, and understanding of the spatial and temporal expression scheme of
miRNAs. Innovative tools (e.g., bioinformatics) can be used to predict, by means of
algorithms, the modulation of miRNA molecules which are part of a very complex
regulatory network of secondary metabolites. Research in this area could help to
understand how biosynthetic pathways are modified by these small molecules that
have particular target genes and that can influence metabolic plant bioengineering,
generating technology to induce the production of secondary metabolites.
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Chapter 6
Natural Compounds Extracted
from Medicinal Plants and Their
Immunomodulatory Activities
Vinod Kumar Gurjar and Dilipkumar Pal
Abstract The fight against cancer cells in the human body involves a defense
system that is comprised of the innate and adaptive immunities which are controlled
by a series of immune responses mediated by different immune cells (ICs) and their
secretory substances including cytokines and chemokines. Natural substances,
synthetic compounds, and antibody elements are used as immunostimulating and
immunosuppressive agents. But here are certain restrictions to the overall use of
these compounds, such as the increased risk of infection and generalized effect
throughout the immune system. The use of plants and plant products as
immunomodulators is still in a developing stage. At non-cytotoxic concentrations,
the phytoconstituents exhibited three types of immunomodulation including type 1
of PHA, ConA, and quercetin (increased lymphocyte activation and IFN-c secre-
tion); type 2 of isopimpinellin (enhanced lymphocyte activation) and type 3 of
rutin, bergapten and xanthotoxin (elevated IFN-c secretion). The augmentation of
lymphocyte proliferation was closely correlated to an increase in the number of
lymphocyte cells including T-helper lymphocytes (CD4+), CD8+ T cells and acti-
vated PBMC, whereas elevation of IFN-c secretion was due to the activated
CD8+ T cells. The present chapter revealed the immunomodulating activity, which
could be explained the traditional use of medicinal plant extract worldwide.
Keywords Immunomodulatory activities T-helper lymphocytes IFN-c

CD8+ T cells CD4+ T cells
V. K. Gurjar
School of Pharmacy, Parul University, P.O. Limda, Ta.Waghodia,
391760 District Vadodara, Gujarat, India
D. Pal (&)
Institute of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya
(A Central University), Bilaspur 495009, Chhattisgarh, India
https://doi.org/10.1007/978-3-030-54027-2_6
198 V. K. Gurjar and D. Pal
Abbreviations
ABCG2 ATP-binding cassette super-family G member-2
ADA Adenosine deaminase
ADCC Antibody-Dependent Cellular Cytotoxicity
AINFLCs Anti-inflammatory Cytokine
AL Alkaline Phosphatase
APCs Antigen-Presenting Cells
BAOECs Bovine Aortic Endothelial Cells
BRMs Biological Response Modifiers
CAMs Cell Adhesion Molecules
CMCs Cortical Microglia Cells
COX-2 Cyclooxygenase
Tc Cytotoxic T Cells
DCs Dendritic cells
DP Dopamine
EGCG Epigallocatechin-3-gallate
ELAM-1 Endothelial Leukocyte Adhesion Molecule
elF 2 Eukaryotic Initiation Factor 2
ERK Extracellular Signal-Regulated Kinases
GM-CSF Granulocyte-Macrophage Colony-Stimulating Factor
HBMECs Human Brain Microvascular Endothelial Cells
HECs Human Mammary Cells
Hsp90 Heat Shock Protein 90
HMECs Human Mammary Epithelial Cells
HSAECs Human Primary Small Airway Epithelial Cells
HUVECs Human Umbilical Vein Endothelial Cells
ICAM) Intracellular Adhesion Molecule
ICAM-1 Intercellular Adhesion Molecule 1
ICs Immune Cells
iNOS Inducible Nitric Oxide Synthase
INFLCs Inflammatory Cytokine
LOXs Lipoxygenases
LPS Lipopolysaccharide
MAPKs Mitogen-Activated Protein Kinase
MCs Mast Cells
MC3T3 E1 Mouse Osteoblastic Cells
MCP-1 Monocyte Chemoattractant Protein-1
MIECs Murine Intestinal Epithelial Cells
MIP-1b Macrophage Inflammatory Protein
MMP-2 Matrix Metalloproteinase-2
MMPs Matrix Metallopeptidases
MNGCs Mesencephalic Neuron Glial Cells
mPGES-1 Microsomal Prostaglandin E Synthase-1
MPO Myeloperoxidase
6 Natural Compounds Extracted from Medicinal Plants … 199
MSU Monosodium Urate

NFAT Nuclear Factor Of Activated T-Cells
NK Natural Killer
NLGP Neem Leaf Glycoprotein
NO Nitric Oxide
NOX NADPH oxidase
NTP Nitro Tyrosine-Protein
PBMCs Peripheral Blood Mononuclear Cells
PBMCs Peripheral Blood Mononuclear Cells
PCNA Proliferating Cell Nuclear Antigen
PINFLCs Pro-inflammatory Cytokine
PINFLMs Pro-inflammatory mediators
PHA Pulmonary Arterial Hypertension
PhI Phagocytic Index
PMA Phorbol 12-Myristate 13-Acetate
PPARs Peroxisome proliferator-activated receptors
RA Rheumatoid Arthritis
ROS Reactive Oxygen Species
SOCS3 Suppressor of Cytokine Signaling 3
STAT3 Signal Transducers And Activators of Transcription 3
TGF-b Transforming Growth Factor
TLR4 Toll-Like Receptor 4
TREM2 Triggering Receptor Expressed on Myeloid Cells 2
VCAM-1 Vascular Cell Adhesion Molecule 1
VEGF-A Vascular Endothelial Growth Factor A
6.1 Introduction
The immune system is the body’s defense mechanism against numerous common
pathogens. The elements which activate the immune system include earlier infec-
tions, vaccination, and several outside stimuli. Also, immunity is accomplished of
discerning between the proteins of the body, cells and foreign invaders. Once the
external element is recognized, the cumulative and corresponding counter of precise
cells and arbitrator against different elements establishes the immune response
(Baxter 2007). Role-based immune mechanisms classified into two main classes,
i.e., nonspecific defense mechanisms and the specific immune system, (Vesely et al.
2011). The physicochemical and microbiological hurdles further seldom counted in
the nonspecific immune system, but, the key intermediaries of the immune mech-
anism which transfer immediate shield comprise macrophages, acute phase pro-
teins, monocytes, cytokines, neutrophils and complement. Entire stages of innate
immunity comprise APCs and macrophages that have a crucial function in
antibody-dependent cellular cytotoxicity (ADCC), the release of cytokines, NO

production and expression of antigen (Ag) molecules, dispensation and phagocy-
tosis. The DCs are important for the stimulation of innate immune memory B and T
cells. Through several stages of DCs’ modification, the modulators of non-specific
immunity along with NKCs are controlled, which regulates the adaptive and innate
immune system by releasing IFN-c, GM-CSF and TNF-a (Jantan et al. 2015). The
integrated or complement system is the tertiary relevant factor of nonspecific
immunity. The key effectors of humoral immunity amongst whole physiological
perturbations of host defenses mechanism. The complement system’s
(CSs) mechanisms/components (C3a and C3b) are triggered by component C9, and
increase and intervene immune response (Oh et al. 2012). Adaptive immunity or
acquired immune system is developed by producing antigen-specific B and T
lymphocytes over a gene reorganization procedure. The introduction of the body to
an antigen (pathogen) to produce an acquired immune response that matures in
weeks or months or maybe long-lasting or even life-long is termed as active
immunity. Active immunity might be natural or artificially developed. The immune
response of higher animals is specially equipped with adaptive immunity. The
antigen-specific immune reactions are intimately involved in the acquired immune
system. The active phagocytic activity of myelogenous cells (MCs) and Tc is
increased by Th1 lymphocytes which release IFN-c, TNF-a, and interleukin-2. The
IL-4, IL-10 and IL-5, are composed of Th2 lymphocytes, classified by B
lymphocytes-stimulated secretion of ABs. The pathogens, toxins, or bacteria are
deactivated when binding with the antibodies (ABs). Besides, ABs can opsonize
various impaired microbes, pathogens, and activate bacteria eradication by
phagocytes via stimulation of various complement proteins (Puri et al. 1994; Jantan
et al. 2015). The type of cells involved in the adaptive and innate immune system
are outline in Fig. 6.1.
6.2 Immunomodulators
In a healthy body, the immune system maintains equilibrium inside the organism
and protect from pathogens. The purpose and efficacy of the immune system are
altered by several endogenous and exogenous influences that produce either
immunostimulation or immunosuppression. Numerous substances having an
activity to regulate or modify pathophysiological progress are termed as
immunomodulators (Jantan et al. 2015). In other words, the biomolecules of bio-
logical origin or synthetic, capable of modulating, or normalizing, suppressing and
stimulating or modify activity of any components of adaptive or innate immunity,
decreasing the inflammatory responses are termed as immunoaugmentors,
immunorestoratives, immunomodulators or biological response modifiers (BRMs).
Immunomodulators are mostly classified into immunosuppressants, immunostim-
ulants and immunoadjuvants in clinical practice. Immunoadjuvants are distinct
immune mechanism stimulators that are added to vaccines, to enhance the immune
Fig. 6.1 Overview of key mediators of innate and adaptive immunity, development, and
signalling
response. Agents that stimulate or induce the mediators or increase the activity of
components of the immune system are called immunostimulants or immunostim-
ulators. Immunostimulators are used to restore the inefficiency in the immune
system as observed in the treatment of diseases. They boost the immune system
(Fig. 6.2).
Fig. 6.2 An overview of the action of immunomodulators
The resistance against autoimmunity, viral infection, tumor, allergy, immunod-

eficiency and the resistance of the body against infections is increased by
immunostimulating agents. Alternatively, immune-suppressing agents are the drug
or compounds that block the immune response, can be used to regulate the
pathological immune response involved in organ transplants (prevent the graft
destructive immune response). Furthermore, these compounds can be used in the
therapy of infections related to autoimmune disorders, hypersensitivity reactions
Fig. 6.3 An overview of various targets and basic principles of functioning of immunomodulators
(HR) and immunopathology (IP) or diseases origin from an autoimmune disorder.

Immunoadjuvants, on the other hand, can enhance the efficacy of vaccines, for
example, Freud’s adjuvant. Immunomodulators can modulate various cellular
activities such as protein synthesis, apoptosis, antigen presentation, etc. and target
various transcription factors and immune mediators (Fig. 6.3).
Some chemically synthesized compounds and monoclonal antibodies (mAb or
moAb) are also being used as immunomodulators. Hence, immunomodulators with
further protection and efficacy are still required. Owing to the incidence of chemical
drugs-associated adverse reactions and effects, natural immunomodulators are the
potential regimens to substitute them in therapeutic immunomodulatory agents
(Puri et al. 1994). Presently, the major drug discovery and advancement aiming at
biochemicals, biologics, or individual molecules as lead molecules that focus on
specific targets thought to be linked to specific diseases. It is difficult to get single
lead compounds with strong efficacy, selectivity, low toxicity and favorable
drug-like properties for molecular and cellular targets and diseases. Thus, the design
and development of a druglike molecule from several traditional or integral and
substitute remedies is getting attention. The inhibition and therapy of various dis-
eases by plant-derived drugs reported for most of human history (Puri et al. 1994;
Mir et al. 2019; Pal and saha 2019; Pal et al. 2006, 2019; Pal and Mitra 2010; Pal
and Dutta 2006; Pal 2015).
The present chapter gives an outline of widely studied plant-origin bioactive
compounds, which displayed potent effects on cellular and humoral immune
response in the pre-clinical evaluation and highlight the clinical potential. The
immunomodulatory properties of plant-derived medicines have seen the interest of
investigators. Advanced technologies and the undue study on plant extracts, natural
immunomodulators, and their active constituents with immunomodulatory proper-
ties, may give us important constituents to get a novel immunomodulators to boost
the current chemotherapies. This chapter an overview of the plant-derived bioactive
immunomodulators which are under clinical studies. Furthermore, the possible
practice as immunomodulators, mechanism of action and plant-derived compounds
of various noteworthy plant-origin lead molecules has also been broadly studied
(Fig. 6.4). Besides these agents, other phytoconstituents including alkaloids,
essential oils, terpenoids, pigments, phenolics, flavonoids and steroids, etc., have
shown valuable noted immunomodulatory activity. Plant-based bioactive agents
display —Úƒd in Table 6.1.
Fig. 6.4 Chemical structures of selected plant-derived immunomodulators in clinical trials

Table 6.1 Plant-based immunomodulator (Jantan et al. 2015)

Chemical category Plant source Mode of action
Alkaloids
Berberine Coptis chinensis Downregulate T-helper
Franch cells, Th2 (IL-4)]
production, and cytokines
TNF-a, Th1, IL-2
Chelerythrine Chelidonium majus L Block PGE2 secretion by
modifying COX-2
activity
Gelselegine Gelsemium elegans Block T lymphocyte
propagation
Koumine Gelsemium elegans Block T lymphocyte
propagation
Leonurine Leonurus japonicas Downregulate IL-6,
Houtt TNF-a, COX-2 and
iNOS, and upregulate
IL-10 by blocking the
appearance of toll like
receptors and the
stimulation of NF-jB
Block the VCAM-1 and
ICAM-1 action
Lycorine Lycoris radiate Block COX-2 and iNOS
action
Piperine Piper longum Linn Decrease count of
PINFCs IL-6, cytokines
TNF-a,IL-1b. Down
regulate expression of
NOS-2, COX-2 and
NF-jB. Block
eicosanoide production by
blocking PLA2 and
TXA2 synthase action
Pseudocoptisine Corydalis Downregulating the
turtschaninovii phosphorylation of p38
Besser and to ERK block NF-jB
stimulation, which leads
in lessening of PRINFL
mediators counts (TNF-a,
COX-2, and iNOS)
Rhynchophylline Uncaria Block phosphorylation of
rhynchophylla(Miq.) MAPK. Block release of
Jack PINFCs, PGE2, NO,
monocyte
chemoattractant protein
IL-1b, MCP-1, TNF-a
(continued)

Sinomenine Sinomenium acutum Block the release of
(Thunb.) Rehd.etWils INFLCs. Block
expression of VCAM-1
Sophocarpine Sophora Block production of NO
alopecuroides L and INFLCs. block
expression of COX-2 and
iNOS
Tetrandrine Matrine Stephania tetrandra Decreased release of ROS
Matrine. Sophora and INFL mediators.
flavescens Ait Block maleic dialdehyde
and myeloperoxidase
function
Essential oils
Z-ligustilide Angelica sinensis Block COX-2 and iNOS
(Oliv.) Diels initiation by controlling
the NF-kB and MAPK
signal pathways
Tetramethylpyra-zine Ligusticum Block INFLCs and ROS
chuanxiong Hort production. Block
macrophages neutrophile
infiltration, chemotaxis,
and NO synthase action
Block the
phosphorylation of p38
MAPK
Flavonoids Chalcone
Butein Semecarpus Inhibit NO production by
anacardium, reducing iNOS
Dalbergia odorifera, appearance. Block
Toxicodendron translocation of NF-jB
vernicifluum
Dihydroxanthohumol Humulus lupulus Block NO production
induced by INF-c and
LPS
Licochalcone E Glycyrrhiza inflata Block release of
PINFLCS blocking the
activity of NF-jB and
activator protein (AP-1)
Mallotophilippens C, D, E Mallotus, Block NO production
Philippinensis induced by INF-c and
LPS. mRNA gene
expression of IL-1b,
COX-2, iNOS and IL-6.
Deactivate NF-jB
Xanthohumol Humulus lupulus Block NO production that
is induced by INF-c and
LPS
(continued)

Flavones
Luteolin Lonicera Japonica Reduce the release of
inflammatory mediators,
reduce ICAM-1
expression COX-2.
Inhibit Hsp90 activity
Apigenin Cynodon dactylon, Downregulate the
Salvia officinalis L., expression of cytokines.
Portulaca oleracea, Decrease response of Th1
Mentha longifolia and Th17 cells.
Downregulate the
expression of iNOS and
COX-2. Reduced
expression of ICAM and
VCAM leading to
lessened neutrophile
Chemotaxis
Chrysin Picea crassifolia Block release of PINFCs
by modulating
intracellular-calcium
decrease histamine release
from MCs
Nobiletin Citrus nobilis Lour, Block proinflammatory
Citrus aurantium L mediators, iNOS and
COX-2 expression by
blocking MAPK and
NF-jB signaling pathway
Baicalein Scutellaria altissima Block mRNA appearance
L.Georgi of TNF-a, COX-2, and
iNOS
Block release of NO and
INFCs by regulating
NF-jB and ER-dependent
pathway
Oroxylin A Scutellariae Block NO production and
baicalensis COX-2 and iNOS
proteins expression via
inhibiting the nuclear
factor-jB pathway
Wogonin Scutellaria Block adhesion and
baicalensis Georgi migration of leukocytes
by Blocking CAMs
expression. Decrease
allergic airway
inflammation
(continued)

Flavonols
Quercetin Dysosma veitchii Reduced expression of
Hemsl. et Wils PINFLCS, iNOS, and
NF-jB
Decrease expression of
E-selectin and VCAM-1
Kaempferol Found in various Decrease COX-2 and
fruits and vegetables. iNOS activity through
e.g., tea, tomato suppression of the
signaling of AP-1,
NF-kappa B, and STAT-1
Reduce the gene
expression of VCAM-1,
ICAM-1, MCP-1
Rutin Ruta graveolens Block leukocyte
relocation. Suppress
release of TNF-a and
IL-6. Reduce the
activation of NF-jB
Flavanols
Epigallocatechin-3gallate Camellia sinensis L Block MAPKs
phosphorylation, ROS
production, adhesion
molecules expression
STAT-3 and STAT-2
Isoflavones
Daidzein Pueraria mirifica, Reduce IL-1b, TNF-a,
Pueraria lobata, MCP-1, iNOS and NO
Glycine max expression at the mRNA
level
Genistein Glycine max Block expression of
COX-2 and iNOS
Decrease TNF-a and
IL-1b production via
activation of PPARs
Puerarin Pueraria lobata Reduce cytokines count.
(wild)Ohwi Block NF-jB and
stimulation of STAT3
Phloroglucinols
Myrtucommulone Myrtus communis L Block the PGE2 release
by blocking the mPGES-1
action automatically
blocking the COX activity
Arzanol Helichrysum italicum Decrease eicosanoids
generation by blocking
LOXs and COX action in
arachidonic acid
metabolism pathways
(continued)

Emodin-8-O-b-D glucoside Polygonum Activate the production
amplexicaule D. Don and separation of
var. sinense Forb osteoblasts
Block PGE2 release by
enhancing ALP
expression in MC3T3-E1
Other
Apocynin Apocynum Block NOX action.
cannabinum L. Suppress PINFLCs, CD4+
(Canadian hemp), and CD8+ T cell release
Picrorhiza kurroa
Royle ex Benth
Stilbenes
Resveratrol Fallopia japonica, Reduce MPO action and
grape, nuts mPGES-1 to basal levels.
Inhibit COX-2 and iNOS
appearance. Decrease
PINFLCs. Increase the
AINFCs IL-10 level
Piceatannol Fallopia japonica, Reduce iNOS expression.
nuts, grape Block transcription
factors stimulation such
as ERK, NF-kB, and
STAT3
Terpenoid
14-deoxy-11,12didehydroandrographolide Andrographis Increase the production of
paniculata lymphocytes. Increase
IL-2 initiation in
lymphocytes
Ginsan Panax ginseng Increase the release of
cytokines and ROS by
macrophages. Activate
the phagocytic property of
macrophages
Oleanolic acid Luffa cylindrica, Decrease the count of
Phytolacca TNF-a IL-1a and IL-6
americana along with their effect on
the complement pathway
by blocking C3
convertase. Blocks ADA
action
Echinocystic acid Luffa cylindrica Block PhI of
macrophages in antibody
and cell-mediated
immune responses
(continued)

Triptolide Tripterygium Blocks lymphocyte
wilfordii stimulation and PINFLCs
gene appearance. Blocks
stimulation of STAT3,
NFAT and NF-kB
Demethylzelasteral Tripterygium Blocks the production of
wilfordii vascular ECs
Celastrol Tripterygium Block expression of
wilfordii PINFLCs, topoisomerase
II and proteasome action,
Asiaticoside Centella asiatica Reduce NO release
Madecassoside Centella asiatica Decrease spleen cell
proliferation Blocking of
PINFLMs. Block release
of COX-2 and PGE2
11-keto-b-boswellic acid Boswellia carteri Reduce PINFLCs through
blocking of NF-kB
stimulation
Flavonoids Chalcone
Butein Semecarpus Inhibit NO production by
anacardium, reducing iNOS
Dalbergia odorifera, appearance. Block
Toxicodendron translocation of NF-jB
vernicifluum
Dihydroxanthohumol Humulus lupulus Block NO production
induced by INF-c and
LPS
Licochalcone E Glycyrrhiza inflata Block release of
PINFLCS blocking the
activity of NF-jB and
activator protein (AP-1)
Mallotophilippens C, D, E Mallotus, Block NO production
Philippinensis induced by INF-c and
LPS. mRNA gene
expression of IL-1b,
COX-2, iNOS and IL-6.
Deactivate NF-jB
Xanthohumol Humulus lupulus Block NO production that
is induced by INF-c and
LPS
Flavones
Luteolin Lonicera Japonica Reduce the release of
inflammatory mediators,
reduce ICAM-1
expression COX-2.
Inhibit Hsp90 activity
(continued)

Apigenin Cynodon dactylon, Downregulate the
Salvia officinalis L., expression of cytokines.
Portulaca oleracea, Decrease response of Th1
Mentha longifolia and Th17 cells.
Downregulate the
expression of iNOS and
COX-2. Reduced
expression of ICAM and
VCAM leading to
lessened neutrophile
Chemotaxis
Chrysin Picea crassifolia Block release of PINFCs
by modulating
intracellular-calcium
decrease histamine release
from MCs
Nobiletin Citrus nobilis Lour, Block proinflammatory
Citrus aurantium L mediators, iNOS and
COX-2 expression by
blocking MAPK and
NF-jB signaling pathway
Baicalein Scutellaria altissima Block mRNA appearance
L.Georgi of TNF-a, COX-2, and
iNOS
Block release of NO and
INFCs by regulating
NF-jB and ER-dependent
pathway
Oroxylin A Scutellariae Block NO production and
baicalensis COX-2 and iNOS
proteins expression via
inhibiting the nuclear
factor-jB pathway
Wogonin Scutellaria Block adhesion and
baicalensis Georgi migration of leukocytes
by Blocking CAMs
expression. Decrease
allergic airway
inflammation
Flavonols
Quercetin Dysosma veitchii Reduced expression of
Hemsl. et Wils PINFLCS, iNOS, and
NF-jB
Decrease expression of
E-selectin and VCAM-1
(continued)

Kaempferol Found in various Decrease COX-2 and
fruits and vegetables. iNOS activity through
e.g., tea, tomato suppression of the
signaling of AP-1,
NF-kappa B, and STAT-1
Reduce the gene
expression of VCAM-1,
ICAM-1, MCP-1
Rutin Ruta graveolens Block leukocyte
relocation. Suppress
release of TNF-a and
IL-6. Reduce the
activation of NF-jB
Flavanols
Epigallocatechin-3gallate Camellia sinensis L Block MAPKs
phosphorylation, ROS
production, adhesion
molecules expression
STAT-3 and STAT-2
Isoflavones
Daidzein Pueraria mirifica, Reduce IL-1b, TNF-a,
Pueraria lobata, MCP-1, iNOS and NO
Glycine max expression at the mRNA
level
Genistein Glycine max Block expression of
COX-2 and iNOS
Decrease TNF-a and
IL-1b production via
activation of PPARs
Puerarin Pueraria lobata Reduce cytokines count.
(wild)Ohwi Block NF-jB and
stimulation of STAT3
Phloroglucinols
Myrtucommulone Myrtus communis L Block the PGE2 release
by blocking the mPGES-1
action automatically
blocking the COX activity
Arzanol Helichrysum italicum Decrease eicosanoids
generation by blocking
LOXs and COX action in
arachidonic acid
metabolism pathways
Emodin-8-O-b-D glucoside Polygonum Activate the production
amplexicaule D. Don and separation of
var. sinense Forb osteoblasts
Block PGE2 release by
enhancing ALP
expression in MC3T3-E1
(continued)

Other
Apocynin Apocynum Block NOX action.
cannabinum L. Suppress PINFLCs, CD4+
(Canadian hemp), and CD8+ T cell release
Picrorhiza kurroa
Royle ex Benth
Stilbenes
Resveratrol Fallopia japonica, Reduce MPO action and
grape, nuts mPGES-1 to basal levels.
Inhibit COX-2 and iNOS
appearance. Decrease
PINFLCs. Increase the
AINFCs IL-10 level
Piceatannol Fallopia japonica, Reduce iNOS expression.
nuts, grape Block transcription
factors stimulation such
as ERK, NF-kB, and
STAT3
Terpenoid
14-deoxy-11,12didehydroandrographolide Andrographis Increase the production of
paniculata lymphocytes. Increase
IL-2 initiation in
lymphocytes
Ginsan Panax ginseng Increase the release of
cytokines and ROS by
macrophages. Activate
the phagocytic property of
macrophages
Oleanolic acid Luffa cylindrica, Decrease the count of
Phytolacca TNF-a IL-1a and IL-6
americana along with their effect on
the complement pathway
by blocking C3
convertase. Blocks ADA
action
Echinocystic acid Luffa cylindrica Block PhI of
macrophages in antibody
and cell-mediated
immune responses
Triptolide Tripterygium Blocks lymphocyte
wilfordii stimulation and PINFLCs
gene appearance. Blocks
stimulation of STAT3,
NFAT and NF-kB
(continued)

Demethylzelasteral Tripterygium Blocks the production of
wilfordii vascular ECs
Celastrol Tripterygium Block expression of
wilfordii PINFLCs, topoisomerase
II and proteasome action,
Asiaticoside Centella asiatica Reduce NO release
Madecassoside Centella asiatica Decrease spleen cell
proliferation Blocking of
PINFLMs. Block release
of COX-2 and PGE2
11-keto-b-boswellic acid Boswellia carteri Reduce PINFLCs through
blocking of NF-kB
stimulation
6.2.1 Plant-Derived Bioactive as Immunomodulators
Plant extracts and phytocompounds are found to fortify the host’s immune system,
and numerous plants have been listed in this category. Phytoimmunomodulatory
agents can increase the body’s immune-responsiveness against pathogens by acti-
vating the immune system in a specific or non-specific manner that includes both
the innate and adaptive immune systems. Immunomodulatory plants play a pivotal
role in the treatment of infection, inflammation, and immunodeficiencies by their
effect on various cell types via cytokines and interleukins. The mode of action could
be as immunostimulators, immunosuppressors, or immunoadjuvants to boost
antigen-specific immune response (Nair et al. 2019). Plant products are widely
considered as immunopotentiators and collectively known as biological response
modifiers (BRMs). Many dietary phytomolecules, vitamins, and minerals have a
protective role in cellular nutrition and the management of diabetic complications.
The immunomodulatory function attributed to BRMs is possibly through modu-
lation of the ICs of the body like the macrophages, the lymphocytes (B-cells and
T-cells), Dendritic Cells (DCs), etc. For example, Concanavalin A lectin, a
carbohydrate-binding protein from Canavalia ensiformis can cross-link glycopro-
teins like TCR/CD3 and thus activate T-lymphocytes. Similarly, Shorea robusta, a
well-known traditional medicine of India can modulate NO, prostaglandins E2,
TNF-a, iNOS expression with anti-inflammatory and wound-healing activity, while
one of its major phytochemical Bergenin enhances T helper-1 responses affording
antimycobacterial immunity by activating the MAP kinase pathway in macrophages
(Nair et al. 2019).
6.2.1.1 Curcumin
Curcumin is one of the natural diarylheptanoid belonging to the group of cur-

cuminoids, which are natural phenols found in the rhizome of turmeric (Curcuma
longa) and other Curcuma species. The medicinal properties of curcumin used for
centuries in Ayurvedic medicine. Curcumin has a variety of therapeutic properties
like anti-proliferative, anti-cancer, pro-apoptotic, anti-angiogenic, antioxidant, etc.
It is one of the most reviewed and studied plant-derived bioactive molecules for its
immunomodulatory activity. Curcumin minimizes the inflammatory responses
(inflammation) by blocking nitric oxide (NO) production, (Surh et al. 2001)
COX-2, iNOS, NK-kB, inducible and LOX in IFN-c and NK cells or
TNF-a-stimulated macrophages. In PMA and hydrogen peroxide activated
human acute myeloid leukemia cell lines, curcumin blocked NF-jB stimulation via
inhibition of breakdown and phosphorylation of I kappa B alpha (IjB-a). The PKC,
which controls the production and survival of the cell, is stimulated by PMA.
Furthermore, lipopolysaccharide (LPS) and TNF-a also stimulate PKC, which then
stimulates NF-jB (Holden et al. 2008). Hence, curcumin might lessen NF-kB
stimulation by the blocking of PKC. The anti-inflammatory property of curcumin
moderately facilitated by preventing the activator protein-1 (AP-1) and NF-jB. The
AP-1 and NF-jB act together and may increase tumor growth. Curcumin lessened
the binding of NF-jB and AP-1 on the treatment of glioma cells (Dhandapani et al.
2007). The AP-1-stimulation also blocked by curcumin in TNF- a-stimulated
bovine aortic endothelial cells (BAOECs).
The stimulated ICs to produce PINFLCs that play an important role in various
inflammation diseases. The expression of PINFLCs like IL-1, IL-6, IL-12 and
TNF-a, blocked by curcumin via LPS or PMA-activated macrophages, monocytes,
splenic lymphocytes and DCs (Gao et al. 2004; Kim et al. 2005). The binding of T
cells to endothelial and APCs is dependent on CAMs. The binding of monocytes to
endothelial cells inhibited by pre-treatment with curcumin. Furthermore, the
appearance of ICAM-1, VCAM-1 and ELAM-1 also reduced in TNF-a-activated
HUVECs via blocking of NF-jB (Kumar et al. 1998). Curcumin improved the
RANKL-mediated differentiation, fusion and development of osteoclasts and has an
immunomodulatory result on macrophage polarization. The defensive action of
curcumin on osteoclast genesis facilitated by decreasing the up-regulation of Akt
and p65 phosphorylation and the stimulation of the downstream transcription factor
NFATc1 (Yang et al. 2020). Curcumin treatment reduced activation of the NFkB,
MAPK, AKT and pBAD pathways either systemically, or within the inflamed
kidneys (Wu et al. 2020). Curcumin is capable of emulating anti-Ab vaccine in
stimulating phagocytic clearance of amyloid by decreasing CD33 and increasing
TREM2 and transmembrane immune signaling adaptor (TyroBP) though improving
neuroinflammatory systems concerned in neurodegenerative diseases. A low dose
of curcumin decreased CD33 (Siglec-3) and increased triggering receptor expressed
on myeloid cells 2 (TREM2) expression and also increased TyroBP, which controls
a neuroinflammatory gene network implicated in AD, in addition to phagocytosis
markers cluster of differentiation 68 (CD68) and Arginase 1. A low dose of
curcumin uniformly reestablished closely associated relations among these genes

expression levels and decreased expression of genes characteristic of toxic
proinflammatory M1 microglia. It stimulated microglial migration to and phago-
cytosis of amyloid plaques both in-vivo and in ex vivo assays of sections of the
human AD brain and mouse brain. Curcumin also reduced levels of miR-155, a
micro-RNA reported driving a neurodegenerative microglial phenotype. Similarly,
it decreased CD33 and increased TREM2. Like curcumin, anti-Ab antibody
increased TREM2 in APPsw mice and decreased amyloid in human AD sections
ex vivo (Teter et al. 2019).
6.2.1.2 Resveratrol
Chemically resveratrol is (3,5,4′-trihydroxytrans-stilbene). Resveratrol is a stil-

benoid polyphenol present in many nutritional foods and floras including red wine
grapevines, and peanuts. Like curcumin, resveratrol shows a broad range of phar-
macological activities like anti-inflammatory, anticancer/proapoptotic, chemopre-
ventive, antioxidant and antimicrobial (Malaguarnera 2019). Resveratrol actively
blocks inflammatory particles. The immunomodulatory actions of resveratrol
comprise the blocking of NF-jB in LPS, PMA or TNF-a-activated epithelial
(HeLa), macrophages, Jurkat, DCs and myeloid (U-937). Resveratrol block NF-jB
stimulation via blocking of IjB kinase (IKK) activity (Holmes-McNary and
Baldwin 2000). Resveratrol also blocks the expression of COX-2 and iNOS in
cytokines excited human primary small airway epithelial cells (HSAECs) although
it also blocks the transcription of COX-2 in HMECs via PMA stimulation.
Resveratrol also significantly blocks the release of TNF-a and NO in
LPS-stimulated N9 microglial and CMCs (Bi et al. 2005), the production of
PINFCs by splenic macrophages and also block lymphocytes (Kowalski et al.
2005). It also actively inhibits C5 anaphylatoxin (C5a)-activated inflammation
in-vivo. The secretion of INFCs in C5a-stimulated human and mouse neutrophils
block by the pre-incubation with resveratrol. Resveratrol also blocks
ERK-phosphorylation, production of glucuronide and C5a mediated oxidative
burst. Additionally, resveratrol blocks the production of INFCs and C5a-stimulated
neutrophil recruitment in the C5a-activated severe peritonitis mouse model (Issuree
et al. 2009). Resveratrol inhibits the expression of CAMs. It is also found that
resveratrol decrease IL-6-activated ICAM-1 appearance in ECs (Wung et al. 2005),
further to the blocking of Porphyromonas gingivalis LPS-activated endothelial
dysfunction in HMECs. Resveratrol also, block VCAM-1 and ICAM-1 appearance
on HMECs by inhibiting the NF-jB activation (Park et al. 2009). Resveratrol acts
by various cellular signaling pathways and shows anti‐inflammatory activity.
Resveratrol can potentiate its tumor-suppressive effect through modulation of the
signaling pathways of cellular components (fibroblasts, macrophages and T cells).
Also, studies have shown that resveratrol can suppress malignant phenotypes of
cancer cells acquired in response to stresses of the tumor microenvironment, such as
hypoxia, oxidative stress and inflammation (Han et al. 2019).
Resveratrol decreases MSU-induced recurrent attacks of gouty arthritis. Despite

its demonstrated anti-inflammatory effects, the mechanisms underlying resveratrol-
mediated repression of IL-1b production in MSU-activated monocytes remain
poorly understood. Resveratrol suppresses the secretion of active IL-1b by human
primary monocytes stimulated with MSU crystals through suppression of Syk
activation (Chung et al. 2019). Antioxidant or cytotoxic properties, of resveratrol,
able to alter the process of differentiation of naive T lymphocytes into Th17 lym-
phocytes through activation in particular of sirtuin-1, and to reduce the production
of pro-inflammatory substances such as interleukins. This action on the cells of the
immune system thus highlights a new property of resveratrol as an immunomod-
ulator that could counteract the occurrence or progression of inflammation in var-
ious pathological processes (Delmas et al. 2019).
6.2.1.3 Epigallocatechin-3-Gallate
Polyphenols are a big family of phytochemicals that includes a wide range of

natural substances with various biological activities. Amongst these activities, the
immunomodulatory property has significant importance due to the central and vital
roles of the immune system in the human body (Pan et al. 2019; Sobhani et al.
2020). It is abundant in phenolic composition of green tea, Camellia sinensis (Fm:
Theaceae). Epigallocatechin-3-gallate (EGCG) is the ester of epigallocatechin and
gallic acid. It has a broad range of reported in-vivo and in-vitro anti-inflammatory,
anti-proliferative, chemopreventive, anti-invasive, and anti-oxidant anti-angiogenic
activity (Singh et al. 2011; Yang et al. 2011). It blocks the stimulation of NF-jB by
blocking the degeneration of inhibitor of nuclear factor kappa B (Muraoka et al.
2002) and also blocks the MAPK pathways. The downregulation of iNOS tran-
scription and NO release in macrophages depend on blocking of NF-jB. EGCG
increases the production of NO to block endothelial exocytosis and to leukocyte
adherence ECs (Yamakuchi et al. 2008). Furthermore, that EGCG blocks NF-jB
stimulation in HECs and therefore it inhibits the expression of MCP-1 (Hong
et al. 2007).
6.2.1.4 Quercetin
The plant flavonol, quercetin, is a polyphenol, describing very broadly spreading

secondary plant metabolites. Food like grapevines, apples, broccoli, red onions,
berries, and tea, capers are a rich source of quercetin. It shows anti-mutagenic,
neuroprotective, anti-oxidative, anti-inflammatory, anticancer/chemopreventive,
antihypertensive activities. It also helps to lower blood-glucose-level. It activates
several kinase enzymes that phosphorylate elF 2, hence blocking cell translation.
The underlying mechanisms behind these activities are broad and extensively
categorized. Quercetin scavenges nitrogen and ROS, targets notable proinflam-
matory signaling pathways including NF-jB, MAPK and STAT1, and block
replication of various forms of viruses and infections of target cells (Boots et al.
2008). It inhibits the action of iNOs and COX-2 by suppressing NF-jB, AP-1 and
STAT-1 signaling in cytokines or LPS-activated macrophages and HUVECs
(Hamalainen et al. 2007). Quercitin decreases the appearance of PINFLCs in cal-
cium ionophore and PMA-activated mast cells. Moreover, quercetin also blocks the
TNF-a-activated NF-jB recruitment to proinflammatory gene promoters in MIECs
(Ruiz et al. 2007; Park et al. 2008). Quercitin reduces the TNF-a- or PMA-activated
expression of ICAM-1 in HECs. Quercetin also inhibits LPS-activated NF-jB and
NO production in mice. It modulates Th1/Th2 cytokine dysregulation in asthma and
types 1 diabetes (Ravikumar and Kavitha 2020). Quercetin, attenuate the
proinflammatory phenotype and function of DCs in-vitro. Quercetin is a potent
immunomodulatory agent to alter human DC phenotype and function, shifting the
immune balance from inflammation to resolution (Jantan et al. 2015; Michalski
et al. 2020).
6.2.1.5 Colchicine
Colchicine is a tropolone derivative and it is the main alkaloid of Colchicum

autumnale (family: Colchicaceae), commonly known as autumn crocus or meadow
saffron. The extracts have been used to treat gout for centuries. Colchicine has
approved by the US FDA for the prevention and therapy for familial Mediterranean
fever and acute gout flares. Colchicine inhibits activation and migration of neu-
trophils to sites of inflammation it also inhibits microtubule polymerization
by binding to its constitutive protein, tubulin (Bhattacharyya et al. 2008; Stanton
et al. 2011).
6.2.1.6 Capsaicin
Capsaicin is a hydrophobic alkaloid chemically it is 8-methyl-N-vanillyl-6-

nonenamide major active phytoconstituents of chili peppers Capsicum sp; belong
to family Solanaceae, and accountable for the typical spiciness/pungency of chili
peppers. It is used in classical and folk medicine as a counter-irritant and topical
rubefacient to reduce the pain of joints and muscles (Caterina et al. 1997).
Capsaicin suppresses the production of cytokines at low concentrations, and it
blocks the release of cytokines at variable concentrations whereas it stimulates IL-6
(Bessler 2016). Furthermore, capsaicin found to inhibit the iNOS countenance and
COX-2 expression in the macrophages in a transient receptor potential vanilloid 1
(TRPV1)-independent way. In an inflammation-based experiment, topical capsaicin
found to be inactive against osteoarthritis (Cameron and Chrubasik 2013).
6.2.1.7 Andrographolide
It is a labdane diterpenoid obtained from Andrographis paniculate (Acanthaceae).

Andrographolide exhibit varied biological activities. Several immunomodulatory
properties of andrographolide studied in-vitro with lessening of cytokines in
macrophages and microglia (Maiti et al. 2006). Andrographolide block the
LPS-activated COX-2 and iNOS countenance in RAW264.7 cells. Andrographolide
also blocks Akt and Erk ½ signaling, subsequently blocking the chemotaxis
movement of macrophages on inflammation site (Tsai et al. 2004; Qin et al. 2006).
Andrographolide blocks the production of ROS in neutrophils (Shen et al. 2002). It
also regulates the secretion of immunoregulatory cytokines and chemokines such as
TNF-a, IFN-c, IL-2 and NK cells. The andrographolide administered at different
doses resulting in the enhanced appearance of CD signs and the release of TNF-a,
thus raising the cytotoxic efficacy of lymphocytes (Rajagopal et al. 2003). It inhibits
IFNc, IL-2, and IL-6 appearance, thus reducing the cellular and humoral acquired
immune response in T cells. It also reduces the antigen-cited action of DCs to T
cells. Andrographolide decreases the serum Ig, ILs, and Th2 cytokine, shown in a
study on the ovalbumin (OVA)-induced asthma rat model. It decreases movement
and ECs proliferation, invasion, and intercellular adhesion molecule 1 (ICAM-1),
suggesting its function in angiogenesis. The activity of NF-jB blocked by several
immunomodulatory reactions and andrographolide block NF-jB binding to DNA
and resulting in decreasing proinflammatory protein expressions (Hidalgo et al.
2005; Iruretagoyena et al. 2005). Andrographolide downregulates iNOS and
COX-2 gene countenance by blocking STAT3 and NF-jB countenance via
inhibiting the appearance of suppressor of cytokine signaling 1 and 3 (Lee et al.
2011; Zhang et al. 2014) shown in a study to control the role of andrographolide on
insulinoma growth. Andrographolide prevents the growth of insulinoma by tar-
geting the TLR4/NF-jB signaling pathway (Zhang et al. 2014). It also inhibits the
growth of cancer cells via several mechanisms, such as cytotoxic activity, induction
of cell cycle arrest, induction of apoptosis, immunomodulatory effect,
anti-inflammatory and anti-angiogenic activities and chemoprotective mechanism.
Furthermore, andrographolide also decreases the expression of Bcl-2 studied by
immunohistochemistry analysis (Rajaratinam and Nafi 2019).
6.2.1.8 Genistein
It is a naturally occurring phytoestrogen, found in soy and soy-derived products.

chemically it is 4,5,7-trihydroxyisoflavone, a prominent tyrosine kinase blocker. It
inhibits IL-1b/IFNc-mediated iNOS and COX-2 expression. Furthermore, pro-
ducing NO and PGE2 in human islets that can stop the pathogenesis of diabetes and
increase insulin resistance. Genistein (Jantan et al. 2015) induces apoptosis similar
to other topoisomerase blockers. It actively blocks angiogenesis, exert a blocking
effect on proliferating cells. It controls vascular action and protects against
atherosclerosis (Si and Liu 2007). Genistein blocked the countenance of
TNF-a-stimulated cell adhesion molecule CD62E and CD106 and monocyte

adhesion when administered in HBMECs and HUVECs (Lee and Lee 2008). It also
decreases the interaction among the ECs and monocyte via stimulation of PPARs
that decrease monocyte adhesion in culture cells and animals. Genistein blocks the
LPS-stimulated release of MCP-1 from macrophages that activated in reduced
migration of monocyte in-vitro (Nagarajan et al. 2008). It blocks the LPS-mediated
countenance of NTP and iNOS in vascular tissue that blocks vascular changes and
hypotension. It exerts a potential effect on neurodegenerative diseases, diabetes
mellites (DM), metabolic syndromes, rheumatoid arthritis (RA) and chronic colitis
by modulating inflammatory response (Jantan et al. 2015). Genistein modulated the
Th1-dominant immune reaction by rising IL-4 secretion and inhibit the release of
IFN-c in a collagen-mediated rheumatoid arthritis rat (Wang et al. 2008).
Nonalcoholic fatty liver disease (NAFLD) is an obesity-related fatty liver disease
initiated by PINFLCs and TNF-a and leads to rising fatty acid uptake and the
defunctions of hepatic cells. It decreases the high fat diet-stimulated hepatic
steatosis by enhancing liver performance and reducing the level of plasma TNF-a
count in rats (Yalniz et al. 2007). Furthermore, genistein decreased LPS-induced
DP uptake loss in rat MNGCs by reducing the release of NO, TNF-a and microglia
stimulation this might prevent the pathogenesis of Parkinson’s disease produced by
dopaminergic neuron damage. The growth of astrocytes at Ab accumulation sites is
the early neuropathological change that initiates inflammation in Alzheimer’s dis-
ease (AD). The NOD, LRR- and pyrin domain-containing protein 3 (NLRP3)
inflammasome reported being associated with inflammatory bowel disease
including colitis due to its potential ability to induce IL-1b secretion. Genistein
blocks NLRP3 inflammasome via TGR5-cAMP signaling in macrophages and it
can be a possibly effective therapeutic drug for inflammatory bowel diseases (Chen
et al. 2019).
6.2.2 Classification of Immunomodulators
Several factors play a deciding role in determining the efficacy of an

immunomodulatory compound such as its mechanism of action structural con-
formation, molecular weight, and solubility Table 6.2.
6.2.2.1 Based on the Mechanism of Action
See Table 6.2.

Table 6.2 Classification of immunomodulators (Konidena et al. 2017)

Immunosuppressants Immunostimulants
Mechanism of action Examples Types Examples
Blocking of lymphocyte Glucocorticoids Vaccine Bacillus
gene expression classic Calmette Guerin
(immunosuppressants) (BCG)
Blocker of lymphocyte a. Calcineurin Antihelminthics Levamisole
signaling (Steroid blocker
sparingdrugs) (cyclosporine,
tacrolimus)
b. mTOR inhibitors
(sirolimus,
everolimus)
Cytotoxic agents (Steroid Antimetabolites Thalidomide
sparing drugs) (azathioprine,
methotrexate,
leflunomid,
mycophenolate
mofetil)
Alkylating agents
(cyclophosphamide)
Cytokine inhibitors a. TNF-a blockers Recombinant Interferons (a, b,
(Anticytokine-antibodies) (etanercept, cytokines c)
[Biologics] infliximab, Interleukins
adalimumab) (aldesleukin,
b. IL-1 blockers des-alanyl-1,
(anakinra) serine 125 human
c. IL-2 blockers IL 2) Colony
(daclizumab, stimulating factors
basiliximab) [filgrastim
(r metHuG CSF)]
Antibodies against a. Polyclonal Isoprinosine
specific immune cell antibodies
molecules [Biologics] [antithymocyte
globulin (ATG)]
b. Monoclonal
Antibodies
[alemutuzmab
(antiCD-52
antibodies)
c. Muromunab
(antiCD-3
antibodies, OKT-3)]
Blockers of immune cell Efalizumab (LFA-1 Immunocynin
adhesion [Biologics] inhibitor)
Tolerogens or blockers of Rh (D) immune
immune cell costimulation globulin
Miscellaneous
6.2.2.2 Based on Molecular Weight
Based on the molecular weight, immunomodulators are classified into low

molecular weight and high molecular weight compounds, both exhibiting
immunostimulatory properties.
6.2.3 Low Molecular Weight Immunomodulators
Several low molecular weight compounds have been reported for immunomodu-
latory properties. Alkaloids, Aristolochic acid obtained from the plant Aristolochia
clematitis exhibited immunostimulatory property. Aristolochic acid enhanced the
phagocytic activity of peritoneal macrophages and leukocytes, but due to its car-
cinogenic property, its use as an immunostimulant is limited. Other bioactive
immunostimulatory alkaloids obtained from Actinidia macrosperma, Cissampelos
pareira, Achillea millefolium, and Murraya koenigii. Cepharanthine isolated from
Stephania cepharantha and Vincristine from Catharanthus roseus stimulate the
production of antibodies. Cepharanthine also counteracts the effect of cytostatic
agents on hematopoiesis. Lower molecular weight compounds like Vincristine and
Staurosporine show immunomodulation in a dose-dependent manner, lower doses
used as an immunostimulant while higher doses used as an immunosuppressor. The
alkaloids isolated from Uncaria tomentosa increased the number of granulocytes.
A beta-carboline indole alkaloid harmine, isolated from Ophiorrhiza nicobarica,
inhibits lysine-specific demethylase-1 during immediate-early transcription of her-
pes simplex virus (HSV) 1 and 2 in-vitro and diseased animal. The interference on
early transcription, a decisive factor for HSV lytic cycle or latency, reveals an
epigenetic target that may help to develop a nonnucleoside antiherpesvirus drug.
Thiosulfinate obtained from Allium hirtifolium are potent adaptogens and
immunomodulators. Naphthoquinones also showed similar effects on lymphocytes
and granulocytes. Plumbagin, a quinoid compound isolated from the roots of
Plumbago zeylanica, reported for inhibitory action on the growth of
hormone-refractory prostate cancers and restrict the growth of Staphylococcus
aureus. Terpenes and its oxygenated derivatives terpenoids, like Amyrine from
Bauhinia variegata Eugenol from Ocimum sanctum, a diterpene from
Andrographis paniculata, Achillea millefolium, Alternanthera tenella and penta-
cyclic triterpene from Cecropia telenitida showed immunomodulatory activity. The
sapogenins triterpenoids and diterpenoids from Gymnema sylvestre have diverse
immunomodulatory potentials. Immunostimulatory phorbol esters (derivatives of
tetracyclic diterpenoids phorbol) shoed anticancer properties at lower doses.
Cichoric acid isolated from Echinacea purpurea activated phagocytic cells both
in-vitro and in-vivo (Nair et al. 2019).
Plant-derived glycosidase yields sugar on acid or enzymatic hydrolysis. These
include iridoid glycosides of Picrorhiza scrophulariiflora and anthraquinone gly-
cosides of Andrographis paniculata. The Chinese medicinal plant Dendrobium
nobile yields sesquiterpene glycosides Dendroside A, Dendronobilosides A, and B

that exerts proliferative effects on lymphocytes. Flavonoids are another class of
phytocompounds that exerts immunomodulatory effects. Lasure et al. (1994)
reported the effect of flavonoids on the activation of complements using hemolytic
assay and found that quercetin, quercitrin, rutin, myricetin, taxifolin, pelargonidin
chloride, and cyanidin chloride inhibited the classical pathway, while hyperoside,
myricetin, baicalein, and pelargonidin chloride inhibited the alternative pathway in
a dose-dependent manner. Besides other flavonoids viz apigenin, anthocyanidins,
flavones, isoflavonoids, and oligomeric proanthocyanidins found in plants like
Terminalia arjuna exhibit immunomodulatory activity. Furthermore, these phenolic
compounds isolated from (Euphorbiaceae) family also stop the classical pathway of
complement activation. Coumarin glycosides isolated from Achillea millefolium,
Citrus natsudaidai and Heracleum persicum revealed immunomodulatory proper-
ties. Hydroxycoumarins like Esculin, Esculetin, and Scopoletin improve
complement-mediated hemolysis. Esculentin a class of 6,7-dihydroxycoumarin
isolated from Euphorbia lathyris, Citrus limonia, and Artemisia capillaris attrib-
uted to a wide range of immunomodulatory properties like free radical scavenging,
protecting DNA against oxidative damage (Leung et al. 2005). It also shows cancer
chemopreventive, antitumor, lipoxygenase-inhibitory activity, and tyrosinase-
inhibitory activity. Certain plants and plant-derived bioactive compounds like
polysaccharides and lipopolysaccharides can activate the alternative pathway of
complement activation and hence play a key role in the regulation of inflammation.
Moreover, bioactive Rosmarinic acid from Rosmarinus officinalis L. inhibits the
complement system via blocking both the classical and alternative pathways of
complement activation (Fig. 6.5).
6.2.4 High Molecular Weight Immunomodulators
The compound having High molecular weight like polysaccharides show

immunomodulatory action primarily on the innate arm of the immune system and
specifically by enhancing the phagocytosis of granulocytes and affecting the
macrophages functions. Polysaccharide obtained from the plant Cistanche deser-
ticola enhanced the proliferation of murine thymus lymphocytes. Compounds
isolated from Salicornia herbacea show anticancer properties via activating
monocytic cells and inducing differentiation of monocytes into macrophages (Rios
2010). Axillary mode of action includes induced interferons, interleukins, and
complement activation. The binding sites for several polysaccharides to immune
components might vary. Xyloglucans (doRosa’rio et al. 2011), glucuronic
acid-containing arabinogalactan and methyl glucuronoxylans, play a critical role in
stimulating the phagocytes (Luettig et al. 1989). The complement-activating
polysaccharides comprise acidic polygalacturonans isolated from E. purpurea.
Polygalacturonans are also active for activating macrophages to counteract tumor
cells and to destroy intracellular pathogens like Candida albicans, Leishmania
Fig. 6.5 Structure of low molecular weight immunomodulators
enrietti, and Listeria cytogenes. Glycanogalacturonans from Achyrocline sat-

ureioides enhance the phagocytic potential of macrophages and granulocytes and
exhibit strong anti-complement and anti-inflammatory activities. Polysaccharides
are also known to develop chemotaxis, for instance, the rhizome of Urtica dioica
produces a-glucan that can stimulate leukocyte migration. Lectins isolated from
Lens culinaris, Canavalia ensiformis, Ricinus communis, Viscum album, Phaseolus

vulgaris, and Phytolacca americana challenged mitosis, inhibited protein synthesis,
bind to the lymphocytes, and agglutinate malignant cells. Certain lectins having
interferon-inducing properties were isolated from Viscum album and Urtica dioica
(Peumans et al. 1984). Not only lectins but also proteins capable to induce inter-
ferons were isolated and analyzed from Artemisia princeps. N-Glycosidase that
recognizes a conserved stem-loop structure in 23S/25S/28S rRNA and irreversibly
block protein translation is known as Ribosome- inactivating proteins (RIPs). RIPs
have been reported from over 50 plant species including (Cucurbitaceae) (Zhang and
Halaweish 2007), (Euphorbiaceae) (Wu et al. 2015), and (Poaceae) (Loss-Morais
et al. 2013). RIPs extracted from Trichosanthes kirilowii (Trichosanthin) was found
to decrease the retroviral protein and RNA levels in acutely infected T lymphoblast
and monocyte/macrophage (Shaw et al. 2005). Other known RIPs are isolated from
plants such as Cucurbita pepo, Ecballium elaterium, Bryonia cretica, etc. Saponins
derived from the plant Quillaja saponaria of the family (Rosaceae) are potent
immunoadjuvants. Quil A, a purified form of Quillaja extract, is used in many
veterinary vaccines (e.g., rabies, and foot and mouth disease), but its adverse
reactions makes it unfit for use in humans (Rajput et al. 2007) (Fig. 6.6).
Fig. 6.6 Structure of high molecular weight immunomodulators

6.2.5 High Throughput Screening (HTS) for Plants

and Bioactive Compounds
Some medicinal plants are currently under high throughput screening for a quick
assessment of pharmacologically important hit molecules that could be utilized as a
lead molecule in drug development. Some of the medicinally important plants
currently under research for their immunomodulatory utility are given in Tables 6.3
and 6.4.
Table 6.3 List of plants with immunomodulatory potential (Nair et al. 2019; Kumar et al. 2012)
S. No. Name of the plant Parts used Compounds isolated
1 Acanthopanax sessiliflorus Root, berry, leaf Saponins, coumarins, lignans,
(Rupr. and Maxim.) syringin, eleutheroside
(Araliaceae) syringaresinol, phenylpropane,
flavones, beta-sitosterol
2 Achyranthes bidentate Root, leaf, seed Triterpenoid, alkaloids, ketosteroids,
(Amaranthaceae) saponins, sterols, flavonoids,
anthraquinones, organic acids
3 Achyrocline satureioides Flower Luteolin, 3-O-methyl quercetin,
(Asteraceae) flavonoids quercetin
4 Aconitum carmichaeliim Root Unesterified-diterpene
(Ranunculaceae) diester-diterpene alkaloids, alkaloids,
monoester-diterpene alkaloids
5 Actinidia macrosperma C. Berry Phenolic compounds, alkaloids
F. Liang Actinidiaceae
6 Aeginetia indica Whole plant Glycoproteins, terpenoids, cichoric
(Orobanchaceae) acid, macrocyclic lactones,
polysaccharides, alkylamides
7 Albizia julibrissin Bark, flower Aromatic ethyl esters, dicarboxylic
(Fabaceae) esters
8 Aloe vera Tourn. ex Linn. Leaf Glucomannans, sterols,
(Liliaceae) anthraquinones, lipids, vitamins,
amino acids, glycosides
9 Alsophila spinulosa Stem, leaf Diploptene, glucopyranose,
(Cyatheaceae) beta-sitosterol, astragalin
10 Angelica acutiloba Root, aerial part Ferulic acid, Z-ligustilide,
(Apiaceae) tokiaerialide, bergaptol
11 Angelica sinensis Root Polysaccharides, ferulic acid,
(Apiaceae) Z-ligustilide
12 Aralia mandshurica Root Aralosides (A, B, C), flavonoids,
(Araliaceae) polysaccharides, saponins, sterols
terpenoid acids, terpenoid
13 Arnica montana Flower Coumarins, lignans, phenolic acids,
(Asteraceae) oligosaccharides, sesquiterpene
lactones, flavonoids, alkaloids,
carotenoids, pyrrolizidine
(continued)

14 Artemisia capillaris Leaf Capillin, isochlorogenic acid,
(Asteraceae) chlorogenic acid, esculetin,
scoparone, scopoletin, artepillin,
isorhamnetin-1,6-diglucoside,
hyperin
15 Artemisia iwayomogi Aerial parts Scopoletin, scopolin, b-sitosterol,
(Asteraceae) esculetin 6-methyl ether,
quebrachitol, chlorogenic acid,
16 Asarum europaeum, Root, leaf Trans-isoasarone, methyl ether,
(Aristolochiaceae) trans-isoeugenol
17 Astragalus embranaceus Whole plant Formononetin, Astragaloside IV
(Fabaceae)
18 Atractylodes lancea Root Phenolic acids, polyacetylenes,
(Asteraceae) sesquiterpenoids, steroids,
atractylodin, b-eudesmol,
monoterpenes
19 Azadirachta indica Whole plant Azadirachtin, quercetin, nimbin, ß-
(Meliaceae) sitosterol, nimbidin
20 Benincasa cerifera Fruit Isovitexin, cucurbitacin
(Cucurbitaceae) B,b-sitosterol, lupeol
21 Bryonia dioica, Root Carbohydrates, triterpenes,
(Cucurbitaceae) polyphenols, sterols alkaloids,
c-heterosides, saponins
22 Bupleurum chinense Root Polysaccharides
(Apiaceae)
23 Caesalpinia sappan Heartwood, Brazilin, diterpene caesalsappanin,
(Fabaceae) seed secang, brazilein
24 Calendula Officinalis L. Flower Quercetin (1), isorhamnetin (2)
(Asteraceae)
25 Camellia sinensis L. Leaf Theophylline, catechins, flavonoids,
(Theaceae) saponins
26 Carthamus tinctorius Flower, seed Safflower polysaccharide, alkaloids,
(Asteraceae) flavonoids,
27 Caulophyllumthalictroides Root, rhizome Triterepenes saponins, alkaloids
(Berberidaceae)
28 Chelidonium majus Whole plant Isoquinoline alkaloids, coptisine,
(Papaveraceae) sparteine, chelerythrine,stylopine,
chelidonine, bebeerine
29 Choerospondias axillaris Folium, fruit Flavones, proanthocyanidins
(Anacardiaceae) peel
30 Centaurea macrocephala Flower Anthocyanins, cyanidin glycoside,
(Asteraceae) cyanidin, patuletin, flavonoids,
kaempferol glycoside
31 Cimicifuga simplex Root Triterpenoid glycosides, phenolic
(Ranunculaceae) compounds
32 Cinnamomum cassia Twig Coumarin, cinnamate, cinnamyl
(Lauraceae) alcohol, cinnamic acid
(continued)

33 Cistanche salsa Whole plant Iridoid glycosides,
(Orobanchaceae) phenylpropanoid-substituted
diglycosides (Cistansalside A)
34 Cnidium officinale Root, rhizome Falcarindiol
(Apiaceae)
35 Coffea arabica Berry, leaf Caffeine, ferulic acid,
(Rubiaceae) 5-caffeoylquinic acid, vanillic,
protocatechuic acids, p-coumaric
36 Combretum micranthum Leaf Kinke´loids A, B, C, and D, sorbitol,
(Combretaceae) inositol, vitexin, orientin, myricetin,
isovitexin
37 Cordycepssinensis Whole plant Polysaccharides, sterols, nucleosides,
(Ophiocordycipitaceae) amino acids
38 Curcuma longa Root Curcuminoids
(Zingiberaceae)
39 Daucus carota (Apiaceae) Root, seed Anthocyanin, b-cyclodextrin,
Cyaniding 3-xylosyl
40 Echinacea purpurea Whole plant Alkamides, polysaccharides caffeic
(Asteraceae) acid derivatives
41 Echinosophora koreensis Root Prenylated flavonoids, alkaloids
(Leguminosae)
42 Epimedium alpinum Root, rhizome Glycosides, lignans, flavonoid,
Berberidaceae terpenoids, polysaccharides,
alkaloids
43 Eupatorium cannabinum Aerial parts Flavonoids, pyrrolizidine,
(Asteraceae) terpenoids, polysaccharides,
sesquiterpene lactones, benzofurans,
alkaloids
44 Fagopyrum cymosum Rhizome Flavonoids, luteolin, b-sitosterol,
(Polygonaceae) phenolic compounds
45 Forsythia koreana Fruit, leaf Lignans (arctigenin)
(Oleaceae)
46 Geranium macrorrhizum Leaf Gallic acid, ellagic acid, quercetin
(Geraniaceae)
47 Glycyrrhiza glabra Root Glycyrrhetinic acid, glabridin,
(Fabaceae) glycyrrhizin, isoflavones, flavonoids,
beta-chalcones, triterpenoid
48 Gymnema sylvestre Leaf Triterpene, saponins, flavonoids,
(Apocynaceae) coumarins, tannins
49 Gynostemmapentaphyllum Whole plant Saponins, flavones, polysaccharides
(Cucurbitaceae)
50 Hedysarum polybotrys Radix Heteropolysaccharide,
(Fabaceae) proanthocyanidin
51 Herpestis monniera Whole plant Glycosides (bacosides A and B)
(Plantaginaceae)
(continued)

52 Morus alba (Moraceae) Berry,Leaf Flavones, Anthocyanin,
oxyresveratrol, flavonoids, pyrrole
alkaloids, lignans, fatty acids,
polyphenols
53 Nyctanthes arbor tristis L. Flower, leaf Ursolic acid, flavonoids, phenol
(Oleaceae)
54 Ocimum sanctum Linn. Whole plant Carvacrol, eugenol, ursolic acid,
(Labiateae) eugenic acid, estragol, anthocyans,
sitosterol, linalool
55 Ophiopogon japonicus Root Galactan, flavonoids,
(Asparagaceae)
56 Paeonia albiflora Root Monoterpenes, glucosides
(Paeoniaceae)
57 Panax ginseng Wall. Whole plant Ginsenosides, polyacetylenes,
(Araliaceae) saponins
58 Petiveria alliacea Leaf S-Propyl propanethiosulfinate,
(Petiveriaceae) S-benzyl phenylmethanethiosulfinate
59 Phellodendron (Rutaceae) Bark, root Alkaloids, limonoids, phenolic
compounds
60 Picrorhiza kurroa Rhizome Iridoid glycoside (picroside I,
(Plantaginaceae) picroside II)
61 Pinellia ternate (Araceae) Tuber Ferulic acid, lectins, coniferin,
p-coumaryl alcohol, lariciresinol,
dihydroxy-cinnamyl alcohol
62 Pinus strobus(Pinaceae) Whole plant Dihydrobenzofurans, xanthenes
63 Polygala tenuifolia Roots Polysaccharides
(Polygalaceae)
64 Potentilla tormentilla Rhizome, root Procyanidin, monogalloylquinic
(Rosaceae) acids, polyphenols
65 Pseudostellaria Whole plant Pectic polysaccharide, peptides,
heterophylla saponins amino acids
(Caryophyllaceae)
66 Quillaja Saponaria Bark Triterpenoid saponins
(Quillajaceae)
67 Rehmannia glutinosa Whole plant Polysaccharides, acetoside
(Orobanchaceae)
68 Sapium sebiferum Leaf Gallic acid, astragalin, kaempferol,
(Euphorbiaceae) quercetin, b-sitosterol glycoside,
6-O-galloyl-d-glucose, methyl
gallate,
methyl-3,4,5trihydroxybenzoate
69 Schisandra chinensis Fruit Schisandrin A, B, C, lignans
(Schisandraceae)
70 Serenoa repens Arecaceae Whole plant Linoleic acid, myristic acid oleic
acid, lauric acid
(continued)

71 Solenostemma argel Leaf Solenoside A, Stemmosides E K,
(Apocynaceae) kaempferol-3-O-glucoside,
kaempferol-3-Orutinoside
72 Taraxacum platycarpum Aerial parts Sesquiterpene glycoside,
(Asteraceae) polysaccharides, desacetylmatricarin,
triterpenes
73 Tinospora cordifolia Stem, Root, Polysaccharides, steroids,
(Menispermaceae) Leaf glycosides, alkaloids, peptides
74 Trichosanthes kirilowii Fruit peel Polysaccharide, cucurbitacin B
(Cucurbitaceae)
75 Tripterygium wilfordii Root Glucosides, triptolide, nerolidol-type
(Celastraceae) sesquiterpene
triptergosidols A-D, triptonide,
celastrol, tripdiolide
76 Viscum album Whole plant Viscotoxins, lectins, flavonoids,
(Santalaceae) phenylpropanoids, triterpene,
phenolic acids, phytosterols
77 Uncaria tomentosa Bark, leaf Procyanidin, hydroxybenzoic acid
(Rubiaceae) propelargonidin dimers, flavanols,
flavalignans, cinchonains,
hydroxycinnamic acids, alkaloids
78 Zea mays (Poaceae) Seed Diterpenoids, flavonoids, phenolic
compounds
79 Zingiber officinale Rhizome 6-Gingerol
(Zingiberaceae)
80 Ziziphus jujube Aerial parts Phenolics, vitamin C,
(Rhamnaceae) polysaccharides, triterpenic acids
81 Achillea millefolium Leaves Alkaloids, Flavonoids,Coumarins,
(Compositae) triterpenes
82 Aloe vera Tourn. ex Linn. Gel from leaves Anthraquinone glycosides
(Liliaceae
83 Andrographis paniculata Leaves Diterpenoids, andrographolide,
Nees (Acanthaceae) flavonoids
84 Asparagus racemosus Roots Steroidal Saponins, Isoflavones,
Wild. (Liliaceae) racemosol, asparagamine,
polysaccharides, Vitamins
85 Abutilon indicum linn. Whole plant Flavonoids triterpenoids
(Malvaceae)
86 Alternanthera tenella Colla Herb Flavonoids, triterpenes
(Amaranthaceae
87 Actinidia macrosperma C. Fruits Alkaloids, Flavonoid, saponins
F.Liang (Actinidiaceae
88 Acacia catechu Willd. Leaf Flavonoids, tannins, quercetin,
(Leguminosae) alkaloids, and
89 Allium hirtifolium Boiss. Herb Thiosulfinates, flavonoids
(Alliaceae)
(continued)

90 Acanthopanax sessiliflorus Shoots and Biopolymers, eleutherosides,
(Araliaceae) roots flavonoids, phenylpropionic acids,
and triterpenic acids
91 Agelas mauritianus Sponge Glycolipid
(Porifera)
92 Aphanothece halophytica Cyanobacterium Exopolysaccharide
(Chroococcales)
93 Apium graveolens Linn. Leaves, seeds Flavonoids, coumarins
(Apiaceae)
94 Artemisia annua Linn. Herb Artemisinin
(Compositea)
95 Bauhinia variegata Linn. Roots, bark, Flavonoids, lupeol, betasitosterol
(Caesalpiniaceae) buds
96 Botryllus schlosseri Tunicates Cytokines
(Botryllidae)
97 Bidens pilosa L. Flowers, leaves Polyacetylenes
(Asteraceae)
98 Boerhaavia diffusa Herb Alkaloid
(Nyctaginaceae)
99 Bugula neritina L. Marine Macrocyclic lactones, cholesterol
(Bugulidae) invertebrates
100 Byrsonima crassa Nied. Leaves Flavonoids, Monoterpenoids tannins,
(Malpighiaceae) Triterpenoids, Iridoid Glycosides and
Phenolic Compounds
101 Couroupita guianensis Fruits, flowers Steroids, phenolics, flavonoids
Aubl. (Lecythidaceae)
102 Cleome gynandra Linn. Leaf, seeds, rots Flavonoids, terpenoids, alkaloids,
(Capperdiceae) and steroids Hexacosanol,
kaempferol
103 Citrus natsudaidai Hayata Fruits Auraptene, flavonoids
(Rutaceae)
104 Calendula Officinalis L. Flowers Polysaccharides, fatty acids,
(Asteraceae) proteins, carotenoids
105 Cistanche deserticola Herb Polysaccharide
(Orobanchaceae)
106 Cliona celata (Clionaidae) Sponge Clionamide, dehydrodopamine
107 Cordyceps militaris L. Fungus Cordycepin, cordyceps acid, free
(Clavicipitaceae) amino acid
108 Crinum latifolium Andr. Herb Alkaloids
(Amaryllidaceae)
109 Cordia superba Cham. and Bark, leaf, fruit, Quercetin, Alpha-amyrin, Linolenic
C. rufescens A. DC. acid
(Boraginaceae)
110 Cissampelospareira Linn. Roots Hayatine alkaloids
enispermiaceae)
(continued)

111 Chlorophytum Roots Sapogenins
borivilianum Sant.
F (Liliaceae)
112 Camellia sinensis L. Leaves Alkaloids, glycosides, terpenoids (-)
(Theaceae) Epigallocatechin gallate, quercetin,
gallicacid
113 Cannabis sativa Leaves Cannabinoids,
(Cannabaceae) volatile terpenes and sesquiterpenes
114 Carpobrotus edulis L. Flowers, fruit Alkaloids, flavanoids
(Aizoaceae) proanthocyanidins, tannins
115 Centella asiatica Linn. Herb Triterpenoid saponins
(Umbelliferae),
116 Dracocephalum Kotschyi Herb Essential oil, flavonoids, terpinen
(Lamiaceae)
117 Echinacea angustifolia Flowers Polysaccharide, Phenolic
(Asteraceae) compounds, alkamides
118 Eclipta alba L. Leaves Triterpenoid glucoside,
(Compositae) wedelolactone, eclalbasaponins,
ursolic acid, oleanolic acid
119 Euphorbia hirta Linn. Herb Quercitol, gallic acid, myricitrin,
(Euphorbiaceae) alkanes, triterpenes, tannins,
polyphenols phytosterols
120 Evolvulus alsinoides Linn. Herb Alkaloids
(Convolvulaceae)
121 Ganoderma lucidum (Fr.) Whole plant Flavonoids, polysaccharides,
P. Karst. (Polyporaceae) triterpenes
122 Genus Ardisia Shrub, Leaves Peptides, Isocoumarins alkyl phenols
(Myrsinaceae) saponins
123 Genus Aristolochia Leaves Aristolochic acid
(Aristolochiaceae)
124 Genus Aspergillus Fungus Polyene triazole
(Trichocomaceae)
125 Hibiscus rosa sinensis Flowers Cyclopropanoids
Linn. (Malvaceae)
126 Hyptis suaveolens (L.) Leaf, flowers Lupeol, beta sitosterol
Poit. (Lamaceae)
127 Heracleum persicum Desf. Shurb Flavonoids furanocoumarins
(Apiaceae)
128 Inonotus obliquus Pers. Mushroom Polysaccharide
(Hymenochaetaceae)
129 Larrea divaricata DC. Herb Lignans
(Zygophyllaceae)
130 Lycium barbarum Linn. Fruits Lycium barbarum polysaccharides,
(Solanaceae) (LBPs),
131 Matricaria chamomilla Flowers Protein
(Rhabdoviridae)
(continued)

132 Mollugo verticillata L. Herb Quercetin, triterpenoid glycosides
(Molluginaceae)
133 Moringa oleifera L. Leaves Vitamin A carotenoids saponins
(Moringaceae)
134 Nyctanthes arbor tristis L. Leaf, seeds Iridoid glucosides
(Oleaceae)
135 Ocimum sanctum Linn. Entire plant Eugenol, cavacrol
(Labiateae)
136 Piper longum L. Fruits Alkaloids
(Piperaceae)
137 Panax ginseng Wall. Fruits, root Saponins (ginsenosides), panaxtriole
(Araliaceae) panaxdiol,
138 Picrorhiza Roots Iridoid glycosides, amphicoside
scrophulariiflora Benth.
(Scrophulariaceae)
139 Rhodiola imbricate Gray. Rhizomes Phenolics
(Crassulaceae)
140 Randia dumetorum Lamk. Fruits Saponins triterpenes Chlorosis
(Rubiaceae)
141 Silybum marianum L. Flowers Flavonoid
(Asteraceae)
142 Salicornia herbacea Herb Polysaccharides
(Chenopodiaceae)
143 Momordica charantia L. Leaf Momordicolide,dihydrophaseic,
(Cucurbitaceae) onordicophenoide
144 Apios americana Flowers Polysaccharide
(Fabaceae)
145 Leucas aspera(Willd.) Whole Plant Triterpenoids, Diterpenes, Ursolic
Linn.(Lamiaceae) Acid, Nicotine
146 Eleutherococcus senticosus Root, Stem Syringin, Caffeic Acid, oleanolic
(Araliaceae) Bark acid, Isofraxidin
147 Ziziphora tenuior Aerial parts Essential oil
(Lamiaceae)
148 Litchi chinensis Fruit Flavonoids
Sapindaceae
149 Terminalia chebula Fruit Alkaloids
(Combretaceae)
150 Trichilia glabra Leaf Trichin, Monadelphin
(Meliaceae)
Table 6.4 Indian medicinal plants used for immunomodulatory activity (Kumar et al. 2012)
S. No. Plant name Parts used Chemical constituents
1 Glycyrrhiza uralensis Fisch Dried roots Polysaccharides
(Leguminosae)
2 Aesculus indica Leaf Alkaloids, tannins saponins
(Sapindaceae)
3 Argyreia speciosa Roots Glycosides
(Convolvulaceae)
4 Abrus precatorius (Fabaceae) Seeds Alkaloids, saponins phenolics, tannins
5 Adhatoda vasica Linn. Leaf Quinazoline vasicinone and essential
(Acanthaceae) oils
6 Balanites roxburghii Leaf Alkaloids, saponins, tannins, and
Zygophyllaceae flavonoids
7 Clitoria ternatea (Linn.) Aerial Part b-sitosterol and kaempferol
(Fabaceae)
8 Citrus aurantifolia (Rutaceae) Fruits Volatile oils
9 Capparis zeylanica Capp Leaf Flavonoids
(Araceae)
10 Caesalpinia bonducella Seeds Flavonoids, amino acids alkaloids, and
(Fabaceae) tannins
11 Habenaria intermedia Tubers Alkaloids and phenolic compounds
(Orchidaceae)
12 Murraya koenigii (Rutaceae) Leaf Coumarins, glucoside, carbazole
alkaloids,
13 Mangifera indica Stem bark Alkaloids, flavonoids, tannins
(Anacardiaceae)
14 Nyctanthes arbortristis Leaf Iridoid glucosides
(Oleaceae)
15 Salacia chinensis Root Flavonoids, alkaloids, carbohydrates,
(Celastraceae) tannins
16 Syzygium cumini (Myrtaceae) Seeds Alkaloids, phytosterols, glycosides,
flavonoids
17 Terminalia arjuna Leaves and Flavonoids, tannins, and oligomeric
(Combretaceae) bark proanthocyanidins
18 Trapa bispinosa (Lythraceae) Fruits Flavonoids, carbohydrates, proteins,
19 Tridax procumbens Aerial Tannins, steroids, flavonoids and
(Asteraceae) parts alkaloids
20 Urena lobata (Malvaceae) Fruits Flavonoids and glycosides
21 Withania somnifera Roots Withanolides
(Solanaceae)
6.3 Immunomodulatory Plants
6.3.1 Acacia Catechu/Senegalia Catechu

(Family: (Fabaceae))
This plant commonly known as Cutch Tree, Black Catechu, Cachou, found in Asia.
The heartwood and bark are used in traditional medicine to treat sore throat and
diarrhea. Several bioactive compounds are isolated from A. catechu like free radical
scavenging catechin (polyhydroxylated benzoic acid), rutin, isorhamnetin
(Li et al. 2011), and other compounds like epicatechin, epicatechin-3-O-gallate,
epigallocatechin-3-O-gallate (Stohs and Bagchi 2015), 4-hydroxybenzoic acid,
ophioglonin, quercetin, afzelechin, kaempferol, 3,4,7-trihydroxyl-3,5-dimethoxy-
flavone, epiafzelechin, mesquitol, aromadendrin, and phenol (Li et al. 2010). Naik
et al. (2003) demonstrated that the aqueous extracts of A. catechu, in rat liver
microsomal preparation, could inhibit radiation-induced lipid peroxidation.
Flavonoids isolated from A. catechu reduce the production of pro-inflammatory
eicosanoids (Burnett et al. 2007). Of note, 70% methanolic extracts of A. catechu is
found to have DNA protective properties. The acetone, ethyl acetate, and
methanolic extracts of the heartwood, leaves, and bark of A. catechu not only
scavenges free radicals but also protects DNA against strand breaks (Guleria et al.
2011) and has antimicrobial properties (Negi and Dave 2010). A. catechu shows its
immunomodulatory effect on both cell-mediated and humoral immunity. The
aqueous extract of A. catechu exhibited an increase in the neutrophil adhesion to the
nylon fibers, produced a significant increase in the phagocytic index, and significant
protection against cyclophosphamide-induced neutropenia indicating its effect on
cell-mediated immunity. A. catechu extract produced a significant increase in the
serum immunoglobulin levels, an increase in the hemagglutination titer values, and
a decrease in the mortality ratio in mice, suggesting enhanced humoral immunity
(Ismail and Asad 2009).
Methanolic and hexane extracts of A. catechu bark have reported for antipro-
liferative, cytotoxic, and anticancer properties against various cancer cell lines but
do not show any effect on human peripheral lymphocytes (Nadumane and Nair
2011). Methanolic extract of A. catechu heartwood exhibited cytotoxic activity in
breast cancer cell line MCF-7, which is due to the enhancement in Bax/Bcl2 ratio
leading to the activation of caspases and subsequent cleavage of polyadeno ribose
polymerase (Ghate et al. 2014). The aqueous extracts of heartwood also show
antidiabetic and antinociceptive action in a dose-dependent manner (Rahmatullah
et al. 2013). Various extract of A. catechu has a chemo-protective role in chemical
(carbon tetrachloride, t-butyl hydrogen peroxide, 7,12-dimethylbenz[a]anthracene,
DMBA) induced hepatocytic damage, breast and squamous cells cancers (Monga
et al. 2011).
The bark extracts of A. catechu showed potent anti-HIV effects, owing to its
effect on viral protease and via hampering the interaction of Viral Tat protein to its
HIV-1 promoter sequence of LTR (Modi et al. 2013). The antiviral compounds
isolated from A. catechu can overcome the conventional problem of generation of a

drug-resistant HIV-1 strain (Ma’rquez et al. 2005) has hence is a promising can-
didate in drug discovery.
6.3.2 Acorus Calamus (Family: Acoraceae)
This plant is commonly known as Sweet Flag, Calamus, found in Central Asia,
Southern Russia, Siberia and Eastern Europe. Acorus calamus shows varied
pharmacological properties including antibacterial, insecticidal, anti-ulcerative, etc.
(Pandit et al. 2011). It is a potent adaptogenic drug. The key bioactive compounds
present in A. calamus are flavonoid, monoterpene, quinone, sesquiterpene, and
phenylpropanoid. The ethanolic extract has antiproliferative and immunosuppres-
sive properties and is found to inhibit the growth of murine and human cell lines,
inhibit mitogen-induced proliferation of peripheral blood mononuclear cells
(PBMCs), and the generation of IL-12 and TNF-a (Mehrotra et al. 2003). The
volatile oil, petroleum ether, and alcoholic extracts of the leaves of A. calamus
enhance the phagocytic activity of neutrophils (Ravichandiran and Patil Vishal
2015). A D-galacturonic acid-containing pectic polysaccharide isolated from the
rhizomes of A. calamus at low concentrations can stimulate murine macrophages to
produce NO and IL-12 similar to those induced by LPS, thus promoting a Th1 and
suppressing the Th2 response. It also lowers serum levels of IgG1 and IgE and
induces the secretion of TNF-a secretion by human PBMCs. Thus, the polysac-
charide activates the macrophages into M1 type (classically activated macrophages)
(Belska et al. 2010). The polysaccharide possibly acts via binding to certain
receptors on APCs, releasing immunoregulatory cytokines, and adhesion molecules
(Retini et al. 2001). Its properties can be used for treating oncological and allergic
diseases. A. calamus exhibited hepatoprotective activity, it restores the hepatic
enzymes in acetaminophen-induced liver damage and lowers free radical-induced
oxidative stress (Palani et al. 2011). Chronic stress can be detrimental to the
immune system. Noise can activate the pituitary-adrenal-cortical axis and the
sympathetic adrenal medullary axis thereby increasing the secretion from adrenal
glands that directly correlate to stress (Babisch 2003) and hence can have detri-
mental effects on the immune status of the body. Noise-induced stress diminishes
the number of CD4+ and CD8+ T-cells, which is reversed by A. calamus and its
active compound a-asarone. The free radical-induced oxidation of lipids is also
prevented by the extract of A. calamus and a-asarone (Dharini et al. 2012).
That the leaf extracts inhibit inflammatory reactions in HaCaT cells via various
mechanisms (Kim et al. 2009). Also, b-asarone showed a neuroprotective role. It
suppresses neuronal apoptosis by downregulating Bcl2, Bcl-w, and caspase-3 and
preventing JNK phosphorylation. It is currently under investigation as a drug in rat
models of Alzheimer (Geng et al. 2010). Lectins isolated from the roots of
A. calamus are potent mitogenic agents for human lymphocytes and murine
splenocytes. These lectins significantly inhibited the growth of a J774, a murine

macrophage cancer cell line, and B-cell lymphoma (Bains et al. 2005).
6.3.3 Allium Sativum (Family: Amaryllidaceae)
This plant is commonly known as garlic found in worldwide. Garlic is an essential

medicinal spice and dietary element attributed to immunomodulatory properties
having certain proteins mainly agglutinins, Alliinase, antifungal Allivin and the
antimicrobial protein Alliumin. The aged garlic constituents show antiallergic and
antitumor properties (Kyo et al. 2001). Alliin isolated from A. sativum enhances the
expression of PINFCs genes like IL-6, MCP-1, and EGR-1 in LPS-stimulated
3T3-L1 adipocytes. It also modulates the cytokine generation. Low doses of garlic
extract increase IL-10 while decreasing the IL-12. IL-1a, IFN-c, TNF-a, IL-6, and
IL-8 on treatment with the extract (Quintero-Fabia’n et al. 2013). Other A. sativum
derived bioactive compounds like allitridin, S-allyl-L-cysteine, Caffeic acid, uracil,
and diallyl sulfide inhibit transcription of several PINFLCs genes like IL-6, MCP-1,
TNF-a, IL-1b, and IL-12 by inhibiting the transcription factor NF-jB (Fu et al.
2015). The lectin isolated from garlic non-specifically activates mast cells and
basophils. Thus, lectins and agglutinins are potent mitogens and have potential
utility in immunomodulation (Clement et al. 2010). Allicin reduced parasitemia
when administered in Plasmodium yoelii-infected mice due to the generation of
PINFLCs like IFN-c. Allicin treatment also activated the macrophages and stim-
ulates the expansion of CD4+ T-cells (Feng et al. 2012). Not only the bioactive
compounds of A. sativum affect the T-cells but also oil macerated extracts con-
taining active ingredient Z-ajoene affects the B-cells and increased the levels of IgA
and interleukins (Washiya et al. 2013). Allicin promotes the maturation of DCs by
increasing the expression of CD40: a costimulatory molecule, thus inducing a
proinflammatory response in rodent malaria models. Aged garlic soaked, sliced, and
extracted (AGE) in ethanol inhibit the antigen-specific generation of histamines in
rat basophil cell line RBL-2H3. Orally administered AGE significantly decreased
the IgE-mediated skin reactions (Kyo et al. 2001) and induced the PINFLCs IL-12,
INF-c, and iNOS in Leishmania-infected murine macrophages (Gharavi et al.
2011). Differing to this, AGE upregulated IL-10 and decreased IL-12 production in
PBMCs, which in effect reduced IFN-c, IL-2, TNF-a, and IL-6 (Oft 2014;
Gazzinelli et al. 1992). Garlic extracts exert an anti-inflammatory effect on
monocytes and lymphocyte proliferation by upregulating IL-10 and downregulating
the production TNF-a on LPS stimulation. Diallyl disulfide from A. sativum
decreased PINFLCS, NO production in murine macrophages, and leukemic
monocyte cell lines (Shin et al. 2013). AGE also affects NK cells and increases its
activity against various cancer cell lines. Fructooligosaccharides present in AGE
show mitogenic potential, activated macrophages and increased phagocytic activity,
comparable to effects shown by mitogens like zymosan and mannan
(Chandrashekar et al. 2011). Immunoproteins isolated from garlic-like lectins and
agglutinins are also known for their mitogenic properties similar to those of ConA
and PHA (Clement et al. 2010). AGE also affects the unique subset of the T-cell
population by increasing the proliferation of cd-T-cells that plays a crucial role in
the recognition of pathogen-associated molecular patterns (Nantz et al. 2012).
Bioactive compounds isolated from garlic also exhibit antiviral activity. Allitridin
or diallyl tri-sulfide inhibits T-reg cells in-vivo (Li et al. 2013) and thus mounts an
antiviral immune response against murine cytomegalovirus. Fresh garlic extracts
stimulate the proliferation and activation of CD8+ T-cells and cause a delayed-type
hypersensitivity (DTH) response (Ebrahimi et al. 2013).
6.3.4 Andrographis Paniculate (Family: Acanthaceae)
This plant is commonly known as King of Bitters, Kalmegh and found in Southeast
Asia, China, America, West Indies. Andrographis paniculata, a well-known
medicinal plant in various parts of the world, contains polyphenols, terpenoids
(diterpene lactones, entalabdane) xanthones, nocardioides, and flavonoids (fla-
vones) as key bioactive compounds. Ethanolic (Pongiuluran and Rofaani 2015) and
dichloromethane extracts (Chao and Lin 2010) of A. paniculata augment the pro-
liferation of lymphocytes at low concentrations. Andrographolide has diverse
immunoregulatory effects. On administration of Andrographolide in animals
bearing metastatic tumors, antibody-dependent cytotoxicity (ADCC) enhanced
when compared to untreated controls. Serum from andrographolide treated mice in
the presence of complement showed higher cytotoxicity suggesting that the extract
can activate the humoral immune system and generate tumor-specific antibodies
that can mediate ADCC (Sheeja and Kuttan 2010). Andrographolide significantly
increased the mean CD4+ T-cells and inhibited HIV-induced dysregulation of cell
cycle tested against HIV-1 infection. Andrographolide and its derivatives also
inhibited the fusion of HL2/3 cells with TZM-bl cells, which occurs via the
interaction of gp120 with CD4 and CCR5, CXCR4, thus inhibiting the HIV
(Uttekar et al. 2012). Andrographolide also inhibits the expression of Epstein-Barr
virus (EBV) (Lin et al. 2008) and HSV-1. It increases secretion of IL-2, IFN-c by
T-cells and the activity of NK cells, thus inhibiting the growth of the tumor. It also
plays a role in autoimmune disorders such as encephalomyelitis, wherein it inter-
feres with the maturation of DCs. Andrographolide can modulate the innate
immune response, regulate the production of antibodies and the generation of
antigen-specific splenocytes, and can activate macrophages both via classical and
alternative pathways (Wang et al. 2010). The immunomodulating property of the
purified andrographolide and neoandrographolide is low than that of ethanolic
extracts this finding reveals that multiple components may bring about such com-
binatorial immunomodulatory effects (Puri et al. 1993). Andrographolide,
7-O-methylwogonin is andrographolide, and skullcapflavone-1 significantly
inhibited INFLCs IL-6, NO, IL-1b in LPS-stimulated macrophages, and inflam-

matory mediators like PGE2 and TXB2 in activated promyelocytic leukemia cells.
Andrographolide, dehydroandrographolide, and neoandrographolide exhibit its
anti-inflammatory effect by inhibiting the cyclooxygenase (COX) enzyme
(Parichatikanond et al. 2010). One more bioactive compound Andrograpanin
extracted from the leaves of A. paniculate also inhibits PINFLCs in LPS-stimulated
macrophages (Liu et al. 2008). A. paniculata is also known for its antioxidant
properties. Andrographolide and 14-deoxy-11,12-didehydroandrographolide
exhibited free radical scavenging effect and inhibited lipid peroxidation under
DPPH-induced oxidative stress (Akowuah et al. 2009).
6.3.5 Azadirachta Indica (Family: Meliaceae)
This tree is commonly known as the Neem tree found in Asia. Azadirachta indica
extract found to increases the number and the activity of peripheral blood lym-
phocytes in various studies. It also increases the CD4+, CD8+ T-cells, and the
markers for T-cell and macrophage activation, namely, CD25 and MAC-3,
respectively. A. indica extracts also resulted in lesser lung and liver metastases in
the sarcoma model of Balb/c mice (Beuth et al. 2006). The nimbolide a triterpenoid
isolated from the leaves exhibits antiapoptotic and antiproliferative properties via
downregulation of bcl2/bax and upregulation of Apaf-1 and caspase-3 (Kumar et al.
2006). Nimbolide inhibits tumor cell proliferation and exerts its growth inhibitory
effects through alterations of cyclins, cdks, PCNA and p53 levels. Nimbolide also
retards tumor cell migration, invasion and angiogenesis by downregulating MMP-2/
9, VEGF-A expression via inhibiting ERK1/2, reducing the nuclear translocation
and DNA-binding activity of NF-jB in cancer cells (Bodduluru et al. 2014). A.
Indica shows anticancer properties, and it fortifies the body’s immune system.
Neem leaf preparation (NLP) also acts as an adjuvant to generate antigen
(B16MelSAg) specific antiserum in C57BL/6 mice (Baral and Chattopadhyay
2004). NLP also stimulates the CD40-CD40L interaction that leads to the activation
of p38MAPK and generation of PINFLCs IL-12, thus the activation of NK cells
(Bose and Baral 2007). Glycoprotein isolated from A. Indica leaves enhances the
expression of IFN-c that downregulates CXCR3B (responsible for apoptosis in
lymphocytes) and thus restored the impaired chemotactic movement of PBMCs
toward tumor (Chakraborty et al. 2008). NLGP leads to the activation of T-cells and
generates a Th1 type of cytokine IFN-c and can effectively activate erythroleukemia
and oral cancer cells (Bose et al. 2009). Administration of aqueous extracts of A.
Indica significantly enhanced the activity of macrophages and facilitates tumor
antigen presentation by macrophages and DCs to T and B-lymphocytes and gen-
eration of an effector and memory response (Tsang et al. 2011).
6.3.6 Boerhavia Diffusa (Family: Nyctaginaceae)
This plant is commonly known as Tarvine, Punarnava, Red Spiderling found in

Asia, Africa, North America, Caribbean, South America, South Pacific region.
Major compounds isolated from Boerhavia diffusa are Boerhavia acid, iso-
flavonoids (rotenoids), Punarnavine, sitosterol, Boeravinone, palmitic acid, steroids
(ecdysteroid), lignan glycosides, and esters of sitosterol. Boerhavia diffusa showed
anti-inflammatory and antioxidant properties and scavenges free radicals during
oxidative stress (Gacche and Dhole 2006). Two well-characterized immunostimu-
lants isolated from the roots of B. diffusa are Punarnavine and syringaresinol.
Aqueous extracts exhibited significant leukocytosis and lowered the mortality in
E. coli induced abdominal sepsis in mice and reduced stress-induced increase in the
level of glucose and cholesterol. Alkaloid fraction normalizes the plasma cortisol
levels and reduced DTH reactions in animals (Mungantiwar et al. 1999). B. diffusa
extracts enhanced the phagocytic activity of macrophages (Sumanth and Mustafa
2007). B. diffusa is also shown certain adaptogenic effects. The immunomodulatory
activity is attributed to compounds like quercetin, Punarnavine, syringaresinol
mono-b-D-glucoside, etc. Ethanolic extracts of B. diffusa exhibit immunosuppres-
sive properties and reduces the production of TNF-a and IL-2 in human PBMCs,
suppresses human NK cells and the generation of NO in murine macrophages
(Mehrotra et al. 2003). Immunosuppressive action is possibly due to the alkaloid/
lignin compounds. The chloroform and ethanolic extract treatment in RAW 264.7
cell line under LPS stimulation induced NO production, inhibited PHA-induced
proliferation, and production of TNF-a and IL-2 from PBMCs. Eupalitin-3-O-b-D-
galactopyranoside the bioactive compound isolated from ethanolic extracts found to
be most effective (Pandey et al. 2005).
Antiosteoporotic properties are also attributed to Eupalitin (O-methylated
flavone) in addition to its anti-inflammatory and immunosuppressive effects.
Therefore, its anti-inflammatory effects are extrapolated for the treatment of rheu-
matic disorders. Various studies have been performed that show the anticancer
properties of B. diffusa extract. Punarnavine downregulates the expression ERK-1/
2, MMP-2, MMP-9, and VEGF (Manu and Kuttan 2009) and prophylactic as well
as simultaneous administration of punarnavine-reduced lung melanoma metastasis.
It also enhances the production of IL-2 and IFN-c; the activity of NK cells and
showed enhanced ADCC. The level of PINFLCs like IL-1b, IL-6, and TNF-a was
reduced on Punarnavine administration. Extracts of B. diffusa also showed
Cytotoxic effects in tumor cells line Hela (Srivastava et al. 2011). Methanolic
extracts of the whole plant reduced cell viability in MCF-7 cell line, and the cells
were arrested in G0-G1 phase (Sreeja and Sreeja 2009). The methanolic extracts
also inhibit metastasis in B16F10 melanoma in C57BL/6 mice and reduced the
serum parameters of metastasis. Retinoids isolated from the root; Boeravinones G
and H are efflux inhibitors of cancer-resistance protein (ABCG2) (Ahmed-
Belkacem et al. 2007).
6.3.7 Clerodendrum Splendens (Family: Lamiaceae)
This plant is commonly known as Flaming Glorybower, Pagoda Flower, Bleeding

Heart Vine and found in tropical Western Africa. The type II arabinogalactan
isolated from Clerodendrum splendens exhibited potent immunomodulatory
activity. Specifically, the high molecular weight monosaccharides induced NO and
cytokine [interleukin (IL)-1a, -1b, -6, -10], TNF-a, and GM-CSF production by
human peripheral blood mononuclear cells (PBMCs) and monocyte/macrophages.
CSP-AU1-induced secretion of TNF prevented by Toll-like receptor 4 (TLR4)
antagonist LPS-RS, indicating a role for TLR4 signaling. It also induced phos-
phorylation of many MAPKs in human PBMC and activated AP-1/NF-jB. In-vivo
administration in mice resulted in increased serum IL-6, IL-10, TNF, monocyte
chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1a/
CCL3, and MIP-1b/CCL4. It also significantly reduced disease severity in this
experimental model of multiple sclerosis. Levels of IL-13, TNF, interferon (IFN)-c,
IL-17, and GM-CSF also significantly decreased, whereas transforming growth
factor (TGF)-b increased in LN cells in mice (Kouakou et al. 2013).
6.3.8 Curcuma Longa (Family: Zingiberaceae)
This plant is commonly known as Turmeric mainly found in Southeast Asia. The
main bioactive compounds isolated from C. longa are Curcuminoids, sesquiter-
penoids, and turmerones. The active component curcuminoid, a mixture of
demethoxycurcumin, curcumin, and bisdemethoxycurcumin, having anti-
inflammatory activity. It has various activity and block COX-2 (Plummer et al.
1999) and stimulating factor NF-jB (Singh and Aggarwal 1995). Extracts of
C. longa increases the action of NK cells and increases the amount of TNF-a and
IL-6 (Xia et al. 2006). It also regulates the propagation and sensitivity of NK cells
macrophages, lymphocytes, and DCs (Jagetia and Aggarwal 2007). Curcumin
exhibits immunosuppressive activity and blocks PMA propagation of human
spleen-derived lymphocytes and IL-2, accountable for leading this circulation
(Ranjan et al. 2004). Immunosuppressive activity of curcumin also examines in
human PBMCs, where it blocks expression of NF-jB, IL-2 and PHA-induced
propagation (Yadav et al. 2005). Curcumin also influences the phosphantigen-
mediated human Vc9Vd2 T cell propagation (Cipriani et al. 2001). Polar extracts of
C. longa primarily contain polysaccharides (lack curcuminoids) that have mitogenic
activity and increases the splenocytes count equivalent to LPS and ConA. The
NR-INF-02 increases the IFN-c, TNF-a, NO, IL-2, IL-6 and MCP-1 in deactivated
splenocytes and murine macrophages (Chandrasekaran et al. 2013).
6.3.9 Cynodon Dactylon (Family: Poaceae)
This plant is commonly known as Bermuda Grass Mainly found in Tropical and
Subtropical Regions. The active compound isolated in ethanolic extracts of C.
dactylon, are, Cynodin, beta-carotene, hydrocyanic acid, and triticin show powerful
antioxidants activity (Santhi and Annapoorani 2010; Pal et al. 2008; Pal 2008,
2009; Pal and Pandav 2010). Intraperitoneal administration of C. dactylon extract
significantly increased the adhesion of neutrophils to nylon fibers. Furthermore, the
antibody responsiveness in mice to sheep red blood cells (SRBC) significantly
increased owing to heightened responsiveness of macrophages and
antibody-synthesizing B-cells (Mungantiwar et al. 1999). Not only humoral but also
cellular immunity affected the administration of protein fraction of C. dactylon.
DTH that correlates to cell-mediated immunity increased due to increased sensi-
tivity of T-cells (Santhi and Annapoorani 2010). Oral administration of C. dactylon
juice increased humoral antibody response against an antigen. C. dactylon inhibits
the release of TNF-a, IL-6, and IL-1 and promoted anti-inflammatory IL-10 that in
turn inhibits PINFLCs (Moore et al. 2001). Studies in Catla catla showed that
ethanolic extracts of C. dactylon incorporated into the diet of the fish activated
nonspecific immune mechanism and afforded resistance against A. hydrophila
infection (Kaleeswaran et al. 2011).
6.3.10 Ficus Benghalensis (Family: Moraceae)
This tree is commonly known as Banyan Tree, Banyan Fig mainly found in Indian
Subcontinent. The root extract used in medicine since ages to boost the immune
system. The active phytoconstituents isolated from F. benghalensis contain glu-
cosides (Bhattacharjee 2008), flavonoids, etc. The aqueous extract (methanolic and
water) shows immunostimulatory activity and increases the phagocytic action of
peripheral blood mononuclear cells (PBMC). It also activates the proliferation of
lymphocytes and thus the production of cytokines that activate further ICs (Gabhe
et al. 2006). The hydroalcoholic leaf extracts significantly increase the phagocytic
action of neutrophils and thus engulfment and clearance of microbes by leukocytes,
together with free radical scavenging activity and decrease of oxidative
stress, exhibited immunomodulatory and antioxidant action (Bhanwase and
Alagawadi 2016).
6.3.11 Glycyrrhiza Uralensis Fisch (Family: Fabaceae)
This plant is commonly known as Chinese liquorice and found in Europe, Asia,
Russia and Turkey. Polysaccharides isolated from Licorice exhibited
immunomodulatory activities in CT 26 tumor-bearing BALB/c mice. The

polysaccharides significantly suppressed tumor growth and increased immune
organ index. Furthermore, the immunomodulatory effect was evident with the
activation of CD4+ and CD8+ ICs population. The polysaccharides also affected the
production of various cytokines, by increasing IL 2, IL 6, IL 7 levels and decreasing
TNFa levels. Low molecular weight exhibit polysaccharides anticancer and
immunomodulatory activities by suppressing tumor growth and improving the
general health of mice. They also augment the thymus/spleen index and population
of T lymphocytes. Furthermore, the polysaccharides enhance the levels of serum
antitumor cytokines, IL 2, IL 6 and IL 7 while decreasing pro-tumor cytokine TNFa
(Ayeka et al. 2017).
6.3.12 Murraya Koenigii (Family: Rutaceae)
This plant is commonly known as Curry Leaves found in Asia and South Africa,
Murraya koenigii has anti-inflammatory, antioxidant, antitumor properties due to
the presence of bioactive ingredients like carbazole alkaloid. Many bioactive
compounds are present in M. koenigii like koenigin, koenine, koenimbine, girin-
imbin, iso-mahanimbin, koenidine, cyclomahanimbine, tetrahydromahanimbine,
carbazole alkaloids, murrayazoline, mahanimbine, murrayastine, etc. Aqueous
extracts of M. koenigii exhibit antioxidant activity and protects cardiac tissue
against cadmium-induced oxidative stress (Mitra et al. 2012). Leaf extracts also
reduced lipid peroxidation in ethanol-induced toxicity in hepatocytes (Sathaye et al.
2011). Methanolic extracts reduce carrageenan-induced paw edema in albino rats,
(Bhandari 2012). Methanolic extracts of M. koenigii also significantly increased the
NO production from macrophages, hence an indicator of its enhanced cytotoxic
activity. It also increased the phagocytic index in the carbon-clearance test.
Moreover, the humoral antibody response to ovalbumin also increased on treatment
with the methanolic extract, thus indicating an overall elevation in humoral
response and immunostimulatory effect of the extract on B-cells (Shah et al. 2008).
6.3.13 Ocimum Sanctum (Family: Lamiaceae)
This plant is commonly known as Holy Basil mainly found in Asia, Europe and the
USA. O. sanctum has anti-inflammatory, analgesic, immunostimulatory activity
and has multidirectional therapeutic uses. Key active compounds are isolated are
eugenol-2 and its methylated derivatives, sitosterol ursolic acid, limatrol, methyl
carvicol, etc. Aqueous extract of O. sanctum mainly contains tannins, flavonoids,
and alkaloids (Gupta et al. 2002). Leaf extract O. sanctum shows immunostimu-
latory activity. It induces an innate or nonspecific immunity, increases the counts of
WBCs, RBCs, phagocytes and lymphocytes and therefore can be used to manage
the immunity against several diseases (Nahak and Sahu 2014). A study demon-
strates that O. sanctum rise a innate immune system against A. hydrophilia con-
tamination and increases the total Ig count and lysosomal action, and having a
specific activity on biochemical and hematological parameters (Das et al. 2015).
The immunostimulatory property of the leaf extract is likely owing to the presence
of bioactive constituents like methyleugenol, eugenol, ursolic acid, caryophyllene,
salrigenin and oleanolic acid (Mukherjee et al. 2005). The aqueous leaf extracts
exhibit immunomodulatory property in subclinical trails in bovines and rise the
count and action of lymphocytes and neutrophils and decrease the count of bacteria.
O. sanctum regulates several cytokines such as TNF-a, IL-2 and IFN-c, at the time
of S. typhimurium contamination which blocks bacterial population and is required
for stimulation of macrophages and therefore supporting unproductive clearance of
the organism (Goel et al. 2010). Oil of O. sanctum seed modulates both humoral
and cell-mediated immune reactions induced by the GABAergic pathway
(Vaghasiya et al. 2010). It increases the count of antibody, number of RBCs,
WBCs, and hemoglobin (Jeba et al. 2011). It exhibits free radical scavenging
activity and restricts certain categories of cancer cell growth. The active component
of ursolic acid show anti-inflammatory and anticancer activity and block COX
enzyme action. Alcoholic and aqueous extracts O. sanctum shows antitumor
activity and decreases the size of Sarcoma-180 solid tumors (Prashar et al. 1998).
6.3.14 Panax Ginseng (Family: Araliaceae)
This plant is commonly known as Ginseng found in Asia. Panax ginseng is a

well-known traditional medicine and immunomodulator. The main isolated com-
ponents are saponins, polyphenolic compounds, polyacetylenes, triterpenoids, etc.
Root, stem, and leaves are commonly made use of to boost the immune system.
Ginseng extracts containing polysaccharides increased the phagocytic activity of
macrophages. On treatment with red ginseng acidic polysaccharides (RGAPs),
peritoneal macrophages showed increased phagocytic activity (Shin et al. 2002).
Ginseng enhances the generation of NO from activated peritoneal macrophages and
RAW 264.7 cells that aids in microbe clearance. Co-treatment of macrophages with
RGAPs and IFN-c further leads to an increase in the production of IL-1b, TNF-a,
and NO (Choi et al. 2008). Polysaccharides obtained from P. ginseng enhanced the
excretion of factors like TNF-a and IL-1b from macrophages in-vitro (Lim et al.
2002). Treatment of murine macrophage cells J774A.1 with ginseng extract
increased the production of IL-12 (Wang et al. 2003). P. ginseng also has
immunostimulatory effects on DCs (Kim et al. 2009). Administration of ginseng
aqueous extracts has immunostimulatory effects on NK cells in both immuno-
competent and immunosuppressed mice (Jie et al. 1984; Kim et al 1990) A study
using blood samples of immunodeficient patients (AIDS and chronic fatigue syn-
drome) showed that P. ginseng enhanced the NK cell activity in PBMCs (See et al.
1997). The metabolic end products (M1 and M4) of steroidal ginseng saponin, can
drive DCs into maturation and increased the expression of cell-surface markers like
MHC class II, CD80, CD83, and CD86. M4 primed mature DCs boosted
antigen-presentation ability as evident from the production of IFN-c and 51Cr by
the DCs. A reciprocal effect is seen on the treatment of dendritic cells with whole
saponins of Pinex ginseng followed by oxidized LDLp. It inhibits the maturation of
DCs and downregulates maturation markers like HLA-DR, CD1a, CD40, CD86,
etc. It also inhibits TNF-a and IL-12. These reciprocal effects of P. ginseng might
be due to the activation of different signaling pathways (Su et al. 2010). Ginsan also
shows immunostimulatory effects on the maturation of DCs (Kim et al. 2009).
Ginsan not only modulates innate immune response but also affects adaptive
immune response. Orally administered with ginseng before infection with
Salmonella in mice showed higher serum IgG1 and IgG2 and IgA against
Salmonella (Na et al. 2010). Ginsenoside injected subcutaneously increases serum
antibodies specific against Toxoplasma gondii and ginsenoside likewise accord-
ingly increase serum special immunoglobulins like IgG1, IgG, IgG2a and IgG2b
mechanism against H3N2 influenza virus (Yoo et al. 2012). Both ginseng and
ginsenoside stimulated T-cell proliferation and affect cell-mediated immune
response. It was also found that splenocytes cultured with ginseng produce
pro-inflammatory IL-2, IFN-c, IL-1a, and GMSF (Kim et al. 1998). This effect was
reversed in mice infected with S. aureus. Macrophages treated with ginseng radix
extract (GRE) produce PINFLCs such as IFN-c, IL-1b, TNF-a, and IL-6 in-vitro
(Liou et al. 2006) possibly through activation of TLR4 signaling (Nakaya et al.
2004) through a non-LPS agent present in GRE. The active compound of ginseng
can also downregulate TLR2 and its downstream Myd88 and inhibit PINFLCs
production (Ahn et al. 2005).
6.3.15 Picrorhiza Scrophulariiflora (Family:

Scrophulariaceae)
This plant is commonly named as Kutki found in Alpine Himalayas, Tibet. The
rhizomes are rich in iridoid glycosides phenolic glycosides, phenylethanoid gly-
cosides and terpenoids. Extracts of P. scrophulariiflora inhibits lipid peroxidation
and possess free radical scavenging activity. Scrocaffeside A isolated from
P. scrophulariiflora is an immunostimulant. In response to Con A and LPS, it
significantly increased the number of splenocytes. It also enhanced the activity of
macrophages and NK cells even in the absence of stimulation (Coico et al. 2015).
The number of mature CD4+ and CD8+ cells and both Th1 and Th2 cytokines also
significantly increased. Activated CD4+ T-cells differentiate either into Th1 type
cells and secrete TNF-a, IL-12, IL-2, IFN-c, or into Th2 cells and secrete IL-5,
IL-10, IL-4, IL-13, and this balance is critical in deciding the immune response and
ultimately the disease outcome. Picrogentiosides isolated from P. scrophulariiflora
enhanced the proliferation of splenocytes comparable to Con A and LPS and
enhanced both humoral and cell-mediated immunity (Zou et al. 2010). Picracin and
deacetylbaccatin isolated from P. scrophulariiflora has antiproliferative effects and
inhibited proliferation of T-cells that inhibits the release of IL-2 and subsequent
IL-2-IL-2 receptor interaction. Picracin inhibited the release of PINFLC such as
IL-1b and TNFa in human monocytes, whereas deacetylbaccatin failed to do so.
Moreover, systemic administration of both picracin and deacetylbaccatin decreased
paw edema, suggesting its immunosuppressive activity. These compounds have
dualistic effects and can induce an inflammatory response on local administration
whereas an immunosuppressive effect on systemic administration.
6.3.16 Syzygium Aromaticum (Family: Myrtaceae)
This plant is commonly known as clove and used as a spice and commercially
found in Indonesia, as well as in India, Pakistan, Sri Lanka, Comoro Islands,
Madagascar, Seychelles, and Tanzania. Eugenol (4-allyl-2-methoxyphenol) is a
phenolic compound representing the most important component of the clove.
Eugenol inhibits IL-1b, IL-6 and IL-10 production and exerts an efficient action
with lipopolysaccharide (LPS) challenge for cytokines. Eugenol affects IL-1b
production but inhibits IL-6 and IL-10 production. The action of eugenol on IL-6
production prevented efficient effects of LPS either before or after its addition,
whereas on IL-10 production it counteracted significantly LPS action when added
after LPS incubation (Bachiega et al. 2012).
6.3.17 Terminalia Arjuna (Family: Combretaceae)
This plant is commonly Arjuna found in the Pantropical region. T. arjuna is com-
monly used in the traditional medicinal system to treat cardiac ailments. Treatment
of peritoneal macrophages with various doses of T. arjuna methanolic bark extracts
and gemmo-modified (embryonic tissue) extracts increased its phagocytic potential
as evaluated by increased sheep red blood cells (sRBC) engulfed by macrophages.
Both the extracts also enhanced the antibody-mediated humoral immune response
and cell-mediated phagocytosis. This immunomodulatory property of the T. arjuna
extract is attributed to the presence of polyphenols and flavonoids (Chiang et al.
2003). Polyphenols attenuate PINFLCs, MMPs, SOCS3 and downregulate TLR2
and NLRP1-an inflammasome component-and upregulate AINFLCs, thus inhibiting
the proliferation of lymphocytes and reducing the activity of NK cells (Jung et al.
2012). In a study, the T. arjuna extracts significantly decreased formalin-induced
paw edema due to its anti-inflammatory properties (Halder et al. 2009). Arjunolic
acid a triterpene is a strong cardioprotective and antioxidant in rat models. Ethanolic
extracts of T. arjuna decreases the level of endothelin-1 and INFLCs like IL-6 and
TNF-a in diabetic rats (Khaliq et al. 2013).
6.3.18 Tinospora Cordifolia (Family: Menispermaceae)
This plant is commonly known as Guduchi, Moonseed, Giloy found in Indian

Subcontinent, China. Tinospora cordifolia has a broad spectrum of immunothera-
peutic properties ranging from antipyretic anti-inflammatory, antiallergic antidia-
betic, antihepatotoxic, antibacterial properties and has relatively low toxicity. It
also helps the cell repair and rejuvenation process (Sharma et al. 2020). The key
active components isolated from the plant include phenyl propanoid glycosides
such as Cordifolioside A, Cordifolioside B and syringin (Maurya et al. 1996),
and immunostimulatory compound-D-glucan (Nair et al. 2004, 2006). The
immunomodulatory property of the Guduchi extract was validated by assessing its
effect on activating resting macrophages and by estimating the generation of
secretory factors like NO and lysosomes (More and Pai 2011). Water and ethyl
acetate extracts of T. cordifolia stem increased the phagocytic activity of human
neutrophils. The immunostimulatory action may be attributed to the synergistic
action of two or more than two active components like Cordifolioside A and
syringin. T. cordifolia extract is also shown to stimulate the proliferation of stem
cells. It increases the total number of WBCs and alpha-esterase-positive cells an
indicator of an increase in bone marrow cells. The extract also increases the number
of antibody-producing cells, hence implying its role in fortifying the humoral
immune system. The extracts of T. cordifolia are also found to be effective against
tumors and reduced tumor growth comparable to that of treatment with
cyclophosphamide (Mathew and Kuttan 1999).
6.4 Conclusion and Future Perspective
Phytochemicals and phytomedicine have been widely used in medicine for cen-
turies. Various traditional and folk medicines were based on the unexplored char-
acter of these phytochemicals. Scientific investigations in the last several decades
have uncovered the molecular roles of these phytochemicals. Recently in-vitro and
in-vivo studies have highlighted their role in immune system modulation. The role
of phytochemicals in treating infectious, acute as well as chronic diseases was
studied extensively. Though, their efficacy, biosafety and bioavailability have not
been fully studied. Application of a systems biology approach to model the mul-
tidimensional targets of these chemicals would be a much effective approach. This
would further pave the path for the application of phytochemicals in personalized
medicine to minimize the adverse/side effects and maximize effectiveness.
Immunomodulatory therapeutics are the agents that could modulate the immune
system of an organism if it increases or stimulate the immune response are called as
immunostimulants or if it decreases or suppresses immune response are called
immunosuppressants. These drugs are most commonly used in autoimmune dis-
eases, inflammatory conditions, cancer, allergic reactions, AIDS, and some viral
infections. Only a few plants have been screened for immunomodulatory activities.
From the above chapter, it is evident that there are several medicinal plants and
marine products which have immunomodulatory activity but insufficient evidence
does not allow their use in clinical practice. Therefore immunomodulatory agents
will gain more importance in the future research of herbal medicine.
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Chapter 7
Antibacterial and Antifungal Plant
Metabolites from the Tropical Medicinal
Plants
Luiz Everson da Silva, Camila Confortin,

and Mallappa Kumara Swamy
Abstract A wide-ranging organism, such as plants, fungi, insects, marine organ-

isms, and bacteria are the main sources of bioactive substances. Among them,
medicinal plants offer harmless elusive means to improve our health conditions.
Moreover, plant-derived bioactive is an inspiration for developing several thera-
peutic agents having extensively diverse chemo-structures and exhibit superior
biological properties like antimicrobial, anticancer, and antioxidant, anti-
inflammatory properties. Hence, many of them are used in therapeutic applica-
tions to treat various human ailments. Markedly, tropical countries harbor the
maximum global biodiversity and constitute several vital plant species with phar-
macological potential. Hence, they are being explored for bioactive compounds,
and other raw materials to manufacture herbal medicines or chemo-drugs. In the
present time, the emergence of microbial resistance to conventional antibiotics has
become a major threat in treating microbial infections. Further, side effects caused
by classical antibiotics have forced scientists to work toward exploring novel
antimicrobial agents to overcome these hitches. Phytoconstituents have been shown
to have prospective antimicrobial properties against both sensitive and resistant
pathogenic microbes by exerting diverse mode of action. In this chapter, the
potential of tropical plant species with antibacterial and antifungal activities are
reviewed in detail.

Keywords Tropical medicinal flora Natural products Antifungal and

antibacterial activities Plant metabolites Phytotherapies
L. E. da Silva (&) C. Confortin

Post-Graduate Program in Sustainable Territorial Development,
Universidade Federal do Paraná, Matinhos 83260-000, Brazil
e-mail: luiever@gmail.com
M. K. Swamy
Department of Biotechnology, East West First College, Bengaluru, Karnataka 560091, India
https://doi.org/10.1007/978-3-030-54027-2_7
264 L. E. da Silva et al.
7.1 Introduction
Many medicinal plants contain large amounts of bioactive compounds, such as

phenolic compounds, terpenoids, nitrogen compounds, vitamins, and various other
secondary metabolites that are being used for various therapeutic purposes, since
the dawn of mankind (Arumugam et al. 2016; Swamy et al. 2017; Kirubakari et al.
2019). The nature is a treasure of several medications, and provides assistance in
the treatment of different health issues, affecting humans. Indian, Egyptian, Roman,
Chinese, and Greek traditional medicines have documented the use of herbs against
numerous health problems. Currently, despite the development of medicine, tech-
nological progress, and the creation of new synthetic substances with medicinal
properties, the use of natural products is gradually growing, mainly in developed
countries. Thus, medicinal plants are present as an alternative in health care (Pandey
and Kumar 2013; Swamy et al. 2016; Mohanty et al. 2017; Anand et al. 2019).
Natural products contribute immensely to the development of numerous drug
molecules having several therapeutic applications (Beutler 2009). A wide-ranging
organism, such as plants, fungi, insects, marine organisms, and bacteria are the
main sources of natural products. Among them, medicinal plants offer harmless
elusive means to improve our health conditions. Moreover, plant-derived bioactives
are an inspiration for developing several therapeutic agents having extensively
diverse chemo-structures and exhibit superior biological properties like antimi-
crobial, anticancer, antioxidant, and anti-inflammatory properties. Hence, many of
them are used in therapeutic applications to treat various human ailments (Swamy
et al. 2016; Kirubakari et al. 2019).
In this context, the tropical region is home to almost a third of the world’s flora
represented in many different biomes with exuberant biodiversity (Brooks et al.
2002; Sobrinho et al. 2014). However, even in the face of the vast biodiversity and
even having great potential for study and knowledge of new perspectives on the
issue of genetic resources, the environmental degradation and the intrusion of new
cultural elements accompanied by the breakdown of traditional life systems
threaten, in addition to a collection of empirical knowledge, the heritage invaluable
genetics for future generations (Chazdon 2019). Therefore, it is essential that sci-
entific and technological innovation provides advances in order to add value to the
products of the tropical biodiversity, and we can make use of these assets ensuring
the sovereignty and vanguard of our nations.
At present, microbial infections due to pathogenic microbes, in addition to their
resistance to conventional antibiotics are posing a major challenge and threatening
the health of humans and other animals. Globally, microbial infections cause mil-
lions of human demises, and are increasing every year (Rudramurthy et al. 2016;
Khameneh et al. 2019). Bacterial and fungal infections, in general, have increased
significantly at an accelerated rate. They are being considered as a wide public
health problem that affects the whole world, affecting countless people. Due to this
advancement, it is necessary to put aside many conventional methods of combating
such diseases, which are often not having the expected effect, and to seek new
7 Antibacterial and Antifungal Plant Metabolites … 265
sources or methods that can significantly affect these infections (Swamy and
Rudramurthy 2016; Anand et al. 2019; Kirubakari et al. 2019). Plant-derived
bioactives are known to be relatively safer compared to the presently available
antibiotic agents. Further, phytocompounds exert numerous tonic advantages linked
with their extraordinary effectiveness. Various plant metabolites have demonstrated
synergistic bioactivity when combined with antibiotic agents against several
multi-drug resistant pathogenic microbes. These plant metabolites comprise phe-
nolics, flavonoids, alkaloids, tannins, terpenoids, and many more (Arumugam et al.
2016; Mohanty et al. 2017; Shin et al. 2018; Kirubakari et al. 2019).
In view of these aspects, the discovery of new active substances from natural
sources is the most priority to researchers, considering several advantages. The
pharmacological potential of medicinal plants occurring in the Tropical region has
attracted to develop novel antimicrobials (Calixto 2019). Further, side effects
caused by classical antibiotics have forced scientists to work toward exploring
novel antimicrobial agents to overcome these hitches. Phytoconstituents have
shown to have prospective antimicrobial properties against both sensitive and
resistant pathogenic microbes by exerting diverse mode of action. In this chapter,
the potential of tropical plant species with antibacterial and antifungal activities is
reviewed in detail.
7.2 Target for Antimicrobial Agents and Resistance

Mechanisms
Microbial infections are affecting the lives of several thousands of populaces around
the globe, and are accounted for a major cause of human deaths. Nearly 17% of
total deaths in the year 2013 were because of microbial infections. Further,
microbes are evolving toward resistance to existing antimicrobials, making the
present way of treating pathogens less effective. In recent times, clinically patho-
genic bacteria and fungi are being classified on the basis of microbial resistance to
either a single or multi-drug. Majorly, microbial resistance is due to many reasons,
including overdose, misuse, and continuous use of antibiotics (Swamy and
Rudramurthy 2016; Rudramurthy et al. 2016; Khameneh et al. 2019). On the other
hand, bacterial resistance has become an increasing health problem with great
impact on the pharmaceutical industry, as many antibiotics have become resistant.
More recently, different approaches are being recommended to overcome the
multi-drug-resistance mechanisms by pathogenic microbes. One such endorsed
approach is the combination therapy, involving two or more molecules with the
weakened antibiotics (Inui et al. 2007; Kirubakari et al. 2019). These molecules can
be other than antibiotic drugs having antibacterial potentials, for example, phyto-
chemicals. Particularly, these molecules function either individually or together
with antibiotic drugs to improve the antimicrobial property, and thus can be
very effective against wide-ranging bacterial pathogens (Khameneh et al. 2019).
This approach effectively restores the desired antimicrobial property (Brown 2015;
Fazly Bazzaz et al. 2018; Rana et al. 2018).
Antimicrobial agents exhibit their actions through various ways, including the
interfering with the biosynthesis or inhibiting bioactivities of bacterial components.
The established targets for antibiotics may include (a) biosynthesis of bacterial
proteins; (b) biosynthesis of bacterial cell wall; (c) damage of bacterial cell mem-
brane; (d) interfering with bacterial DNA replication and/or repair mechanisms, and
(e) suppressing metabolic pathways. Some classes of antibiotic drugs like tetra-
cyclines, macrolides, and aminoglycosides exhibit their antibacterial properties,
which particularly inhibit protein biosynthesis via targeting the ribosomal subunits
(Swamy et al. 2016; Khameneh et al. 2019). A protein is generally synthesized
inside the cells by several means of the molecular processes, including initiation,
elongation, termination, and assembly of protein-mediated by ribosomes. Hence,
bacterial pathogens can be targeted by inhibiting the actions of ribosomes and
affecting protein synthesis (Walsh 2003). Further, some antibiotics can modify the
permeability of the bacterial exterior cell membrane, and later disrupt the structural
alterations of cell membrane, leading to a rapid bacterial death by creating osmotic
imbalances. The polymyxin class of antibiotics attach to the lipid A constituent of
lipopolysaccharide, and results to cause cell membrane structural modifications
(McBain et al. 2003; Tenover 2006). Several classes of antibiotics inhibit cell wall
synthesis. Bacterial cell wall has covalently cross-linked strands of peptide and
glycan, and can be responsible for increased mechanical strength, and prevents cell
lysis due to osmotic pressure. The enzymes transglycosylases and transpeptidases
are responsible for forming this layer. Antibiotics such as penicillins and cepha-
losporins are shown to target the cell wall assembly, and exhibit bactericidal
properties. Antibiotics such as vancomycin is proved to, particularly disrupting the
peptidoglycan layer, and weaken the cell wall structure to result in bacterial cell
death (Schneider and Sahl 2010). The DNA gyrase enzyme is accounted to perform
the uncoiling and supercoiling of DNA strands, and controls DNA replication
process. Thus, targeting this enzyme can be another target for antibacterial drugs/
antibiotics. The antibiotics, ciprofloxacin (a fluoroquinolone), and nalidixic acid
suppress the replication of DNA by attaching to DNA Gyrase enzyme bound DNA
complex (Maxwell 1997). Bacteria might exhibit drug-resistance property to one or
more antibiotics via different types of mechanisms. The resistance property may
vary from one bacterial species to another and to different classes of antimicrobial
agents. So, knowing about the resistance mechanisms exhibited by bacterial strains
can be very useful in designing novel antibacterial drugs (Walsh 2003; Tenover
2006; Khameneh et al. 2016).
Noteworthy to mention here that the drug-resistance could be linked to a single
or more types of mode of actions together (Fig. 7.1). Some of the major proved
mechanisms of antibacterial drug-resistance by bacteria includes the destruction of
the antibiotic drugs by producing damaging enzymes, modifying antibiotic drugs by
secreting modifying enzymes, stimulation of efflux pumps, and changing the
structure of target in the bacterial cells, so that it will have less affinity for recog-
nizing antibacterial agents (Khameneh et al. 2016; Munita and Arias 2016; Peterson
Fig. 7.1 Schematic representation of different antibiotic resistance mechanisms in bacteria, shown
with examples. A Antibiotic modification involves the addition of acetyl, phosphate, or adenyl
groups to aminoglycosides by N-acetyl transferases (AAC), O-phosphotransferases (APH), and O-
adenyltransferases (ANT). Other examples include chloramphenicol acetyl transferases (CAT) and
bleomycin N-acetyltransferases (BlmB). B Antibiotic degradation is observed with b-lactamases,
which hydrolyze the antibiotic. C Antibiotic efflux pumps remove the antibiotic from the cell using
energy from ATP hydrolysis in ABC pumps like DrrAB, OtrC, TlrC, and MlbYZ, or proton
gradients in MFS, MATE, SMR, and RND family pumps. D Target modification includes various
target alterations, such as 23S rRNA or 16S rRNA methylation, alterations in the peptidoglycan
precursors (for example, in the case of glycopeptides), or synthesis of alternate low-affinity targets
(PBPs) that reduce or completely block antibiotic (penicillins) from associating with the target.
E Antibiotic sequestration involves proteins that can associate with the antibiotic and block them
from reaching their targets. F Target bypass involves generation of additional antibiotic targets or
subunits that are not susceptible to binding of the antibiotic. Meth, methylation ( Adapted from
Peterson and Kaur 2018)
and Kaur 2018; Khameneh et al. 2019). In general, antibacterial drugs will be more
effective at definite concentrations and after it reaches to the precise site of action.
Efflux pumps may function by acting as an efflux or transfer system, where
antibacterial drugs are quickly pushed out of cells than the time required for drug
molecule to get diffused into the bacterial cell. This condition will subsequently
lead to a condition, where the intra-bacterial concentration will be much lesser as
compared to the concentration required for effective actions. For instance, the
cytoplasm consists of ribosomes required for proteins. When protein synthesis
inhibitors, including antibiotics are treated, they are enforced to pass through the
cell membrane, and later accumulate at a higher level to inhibit protein synthesis
(Levy 1992; Paulsen et al. 1996; Munita and Arias 2016; Shriram et al. 2018).
A wide range of pathogenic bacterial species, including Staphylococcus aureus,
Acinetobacter baumannii, P. aeruginosa, in addition to fungal species, Candida
albicans show antibiotic resistance through this mechanism. Efflux pumps can
eliminate the antibiotic agents from bacterial strains, for example, trimethoprim and
fluoroquinolones resistance in Pseudomonas aeruginosa. Further, making use of
efflux pump inhibitors along with antibacterial drugs could be a better option of
treatment in overcoming the threatening microbial infections due to multi-drug
resistant microbes (Blair et al. 2015; Munita and Arias 2016; Khameneh et al.
2019). The structural modifications of porins (protein channels) can control the
diffusion of intracellular molecules, including antibiotics. Through this way, few
bacterial strains prevent the antibiotics influx by altering the structure of porins, and
also membrane permeability to exhibit resistance mechanism. This mechanism of
antimicrobial resistance is mediated by several microbes, including gram-negative
pathogenic Pseudomonas spp. and Acinetobacter spp. (De et al. 2001; Vila et al.
2007; Pages et al. 2008). Bacterial species belonging to Enterobacteriaceae can
degrade the b-lactam antibiotics (cephalosporins, penicillins, and carbapenems)
(Olsen et al. 2015; Blair et al. 2015). P. aeruginosa produces modifying enzymes
that destroy or alter the structure of antibiotics, including chloramphenicol and
fosfomycin (Munita and Arias 2016; Khameneh et al. 2019). In addition, a study
carried out with a-Bisabolol isolated from Matricaria chamomilla L in single and
complex form with b-Cyclodextrin as TetK and NorA efflux pump inhibitors
in Staphylococcus aureus strains. a-Bisabolol potentiated the action of tetracycline
and reduced the MIC of norfloxacin to a clinically relevant concentration. The
complexed substance showed synergism; however, the effect of the isolated
a-Bisabolol was superior to the complex. These results indicate a-Bisabolol is a
potential substance to be used as an efflux pump inhibitor (Cruz et al. 2020).
The continued use of medicinal plants and the empirical knowledge of the
communities about them has aroused interest in pharmacological research related to
plants. Many botanical families are being studied with the purpose of finding
biochemical substances in these plants against bacteria and fungi that are resistant to
multiple drugs that are currently available in the market. The combination of tra-
ditional knowledge about medicinal plants, together with technology and science
has enabled countless advances in research, aimed at the search for new alternatives
in the treatment of bacterial and fungal infections. Of the native families of the
tropical flora, the family Myrtaceae, Asteraceae, Piperaceae, Lauraceae,
Verbenaceae, Lamiaceae, among many others stand out to be very important. These
tropical plants solvent extracts, decoctions or crude extracts and essential oils have
been researched extensively in relation to their bioactive potentials.
Due to the difficulty in the treatment of multi-resistant bacteria, it is notoriously
necessary to find new substances that have antimicrobial actions to be used in the
combat of these microorganisms. In this context, photodynamic therapy emerges as
a new alternative in this scenario. Light Emitting Diodes (LED) has been a very
promising resource, although infrequent in clinical practice, but it has already

proven to be very effective in antibacterial action (Gwynne and Gallagher 2018). It
is characterized by producing an absolutely safe irradiation power, consumes little
energy, extremely long-life span, good power, and low intensity. Macedo da Silva
et al. (2020) reported antibiotic-modulating activities of the essential oils
of Eugenia brasiliensis Lam and Piper mosenii C. DC singly or in association with
blue LED (Light-emitting diode) light. They concluded that the association of
aminoglycosides with the blue LED light and essential oils represents an effective
modulatory potential against resistant bacteria such as multi-resistant strains
of Escherichia coli and Staphylococcus aureus. Piper aduncum essential oil was
evaluated in a modulatory experiment associated with blue LED light. The com-
bination of volatile oil with antibiotics showed synergistic effect against S. aureus
and E. coli which was potentiated in the presence of blue LED. The results obtained
in this study showed that the essential oil obtained from Piper aduncum inter-
feres with the action of antibiotics against bacteria exposed to blue LED (Barbosa
et al. 2018).
7.3 Tropical Plants with Relevant Antibacterial Activities
Due to increased bacterial resistance to multiple drugs, antimicrobials arise to

concern and the search for new alternatives therapeutic plants, with medicinal
plants representing an important source to obtain these medicines. The antimicro-
bial activity of extracts and oils essential of medicinal plants has been proven in
several studies conducted in countries that have a diversified flora (Lautié et al.
2020). In South America, with a wide variety of medicinal plants, research showing
that these may be sources of antimicrobial substances has been frequently reported
in recent years (Spézia et al. 2020). According to Michelin et al. (2005), plant
antibiotics have a chemical structure that differs from that of antibiotics derived
from microorganisms, and may regulate the intermediate metabolism of pathogens,
activating or by blocking reactions and enzymatic synthesis or even changing the
structure of membranes. However, since the advent of antibiotics, the use of plant
derivatives as antimicrobials has been little explored (Cowan, 1999).
The acquisition of resistance to antimicrobials is a phenomenon genetic, related
to alteration of genes contained in micro-organisms, which codify different bio-
chemical mechanisms that impede the action of drugs, These mechanisms of action
can be interference with cell wall synthesis; inhibiting protein synthesis; interfering
with nucleic acid synthesis; decreasing permeability to the antimicrobial agent; and
destructing the structure of the cell membrane (Tenover 2006). Bacterial resistance
can arise by acquisition of mutations or by acquisition of genetic material from
other bacteria. Genes encoding proteins involved in resistance mechanisms can be
located on the chromosome or on extra-chromosomal elements, such as plasmids
and transposons, which move easily from one strain to another, from one species
to another, or even from one genus to another (Sultan et al. 2017). Despite the
availability of new antibiotics, bacterial resistance occurs at an increasingly rapid

rate in the different Gram positive and Gram and represents a major therapeutic
challenge (Hayes et al. 2020). In recent decades the focus given to the control of
bacterial infections, may have contributed to the emergence of bacteria multi-drug
resistant, mainly methicillin-resistant Staphylococcus, Penicillin, and erythromycin-
resistant Pneumumococcus, Enterococcus resistant to vancomycin and also
Pseudomonas aeruginosa and Klebsiella pneumoniae resistant to b-lactams and
carbapenens (Ventola 2015).
Considering the increase of multi-resistant bacteria due to the use of antimi-
crobial agents, The World Health Organization (WHO) has recognized the
importance of the potential therapeutic power of plants, and that the associated use
of medicinal plants and/or their by-products with antimicrobial drugs can inhibit or
intensify the therapeutic effect of conventional medicines. In this context, we can
observe some advancements with the products from some species (Table 7.1),
which are already used in the medical clinical routine as antibacterial herbal
medicines.
7.4 Synergisms of Phytochemicals and Conventional

Antimicrobial Drugs
Research into natural products with modulating properties has been widely dis-
seminated. Many of these plant products have considerable synergistic effects,
positively altering the effect of antibiotics and providing an important alternative to
combat increased microbial resistance. The synergistic effects that are usually
obtained by associating natural products with antibiotics are related to the increase
in the influx of the drug, which alters the permeability of the cell membrane,
favoring the penetration of antibiotics and potentiating their effect. Thus, studies on
the association of natural products and synthetic drugs are still promising in an
attempt to discover new chemical classes with antibiotic potential (Cheesman et al.
2017; Khameneh et al. 2019).
Increasing microbial resistance to existing drugs is a problem that is a serious
health issue, and therefore there is a pressing need to look for new classes of
antibacterial substances, especially from natural sources. A therapeutic alternative
for the treatment of micro-organisms resistant to antibiotics is the use of plant
extracts. There are many advantages in using antimicrobial compounds from
medicinal plants, as fewer side effects, better patient tolerance, more economical,
better acceptance due to long history of use, and being renewable because it is
available in nature (Parekh and Chanda 2007). Unlike synthetic drugs, antimicro-
bials of plant origin are not associated with side effects and have a large therapeutic
potential for many infectious diseases (Chanda et al. 2010; Habbal et al. 2011).
Numerous studies have proven the antimicrobial activity in vitro plant. However,
the problem of drug resistance continues to grow. Thus, the need of the moment is
Table 7.1 Some of the plant products with antimicrobial activity

Common Scientific name Compound Active against Dosage form
name
Aloés Aloe vera (L.) Alloin Bacteria Capsule 60 mg
Burm.f
Barberry Berberis Berberine Bacteria, Soft gel 1000 mg
vulgaris protozoa
Black Piper nigrum Piperine Fungi,
pepper lactobacillus,
micrococcus
Calêndula Calendula Triterpenoids Bacteria, fungi Tinture
officinalis
Canela da Cinnamomum Eugenol Bacteria, fungi, extracts 250 mg and
índia zeylanicum 500 mg
Burdock Arctium lappa Tannins Bacteria, fungi, Capsule 475 mg
viruses
Caraway Carum carvi Carvone, Limonene Bacteria, fungi, Capsule 1000 mg
viruses
Cascara Rhamnus Tannins Bacteria, fungi, Capsule 425,
sagrada purshiana viruses 450 mg
Chamomile Matricaria Anthemicacid M. tuberculosis,
chamomilla S. typhimurium,
S. aureus
Clove Syzygium Eugenol General Capsule 500 mg
aromaticum
Cranberry Vaccinium spp. Fructose Bacteria Capsule 500 mg
Croton CrotonsppL proanthocyanidinsand/ General Teaandpills
oralkaloids
Erva de Achillea Lactones Bacteria extracts
Carpinteiro millefolium L
(mil folhas)
Eucalyptus Eucalyptus Tannin Bacteria, viruses Inhalerand tablet
globulus
Garlic Alliumsativum Allicin, ajoene General Tablet
Ginger Zingiber Gingerol Bacteria Capsule 100 mg
officinale
Goldenseal Hydrastis Berberine, hydrastine Bacteria, Solution, 500 mg
canadensis Giardiaduodenale per dosage
Green tea Camelliasinensis Catechin General
Grumixama Eugenia Tannin General An infusion of 10 g
brasiliensis of leaves or bark in
300 ml water
Licorice Glycyrrhiza Glabrol S. aureus, M. Capsule 450 mg
glabra tuberculosis
Mentha Mentha L Menthol General Infusionandtinture
(continued)

Common Scientific name Compound Active against Dosage form
name
Oak Quercus rubra Tannins Capsule 500,
650 mg
Allium cepa Quercetin
Onion Allium cepa Allicin Bacteria,
Candida
Oregon Mahonia Berberine Plasmodium Capsule 500 mg
grape aquifolia
Pata de Bauhinia L Ellagicacid Bacteria Infusion/decoction:
Vaca 2–3 cups of tea a
day, preferably after
the meals
Romã Punica peletierina, Fungi—Candida Infusion, decoction
granatumLinn isopeletierina,
methylpeletierina
Salvia V. curassavica a-Humuleno Bacteria Fungi Tinture
Senna St. Hypericum Hypericin, others General Table 450 mg
John’s wort perforatum
Thyme Thymusvulgaris Caffeicacid Viruses, bacteria, Capsule 450 mg
fungi
Turmeric Curcuma longa Curcumin, Bacteria,
Turmericoil protozoa
Adapted from Khameneh et al. 2019
to develop useful or new antimicrobial agents’ ways to treat the resistant

microorganism (Negi and Dave 2010). In that case, a new form of therapy would be
the use of the combination of synergistic antimicrobial therapy between antimi-
crobial agents known and bioactive plant extracts. The combination therapy
between plant extracts and antibiotics can expand the antimicrobial spectrum,
prevent the emergence of mutant resistance, and minimize toxicity. Sometimes the
use of a single antibiotic does not produce the inhibitory effects desired effective-
ness, but a combination of drugs often has an effect synergistic that surpasses your
individual performance. The synergistic effect can be due to the formation of the
right complex that becomes more effective in inhibiting a particular species of
microorganism, either by inhibition of the synthesis of the cell or causing its lysis or
death (Chanda and Rakholiya 2011). Thus, the most economically viable combi-
nation of natural products associated with available antibiotics shows as an alter-
native, since the synergistic effect between the two can provide an increased
antibacterial activity against sensitive and resistant microorganisms. Therefore, the
potentiated effect of these associations can serve as a new infection treatment
strategy, enabling drug use antimicrobial when it alone is not effective on certain
bacterial strains. The association of extracts from various plants with ampicillin,
chloramphenicol, and tetracycline against sensitive bacteria (S. aureus, Salmonella
choleraesuis, P. aeruginosa, Bacillus subtilis, Proteus spp) and resistant bacteria

isolated from hospital environment (K. pneumoniae, Shigella spp, Proteus spp,
P. aeruginosa, Enterobacter aerogenes, E. coli and S. aureus) showed that in some
cases it occurred synergism enabling already ineffective antibiotics to act on these
bacteria (Liu et al. 2017).
7.5 Antibiotic Resistance—Combination Therapy

(Antibiotic + Phytochemical/Plant Extract)
It is estimated that the herbal medicine market moves up to US$ 300 billion around
the world and the traditional pharmaceutical market is growing at an annual rate of
3% to 4% worldwide, while that of herbal medicines rises from 6 to 7% (Calixto
2019). On the other hand, the World Health Organization (WHO) recognizes the
importance of the therapeutic potential of plants, but does warnings about improper
use and preparation, and recommends caution if they consider the lack of knowl-
edge about possible side effects with a joint administration of prescribed drugs. In
any case, what occurs in most societies today is a complementarity between
allopathy and the use of medicinal plants. In recent years and in most developing
countries, people have resorted to self-medication, which has often led to increased
microbial resistance to the drug. However, the trend in drug discovery is gradually
returning to natural products because of the constant development of microbial
resistance to synthetic drugs. The large-scale use of antibiotic therapy in recent
decades is largely a result of the aging process, which influences the increase of
infections associated with chronic age-related diseases such as metabolic, cardio-
vascular, and neoplastic diseases. These conditions are often associated with
oxidative stress and act directly on the global pharmaceutical burden. Resistance to
antimicrobials represents a growing challenge for modern science. Especially in a
hospital environment, the use of powerful, broad-spectrum antibiotics, inadequate
or sub-therapeutic treatment, as well as the lack or deficiency of preventive mea-
sures contribute to the development of resistance to these drugs. To this end,
strategies that prolong the effectiveness of currently available antibiotics, encourage
their rational use and reduce or avoid the spread of multi-drug-resistant microor-
ganisms (MDRs) are sought. Understanding the mechanisms of antibiotic resistance
is a fundamental step toward the discovery of effective therapeutic measures (Rice
2018; Yelin and Kishony 2018).
Extracts from Piper species have been reported to be effective against
Gram-positive and Gram-negative bacteria and could be a potential lead to the
discovery of new antimicrobial drugs (Mgbeahuruike et al. 2017; Salehi et al.
2019). Antimicrobial photodynamic therapy has grown considerably in recent
decades as an effective and inexpensive alternative. Piper aduncum is popularly
known as monkey pepper with antibacterial activity. This study aimed to evaluate
the antibacterial and modulatory activity of Piper aduncum essential oil (EOPad)
associated with blue LED light, which was exposed to the LED for 20 min. The
Germacrene A was identified as the main component. The OEPad presented
MIC 1024 lg/mL against S. aureus and E. coli. The combination of EOPad
with antibiotics showed synergistic effect against S. aureus and E. coli which was
potentiated in the presence of blue LED. The results obtained in this study showed
that the essential oil obtained from Piper aduncum interferes with the action of
antibiotics against bacteria exposed to blue LED (Barbosa et al. 2018).
The antimicrobial potential of leaf of Piper caldense essential oil was investi-
gated, and its potential was investigated. The minimum inhibitory concentration
(MIC) and minimum fungicidal concentration (MFC) was determined. Besides the
essential oil was also tested as a modulator for several antibiotics, and its effect on
the morphology of Candida albicans (CA) strains was also investigated. The
essential oil modulated the activity of fluconazole against CA URM 4387 strain,
which was demonstrated by the lower IC50 obtained, 2.7 lg/mL, whereas
fluconazole itself presented an IC50 of 7.76 lg/ml. No modulating effect was
observed in the MFC bioassays. The effect on fungal morphology was observed for
both CA INCQS 40,006 and URM 4387 strains. The results demonstrated that the
oil has potential as an adjuvant in antimicrobial formulations (Bezerra et al. 2020).
A study was carried out with essential oil from the fresh leaves of Hyptis
martiusii to investigate the modulating activity in association with different
antibiotics against the bacteria Staphylococcus aureus, Escherichia coli, and
Pseudomonas aeruginosa, and to evaluate the cytotoxic activity of the species. The
research has shown that the essential oil of Hyptis martiusii Benth (OEHM) leaves
presents synergism only in association with gentamicin antibiotics and imipenem
against the bacteria Pseudomonas aeruginosa and Escherichia coli. However, it
presents antagonism in association with amikacin, gentamicin, and imipenem
against the three bacteria studied. Apart from ciprofloxacin, no relevant results were
demonstrated. In relation to the cytotoxic activity, the mean lethal concentration
(lc50) exposed a value of 263.12 lg/ml. The results reveled that H. martiusii
presents synergistic cytotoxic activity against the evaluated bacteria (Figueiredo
et al. 2018).
A study carried out by Ferreira et al. (2019) identified the chemical composition
of the Senna spectabilis species, and they analyzed the antimicrobial potential
in vitro and its modulating effect on antibiotic activity. The antibiotics used in the
experiment were aminoglycosides (amikacin and gentamicin), lincosamides (clin-
damycin), cephalosporins (cephalexin), and azithromycin. The Staphylococcus
aureus, Escherichia coli, Pseudomonas aeruginosa, and Salmonella enterica
strains were used in this approach. The results have shown that the antimicrobial
action of the extract of S. spectabilis is only regular; however, in modulation, the
extract in combination with the tested antibiotics showed a synergistic effect against
most of the tested bacteria, potentiating the effect of the antibiotics used.
Ferreira and Costa evaluated the antimicrobial activity of ethanolic extracts of
Banisteriopsis anisandra (Malpighiaceae) and drug interactions with drugs
widely used in the field of oral health. They obtained the leaf extract and performed
serial dilutions, which were tested against Candida albicans (ATCC 10,231),
Streptococcus mutans (ATCC 25,175), Streptococcus salivarius (ATCC 7073), and

Staphylococcus aureus (ATCC 6538). As positive control, antibiotics ampicillin
(10 µg/ml), amoxicillin (10 µg/ml), and penicillin (30 µg/ml) were used for bac-
terial strains; and the antifungal ketoconazole (2 mg/ml), for yeast. There was a
positive drug interaction of the extracts with antibiotics and ketoconazole in con-
centrations of 1: 2 for the extract. The extract of B. anisandra has an antimicrobial
potential and drug interaction with drugs related to oral health. Studies on the
pharmacological properties of the species in question are rare and the results are the
first reports on its antimicrobial activity in association with antibiotics and
antifungals.
The positive results found not only in these reported studies, but also in
countless other studies, with different species increasingly show the importance of
the search for new metabolites that can help and reinforce drugs that no longer play
the role of eliminating pathogenic microorganisms.
7.6 Essential Oils with Antimicrobial Activity

from Tropical Medicinal Plants
In nature, EOs play an important role in providing plant protection against patho-
genic bacteria, viruses, and fungi and preventing the attack by insect pests. In
addition, EOs can attract or repel insects when present in pollen and seeds. To
protect chemical compounds’ ecological equilibrium, the use of EOs in pharma-
ceutical, food, bactericidal, and fungicidal is becoming more prevalent in recent
times (Swamy et al. 2016). EOs yielding medicinal and aromatic plants are nor-
mally native to warm countries, where they represent an important traditional
pharmacopeia.
Apiaceae, Lamiaceae, Myrtaceae, Poaceae, and Rutaceae families are of par-
ticular importance for medicinal applications. For example, some of the EOs, like
anise, caraway, black caraway, clove, oregano, cumin, coriander, sage, basil, dill,
lemon balm, peppermint, thyme, and tea oils, already have widespread medicinal
applications. Some of the essential oil containing plant families, like Liliaceae,
Fabaceae, Pinaceae, Piperaceae, Cupressaceae, Asteraceae and Hypericaceae, also
exhibit a considerable medicinal potential.
The Apocynaceae family is considered to be dicotyledonous characterized usually
by the presence of latex, consisting of about 155 genus and 2000 species, with
distribution in Tropical and Subtropical regions (Lorenzi 1998). This family can be
considered one of the most important plant sources of chemical constituents with
therapeutic utility in modern medicine. The most important genres in this family are
Alstonia, Aspidosperma, Vinca, Tabernaemontana, Mandevilla, Hancornia, Nerium,
Strophantus, Catharanthus, Allamanda, Thevetia, Wrightia, Plumeria, Himatanthus
and Rauvolfia. The Apocynaceae family is chemically characterized by structures
alkaloids mainly monoterpenic. Several indolic alkaloids have already been isolated
from this family, especially from the Aspidosperma and Geissospermum genus
(Ekalu et al. 2019). In addition to representatives with numerous medicinal properties,
the family Apocynaceae is an important source of economic resources. Rubber is
derived from the latex of several species, markedly inferior in quality to that of the
extracted from Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg. the rubber tree. At
African countries and indigenous peoples of South America, toxic species are used to
poison arrows in animal hunting and fishing. Other species provide excellent quality
wood for the manufacture of furniture such as case of the genus Aspidosperma,
whose most common representative is A. peroba Allemão ex Saldanha, known as
“peroba-rosa” (Baratto et al. 2010).
The therapeutic potential of the Himatanthus drasticus (Mart.) Plumel,
Apocynaceae was assayed. This specie presents triterpene-rich fraction which the
anti-inflammatory activity is mediated by NF-alpha, iNOS, COX-2 and NF-kB
(Almeida et al. 2017). The essential oil of Mikania cordifolia and its major con-
stituent limonene was investigated alone or associated against Pseudomonas
aeruginosa, Escherichia coli, and Staphylococcus aureus. The antibiotic-
modulating activity was determined in combination with conventional antibacte-
rial drugs. The results demonstrated no relevant antibacterial activity; however, a
modulatory effect was observed against P. aeruginosa, presenting synergistic
effects when associated with gentamicin and norfloxacin. In addition, the oil
reduced the MIC of norfloxacin against E. coli as well as reduced the MIC of
gentamicin against S. aureus. The best effect of limonene was obtained against S.
aureus. It is possible to conclude that the Mikania cordifolia essential oil and the
isolated compound limonene modulate the action of antibiotics against MDR
bacteria (Araújo et al. 2020). The essential oil bacterial pathogens action is asso-
ciate to capacity to destabilize the cellular machinery, leading to breakdown of
membrane wall, disrupting many cellular activities including energy production and
membrane transport. The lipidic components of essential oils induce membrane
rupture leading to leakage of cellular components and loss of ions (Fig. 7.2)
(Swamy et al. 2016; Tariq et al. 2019; Basavegowda et al. 2020).
7.7 Plant-Derived Natural Products with Antifungal

Activity
The resistance of microorganisms to therapeutic agents currently used has caused

economic and social impact. Fungal resistance has been somewhat neglected when
compared to the great repercussion that bacterial resistance has reached. One is
strictly related to the other, since the intensive use of antibacterial antibiotic therapy
reduces competitiveness in human biota and favors the growth of yeasts of Candida
spp. which in due course change from diners to pathogens. In addition, other factors
contribute to increased resistance, such as the significant increase in the number of
immunosuppressed patients and the prolonged use of surgical devices, considered
resistance drives.
Fig. 7.2 Antimicrobial mechanisms of essential oils on microbes (Adapted from Swamy et al.
2016)
Infections caused by Candida spp. have shown a high level of incidence, where
Candida albicans and Candida tropicalis stand out, with notoriety for the former,
as agents capable of causing invasion. Their virulence factors provide adaptability,
since they allow the fulfillment of important criteria for survival, such as having
tolerance to high temperatures and invasive potential, promoting lysis and
absorption of human tissues and resisting immunological defenses. The current
context requires searches for bioactive substances with antifungal potential. New
therapeutic agents may be contained in plants and consequently, their extracts and
essential oils may serve as subsidies for in-depth studies and research that con-
template and target the formulation of new drugs. Research has been conducted to
promote the combination of commercial drugs, for which resistance phenomena
have already been verified, with natural products in the form of extracts, fractions,
essential oils or isolated constituents, in an attempt to circumvent the resistance of
Candida spp.
Recently, a medicinal plant Mesosphaerum suaveolens aqueous extracts of
leaves (AELMs) and aerial parts (AEAPMs) was evaluated against strains of the
genus Candida. The modulatory effect was observed with the drug fluconazole. The
AELMs have shown good results since modulated fluconazole activity decreased
fluconazole’s IC50 from 7.8 lg/mL to an IC50 of 4.7 lg/mL (CA LM 77 strain)
and from 28.8 lg/mL to 18.26 lg/mL (CA INCQS 40,006 strain) for the
C. albicans strains. For the C. tropicalis LM 23 strain, the AEPMs obtained an
IC50 of 25 lg/mL and the AELMs an IC50 of 359.9 lg/mL. The AEAPMs as well
as the AELMs presented clinically relevant activities for C. tropicalis strains (Costa
et al. 2020).
A modulatory effect of Psidium guajava L., (known as goiaba) an important

medicinal plant found in tropical regions, was assayed against Candida albicans,
Candida tropicalis and Candida krusei in association with fluconazole. The syn-
ergistic effect varied from 925.56 to 1.57 lg/mL. The flavonoid and tannic frac-
tions, rich in phenolic compounds, potentiated the action of Fluconazole, reducing
its concentration and impeding morphological transition, one of the virulence
factors of the genus (Bezerra et al. 2018).
7.8 Fungal Resistance and Modulatory Potential

of Extracts, Fractions and Essential Oil from Tropical
Medicinal Species
The involvement of fungal infections in humans has gained prominence in recent

years, and among the most common fungi infections, those of the genus Candida
stand out, being among the species causing opportunistic infections. This is justified
by the fact that they are very well adapted to the human body, being able to
colonize it without producing signs of disease under conditions of physiological
normality. However, they can become pathogenic in immunosuppressed patients,
generating infections that are difficult to diagnose and are associated with high
mortality and morbidity. As for clinical manifestations, we can divide them into
cutaneous, systemic, and allergic. The treatment of these pathologies is performed
with synthetic drugs with systemic action such as amphotericin B, fluconazole,
itraconazole, voriconazole and echinocandins, azoles being the most used, due to
their low cost and low toxicity.
The availability of antifungal agents for the treatment of systemic infections is
somewhat reduced, becoming a problem with multi-drug resistant yeasts. In addi-
tion, one of the stalemates today is resistance to antifungals and difficulties in
effective treatment. As a way to ease this resistance and difficulty in treatment,
medicinal plants are strongly used by the population for the treatment of infections,
and this fact has aroused interest from several research centers for the study of this
activity, with the aim of isolating new molecules with antimicrobial activity.
Natural plant products have been evaluated not only for their direct antimicrobial
activity, but as a modification agent of resistance to antifungals, which can be a
synergistic or antagonistic effect. Thus, the associated use of medicinal plants or
their derived compounds may interfere, inhibiting or intensifying the therapeutic
effect of conventional antimicrobials. In this context, the synergistic effect of Piper
mikanianum essential oil species rich in safrole was investigated against Candida
strains. An interesting result was observed where the oil was combined with gen-
tamicin against the multi-drug resistant E. coli strain and also associated with
fluconazole against fungal strains. The oil presented a fungistatic effect. The
P. mikanianum essential oil has shown an inhibitory effect of one of the main
virulence factors of the Candida genus, morphological transition, which has been
previously shown to be responsible for causing invasive infections in human tissues

(Carneiro et al. 2020). The mechanism can be associate with membrane potential
across cell wall and disrupt ATP assembly, leading to cell wall damage. Essential
oils can also disintegrate mitochondrial membrane interfering with the electron
transport system (ETS) pathway (Tariq et al. 2019). Several species of Asteraceae
are producers of essential oil of commercial importance. The genus Baccharis is
represented by more than 500 species, distributed predominantly in South America.
In Brazil, 120 species are described, distributed in greater concentration in the
South Region of the country (Giuliano 2001). This genus produces many secondary
metabolites, and in general, the compounds that stand out the most are flavonoids
and terpenoids, especially monoterpenes, sesquiterpenes, diterpenes and triterpenes
(Trombin-Souza et al. 2017).
Recent studies have shown the biological activities of the Baccharis genus, such
as antioxidant (Zuccolotto et al. 2019), antiparasitic (Budel et al. 2018), cytotoxic
activity (Fukuda et al. 2006), as well as acts in inflammatory processes (Florão
et al.2012). A study carried out with Baccharis reticulata demonstrated an
antibiotic-modulating activity against both Gram-positive and Gram-negative bac-
teria. The best result was observed with the volatile oil in association with nor-
floxacin and gentamicin against the multiresistant strain of S. aureus. In addition,
the oil exhibited a synergistic effect for Gram-positive and Gram-negative bacteria
(Freitas et al. 2020). Evidence of in vitro synergism with MDR strains provides a
higher probability of successful in vivo therapy. Thus, these associations can be
useful in selecting the most promising combinations of antimicrobials for practical
therapy of severe bacterial infections.
Licania rigida Benth (known as “oiticica”), belongs to the Chrisobalanaceae
family and it is distributed in tropical and subtropical regions. A study was carried
out with leaf ethanolic extract to evaluate the virulence factor associated with the
ability to form biofilms on biotic and abiotic surfaces in association with fluo-
conazole since its action on biofilms is described. The study demonstrated that
biofilms formed by Candida sp. isolated in acrylic resin discs reduced biofilm
formation. It is associated with the reduction of hydrophobicity mechanisms of the
cell surface, which reduce the aggregative potential and the formation of the biofilm
(Freitas et al. 2019).
These outcomes of the use of medicinal plants by different populations, a fact
that passes through generations, have highlighted species that present active prin-
ciples of recognized clinical relevance, which in some cases have been transformed
into modern medicines. Successful therapeutic evidence observed from the daily
life of traditional or local communities has inspired pharmacological research, and
this forms the basis of the ethnobiological approach, which is guided by popular
practices and knowledge, consolidated through time, culture and history of several
people, contributing to the discovery of agents with therapeutic potential or even a
new drug, more specifically.
7.9 Conclusions and Future Prospects
The valorization of biological and cultural diversity, as well as the knowledge

acquired by several traditional practices, becomes a necessary tool to know and
classify the use of natural resources. In this context, empirical knowledge not only
has great relevance in the discovery of new medicinal plants, suppliers of bioactive
chemical substances originated mainly from the secondary cellular metabolism of
the plant, but also are focused on the planning of actions related to the sustainability
of natural resources managed. It is known that in tropical regions some social
groups have vast traditional knowledge about the different forms of exploitation and
management of natural resources, mainly about plant species. Several ethnic groups
have created mechanisms and strategies for disease treatment and prevention. The
accumulation of knowledge and practices form empirical medical systems often
based on the use of natural plant resources.
The study of these mechanisms provides ethnopharmacology with a supposed
efficacy of plants that are selected depending on the concept of disease and health
by these groups, and this identification also helps in the improvement of other
approaches that use medicinal plants as the object of study. Obtaining new alter-
natives for the development of new products of natural origin, it can be charac-
terized as a new chemically defined compound with determined pharmacological
activity (pharmacotherapeutic) or a simple traditional preparation from parts of a
medicinal species (phytotherapeutic) that has evaluated its efficacy.
The discovery of compounds with biological activity may be more effective from
active extracts of perennial plants. This is because these plants invest effectively in
lifelong chemical defenses and have higher levels of compounds. One of the main
advantages of using natural products in the search for new drugs, compared to
synthetic compounds, is the high probability of biological activity. Secondary
metabolites are synthesized and optimized through the evolution of specific bio-
logical interactions between the producer and the environment. In this sense, the
chemical diversity of these substances becomes unequalled.
Thus, it is fundamental to understand how medicinal plants behave in the
environment, how these resources are used and how this information can contribute
to sustainable use strategies. From this conception of traditional knowledge, it is
possible to design strategies that lead to alternatives that respect the need for
conservation, together with the traditions of the people who use these resources and
the scientific knowledge, being able to provide information for a better time of
collection and a better period in which it produces high yields of essential sub-
stances for medicinal treatment and subsequent pharmacological uses.
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Chapter 8
Capillary Electrophoresis: A New
Evolutionary Platform of Plant
Secondary Metabolites
Dilipkumar Pal and Souvik Mukherjee
Abstract Capillary electrophoresis (c.e) is a modern Spt technique other than any
other chromatographic technique for the detection of plant secondary metabolites
(Psm). There are two types of c.e such as capillary zone electrophoresis (c.z.e) and
micellar electro kinetic (M.e.k.c.c). All kinds of secondary metabolites (Sm) (fla-
vonoids, cardiac glycosides, aglycones, steroids, diterpene, saponin, etc.) are not
possible to isolate with the help of HPLC and Gas chromatography. But such type
of metabolites may possibly be isolated with the help of c.e smoothly and easily.
However, in c.e charged molecules are transferred to the opposite charged mole-
cules in the presence of the electrical field. Very low solvent, low price silica
columns, and small amount of samples are needed to run the c.e. There are various
characteristics such as voltage (Vtg), temperature, electrolyte concentration, BF pH,
micelle concentration, and organic modifiers may influence the Spt of different Sm.
In this book chapter we will describe the different parameters of c.e like method-
ology, detector sample analysis, and combination of other hypnated technique for
the detection of metabolites.

Keywords Capillary zone electrophoresis Micellar electro kinetic capillary

chromatography Phenolics Alkaloids Terpenoids
Abbreviation
ANT Acetonitrile
Alkd Alkaloid
AACT Ammonium acetate
BRT Borate
BF Buffer
BFS Buffer solution
C.e Capillary electrophoresis
C.w Capillary wall
D. Pal (&) S. Mukherjee

Department of Pharmaceutical Sciences, Guru Ghasidas Viswavidyalaya
(A Central University), Bilaspur, Chhattisgarh 495 009, India
https://doi.org/10.1007/978-3-030-54027-2_8
288 D. Pal and S. Mukherjee
C.z.e Capillary zone electrophoresis

C.n Coumarin
Esp Electrolyte solution parameter
ESOP Electro-osmotic pressure
E.c Eukaryotic cell
Fvd Flavonoid
F.s.c.c Fused silica capillary column
I.p Instrumental parameters
M.m Metabolism
Mtd Method
M.e.k.c.c Micellar electro kinetic
M.C.L Micelle
NI Negative ion
Ps Plant species
PI Positive Ion
P.c Prokaryotic cell
Qn Quinone
Spt Separation
Tps Terpenoids
Vtg Voltage
8.1 Introduction
Metabolism (M.m) is a magical strategy for the formation of various cellular

function. It is also observed that M.m is created either the beginning of eukaryotic
cell (e.c) and prokaryotic cell (p.c) (Harstad et al. 2016). If M.m does not occur, all
the functions related to natural cellular works of a normal cell would be lost. Energy
is required for conducting the formation of the cell. This energy is derived from
food, cellular function is switched on when energy is derived from food (Han et al.
2017). There are two types of M.m such as catabolism (compound breaking) and
anabolism (compound making). The metabolite products (m.ps) of e.c are protein,
carbohydrate, lipid, and nucleic acid which are also known as a primary metabolite
(Zhang et al. 2017). Besides, m.ps of p.c (plant, bacteria, fungus, etc.) are alkaloid,
glycoside, terpenoids, saponin, etc. However, there are different Mtd (various
spectroscopic technique and chromatographic technique) in regards knowing for
what types of P are created in e.c (Junger et al. 2019). By the application of external
electrical field, if a sample contained positive (PI) and negative ion (NI), then if
these PIS are transferred into the negative end and NIS are positive end then this
phenomenon is called as electrophoresis (Fig. 8.1). There are various Mtd involved
in this technique (Fig. 8.2). So, Capillary electrophoresis (c.e) is a new hyphenated
separation (Spt) technique (Fig. 8.3). It was first invented by Herten in 1967,
8 Capillary Electrophoresis: A New Evolutionary … 289
thereafter, it was recognized through Jorgensen and Lukas in 1981 (Lu et al. 2018).
Based on these techniques there are created two processes such as c.z.e and
Micellar electro kinetic capillary chromatography (m.e.k.c.c). At the first time these
technologies used for the detection of amino acid, oligonucleotides, nucleotides,
DNA, protein, etc., but at present, it is used for the detection of Psm (Voeten et al.
2018).
8.1.1 Principle of the Technique
This c.e method (Mtd) is completely different from any other Spt technique.
Suppose we have various particles containing colloidal solution. Now, if these
particles are dynamic by an electrical field, then positive charges are accumulated in
cathode and negative charge in anode (Aiken and Hue 1993; Singh et al. 2017).
Because, during the particles dynamic movement, an electrical potential is gener-
ated into the solution. It is the main principle of c.e. However, managing this
technique requires a lot of things such as (a) capillary tube, (b) siphon tube (c) UV
detector. 50–100 µm diameter and 1 m length containing fused silica capillary
column (F.s.c.c) is used this instrument. F.s.c.c is cover by polyamide coat
(Rahman 2018). The sample is loaded in the end part of anode. Though there are a
various technique for sample loading such as (a) pressure differential (b) Siphoning
(c) Split flow injection (d) Electrophoretic injection. µg/ml and Nano/ gm. (Zhang
et al. 2020). Concentration containing sample is used for better Spt. At the first stage
of Spt electrical Vtg (maximum 50 kV Vtg but 30 Vtg is needed) is applied as a
result solution containing whole particles are accumulated in cathode by water
electro-osmotic pressure (ESOP). ESOP is the main driving force in this technique
(Issaadi et al. 2017). The water flow depends on the principle of (a) electro potential
of capillary and (b) nature of capillary. In neutral or alkaline PH medium the ESOP
is maximum. The silanol group of a capillary wall (C.w) is being polar because of
the highest ESOP (Ribeiro et al. 2018). The surface of the C.w is surrounded by the
negative charge. When these counter ions are attached with solution then a positive
Applied electrical field
Fig. 8.1 Gel electrophoresis

Fig. 8.2 Various techniques for electrophoresis
Fig. 8.3 Brief summary of capillary electrophoresis
charge is formed into the (C.w). C.w has formed a doubled layered wave due to
applied Vtg attachment of water molecule with various ion (Zhao et al. 2016).
8.1.2 Nature of Spt
Based on the C.e principle, there are two types of other Spt technique such as
capillary zone electrophoresis (C.z.e) and Meccc. Now, we will discuss these types
of Spt technique (Lima et al. 2019).
(a) C.z.e: It is also known as free solution capillary electrophoresis. It depends on

charge mass ration of the molecule. This Spt is started when PH value is above
4. If the mass of the element is small and higher negative value then migration
time is also increased. Complex sugar molecule and positive, negative neutral
molecules are also separated by this technique (Wu et al. 2018).
(b) Meccc: Neutral and hydrophobic molecule are separated in this analytical Mtd.
A unique incident occurs while it is processing. Micelle (M.C.L) is activated in
the opposite direction of the electrical osmotic pressure when micelle is used
(Miyagi et al. 2017). When micelle M.C.L is attached with anionic surfactant
then M.C.L is accumulated in anode and E of is transfer into the cathode.
Thereafter, cationic surfactant is attached with M.C.L then E of is entered into
the anode and M.C.L in cathode (El et al. 2017).
8.2 Factors Effecting Spt
There are various factor effect the Spt process. These factors are divided in two
section such as (1) Instrumental parameters (I.p) and Electrolyte solution parameter
(Esp.). I.p are four types (a) Vtg (b) polarity (c) Temperature (d) Capillary wall and
I.ps are (a) type and concentration of buffer (BF) (b) PH of the BF (c) Organic
modifiers (d) Additives (Gomes et al. 2018).
8.2.1 I.P
(a) Voltage: For the application of the Vtg in this instrument, Jules law must be
followed. The disunion time is inversely proportional to an applied Vtg. When
the Vtg is increased then more amount of heat is produced and increased the c.w
temperature (Dresler et al. 2017a, 2017b). As a result resolution of the Spt
process is also reduced by the help of viscous BF of c.w.
(b) Polarity: There are two electrodes such as (a) Cathode (outlet) and (b) Anode
(Inlet). ESOP is situated far from the outlet. When the charge of the molecules
are highest than the ESOP then molecules are easily accumulated in outlet
(Kelley et al. 2019).
(c) Temperature: This parameter is also affected by the Spt process. If increased the
C.w temperature then changed the conformation of the molecule (Cong et al.
2017).
(d) Wall of the capillary: During capillary length and internal diameter analysis, the
capacitance influences the Spt efficiency and load. Depending on the volume of
the capillary and the injected sample, the detection system also affects the
detection limit. Limits the adsorption efficiency of the sample material on the
capillary wall; Therefore, Mtd s to avoid these interactions should be considered
in the development of segregation Mtd s (Yang et al. 2019).
8.2.2 Esp
(a) Type and conc of buffer: BF suitable for c.e have appropriate BF capacity at pH
range. Choice and low mobility to slow down the current generation (Sharma
et al. 2018).
(b) PH of the BF: It can affect the Spt by altering the charge of the analytes or
additives ESOP (Huang et al. 2019).
(c) Organic modifiers: We need for operating this process to reduce the either
velocity of ESOP or adjustment. For maintaining this system various organic
modifier (Methanol and Acetonitrile [ ANT]) are added the BF solution to
increase the solubility (Zhao et al. 2018).
(d) Additive: To separate the optical isomers, the chiral selective Spt buffer is
additional. Mostly Chiral selectors usually use cyclodextrin, though in some
cases crown ethers, bound polysaccharides, or possibly proteins can be used
(Dresler et al. 2017a, 2017b), (Rama and Vinutha 2019).
8.3 Instrumentation
The main components for running the C.e technique such as (a) To keep the sample
in a vial (b) Assemble vial (c) Buffer solution (BFS) (d) Capillary tube (e) Vtg
(Electricity or current) (f) Detector (g) Computer system (Sharma et al. 2017).
Firstly, the BFS is made by processing in which type of compound is detected
based on chemical nature and PH range. Thereafter, the BFS solution is poured into
the vial, assembling vial and capillary (Jones et al. 2018). Then the tip of the
capillary is made entered into the vial. The sample goes towards the capillary side
by the help of capillary force and siphoning force and returned into the reverse way.
It is pertinent to mention here, that main cause for going towards the capillary side
an electrical field is generated between the vial and assembling vial (Mendoza and
Silva 2018). As a result, positive and negative ions of the samples are accumulated
into the middle part of the capillary by an electrical force. At the end samples being
isolated into the end part of capillary and assemble into a vial (Memon et al. 2017).
Thereafter, detectors identify the chemicals which are existing with the sample
through the computer system. The capillary wall is covered with polyamide or
Teflon so that smoothly detect the sample (Dresler et al. 2018). The sample is
detected by the help of Beers and lamberts law. There are various detector used
namely (a) UV (b) UV-Visible (c) 0.01attomole containing lase induced fluores-
cence detector (d) potentiometric detector (d) Conduct metric detector. But UV or
UV-Visible detector is mostly used (Ngoc et al. 2019).
8.4 Difference Between C.e and Others Spt Technique
Some differences are observed between C.e techniques with any other Spt Mtd.
Especially seen in High-performance Liquid Chromatography (HPLC). To operate
the C.e some hindrances are facing such as (a) Uncleanliness of C.e.w (b) Diameter
of C.e (c) Reaction between silica-containing C.e.w and basic BF, (Spisso et al.
2018).
To develop the Mtd of C.e scientists are frequently trying with best efforts. To
clean the C.e.w water, sodium hydroxide and any other solvents are used. But the
cleaning position does not exist during more times if such solvents are used (Xiao
et al. 2018). Let us we discussed about the C.e techniques with others Spt technique.
To keep power a compound can be separated by using C.e by using 1–30 min time,
high amount of Vtg and using minimal sample. C.e can separate 87,000–120,000
sample plate within minimum time. On the other hand, HPLC can separate the
2600–18,000 plate (Nimse and Pal 2015). Very less amount sample and minimum
samples are separated in HPLC technique. To process the HPLC a very good
knowledgeable person is required, but besides in the case of C.e a good person is
not required and even glass room is not needed (Pal et al. 2006).
8.5 Application
Alkaloid (Alkd) is a nitrogenous basic element. It is also known as PSM. Seens

Alkd is a positive charge in nature and less value of PH, It is easily separate by C.e
(Pal et al. 2006). Various alkaloidal compounds are separated by C.e (Pal et al.
2005). For example quaternary alkaloidal compound (Fig. 8.4), (Table 8.1).
Fig. 8.4 Schematic representation of capillary electrophoresis

294
Table 8.1 Application of C.e in Alkd

Name of Structure Solvent Diameter of column Vtg Detector References
The
alkaloid
Purine O H N O 20 mM borate, pH 9.2 50 cm x 50 um 10 kV 254 nm (Mukherjee
N NO N N N N
NH N et al. 2018)
N O N N O
O
Ephedra OH H 20 mM isoleucine, 5 mM barium hydroxide 60 cm 75 pm (52.5 cm 27 20–28 185 nm (Chahel
N (pH 10.0 with ammonia) methanol, 10 mM °C 60cm 75pm (52.5 25 ° et al. 2019)
phosphate (1:4); 10 mM borate (pH 9.0), C 48 cm 50 pm (26 cm
100 mM SDS 86) 30 °C
Imidazole O 100 mM phosphate PH 6.9, 10 mM 50 cm 50 pm (40 um to 8 217 nm (Badria and
N O B-cyclodextrin detector) Aboelmaaty
N 2019)
Oxindole N 20 mM phosphate (PH 5.6) 57 cm 75 pm (50 cm to 10 254 (Kolrep
O detector) 25 °C et al. 2019)
O
N
H
D. Pal and S. Mukherjee
Methodology:
8.5.1 Quaternary Alkaloidal Compounds
At first, 100 mM sodium acetate is made into a 100 ml volumetric flask and make
up to 100 ml. Then withdraw 85 ml solution from this 100 ml solution as a result
BSF (For better resolution) is made (Pal and Verma 2013). Then 15 ml methanol is
added into the BF for sharp peak. In this Mtd 100 cm * 100 µm column is used at 28
°C. 25 kV Vtg is applied in this technique (Sannigrahi et al. 2009). The Spt power of
this Alkd is completely depended on which substituted are attached with aromatic
ring, So, the serial of separating power is Methylene dioxy < two methoxy
group > four methoxy group < one aliphatic hydroxy group < two aromatic
hydroxy group (Gupta et al. 2003).
8.5.1.1 Opium
Particularly this compound (Fig. 8.5) is separated by the help of M.e.k.c.c. But c.z.e
technique is used mostly (Pal and Mitra 2010). 25 mM citrate BF and 50 cm *
75um fused silica column are used for C.z.e at PH 4.2 to detected this type of Alkd
in UV range 214 nm (Mir et al. 2019). However 20 kV Vtg is also used. 10 mM
borate and 50 mM SDS are used in PH 9.2 in M.e.k.c.c. Here 50 cm * 50um
diameter Coolum and 20 kV Vtg are needed. The UV range of this compound is
214 nm (Pal et al. 2019) (Figs. 8.6, 8.7, 8.8 and 8.9).
8.5.2 Flavonoid
Three carbon phenolic hydroxyl aromatic ring containing Psm is called flavonoid
(Fvd). There are 4000 Fvd is discovered till date. Fvd is available in different kinds
of fruit, seed, and vegetables. This Fvd is a most important part of our daily life for
the prevention of various diseases (Jorge et al. 2019; Pal et al. 2006; Pal and Saha
2019). To know what type or how quantity is contained in a natural product there
are various technique are available (GC, GC–MS, TLC, HPLC) but these are more
time limited (Soldatou et al. 2019). As a result there is a new introduced technique
for the Spt of Fvd which is known as C.z.e (Alagoz et al. 2020). A specific time of
19 min is required for the detection of Fvd with the help of 229 C.z.e. In this field
various column (25, 50, 60, 75, 100, and 200um * 68 cm) and 230 various BFS (10
mM NaH2Po4, 50 mM Na2B4o7) are used for Spt of Fvd in PH range of 9.0–9.6.
(Poliwoda et al. 2020). In case of polyphenolic flavonoidal compound 20 mM
borate (BRT) BF 2 mM Beta-cyclodextrin (additive) and 7% methanol are used
(Brüggemann et al. 2019). 25 mM phosphate BF is used for the detection of
Fig. 8.5 Equation of

injection
Fig. 8.6 Parameters of c.e
Epicatechinand Catechin in PH 7.0. 0.20 mm and 300 µm OD containing carbon

electrode are used for the detection of which types of Fvd are present in caffeine
molecule (Wu et al. 2019). There after here 5 mM ammonium acetate (AACT), BF
and 80 ml water and 20 ml ANT are used in PH 4 and temperature 25 °C (Nawabi
et al. 2019).
Fig. 8.7 Migration current
Fig. 8.8 Structure of

morphine
8.5.3 Terpenoids
C.e is generally used in the field of almost Psm. But in case of terpenoids (Tps), it is
not used prevailing system. Yet C.e is used some of the Tps such as mono Tps, di
Tps, tri Tps (Shihabi and Oles 1994).
8.5.3.1 Mono Tps
Which types of monoterpenes are detected by C.e technique, which has displaced in
Fig. 8.10.
Fig. 8.9 C.e of quaternary alkaloid compounds
Fig. 8.10 C.e for flavonoids

8.5.3.2 Di-Tps
It is reminded from the statement of Gibberellin (plant hormone) (Fig. 8.11), that
when it was separated by C.e technique where using boro sulfate BF, para/ gamma
cyclodextrin at PH 7.5. This isolation technique is depended on such factors as:
Hydrogen bond, Vander wall force, and solution power (Weegels et al. 1995),
(Arndt et al. 2008).
8.5.3.3 Tri-Tps
Cardiac glycoside (Digitalis) (Fig. 8.12) is a triterpenoid which was formerly

invented by the help of C.e techniques, where was used BRT, SDS, cyclodextrin at
PH 9.3. But some hindrances have occurred when cyclodextrin used as an additive
for isolating this compound (Helander et al. 2005). In this reason, scientists have
used a mixture of additives such as cyclodextrin and akylodextrin for the Spt of Tps.
25% ACN + 75 Mm Chlorate and BRT are used for the Spt of saponin at PH 7.
Then, SDS + 5% methanol and phosphate BF are used for isolation of steroidal
triterpene at PH 9.4. This isolation is occurred in 7.5 min (Lee and Ong 2000)
(Table 8.2).
8.5.4 Coumarin Derivatives
Coumarin (C.n) is a very important Psm. The existence of various structural con-
taining C.n is observed from the plant to fruit. Cn plays a vital role in preventing
various human diseases (Nayak and Pal 2017). Structurally there are 3 types of Cn,
which has been shown in Fig. 8.13. In the past situation, there are various chro-
matographic techniques (HPLC, UPLC, RP–HPLC, TLC, etc.) used for the
determination of what types of Cn exist in plant sample (Mandal et al. 2015), (Fiehn
et al. 2008). But in the present situation, C.e technique stands a special field of a
scientist for Cn Spt. Now we will discuss about the how and what types of Cn are
separated by C.e in this section Fig. 8.14.
Fig. 8.11 Monoterpene by capillary electrophoresis


Gibbgerellin
Table 8.2 Application of C.e in terpenoids

Name Mtd Solvent PH Diameter Vtg Wave References
Of column (kv) length
Terpenoids C. 100 mM borate 10.5 80 cm 50 20 254 (Troujman
(Monoterpene, z.e um and
Diterpene, Chottard
Triterpene) 1997)
Gibberellins C. 50 mM 7.54 60 cm 50 15 196– (Pal et al.
z.e phosphate, um 210 2019)
100 mM borate,
75 mM
Cyclodextrin
Digitalis M. 30 mM borate, 9.3 80 cm 50 20 225 (Nayak
e.k. 7 mM urea or um et al. 2010)
c.c sodium cholate
Phyto-ecdystereoid M. 40 mM 9.4 72 cm 50 20 240 (Pal and
e.k. phosphate, um Nayak
c.c 20 mM borate, 2012)
20 mM SDS,
5% methanol
Saponins M. 20 mM 7.0 102 cm 50 30 200 (Pal2015)
e.k. phosphate, 25% um
c.c CAN, 75 mM
cholate
Mtd of Spt: Firstly, plant species (ps) will be collected from any region of the
forest. Then the collected ps will be authenticated by the help of botanist and kept in
a hot air oven and shade dryer for removing the moisture (Xiang et al. 2011). Then
the dry plant material will be cut into small pieces and powdered by the help of a
mixer grinder or ball mill. Then the powder material will be weight and extraction
process will be done by the various solvent system (Tolstikov et al. 2003). Now
extract materials will be diluted with Me OH or EtOH and transferred into a various
conical flask. Then the 24–30 kV electronic Vtg 50 mbar pressure, and 50um fused
silica capillary will be used in extract material for processed the C.e technique
(Sugimoto et al. 2012). At the end of the process, the compound will be detected by
the help of UV detector at 190–600 nm (Ward et al. 2007).
Fig. 8.13 Structure of digitalis derivatives
Fig. 8.14 C.n derivatives
8.5.5 Quinones
Quinone (qn) molecules are especially displayed in the whole plant world. In 1992,
the first qn was separated by the help of C.e. Hqn and Aqn were separated by C.e
and M.e.k.c.c Fig. 8.15. 1OMm BRT, 75 Mm SDS and 10% methanol, 20 kV Vtg
was used for the detection of Aqn methyl ester at PH 9.5. 50 Mm BRT, 15 kV Vtg
was used for the Spt of Hqn mono sulphate at PH 10 (Lie et al. 2008) (Fig. 8.16).
8.5.6 Polyamine
It is a stress resulting metabolite product, which has been shown in Fig. 8.17. These
type of compounds are easily separated by C.e technique. 8 mM quinine sulphate,
20% ethanol, quinine sulphate as an electrolyte, and HMC are used for this tech-
nique (Kim and Verpoorte 2010).
Fig. 8.15 Spt of C.n.s by C.e

quinone derivatives

various polyamines
8.6 Capillary Electrophoresis Non Aquas (C.eN)

for Bioactive Compound
The meaning of C.eN, where does not use any water or moisture containing system.
This method is first developed in 1984. This Mtd is continuously used either
beginning of Psm or differentiation of small molecule (Sun et al. 2012). There
various BF systems are used as a result increased the electrical density and power.
Even very small pole containing substances are easily soluble in the BF system. In
past, heteroconjugated compounds were not separated in this method but recently, it
has been recognized for C.eN (Pomponio et al. 2003). In case of Alkd by the help of
C.eN here 75 ml methanol, 25 ml ANT, and 50 Mm AACT are used for preparing
the BF. For Di–Tps 250 Mm AACT is used. For Aqn and C.n there various BF are
used such as 50–60 Mm SCH, and 20 Mm AACT (Huang et al. 2005).
8.7 Online C.e Concentration
The detector is frequently used for Spt of the compound in C.e because of the
shortest length of C.w, the smallest rate of injection and used picogram amount
exam substance (Vanhoenacker et al. 2000). For removing this above problem
research invented Mtd has been progressed, which is known as C.c Ms online CNC
Mtd, where the detector is not used frequently (Sun and Yeh 2005). It is also called
as sweeping. Here Nano gram amount sample and very complex molecules are
detected very easily in this Mtd. Firstly, this Mtd was applied for Spt Fvd and
Riboflavin. This examination described that C.e Mtd is better than general C.e.
Here, 50 Mm borate BF and 50 Mm phosphate is used in PH 9.5 (Zang et al. 2004).
8.8 C.e Versus C.e MS
In 1987, a joint Mtd is introduced for high-velocity compound detecting power,

determination of molecular weight in minimal time, and used a small amount of
sample. This Mtd is known as C.e MS. As a result, the liquid smoothly flows through
C.w. In the past situation, this Mtd was introduced for qualitative analysis of Alkd
(Gao et al. 2004). Firstly, filodren Alkd is produced with the help of C.eMS.
However, this Mtd is not limited to Alkd. Now, we will discuss about the difference
between C.e and C.e MS. In the case of C.e ions are transferred into one end to
another end with the help of various factors such as high electrical field, arresting
liquid flow, and shape of ions. In the other hand in the case of C.e MS, compounds
are detected depending on ratio between charge and mass (Bo et al. 2003). Firstly,
ions are emitted from C.e then they are transferred into the mass instrument where
these ions are formed into small pieces with the effect of electrical and magnetic
field. However, the main reason of C.e Mtd is here compound is not easily detected.
In this above point of view, MS is attached with C.e via various technique
TOF-MALDI, FAB etc. (Scherz et al. 2007). To know what types of phenolic
compounds (pc) have existed in red wine (rw), firstly it was extracted with diethyl
ether and analyzed with this Mtd through ESI ion mode. In spite of the existence of
high power of isolation in C.e than HPLC, but C.eMS was stronger because of the
best selection and taking better response.13 pc were analyzed with this Mtd out of
23. With removing the borate ACT here, AACTBF was used in this Mtd in PH 9.3. As
a result, it was frequently affected by the hydroxyl group of rw is attached with
expired liquid of methanol and BF. Adverse position of fingerprint analysis of the
natural substance, this instrument shows less response comparatively so, the ion
trapping Mtd is used for Psm determination (Lai and Dabek-Zlotorzynska 1999).
Extension of the plant is a very controlled Mtd. Various examination proves that this
tragedy is controlled by various substances. These substances are called
Phytohormones. It is synthesized in first leaf, bud, flower, and phloem tissue. Auxin
(AN), gibberellin, cytokinins are plant hormone (PHR). However AN is an essential
PHR. AN contained indole. So, we will discussed about how this substance is
separated by C.e Mtd (Li et al. 2000), (Ganzera et al. 2003), (Lurie et al. 1998),
(Desiderio et al. 2005), (Starkey et al. 2002).
8.9 Conclusion
In view of all aspect, we have decided that the C.e technique will open a new door
of scientists for Psm detection, especially on the complex molecular structures
which are not detected by HPLC. Thereafter, there are various disadvantages in this
technique. If we get rid of these disadvantages, as a result, this C.e Mtd will open a
new research area for the analytical chemistry industry. However, it may be used in
the field of drug development from a plant source. In the other hand, various
applications have existed in this C.e Mtd except for bioactive compound. Such as,
what types of ions are present in our saliva, to know the characters, and movement
of DNA pieces which are derived from polymerase chain reaction as this method
act as a gatekeeper for the forensic scientist. DNA is not separated without an
electrical field so, C.e method is essential. So that activated liquid is easily passed
throughout the C.w so, C.e is needed. One profile is created from a high polymeric
genetic marker with the help of C.e. Nowadays finding ink relation is very
important for forensic lab because normal ink cannot find out the accurate relation,
hence C.e method is essential. In other ways, DNA sequencing is made possible in
540 s with the help of C.e.
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Chapter 9
Camptothecin: Occurrence, Chemistry
and Mode of Action
Mallappa Kumara Swamy, Boregowda Purushotham,

and Uma Rani Sinniah
Abstract Camptothecin (CPT), a planar pentacyclic ring system, constituting a

pyrrolo(3, 4-b)-quinoline group along with a-hydroxy lactone is observed mainly
from the plants that belong to the genus, Camptotheca. They tend to exhibit a
broad and significant clinical impact including antitumor, antiviral, antibacterial,
antipsoriasis, antirheumatic, antispasmodic, neurotropic, antimalarial activities, etc.
Due to their unique mode of action, CPT has a broad clinical interest. These agents
turn topoisomerase-I (top-I), an enzyme that exerts the torsional stresses of
supercoiled DNA into an intracellular poison. The CPTs stabilize the covalent
binding of top-I to its DNA substrates and the formation of these complexes tends
to the formation of reversible, single-strand nicks producing potentially lethal
double-strand DNA breaks resulting in apoptosis. Owing to high market value,
CPT-producing plant species have been scientifically explored in recent years. The
present chapter offers a comprehensive info on the sources, chemistry, and mode of
action of CPT.

Keywords Camptothecin Nothapodytes nimmoniana Anticancer compounds

Cytotoxicity Mode of action
9.1 Introduction
Bioactive natural compounds obtained from a wide-ranging medicinal plants have

gained more attention in the last few decades for treating numerous human health
issues. Several types of plant-derived compounds like phenolics, alkaloids, ter-
penoids, polyketides, and others are being already endorsed to have worthy clinical
M. K. Swamy (&) B. Purushotham

Department of Biotechnology, East West First Grade College, Bengaluru, India
e-mail: swamy.bio@gmail.com
U. R. Sinniah
Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, Darul Ehsan,
43400 Serdang, Malaysia
https://doi.org/10.1007/978-3-030-54027-2_9
312 M. K. Swamy et al.
perspective, including anticancer, antimicrobial, anti-inflammatory, antioxidant,

antiinsecticidal activities to name a few (Lodh and Swamy 2019). In recent times,
cancer is affecting the lives of many people in the society and is one among the
highest death instigating menace around the globe (Ravichandra et al. 2018; Akhtar
and Swamy 2018, b; Sasidharan and Saudagar 2019). Herbs are being utilized since
from the history in different folk medicines in India, China, Indonesia, Malaysia,
etc., for treating several human health issues. The occurrence of chemically diverse
biologically active phytoconstituents is accounted for the impartment of medicinal
values to plant species. Many such biologically active constituents have been
isolated and documented with experimental proofs on their pharmacological
activities (Lodh and Swamy 2019). Some of the important medicinal plants having
bioactive compounds include Camptotheca accumulata, Catharanthus roseus,
Coleus forskohlii, Tinospora cordifolia, Centella asiatica, Gloriosa superba,
Oplopanax horridus, etc. Today, several well-known therapeutically much-admired
compounds, such as camptothecin (CPT), taxol, podophyllotoxins, luteolin, ros-
marinic acid, vincristine, ginsenosides, homoharringtonine, and many more are
being sequestered and quantified from several herbs. Also, plants are the major
natural resources for recovering anticancer agents (Lee et al. 2018; Akhtar and
Swamy 2018a, b).
The innovation of an important antitumor alkaloid, CPT has prospered the
interest among plant scientists, phytochemists, biologists, medical doctors, drug
discovery institutions, and pharmaceutical industries. CPT is a highly valued
compound because of its unique therapeutical potential especially in treating var-
ious cancers (Rahier et al. 2005; Venditto and Simanek 2010; Kacprzak 2013;
Prakash et al. 2016; Pu et al. 2019). The presence of CPT was firstly revealed from
the Camptotheca acuminata Decne. (Nyssaceae) tree species, available in China.
Soon after that CPT was also found in other plant sources such as Nothapodythes
nimmoniana, Chonemorpha grandiflora, Ophiorrhiza mungos; Ophiorrhiza pumila
and Ophiorrhiza filistipula, etc. Also, some of the entophytic fungi such as
Entrophospora infrequens, Fusarium solani, etc. associated with these plant species
were shown to secrete CPT in low amount (Kaushik et al. 2015; Raveendran 2015;
Prakash et al. 2016; Pu et al. 2019). However, the yield of CPT is very low in all
these natural resources. Globally, the annual production of CPT from plant sources
is only about 600 kg, while the demand is assessed to be roughly 3000 kg per
annum in the world market. Thus, alternative approaches such as biotechnology or
chemosynthesis must be adopted.
Though, this natural compound is effective in exhibiting cytotoxicity, however,
numerous critical issues are presented by CPT including less water solubility,
stability in addition to unpredictable adverse effects in patients such as vomiting,
myelosuppression, diarrhea, etc. (Martino et al. 2016). For overcoming these issues,
several attempts are being made to develop new chemical derivatives, which are
readily soluble through semisynthetic approaches involving the basic structure
modifications (Venditto and Simanek 2010). Over a time, the chemical exploration
of CPT has resulted in few major anticancer chemotherapeutic drugs such as
irinotecan and topotecan. Presently, irinotecan, topotecan, 9-aminocamptothecin,
9 Camptothecin: Occurrence, Chemistry and Mode of Action 313
and 9-nitrocamptothecin are the widely used water-soluble semisynthetic analogues

of CPT for treating cancers all over the world. (Deorukhkar et al. 2007; Ramawat
and Goyal 2009; Raveendran 2015). Presently, they represent a distinctive class of
clinically approved chemo-drugs of modern medicines (Lee 1999; Ramawat and
Goyal 2009). The anticancer mechanism of action of these chemo-drugs is through
causing cellular damages by apoptosis intermediated by the inhibition of Top-I
(Topoisomerase I) enzyme structure and activity (Wright et al. 2015; Sadre et al.
2016). Though many leads are presently in clinical trials against cancers, only
topotecan and irinotecan are permitted for clinical uses (Martino et al. 2016; Pu
et al. 2019).
This chapter summarizes the occurrence of CPT in natural resources, chemistry,
and its mode of action. Overall, this comprehensive review encourages research
interest among chemists and biologists to explore more on this novel compound.
9.2 Camptothecin Discovery and Its Chemistry
In 1955, to evaluate the anticancer compounds from nature, Cancer Chemotherapy

National Service Center (CCNSC) was created by the National Cancer Institute
(NCI). It was initially aimed to screen unknown chemical compounds for
chemotherapeutic uses. However, later in the year 1960, the screening program was
extended to include different natural resources, mainly plant species and their
potential anticancer compounds. In this collaborative work, many pharmaceutical
companies and research institutions, including The United States Department of
Agriculture (USDA) submitted numerous potential plant species and phytocom-
pounds to be screened for their antitumor activities (Hartwell 1970; Perdue et al.
1970; Moore et al. 1970; Raveendran 2015). The NCI screening work was led by a
well-known organic chemist, named Jonathan Hartwell, and it also included data
from the documents of traditional medical practices of Egypt, Greece, China, and
Rome that employed a wide range of medicinal plant species and their preparations
against cancer cure. Different plants species extracted initially at Wisconsin Alumni
Research Foundation was directed to many other labs for evaluating their potential
of killing cancerous cells. Primarily, the anticancer effects of all samples were
evaluated in oral epidermoid carcinoma (KB) cell culture. Also, the samples were
screened using tumor xenograft mouse models, i.e., CA-755, an adenocarcinoma;
S-180, a sarcoma; and L1210, a lymphoid leukemia (Kessel 1971a,b; Raveendran
2015). From these results, the crude extracts showing active antitumor activities
were later subjected to different separation techniques to isolate the active com-
pounds at different laboratories, including Research Triangle Institute (RTI), North
Carolina. More than 1000 plant compounds were isolated from different plant
species solvent extracts, and among them, only Camptotheca accuminata, a tree
species bark and stem extract disclosed noteworthy anticancer activity. Further
screening and isolation techniques have led to the identification of novel compound,
which was named as CPT in 1966, and the new sequestration of this anticancer
compound from C. acuminata extracts was carried out by Keith Palmer and Harold
Taylor. CPT, which is an indole alkaloid was effective against both L1210 and p338
leukemic cells (Wall et al. 1996; Raveendran 2015). C. accuminata is used in the
traditional medical practices to treat leukemia, psoriasis, liver diseases, spleen, and
digestive tract (Govindachari and Viswnathan 1972; Efferth et al. 2007). CPT
displayed extraordinary anticancer action in the initial clinical trials. However, it
showed poor water solubility, which restricted its ready usage in cancer
chemotherapy (Kehrer et al. 2001; Li et al. 2006). Thus, medicinal and synthetic
chemists have established several blends of CPT, and several CPT-derivatives were
developed to increase its anticancer potentials with good results. So far, four CPT
analogues are being permitted by the FDA (Food and Drug Administration), USA
to treat various cancer types. These include irinotecan, topotecan, belotecan, and
fam-trastuzumab deruxtecan (Wall et al. 1996; Samuelsson 2004).
The chemical structure of CPT comprises a planar pentacyclic ring configura-
tion, encompassed with a pyrrolo[3,4-b]-quinoline moiety (rings A, B, and C),
conjugated pyridone moiety (ring D) and one chiral center (positioned at C20)
within the a-hydroxy lactone ring having (S) configuration (E-ring) (Fig. 9.1). The
planar arrangement is believed to be the major factor that inhibits topoisomerase
enzymes activity (https://en.wikipedia.org/wiki/Camptothecin). Some of the prob-
lems associated with parent CPT is its susceptibility to hydrolysis, which is mainly
because of the lactone ring structure, as well as considerable toxicity. The
a-hydroxy lactone ring having (S) configuration (E-ring) opening under biological
conditions leads to form the carboxylate open form of CPT. Later, sodium salt of
CPT was proposed and reported with higher solubility than the parent CPT.
Unfortunately, clinical investigations showed reduced efficiency against cancers
associated with severe adverse effects, for example, myelotoxicity and hemorrhagic
cystitis (Adams and Burke 2005; Kacprzak 2013). These drawbacks were later
addressed by many researchers, and eventually, semisynthetic and total synthetic
approaches were used to yield many analogues of CPT. Through the structure
activity relationship (SAR), a detailed understanding on CPT structure was made
and developed few soluble and very active antitumor drugs, namely irinotecan,
topotecan, belotecan, and an active metabolite of irinotecan, named as SN-38
(Fig. 9.2). These aspects are described in detail by a number of review articles
(Zunino et al. 2002; Sriram et al. 2005; Kacprzak 2013; Li et al. 2017; Amin et al.
2018), and readers may refer them for better understanding.

camptothecin showing a
planar pentacyclic ring
structure
Fig. 9.2 Camptothecin and its major chemical analogues, approved for cancer chemotherapy
9.3 Natural Sources of Camptothecin
9.3.1 Camptothecin from Plants
The botanical classification assists in understanding the presence of phytocom-

pounds in plant species (Larsson 2007). Plant-origin secondary metabolites with
similar chemo-structures can be present in dissimilar families of the plant kingdom.
Likewise, CPT occurs in different plant species of the distinct orders and families of
angiosperms. Owing to high market value, CPT-producing plant species have been
scientifically explored in recent years. Further, plant-based CPT biosynthesis still
remains as the best and suitable route for production, because of its limited natural
sources and productivity. The occurrence of CPT is commonly distributed among
angiosperms (Wink 2003; Oberlies and Kroll 2004; Pu et al. 2019). The isolation
and characterization of CPT was performed firstly by Wall et al. (1996) from C.
acuminata. Later, it was re-isolated from a medicinal plant, O. mungos L. that
belongs to the Rubiaceae family (Tafur et al. 1979). Remarkably, C. acuminata and
O. mungos belongs to totally dissimilar phylogenetic clades, however, they pos-
sessed similar biosynthetic competency for CPT (Pu et al. 2019). This discovery
revitalized the knowledge of distribution patterns of CPT in plants. Over the next
two decades, CPT-producing plant species were identified and isolated from a few
other plant species that belong to different plant families, i.e., Icacinaceae,
Apocynaceae, and Rubiaceae. These plants include Mostuea brunonis Didr.
(Loganiaceae), Tabernaemontana alternifolia L. (Apocynaceae; syn. Ervatamia
heyneana (Wall.) T.Cooke); O. filistipula Miq. and O. pumila Champ. ex Benth.
(Rubiaceae); Nothapodytes nimmoniana (Grah.) Mabb., Nothapodytes obtusifolia
(Merr.) R. A. Howard., Nothapodytes obscura C.Y. Wu. and Merrilliodendron
megacarpum (Hemsl.) Sleumer. (Icacinaceae). These plants sources have the
potential to serve as alternatives to obtain CPT. Nevertheless, only in two species,
namely C. acuminata and N. nimmoniana, the basic accumulation patterns of CPT
is detailed so far (Gunasekera et al. 1981; Arisawa et al. 1981; Arbain et al. 1993;
Liu et al. 1998; Ramesha et al. 2008; Bai and Song 2014; Upadhya et al. 2014;
Kaushik et al. 2015; Isah and Mujib 2015; Prakash et al. 2016; Pu et al. 2019). CPT
occurs in leaves, bark, seeds, fruits of C. acuminata, however the maximum
quantity can be obtained from young leaves of C. acuminata, which is about 5 mg/
g DW (dry weight). The matured leaves contain minimum levels of CPT, which is
250% lesser than that can be obtained from the bark, and ten-fold lesser than that
can be obtained from young leaves (Lopez et al. 1994; Li et al. 2000; Yan et al.
2003; Kaushik et al. 2015; Prakash et al. 2016).
The increased exploration of plant resources for CPT-producing capability have
resulted in the documentation of 34 new plant species that belong to Apocynaceae,
Rubiaceae, Gelsemiaceae, Betulaceae, Icacinaceae, Nyssaceae, Meliaceae, and
Violaceae. Among them, the majority of plant species belong to Rubiaceae and
Icacinaceae family. Importantly, most of these described plant species have their
natural distribution in the South-East Asian nations, exclusively in the south part of
China and Western Ghats of India (Pu et al. 2019). The herbaceous species of
Ophiorrhiza are mainly spread across the Western Ghats of India, particularly at
Travancore, Wynaad, Anamalai, and Kemmannugundi. Besides, these species
occur in the regions of Assam, Andamans and Nicobars islands, Srilanka, Thailand,
Philippines, Malaysia, and China (Namdeo et al. 2012; Kaushisk et al. 2015). The
organs of Ophiorrhiza species produce low levels of CPT content. Similarly,
CPT-yielding C. grandiflora (Apocynaceae), which is a latex-bearing shrubby
climber has been observed by Kulkarni et al. (2010), and this plant is prevalent in
the regions of Kerala and Karnataka in India. The content of CPT in these plants
ranges between 0.600 and 1.350%. In C. acuminata, CPT content ranges from
0.012 to 0.236%, while in N. nimmoniana, it ranges from 0.081 to 0.775%,
depending on different organs, and these plant species are the chief sources for CPT
extraction in large scale (Liu et al. 1998; Lorence and Nessler 2004; Ramesha et al.
2008; Khan et al. 2013; Isah and Mujib 2015). In C. acuminata, the accretion of
CPT largely depends on branch, age of the tree, and seasons. Moreover, the
increased shade enhances CPT accumulation (Liu et al. 1998). It was established by
a study that N. nimmoniana inner root bark and stem bark contained with the CPT
amount of 0.23 and 0.33%, respectively (Uma et al. 2008; Isah and Mujib 2015).
9.3.2 Camptothecin from Endophytes
The plant-derived CPT cannot meet the global demand, and hence constant efforts
are being carried out to recognize novel alternative resources of CPT. In this regard,
endophytic microbes can be a choice for obtaining CPT (Uzma et al. 2018). There
are limited reports on the CPT-yielding endophytic microorganisms that were
identified, associated with CPT-producing plants. In a study, an endophytic fungus
(Entrophospora infrequens) was isolated from N. foetida, growing in the coastal
regions of India (Puri et al. 2005). The endophytic fungus was cultured in
Sabouraud broth, a synthetic liquid media having dextrose (4%) and peptone (1%)
under shaking conditions. Cultures, producing CPT was identified using both
chromatographic and spectral evaluations. Further, they reported that CPT obtained
from fungal cultures with anticancer activity against cancer cells, namely A-549 (a
lung cancer), HEP-2 (a liver), and OVCAR-5 (ovarian) cancer cell lines. They
testified the sequestration of about 18 lg/mg CPT content from the E. infrequens
chloroform extract. Likewise, another study by Amna et al. (2006), used a biore-
actor for culturing E. infrequens, and they found the synthesis of high levels of CPT
(4.96 mg/100 g of dry mass) after 48 h. The optimum conditions, i.e., pH of 5.6,
temperature of 28 ± 2 °C, agitation rate of 200–230 rpm, and aeration rate of 1
vvm were found optimal for the growth of E. infrequens in bioreactor culture, and
to produce higher levels of CPT. The above studies clearly suggest the possible use
of E. infrequens endophyte to produce CPT on a large-scale by employing
bioreactors.
Rehman et al. (2008) identified an endophyte (Neurospora crassa), and desig-

nated it as ZP5SE from N. foetida seeds, and was able to produce CPT, when
cultured in Sabouraud Dextrose Liquid Medium under the shake flask culturing
environments. This endophyte was capable to propagate in Sabouraud broth
(100 ml) poured in a 500 ml Erlenmeyer flask, and grown on a shaking incubator,
agitated at 220 rpm for a duration of 10 days at a temperature of 28 ± 2 °C. From
the growth kinetics study, it was evidenced that this fungal species, showing the
exponential phase of growth up to 7 days of culturing. The produced CPT content
by this endophyte was analyzed using High Performance Liquid Chromatograhy
(HPLC) followed by Liquid chromatography–mass spectrometry (LC/MS) and
Tandem mass spectrometry (MS/MS). The obtained CPT was tested against the
growth of human lung cancer (A-549) and ovarian cancer (OVCAR-5) cells. The
results were positive with effective inhibition of the growth of cancer cells, and the
observed data were comparable to the data obtained for the standard drug, CPT.
Another endophyte, Nodulisporium sp. was isolated from N. nimmoniana inner
barks by Rehman et al. (2009). This fungal species possessed thin hyphae, which
was found to extend up to 6.4 lm in diameter, and profuse conidiophores that were
branched, verticillately. The fungus, when grown on liquid culture (Sabouraud
broth) media produced CPT up to 5.5 lg/g DW of mycelia after one week. Further,
the authors have explored the possibility of its culturing in an airlift bioreactor with
5 to 18 l volume. They found that mycelium growth initiated within 5 h of inoc-
ulation, and the maximum growing was documented on Sixth day of culturing in a
bioreactor with the yield of 45 lg/g DW of CPT.
From the plant, Apodytes dimidiata, 2 strains of F. solani (MTCC 9667 and
MTCC 9668) were isolated, and these strains were shown to produce both CPT in
their mycelia. Interestingly, MTCC 9668 strain was also shown to produce
10-hydroxycamptothecin, however in low quantities in mycelia. These endophytes,
MTCC 9668 and MTCC 9667 after culturing on a liquid broth for 4 days could able
to produce 53 and 37 lg CPT/100 g DW of mycelia, respectively (Shweta et al.
2010). Likewise, the produced quantity of 9-methoxycamptothecin and
10-hydroxycamptothecin by MTCC 9668 was observed to be 44.9 and 8.2 lg/
100 g DW, correspondingly.
A study carried out by Musavi et al. (2015) identified the occurrence of an
endophyte, F. oxysporum NFX06 associated with N. nimmoniana. They evaluated
the production of cell-allied CPT from this endophytic fungus, and proposed its
growth kinetics. To determine the consequence of substrate levels, a model was
constructed by applying response surface methodology on the basis of central
composite design. They used dextrose, magnesium sulfate, and peptone as inde-
pendent variables to evaluate the production of CPT under sub-merged fermenta-
tion conditions. The highest CPT yield noticed from the central composite design
was found to be 598 ng/g DW of mycelia. Also, the model-endorsed experimental
yield of CPT and the optimal expected yield of CPT from dried mycelia were
observed to be 610 ng/g and 628 ng/g DW at the substrate levels of 9.2 g/l peptone,
42.6 g/l dextrose, and 0.26 g/l MgSO4, correspondingly.
Likewise, new endophyte, Xylaria sp. occurring in C. acuminata was isolated,

and it was noticed that this fungus could produce 10-hydroxy-CPT, but failed to
produce CPT (Liu et al. 2010). Endophytic microbes that can biosynthesize CPT
have been witnessed by many researchers, however, attenuation of CPT synthesis is
one of the chief problems in this area of studies.
Later, study by Shweta et al. (2013), described 3 endophytes, namely Phomopsis
sp., Fomitopsis sp., and Alternaria alternate that occurred in the shrub species,
Miquelia dentata Bedd. These fungi produced CPT, 9-methoxy-CPT, and
10-hydroxy-CPT. The obtained yield of CPT from these species was found to be
about 42, 73, and 55 lg/g DW, respectively for Phomopsis sp., Fomitopsis sp., and
A. alternate. About 161 endophytic fungal species were isolated from C. acumi-
nata, and among them, only one fungus, i.e., Botryosphaeria dothidea X-4 was
shown to be capable of producing 9-methoxy-CPT (Ding et al. 2013).
Likewise, 3 new endophytic fungi (Aspergillus sp. LY355, Aspergillus
sp. LY341, and Trichoderma atroviride LY357) were isolated from C. acuminata.
These species yielded CPT content of 42.9, 7.9, and 197.8 lg per liter of broth
culture, respectively. However, repeated sub-culturing of these fungal strains were
reported to lead to the diminishing of CPT-yielding ability. However, there was a
persistent production of CPT by Aspergillus sp. LY357 strain until the eighth
generation of sub-culturing (Pu et al. 2013; Kai et al. 2015; Uzma et al. 2018).
An investigation of Soujanya et al. (2017) reported the association of 4 new
bacterial strains with the stems, fruits, and leaves of Pyrenacantha volubilis,
belonging to Icacinanceae plant family. The identified bacteria included B. amy-
loliquefaciens (KY741854), Bacillus sp. (KP125956), Bacillus sp. (KP125955),
and B. subtilis (KY741853). All these isolates were reported to produce CPT as
revealed by the use of electrospray ionization mass spectrometry (ESI–MS) and
nuclear magnetic resonance spectroscopy (NMR) analysis. The crude extract of
these bacterial isolates were analyzed for their antitumor activity on colon cancer
cells, and the results have shown that crude extracts exhibiting effective cytotoxi-
city. Also, their study evaluated the part played by plasmid in producing CPT. They
proposed the potential role of a plasmid (5 kbp) in bacteria to biosynthesize CPT.
However, the exact mechanisms are yet to be revealed. B. subtilis, KY741853
strain, producing CPT was attenuated after sub-culturing in the culture medium.
In another study, solid-state fermentation was utilized to culture the endophytic
fungus, Fusarium oxysporum, and evaluated for its capability to produce CPT
(Bhalkar et al. 2016). According to them, soybean meal, among different substrates
utilized was observed to be the best medium for producing CPT effectively by F.
oxysporum under optimized solid-state fermentation conditions. The use of soy-
abean meal substrate was effective in yielding CPT content of about 128 mg/l dry
weight after 3 days of growth in a bioreactor. In addition, the use of whey (the
liquid remaining after milk) as a moisture source in solid-state fermentation played
an important role in augmenting the yield of CPT. The application of this developed
process can be environmentally very effective, and it is evidenced by the fact that
after fermentation stage, there was a significant reduction in the levels of biological
oxygen demand (BOD), chemical oxygen demand (COD), total soluble solids
(TSS), total dissolved solids (TDS), and total protein content of whey effluent.
Hence, this simple solid-state fermentation approach, employing the endophytic
fungus and cheaper substrates, such as whey and soybean meal may be useful in
producing valued anticancer pro-drug, CPT.
Lately, Clarance et al. (2019), reported the endophyte, F. solani strain ATLOY-8
from Chonemorpha fragrans (Moon) Alston., and it was capable of producing
CPT. The optimized process to yield camptothecin was designed using the con-
ventional approaches and analytical methods. For improved biomass and yield of
CPT, the Box–Behnken design matrix (n = 17) and one factor at a time method was
used to identify and select optimized variables. According to them, the Box–
Behnken design matrix method in the basal media with 1% glucose, 5% absolute
ethanol, and 0.03% precursors (a mixture of geraniol, tryptophan, and tryptamine)
could increase the CPT production up to 1.4 fold increase and biomass up to 1.2
fold in comparison to one factor at a time method.
9.4 Mode of Action of Camptothecin
Early researchers have stated the antineoplastic activity of CPT mainly mediated by
inhibiting nucleic acids synthesis in both tumor (L1210) and normal cells (HeLa)
(Kessel 1971a,b; Horwitz et al. 1971). This inhibitory effect of CPT over macro-
molecules synthesis was observed to be transient with the removal of the drug.
However, its prolonged treatment period may completely suppress nucleic acids
synthesis leading to cell death. Also, CPT-induced rapid fragmentation of nucleic
acids at concentrations that do not primarily impact on the synthesis of protein
(Horwitz et al. 1971). In intact cells, the suppression of RNA synthesis can be fully
reversed by removing CPT. Nevertheless, CPT failed to block the activities of
purified enzymes (DNA and RNA polymerases). CPT exerts its cytotoxicity by
inhibiting topoisomerase enzymes that relieve the topological strains created during
the process of chromosomal recombination, replication, and transcription
(Champoux 2001; Das et al. 2016). Topoisomerases are of two types (topoiso-
merase 1 and topoisomerase (2) classified based on their mode of action, i.e.,
whether they cleave either a single or double DNA strands (Raveendran 2015).
Typically, Topoisomerase 1 (Top 1) enzyme relaxes the DNA supercoiling that
involves the following steps: (a) cleavage of one of the two strands of DNA after
the attachment, (b) relaxation of the strand, and (c) re-annealing of the strand. The
incubation of CPT together with isolated DNA results in intact binding and cause
fragmentation of DNA. Further, molecular interaction studies using X-ray crys-
tallography has witnessed the establishment of a Topo1-DNA complex with
numerous interactions (Redinbo et al. 1998; Liu et al. 2000). This forms the target
for many anticancer drugs to induce cell death by trapping this covalent complex
formed by Top-I and II enzymes (Zhang et al. 2011). As stated earlier, the
obstruction of DNA synthesis and DNA cleavage is because of the suppression of
DNA Top 1 enzyme (Hsiang et al. 1985). Top 1 inhibition is mainly mediated by
the formation of reversible CPT-induced Top 1-DNA cleavable complex. In par-

ticular, CPT attaches to the Top 1-DNA complex and forms the reversible Top 1–
CPT–DNA covalent tertiary complexes that inhibit the Top 1-religation reaction
(Redinbo et al. 1998; Zhang et al. 2011; Das et al. 2016). Further, it was shown that
CPT-induced cytotoxicity is directly related to the CPT-mediated assembly of Top
1-DNA cleavable complexes (Hsiang and Liu 1988; Hsiang et al. 1989; Liu et al.
2000; Pommier 2006). The interaction of CPT with the DNA occurs by interca-
lating at the cleavage site of the enzyme. Also, it has been suggested that other
interactions might occur between CPT and Top 1, as well as CPT and a flipped base
of DNA at the +1 site (Liu et al. 2000). However, these molecular interactions at
Top 1-CPT-DNA complex are yet to be understood in detail. The cell cytotoxicity
effect of CPT is mostly mediated during S-phase (synthetic phase) of the cell cycle
(Cliby et al. 2002; Pommier 2006; Raveendran 2015). Based on the understanding
of S-phase-specific cytotoxicity of CPT, researchers have proposed a replication
fork collision model. According to this model, Top 1-CPT-DNA tertiary complexes
are reversible and non-lethal by themselves but, after colliding with the replication
forks causes DNA strand break, leading to apoptosis (Hsiang et al. 1989; Liu et al.
2000; Pommier 2006). After the collision of replication fork, the following events
are observed: (1) double-strand breakage, (2) replication fork drive arrest, and
(3) the establishment of Top 1 induced DNA breaks at the collision site. However,
these biomolecular interactions leading to cell toxicity are yet to be understood in
detail (Liu et al. 2000; Raveendran 2015). Interfacial inhibitor concept has been
critically reviewed for CPT by Pommier (2006) and Pommier (2009). Accordingly,
CPT binds at the Top 1-DNA interface and trap cleavage complexes. It is proposed
that these topoisomerase inhibitor drugs stack between the DNA base pairs
adjoining the cleavage site due to their aromatic nature (Pommier 2009; Koster
et al. 2007; Pommier and Marchand 2012). CPT obstructs the rotation of DNA after
intercalating at the enzyme–DNA complex through the interaction of p-p electrons
between the nucleotide bases flanking the cleavage site and the aromatic structure
of the drug (Marchand et al. 2006; Koster et al. 2007). This interference formation
inhibits the release of torsional stress and when the replication fork progresses
further, it collides with Topo 1-CPT-DNA tertiary complex inducing DNA strand
breakage and cell death. CPT, at higher concentrations can also destroy
S-phase-independent cells through transcriptionally mediated DNA damage and
apoptosis (Morris et al. 1996). Another prominent way of CPT-induced cell toxicity
is by arresting RNA synthesis during transcription elongation process (Ljungman
et al. 1996; Liu et al. 2000). It is also reported that the Top 1 cleavable complex is
attached by ubiquitin/26S proteasome complex that blocks the re-ligation step of
the Top 1 reaction (Desai et al. 1997; Liu et al. 2000). CPT, a Top 1-specific poison
induces the attachment of SUMO-1 (Small Ubiquitin-like MOdifier) to Top 1 and
facilitate its degradation (Mao et al. 2000; Rallabhandi et al. 2002). However, the
exact function of SUMOylation of Top 1 in response to CPT is not clear. It is
believed that SUMO-1 regulates the cellular localization of Top 1 as it contains
nuclear localization signals (Desai et al. 1997, 2001; Mo et al. 2002). On the other
hand, Poly(ADP-ribose) polymerase-1 (PARP 1), a chromatin-associated enzyme
interacts with various proteins involved in DNA repair mechanisms destabilizes the
Top 1-CPT-DNA complex by interacting with the NH2-terminal domain of Top 1
(Park et al. 2005; Das et al. 2014).
9.5 Conclusions
CPT and its derivatives are important natural products, and are used to cure dif-
ferent types of cancers. CPT compound also possesses antiprotozoal, antiviral, and
insecticidal activities. Since its discovery, numerous studies were made to evaluate
its anticancer potential, which can be evidenced by the several hundreds of pub-
lished research works each year. Though many plant species have been documented
to produce CPT, its basic accretion configurations have been recognized in detail
only in C. acuminata and N. nimmoniana. Several endophytic microbes have been
identified from CPT-producing plant species, and they have shown to synthesize
CPT, however, in small quantities. Semisynthetic CPTs like topotecan and
irinotecan exhibit strong anticancer effects, and hence are widely used in cancer
therapy. Likewise, several other medicine leads are being established, and some of
them are being investigated for anticancer activities. However, many of them are
yet to pass through clinical trials. Effective drug delivery, biological stability, and
low toxicity of CPTs are some of the areas, which have to be investigated in detail.
As research studies are actively involved in these areas, success can be expected in
the coming days. Importantly, effective delivery of CPTs via new formulations
involving nanotechnology is also being investigated in the present time. Due to
higher demand and complexity of chemosynthesis of CPTs have prompted to look
into simple and sustainable approaches of its production in large-scale. In this
regard, biotechnological approaches, involving plant cell culture, endophytic
microbial fermentation processes/techniques are more productive. CPTs production
in heterologous systems/organisms and the use of metabolic engineering approa-
ches can be very useful. Hence, future research should focus more on these aspects
to fulfill the ever-growing demand for treating different types of cancers throughout
the world.
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Chapter 10
Secondary Metabolites from Plant
Sources
Chandi Charan Kandar
Abstract A Plant can be considered as a storehouse of a huge number of chemicals

biosynthesized by many metabolic pathways like photosynthesis, glycolysis, Kreb’s
cycle, shikimic acid pathway, mevalonic acid pathway, etc. Primary metabolites
include sugars, citric acid, Kreb's cycle intermediates, amino acids, protein, nucleic
acid, and polysaccharides. Primary metabolites are identical in all living plant cells
and they carry out basic life activities like growth, cell division, storage, respiration,
and reproduction. On the other hand, the secondary plant metabolites, well-known
as phytoconstituents are derived from primary metabolites by the influence of
various surroundings stress like light, temperature, and different metals with the
help of several metabolic pathways. The formation of secondary metabolites is very
much specific to the plant family concern. By using similar primary metabolites,
plants of various families produce a large number of different secondary metabolites
having various pharmaceutical values. Generally, secondary metabolites of the
plant have a great role to defense from herbivorous and pathogens, attract other
animals and protect from UV radiation. Moreover, secondary metabolites show a
lot of importance in the pharmaceutical application as medicines used for the
treatment of various diseases in the folklore medicine as well as traditional medi-
cine. They are also used as flavors in pharmaceutical ingredients, perfumes in
pharmaceutical and perfumery industry, insecticides, dyes, polymers used for the
preparation new drug delivery systems and therefore, they have a great value to
economic concern.

Keywords Secondary metabolites Biosynthesis Phenolic compounds

Alkaloids Glycosides Terpenes Saponins Tannins
C. C. Kandar (&)
Department of Pharmaceutical Chemistry, Institute of Pharmacy, Government of West
Bengal, Jalpaiguri, West Bengal 735 101, India
e-mail: cckandar@rediffmail.com
https://doi.org/10.1007/978-3-030-54027-2_10
330 C. C. Kandar
10.1 Introduction
A plant may be regarded as a large biosynthetic laboratory not only for the
preparation of primary metabolites but also for a huge number of secondary
metabolites having pharmaceutical importance. Primary metabolite synthesized by
green plant includes glucose prepared by photosynthesis process, citric acid,
phosphoenolpyruvate from glycolysis, acetyl CoA, citric acid, a- keto glutaric acid
as Kreb’s cycle intermediates, erythrose-4-phosphate from pentose phosphate
pathway, amino acids by transaminase enzyme, protein, nucleic acid by de novo
synthesis process, and polysaccharides. The basic roles of primary metabolites are
general growth and physiological development, involvement in respiratory, storage,
and reproductive system. Practically, primary metabolites are identical from lower
to higher plant systems (Seigler 1995; Kokate et al. 2005).
Secondary metabolites biosynthetically produced from primary metabolites are
considered as chemical adaptation due to environmental stresses such as light,
temperature, and different metals but their distribution is mostly limited, generally
restricted to a taxonomical group (Fig. 10.1). A particular family generates a similar
group of phytoconstituents due to the presence of the definite enzymatic system in
those plants. The different biosynthetic processes occurring in plant cells are
dependent on enzymes that act as catalysts for such reactions. As it occurs by the
control of enzyme activity, it is always directed into a specific pathway resulting
formation of a definite phytoconstituent. For example, tropane alkaloids are
obtained from the Solanaceae family, volatile oils from the Umbelliferae family.
The idea of the secondary metabolite was primarily given by Albrecht Kossel, who
got Nobel Prize for physiology or medicine in 1910, for his contribution (Jones
et al. 1953). Later, Czapek described the secondary metabolites as ultimate products
and also regarded as waste products or secretory substances of plant metabolism but
very much essential for all the animals in the World (Bourgaud et al. 2001). These
products are synthesized by nitrogen metabolism, i.e., secondary modification like
deamination as per the opinion of the scientist. The development of analytical
chemistry in the mid of the previous century become easier to recover of more and
more of these phytoconstituents, and ultimately, this was the pillar for the devel-
opment of the well-known discipline of phytochemistry. Secondary metabolites are
very much expensive to produce, as well as accumulate in the different plant parts
and that is why they are available in much smaller quantities than primary
metabolites. Nowadays, the extraction of a few secondary metabolites has become
costly due to their less availability (Kokate et al. 2005; Bourgaud et al. 2001).
The isotopically labeled markers are used to elucidate the biosynthetic pathways
in plant cells for the manufacturing of numerous plant metabolites. With the help of
radioactive carbon (C14), hydrogen (H3), and in few cases phosphorus (P32), sul-
phur (S36), the biosynthetic pathways are established and become easy to under-
stand the different chemical steps. The specific information regarding biosynthetic
pathways of alkaloid, proteins, and amino acids was achieved by using labeled
nitrogen atom (N14) (Kokate et al. 2005).
10 Secondary Metabolites from Plant Sources 331
Glucose
(C6H12O 6)
Pentose phosphate pathway

Glycolysis
process
Phosphoenol pyruvate
(C3H3O 6) Erythrose-4-phosphate ( C4H7PO 7)
Polysaccharide (C6H5O 5)n
Nucleic acid
6- Deoxyxylulose
Pyruvic acid (C 3H4O 3) Shikimic acid pathway
Aliphatic
amino Hydroxy-
Aromatic
acids amino benzoic acid
TCA cycle acids
GLYCOSIDES
ALKALOIDS PHENYL
Acetyl CoA PROPANOIDS
SUGAR DERIVATIVES
Mevalonic acid Malonyl CoA COMPLEX

ALKALOIDS
FLAVONOIDS
ISOPRENOIDS
(C5H8)
POLYKETIDES COMPLEX
COMPLEX FLAVONOIDS
ISOPRENOIDS
The primary and secondary metabolites obtained by carbon metabolism in plants
Fig. 10.1 The primary as well as secondary plant metabolites obtained from metabolism in plants
(Kokate et al. 2005)
Secondary metabolites have a great importance to exhibit the pharmacological

activities which is established by the application of the extracts of medicinal plants
in the primitive peoples and animals to cure or treatment for various illness that is
published in different books and journals. Phytoconstituents provide various
activities such as analgesic, anti-inflammatory, antidiabetic, antimalarial, antihy-
pertensive, antiprotozoal, antibacterial, antifungal, antiviral activity, etc. It has been
observed that some grasses such as alfalfa show biological activity like estrogen
that provides an impact on the fertility of animals (Bennets et al. 1946).
Secondary plant metabolites are grouped on the basis of their chemical structures
into several classes. This chapter deals with the comparison of secondary
metabolites with primary metabolites, description, and structure of various sec-
ondary plant metabolites, their biosynthesis, and their pharmacological activities.
Stress compounds are generally produced in plants due to several factors such as
injury in the plant and metabolic disturbance. These compounds are the products of
either primary metabolism or secondary metabolism in plants. These products are
available in the undeveloped flower buds, sub-epidermal glands, and seed kernels
of Gossypium hirsutum (cotton plant). The synthesis of such compounds is
332 C. C. Kandar
developed by different biological and environmental conditions such as dehydra-

tion, chemical treatment, microbial infection, mechanical injury to the plants, and
UV irradiation. They are manufactured for pharmaceutical benefits, for example, as
oleoresins and gum, e.g., gossypol. Gossypol is useful for male antifertility activity
and insecticidal activity (Kaur 2010).
10.1.1 Comparison of Secondary Metabolites with Primary

Metabolites
Primary metabolites are available in all types of plants and they perform all essential
metabolic reactions by sharing in nutrition, as well as reproduction (Croteau et al.
2000). In some cases, it is difficult to differentiate primary and secondary metabolites
of plant origin. Under the class of terpenoids, both primary and secondary metabolites
are available. The same phytoconstituent exhibits the role of both primary and sec-
ondary metabolites. Actually, secondary metabolites contain a lot of phytocon-
stituents derived from various plant families in environmental stress conditions that
show varieties of activity. Cell pigmentation in seed and flower provided by flavo-
noids and carotenoids (basically secondary metabolites) shows activities like primary
metabolites such as the attraction of pollinators and dispersion of seed and they have,
therefore, also involved in plant reproduction (Winkel-Shirley 2001). Plant primary
products are mainly glucose, nucleic acids, amino acids, proteins, carbohydrates, fats,
and lipids and they are concerned with the structure, physiology, and genetics of the
plant, which indicate their significant role in plant development. On the other hand,
secondary products are available in very few numbers and also in less concentrations
with comparison to primary metabolites. The production of carboxylic acids of the
Krebs cycle is under the involvement of primary metabolism. In contrast, secondary
metabolites are involved in providing fitness for survival to the plant species. The
particular phytoconstituents in a certain species have been used to determine sys-
tematically the groups of secondary plant products that are used to classify the plants
on the basis of the chemotaxonomic process (Winkel-Shirley 2001).
Plants produce an amazing diversity of low molecular weight compounds.
Among the estimated 400,000–500,000 plant species in World, only a small per-
centage of plants have experimented phytochemically and a small fraction is sub-
jected to biological or pharmacological screening. The capability to produce
secondary plant products has been determined through the evolution of different
plant progeny when they faced surrounding stresses:
(a) Floral scent volatiles and pigments have developed to attract insect pollinators
and thus enhance fertilization that is involved in reproduction.
(b) Preparation of toxic chemicals to safeguard the pathogens and herbivores or to
inhibit the development of neighboring plants.
(c) Chemicals found in fruits inhibit spoilage of fruits and provide signals in the
form of color, aroma, and flavor to prove the presence of significant materials
such as sugars, vitamins, and flavoring agents. Animals eat fruit and thereby
help in the dispersion of seeds.
(d) Some chemicals are involved in cellular functions which are unparalleled to
certain plants such as resistance to salt or resistance to drought.
The various classes of secondary plant metabolites include:
(1) Alkaloids
(2) Saponins
(3) Phenolics
(4) Terpenes
(5) Carbohydrates
(6) Lipids
(7) Glycosides.
10.2 Alkaloids
Alkaloids are organic heterocyclic compounds having minimum one nitrogen atom
in the ring or outside the ring. The term alkaloid is originated from “alkali-like” and
hence, they show alkaline character due to the presence of lone pair electrons on the
nitrogen atom. They become like the characteristics of some naturally derived
complex amines. They include greater than 6000 nitrogenous compounds in about
15% of vascular terrestrial plants and spread in more than 150 plant families. The
proper definition of alkaloids may not be described because they exist as a variety
of group of compounds (Giweli et al. 2013). The only similarity in these com-
pounds is that all these compounds have nitrogen atom. All alkaloids are not
defined by a common definition. A huge number of alkaloids are isolated from
different plant sources. Since alkaloids have a vast variation with respect to
chemical structure present in these compounds, botanical sources, physiological
responses, similar biochemical origin in spite of different taxonomic distribution
and pharmacological activities, and therefore, they are classified chemically, tax-
onomically, pharmacologically and biosynthetic way, respectively (Tadeusz 2015;
Nicolaou et al. 2011; Aniszewski 2007).
Chemically alkaloids are subclassified into three groups (Kokate et al. 2005;
Clarke1970).
a. True alkaloids (Heterocyclic alkaloids): they are originated from amino acids
and contain a heterocyclic ring.
b. Proto alkaloids (non-heterocyclic alkaloids): They are originated from amino
acid but the nitrogen atom is not present in the ring structure
c. Pseudo alkaloids: They are not originated from amino acids but from purine or
terpenoids. They have a nitrogen-containing heterocyclic ring (Table 10.1).
They are classified as follows:
334 C. C. Kandar
Table 10.1 Ring structure present in alkaloidal drugs

Heterocyclic Alkaloidal drugs Ring structure
ring
True alkaloidal drugs (Heterocyclic alkaloids)
Indole Reserpine, Ergotamine, Ergometrine, Physostigmine,
Strychnine, Vincristine, Vinblastine
N
H
Imidazole Pilosine, Pilocarpine, N
N
H
Isoquinoline Morphine codeine, Papaverine, Narcotine,
d-tubocurarine, Emetine, Cephaeline
N
Norlupinane Lupanine, Sparteine
N
Pyrrole and Cocoa, Hygrine

pyrrolidine N N
H H
Pyridine and Arecoline, Conine, Lobeline
piperidine
N N
H
Pyrrolizidine Senecionine, Symphitine
N
Quinoline Quinine, Quinidine, Cinchonine, Camptothecin
N
Steroidal Conessine, Funtumine, Solanidine, Protoveratrine,
Withanine
Tropane Atropine, Hyoscine, Cocaine, Meteloidine

H3C N
Proto alkaloidal drugs (non-heterocyclic alkaloids)

Alkylamine Ephedrine, Colchicine, Pseudoephedrine, Aconite R-NH2
(Amino
alkaloid)
Pseudo alkaloidal drugs
Diterpenes Aconitine, Aconine C10H16
Purine Caffeine, theophylline, theobromine O
HN NH
O
N N
H H
Although human beings have been using different parts of plants from the ancient
age as drinks like tea, or as medicines like antimalarial, narcotic analgesics, but these
alkaloidal substances were not separated and identified from the plant sources up to
the beginning of the nineteenth century due to unavailability of modern analytical
instruments (Seigler 1995). Alkaloids are not present in the lower plants. Lysergic
acid derivatives (LSD) and gliotoxins (alkaloids having sulphur atom) are available
in fungi. Other kinds of alkaloids such as ephedra, taxol, and lycopodium present in
pteridophytes and gymnosperms are reported for their medicinal values. Based on
the taxonomic characteristics, the alkaloids are classified as (a) Centrospermae
belonging to the family Chenopodiaceae, (b) Magnoliales belonging to the families
Lauraceae, Magnoliaceae, (c) Ranunculales belonging to the families
Berberidaceae, Menispermaceae, Ranunculaceae, (d) Papaverales belonging to the
families Papaveraceae, Fumariaceae, (e) Rosales belonging to the families
Leguminosae, subfamily Papilionaceae, (f) Rutales belonging to the family
Rutaceae, (g) Gentiales belonging to the families Apocynaceae, Loganiaceae,
Rubiaceae, (h) Tubiflorae belonging to the families Boraginaceae, Convolvulaceae,
Solanaceae (i) Campanulales belonging to the family Campanulaceae having
subfamily Lobelioideae, and another family Compositae having subfamily
Senecioneae. But yet, no such scientific proofs are available to show the availability
of alkaloids in the plants of various dicot orders such as Salicales, Cucurbitales,
Fagales, and Oleales (Evans 2009).
Chemicals found in fruits inhibit spoilage of fruits and provide signals in the
form of color, aroma, and flavor to prove the presence of significant materials such
as sugars, vitamins, and flavoring agents. Alkaloidal substances exhibit a lot of
biological properties such as a) analgesic or painkiller, i.e.,morphine, colchicine; b)
local anesthesia, i.e., cocaine; c) respiratory stimulation and relaxation, i.e.,
arecoline, lobeline; d) vasoconstriction, muscle relaxation, i.e., d-tubocurarine; e)
anti-neoplastic, i.e., vincristine, vinblastine; f) hypotensive properties, i.e., reser-
pine, protoveratrine, and g) cardiac stimulation, i.e., ephedrine. The alkaloids also
show another type of biological properties mentioned in the various journals like
protection of plants from the herbivorous animals, arrest the growth or killing the
bacteria, anticancer property, action against fungi and viruses, causing cancer.
Some of the alkaloids are so toxic that they can even cause the death of animals.
Several alkaloids like as nicotine and anabasine are useful as insecticides (Seigler
1995; Hoffmann 2003).
Various categories of alkaloids having different taxonomic distribution and
pharmacological activities can be brought under the same category. This classifi-
cation provides the significance of the precursor from which the alkaloids are
biosynthesized in the plant. The alkaloids are biosynthetically classified on the basis
of their amino acid precursor like lysine, tyrosine, phenylalanine, ornithine, tryp-
tophan, etc. (Kokate et al. 2005).
The chemical structures of some major amino acid precursor are represented
below (Fig. 10.2).
The chemical structures of some well-known alkaloids (Fig. 10.3), are as follows
(Kokate et al. 2005; Tyler et al. 1988).
336 C. C. Kandar
O O
O O
OH OH
OH
H2N OH H2N
H2N H2N
H2N NH2 OH
Ornithine Lysine Phenyl alanine Tyrosine
Fig. 10.2 The structures of amino acid precursors of alkaloidal drugs
The schematic diagram of the biosynthesis of different alkaloids from various

amino acids (Figs. 10.4, 10.5, 10.6, 10.7) is depicted below (Kokate et al. 2005; Ali
2019, Evans and Mitch 1982).
10.3 Saponins
Plants having saponin glycosides are of economical importance, as well as

medicinal value. Saponins are those compounds which contain a polycyclic agly-
cone moiety known as a sapogenin. The aglycone part is either a steroidal nucleus
also called tetracyclic triterpenoid saponins or triterpenoid which contains five rings
also popular as pentacyclic triterpenoid saponins (Fig. 10.8). Pentacyclic saponins
are further differentiated into (a) a-amyrin type (b) b-amyrin type, and (c) lupeol.
The aglycone part is attached to a carbohydrate unit that may be a monosaccharide
unit like glucose, mannose, galactose, etc., or sometimes oligosaccharide chain like
glucose-gulose-mannose-rhamnose. This glycone part comprises various pentoses
and, hexoses sugars, or uronic acids. This hydrophobic-hydrophilic balance made
by these types of compounds lower the surface tension between two immiscible
liquids and also make lather in their aqueous solutions and hence behave like a
soap. Another important property of saponin is the ability to cause hemolytic of red
blood corpuscles (RBC) in vitro. The non-sugar part of the saponin compound is
known as genin or especially sapogenin due to the ability of the formation of foam.
Saponins are found widely among plants and reported more than 500 plants from
about 90 different families. These substances are extracted from most of the plant
parts like roots bulbs, leaves, stems, flowers, and fruits. The literature survey
reveals that saponins are usually found in the roots of various plants like
Chlorophytum borovillianum known as safed musali, Dioscorea deltoidea popular
as wild yam, Eleutherococcus senticosus called as Siberian ginseng, Asparagus
racemosus popular as Shatavari, Gentiana lutea generally called gentian,
Glycyrrhiza glabra popular as licorice and Panax ginseng called as Korean ginseng
(Assa et al. 1973).
H H CH2 H CH2
HO
HO N N
R H
R
N N
Quinine R= OCH 3 ( 8S, 9R) Quinidine R= OCH 3 ( 8R, 9S)
Cinchonidine R= H Cinchonine R= H
N
H3CO N
H H H
O
H
OCH3
H3COOC O
CH3 OCH3
Reserpine OCH3
N
H3CO N
H H H
O
H
OCH3
H3COOC O
CH3 OCH3
Rescinnamine OCH3
O CH2OH
H3C N
H
H O
( -) - Hyoscyamine
+
( _) - Hyoscyamine = Atropine
Fig. 10.3 The structure of some important alkaloidal drugs

338 C. C. Kandar
COR COR
NH NH
N N
CH3 CH3
R= OH Lysergic acid Isolysergic acid

R= NH2 Lysergic acid amide Isolysergic acid amide
R= NHCH(CH3)CH2OH Ergometrine Ergometrinine
R=N(C2H5)2 Lysergic acid diethylamide (LSD)
OH
N CH 2 CH 3
N
H COOCH 3
CH 2 CH 3
H H
H 3 CO N OCOCH 3
HO
COOCH 3
R
Vinblastine R= CH 3
Vincristine R = CHO
O CH2OH
H3C N
O H
H O
( -) - Scopolamine ( Hyoscine)
Fig. 10.3 (continued)
Saponin ¼ Sapogenin þ Sugar
Sapogenin has a steroidal nucleus (neutral saponins) and a pentacyclic triter-

penoid nucleus (acid saponins). Sapogenin includes diosgenin, asiatic acid,
panaxadiol, glycyrrhetinic acid, carbenoxolone, brahmic acid, and shatavarin I–IV,
etc. (Fig. 10.9). Sugars include glucose, xylose, glucuronic acid, arabinose, rham-
nose, mannose.
COOH Hygrine Pyrroline Nicotine

(C4H7N)
NH2
Phenyl alanine
Tropic acid Ornithine

Cocaine
(C9H10O 3) (C5H12N2O 2)
Scopolamine
Atropine
Tropine
(C17H23NO3) (C8H15NO)
Fig. 10.4 Schematic diagram of biosynthesis of alkaloids derived from ornithine
Lupinine
Lysine
Isopelletierine (Amino acid)
Anabasine
Fig. 10.5 Schematic diagram of biosynthesis of alkaloids derived from lysine
Saponins have shown a lot of pharmacological activity. Some saponins have

hypoglycaemic, antitumor, piscicidal, diuretic, molluscicidal, nervine tonic, adap-
togen, spermicidal, galactagogue sedative, stimulant expectorant, and analgesic
activities. Glycyrrhizin from G. glabra belonging to the family Fabaceae is useful
as an expectorant, antitussive agent, and for the treatment of peptic ulcer. Saponin is
also useful for the treatment of chronic hepatitis, as well as cirrhosis. The saponins
obtained from Bupleurum falcatum belonging to the family Apiaceae exhibit
potential anti-inflammatory action in the rat paw edema model. In Korean medicine,
the saponin derived from the roots of Phytolacca americana also shows
anti-inflammatory activity. Another saponin, aescin, showing anti-inflammatory
activity is obtained from the plant, Aesculus hippocastanum popular as horse
chestnut. Aescin has been reported 600 times more potent anti-inflammatory action
in comparison to another standard molecule, rutin, in rat paw edema model
(Guclu-Ustundag et al. 2007).
A list of saponin drugs with their active constituents and uses (Table 10.2), are
given below (Kokate et al. 2005, Sparg et al. 2004).
Some chemical structures of important saponins are given below in Fig. 10.9
(Kaur 2010; El Aziz et al. 2019).
The sapogenins are biosynthesized by a mevalonic acid pathway from acetyl
CoA to squalene, a 30 carbon-containing compound, with the help of several steps.
340 C. C. Kandar
COOH Ephedrine
NH2
Phenyl alanine
Mescaline
COOH
HO
Papaverine
NH2
Tyrosine
Hydroxylation
Thebaine Codeine Morphine
HO
Emetine
COOH
HO
NH2 Colchicine
DOPA
Fig. 10.6 Schematic diagram of biosynthesis of alkaloids derived from tyrosine and phenyl
alanine
N-Methyl dimethyl allyl Dimethylallyl

tryptophan Tryptamine
tryptophan Secologanine
Chanoclavine Tryptophan Vincoside
Agroclavine Quinine
Elymoclavine Lysergic acid
Fig. 10.7 Schematic diagram of biosynthesis of alkaloids derived from tryptophan
Squalene on further structural modification produces various aglycone namely,

diosgenin, yamogenin, tigogenin, etc. (Fig. 10.10).
10.4 Phenolic Compounds
Among all the secondary plant products, phenolic compounds have created great
importance in plant systems. The color, flavor, and taste of the plant parts, foods,
and drinks are due to the presence of phenolic compounds such as flavonoids,
coumarins, and tannins, etc. Therefore, phenolic compounds have become the
H3C CH3
H3C
CH3 CH3
CH3 CH3 CH3

CH3
CH3
Sugar-O Sugar-O
Steroid pattern of saponins Pentacyclic triterpenoids
CH3 H3C CH3 H2C CH3

H3C
CH3 CH3 CH3 CH3 CH3 CH3

CH3 CH3 CH3
H H CH3 H
CH3 CH3
HO H HO HO
H3C H3C CH3 H
CH3 H3C
CH3
Alpha- amyrin Beta- amyrin Lupeol
Fig. 10.8 The structure of the skeleton of sapogenins
largest group of secondary plant products. The common characteristic of such a

group is the presence of one or more phenol groups. The compounds are available
either as a very simple aromatic structure or high molecular complex structure due
to polymerization. Phenolic compounds manufactured by vegetables, leaves, fruits
cocoa, tea, and various plants exhibit definite health well-being. Some phenolic
compounds exhibit a variety of pharmacological properties like anti-inflammatory
properties, e.g., quercetin and hepatoprotective potential, e.g., silybin. Some other
compounds show estrogen-like action, e.g., genistein and daidzein are also active
against insects, for example, naringenin. Phenolic molecules also act as potent
antioxidants and free radical scavengers, especially flavonoid compounds
(Golawska et al. 2014).
Other literature revealed that they exhibit anti-carcinogenic and other biological
properties along with antioxidant and anti-inflammatory activities (Park et al. 2001).
Simple phenolic compounds are antiseptic at low concentration, whereas disin-
fectant at higher concentration and act against different helminths. Phenol itself
shows as an antimicrobial agent (Pengelly 2004). Phenolic compounds are wide-
spread in almost all the plants and involved in various studies in different fields such
as biological, chemical, agricultural, and pharmaceutical fields (Dai et al. 2010;
Herrmann et al. 1989).
Phenolic compounds may be classified according to their chemical structure or
biosynthetic precursor (Fig. 10.11). Based on their structures, phenolic compounds
can be classified into (a) Simple phenolics (b) Coumarins (c) Flavonoids
(d) Chromones xanthones (e) Tannins (f) Lignans, and (g) Stilbenes.
342 C. C. Kandar
H3C COOH
CH3
CH3 CH3
H
OO O
CH3 CH3 CH3
CH3
CH3
HO HO
Diosgenin H3C CH3 Glycyrrhetinic acid
CH3
H3C COOH H3C
CH3
O CH3 COOH
CH3
CH3 H HO
CH3
H CH3
CH3 HO
HOOC-H2C-H2C-COO R
HOH2C CH3 Asiatic acid R=H
Carbenoxolone
H3C CH3 Brahmic acid R= OH
H3C CH3
O
OH H3C
CH3 CH3
CH3
CH3 OO
CH3
CH3
CH3
HO
Panaxadiol Glu-Glu-(Rhamn)-Glu-O
H3C CH3 Satavarin I
Fig. 10.9 The chemical structures of important saponins
10.4.1 Simple Phenolics
Phenolic substances are universal in plants but the availability of free phenol (also
known as carbolic acid) in plants is very rare. Gallic acid (3,4,5-trihydroxy benzoic
acid), a phenolic acid is found in the plants as gallotannins. This compound shows
astringent property due to its ability to precipitate proteins in cases of internal
hemorrhage and also exhibits several pharmacological activities, for examples,
anti-inflammatory activity to inhibit inflammation, antiviral, widely used in fungal
infection and viral infection, shows cytotoxicity against cancer, some time to treat
anaphylactic shock, antibacterial, antimutagenic, choleretic, and bronchodilatory
actions. Gallic acid is also used to treat diabetes by inhibiting insulin degradation
and enhances the relaxation of smooth muscles (Harborne et al. 1993).
Table 10.2 The uses and active constituents of saponin obtained from plants
Sl. Drug and Active constituents Uses
No synonym
1 Dioscorea Diosgenin, precursor of Tonic, anti- rheumatoid arthritis, for
(Yam) prostaglandin synthesis of oral contraceptives, cortisone,
prednisone, progesterone
2 Shatavari Shataverin I and Galatogogue (increase production of milk),
(satamuli) Shataverin II diuretic, tonic, anti-oxytocic, anti-
rheumatoid
3 Brahmi Bacoside A and Nerve tonic (increase memory)
Bacoside B
4 Liquorice Glycyrrhizin, Treatment of peptic ulcer, expectorant and
Carbenoxolone, demulcent, antispasmodic, addison’s
Liquiritin, isoliquiritin disease
5 Safed Hicogenin General tonic, sex stimulant
musali
6 Ginseng Panaxadiol, panaxatriol Tonic, sex stimulant, and adaptogen,
ginsenoside, oleanolic cosmetics, immunomodulatory
acid
7 Gokhru Gitogenin, ruscogenin, Aphrodisiac, treatment of gout, calculous
chlorogenin formation and painful micturation, diuretic
8 Momordica Charantin, momordicin Hypoglycaemic agent
Mevalonic acid (C 6) Acetic acid
Squalene (C30) Pentacyclic triterpenoid
Cholesterol (C27) Yamogenin Sarsasapogenin
Diosgenin Tigogenin
Fig. 10.10 The Schematic diagram of the biosynthesis of different sapogenins (Kokate et al. 2005,
Mugford et al. 2013)
The simple phenolic compounds vary on the basis of the presence of their
functional groups, such as hydroxyl (–OH), aldehyde (–CHO), or carboxylic
(–COOH) group; these are a phenolic aldehyde, e.g., vanillin, phenolic acids, e.g.,
salicylic acid, ferulic acid, and caffeic acid and a phenolic phenylpropane, e.g.,
eugenol. Hydroquinone (4-hydroxy phenol) is abundantly found as simple phenols,
344 C. C. Kandar
Phenolic compounds
Simple phenolic Flavonoids Tannins Stilbenes

C6 / C 6 - C 1 / C6 - C 3 - C 6 C6 / C 6 - C 1 / C6 - C 2 - C 6
C6 - C 2 / C 6 - C 3 Benzo-gama-pyrone diester of C6 - C 6
Coumarins Chromones /xanthones Lignans

C6 - C 3 O Benzo-gama-pyrone &
C6 -C3 O / C6 - C 4 - C 6
Benzo-alpha-pyrone
dibenzo-gama-pyrone
Fig. 10.11 Classification and carbon skeleton of phenolic compounds of plant sources
in arbutin, a glycoside (Fig. 10.12). The well-known glycoside coniferin and many
related substances derived from phenolic cinnamic alcohols are considered as an
ingredient for the biosynthesis of lignin (Evans 2009; Hoffmann 2003).
Simple phenolic substances show various pharmacological activities such as the
urinary tract infection exhibited by arbutin (Zbigniew et al. 2014), as well as
anti-inflammatory action shown by salicylates (Amann and Peskar 2002). It is
popular that all phenols at low concentrations act as antimicrobial. In fact, phenol
was considered as the first antiseptic used in surgical operation (Pelczar et al. 1988).
The chemical structures of some important simple phenolic compounds are
represented below in Fig. 10.12.
COOH CHO
OH
COOH
HO OH OCH3 OH
OH OH
Phenol Gallic acid Vanillin Salicylic acid
COOH
CH2 COOH
COOCH3
OH
OCH3 OCH3
OH OH
OH OH
Caffeic acid Eugenol Ferulic acid Methyl salicylate
Fig. 10.12 The chemical structures of simple phenolic compounds derived from plants
10.4.2 Coumarins
Coumarins and its derivatives are found in so many plants. Coumarin is ubiqui-
tously available in about 150 species of 30 different families. They are available in
the families mainly Solanaceae, Rubiaceae, Leguminosae, Oleaceae, Umbelliferae,
Caprifoliaceae, etc. The important source of coumarin compounds is sweet clover
belonging to Melilotus spp., tonka bean (scientific name: Dipteryx odorata), and
sweet woodruff (scientific name: Galium odoratum) (Hoffmann 2003). They are the
derivatives of benzo-a-pyrone, also known as lactone of o-hydroxycinnamic acid,
i.e., cyclic ester of phenolic compound (Hoffmann 2003). Aesculetin, aesculin,
herniarin, fraxin, umbelliferone, scopolin, and scopoletin are popular coumarin
compounds having benzo-a-pyrone nucleus available both in the free state and as
glycosides also (Fig. 10.13). Plants having coumarin derivatives are belladonna
(Atropa belladonna), Datura (Datura stramonium) belonging to the family
Solanaceae, February daphne (Daphne mezereum belonging to the family
Thymeliaceae), common rue (Ruta graveolens belonging to the family
Umbelliferae), and Horse chestnut (A. hippocastanum belonging to the family
Hippocastanaceae) and certain Rosaceae (Evans 2009).
Furanocoumarin derivatives are also available in bergamot oil, bael fruit, pso-
ralea, etc. These compounds are formed by the fusion of furan ring with coumarin at
6 and 7 positions and 7 and 8 positions, respectively (Bruni et al. 2019). These
5 4
3 HO H3CO
6
7 CO CO CO
O 2 HO HO O
8 O
1 O-Glu
Coumarin Aesculetin Fraxin
H3CO
CO CO CO
H3CO HO HO O
O O
Herniarin Scopoletin Umbelliferone
HO O O
H3C
H3C
O
Psoralidin CH3
Fig. 10.13 The chemical structures of some important coumarin drugs of plant sources
346 C. C. Kandar
compounds show various pharmacological activities like anticoagulant, anticancer,

anti-Alzheimer, and anti-inflammatory activities (Xu et al. 2015).
The structures of some important coumarins are shown below in Fig. 10.13.
10.4.3 Flavonoids
Among the normally occurring phenolic compounds, flavonoids have taken a

maximum place in the plant kingdom. Out of about 2000 of these known com-
pounds, around 500 compounds are found as a free state (Evans 2009). The most
available flavonoids include flavones, flavonols, and anthocyanins. The flavones
and their derivative are generally yellow in color (Latin flavus, meaning yellow).
These compounds are ubiquitously distributed worldwide but they are mainly
available in young tissues and in the higher plants. They are found in Pteridophyta
(mainly fern and related plants), Gymnosperma (conifer), monocots, and dicots.
Among the dicot families, plants belonging to the families Leguminosae,
Polygonaceae, Rutaceae, Umbelliferae, and Compositae are a rich source of fla-
vonoids. Researches worldwide have reported the medicinal activities of drugs
containing flavonoids. For examples, milk thistle (Silybum marianum belonging to
the family Compositae), liquorice root (G. glabra belonging to the family
Leguminosae), Roman chamomile (Chamaemelum nobile, Buck–wheat
(Fagopyrum esculentum belonging to the family Polygonaceae), and ginkgo (
Ginkgo biloba Linn. belonging to the family Gingkoaceae). Many herbs having
flavonoids have now been incorporated in the British Pharmacopeia (B.P.). For
example, Birch Leaf (Betula pendula), Calendula flower (Calendula officinalis),
Horsetail (Equisetum ramosissimum), Elderflower (Sambucus nigra), Motherwort
(Leonurus cardiac), Lime Flower (Tilia cordata), and passionflower (Passiflora
edulis) are well-known plants. The flavonoid compounds have been shown phar-
macological properties like anti-inflammatory, antiallergic, antithrombotic, vaso-
protective, antitumor, antifungal, hepatoprotective, and gastric mucosal protective
activities (Table 10.3) (Montanher et al. 2007; Serafini et al. 2010). Flavonoids have
a 15 carbon skeleton arranged as C6–C3–C6 in which two phenyl rings are linked by
a three-carbon chain. A carbonyl group is present at one end of a three-carbon
chain. This three-carbon chain may be either open in case of chalcone and dihy-
drochalcone (simplest naturally occurring flavonoid) or as a part of the heterocyclic
ring and the heterocyclic ring fused with benzene ring forms chroman ring. Flavone
and flavonol are common flavonoids in which B-ring is attached at 2 position of
C-ring, on the other hand, in isoflavone structure, the B-ring is attached to 3
position of C–ring. Flavonoids exist as free molecules or as glycosidic forms
(Fig. 10.14). The glycosidic form of flavonoids is generally O-glycosides and few
are C-glycosides. Chemically, flavonoids are also known as benzo-c-pyrone
derivatives. Biflavonoids are also available in which two flavonoids molecules are
joined through either C–C linkage or C–O linkage. Anthocyanin present in most
plants is responsible to provide red, blue, purple color to the flower. Anthocyanin
Table 10.3 The uses of some flavonoid compounds of plant sources

Compound or plants Pharmacological activities
Silymarin Hepatoprotective, chronic inflammatory hepatic disorder
Baicalin Anti- HIV, antihypertensive, anti-inflammatory
Genistein Anticancer, cosmetic
Isoflavone of Erythrina Treatment of fungal infection, antibacterial, anti-venom,
sigmoidea antirheumatic, kidney treatment
Formononetin Estrogenic activity, oral contraceptive, antioxidant, treatment of
Alzheimer’s disease
Coumestrol Estrogenic activity, breast cancer treatment
Gingko Anti-Alzheimer’s disease, Raynaud’s disease, ant- PAF,
acrocyanosis, anti-sepsis
Buck wheat Treatment of retinal hemorrhage
Quercetin Anti-oxidant, estrogenic activity
exists as flavylium ion in which a positive charge is present on O–atom of chroman

ring (Kokateet al. 2005; Wang et al. 2018).
The pharmacological uses of some significant flavonoid compounds are repre-
sented in the following Table 10.3 (Wang et al. 2018).
The structures of some important flavonoids are shown below in Fig. 10.14
(Kokate et al. 2005; Panche et al. 2016).
10.4.4 Chromones and Xanthones
Chromones and xanthones are also structural derivatives of benzo-c-pyrone

(Figs. 10.15 and 10.16). The biological importance of these compounds is less.
Only a few compounds have medicinal importance, for example, eugenin is
obtained in the clove plant and khellin is produced from mustard seeds (Bagci et al.
2010). More complex structures of chromone are furanochromones, the active
constituents from Ammi visnaga.
Xanthones compounds are available mainly in the families of Gentianaceae, as
well as Guttiferae, otherwise spread scattered over the plant kingdom in the families
of Moraceae, as well as Polygalaceae. Mangostin isolated from mangosteen tree
(Garcinia mangostana) has been reported for several biological activities like
antibacterial, anti-inflammatory, antioxidant, and anti-cancer properties (Jung et al.
2006). Polygala nyikensis is mainly used for the treatment of fungal infection by the
highlanders of Malawi and bordering countries. Xanthones are also useful as a
larvicide. The root of the plant exhibits antifungal activity due to the presence of
xanthones (Susana et al. 2011).
348 C. C. Kandar
O O
Dihydrochalcone Chalcone
3' OH
2' 4' OH
8 1
B
O 2 HO O
7 5'
A C 6'
6 3 OH
5 4
O OH O
Flavone Flavonol (Quercetin)
OH OH
HO O HO O OH
OH
OH O OH
Flavone (Apigenin) Catechin
OH
OH
+ HO O
HO O
OH
OH O
OH OH
Anthocyanin ( Cyanidin) Isoflavone ( Genistein)
OH
O
OH OH
HO O HO O OCH3
O
O-Rham- Glu OH OH
OH O OH O Silybin
Rutin
OH
HO O
OH
OH O
Flavone (Kaemferol)
Fig. 10.14 The structures of some important flavonoids derived from plants
OCH3
O H3CO O CH3
O O CH3
O OH O
OCH3 O
Chromone Eugenin Khellin ( Furanochrome)
Fig. 10.15 The structures of chromones obtained from plants
O HO O OH
H3C OCH3
O CH3 OH O CH3
Xanthone CH3
Mangostin
Fig. 10.16 The structures of xanthones obtained from plants
10.4.5 Tannins
Tannins are the most widely distributed natural compounds found in various
families of higher plants. They are non-crystalline substance and make colloid when
mixed with water. Tannins are available in vacuoles and cell sap. Chemically they
are a mixture of various complex organic compounds containing polyphenols
mainly 1,2-dihydroxy benzene (catechol) or 1,2,3-trihydroxy benzene (pyrogallol).
Tannins are soluble in dilute alkalies, glycerine, and alcohol due to the formation of
hydrogen bonds. Though they are organic compounds, basically they are insoluble
in organic solvents except for acetone. Tannins show some characteristic reactions
such as precipitation of alkaloids and gelatin, precipitated by copper, lead, and tin
salts, as well as concentrated potassium dichromate solution, bluish-black, or
brownish-green color with ferric chloride (Sieniawska et al. 2017).
Tannins containing polyphenols have the capability to precipitate protein. Due to
its ability to convert raw animal skins into leather, and therefore, they are exten-
sively used in leather technology for decades. Tannins crosslink the proteins of
animal hides and therefore hides become more resistant to bacteria, as well as
fungus, resulting in the prevention of putrefaction (Hagerman et al. 1981). Based on
their chemical characteristic and behavior on dry distillation, tannins are generally
divided into two major groups: (a) hydrolyzable tannins (b) condensed tannins or
non-hydrolyzable tannins. The hydrolytic product of hydrolyzable tannins is gen-
erally gallic acid (3,4,5-trihydroxybenzoic acid) or ellagic acid (di ester of hex-
ahydroxydiphenic acids) which is combined by ester linkage with –OH group of a
glucose molecule. They are hydrolyzed quickly by acids or enzymes. Two main
types of hydrolyzable tannins are available such as gallotannins and ellagitannins
derived from gallic acid and ellagic acid, respectively. Ellagitannins have the
350 C. C. Kandar
COOH OH
HO HO OH OH
OH
OH HO
HO
CO- O-Glu OH
OH
Gallic acid Catechol Glucogallin Pyrogallal
OH
CO O
HO OH
OH COOH
HO O CO OH
OH COOH
O COOH
O
Ellagic acid
Chebulic acid
OH
HO O OH
OH
OH
Catechin (Condensed tannin)
Fig. 10.17 The structure of some hydrolyzable tannins
medicinal interest, for this reason, the structures of ellagitannins have been eluci-
dated by analytical techniques (Fig. 10.17). The elucidated structures comprise
geraniin sequestered from Geranium robertianum known as Herb Robert and also
Geranium maculatum called as American cranesbill (Catarino et al. 2017) and
tellimagrandins 1 and 2 sequestered from Quercus alba called as Oak bark, Punica
granatum known as pomegranate and Filipendula ulmaria called as Meadowsweet
(Yi et al. 2004).
Condensed tannins, or proanthocyanidins, or non-hydrolyzable tannins are very
much reverberating to hydrolysis. These substances are oligomeric flavonoids
derived from flavones like flavan-3-ol, flavan 3,4-diol, or catechin. The character-
istics of such types of tannins depend upon four parameters namely, the type of
linkages between flavonoid units; hydroxyl group arrangement, or the enantiomeric
effect of the carbon atoms situated at 2, 3, 4 positions of pyran ring and the effect of
substituents on the ring structure. Few substances contain both the hydrolyzable and
non-hydrolyzable tannins, e.g., bark and leaves of Hamamelis virginiana and
leaves of Camellia sinensis popular as a tea (Goenka et al. 2013).
The drugs having tannins have a great medicinal value. They are useful for the
treatment of diarrhea, to check small hemorrhage in the gastrointestinal tract (g.i.t.),
as a mild antiseptic, and as antidotes in poisoning by heavy metals like lead, tin, and
alkaloids by precipitating them. It has reported that Epigallocatechin-3-gallate
obtained from tea, shows antiangiogenic activity in mice. The juice of Vaccinium
oxycoccos (cranberry) is used for long times as urinary antiseptic which is also
experimentally proven (Jepson et al. 2008).
10.4.6 Stilbenes
Stilbenes are another important phytoconstituents broadly distributed worldwide.

They are available as heartwood constituents of plant species. They are generally
found in the heartwood of trees like as, Eucalyptus belonging to the family
Myrtaceae, Madura belonging to the family Moraceae, and Pinus belonging to the
family Pinaceae (Seigler 1995). Resveratrol (3,5,4′-trihydroxy-trans-stilbene), a
type of natural phenolic compound is widespread in different plants (Fig. 10.18).
Resveratrol exhibits estrogen-like property and available in Picea, Fabaceae,
Myrtaceae, and Vitaceae (Gehm et al. 1997).
10.4.7 Lignans
Lignans (C18) are 18 carbon-containing compounds that are biosynthetically

manufactured by dimerization of two units containing C6–C3 linked at b-carbons of
the side chain. Neo-lignans are not linked by b-b’ (or 8–8′) -carbon bonds and are
derived by the union of the head to tail of two C6–C3 moieties. On the basis of
structural types, they subclassified as Aryl tetrahydronaphthalene, arylnaphthalene,
dibenzocyclooctadiene, cyclobutane, dibenzylbutane, dibenzyl-butyrolactone,
tetrahydrofuran, and furofuran (Fig. 10.19). The plants having medicinally
Fig. 10.18 The structure of OH

the stilbene derivative
HO
OH
Resveratrol
352 C. C. Kandar
CH3
O O
CH3
O O
Aryl tetrahydronaphthalene Aryl naphthalene Dibenzylbutane
H3C CH3 H3C CH3

O
O
O
Dibenzyl butyrolactone Diphenyl cyclobutane Diphenyl tetrahydrofuran
O
O
O
Dibenzo-cyclooctadiene Diphenyl furofuran
H3CO H3CO
O O
HO HO
HO HO
OH OCH3
OH OH
(-)-Lariciresinol
(-)-Taxiresinol
OH
H3CO O
OH
OH O
HO
O
O
OCH3 H3CO OCH3

OH
OCH3
(-)-Isolariciresinol Podophyllotoxin
Fig. 10.19 The structure of lignan derivatives derived from plant sources
important lignans include Taxus baccata linn., Podophyllum hexandrum,

Schisandra chinensis, Linum usitatissimum, and S. marianum. It is reported that
taxiresinol and lariciresinol are known as flavonoid lignins whereas isolariciresinol
and demethyl-isolariciresinol called as dibenzyl butane lignans, all these lignans
show anti-ulcerogenic activity. Taxiresinol, and demethyl-isolariciresinol exhibit
antifungal property. Podophyllotoxin is reported as an anticancer agent.
Schisandrin A, B, C show hepatoprotective activity. They are also used as
antioxidants, tonifier, anti-fatigue anti-inflammatory agents. Silymarin is useful for
liver protection and shows lipid-lowering activity. Lignan obtained from linseed is
reported to reduce colon and mammary cancer (Cunha et al. 2012).
Various structural nuclei of lignans are given below (Cunha et al. 2012).
The biosynthesis of different phenolic compounds from shikimic acid is shown
in the schematic diagram (Fig. 10.20).
10.5 Terpenes and Terpenoids
Terpenes and terpenoids comprise one of the largest group of secondary plant
metabolites. The term “terpene” is originated from the word “turpentine”, meaning
resin. Terpene represents hydrocarbon (C5H8)n and terpenoids comprise hydro-
carbon and their oxygenated products (Perveen 2018). They are also known as
volatile oils or ethereal oils. Most of these compounds are employed in the appli-
cation of the pharmaceutical industry as pharmaceutical dosage forms or flavoring
agents and as perfumes in cosmetic and perfumery industries. Many of these
compounds are used as insect repellants, fungicides, as well as waterproofing
substances. They mediate electron transport processes in respiration and photo-
synthesis. They are also used for the treatment of various diseases (Perveen 2018;
Lalonde 2005).
Terpenoids are soluble in ether, alcohol, and lipid solvents and basically
insoluble in water. They are generally lighter than water and high refractive index.
They are mostly optically active either dextrorotatory or levorotatory. These
compounds are obtained from the duct, cell, trichomes, and lysigenous or
schizogenous glands. They are generally found in the families of Umbelliferae,
Labiatae, Myrtaceae, Zingiberaceae, Lauraceae, Rutaceae, and Piperaceae (Perveen
2018; Lalonde 2005). The structure of isoprene (C5H8) unit is given below
CH3
1 3
CH2 CH
[ CH3
H3C 4 ]n
C
2 CH2
Isoprene unit ( 2-Methyl 1, 3-butadiene]
Based on the number of isoprene (C5H8) units, terpenes are divided into the
following classes (Table 10.4).
354 C. C. Kandar
COOH
Shikimic acid NH2

Phenyl alanine
COOH
Malonyl CoA ( 2 units) Cinnamic acid
+
Acetyl CoA
C15 intermediate
OH
OH
OH
OH
+
HO O
HO O
OH
OH
OH O
OH
Quercetin
Cyanidin
COOH
Shikimic acid
NH2
Phenyl alanine
COOH
Cinnamic acid
O-coumaryl glycoside Hydroquinone Helicin
5 4
OH CH2OH
6 3 CHO
O-Glu
7 CO HO
8
O 2
1 OCH3
Coumarin O-Glu
Vanillin Salicin
Arbutin
Fig. 10.20 Biosynthesis of various phenolic compounds (Santos-Sanchez et al. 2019)

Table 10.4 The formula and examples of different classes of terpenoids of plant sources
Number of isoprene Class ( formula) Examples of compounds/drugs
units
1 Hemiterpene (C5H8) Isoprene, prenol, isovaleric acid,
tiglic acid
2 Monoterpenes Limonene, eucalyptol, pinene
(C10H16)
3 Sesquiterpenes Abscisic acid ( ABA)
(C15H24)
4 Diterpenes (C20H32) Gibberellin
5 Sesterterpenes Dysidiolide
(C25H40)
6 Triterpenes (C30H48) Brassinosteroids, squalene, lanosterol
8 Tetraterpenes Carotenoids, lycopen
(C40H64)
>8 Polyterpenes Ubuquinones, rubber, cytokonines,
(C>40H>64) vitamine E
COOH CH3 CH3

H3C
COOH CH2OH
CH3 H3C H3C
Angelic acid Isovaleric acid ( 3-Methylbutanoic acid} Prenol
Fig. 10.21 The structure of hemiterpenes derived from plant sources
10.5.1 Hemiterpenes
Hemiterpenes are the simplest among the terpenoids. They consist of only one
isoprene unit. Isoprene itself is considered as hemiterpene, but oxygen-containing
isoprene compounds like angelic acid extracted from Angelica archangelica, iso-
valeric acid isolated from Vaccinium myrtillus and prenol obtained from citrous
fruit, tomato, cranberry are hemiterpenoids (Fig. 10.21) (Seigler 1995; Perveen
2018).
10.5.2 Monoterpenes
Monoterpenes are made up of two isoprene units and 10 carbon backbone structure
having molecular formula C10H16. They are important constituents of essential oils
or volatile oils of plant origin. They are found in certain plant families, such as
Lamiaceae, Rutaceae, Apiaceae, and Pinaceae. Few compounds, like geraniol, are
almost universal and are available in minute quantity in the volatile excretions of
356 C. C. Kandar
the plants. They are divided into three groups (a) acyclic monoterpenes, e.g., citral,
geraniol, citronellal, Linalool (b) monocyclic monoterpenes, e.g., menthol, men-
thone, limonene, carvone, (c) bicyclic monoterpenes further grouped as (i) Chass I
or (6 + 3) membered ring systems: thujane type – a-thujene, sabinene and carane
type – carone, car-3-ene, car-2-ene (ii) Class II or (6 + 4) membered ring systems or
pinane type ring system: a-pinene, b-pinene, myrtenol, pinocarvone (iii) Class III or
(6 + 5) membered ring systems: camphor, camphene, borneol, isoborneol, fen-
chone (Fig. 10.22). Monoterpenes show a lot of pharmacological applications
(Table 10.5). The substances like camphor, as well as menthol are useful as
counterirritants, painkillers, and also act against skin infection like itching. They are
also used as anthelmintics. A group of monoterpene glycosides has reported
showing a vasodilation effect on coronary vessels, as well as the femoral vascular
bed (Kaur 2010, Lalonde 2005, Bergman 2019). The pharmaceutical uses of
monoterpenes are listed below in Table 10.5.
The chemical structures of different classes of monoterpenoids are provided
below in Fig. 10.22.
10.5.3 Sesquiterpenes
Sesquiterpenes composed of three isoprene units combined together in a head to tail

fashion and the molecular formula is C15H24. On the basis of biogenetic source,
about 200 various structural types of sesquiterpenes and many such compounds are
reported. Sesquiterpenes can be classified into four different classes in accordance
with their structure: (a) acyclic sesquiterpenes (e.g., a-farnesene, b-farnesene,
nerolidol), (b) monocyclic sesquiterpenes further subclassified as per basic skeleton
(i) Bisabolane type, e.g., zingiberene, b-bisabolene, (ii) Elemane type, e.g., abscisin
II, elemol (iii) Humulene type, e.g., humulene (iv) Germacrane type, e.g., germa-
crone aristolactone, (c) bicyclic sesquiterpenes are further subclassified into
(i) Cadinane type, e.g., a-cadinol, d-cadinolii) Eudesmane type, e.g., b- eudesmol,
santonin (iii) Perhydroazulene type, e.g., guaicol, aromadendrene (d) tricyclic
sesquiterpenes are further subclassified into (i) Cedrene type, e.g., cedrol, cedrene
(ii) Longifolene type, e.g., longifolene (Fig. 10.23) (Seigler 1995; Lalonde 2005;
Hikino 1985).
A group of sesquiterpene exhibit antimalarial, antibacterial, anthelmintic, anti-
fungal, anti-inflammatory, antirheumatic, adaptogen, flavoring agent, counterirri-
tant, and antiprotozoal activities (Table 10.6). The list of sesquiterpenes and their
uses are mentioned below in Table 10.6 (Kaur 2010; Lalonde 2005).
The chemical structure of sesquiterpenes is represented below in Fig. 10.23.
a) Acyclic monoterpenes:
CH3 CH3 CH3 CH3
OH
CHO CH2OH CHO
CH2
H3C H3C CH3 H3C CH3 H3C CH3

CH3
Citral Geraniol Citronellal Linalool
b) Monocyclic monoterpenes
CH3 CH3 CH3 CH3
O
OH O
H3C CH3 H3C CH3 H3C CH2 H3C CH2
Menthol Menthone Carvone Limonene

c) Bicyclic monoterpenes
i) Class –I type:
CH3 CH2 CH3 CH3
H3C CH3 H3C CH3

H3C CH3 H3C CH3
alpha-Thjene ( ) - Sabinene Car-2-ene Carone
ii) Class –II type:

CH3 CH2 Ch2OH CH2
O
alpha - Pinene beta - Pinene Myrtenol Pinocarvone

iii) Class –III type:
CH3 CH3 CH3

CH3
OH O
O
Camphor Camphene Borneol Fenchone
Fig. 10.22 The structures of different classes of monoterpenes of plant sources

358 C. C. Kandar
Table 10.5 The pharmaceutical uses of some important monoterpenes of plant sources
Sl. Name of the Uses
No drugs
1 Menthol Antipruritic, counterirritant, antiseptic, carminative, flavoring agent,
and stimulant
2 Thymol Antibacterial, and antifungal
3 Limonene Flavoring agent, stimulant, stomachic, and carminative
4 Camphor Rubifacient, counterirritant, antiseptic, antipruritic, carminative,
masking agent in perfumery industry, and insect repellant
5 Eugenol Analgesic in dental products, flavoring agent, stimulant, antiseptic,
and condiment in cooking
6 Methyl Counter-irritant, antirheumatic, antiseptic, and flavoring agent
salicylate
7 Cineole Analgesic in nasal inhaler and spray, antiseptic in mouthwash,
diaphoretic, and expectorant
8 Terpinol Stimulant and expectorant
10.5.4 Diterpenes
Diterpenes consist of four isoprene units combined together in a head to tail fashion
and the molecular formula is C20H32. Diterpenes can be divided into different
classes on basis of the number of rings present in the compounds such as (a) acyclic
diterpenes, e.g., phytol (b) monocyclic diterpenes, e.g., retinol (vitamin A) (c) tri-
cyclic diterpenes, e.g., abietic acid (d) tetracyclic diterpenes, e.g., gibberellins
(Fig. 10.24). Diterpenes contain several rings having various sizes. They may be
6-membered ring structures and also they may contain fused 5- and 7-membered
ring structures. Many diterpenes have also additional ring systems. The additional
ring is present in the side substitutions as esters or epoxides [14]. The phytocon-
stituents containing diterpenoids have great importance for medicinal values.
Phylloquinone, also known as Vitamin K1 having an antihemorrhagic property is a
diterpene that was first discovered in plants in 1929. Vitamin A1, chemically called
retinol is also another diterpenoid that is prepared from a tetraterpenoid, carotene
found in carrot. The bitter principles of Jateorhiza palmata (calumba root) are
under to furanoditerpenes. The diterpenes obtained from Teucrium chamaedrys
(wall germander) and T. scorodonia (wood sage) belonging to the family Labiatae,
both the products show diaphoretics and antirheumatics activity (Papanov et al.
1980). Like all other classes of terpenes, diterpenes have been reported to exhibit a
wide variety of pharmacological activities like as analgesic, antibacterial, antifun-
gal, anti-inflammatory, antineoplastic, antiprotozoal activities and for the treatment
of asthma, glaucoma, as well as congestive cardiomyopathy(Winkel-Shirley.2001).
Few diterpenes isolated from Kalmia latifolia belonging to the family Ericaceae
exhibit antifeedant activity. The gibberellins, plant hormones, first isolated from a
fungus belonging to genus Gibberella and also available in higher plants, are
a) Acyclic sesquiterpenes
CH3 CH3
CH3
HO
H3C H2C H3C
H3C CH3 H3C CH3

H3C CH3
alpha- Farnesene beta- Farnesene Nerodiol
b) Monocyclic sesquiterpenes
CH3 CH3 CH3 CH3
O
CH3
CH3
H3C
CH3
CH3
CH3 H3C OH CH3
H3C CH3
Zingiberene Elemol Humulene Germacrone
c) Bicyclic sesquiterpenes
H3C OH
H3C CH3 CH3

CH2 H3C OH H3C OH
CH3
H3C CH3
alpha- Cadinol beta- Eudesmol Guaicol
d)Tricyclic sesquiterpenes
H3C CH3
CH3 CH3
OH
CH2
CH3
Cedrol CH3 Longifolene

CH3
Fig. 10.23 The structure of various classes of sesquiterpenes derived from plant sources
tetracyclic diterpenoid used for the growth of seedlings in the agricultural field
(Evans 2009). The chemical structures of sesquiterpenes are provided below in
Fig. 10.24.
10.5.5 Sesterterpenes
The meaning of the term “sester” is half to three, i.e., two and a half. Sesterterpenes
having five isoprene units containing 25 carbon atoms are rarely available. Geranyl
farnesol, a sesterterpenoid, extracted from seeds of the plant, Camellia sasanqua
360 C. C. Kandar
Table 10.6 The pharmaceutical uses of some important sesquiterpenes

Sl.No Sesquiterpenes Pharmaceutical uses
1 Artimimisinin Antimalarial, antiprotozoal
2 Santonin Anthelmintic
3 Gossypol Male antifertility activity, insecticidal, used in menorrhoea
4 Zingiberene Antibacterial, antiulcer. antifungal
5 Helenalin Cardiotonic
6 Farnesene Antibacterial, anti-inflammatory
7 Guaiol Anti-rheumatic, food additives
8 Cedrol adaptogen
9 Vetivone Treatment of burn, sore and stimulant and diaphoretic
10 Caryophyllene Antiseptic, counterirritant, carminative
called sasanqua and another plant, Camellia japonica called as camellia both
belonging to the family Theaceae exhibited cytotoxic activity in mouse leukemic
M1 cells(Akihisa et al. 1999; Ishikura et al. 1984). Another sesterterpene, dysidi-
olide was isolated from the Caribbean sponge. Ophiobolin, a cytotoxic agent, is
produced through various diverse cyclization (Au et al. 2000).
CH3
O H3C CH3
O
O
CH3
OH
H3C Ophiobolin
10.5.6 Triterpenes
Triterpenes having six isoprene units containing 30 carbon atoms are found
abundantly in nature. Triterpenes may be linear, tetracyclic, or pentacyclic. The
linear triterpene, squalene, derived from the coupling of two molecules of farnesyl
pyrophosphate by mevalonic acid pathway is found in the animal source, e.g., shark
liver oil and plant sources, e.g., olive oil, arachis oil. Triterpenes are mostly lipid
substances of all plants and about 4000 triterpenoids have been identified and
extracted. These compounds are required to prepare animal steroids, as well as plant
steroids. Both triterpenes and steroids (tetracyclic triterpenoids) exist as a free
a) Acyclic diterpenes
CH3 CH3 CH3
CH3
CH2OH
H3C
Phytol
b)Monocyclic diterpenes
CH3 CH3
CH3
CH2OH
CH3
Vitamin A 1 (Retinol)
CH3
c)Tricyclic diterpenes
CH3
CH3 CH3
H3C COOH
Abietic acid
d)Tetracyclic diterpenes
OC
OH
H3C
CH3 CH2
COOH
Gibberellic acid ( GA 3)
Fig. 10.24 The structures of lignan derivatives derived from plant sources
molecule, as glycosides form or in other combinations (Seigler 1995). The

oleo-gum-resin of Boswellia carterii contains two types of triterpenoids namely,
one is b-Boswellic acids (ursane-type triterpene) and anther one is a-boswellic
acids (oleanane-type triterpene). These compounds exhibit anti-inflammatory, as
well as antirheumatic properties. Limonoids are tetracyclic triterpenoids but triter-
penoid saponins are pentacyclic triterpenoids. An example of medicinally important
triterpenoid is cardiac glycosides or steroid glycosides used as cardiotonic by
increasing the force of systolic contraction in congestive cardiac failure (CCF).
Another important class of compounds, quassinoids is isolated from Quassia
amara. Quassinoids are compounds having less than 30 carbons and are
362 C. C. Kandar
synthesized by the degradation of triterpenoids along with the rearrangement of

degradable products. Quassia has great biological importance and is used as an
insecticide, as a bitter tonic and as anthelmintic for the expulsion of threadworms
from the intestine (Culioli et al. 2003).
Squalene
10.5.7 Sesquarterpenes
Sesquarterpenes contain seven basic units, i.e., isoprene (C5H8) having molecular
formula C35H56. They are actually diterpene-sesquiterpene derivatives.
Plagiospirolide A, B, C, D are obtained from the liverwort of Plagiochila mor-
itziana. Ferrugicadiol and ferrugieudesmol are obtained from the bark of
Calocedrus macrolepis. C-35 terpene having pentacyclic structure is obtained
biosynthetically from Bacillus subtilis via cyclization with the help of an enzyme.
H3C CH3
CH3
CH3
CH3 CH3
CH3
H3C CH3
Sesquarterpene obtained from Bacillus subtilis
10.5.8 Tetraterpenes
Tetraterpenes are 40 carbon (C40) containing compounds. In this class, the

medicinally important compounds are well-known carotenoids. They are colored
substances usually red, yellow, or orange in color. They are used as food colorants
over synthetic color due to their non-toxic and stable nature. The coloring agents
are used as pharmaceutical aids. The examples of carotenoids are b-carotene,
capsanthin, capsorubin, lycopene, zeaxanthin, lutein, etc. (Lalonde. 2005).
H3C
CH3 CH3
H3C CH3
H3C CH3
CH3 beta - Carotene CH3 CH3
10.5.9 Polyterpenes
Polyterpenes consist of more than eight isoprene units. They are available in the
plants belonging to the family Euphorbiaceae, Moraceae, Apocynaceae, and
Asclepiadaceae.
Pure rubber chemically polymer of cis form of isoprene is obtained from Hevea
brasiliensis belonging to the family Euphorbiaceae. Gutta-percha is chemically a
polymer of trans form of isoprene (Lalonde 2005).
CH3 CH3 CH3
H3C CH3
n
Structure of natural rubber
Terpenoids are a large group of secondary metabolites having various classes of

compounds. They are biosynthesized via mevalonic acid pathway from simple
compound, acetyl CoA (Fig. 10.25).
10.6 Carbohydrates
Carbohydrates are ubiquitously found in living beings on the Earth. Carbohydrates

are sugars, starches, and fibers that are available in grains, vegetables, and milk
products. Carbohydrate, e.g., glucose is the first product manufactured by photo-
synthesis from atmospheric carbon dioxide (CO2) and water from the soil in the
presence of sunlight with the help of chlorophyll, a green pigment. Glucose is the
beginning material for the preparation of all phytoconstituents and also, for the
preparation of all animal biochemicals. More carbohydrates occur in nature. The
abundance of carbohydrates is maximum among all other kinds of natural products.
Cellulose, a polymer of glucose combined by b-glycosidic linkage is the principal
structural component of plants and has taken top-ranked abundance as a single
364 C. C. Kandar
Acetyl CoA (C 2) Acetoacetyl CoA (C4) HMG CoA (C6)
DMAPP (C5) IPP (C 5) Mevalonic acid (C 6)
GPP (C 10) Monoterpenoids (C10)
Isopentenyl pyrophosphate Sesquiterpenes (C15)
C30 PP Squalene (C30)

FPP (C 15)
Steroids
Isopentenyl pyrophosphate
Diterpenoids (C20)
GGPP (C 20) Carotenoids (C40)
Polyterpenoids (C >40)
Isopentenyl pyrophosphate
GFPP (C 25) Sesterterpenoids (C25)
HMG CoA Hydroxy methyl glutaryl CoA IPP Isopentenylpyrophosphate GPP Geranyl pyrophosphate
DMAPP Dmethylallyl pyrophosphate FPP Farnesyl pyrophosphate GGPP Geranylgeranyl pyrophosphate
GFPP Geranylfarsenyl pyrophosphate
Fig. 10.25 Flow chart of the biosynthetic pathway of terpenes (Kokate et al. 2005; Lalonde 2005)
organic substance. Although carbohydrates are primary metabolites, they become

part of the huge number of secondary metabolites through glycosidation linkages.
Glycosides consist of a sugar part known as glycone. The glycone sugars are mainly
glucose, galactose, rhamnose, mannose, digitoxose, cymose, etc. Mucilages and
gums are the polymeric product of simple sugars and uronic acids (Asif et al. 2011).
Carbohydrates can be defined as carbon, hydrogen, and oxygen-containing
polyhydroxy aldehydes or ketones that on hydrolysis produce simple sugars. They
are classified into monosaccharides, disaccharides, oligosaccharides (trisaccharides,
tetrasaccharides, etc.), and polysaccharides. Carbohydrates are classified based on
their presence of saccharide units as follows:
Carbohydrates
Monosaccharides Disaccharides Trisaccharides Tetrasaccharides Polysaccharides
Homo-polysaccharides Hetero-polysaccharides
Depending upon reducing property, they are divided into two groups such as
(a) reducing sugars, e.g., all monosaccharides (e.g., glucose, fructose, galactose,
mannose, gulose, sorbose, etc.) and disaccharides (e.g., maltose, lactose) except
sucrose
(b) non-reducing sugars, e.g., oligosaccharides, polysaccharides.
Depending upon the hydrolytic product, they are grouped as pentosan (e.g.,
xylan) and hexosan (e.g., starch, cellulose, and inulin).
Cellulose consists of glucose units combined by b-1,4-glycosidic linkages which
are hydrolyzable by cellulase enzyme available in herbivorous animals or cattle
class.
Monosaccharides have three-carbon atoms to nine-carbon atoms and accord-
ingly, they are known as triose, tetrose, pentose, hexose, heptose, etc. The simplest
carbohydrate is glyceraldehydes containing three-carbon atoms. But those
monosaccharides having five carbon atoms like pentoses, C5H10O5, and six carbon
atoms like hexoses, C6H12O6 ( Fig. 10.26), are collected in plants in the largest
amount (Kokateet al. 2005, Morrison et al. 2004).
Depending upon the presence of functional groups (aldehyde or ketone), they are
divided into aldose sugar and ketose sugar. For example, glucose is aldohexose,
whereas fructose is ketohexose.
Gum, mucilage, pectin, guaran, tragacanthin, and alginic acid are the important
polysaccharide derivative having pharmaceutical values. They are used as emul-
sifying agents, binding agents, suspending agents, thickening agents, adsorbent,
laxatives, and pharmaceutical aids. Gums are pathological products of poly-
uronides, on hydrolysis they produce the mixtures of uronic acids and sugars.
Whereas mucilages are the physiological product mainly of sulphuric acid esters.
OH H OH
OH
O OH OH
H OH H HO
H H OH H
OH H H OH H HH H
HO OH OH OH HO OH H OH
HO OH H OH OH OH
H OH H H
alpha-D-Glucopyranose alpha-D-Mannopyranose alpha-D-Sorbopyranose alpha-D-Gulopyranose
Fig. 10.26 The structure of various hexose monosaccharides

366 C. C. Kandar
On decomposition, cellulose produces gum and mucilage. Pectin, methoxy ester of

aldobionic acid and pectic acid, is obtained from the inner portion of the skin of
citrus fruits, pectins are polyuronides and made of pectic substances such as pec-
tinic acid, protopectin, and calcium pectate. Another viscous sticky material known
as mucilage obtained from maximum plants protect the plant by thickening the
membranes in plants. It helps to store water and food and also helps in the ger-
mination of seed. It is chemically a polar glycoprotein and an exo-polysaccharide.
Mucilage mainly acts as a demulcent. The important sources of mucilage are Cactus
(and other succulents) and L. usitatissimum (flax seeds). The extract of the
mucilaginous root of the marshmallow plant exhibits a demulcent effect and hence
used as a cough suppressant in respiratory tract infection. When mucilage comes in
direct contact with the mucous membrane surface, it acts as an emollient (Kokate
et al. 2005; Khowala et al. 2008).
The water-soluble portion approximately 85% of guar gum obtained from the
powder of the endosperm of Cyamopsis tetragonolobus is called guaran. On
hydrolysis, it gives 65% of galactose and 35% of mannose. Honey, a saturated
solution of sugar, deposited in the honeycomb by Apis mellifera (bees) is used as a
demulcent, antiseptic to burns and wounds, and also as a sweetening agent.
Tragacanth, obtained by incision from branches of Astragalus gummifer belonging
to the family Leguminosae, has pharmaceutical importance as a suspending agent,
the binding agent in tablets, emollient in cosmetics. Chitin is also a polysaccharide
derivative having acetyl and amino groups and are found in skeletal of inverte-
brates. Chitin is employed in wound healing products and is used in water treatment
plants. Agar, also known as vegetable gelatin, obtained from Gelidium amansii has
two polysaccharides such as agarose and agaropectin. Agarose provides gel strength
of the agar, whereas agaropectin of agar provides viscosity when added to the
solution. Carrageenan, a sulphated polysaccharide, extracted from seaweed (car-
rageen) is used as an emulsifying agent and gelling agent. Carrageenan is useful as
an experimental tool to produce inflammation in the rat paw edema model of the
anti-inflammatory test. Inulin is a polysaccharide which is obtained from the bulbs
of dahlia, Inula helenium. Inulin is chemically 35–50, 1, 2-linked fructofuranose
units, terminated by one glucose unit. Dextrin, an incomplete hydrolytic product of
starch with dilute mineral acids, has pharmaceutical value as tablet excipients.
Whereas dextran is another polysaccharide that is generated by growing bacteria on
sucrose solution. Dextran is a chemical polymer of D-glucose linked by a (1,6)
glycosidic linkage (Kokate et al. 2005; Anbalahan 2017).
10.7 Lipids (Fixed Oil, Fats, Waxes and Phospholipids)
Lipids consist of a large number of natural products such as fixed oils (triglyc-
erides), waxes (ester of long-chain fatty acid and long-chain alcohol), volatile oils,
steroids (having cyclopentanoperhydrophenanthrene ring), fat-soluble vitamins,
important ingredients of foods like vit. A, vit. D, vit. E, vit. K, phospholipids, and
LIPIDS
Simple lipid
Compound lipid Derived lipid
Fats & Oils Waxes
Phospholiipids Glycolipids Lipoproteins
Steroids Terpenes Carotenoids

Vit. D Lycopene
Bile acids
or isoprenes Xanthophill Fatty acids
Sex hormones
Saturated Unsaturated
Poly unsaturated Mono unsaturated
Leucotrienes Thromboxanes Prostaglandins Prostacyclins
Fig. 10.27 The classification of lipids
other compounds (Fig. 10.27). They exhibit various biological properties such as
the main structural components of all biological membranes which contain
lipid-protein lipid layer, as energy reservoirs that provide 9.3 kcal. per mole of fat,
as fuel for cellular functions along with other food components such as vitamins
and as coordinating compounds, hormones (Fahy et al. 2009; Subramaniam et al.
2011). Lipids are regarded as primary plant metabolites, but lipids have been
reported to exhibit several pharmacological actions, and therefore, they considered
as phytoconstituents. Lipids are classified as follows on the basis of their structure
(Fig. 10.27).
10.7.1 Fixed Oils
Fixed oils obtained from plant sources are available in the seed of the plants. Fats
and oil are esters of glycerol and long-chain fatty acids. Fatty acids can be divided
into two groups such as saturated fatty acids in which long-chain aliphatic acid
368 C. C. Kandar
contains no double bond and unsaturated fatty acids which contains one or more
double bonds. High molecular saturated long-chain fatty acids include palmitic,
stearic, arachidic lignoceric acids, whereas unsaturated long-chain fatty acids are
oleic acids, linoleic, linolenic, and erucic acids. Fixed oils are liquid in nature at
room temperature and have a comparatively high percentage of glycerides con-
taining polyunsaturated fatty acids such as glycerin oleate, whereas fats are solid in
nature at room temperature and contain glycerides of saturated fatty acids such as
glycerin stearate (Fahyet al. 2009). Fixed oils show a lot of pharmaceutical
importance. Arachis oil obtained by expression of the seed kernel of Arachis
hypogea is used as a solvent for intramuscular injection, for the preparation of
liniment and soap. Castor oil obtained by cold expression of seeds of Ricinus
communis contains ricinoleic acid which exerts laxative property due to its irritant
action. Chaulmoogra oil is another fixed oil obtained by cold compression of ripe
seeds of Hydrocarpus anthelmintic. Chaulmoogra oil is used for the treatment of
leprosy due to its strong bactericidal activity. Linseed oil obtained by compression
of seeds of L. usitatissimum, family Linaceae, is recommended for external
preparation as lotions, liniments. Polyunsaturated fatty acids (PUFA) present in few
fixed oils can cause the reduction of excretion of lipid peroxidation products and
hence they are recommended as good antioxidant potential and also act against
inflammation agents. Polyunsaturated fatty acids are generally used to reduce the
risk of atherosclerosis disease and for the treatment of other cardiovascular diseases
(Kokate et al. 2005; Wallis 2005). The hydrolytic products of glyceryl stearo oleo
linolein, a fat, is shown as follows:
H
H O-OC(CH2)16CH3 On hydrolysis
Glycerol + Stearic acid +
H O-OC-(CH2)7-CH CH(CH2)7CH3
Oleic acid + Linoleic acid
H O-OC-(CH2)7-CH CHCH2 CH CH (CH2)4 CH3
H
Glyceryl stearo oleo linolein (Fat)
10.7.2 Waxes
Waxes are fusible, oily, viscous solid substances with a waxy luster. They are ester
of long-chain monohydric alcohols with long-chain fatty acids. High molecular
weight monohydric alcohols include cetyl alcohol, cholesterol, and melissyl alco-
hol. Natural waxes consist of unsaturated bonds and several kinds of functional
groups like as primary and secondary alcohol, aldehydes, ketones, aromatic com-
pounds, and ester of fatty acids, whereas synthetic waxes contain aliphatic hydro-
carbons having no functional groups. Waxes are comparatively more resistant to
saponification than oils and fats. They are available from plant and animal sources.
The vegetable waxes include bayberry wax, seasal wax, carnauba wax, etc.,
whereas animal waxes include beeswax, wool, spermaceti wax. The vegetable
waxes contain more amount of the mixture of unesterified hydrocarbons over
esterified hydrocarbons. The composition of wax depends upon the species of the
plants, as well as the geographical location of the plants (Baker 1982). Jojoba wax,
a mixture of liquid wax obtained from seeds of Simmondsia chinensis, consists of
ester of C-18, C-20, C-22, and C-24 carbon chain monounsaturated acids with
alcohols (Wilhelm et al. 2012). Jojoba wax shows various pharmacological activ-
ities such as anti-aging, wound healing activities, as well as anti-inflammatory
property, and it is also used in several skin diseases due to wound healing property.
Jojoba wax has pharmaceutical applications in the preparation of topical medica-
ments to increase the absorption of drug molecules, i.e., to be a good carrier in
dermatological products. It is also used in cosmetics products such as sunscreens
and moisturizers. Carnauba wax, the hardest wax, is the exudates of leaves of the
Brazilian palm tree, Copernicia prunifera, and Copernicia cerifera belonging to the
family Palmae. Carnauba wax consists of carnaubic acid, cerotic acid, and melissyl
cerotate. It is useful for the preparation of cosmetic products and deodorant sticks. It
is applied as automobile wax and high-quality shoe polish. It is employed for the
coating of tablets (Kokate et al. 2005; Wallis 2005).
10.7.3 Phospholipids
Lecithin, an important phospholipid, is obtained from soyabean oil and also

available in vegetable seeds and corn. Lecithin consists of glycerin, phosphoric
acid, choline, and fatty acids. Generally, a saturated fatty acid is present at a-
position of lecithin, and an unsaturated fatty acid is present at b-position of it.
Lecithin is combined with protein to form lipoproteins of cells and plasma. It has
great importance to transport and utilization of fats, and therefore, it prevents the
accumulation of fat in the liver, i.e., fatty liver. It decreases the surface tension of
lung alveoli and so becomes easier to expel the liquid cough. It is industrially useful
as a lubricant for the textile and petroleum industry (Kokate et al. 2005; Wallis
2005).
O
H2C O R1
O
HC O R2
O
P CH3
H2C O O
+
N
OH CH3
Lecithin CH3
370 C. C. Kandar
10.7.4 Fatty Acids
Fatty acids are also found in plant origin. Evening primrose oil, obtained from dried
seeds of Oenothera biennis, contains 9% unsaturated fatty acid called c-linoleic
acid. It is useful as a prostaglandin precursor and to control the severity of the
premenstrual syndrome and for the treatment of eczema (Kokate et al. 2005; Wallis
2005). The sources and structures of saturated fatty acids, as well as unsaturated
fatty acids derived from plants, are depicted as follows (Tables 10.7 and 10.8):
10.8 Glycosides
Glycosides are widely available in plant kingdom throughout the world and become
the largest group of secondary plant metabolites. Glycosides contain a sugar part
called as glycone and a non-sugar part known as aglycone. Chemically glycosides
are acetal which is formed by condensation between the hydroxyl group of glycone
(exists as hemiacetal) and another hydroxyl group of aglycone part. The sugar part
attached to glycosides is either a monosaccharide such as glucose, mannose,
rhamnose, etc., or deoxy sugar such as cymarose, digitoxose, etc. One or more
number of monosaccharides molecules are attached to the aglycone moiety.
Aglycone moiety includes a wide variety of chemical classes such as anthraqui-
none, steroid, coumarin, chromone, cyanogenic, flavonoid, phenolic, saponins,
aldehyde, etc., which is discussed above of this chapter. On acid or enzymatic
Table 10.7 The sources and structures of saturated fatty acids derived from plants
Saturated fatty acid (IUPAC name) Natural source Chemical structure
Butyric acid (Butanoic acid) Butter fat, Cow’s milk CH3 ðCH2 Þ2 COOH
Caproic acid (Hexanoic acid) Palm kernel oil, butter CH3 ðCH2 Þ4 COOH
Caprylic acid (Octanoic acid) Coconut oil, palm oil CH3 ðCH2 Þ6 COOH
Capric acid (Decanoic acid) Palm oil, coconut oil, milk CH3 ðCH2 Þ8 COOH
Lauric acid Dodecanoic acid) Coconut oil, palm oil CH3 ðCH2 Þ10 COOH
Myristic acid (Tetradecanoic acid) Palm oil, milk fat CH3 ðCH2 Þ12 COOH
Palmitic acid (Hexadecanoic acid) Sesame oil, Arachis oil CH3 ðCH2 Þ14 COOH
Stearic acid (Octadecanoic acid Arachis oil, Sesame oil CH3 ðCH2 Þ16 COOH
Arachidic acid (Eicosanoic acid) Peanut oil, Mustard oil CH3 ðCH2 Þ18 COOH
Behenic acid (Docosanoic acid) Peanut oil, Rapeseed oil CH3 ðCH2 Þ20 COOH
Lignoceric acid (Tetracosanoic acid) Peanut oil CH3 ðCH2 Þ22 COOH
Cerotic acid (Hexacosanoic acid) Bees wax, Carnauba wax CH3 ðCH2 Þ24 COOH
Montanic acid (Octacosanoic acid) Fruit skin, bees wax CH3 ðCH2 Þ26 COOH
Melissic acid (Triacontanoic acid) Cotton wax CH3 ðCH2 Þ28 COOH
Table 10.8 The sources and structures of unsaturated fatty acids derived from plants
Unsaturated Natural source Chemical structure
fatty acid
Palmitoleic Cotton seed oil CH3 ðCH2 Þ5 CH ¼ CHðCH2 Þ7 COOH
acid
Oleic acid Corn oil, Safflower CH3 ðCH2 Þ7 CH ¼ CHðCH2 Þ7 COOH
oil
Linoleic acid Sun flower and CH3 ðCH2 Þ4 CH ¼ CH CH2 CH ¼ CHðCH2 Þ7 COOH
sesame oil
Erucic acid Rapeseed oil CH3 ðCH2 Þ7 CH ¼ CHðCH2 Þ11 COOH
Ricinoleic acid Castor oil CH3 ðCH2 Þ5 CH(OH) CH 2 CH ¼ CHðCH2 Þ7 COOH
Chaulmoogric Chaulmoogra oil
(CH2)12 COOH
acid
hydrolysis, glycosides provide glycone or sugar part and aglycone or non-sugar part
(Kaur 2010, Cheeke 2001).
Glycone part is responsible to show the solubility character of the glycosides
whereas the aglycone part shows the therapeutic activity. Glycosides exhibit.
Several varieties of pharmacological activities such as purgative, antidepressant,
cardiotonic, nervine tonic, demulcent, sedative, counterirritant, rubefacient, hep-
atoprotective, diuretic, antifungal, etc.
On the basis of the chemical nature of the aglycone part, glycosides are divided
as follows:
1. Anthraquinone glycosides, e.g., aloe leaves, rhubarb
2. Steroidal glycosides or cardiac glycosides, e.g., digitalis leaves, squill
3. Coumarin glycosides, e.g. cantharides, psoralea
4. Chromone glycosides, e.g., hypericum
5. Cyanogenic glycosides, e.g., bitter almond, wild cherry bark
6. Flavonoid glycosides, e.g., silymarin, ginkgo
7. Phenolic glycosides, e.g., bearberry
8. Saponin glycosides, e.g., Ginseng, dioscorea, gokhru, licorice
9. Aldehyde glycosides, e.g., vanilla pods
10. Steviol glycosides, e.g., stevia
11. Iridoid glycosides, e.g., nux vomica seed
12. Thioglycosides, e.g., black mustard
13. Steroidal glycol-alkaloids, e.g., solanum.
The biological sources, chemical structure and pharmacological activities of
various kinds of glycosides (Table 10.9), are given below (Kokate et al. 2005;
Hossain et al. 2019).
372 C. C. Kandar
Table 10.9 The biological sources, structure, and uses of various kinds of glycosides
Type of glycosides Biological Chemical Structure Therapeutic uses
source
1. Anthraquinone Dried leaflets of C6H11O 5O O OH Laxatives,
glycosides e.g., Cassia purgative
Sennoside A and B angustifolia COOH
COOH
C6H11O 5O O OH
Sennoside A
2. Steroidal Dried leaves of O

O
Cardiotonic,
H3C
glycosides or Digitalis CH3 cardiac stimulant
CH3
cardiac glycosides purpurea
e.g., digoxin OH
(Digitoxose)3
H Digoxin
3.Coumarin Ripe fruits of HO O O Anthelmintic,

glycosides Psoralea laxative, treatment
H3C
e.g., psoralidin corylifolia of leucoderma,
H3C
O leprosy
Psoralidin CH3
4. Chromone Whole plant of CH3 CH2 Anti- H pyroli

glycosides e.g., Hypericum activity
O
hyperimone erectum
HO O CH3
C6H11O 6
Hyperimone
5. Cyanogenic Ripe seeds of O-C6H10O 4-O-C6HH11O 5 Sedative,

glycosides e.g., Prunus demulcent skin
CN
amygdalin amygdalus lotion
Amygdalin
6. Flavonoid Ripe seeds of O Hepatoprotective,
OH
glycosides e.g., Silybum HO O OCH3
antioxidant
O
silybin marianum
OH OH
OH O Silybin
7.Phenolic Dried leaves of OH Diuretic, astringent

glycosides e.g., Arctostaphylous
arbutin uva-ursi
O-Glu
Arbutin
8. Saponin Dried tuber of CH3 Precursor of

CH3 CH3
glycosides e.g., Dioscorea steroids, treatment
OO
diosgenin deltoidea CH3 of rheumatic
arthritis
HO
Diosgenin
9.Steroidal Dried berries of H3C Sex hormone, oral

CH3
glycol-alkaloids Solanum CH3
O
NH contraceptive
e.g., solasodine khasianum CH3
Solasodine
(continued)

Type of glycosides Biological Chemical Structure Therapeutic uses
source
10. Aldehyde Unripe fruits of CHO Flavoring agent
glycosides e.g., Vanilla
vanillin planifolia HO
OCH3
Vanillin
11. Steviol Leaves of Stevia O 5H11C6-O-O 4H10C6-O Sweetening agent

glycosides rebaudiana H3C
CH2
H3C CH3
e.g., stevioside
H
O 5H11C6-O H
CH3
Stevioside
CH2
12. Iridoid Seeds of HO Neuroprotective

HO
glycosides Strychnos O and
H
e.g., loganin nuxvomica HO O
anti-inflammatory
H CH3
OH
O
properties
OH
Loganin H
COOCH3
13. Isothiocyanate Ripe seeds of S-C6H11O 5 Emetic,

glycosides e.g., Brassica juncea H2C counterirritant,
sinigrin N O-SO 3K rubifacient
Sinigrin
14, Alcohol Bark of Salix CH2OH Analgesic,
glycoside e.g., babylonica O-Glu antipyretic
salicin (Willow) anti-inflammatory
Salicin
10.9 Conclusion
As the plant can be considered as a storehouse of the various kinds of secondary

metabolites and the production of secondary substances of plant origin fully
depends upon the selectivity of the species of a family, i.e., the plants belonging to a
particular family provide most specific types phytoconstituents. A huge number of
different classes of plants and various types of phytoconstituents are found
worldwide but only very few of them are identified, extracted, isolated, elucidated
for the chemical structure, and evaluated for their pharmacological activity.
To overcome this, the knowledge regarding various fields of science such as
Botany, Organic Chemistry, Biochemistry, Analytical Chemistry, and
Pharmacology is needed. A plant extract contains several phytoconstituents
showing various biological activities due to complexity of the secondary metabo-
lites in which a component may synergic or counteract the activity, and therefore,
specific research or study for gathering more information of the phytoconstituents
to develop a new drug or a new combination of drugs having less toxic and more
potent is highly essential. Since the chapter summarized the description and
374 C. C. Kandar
structure of various secondary plant metabolites, their biosynthesis and their

pharmacological activities may be considered as an informative tool for the
development and research purpose of phytoconstituents. Secondary metabolites are
available in much smaller quantities than primary metabolites and are very much
expensive to produce or extract them. In spite of different difficulties, the herbalist
and scientists are giving much effort to discover new molecules for relief and cure
of various diseases.
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Chapter 11
Pharmaceutical and Therapeutic
Applications of Fenugreek Gum
Purusottam Mishra, Amit Kumar Srivastava, Tara Chand Yadav,

Vikas Pruthi, and Ramasare Prasad
Abstract Plant-derived gums play an essential role in the biopharmaceutical field

due to their advantageous physicochemical properties, nutritional value, ease of
availability and biodegradability. Fenugreek gum extracted from the seeds consists
of galactomannan with galactose to mannose ratio as one where higher number of
galactose enhances the water solubility of fenugreek gum. Exciting contemporary
research shows enormous possibilities of fenugreek gum in biopharmaceutical
landscape as a natural excipient and therapeutic agent. Since last decade, fenugreek
gum has been explored as retarding, mucoadhesive and disintegrating agent for
various biopharmaceutical applications. Moreover, seed extract of fenugreek gum
has been extensively studied as an antidiabetic, anti-carcinogenic, anti-inflammatory
and hepatoprotective agent. This review aims to highlight the applications of fenu-
greek gum as a natural pharmaceutical excipient and its extensive role in drug
delivery systems. Further, physicochemical properties, toxicological aspects and
therapeutic efficacy of fenugreek gum has been also discussed.
Keywords Antidiabetic Exicipient Fenugreek gum Galactomannan

Mucilage
11.1 Introduction
Since the last few decades, we have witnessed a substantial development in the field
of drug delivery using a diverse range of excipients (Raghuwanshi et al. 2017).
These excipients have been extensively used in the conventional dosage forms for
their multiple pharmaceutical purposes such as glidant, binder, sweetner and
thickening agent (Rowe et al. 2009), which may alter the physicochemical properties
P. Mishra A. K. Srivastava T. C. Yadav V. Pruthi R. Prasad (&)

Department of Biotechnology, Indian Institute of Technology Roorkee,
Roorkee 247667, Uttarakhand, India
e-mail: ramasare.prasad@bt.iitr.ac.in
https://doi.org/10.1007/978-3-030-54027-2_11
380 P. Mishra et al.
of the final formulation. Besides this, they facilitate the regulation of pharmacody-
namic and pharmacokinetic properties of the drug in the physiological system. As
per the conventional hypothesis, excipients used in the pharmaceutical preparations
do not have a prime role in the treatment of disease and are thought to be inert as
well. Till date several polymers have been used as excipients for the development of
polymer-based drug delivery system with the aim of targeted delivery of the active
therapeutics to the specific tissues (Ulbrich et al. 2016). Polymers can play a vital
role in disease management as well as they can also modulate the behaviour of the
drug in the physiochemical system. Although significant research has been done to
investigate the mechanistic behaviour of these polymers when used alone or as a
polymer-drug conjugate for the purpose of drug delivery. However, evaluation of the
physicochemical properties of the polymer prior to pharmaceutical application is a
task of priority (Singh and Pai 2015). A non-toxic, biocompatible and biodegradable
polymer is always a need for a biologically safe and effective pharmaceutical dosage
form. In recent years, demand of natural polymers has taken a quantum leap in
pharmaceutical, food and cosmetic industries than its synthetic counterpart (Zia et al.
2017). Synthetic polymers are used for drug delivery and development of biomedical
devices and implants. Though synthetic polymers exhibit high chemical, physical
and mechanical stability as well as flexibility to bind with diverse range of thera-
peutics, bio-incompatibility and cellular toxicity illustrated by them are always a
concern for drug delivery purpose (Nair and Laurencin 2007). Natural polymers
such as polysaccharides and proteins have been extensively used to develop
numerous biomedicines as they possess mighty biocompatible, biodegradable and
therapeutic properties. Moreover, natural polymers are more also easily assessable
and lower in cost compared to their synthetic counterparts (Li et al. 2015). These
natural polymers based drug delivery systems possess several advantages such as
high drug pay load, presence of diverse surface functional groups for drug binding
and low toxicity (O’Elzoghby et al. 2016).
11.1.1 Natural Gums
Gums are complex hydrocolloid biopolymers composed of polysaccharide con-

sisting of one or more monosaccharides or their derivatives linked with chemical
linkages (Prajapati et al. 2013a, b). Natural gums can be classified on the basis of
their origin, e.g. marine origin (alginic acid, agar and carrageenans), animal origin
(chitin), microbial origin (gellan gum, xanthum gum and lentinan) and plant origin
(gum tragacanth, guar gum and cellulose) (Prajapati et al. 2013a, b; Choudhary and
Pawar 2014). Natural gums produced by higher plants are consequence of injury or
can be obtained from exudates of different parts of the plant. Easy availability,
non-toxicity, cost effectiveness and biocompatibility are some of the advantages of
natural gums which makes them a better alternative than their synthetic counterparts
11 Pharmaceutical and Therapeutic Applications of Fenugreek Gum 381
(Prajapati et al. 2013a, b). Complicated macromolecular architecture and water

solubility offers natural gums adhesiveness and cohesiveness which render natural
gums a biopolymer of choice to be used as stabilizing agent, gelling agent, binder,
disintegrant and suspending agent in the pharmaceutical preparations (Prajapati
et al. 2013a, b). Gums tend to work efficiently at a higher concentration in the drug
delivery systems and high swellability possesses challenges. Apart from these
above described versatile properties of natural gums, certain drawbacks associated
with them are environmental and microbial contamination during procurement and
storage, loss of viscosity on long-term storage and high degree of hydration
capacity. Chemical modification and copolymer grafting are the two major methods
which are followed to neutralize the above-mentioned issues with natural gums
(Prajapati et al. 2013a, b).
Various plant-derived gums such as guar gum, locust bean gum, acacia gum and
gum karaya are used as natural excipient and therapeutic agent for application in
diverse drug delivery systems (Castro et al. 2018; Prajapati et al. 2014; Singh
2018). Fenugreek gum hosts enormous biopharmaceutical possibilities as an
excipient and therapeutic agent due its unique physiochemical properties such as
low cytotoxicity, water solubility and biocompatibility like other natural
plant-derived gums. This article attempts to discuss various physiochemical prop-
erties and biosafety issues of fenugreek gum which play a critical role in selection
of a polymer as an excipient to develop a formulation. The latest developments in
drug discovery using fenugreek gum are surveyed to illustrate areas of research
advancing the frontiers of biomedicine.
11.1.2 Fenugreek Gum
Fenugreek (Trigonella foenum-graecum) belongs to family Leguminosae

(Fabaceae) and known as one of the oldest medicinal plant cultivated around the
globe (Goyal et al. 2016; Ouzir et al. 2016). Both leaves and seeds of fenugreek
have been traditionally used as flavuor enhancers in foods and medicinal products.
Seeds of fenugreek contain a gummy substance, i.e. mucilage, and consists of
galactomannan which is a polysaccharide (Sindhu et al. 2012). Apparently,
galactomannan of various gums differs in the physiochemical properties as the ratio
of mannose to galactose varies. The higher number of galactose in fenugreek gum
reduces this ratio to approximately one as a result, the water dissolving capacity
increases (Iurian et al. 2017). Due to the high water solubility of fenugreek gum
(Doyle et al. 2009; Kamble et al. 2013) numerous properties such as disintegrating
ability (Kumar et al. 2009), suspending capacity (Nayak et al. 2013a, b),
mucoadhesiveness (Nayak et al. 2013a, b; Nayak and Pal 2014) have been inves-
tigated extensively.
11.1.3 Chemical Composition of Fenugreek Seed
Fenugreek seeds possess a variety of phytochemicals present therein including

alkaloids, polyphenols, saponins, carbohydrates, proteins, fibres, amino acids and
lipids, (El Nasri and El Tinay 2007; Kakani and Anwer 2012). Potassium and
manganese are known to be the major metals present in seeds of fenugreek (Kakani
and Anwer 2012). Lipid composition of fenugreek seeds comprise natural lipids
(85%), phospholipid (10%) and glycolipid (5%) while its fatty acid profile is
dominated by unsaturated fatty acid such as oleic acid and linoleic acid (Kakani and
Anwer 2012). Diosgenin, gitogenin, yamogenin, rigogenin, tigogenin and neori-
gogenin are the major saponins present in fenugreek seeds (Kakani and Anwer
2012; Dawidar et al. 1973). Sesquiterpenes, n-alkanes and non-alactone are the
reasons behind the aroma of the fenugreek seeds (Kakani and Anwer 2012).
Gentianine, carpaine and trigonelline are the major alkaloids present in the endo-
sperm of fenugreek seeds (Garg 2016). Besides all these phytochemicals, galac-
tomannan extracted from the seeds of fenugreek is also counted as a major bioactive
component. The detailed chemical constituents of fenugreek seeds are listed in
Table 11.1.
Table 11.1 Chemical composition of Fenugreek seeds

Class Chemical constituent References
Saponin Diosgenin, trigogenin, gitongenin, fenugrin B, Kakani and Anwer
yamogenin, rigogenin and neorigogeninetc (2012), Dawidar et al.
(1973)
Alkaloids Trigonelline, gentianine and carpaine Garg (2016)
Polyphenols Rhaponticin and isovitexin He et al. (2015)
Volatile oils olfactometry diacetyl, 1-Octene-3-one, sotolon, Blank et al. (1997)
and fixed oil acetic acid; 3-Isobutyl-2-methoxypyrazine,
butanoic acid, isovaleric acid,
3-isopropyl-2-methoxypyrazine, caproic acid,
eugenol, 3-Amino-4,5-dimethyl-3, linalool, (Z)-
1,5-Octadiene-3-one and 4-dihydro-2(5H)-
Furanone
Amino acid Glutamic acid, aspartic acid, phenylalanine, Wani and Kumar
leucine, lysine, valine, glycine, isoleucine, serine, (2018)
proline, alanine, threonine, tyrosine, histidine,
phosphoethanolamine and methionine
Metals Manganese, potassium, phosphorus, copper, Kakani and Anwer
calcium, sodium, iron and zinc (2012)
11.2 Galactomannan: The Chief Constituent

of Fenugreek Gum
11.2.1 Structural Properties
Galactomannan is the chief constituent of fenugreek seeds composed of b-(1,4)-D-

mannose and a-(1,6)-D-galactose subunits (Cerqueira et al. 2011). It is extracted
from fenugreek seeds and have an approximately equal number of mannose and
galactose residues which makes it readily soluble in water. Generally, the existence
of 180-190 number of monosaccharides (both galactose and mannose) provide a
molecular weight of approximately 30 KDa to this natural polymer (Prajapati et al.
2013a, b). The backbone of galactomannan consists of 90–95 residues of b-1,4
linked mannopyranosyl units and each backbone monomer comprise an a-1,6
linked galactopyranosyl unit (Prajapati et al. 2013a, b). X-ray diffraction studies
show the orthorhombic lattice of hydrated fenugreek gum with a, b and c value of
0.912 nm, 3.335 nm and 1.035 nm, respectively (Song et al. 1989). Gas
chromatography-mass spectroscopy (GC-MS) studies of partially methylated
alditol acetates gum extracted from fenugreek showed it to be a polysaccharide
consists of 1,4 linked mannose having partially substituted galactose at C6 position
(Brummer et al. 2003). 1H NMR and 13C NMR studies elucidated the structural
characteristics of galactomannan and showed that it consists of b-(1,4)-linked D-
mannopyranose and a-(1,6)-linked D-galactopyranose. Although the ratio of
mannose to galactose residues varies from species to species, it was observed that
the polymer backbone consists of mannose residues and about 83% of backbone is
substituted by galactose at C6 position of mannose. Coupling constant analysis
revealed that D-mannose and D-galactose possess b and a linkage, respectively
(Jiang et al. 2007) (Fig. 11.1).
11.2.2 Physicochemical Properties
Galactomannans extracted from the fenugreek seeds are neutral polysaccharides

with highly substituted galactose residues on mannose backbone (Jiang et al. 2007;
Cui 2001; Hefnawy and Ramadan 2011). Extremely branched structure and high
molecular weight of fenugreek gum are responsible for its lower viscous nature
among other industrially used gums such as locust bean gum and guar gum
(Brummer et al. 2003, Jiang et al. 2007; Cui 2001). Fenugreek gum solution
appears to be more transparent than the guar gum solution due to the presence of
less water insoluble constituents (Jiang et al. 2007). The higher degree of galactose
substitution on the mannose backbone has been claimed as the prime reason for this
behaviour (Brummer et al. 2003). Viscosity of aqueous fenugreek gum solution
depends upon shear stress, temperature and concentration (Wei et al. 2015).
Increase in the fenugreek gum concentration enhances the viscosity of solution

galactomannan derived from
fenugreek gum
while increasing shear stress and temperature which cause a decline in the viscosity
of solution (Wei et al. 2015). Wei et al. observed a drop in viscosity of aqueous
fenugreek gum solution in steady shear flow conditions (Wei et al. 2015).
Disentanglement of the polymer chain and distortion of the supramolecular struc-
ture of gum which might be responsible for this physical change (Singh et al. 2009;
Jian et al. 2014). Fenugreek gum, when dissolved in water, undergoes sol–gel
transition with an increase in gum concentration (Wei et al. 2015). Fenugreek gum
at a concentration of 0.05% (w/v) evinces sol–gel transition whereas gel-like
properties have been observed above 1% (w/v) concentration (Wei et al. 2015).
Thermogram analysis showed that biopolymer extracted from the fenugreek seeds
has a melting range of 66−139 °C and degradation temperature of about 296.45 °C
(Rashid et al. 2018). Previous study reported that galactomannan extracted from
fenugreek seeds can be effectively recovered from aqueous solution using less
amount of isopropanol than ethanol due to its lower dielectric potential (Rashid
et al. 2018). It was also observed that precipitation of galactomannan-based gums in
polar organic solvents depends on their molecular weight and high density of
hydrogen bonding due to large number of galactose substitution (Jian et al. 2014).
Viscosity of fenugreek gum rises as hydration time extends to a certain point (12 h)
and after that it starts decreasing (Jiang et al. 2007); whereas intrinsic viscosity of
fenugreek gum solution decreases with an increase in alkaline environment (Doyle

et al. 2009). It was proposed that there could be two reasons for this effect,
(a) increment in negative charge along the polymer chains eradicate the hyper
entanglement and (b) association between charged chain continues due to enhance
ionic strength. However, viscosity of the fenugreek solution is recovered by neu-
tralizing the solution with the addition of salt (Doyle et al. 2009). Previously, flow
behaviour of fenugreek gum as a function of concentration and shear rate was
examined and it was observed that the shear thinning behaviour was present in all
concentration ranges demonstrating pseudoplastic behaviour (Wei et al. 2015). At
low shear rates and concentration (<1%, w/v) the apparent viscosity displayed
narrow range newtonian plateau as concentration of fenugreek gum increased, while
newtonian plateau disappeared at 1% (w/v) concentration (Wei et al. 2015).
Pseudoplastic behaviour of fenugreek gum explains the existence of interlacing
molecular interaction between polymer chains (Wei et al. 2015). Galactomannan
exhibits coiled conformation at small shear rates and high apparent viscosity may
induce the interlocking between these coiled complexed macromolecules (Singh
et al. 2009). Deformation in interlocking conformation of complexes due to shear
rate induction leads to decline in solution viscosity (Wei et al. 2015; Jian et al.
2014). Shear strain also changes the orientation of galactomannan to an aligned
fashion, which may reduce the viscosity of solution (Adeli et al. 2015). Fenugreek
gum solution at high concentration display elastic behaviour as the thixotropic
property enhances with an increment in polymer concentration (Wei et al. 2015).
Numerous physical properties of fenugreek gum make it as an ideal excipient to
be used in pharmaceutical industries such as viscosity of fenugreek gum changes
with time, which affects the release profiles of loaded therapeutic agents (Jiang et al.
2007; Lee et al. 2009). Viscosity of fenugreek gum also varies with pH, which gives
it the pharmacological efficacy to be applied in vaginal and ophthalmic drug
delivery (Doyle et al. 2009; Lee et al. 2009).
Thixotropic property of polymer has a vital impact on the therapeutic efficacy of
the formulations by affecting their retention time at the administered site and
enhancing the bioavailability. Thixotropic behaviour of fenugreek gum at higher
concentration might enhance its ability to be used as a smart excipient for numerous
formulations such as ointment, hydrogel, emulsions and suspensions through dif-
ferent routes like oral, topical, ophthalmic and mucosal (Lee et al. 2009).
Smart sun screen formulations must have the pseudoplastic behaviour so that
they can form coherent protective layer over screen to eradicate the hazardous effect
of UV light (Lee et al. 2009). Pseudoplastic behaviour of fenugreek gum at all
concentration range makes it a cost effective candidate to develop sunscreen lotions
(Wei et al. 2015). These rheological properties make fenugreek gum as flexible and
cost effective excipient for various pharmaceutical formulations.
11.2.3 Biosafety and Toxicological Studies
Utilization of natural polymers for biomedical applications is continuously

increasing owing to their minimal cost, biocompatibility and minimal side effects. It
is of utmost importance to evaluate the probable toxic effects of these polymers
before their preclinical and clinical applications. Previous toxicological studies
affirm that seed extract/seed powder of fenugreek is non-toxic to experimental
animals at a single oral dose of 5 g/kg. Opdyke et al., showed that acute oral LD50
of fenugreek was more than 5 g/kg in rats (Opdyke 1978) while the LD50 for acute
dermal toxicity in rabbits was found to be 2 g/kg. Repeated dose toxicity studies for
90 days concluded that debitterized fenugreek was safe to weaning rats and no
significant toxic effects were noticed in behavioural pattern, food intake and growth
of animals (Narasimhamurthy et al. 1999). Trigonelline, an alkaloid present in the
seeds of fenugreek, was also found to be not altering the weights of mice thymus,
kidney, liver, thyroid, adrenals, uterus or ovaries when fed for three weeks. Oral
uptake of fenugreek seed extract for six months did not cause any clinical, hepatic,
renal and hematological irregularities in diabetic human patients (Sharma et al.
1996a, b). On the other hand, in a randomized, double blinded, placebo-paralleled
trial type II diabetes melitus patients suffered from stomach discomfort, nausea and
diarrhea due to subchronic administration of saponins derived from fenugreek
seeds. Patients were provided with 2.1 g of saponins thrice a day for a period of
three months (Lu et al. 2008).
Subchronic oral administration of fenugreek seed powder acts as an antifertility
agent in rodents by altering the weight of the reproductive tissues, sperm count and
morphological irregularities in sperm cells (Al-Ashban et al. 2010; Al-Yahya 2013).
Sharma and Bhinda. observed decline in weights of ovaries and uterine due to
administration of steroidal extract of fenugreek (100 mg/day/rat for 15 days) to
adult female rats (Sharma and Bhinda 2005). Elbetieha et al. (1996) suggested
fenugreek seed powder induced abnormalities in fertility of female rats due to its
estrogenic activity that distorts endothelial lining and interfaces of fetal develop-
ment (Elbetieha et al. 1996).
The toxicological properties of fenugreek seed powder were observed by various
animal models and human trials. From the above observations, it was clear that
fenugreek act as antifertility agent in both male and female animals. But particular
molecule and cellular mechanism responsible for antifertility of fenugreek is still in
debate. Further investigations are needed to identify the compositional element of
fenugreek seed, which acts as toxic agent. Toxico-metabolomics and metabolic flux
analysis experiments may be helpful to understand the cellular metabolism and
detoxification pathways (Table 11.2).
Table 11.2 Toxicological aspects of fenugreek seed powder in various animal studies
Type of Acute oral toxicity Acute Subchronic toxicity Chronic toxicity
toxicity dermal (90 days exposure) (exposure over
toxicity 24 weeks)
Dosage LD50 > 5 g/kg (rat) >2 g/kg NOAEL= 10% (rat) 25 g/day (diabetic
Opdyke (1978) (rabbit) Diet (*2 g/day) patients)
LD50 > 5 g/kg Opdyke Opdyke (1978) No toxic effects
(rat) (1978) NOAEL= 20% Sharma et al. (1996a,
(Narasimhamurthy (rat)87 b)
et al. 1999) Diet (*4 g/day)
LD50 > 2 g/kg Rao et al. (1996)
(mice)
Narasimhamurthy
et al. (1999)
LD50—Dose that is expected to be lethal in 50% of test subjects
NOAEL—No Observed Adverse Effect Level
11.3 Drug Delivery Applications of Fenugreek Gum
11.3.1 Ophthalmic Drug Delivery
Eyes are delicate and sensitive organs of the human body so ophthalmic formu-
lations strictly need to be non-irritant when applied in the ocular area. Cataracts,
macular degeneration, glaucoma, diabetic retinopathy, dry eye syndrome and ocular
allergies are the major challenges in the ophthalmology arena. The currently
available drugs to treat these diseases are associated with four major problems like
poor bioavailability, prolong drug release characteristics, higher dosage and repe-
ated administration. Novel polymer-based drug delivery systems such as liposomes,
hydrogels and nano/microparticles addressed these drawbacks mentioned above.
Polymers of natural origin proved to be the alternative candidate for ophthalmic
drug delivery systems due to less toxic nature, ease of availability and low cost.
Pathak et al. developed nanoparticulate system for ocular delivery using chitosan
and fenugreek seed mucilage. Haemocompatibility studies of developed chitosan/
fenugreek mucilage nanoparticles showed that it was haemocompatible up to
2000 µg/ml. Acute ocular irritation study of nanoparticle-based system at different
concentration ranges (50–2000 µg/ml) was examined using Draize’s test in New
Zealand white female rabbits. Ocular safety studies concluded that the developed
ocular delivery system was non-irritant and can be used for human eyes safely
(Pathak et al. 2014).
Mucoadhesive nature of fenugreek mucilage may be were going to enhance
pre-cornical drug retention which will provide prolonged drug release to treat back
eye diseases. Numerous formulations like viscous polymer vehicles and receptor/
transporter targeted nano-molecules can be developed using fenugreek mucilage to
build smart ophthalmic drug delivery systems. Concepts of micro-dialysis and
imaging technology can be applied to modify animal models to develop

fenugreek-based proactive ocular drug delivery systems.
11.3.2 Gastroretentive Drug Delivery
Gastric retention has been known as a key approach to increase the efficacy and
bioavailability of drugs having a slender absorption frame. Numerous drug delivery
strategies such as swellability, high density, floating and mucoadhesive machineries
have been fabricated to enhance the absorption and bioavailability of poorly gas-
troretentive drugs (Hao et al. 2014; Bera et al. 2015a, b; Deshpande et al. 1997).
Floating drug delivery system is one the most used strategy for gastorententive
drug delivery; however, one major disadvantage of this drug delivery system is its
dependency on gastric emptying/transit time (Deshpande et al. 1997). Different
strategies have been utilized to address this issue such as combining floating and
swelling approaches together (Chen et al. 2013). Floating and swelling behaviour of
drugs could be upgraded by applying polymer membrane on the gastroretentive
matrix (Deshpande et al. 1997). Polysaccharides extracted from the plants are used
as coating agents in gastroretentive drug delivery system as they possess key
pharmaceutical properties like stability, swelling and regulated drug release rate.
In this context, an efficient drug delivery system consisting of alginate-fenugreek
gum gel membrane covered with hydroxy-propyl-methyl-cellulose (HPMC) based
matrix containing quetiapine-fumarate (QF) drug for intra-gastric delivery was
developed. This formulation possesses improved buoyancy, prolonged drug release
and better swelling ability (Bera et al. 2015a, b). The presence of fenugreek-alginate
gel membrane on the surface of the tablet blocks many channels of core tablet,
which slow down the drug release rate (Bera et al. 2015a, b). Presence of an air
compartment between the coating layer and core tablet might be the reason for the
superior floating capability of the alginate-fenugreek gum-based formulation (Bera
et al. 2015a, b). Drawbacks such as weight gain and hyperglycemia associated with
QF therapy can be avoided using fenugreek gum (Bera et al. 2016). This study not
only proved that fenugreek gum as a potential natural polymer to be used in
targeted drug delivery system but also proposed an alternative approach to regulate
the disadvantages associated with QF therapy.
Though there are significant developments in gastro retentive drug delivery
systems formulations with meal independent gastric retention capacity, floating
behaviour is yet to be achieved. Fenugreek-based bioadhesive systems, super
porous hydrogels, floating systems, high density-based formulations and expand-
able systems may be acting as game changers in gastro retentive drug delivery
arena. Complex in vitro experiments can be designed to evaluate the efficiency of
fenugreek-based gastro retentive drug delivery systems by varying the density/
viscosity of gastric contents and providing a different degree of contractions in the
presence of food. The impact of pharmacokinetic property on gastric retention
ability can be observed using sophisticated in vivo imaging techniques.
11.3.3 Colon Drug Delivery
Orally administered colon-targeted drugs are expected to protect the therapeutic

agent release and degradation in the stomach as well as in the small intestine and
able to deliver it in colon. However, the acidic environment of upper gastroin-
testinal tract induces the degradation of drugs. This obstacle has been addressed by
designing drug delivery system that facilitates the release of therapeutic agent in
neutral pH. But the difference in the pH of intestine (7.4) and colon (6.8) region
adversely affected the ability of pH-dependent drug delivery system to deliver the
drug to the colonic region. In order to overcome this pH associated issue, drug
encapsulated with polysaccharides offers advantages due to their stability in
divergent pH conditions (Park et al. 2010). Natural polysaccharides found to be
alternative candidates for the fabrication of colon specific drug delivery systems as
colonic bacteria able to degrade them effectively (McConnell et al. 2008). Inter
polymer complexes composed of chitosan and carboxymethyl fenugreek gum with
tamoxifen was fabricated by Randhawa et al. Drug coated with polymeric system
were able to protect the release of bioactive compound in stomach as well as in
small intestine, and in the presence of rat cecal content 91% of tamoxifen released
was observed from in vitro and in vivo experiments (Randhawa et al. 2012).
Easy availability, low cost, cytocompatibility and mucoadhesive nature of
fenugreek gum make it as a gold standard natural polymer-based excipient for colon
specific drug delivery. But research involving fenugreek gum based excipients for
colon drug delivery is still in nascent stage, and therefore pressure controlled colon
specific capsules and osmotic controlled drug delivery can be developed using
fenugreek gum. These colon specific drug delivery systems can be evaluated by
mimicking various pH conditions of gastric fluid, jejunum, small intestine and
ileum. The efficacy of drug delivery system could be observed by incubating it with
buffer medium containing ezypectinase and dextranase. Numerous animal models
involving dog, guinea pigs and rats can be designed to evaluate the activity of
formulation in complex cellular conditions. The absorption and distributional
behaviour of fenugreek-based drug delivery system can be evaluated using gamma
scintigraphy and high frequency techniques.
11.3.4 Vaginal Drug Delivery
Self-cleansing property of vaginal tract has always been a problem for the estab-
lishment of drug in the area of infection. Dynamic vaginal delivery system is
expected to deliver the therapeutic agents to the site of infection or injury for a
longer time span (Pavelić et al. 2001). Vaginal films comprising of polymers loaded
with bioactive agents proved to be the best candidate as it offers stability, ease of
administration and longer retention (Garg et al. 2005). Bioadhesiveness and bio-
compatibility of natural polymers draw interest of researchers for their application
in vaginal drug delivery system. Polymeric films composed of aminated fenugreek

gum and glycerol, loaded with therapeutic agent nystatin were developed for the
treatment of vaginal candidiasis. These bioactive films were able to deliver thera-
peutic agents in 8 h, however, these films cured vaginal candidiasis in rats which
were evident from histopathological studies (Bassi and Kaur 2015a, b). Enhanced
bioadhesiveness of these films might be due to the interaction between amine
groups of aminated fenugreek gum and negatively charged mucin chains through
hydrogen bonding or electrostatic interaction (Kaur et al. 2013). In another report
Bassi and Kaur (2015a, b) prepared bioengineered films using carboxymethyl
fenugreek gum and glycerol functionalized with nystatin which have delivered
100% drug in 5 h. This drug delivery system is found to be compatible with vaginal
mucosa which was inferred from the in vivo studies. Derivatives of fenugreek gum
(aminated, carboxymethylated) make them as pivotal macromolecule to be explored
in the field of targeted drug delivery by enhancing its bioadhesive and drug delivery
properties (Bassi and Kaur 2015a, b). Smart nanofibrous mats can also be fabricated
using fenugreek-based biopolymer for numerous biomedical applications (Yadav
et al. 2019). Mucoadhesive nature of fenugreek gum can be applied to fabricate
various potent formulations such as nanoparticle/emulsions, vaginal inserts and
hydrogels to treat bacterial vaginosis, vulvo vaginal candidiasis, trichomoniasis,
urinary tract infections and aerobic vaginitis.
11.3.5 Aerogels
Aerogel drug delivery system has become the prime area of research in biomedical
and pharmaceutical terrain due to its porous structure and large surface area.
Aerogels composed of plant-based polysaccharide are biocompatible in nature, and
therefore it can be applied as drug carriers. It was shown that aerogel made up of
laccase oxidized fenugreek galactomannan act as a hydrolytic glycosidase agent
when loaded with lysozyme. Developed drug loaded aerogels were capable of
uptaking organic and inorganic solvents 20 times of its own weight. Hydrolytic
activity and clemency of lysozyme was noticed when biomaterial was kept on agar
plate containing M. lysodeikticus cells (Rossi et al. 2016). Chemical composition of
galactomannan plays a vital role in enhancement of overall efficiency of
aerogel-based drug delivery system. This study compared the aerogels obtained by
enzymatic lyophilization of galactomannans of fenugreek, guar and sesbania loaded
with therapeutic agents like lysozyme, nicin and polymyxin B. Fenugreek-based
aerogels expressed better mechanical properties than guar and sesbania. The highest
amount of galactose on the mannose backbone in fenugreek-based galactomannan
which could be the possible reason for the better mechanical strength (Campia et al.
2017). Excellent biocompatibility, biodegradability, easy availability, low cost and
particularly high porosity with open pore structure make fenugreek-based galac-
tomannan as a potential candidate to synthesize robust aerogel delivery systems for
biopharmaceutical industries (Fig. 11.2).
Fig. 11.2 Numerous applications of fenugreek gum
11.4 Fenugreek Gum as a Pharmaceutical Excipient
11.4.1 Retarding Agent
The release of therapeutic agent from the oral delivery system depends on the
physicochemical properties of both polymer and drug used in the formulation. The
drug release rate can be manipulated by using a combination of hydrophilic or
hydrophobic polymers and varying their concentrations (Gade and Murthy 2014).
Features of fenugreek gum as a retarding polymer was evaluated by Sav et al. 2013.
Both fenugreek gum and its hydrophobic derivative octenylsuccinate anhydride
fenugreek gum was used to prepare extended release tablets of metoprolol succinate
and carbamazepine as hydrophilic and hydrophobic drug, respectively. The
development of extended release tablet formulations displays similar drug release
kinetics like marketed formulations, viz., Seloken® XL 50 mg and Tegretal® 200
CR (Sav et al. 2013). It is imperative to mention that controlled drug release can be
accomplished not only by chemical alteration of fenugreek gum but also by using
various copolymers to enhance unchanged drug concentration in the systemic cir-
culation of poorly bioavailable drugs.
11.4.2 Super-disintegrating Agent
Oral route of administration plays a pivotal role in drug delivery due to simple
administration, less patient complication and efficient dosing (Bhowmik et al.
2009). Tablets and capsules are common dosage form to be administered through
oral route but physiological changes in paediatrics and elderly cause complications
in swallowing the tablets. This kind of obstacle can be addressed by engineering
fast dissolving tablets (Karthikeyan et al. 2012). Fast dissolving tablets quickly
disintegrate within few seconds by saliva when it is placed in oral cavity (Puttewar
et al. 2010). Super-disintegrants induce the rate of dissolution by breaking up the
tablets into small particles.
Fenugreek gum was subjected as effective disintegrants and compared to con-
ventional disintegrants like croscarmellose and sodium starch glycolate. Fast dis-
solving tablets were loaded with diclofenac sodium fabricated by direct
compression method using fenugreek gum of different concentration (1–6%, w/w).
It was observed that an increase in concentration of fenugreek gum enhanced the
rate of drug release and reduced the disintegration time. Application of fenugreek
gum in the formulation caused significant reduction of inflammation and diclofenac
concentration in the formulation. This reduction of inflammation may be due to
synergistic activity of diclofenac and fenugreek gum in curbing the inflammatory
mediators like leucocytes migration, cytokinins production and prostaglandin
synthesis. Therefore, use of fenugreek gum as a superdisintegrant could minimize
the dose related side effects of diclofenac (Kumar et al. 2014).
11.4.3 Mucoadhesive and Bioadhesive Agent
Moderate water solubility, short half life and slow dissolution rate are the major
pitfalls associated with conventional antidiabetic drugs such as Metformin HCl
(MeHCl) and Glimpiride (Ahmed et al. 2016). Therefore, designing of smart drug
delivery system which can overcome these pitfalls is a prime area of research in the
pharmaceutical domain. Polymeric beads composed of natural or synthetic polymer
loaded with therapeutic agents offer great success as efficient drug carriers (Nayak
et al. 2013a, b). Easy availability, low cost, biocompatibility, biodegradability and
environment friendly nature of plant-derived polymer and mucilage attracted
researchers to use them as bead forming agent for therapeutic applications (Avachat
et al. 2011). It was observed that beads consisting calcium pectinate and fenugreek
seed mucilage embodied with MeHCl of size range 1.47–2.08 mm shows
pH-dependent mucoadhesive and swelling properties. Formulated beads exhibited
significant excellent hypoglycemic effect in diabetic rats after oral administration
(Nayak et al. 2013a, b).
Nayak et al., developed sodium alginate and fenugreek seed mucilage based
mucoadhesive beads using MeHCl as a therapeutic agent (Nayak et al. 2013a, b). In
vivo studies revealed mucoadhesiveness and prolonged drug delivery capacity of
the developed mucoadhesive beads. In another report, Nayak and Pal used iono-
tropic gelation technique to fabricate mucoadhesive beads composed of gellan gum
and fenugreek seed mucilage functionalized with MeHCl. Release of MeHCl from
the drug followed zero-order model with super case-II mechanism. Excellent
hypoglycemic effect was noticed when fenugreek-based beads were administered
orally to alloxan-induced diabetic rats. Mucoadhesive nature of these system could

be due to the OH groups in both of the biopolymers which lead to form hydrogen
bonding with mucous biological membranes. Excellent ability to induce chemical
interaction with mucous membrane, fenugreek seed mucilage based formulations
might enhance bioavailability of drugs, higher gastric retention and effective contact
between mucous membrane and the therapeutic agent resulting in number of
re-administration of the drug (Nayak and Pal 2014). Glimepiride-loaded mucoad-
hesive composite beads are made up of carboxymethyl fenugreek galactomannan,
gellan gum and calcium silicate. Enhanced drug entrapment efficacy, constant drug
release and hypoglycemic activity of glimepiride beads were confirmed from
in vivo and in vitro studies (Bera et al. 2018).
Momin et al. 2015 developed a bilayer tablet of venlafaxine. Sustained and
bioadhesive layers of tablet were formed using combination of fenugreek seed
mucilage (FNM) and xanthan gum, carbopol, hydroxy propyl methyl cellulose
(HPMC). The batch containing FNM:HPMC (80:20) displayed substantial bioad-
hesive force and tablet adhesion retention time as 2.4 ± 0.028 g and 24 ± 2 h,
respectively. Optimized formulation obtained from this study showed enhanced
drug release profile than marketed formulation Venlor XR. It was observed that
enhanced concentration of fenugreek seed mucilage favoured bioadhesion and
bioadhesive rention time of the formulations (Momin et al. 2015).
From the above studies it is clear that there were no significant alterations in
drug release rate when fenugreek mucilage/gum is added with any other natural/
synthetic polymers. This property promotes the ability of fenugreek mucilage/gum
to be used as a potential biopharmaceutical excipient for oral route of
administration.
11.4.4 Matrix Forming Agent
Application of polysaccharides extracted from fenugreek seed in matrix formulation

containing propranolol hydrochloride was evaluated by Nokhodchi et al. (2008). In
this investigation biopolymer extracted from the seeds of fenugreek compared with
Methocel® hypromellose K4M, a conventionally applied matrix forming agent. An
increase in fenugreek seed mucilage concentration in matrix formulation retarded
propranolol diffusion from the matrix. Addition of lactose to the matrix formulation
enhanced the pore size and reduce tortuoisity as a result water diffuses into the
tablet by increasing drug release rate. Fenugreek mucilage observed to be a better
retardant at concentration 66% (w/w) than hypromellose of equivalent content.
Iurian et al., utilizes freeze drying method to develop fenugreek seed mucilage
based oral lyophilisates containing meloxicam as a model drug. It was observed that
rise in mucilage concentration retards disintegration of therapeutic agent from the
matrix. Longer disintegration time and higher crushing strength was noticed in the
case of fenugreek-based tablets than gelatin based tablets produced under same
conditions (Iurian et al. 2017). Enhancement in disintegration time with increase in
fenugreek seed mucilage concentration in the formulation might have resulted due
to the rise in viscosity of liquid medium which ultimately slow down the water
absorption into the matrix. The natural polymer extracted from fenugreek seed can
be further used to develop wound healing lyophilized products and mucoadhesive
films by incorporating synthetic or semi synthetic polymers.
11.4.5 Bioavailability Enhancer
Limited intestinal absorption is one of the major problems to deliver bioactive

agents efficiently. Krishnakumar et al., developed curcumin impregnated particles
of galactomannan derived from fenugreek in the order of 150 ± 20 µm by using
ultrasound mediated gel phase technique. Enhancement in bioavailability was
observed in formulated particles at pH 1.2 and 6.8 from in vitro studies. Further,
prolonged drug release activity was observed in Wistar rats (20 times higher) and
human volunteers (15.8 times higher) in case of new formulation compared to raw
curcumin (Krishnakumar et al. 2012).
In another study ultra-performance liquid chromatography coupled with triple
quadruple electrospray ionization tandem mass spectrometry was used to evaluate
blood brain barrier permeability, pharmacokinetics and bio-distribution of
above-mentioned drug delivery system and free curcuminoids in Wistar rats.
These formulations of curcumin with fenugreek fibre were administered to the
rats with value of 200 mg/kg body weight. Post administration of tissue samples
obtained from rats demonstrated 25 times enhancement in bioavailability of
fibre-based curcumin than the native. Effective blood brain barrier permeability also
noticed from the tissue distribution of free curcuminoids at brain, heart, liver and
kidney than standard curcumin (Krishnakumar et al. 2015). A dynamic fenugreek
galactomannan-based formulation can be developed in near future by performing
various investigations such as drug diffusion, transport, distribution and impact
among various tissues.
11.4.6 Microencapsulation of Probiotic
Survival in gastrointestinal acidic and bile habitat is the prime criterion for an ideal
probiotic. Establishment of an effective drug delivery system which successfully
delivers probiotic by oral route and protects it from acidic environment is necessary.
Encapsulation of probiotic bacteria offers a solution for these problems. Various
polymer such as gelatin, alginate, starch, etc., has been used as matrix forming layer
for the probiotic systems (Sohail et al. 2011). Haghshenas et al. designed a smart
probiotic system containing Lactobacillus plantarum 15HN bacteria encapsulated
with alginate–fenugreek–psyllium polymeric blends (Haghshenas et al. 2015a, b). It
was observed that alginate combined with fenugreek or psyllium offers great
potential as encapsulation matrix for probiotics. These gel formulations were able to
protect bacterial cells not only at low pH and high bile salt conditions but also they
support the growth of bacteria in gastrointestinal environment. In another report
Haghshenas et al. observed that Enterococcus durans 39C encapsulated with
algenic-psyllium blend with fenugreek gum possess excellent encapsulation effi-
cacy, cell viability as well as enhanced release rates (Haghshenas et al. 2015a, b).
Therefore, fenugreek gum can be used as encapsulating agent due to its potential to
act as a prebiotic in the formulations. Low toxicity and affordable cost present
fenugreek-based polymer as better alternative than synthetic polymers currently
being used in probiotic formulations (Table 11.3).
11.5 Therapeutic Applications of Fenugreek Gum
11.5.1 Antidiabetic Property
Diabetes mellitus is a metabolic disorder affecting millions of human being day by

day. Chronic diabetes tends to offer retinopathy, nephropathy, neuropathy and
cardiovascular diseases (Pradeep and Srinivasan 2017; Zarvandi et al. 2017).
Currently used hypoglycemic and hypolipidemic agents for the treatment of dia-
betes offer several side effects such as lactic acidosis, hypoglycemia, abdominal
discomfort, peripheral edema, myopathy and heaptic toxicity (Sorrentino 2012).
Exploration of drugs of natural origin for the treatment of diabetes is an active area
of research and several studies proved their effectiveness as an alternative treatment
option than commonly available synthetic drugs. Fenugreek is a plant of traditional
and ethnopharmacological relevance used by the tribal people for the treatment of
diabetes and other diseases.
Antidiabetic potential of fenugreek seeds has been scientifically validated by
several research groups. Different bioactive compounds have been isolated from the
fenugreek seed and evaluated for their therapeutic application. Galactomannan
extracted from the fenugreek seed also showed its potential for the treatment of type
II diabetes which was evident by the clinical studies conducted on experimental
animals and human volunteers. Sharma et al. clinically evaluated hypoglycemic
effect of fenugreek seed powder in non-insulin dependent diabetic patients. Patients
were fed with two equal doses (12.5 g each) of fenugreek seed powder in diet
(during lunch and dinner) daily for 24 weeks. Reduced levels of fasting blood
glucose, insulin and glycosylated hemoglobin along with enhanced glucose toler-
ance were observed in the patients fed with fenugreek seed powder. This study
showed the potential role of fenugreek seed powder for the treatment of type II
diabetes (Sharma et al. 1996a, b).
Raju et al. evaluated the therapeutic efficacy of fenugreek seed powder in type I
diabetes. In this study, 5% fenugreek seed powder was orally administered with diet
in alloxan-induced diabetic Wistar rats daily for 21 days. Altered activities of
Table 11.3 Applications of Fenugreek seed powder/mucilage in the drug delivery
396
Active constituent Application Formulation Drug delivery Model drug Reference

type type
Fenugreek seed/mucilage Polymer to develop Nanoparticles Opthalmic – Pathak et al.
nanoparticles (2014)
alginate–fenugreek gum Coating gel to matrix Tablet Gastroretentive Quetiapine fumarate Bera et al.
(2016)
Chitosan and carboxymethyl fenugreek Protecting agents Tablet Colon Tamoxifen Randhawa
gum et al. (2012)
Amiated fenugreek gum Bioadhesive agent Vaginal film Vaginal Nystatin Bassi and
Kaur (2015a,
b)
Carboxy-methylated fenugreek gum Bioadhesive agent Vaginal film Vaginal Nystatin Bassi and
Kaur (2015a,
b)
Fenugreek gum Delivery system Aerogel – Lysozyme Rossi et al.
(2016)
Fenugreek gum Delivery system Aerogel – Polymyxin-B, Nisin, Campia et al.
Muraminidase lysozyme (2017)
Octenyl succincate anhydride derivative Retarding agent Tablet Oral Metoprolol succinate Sav et al.
of fenugreek gum (2013)
Octenyl succincate anhydride derivative Retarding agent Tablet Carbamazepine Sav et al.
of (2013)
fenugreek gum
Fenugreek gum Super-disintegrating Tablet Oral Diclofenac sodium Kumar et al.
agent (2014)
Fenugreek seed mucilage and calcium Mucoadhesive agent Beads Oral MeHCl Nayak et al.
pectinate (2013a, b)
(continued)
P. Mishra et al.
11
Active constituent Application Formulation Drug delivery Model drug Reference

type type
Sodium alginate and fenugreek seed Mucoadhesive agent Beads Oral MeHCl Nayak et al.
mucilage (2013a, b)
Gellan gum and funugreek seed mucilage Mucoadhesive agent Beads Oral MeHCl Nayak and
Pal (2014)
Carboxymethyl fenugreek Mucoadhesive agent Beads Oral Glimepiride Bera et al.
galactomannan (2018)
Fenugreek seed mucilage and hydroxy Bioadhesive and Tablet Oral Venlafaxine Momin et al.
propyl methyl cellulose (HPMC) sustained release layer hydroclhoride (2015)
Fenugreek seed mucilage Matrix forming agent Tablet Oral Propranolol Nokhodchi
hydrochloride et al. (2008)
Fenugreek seed mucilage Matrix forming agent Oral Oral Meloxicam Iurian et al.
lyophilisates (2017)
Pharmaceutical and Therapeutic Applications of Fenugreek Gum
397
various enzymes such as gluconeogenic, lipogenic and glycolytic were observed in

both liver and kidney of diabetic rats. However, treated animals showed reduced
fasting blood glucose level as well as eradication of the abnormalities associated
with activities of enzymes (Raju et al. 2001).
Zia et al. 2001 examined hypoglycemic effect of aqueous and methanolic seed
extract of fenugreek in normal mice. More prominent blood glucose lowering effect
was observed in the case of aqueous extract as compared to methanolic extract. This
experiment revealed that the hypoglycemic agent of fenugreek seed extract is polar
in nature (Zia et al. 2001). In another study, it was shown that fenugreek seed
mucilage is able to lower fasting blood glucose level in streptozotocin-induced
diabetic rats (Kumar et al. 2005).
Liver pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (PEPCK) and
glucose transporter (GLUT 4) play a pivotal role in glucose homeostasis and
abnormal expression of these proteins was noticed in diabetes (Board et al. 1990;
Mathupala et al. 1997). Mohammad et al. 2006 explored the ability of fenugreek
seed powder in recovering the altered expression of PK, PEPCK, GLUT 4 (in
skeletal muscle), and lowering the blood glucose level in alloxan-induced diabetes
in rats. Eidi et al. noticed that ethanolic extract of fenugreek seeds was able to lower
serum glucose, total cholesterol, urea, uric acid, triacylglycerol, aspartate amino
transferase, alanine amino transferase, and enhanced serum insulin level when
orally administered to streptozotocin-induced diabetic rats (Eidi et al. 2007).
Srichamroen et al. showed that galactomannan is able to reduce the intestinal
glucose uptake in genetically determined lean and obese rats and concluded that an
increase in viscosity with rise in galactomannan could be the possible reason for
declining uptake of glucose (Srichamroen et al. 2009).
Previous studies showed that an increase in oxidative stress during hyper-
glycemia is a primary reason behind the cardiovascular mortality in diabetic
patients. Upregulation of angiotensin converting enzyme (ACE) and receptor AT1
of renin-angiotensin system (RAS) is the reason behind cardiovascular disease
(Murça et al. 2012). ACE induce gene expression of extracellular matrix
(ECM) components, growth factors and infiltration of inflammatory cells. Due to
increment in ECM components (collagen, fibronectin) cardiac stiffness enhances
and diastolic dysfunction arises (Ban and Twigg 2008; McKarns and Schwartz
2005). Dietary fenugreek seeds and onion powder have been used as therapeutic
agents to treat the cardiovascular damage caused due to diabetes (Pradeep and
Srinivasan 2018). It was observed that fenugreek seed and onion powder were able
to block renin-angiotensin system (RAS) by downregulation of ECM components
in diabetic rats. These observations provided substantial evidences for fenugreek
seed powder as a potential antidiabetic agent and offers advantages over marketed
drugs in terms of reduced side effects.
Combination of fenugreek seed powder and sodium orthovanadate is used to
treat diabetes and long-term difficulties associated with the diabetes like structural
abnormalities in sciatic nerves. It was shown that the combination (sodium ortho-
vanadate + fenugreek seed powder) was able to restore the altered glucose level and
eradicated structural abnormalities in peripheral nerves (Preet et al. 2005). Several
studies reported the defensive role of fenugreek seed against the diabetic
nephropathy. Fenugreek seed extract was able to reduce the thickening of
glomerular base membrane by inhibiting the accumulation of oxidized DNA in the
kidney (Xue et al. 2011). Fenugreek seed powder proved as an effective therapeutic
agent to treat both type I and type II diabetes. But fenugreek seed powder based
prediabetes treatment is still in its infancy. More studies involving animal models
and human volunteers are required to evaluate the efficiency of fenugreek seed
powder as a bioactive agent to treat prediabetes.
11.5.2 Hypolipidemic Potential and Role in Fat

Accumulation
Vijayakumar et al. observed hypolipidemic potential of fenugreek seed extract in

3T3-L1 and HepG2 cells by quantifying fat accumulation by western blot analysis
of lipogenic and adipogenic factors. Fenugreek seed extract decreased the fat
accumulation in 3T3-L1 cells by downregulating adipogenic factors like peroxi-
some proliferators activated-receptors-c (PPAR-c), sterol regulatory element-
binding protein-1 (SREBP-1), CAAT element-binding proteins-a (c/EBP-a) fur-
ther, cellular triglycerides and cholesterol concentrations declined in HepG2 cells
via reduced expression of (SREBP-1) at mRNA and protein level. Seed extract of
fenugreek was also able to upregulate low density lipo-protein receptor (LDLR)
expression resulting enhanced LDL uptake (Vijayakumar et al. 2010).
Diosgenin, a steroidal sapogenin (component of fenugreek seed powder) at
different concentrations (5–10 mmol/l) inhibit triglyceride accumulation in HepG2
cell lines and expression of lipogenic genes. It inhibited fat accumulation by dis-
rupting the transactivation of liver-X-receptor-alpha (LXR-a), a prime regulator of
cholesterol homeostasis in hepatocytes (Uemura et al. 2011).
Kumar et al. fed aqueous extract of fenugreek seed to female Wistar rats for
28 days and it was found to be anti-hyperlipidemic agent as it was able to reduce
white adipose tissue weight, body mass index, weight gain, leptin, lipids, serum
insulin, blood glucose, lipase, and apolipoprotein-B levels (Kumar et al. 2014).
Apart from that rise in antioxidant enzymes (superoxide dismutase, glutathione,
catalase) level were noticed and serum aspartate amino transferase (AST), alanine
amino transferase (ALT), lactate dehydrogenase levels were upregulated due to
feeding of trigonella seed extract. Restored activity of liver and uterine WAT
lipogenic enzymes to normal level suggests fenugreek seed extract could be used as
a bioactive agent to inhibit fat accumulation. Clinical studies can be designed to
observe therapeutic nature of diosgenin to treat hyperlipidemia, and diosgenin
based drugs can be formulated to address these clinical consequences.
11.5.3 Anti-inflammatory Potential
Rheumatoid arthritis is a chronic inflammatory disease which causes multiple

pathophysiological consequences like swelling of joints, cartilage and bone
demolition, synovial hyperplasia, vasculogenesis. This disease causes rise in
inflammatory enzymes like cyclooxygenase, lipoxygenase, myeloperoxidase and
induce free radical formation along with low antioxidant level (Tristano 2009;
Hoozemans and O’Banion 2005). Sindhu et al. evaluated antioxidant and
anti-inflammatory potential of fenugreek mucilage in adjuvant induced arthritis in
rats. Fenugreek mucilage was fed orally to the rats and 73.65% edema inhibition
observed with 75 mg/kg mucilage dose. Fenugreek seed mucilage effectively
reduced the activities of inflammatory enzymes and upregulated the activities of
antioxidant enzymes, vitamin C and reduced glutathione (Sindhu et al. 2012).
Liu et al. investigated lipid peroxidation (LPO) and cyclooxygenase
(COX) enzyme inhibitory activity of fenugreek seed extract in different mediums
such as water, hexane, methanolic and ethyl acetate by MTT, COX-1, COX-2, LPO
enzyme inhibitory assays. These extracts at 250 lg/mL concentration was able to
inhibit LPO, COX-1 and COX-2 (Liu et al. 2012). Non-steroidal anti-inflammatory
drugs (NSAIDs) are the most preferred drugs to address inflammatory diseases but
NSAIDs are associated with certain drawbacks like gastrointestinal ulcerogenicity
and renal morbidity (Pincus et al. 1992). Therefore, long-term use of these con-
ventional drugs need a safe alternative like fenugreek seed mucilage as it suppresses
inflammatory enzymes activity and upregulate the antioxidants. Fenugreek muci-
lage based nano-molecules can be engineered to enhance its anti-inflammatory
properties and further studies can be performed to gain more insight for molecular
mechanism behind its anti-inflammatory properties.
11.5.4 Anticancer Potential
Free radicals generated by inflammatory cells, i.e. neutrophils, eosinophils, macro-

phages, could be the pathogenic factor for tumour formation in some cases (Rosin
et al. 1994). Earlier studies demonstrated, fenugreek seed extract at different con-
centrations (10–15 µg/mL) are able to inhibit the growth of breast, pancreatic and
prostate cancer cell lines but not the healthy cells for 72 h. It was also observed that
the crude fenugreek seed extract was able to differentiate between normal and
cancerous cells; however, its pure agents like diosgenin and sulforaphane did not
differentiate in malignant and normal cells (Shabbeer et al. 2009). In another report, it
was informed that not only fenugreek seed extract but also bioactive components, i.e.
diosgenin and trigonelline act as anticancer agents (Raju and Rao 2012). From the
above-mentioned reports it was observed that there is still a conflict about the par-
ticular therapeutic agent of fenugreek seed mucilage. So there is significant demand
to evaluate the anticancer potential of fenugreek seed mucilage and its components.
Fig. 11.3 Hepato-protective mechanism of fenugreek-based seed extract
11.5.5 Hepatoprotective Potential
Hepatoprotective ability of fenugreek seed extract was investigated for ethanol

induced hepatic injury and apoptosis rats by Kaviarasan and Anuradha (2007).
Upregulation of liver dysfunction markers (alanine aminotransferase (ALT),
gamma-glutamyl transferase (GGT), lactate dehydrogenase (LDH), aspartate
aminotransferase (AST), alkaline phosphatase (ALP) and bilirubin) in plasma and
declined liver glycogen were noticed in rats with chronic ethanol administration
(6 g/kg/day) for 60 days. Ethanol administration altered the activities of alcohol
metabolizing enzymes. Treatment with polyphenolic extract of fenugreek seed
restored the level of liver injury markers to normal levels and this result was com-
pared with silymarin, a hepatoprotective agent (Kaviarasan and Anuradha 2007).
Belaïd-Nouira et al. (2013) observed fenugreek seed powder (5% in diet) effi-
cacy to cure hepatic toxicity due to the oral exposure of AlCl3 (500 mg/kg bw i.g.
for one month then 1600 ppm via drinking water) in Wistar rats by preventing the
liver weight loss and ameliorating liver dysfunction factors. Regeneration of hepatic
cells, management of iron, glucose and lipid metabolism may be the proposed
mechanism behind the efficient activity of fenugreek seed extract to cure the hepatic
disease (Belaïd-Nouira et al. 2013). Fenugreek seed extract stimulate the liver
function and regenerate the liver cells. Thus it can be used as potential pharma-
ceutical alternative to treat hepatic diseases (Fig. 11.3).
11.6 Conclusion and Future Perspectives
In this review, we have outlined numerous applications of fenugreek gum as a

potential excipient and therapeutic agent for various biopharmaceutical usage.
Chemists and biologists can access fundamentals of rheological, toxicological and
bio-macromolecular characteristics of fenugreek gum to design a proactive excip-
ient which can enhance the drug discovery strategies in scalable manner to fabricate
cost-effective lifesaving drugs. Indeed, excellent work of industry-academia
researches have introduced this biopolymer as one of the prime excipient for the
next generation of drugs. In near future, there is a significant demand to transform

fenugreek gum as an effective excipient by applying polymer science,
nano-biotechnology, computational biology and design driven strategies.
One of the major challenges in the field of synthetic excipient discovery are the
long time span required for structure screening, design of scale up strategies and
toxicological testing for drug master file submission. Therefore, fenugreek gum
serves as an effective alternative than its synthetic counterparts due to its bulk
availability, ease of extraction property, low cost and biocompatibility.
Mucoadhesive property, rapid disintegration capability, high water solubility and
controlled release capacity pose it as a smart excipient to develop tangible phar-
maceutical product. Although the application of fenugreek gum in pharmaceutical
terrain is in its infancy today, new concepts of biopharmaceutical science can
increase the possibilities of fenugreek gum in pharmaceutical industries. Strategies
like chemical modification of biopolymer extracted from fenugreek seed mucilage,
blending with copolymer and computational biology can be used to tailor the
physiochemical property of biopolymer as per the formulation. However, complex
in vitro and in vivo models, sophisticated imaging techniques and gamma
scintigraphy could be used to analyze the therapeutic potential of fenugreek gum in
the circulation. Nanomolecule-based drug delivery systems, liposome-based for-
mulations and polymeric micelles can be fabricated using fenugreek gum with
improved solubility, bioavailability, membrane permeation and active targeting.
Recent articles show the huge potential of fenugreek gum as a biopolymer to
address formulation associated challenges with current therapeutic regime to treat
acute and chronic disease worldwide. As a result, scope of performance, cost
effectiveness and accessibility of valuable drugs would increase for growing soci-
ety. Altogether, this review demonstrates the recent advances related to fenugreek
gum aimed at expanding the horizon of drug delivery in the lap of rapidly growing
pharmaceutical arena.
Acknowledgments PM, AS and TCY are thankful to the Ministry of Human Resource
Development, Government of India for Senior Research Fellowship. Authors are thankful to
Indian Institute of Technology Roorkee for providing research facilities for this work.
Conflict of Interest We confirm that there are no known conflicts of interest associated with this
article.
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Chapter 12
Antimicrobial Application Potential
of Phytoconstituents from Turmeric
and Garlic
Shiv Kumar Prajapati, Gaurav Mishra, Akanksha Malaiya,

Ankit Jain, Nishi Mody, and Ashok M. Raichur
Abstract In recent years, natural phytochemicals are gaining much attention for
their antimicrobial potential. Garlic and turmeric are most widely used from the
natural source, and their constituents directly or indirectly provide various health
benefits, especially due to antimicrobial potential. Though the conventional
antimicrobial compounds are effective against various pathogens, up till now there
is a necessity of effective agents against MDR pathogens. Phytochemicals have
been used for their antimicrobial potential from ancient times. These phytochemi-
cals can work by multiple mechanisms, such as by inhibiting target modifying and
drug degrading enzymes or as efflux pump inhibitors. The use of natural phyto-
constituents (e.g., curcumin from turmeric and allicin from garlic) from these two
medicinal plants may be an alternative strategy and can overcome the side effects
associated with antibiotics or other allopathic means of treatment. A wide range of
indications has revealed the therapeutic efficacy of these compounds on bacterial,
S. K. Prajapati A. Malaiya
Department of Pharmaceutical Sciences, Bhagyoday Tirth Pharmacy College,
Sagar 470002, MP, India
e-mail: shivprajapati1992@gmail.com
A. Malaiya
e-mail: jain.akanksha2107@gmail.com
G. Mishra
Faculty of Ayurveda, Department of Medicinal Chemistry, Institute of Medical Sciences,
Banaras Hindu University, Varanasi 221005, UP, India
e-mail: vatsgaurav880@gmail.com
A. Jain (&) A. M. Raichur
Department of Materials Engineering, Indian Institute of Science, Bangalore 560012,
Karnataka, India
e-mail: ankitjainsagar@gmail.com
A. M. Raichur
e-mail: amr@iisc.ac.in
N. Mody
Department of Pharmaceutical Sciences, Dr. Harisingh Gour Central University,
Sagar 470003, MP, India
e-mail: nishimody@gmail.com
https://doi.org/10.1007/978-3-030-54027-2_12
410 S. K. Prajapati et al.
viral, and fungal infections. To improve safety and efficacy, these phytoconstituents
have been delivered using nanoformulations such as liposomes, hydrogels, and
nanoparticles for the treatment against different bacteria, viruses, fungi, and para-
sites infections. This chapter is attempted to discuss phytochemistry, antimicrobial
mechanisms, and application potential of phytoconstituents from turmeric and
garlic.

Keywords Phytoconstituents Antimicrobial activity Turmeric Garlic

Curcumin Allicin Liposomes Nanoparticles Helicobacter pylori infection
Staphylococcus aureus infection
12.1 Introduction
The demand for plant-based antimicrobial agents has been increased day by day
because of the adverse effect of synthetic antimicrobial agents (Venkatesan and
Karrunakaran 2010). Approximately 250,000 to 50,000 plant varieties are available
on the earth in which only 10% are of therapeutic benefit till now (Prakashet al. 2020).
The traditional system of medicines such as Ayurvedic, Unani, Siddha, Homeopathic,
Naturopathy, and Aromatherapy plays an important role to maintain health, hygiene,
proper sanitation, and also increases the longevity of life without affecting their
sensory properties. India is known as the “Kingdom of spices” because it is one of the
biggest producers, exporters, and consumers of herbal medicines (Ansari 2020).
Garlic, turmeric, coriander, black pepper, cardamom, onion, anise, fennel, mustard,
asafoetida, ginger, rauwolfia, basil, nutmeg, ajowan, chirata, long pepper, gokharu,
and pippala, etc., have been directly or indirectly used for the therapeutic benefit
because of the presence of phytochemicals. These species and herbs not only reveal
antimicrobial properties but also used in cancer, diabetes, inflammation, cardiovas-
cular diseases, hypercholesterolemia, arthritis, as carminative, and antioxidants, etc.
(Arora and Kaur 1999). WHO says antimicrobial resistance is one of the major health
threats in the twenty-first century against some pathogens (bacteria, pathogen, fungi,
and virus) and these pathogens are ever-increasing the infections. Antimicrobial
resistance emerges when microbes transform when they are subjected to antimicrobial
drugs (such as antibiotics, antifungals, antivirals, etc.). Such type of microorganism
which develops resistance are generally termed as “superbugs”. Therefore, detection
of natural, resourceful, harmless antimicrobial agents, such as phytoconstituents, has
noticed since the past decades. Among the above-mentioned herbal drugs, both the
turmeric and garlic reveal maximum antimicrobial efficacy against various strains of
microbes (Fig. 12.1).
One of the great challenges is to develop herbal drugs for clinical efficacy due to
poor bioavailability of phytochemicals within the body and various herbal drug
constituents get smashed in the stomach due to high pH before they reached to the
blood (Jain et al. 2019). It can be attributed to their low absorption and high meta-
bolism rate and rapid elimination from the body because of their inability to reach the
12 Antimicrobial Application Potential … 411
Turmeric Garlic
Supportive Compounds Supportive Compounds
Ajoene
Curcumin
Allicin
Demothoxycurcumin
Diallyl sulphide
Bisdemethoxycurcumin
Diallyl disulphide
Active against Active against
Bacteria Parasite Virus Bacteria

Virus Fungus Fungus Parasite
Antimicrobial action Antimicrobial action
The hydrophobic interaction causes breakdown of Garlic extract and its constituents hinder the
cell wall. reproduction of bacteria and viruses which makes
Curcumin can reinstate bacterial susceptibility, it a alternative choice of antibiotics.
impeding development of biofilm. Helps in ROS production, which causes the
It also inhibits in vitro production of aflatoxin. apoptosis in microbes.
Fig. 12.1 Antimicrobial application potential of turmeric and garlic phytoconstituents
target site. Nanocarriers have the potential to carry a significant amount of herbal
drug to their target site. These carriers also cross all barriers such as acidic pH of
stomach and liver metabolites because of the small size of nanocarriers, these
enhance permeability and retention effects which cause very lesser side effects (Jain
and Jain 2014, 2017). Nanocarriers can easily be altered according to need, and to
enhance the blood circulation so that their bioavailability, stability, and solubility of
lipophilic compounds can be augmented. Nanocarriers such as liposomes, micelles,
nanogels, niosomes, solid-lipid nanoparticles (NPs), carbon nanotubes, polymeric
NPs, dendrimers and cyclodextrins are one of the substitutes that deliver the thera-
peutic concentration of phytoconstituents of garlic and turmeric (Jain et al. 2013,
2020; Jain and Jain 2013, 2016b; Paramanya et al. 2020; Saraf et al. 2020; Subramani
et al. 2017; Prajapati and Jain 2020).
12.2 Turmeric
Turmeric or Haridra, i.e., Curcuma Longa is a tropical herb and belongs to the
family Zingiberaceae family (Zorofchian Moghadamtousi et al. 2014). It acts like a
magic bullet in ayurvedic and homeopathic medicine uses externally as well as
internally both (Negi et al. 1999). Several studies have reported that Curcuma longa
rhizome is broad-spectrum antimicrobial activity including antifungal, antiviral,
antibacterial, and antimalarial activities as well as in swellings, stopping pain,
cleaning wounds, helping coloration of the skin and also called “Charaka Samhita”
as protect skin from itching and also protect from leprosy, urinary disorder,
indigestion, blood anemia. Turmeric has shown antiseptic, antimicrobial, and insect
repellent properties. It is effective in respiratory disorders. Its ethanolic extract
showed high antimicrobial activity against Aspergillus oryzae, Escherichia coli,
Staphylococcus aureus, and Saccharomyces sake. Hexane extract is an active
fraction of curcumin and it is the yellow pigment of turmeric prevents against
Bacillus coagulant, Bacillus subtilis, Staphylococcus aureus, B. cereus, Escherichia
coli, and Pseudomonas aeruginosa (Kesharwaniet al. 2020; Negi et al. 1999).
Clinical trial data indicate that turmeric and garlic show potent antimicrobial
activity which protects from very harmful diseases (De and De 2019).
12.2.1 Phytochemistry of Turmeric
Turmeric belongs to the oleoresins consisting of light volatile oil fraction and heavy
yellow-brown fraction. The wide range of advantages is also due to the presence of
a variety of monoterpenes, sesquiterpenes, and curcuminoids. Flavonoid curcumin
is a chief phytoconstituent of turmeric some volatile oils are also present. Turmeric
is a chief source of phenolic compounds, for example, curcuminoid. Curcumin is a
phenolic diketone also known as diferuloylmethane (Nisar et al. 2015). The cur-
cuminoids comprise varying concentrations of, i.e., 77%, 17%, and 3% of curcumin
I, II, and III, respectively (Fig. 12.2) (Lee et al. 2013). Some volatile oil such as
turmerone, zingiberone, and atlantone are also present. Curcuminoids have been
recognized by the US Food and Drug Administration (FDA) has been suggested the
safer doses of curcuminoids between 4 and 8 mg per day (Hewlings and Kalman
2017). Apart from all these some other phenolic diketones such as demothoxy-
curcumin and bisdemethoxycurcumin are also available in the rhizomes. The yel-
low color of curcumin is due to the presence of phenolic diketones (Basnet and
Skalko-Basnet 2011). Curcumin I was considered to have minimum stability, while
curcumin III was proved to be the most stable among all curcuminoids. Not only the
above-mentioned phytochemicals it also consists of some essential oils, for
instance, a-phellandrene, a-pinene, caryophyllene, C8-aldehyde, geraniol methyl
heptanone, linalool, myrcene, pinene, p-cymene, sabinene, and 1,8-cineole.
Curcumin potentially shows antimicrobial activity, anticancer, and antioxidant
activity and extensively used in neurodegenerative ailment, inflammatory, and
cardiac symptoms (Pandey and Dalvi 2019). The nutritional composition of tur-
meric is described in Table 12.1.
12.2.2 Antimicrobial Mechanism of Curcuminoids
The antimicrobial activity depends on curcuminoids depend on the mode of their

delivery (Shlar et al. 2017). Generally, the antimicrobial action depends on the
hydrogen bonding and hydrophobic interaction of various phenolic compounds to
Fig. 12.2 Chief Constituents of Turmeric
Table 12.1 Nutritional composition of turmeric (per 100 g)

Constituent name Proportion Constituent name Proportion
Water (g) 6 Iron (g) 47.5
Calories (Kcal) 390 Sodium (mg) 30
Protein(g) 8.5 Phosphorous (mg) 260
Carbohydrates (g) 69.9 Riboflavin (mg) 0.19
Ash (g) 6.8 Ascorbic acid (mg) 50
Calcium (g) 0.2 Niacin (mg) 4.8
Fat (g) 8.9 Thiamine (mg) 0.09
Potassium (mg) 2000
the membrane proteins, which cause cell membrane distraction and breakdown of
cell wall and impairment of the electron transport chain. The antibacterial efficacy
of aqueous extracts is probably because of the anionic elements present such as
chlorides, nitrate, sulphates, and thiocyanate. The alcoholic extracts display
improved antimicrobial activity might be due to much solubility than the aqueous
extract solution. Curcumin potentially shows antimicrobial efficacy against a wide
range of gram-positive and gram-negative bacteria and it is generally much sen-
sitive for gram-positive species than gram-negatives (Betts and Wareham 2014;
Kaur et al. 2010). This may be due to the specific cell wall structure of
gram-negative bacteria. The lipopolysaccharides of the superficial layer of cell wall
represent the furthest penetrability barrier for a diversity of antimicrobials which are
the main cause of the remarkably leisurely influx of lipophilic molecule in
gram-negative bacteria. Instead, porin proteins entrenched in the superficial sheath
signify the main channels for entry of molecules inside the cells of gram-negative
bacteria. Curcumin can reinstate bacterial susceptibility, impeding the development
of biofilm, and make microorganisms more sensitive to antibiotics. Accordingly, it
has been noticed that its antibacterial activity potentiated once it is allied with other
antibiotics, such as cefixime, tetracyclines, and vancomycin (Alves et al. 2018). The
antimicrobial potential of curcumin can be significantly improved by acquaintance
with light. The broad absorption range (300–500 nm) with a maximum absorption
band at 425 nm makes curcumin a potent photosensitizer, leading to the enhanced
antimicrobial potency (Nikaido 2003). Curcumin also inhibits in vitro production of
aflatoxin, it is produced by mold Aspergillus parasiticus, which may grow and
contaminate poorly preserved foods and it is a potent biological agent causing
injury to the liver.
12.3 Garlic
Garlic, i.e., Allium sativum Linn is a bulbous herb of the Alliaceae family and has
been used traditionally as a spice for thousand years and extensively used for its
medicinal properties such as antibacterial, antiviral, and antifungal properties. The
garlic plant is a bulb growing upward to 25-70 cm with hermaphrodite flowers
(Mikaili et al. 2013). Not only the root bulb of the garlic but leaves and flowers for
medicinal properties for very long. Throughout the time of pre-antibiotics, garlic
bulbs assisted as one of the chief treatment approaches for a wide-ranging illness
due to its broad-spectrum effects (Majewski 2014; Petrovska and Cekovska 2010).
Garlic was also used as a supplement nutrient, as a treatment for weakness and skin
infections. The first indication of garlic for its antimicrobial properties was reported
in France during the plague of Marseilles in 1721 when four men resorted to
consuming a mixture of macerated garlic and wine tincture known as ″vinaigre des
quatre voleurs″ to protect themselves from getting the disease that was afflicting
those around them (Harris et al. 2001). Further, around five years later the garlic
was utilized at a mass rate by the Egyptians to treat the abnormal growth and also
with the circulation problems. Then, North Americans started utilizing this for the
treatment of symptoms like flu. Therefore, these shreds of evidence show that it is a
prime notification toward the accurate research in the field of science before the
development of modern universities and they also develop the attitude to the sci-
entist around the globe. Garlic has a minor, faint smell up until it has been unpeeled.
After it is peeled, cut up, or meshed, it instantly activates to feast a strong smell,
glycosides. The distillation of garlic with water vapor yields etheric oil with its
distinctive sharp smell. The examination of the chemical content of that garlic oil
confirmed the presence of few aliphatic unsaturated sulfur compounds (Petrovska
and Cekovska 2010). Even in dilution of allicin 1: 85,000 to 1: 250,000, allicin
showed strong antibacterial activity against gram-positive and gram-negative bac-

teria (Dervendži and Karanfilov 1992). Another compound called alliin, with
needle-shaped crystals without smell was isolated. Alliin has no antibacterial action
but by adding the enzyme alliinase from fresh garlic, allicin having strong
antibacterial action is produced (Petrovska and Cekovska 2010). The wide spectrum
of pharmacological effects of A. sativum has been reported with the minimum
toxicity. It is also used as a spice and food additive Health benefits of garlic are well
known and studied to cure ailments caused by infections. The active constituents of
garlic showed potential antimicrobial activity due to sulfur compound present
in their chemical structure that has been proved against some microorganisms
such as Escherichia, Helicobacter, Klebsiella, Mycobacterium, Porphyromonas,
Pseudomonas, Salmonella, Streptococcus species (Cellini et al. 1996; Uchida et al.
1975), Candida, Cryptococcus, and Rhodotorula, etc. (Yousuf et al. 2011). Some
fungal infections caused by fungi such as Trichophyton and Aspergillus have also
been cured by the use of garlic constituents (Saif et al. 2020). Calcium hydroxide
show less antibacterial activity compared to garlic. There is also a report that is
utilized for the management of the parasite infection and in diarrhea.
12.3.1 Phytochemistry of Garlic
The bulbs of garlic are known to contain hundreds of phytoconstituents including

0.1 to 0.36% volatile oil and these are generally responsible for their pharmaco-
logical effects. Garlic includes as ammonium of 33 sulfur compounds, for instance,
alliin, allicin (thiosulfinates), ajoenes (E-ajoene and Z-ajoene), allyl propyl, diallyl
disulfide, diallyl trisulphide), sallylcysteine, and vinyldithiines, etc. (Motteshard
2008). After the chopping of the garlic bulb and breaking down the parenchyma,
the cysteine sulfoxide, i.e., alliin in the presence of alliinase enzyme produces
allicin, the chief odoriferous organosulfur compounds are alliin, S-allyl cysteine,
S-ethylcysteine, and S-methylcysteine (Al-Snafi 2013; El-Saber Batiha et al. 2020;
Zeng et al. 2017). During the storage of garlic bulbs at cool temperatures, alliin
accumulates naturally. Allicin can be transformed into stable lipid-soluble allyl-
sulfides such as ajoene. Ajoene from garlic is obtained by the conversion of alliin
into allicin by the alliinase-induced cleavage and then in the presence of a polar
solvent such as water or alcohol, allicin forms ajoene. Ajoene is chemically more
stable than allicin (Viswanathan et al. 2014). Garlic bulbs upon steam distillation
yield oil dimethyl mono to hexasulfides (6% of oil), allyl methyl sulfides (37% of
oil), and diallylsulphides (57% of oil), which are reported for excellent antibacterial
properties (Abubakar 2009; Tripathi 2009). On average, a garlic bulb contains up to
0.9%—glutamylcysteines and up to 1.8% alliin. The pungent offensive odor and its
health benefits are generally due to the presence of sulfur compounds. Chemical
structures of chief constituents are illustrated in Fig. 12.3. Besides these com-
pounds, garlic also contains 17 amino acids such as leucine, methionine,
S-propyl-L-cysteine, S-propenyl-L-cysteine, S-allyl cysteine sulphoxide (alliin),
Fig. 12.3 Chemical structures of phytoconstituents from garlic
S-ethyl cysteine sulphoxide and S-butyl cysteine sulphoxide and their glycosides,
arginine, and others (Shah 2009). Some minerals such as selenium and enzymes
such as myrosinase, peroxidases, and alliinase are also present. The nutritional
composition of garlic is described in Table 12.2.
12.3.2 Antimicrobial Mechanism of Phytochemicals

of Garlic
Among the various phytoconstituents of garlic, allicin shows greater antimicrobial

activity than others and it is due to the interaction with thiol-containing enzymes
(Mikaili et al. 2013). Allicin elicits antibacterial activity for wide-ranging of
gram-negative and gram-positive bacteria’s, antiparasitic effect on human intestinal
protozoan parasites (Giardia lamblia and Entamoeba histolytica), antiviral activity
Table 12.2 Nutritional composition of garlic (per 100 g)

Constituent name Proportion Constituent name Proportion
Water (g) 59 Magnesium (mg) 25
Calories (Kcal) 149 Sodium (mg) 17
Lipids (g) 0.5 Vitamin B6 (mg) 1235
Carbohydrates (g) 3307 Vitamin C (mg) 31
Fiber (g) 2.1 Glutamic acid (g) 0.805
Manganese (mg) 1672 Arginine (g) 0.634
Potassium (mg) 401 Aspartic acid (g) 0.489
Sulfur (mg) 70 Leucine (g) 0.308
Calcium (mg) 181 Lysine (g) 0.273
Phosphorous (mg) 153
and antifungal activity for Candida albicans (Chavan et al. 2016). The garlic and its
constituents have been described to impede various strains of microbes such
as Aeromonas, Aerobacter, Bacillus, Clostridium, Citrobacter, Citrella, Escherichia,
Enterobacter, Klebsiella, Leuconostoc, Lactobacillus, Micrococcus, Mycobacterium,
Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Staphylococcus,
Streptococcus, and Vibrio (Sivam 2001). The constituents of garlic can be blended
with antibiotics to synergize the antimicrobial effect. Consequently, the level of
toxins reduces and prevents their production due to the bactericidal activity of garlic
(Karim et al. 2011). The allicin reacts with the multiple enzymes having thiol
groups such as thioredoxin reductase, alcohol dehydrogenase, and RNA poly-
merase, which can affect the metabolism of cysteine proteinase engaged in the
virulence of Entamoeba histolytica (Ankri and Mirelman 1999) and in amoeba
parasite, allicin hinder the cysteine proteinases by interacting with thioredoxin
reductases and alcohol dehydrogenases. A study reported that allicin showed
antimicrobial activity owing to the inhibition of RNA synthesis. Though, the pro-
tein syntheses and DNA are also inhibited partially, signifying that RNA is the chief
target of allicin. The primary aim of allicin to target RNA then partially inhibits
DNA, protein synthesis, and membrane damage. The ROS production leads to the
DNA and protein damage followed by microbial cell apoptosis (Fig. 12.4). Allicin
is a dose-related biocide that can stimulus vital metabolism of cysteine proteinase,
and thus, kill all eukaryotic cells due to the presence of thiol groups in all living
cells. Ajoene exerts antiparasitic activity by preventing the human glutathione
reductase and Trypanosoma cruzi trypanothione reductase.
Phytochemicals with
Nanocarrier
Phytochemicals
Cell membrane damage due to
Hydrogel Nanoparticle
phytochemical or their
Or Formation of pores
nanocarrier formulation
in membrane
CNTs Micelles
Cell membrane Cytoplasmic

leakage Cytoplasm
Destabilize ribosomes
Generation of Increased
free radicles, ROS ROS level
DNA damage
and oxidative stress
ROS Mitochondrial
Production Oxidation of Damage
cellular
Protein
component
denaturation
Fig. 12.4 Proposed mechanism of action by turmeric and garlic extract

12.4 Applications

from Turmeric
Mariselvam et al. (2012) prepared the natural dye from turmeric to evaluate their
potential for antimicrobial activity and it is evaluated using agar well diffusion
method. The in vitro studies revealed a greater inhibitory effect against Escherichia
coli and Vibrio cholera that the curcumin dye with a zone of inhibition 7 mm to
15 mm and 10 mm to 15 mm, respectively (Mariselvam et al. 2012).
A toxicoproteomics approach was followed by Shlar et al. (2017a, b) to evaluate
the antibacterial mechanism of action of curcumin on Escherichia coli. The inclu-
sion complex of curcumin was developed with the b-cyclodextrin under the light
and dark conditions. When Escherichia coli treated under light, the light-induced
curcumin toxicity was conquered by maladaptive responses. The ROS encouraged
by the treatment of curcumin over the light dominated the cellular adaptive
mechanisms interrupted the metabolism of iron, deregulated the biosynthesis of
iron-sulfur mass, and ultimately resulted in cell death. The curcumin activity was
potentiated in dark by detoxification of free radicals and modulation of cellular
redox status (Shlar et al. 2017). Gopal et al. (2016) prepared the water-soluble
extract of curcumin and compared their antimicrobial activity with the ethanolic
extract. The entrance of nano curcumin particle within the microbial cell (oral
microflora) was markedly improved and was confirmed by the CLSM study. This
might be the result of their nanosized, which increased the interaction with cells and
led to damage of oral microflora cells. The bioactivity was critically affected by its
solubility in water (Gopal et al. 2016). Osteomyelitis is generally caused by
Staphylococcus aureus. For the treatment purpose of osteomyelitis, Zhou et al.
(2017) gave erythromycin in combination with curcumin. The monotherapy
through erythromycin was not found to be efficient to inhibit bacterial growth,
while it reduced the TNF-a and IL-6. Contrary to this, curcumin slightly reduced
the growth of bacteria. Treatment of rat with the blend of erythromycin and cur-
cumin distinctly inhibited bacterial growth significantly lessened bone infection,
and decreased TNF-a and IL-6. The combination of both showed better proficiency
for MRSA induced osteomyelitis (Zhou et al. 2017).
The antivirulence effect of curcumin was investigated by Darmani et al. (2020).
The efficacy was estimated on Helicobacter pylori in the presence and absence of
blue light-emitting diodes (LEDs). In the presence of LEDs, the viability was
markedly reduced along with the decrease in the urease production and motility.
This also enhanced the interruption of developed preformed biofilms of
Helicobacter pylori (Darmani et al. 2020). The in vitro antiparacytic efficacy was
examined by Cervantes-Valencia et al. (2019) for the treatment of besnoitiosis
caused by Besnoitia besnoiti. Functional inhibition assays exposed that curcumin
diminished viability of tachyzoite and encouraged fatal effects in up to 57% of
tachyzoites (IC50 in 5.93 lM). Curcumin treatments only inhibited helical gliding
A B
C D
Fig. 12.5 a–d Effects of curcumin and parasite proliferation in bovine endothelial host cells
(Cervantes-Valencia et al. 2019)
and twirling activities at the same time longitudinal gliding motility was not
markedly affected. Pre-treatments by curcumin of tachyzoites caused
dose-dependent lessening in host cell invasion (Fig. 12.5a–d) (Cervantes-Valencia
et al. 2019). The antifungal efficacy was tested against 23 strains of fungi by
Martins et al. (2009). The antifungal exposure was assessed by broth microdilution
assay. Paracoccidioides brasiliensis isolates were found to be most susceptible to
curcumin while the growth of Aspergillus isolates was not affected. Curcumin
showed 2.5-fold more efficacy than fluconazole at inhibiting the adhesion of
Candida albicans or Candida parapsilosis to buccal epithelial cells (Martins et al.
2009).

of Garlic
The main activity of garlic constituent is allicin activity and there are many pieces
of evidence of allicin for inhibition of different organisms like gram-positive,
gram-negative Escherichia coli and also antibiotic-resistant (Ross et al. 2001).
Staphylococcus aureus, Pseudomonas aeruginosa (Kuda et al. 2004), Streptococcus
mutans, Streptococcus faecalis, Streptococcus pyogenes, Salmonella enterica,
Klebsiella aerogenes (Cutler and Wilson 2004), Vibrio, Mycobacteria, Proteus

vulgaris, and Enterococcus faecalis (Wallock-Richards et al. 2014). There are many
forms of extracts of garlic that were reported for antimicrobial activity with inhi-
bition of different pathogenic bacteria. There is another study revealed that the
ethanolic extract has better inhibition against S. typhi and Escherichia coli then
aqueous extract (Mikaili et al. 2013). Sharma et al. (1977) reported the antibacterial
property of garlic water extract against both gram-positive and negative of the
gastrointestinal tract of chicks indicating its effectiveness and they also reported the
inhibition of some flora which are resistant to few antibiotics. Allicin had shown a
very suggestive response toward methicillin-resistant Staphylococcus aureus
(Sharma and Kumar 1977). Allicin’s which is produced from Alliin showed its
antimicrobial property due to the chemical enzymatic reaction having thiol, viz,
alcohol dehydrogenase, RNA polymerase, and thioredoxin reductase by oxidizing
protein cysteine or glutathione residues under physiological conditions (Gruhlke
et al. 2011). Further due to the presence of thiol within allicin group all living cells
of eukaryotic cells are killed by increasing the rapid metabolism of cysteine pro-
teinase. Broadly we can say that it also shows the antiviral, antifungal, and
antiparasitic activity (Weber et al. 1992). Deeply we can say that the inhibition
within the bacteria by S-allylmercapto modification of thiol-containing protein and
this leads to the shortage of glutathione level further the accumulation of protein
and scratch to the enzyme inactivation (Getti and Poole 2019; Müller et al. 2016).
There is another study revealed that allicin in gaseous form showed antimicrobial
potential effectively toward lung pathogens (Reiter et al. 2017). Further in case of
skin disease caused by methicillin-resistant Staphylococcus aureus showed
improved recovery of the skin with allicin there is one drawback with allicin is its
stability so it may not be used for inhalation purpose (Freeman and Kodera 1995;
Sharifi-Rad et al. 2014). The next compound is a combination of two Ajoenes
(Z-ajoene and E- ajoene) which was obtained by the maceration process of garlic
having active sulfur compound transformed from three allicin molecule (Block
1985). There is another scientific group of Yoshida and his co-workers have
reported the activity against gram-positive and gram-negative bacteria of ajoenes
and they evaluated the minimum inhibitory concentration and they find that
Z-ajoene has greater activity than E-ajoenes (Yoshida et al. 1998). Another group of
Ohata and his team reported that antimicrobial property of three strains of H- pylori
and they found that both forms of ajoenes are having an equal inhibitory activity
(Ohta et al. 1999). Ajoenes are having activity for fungi, i.e., Aspergillus niger and
Candida albicans (Maluf et al. 2008). Further, there is another component which
was obtained by the steam distillation of garlic oil is DASn having very low
potency toward gram-positive and resistant microbes the potency depends upon the
number of sulfur atoms within the compound. It was seen that more than five sulfur
atoms showed more potency than the lower number of atoms (Tsao and Yin 2001).
Some sulfur free garlic compounds show antimicrobial activity. Matsuura et al.
(1989) and Matsuura et al. (1988) isolated new furostanols termed
proto-eruboside-B and satiboside-B from a crude glycoside fraction of garlic. They
also found that these saponins get transformed into spinostanol by endogenous
Fig. 12.6 Effect of garlic TACs on the cytolytic damage caused by giardipain-1 in giardiasis
(Argüello-García et al. 2018)
b-glucosidase during processing period. (Matsuura et al. 1989; Matsuura et al.

1988). Argüello-García et al. (2018) studied the efficacy of thioallyl compounds of
garlic to measure their potential against Giardia duodenalis Trophozoites. Allicin
presented the maximum antigiardial efficacy. Allicin had a cytolytic mechanism in
trophozoites with successive loss of diesterase enzyme and oxidoreductase activi-
ties. These findings were similar to the fresh aqueous extract (AGE). The partial cell
cycle arrest was observed at the G2 phase without any oxidative stress. The
intragastric administration of AGE or allicin lessened the number of parasites and
removed trophozoites in the experimental animals of giardiasis. Giardipain-1 (a
protein) caused a robust demolition of monolayers. While the preincubation of
giardipain-1 with allicin (ALC) and allyl mercaptane (AM) prominently diminished
such outcomes while diallyl disulphide (DADS) lessened the occurrence of cells
presenting apoptotic impairment. In general, the garlic thioallyl compounds showed
proteolytic activity of giardipain-1 (Fig. 12.6) (Argüello-García et al. 2018).
12.4.3 Nanoformulation Based Applications
The phytoconstituents are gaining much attention but some of their drawbacks such
as poor solubility, stability, and inauspicious bioavailability and lack of targeting
ability have constrained their clinical applications. On the other side, nanotech-
nology has been proved to reduce such types of shortcomings by the use of
nanocarriers such as NPs, liposomes, carbon nanotubes, quantum dot, and hydrogel,
etc. These nanocarriers not only reduce the dose but also enhance their targeting
potential including improvement of the solubility and stability (Bishnoi et al. 2014,
2020; Fahimirad and Hatami 2019; Jain et al. 2016, 2018, 2019, 2020; Jain and Jain
2016a, c, 2016; Kumari et al. 2018; Prajapati et al. 2019; Saraf et al. 2020; Verma
et al. 2020). Some potential applications are discussed here and tabulated in
Table 12.3. Many research groups were conducted with clinical trials to evaluate
their actual performance. The clinical trials were taken from the clinicaltrials.gov
(Table 12.4).
12.4.4 Nanoparticles
The NPs due to their smaller size, biocompatibility, and their easy modification has
shown its potential for antimicrobial, drug, gene, and vaccine delivery.
Biodegradable polymeric NPs have shown microbicidal activity (antifungal and
antibacterial efficacy, etc.) by reducing osmotic stability when interacted with
microbial cell and cytoplasm membrane (Landriscina et al. 2015; Prajapati et al.
2020). In this series, Bhawana et al. (2011) prepared the curcumin NPs with very
small size, freely soluble in water, and have very good antibacterial activity.
Therefore due to smaller size, it was penetrated within the cell wall and inhibited
more as compared to the pure. The TEM study revealed that the nano curcumin
invaded the cell by disrupting the cell wall and destroyed the bacterial cell (Bhawana
et al. 2011). Adahoun et al. (2017) prepared curcumin NPs by wet milling apparatus
and the prepared formulation was analyzed in vitro for antimicrobial activity on four
different bacterial strains two gram-positive (Micrococcus luteus ATCC 9341,
Staphylococcus aureus ATCC 29213), two gram-negative (Escherichia coli ATCC
25922, Pseudomonas aeruginosa ATCC 27853) the result showed that the
NPs-containing curcumin was effective and safe toward a different type of bacterial
strain (Adahoun et al. 2017). dos Santos et al. (2016) reported the Polyethylene
glycol containing gold NPs loaded with curcumin and cell viability of different
strains of bacteria like Staphylococcus aureus, Staphylococcus epidermidis,
Pseudomonas aeruginosa, Escherichia coli, Citrobacter freundii, and Klebsiella
pneumoniae was studied using broth dilution assay and the result was enchanting
that the percentage of inhibition was more than 80 percent with 32 mM of the
prepared formulation (dos Santos et al. 2016). In another study, Sharifi-Rad et al.
(2014) used allicin from garlic extract in combination with gold NPs and applied it
against the methicillin-resistant Staphylococcus aureus spp. in the rat model. The
results proved that there was a synergistic effect of allicin with silver NPs and the
inhibition was more in combination as compared to the group treated separately
(Sharifi-Rad et al. 2014). Further, El-Refai et al. (2018) prepared different metallic
NPs like silver, copper, iron, and zinc and these are investigated against the different
microbes such as Staphylococcus aureus, Klebsiella pneumoniae, Candida albicans,
Bacillus subtilis, Erwinia. carotovora, and Proteus vulgaris and it was observed that
silver and zinc NPs with garlic extract were strongest bactericidal with inhibition
Table 12.3 Application of turmeric and garlic phytoconstituents
12
Active Nanocarrier Disease/microbe(s) Remark References

constituent
Curcumin Silica NPs Pseudomonas Bacteria in planktonic condition and bacterial biofilm Mirzahosseinipour
aeruginosa and Staphylococcus production and improved wound healing properties et al. (2020)
aureus
Curcumin Solid lipid Escherichia coli and The antimicrobial efficacy was improved and showed a Jourghanian et al.
NPs Staphylococcus aureus synergistic effect when given with other antibacterial (2016)
compounds
Curcumin Hydrogel Listeria innocua Hydrogel formulation significantly reduced the bacteria level Tosati et al. (2018)
from 4 to 1 log CFU/mL
Curcumin Silver NPs Escherichia coli Pseudomonas MIC showed relatively more efficacy against Gram-negative Alves et al. (2018)
aeruginosa and Staphylococcus bacteria than gram-positive
Antimicrobial Application Potential …
aureus
Curcumin NPs Trichophyton rubrum0 Antimicrobial photodynamic inhibition by curcumin Baltazar et al.
completely inhibited fungal growth by apoptosis via ROS and (2015)
RNS
Diallyl Niosomes Candida albicans Niosome formulation markedly decreased fungal load and Alam et al. (2009)
sulfide mortality when compared with the free diallyl sulfide
Diallyl Liposomes Candida albicans Liposomal formulation significantly reduced residual fungal Maroof et al.
sulfide load when compared with diallyl sulfide (2010)
Garlic Copper Staphylococcus aureus, Extract stabilized the copper NPs. The developed NPs showed Amatya and Joshi
extract NPs Escherichia coli much antimicrobial efficacy toward gram-negative bacteria (2020)
(Escherichia coli)
Diallyl Nanorod Staphylococcus aureus The combination treatment boosted the activity even at a low Rauf et al. (2018)
sulfide concentration and markedly inhibited MRSA biofilm
Allicin – Trichophyton rubrum MIC50 and MIC90 ranged from 0.78–12.5 lg/ml for allicin. Aala et al. (2012)
Allicin showed better antifungal activity than ketoconazole
(continued)
423
424
Active Nanocarrier Disease/microbe(s) Remark References

constituent
Allicin – Trichophyton rubrum Pure allicin extract was more efficient than garlic extract to Aala et al. (2014)
impede the h0yphae cell growth
Allicin – Antibacterial and antifungal The various thiosulfonates analogous of allicin were tested. Leontiev et al.
activity The more volatile compounds exhibited substantial (2018)
antimicrobial properties
Ajoene – Antimycobacterial activity When the ajoene was treated with macrophage. Ajoene Choi et al. (2018)
stimulated the JNK followed by the initiation of ROS
production and accumulation resulted in stimulation of ER
stress and autophagy
Allicin, – Various gram-positive species Both allicin and s-allyl cysteine showed better antimicrobial Uzun et al. (2019)
s-allyl activity to treat ear canal and middle ear infections against a
cysteine, wide range of bacteria
Curcumin – Antibacterial activity against A combination of curcumin with tetracycline showed a Sukandar et al.
Escherichia coli and synergistic effect by rupture of the bacterial cell wall (2018)
Staphylococcus aureus
S. K. Prajapati et al.
Table 12.4 Clinical trials of phytoconstituents of turmeric and garlic

Clinical trial id Disease Drug Phase Status Period Sponsored by
Turmeric
NCT02018328 Helicobacter Curcumin Not Unknown Jan 2014– Gingold Belfer
pylori applicable Dec 2015 Rachel
infection
NCT04012424 Acute Curcumin Not Not yet Aug 2019– Cairo University
Pulpitis applicable required Nov 2020
NCT03790605 Periodontitis Curcumin Phase III Recruiting Sep 2019– KLE Society’s
Dec 2020 Institute of
Dental Sciences
NCT01035580 Uterine Curcumin Phase I Completed Jan 2010– Emory
Cervical Jan 2012 University
Dysplasia
NCT03746158 Metabolites Curcumin Not Not yet Dec 2018– University of
Gut applicable required Apr 2019 Massachusetts,
Microbiota Amherst
NCT03141918 HIV Curcumin Not Completed Sept 2017– Universidade
infection applicable Dec 2017 Federal do Rio
Grande do Norte
NCT03016039 Wound Curcumin Not Unknown Jan 2017– Meir Medical
infection in applicable Oct 2017 Centre
female
NCT04071106 Psoriasis Turmeric Phase II Not yet 19-Sep Zagazig
extract required University
Turmeric Phase III
extract in
olive oil
NCT02152475 Oral Curcumin Phase I Completed 13-May University of
disinfection mouthwash Sao Paulo
NCT02442453 Chronic Curenext Phase IV Completed 14-Jan Tatyasaheb Kore
Periodontitis gel Dental College
NCT04032132 Periodontitis Curcumin Phase IV Completed 16-Aug Ain Shams
paste University
Garlic
NCT03795636 Intracanal Garlic Not Completed Oct 2016– Minia University
irrigant applicable Aug 2018
NCT01198223 Chronic oral Garlic, Phase II Completed July 2010– Qazvin
candidiasis nystatin Oct 2010 University of
Medical
Sciences
NCT03492723 Periodontitis Garlic Not Recruiting Apr 2018– Hadassah
applicable Apr 2020 Medical
Organization
zone 12.6 mm of killing Proteus vulgaris strain (El-Refai et al. 2018). In the next
series of modified silver NPs, Vimala et al. (2011) prepared curcumin fabricated
chitosan polyvinyl alcohol silver NPs and reviewed its antimicrobial activity on
some wound born bacteria and fungi, i.e., Escherichia coli, pseudomonas,
micrococcus, Staphylococcus, and Candida albicans. They found that curcumin

loaded chitosan silver nanocomposite having very remarkable potential to inhibit the
growth of bacterial colonies as compared to other blank NPs and pure curcumin and
this rapid inhibition was noticed for 7 h (Vimala et al. 2011).
12.4.4.1 Nanohydrogels
The need for hydrogel is due to the targeted delivery of the compound and many
more like preserving the activity improvement in the stability taste masking of the
compound and enhances patient compliance. So that delivery of curcumin with
hydrogel was also needed to enhance the biocompatibility. Milk protein is a
macronutrient. Bourbon et al. (2016) reported lactoferrin encapsulated glyco-
macropeptide nanohydrogels loaded with curcumin and caffeine to find its
antimicrobial property against Staphylococcus aureus and Escherichia coli by the
disc agar diffusion test and they found that the lactoferrin encapsulated curcumin
showed a significant increase in antimicrobial activity (p < 0.05). The prepared
hydrogel containing curcumin and caffeine was found to release them based on pH,
the first release at pH 2 was relaxation dependent and at pH 7 it followed Fick’s
diffusion (Bourbon et al. 2016). In another study, Ravindra et al. (2012) slightly
modified curcumin loaded silver hydrogel nanocomposite by redox
co-polymerization method, and the antibacterial activity on nutrient agar medium
was noted excellent due to presence of curcumin which extremely inhibited
Escherichia coli in comparison to the plain hydrogel without curcumin (Ravindra
et al. 2012). Archana et al. (2015) prepared curcumin loaded nano-cubosomal
hydrogel for topical delivery which was self-assembled with size 75.2 nm. They
found the antibacterial activity against Escherichiacoli and reported that the zone of
inhibition of cubosomes was significantly high, i.e., 16.20 ± 4.26 mm than pure
curcumin 11.36 ± 1.14 mm at 24 h (Archana et al. 2015).
12.4.4.2 Quantum Dots
Quantum dots (QDs) reveals size-dependent optical properties and could be the
nanomaterial of choice for the antimicrobial application. Under irradiation, QDs
produce free radicals, of which the quality and the type are regulated by their core
materials. It is well understood that the extra number of free radicals are destructive
to microbes (Ipe et al. 2005; Lu et al. 2008). The crucial role for antimicrobial
activity of QDs is considered to be reactive oxygen species (ROS) (Fig. 12.4). QDs
are usually targeted to the cell wall and cell membrane due to the availability of
phosphatidylglycerol and lipoteichoic acid (Manna et al. 2019). There are many
reports found that there is biofilm formed around the different strain of bacteria, so
to inhibit on a vast scale the QDs with the curcumin was prepared by Singh et al.
(2017) and it was having very good dispersion rate in water and its antibacterial
activity in contrast to the formation of biofilm on gram-positive and negative
bacteria revealed that there was complete inhibition of Escherichia coli, but in case
of Pseudomonas aeruginosa, it was completely resistant side by side to the other
film formed by the bacteria like Staphylococcus epidermis, the destruction was 50%
with the concentration 0.25 mg/ml (Singh et al. 2017).
12.4.4.3 Carbon Nanotubes
Carbon nanotubes (CNTs) have been extensively used for their application in the
delivery of antimicrobials. Length of CNTs potentially affect their antimicrobial
potential, the shorter length of CNTs shows more bactericidal performance. The
open end such type of the CNTs interacts with microorganisms and disrupts the cell
membrane. Antimicrobial activity can be improved by functionalizing with
chemical groups or by coating with metallic NPs (Al-Jumaili et al. 2017; Prajapati
et al. 2020). The antimicrobial application potential of CNTs generally hinges on
numerous operational circumstances such as temperature, pH, retention time, and
solvent (Kassem et al. 2019). Curcumin was delivered by another bio-nano com-
posite system that was prepared and modified by Chegeni et al. (2020). They had
firstly modified the calcium alginate single-walled CNTs which was modified with
the surface by glucose. This carrier was proved to be best in carbon nanotube carrier
due to its maximum therapeutic activity and minimum undesirable effect or toxicity.
The antimicrobial pattern of this modified CNTs loaded with curcumin was eval-
uated against Escherichia coli and Bacillus cereus. Though, inhibition zone diam-
eter showed that the curcumin loaded CNTs were more effective in comparison to
the curcumin alone (Chegeni et al. 2020).
12.4.4.4 Polymeric Micelles
It is a new class of vehicles to deliver poorly soluble drugs which can be modified
according to the need (Khan et al. 2018). Therefore, Margaritova et al. (2019)
prepared the modified copolymeric micelle based on Pluronic (P123 and F127)
having alkylphosphacoline with curcumin to investigate the synergistic antibacterial
effect against Staphylococcus aureus strain and it was seen that 1:1 ratio of P123/
F127 was enhanced dramatically (Margaritova et al. 2019). In another study, Huang
et al. (2017) prepared silver decorated polymeric micelle tethered with curcumin
encapsulated into poly(e-caprolactone) through hydrophobic interaction and it was
tested against Pseudomonas aeruginosa (gram-negative) and Staphylococcus aureus
(gram-positive) and the antibacterial activity was very good and it was observed
that there was a synergistic effect with the inhibition of bacteria when curcumin was
incorporated with the silver decorated micelles in comparison to the micelles
without curcumin (Huang et al. 2017). Mixed polymeric micelles were developed
by Akbar et al. (2018) to compare the antimicrobial efficacy between micellar
formulation and curcumin alone. The found results proposed that curcumin loaded
micelles had significant inhibitory activity toward bacteria and fungi compared to
pure curcumin. It was found that the MICs of curcumin against 10 strains of
Staphylococcus aureus varied between 125 and 250 lg/mL (Akbar et al. 2018).
12.4.4.5 Microemulsions
Because of their exceptional properties of easy production, solubilization,

long-lasting stability, and biocompatibility, microemulsion have ever-increasing as
a looming antimicrobials delivery system, either as a carrier for topical applications
or oral delivery for poorly water-soluble active constituent (Shukla et al. 2018).
Bactericidal microemulsion using myristic acid was developed for the delivery of
curcumin by Liu et al. (2012). Myristic acid is a fatty acid that shows antibacterial
potential. Curcumin exposed superior inhibitory capacity toward Staphylococcus
epidermidis when compared to myristic acid and there were 43 folds reduction in
the IC50 concentration for curcumin was observed compared to azelaic acid.
Curcumin loaded microemulsion at the concentration of 0.86 µg/mL inhibited
bacterial growth by 50% when it was compared with dimethyl sulfoxide solution of
curcumin. The finding revealed the potential of formulation as an antimicrobial and
for acne vulgaris (Liu and Huang 2012). The comparative activity against
Escherichia coli and Staphylococcus aureus was evaluated using garlic oil and their
microemulsion formulation. The garlic oil itself had not shown antibacterial efficacy
for Escherichia coli (gram-negative) while it showed significant results for
Staphylococcus aureus (gram-positive). The microemulsion having the garlic oil
(150 lg/mL) showed marked antibacterial activity (Zheng et al. 2013).
12.5 Conclusion
The research community is attracting toward the phytochemicals due to their

diversity of applications in various diseases as an antimicrobial, anticancer,
anti-inflammatory, antimalarial, and antioxidant agent, etc. Turmeric and garlic
both are full of many therapeutic abilities. These phytochemicals have proved their
potential as antimicrobial not only in the era of nanotechnology but are being used
traditionally for very long without causing cytotoxicity. The nanocarriers have been
proved to improve their antimicrobial performance by enhancing their stability,
bioavailability, and targeting ability. These nanocarriers can easily be modified for
the desired purpose. The phytoconstituents of garlic and turmeric are also given
with other allopathic medicines, which showed the potential to improve their effi-
cacy. Looking at the present, some more research studies must be conducted to
accelerate their therapeutic efficacy, targeting ability so that their side effects and
adverse effects can be diminished.
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Chapter 13
Carvacrol (Origanum vulgare):
Therapeutic Properties and Molecular
Mechanisms
Arijit Mondal, Sankhadip Bose, Kamalika Mazumder,

and Ritu Khanra
Abstract Carvacrol (cymophenol) has been reported to be the major component of

aromatic plant essential oils belonging to the Labiatae family, such as Origanum
vulgare and Thymus vulgaris. It is a phenolic moiety containing monoterpenoid
compound, chemically known as 5-isopropyl-2-methyl phenol. Carvacrol is used in
food products both as a flavoring component and as preservative. Current research is
being directed to establish a potent compound carvacrol with diverse pharmaco-
logical activities, such as antioxidant, antifungal, antimicrobial, anti-inflammatory,
anticancer, hepatoprotective, anti-spasmodic, anti-parasitic, and insecticidal activi-
ties. There are various derivatives of carvacrol, which possess antimicrobial action
against microbial pathogens. The carvacrol phenolic group does have a good
antimicrobial and anti-oxidative function. Its hydrophobic nature is owing to the
existence of benzene ring and methyl and isopropyl substituents helped the moiety to
bind with guanine present in DNA. This review is based upon an evaluation of the
existing data or knowledge regarding the extraction of carvacrol, chemical compo-
sition of the potent derivatives synthesized from carvacrol, their pharmacological and
toxicological effects. This chapter documents the above-mentioned activities and
strived to critically assess the molecular pathway involved in the action of carvacrol.
Keywords Carvacrol Essential oil Antimicrobial Anticancer Toxicity
A. Mondal (&)
Bengal College of Pharmaceutical Technology, Dubrajpur 731123, India
e-mail: juarijitmondal@gmail.com
S. Bose
Bengal School of Technology, Chuchura, Hooghly 712102, India
e-mail: sankha.bose@gmail.com
K. Mazumder
BCDA College of Pharmacy and Technology, Kolkata 700127, India
e-mail: kamalika@bcdapt.com
R. Khanra
JIS University, Kolkata 700109, India
e-mail: ritukhanra@yahoo.co.in
https://doi.org/10.1007/978-3-030-54027-2_13
438 A. Mondal et al.
13.1 Introduction
Essential oils were applied in traditional remedies. It relates to essential oils as

ethereal/volatile oils having an aromatic oily texture, collected from the various
sections of herbs and then used as food flavors. The convenient form of sepa-
rating essential oils is the inexpensive and easy-to-use hydro-distillation
methodology. Terpenes, terpenoids, aromatic, and aliphatic analogs are the vital
components of the essential oils (Marinelli et al. 2018). Carvacrol [2-methyl-5-
(1-methyl ethyl)-phenol] mainly comes from many essential oils of the family
Labiatae/Lamiaceae (Kirimer et al. 1995). It is a phenolic moiety containing
monoterpenoid having a molecular formula C10H14O. It is also found in the
essential oils of oregano (Origanum vulgare, Origanum ehrenbergii, Origanum
syriacum), sage apple (Salvia fruticosa), Calamintha origanifolia, thyme (Thymus
vulgaris), Lepidium flavum, Citrus aurantium, and many more. Friedel-Crafts
alkylation reaction was employed to generate carvacrol using o-cresol and iso-
propanol as reactants and utilizing mesoporous superacidic UDCaT‐5 as catalyst
(Yadav and Kamble 2009). Carvacrol is used primarily in food products for its
flavoring and preservatives nature, and likewise had a significant application in
the pharmaceutical industry (Can Baser 2008). Carvacrol’s anticancer activity has
gained recognition in recent times (Almanaitytė et al. 2020). It possesses other
pharmacological actions such as antioxidant (Han et al. 2017), antimicrobial
(Liolios et al. 2009), antibacterial (Menniti et al. 2010), anti-inflammatory (Landa
et al. 2009), antiviral activities (Pilau et al. 2011), and hepatoprotective properties
(Aristatile et al. 2009). The European Commission (2012) and the Food and Drug
Administration (FDA 2017) have classified carvacrol as a safe substance from a
toxicological perspective and its application is highly attractive as an additive in
foods.
There are only a few previous reports that present an in-depth overview of this
important field of research. Many of the preceding publications focus exclusively
on the compilation of the various pharmacological activities of the secondary
metabolite carvacrol for study on natural products (Mohammad et al. 2017;
Sharifi-Rad et al. 2018; Bayir et al. 2019). This review looks at the pharmacology of
carvacrol, and its analogs concerning their significant potential anticancer and
antimicrobial functionality to become a clinical drug. The data procured covers
published preclinical study, research papers, and review of molecule carvacrol
isolated from a diverse group of plants. This study would investigate the antimi-
crobial and anticancer function of the metabolite carvacrol and the derivatives
synthesized from carvacrol, emphasizing their chemical structure and discovering
the action processes that underlie it, for the discovery of potent metabolites of
therapeutic potential.
13 Carvacrol (Origanum vulgare): Therapeutic Properties … 439
13.2 Literature Search Methodology
In vitro analysis, in vivo experimentation, and human clinical studies which

explored the antibacterial and antiproliferative potency of phytoconstituent car-
vacrol, and its analogs by inhibiting various pathways were screened using
authentic databases, such as ScienceDirect, PubMed, Google Scholar, and Springer
reference. Relevant full articles published until April 2020 in peer-reviewed jour-
nals have been included. Conference abstracts and unpublished findings have not
been included. Only papers published in the English language have been considered
and included in this review. The keywords used for literature search included
carvacrol, antimicrobial, cancer, tumor, proliferation, phytochemicals, terpenoids,
in vivo, in vitro, and clinical studies.
13.3 Extraction and Isolation of Carvacrol
Like for most naturally occurring substances, the isolation and characterization of
carvacrol are based mainly on chromatographic approaches. Carvacrol is an aro-
matic chemical compound bearing a complex composition, isolated from the plant’s
raw material either by steam distillation, dry distillation, or a relevant automatic
process without heating. Carvacrol is extracted from an aqueous environment by a
physical hydro-distillation process which does not adversely impact their compo-
sition utilizing Clevenger equipment fitted with a microwave apparatus (Dhifi et al.
2016, Cáceres et al. 2020). Obeying to this information, many scientists processed
the isolation of the constituent carvacrol from the stems and leaves of many plants,
such as Origanum ehrenbergii, Origanum syriacum, Salvia fruticosa, and
Calamintha origanifolia. To immerse the whole plant in a flask, a specific amount
of water is added and boiled for 3 h. Carvacrol is driven with water vapor into a
condensed form owing to its volatile nature and later recollected which was purified
with anhydrous sodium bicarbonate. It is then stored in the dark at 4°C in a sealed
glass container for further utilization (Galehassadi et al. 2014, Cáceres et al. 2020).
Its quantification can be analyzed by high-performance liquid chromatography
(HPLC). Nonetheless, HPLC approach for carvacrol isolation has now become
commonly adopted. It is very sensitive and has the potentiality to identify extremely
small concentrations of a substance. Carvacrol is the major constituents of
Origanum vulgare with a concentration of 83.7% (Laothaweerungsawat et al.
2020). Another documented report revealed that carvacrol content is 48% in
Origanum vulgare (Bahmani et al. 2019)
Another method widely considered for carvacrol analysis is gas chromatogra-
phy. While this approach is not as common as HPLC, both a qualitative and a
quantitative study of carvacrol were carried out. Excellent performance but with
small productivity and a basic process were the key reasons for its adoption. It was
identified and quantified using the gas chromatography-mass spectroscopic
(GC-MS) analysis (Cáceres et al. 2020). Current techniques for the structural elu-
cidation of carvacrol are spectroscopic instruments including high-resolution mass
spectrometry (MS) and nuclear magnetic resonance (NMR). These approaches may
be used either individually or in combination with chromatographic processes
(Cáceres et al. 2020).
13.4 Biosynthesis of Carvacrol
Carvacrol was biosynthesized via the mevalonate pathway in various plants. At

first, glucose is cleaved to phosphoenolpyruvate and then it is decarboxylated and
acetylated to acetyl coenzyme A (acetyl CoA) and transformed to mevalonic acid.
The mevalonic acid is then converted to gamma-terpinene, which after aromati-
zation produced p-cymene. The hydroxylation p-cymene produced carvacrol which
is shown in Fig. 13.1 (Friedman 2014).
Fig. 13.1 Biosynthesis of carvacrol through the mevalonate pathway

Another literature has shown an increased biosynthesis of carvacrol in shoot

cultures of the Iranian plant Satureja khuzistanica by inhibiting the mevalonate
pathway (Ramak et al. 2013). The methylerythritol-4-phosphate (MEP) pathway
provides isopentenyl diphosphate (IPP) which was the precursor for the biosyn-
thesis of 90% carvacrol. Figure 13.2 showed that carvacrol was biosynthesized
from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMADP)
which were derived via the MEP pathway located in plastids (Rohmer et al. 1993;
Dudareva et al. 2005) of oregano and thyme. IPP is reversibly converted to
DMADP by isopentenyl diphosphate isomerase (IDI). The irreversible conversion
of 1-deoxy-D-xylulose-5-phosphate (DXP) into 2-C-methyl-D-erythritol-4- phos-
phate (MEP) was catalyzed by 1-deoxy-D-xylulose-5-phosphate reductoisomerase
enzyme (DXR) (Takahashi et al. 1998). The condensation of IPP and DMADP
produced geranyl diphosphate (GDP) and that was catalyzed by the geranyl
diphosphate synthase (GDS) enzyme (Lichtenthaler 1999; Burke et al. 1999; Majdi
et al. 2017). The cyclization of GDP by gamma-terpinene synthase produced
gamma-terpinene. The enzymes such as CYP71D178, CYP71D180, and
CYP71D181 of cytochrome P450 (CYP) monooxygenases converted c-terpinene to
yield carvacrol (Crocoll et al. 2010; Crocoll 2011).
13.5 Physical Properties
It is found in liquid form having molecular weight 150.22. Its boiling point is about
237− 238 °C; density is 0.967 and melting point 0 °C. Its kmax value showed
277.5 nm in 95% ethanol in the UV spectrophotometer. Its pKa value is 10.9. It is
insoluble in water, soluble in 95% ethanol. Carvacrol has a pleasant pungent taste
and odor, sweet and spicy like marjoram (Ramak et al. 2013; Friedman 2014).
13.6 Metabolism and Excretion of Carvacrol
Carvacrol’s ADME process in the human body system is very rapid and it follows
two types of pathways. The major metabolic pathway of carvacrol depends on the
phase II metabolism related to the esterification of phenolic group of carvacrol by
glucuronidation and sulfation, but when used in low dose, another minor route of
carvacrol metabolism was also observed. In the minor pathway transformation of
the terminal methyl groups to primary alcohols took place (Alagawany 2015; Dong
et al. 2012).
Austgulen et al. 1987 also showed the pattern of metabolism of carvacrol
(Fig. 13.3) in male albino rats. After administration by oral route at a dose of
1 mM/kg, the major portion of metabolites was excreted through urine where seven
metabolites of carvacrol were identified, such as 2,3-dihydroxy-p-cymene, 2-
(3-hydroxy-4-methyl phenyl) propan-2-ol, 2-(3-hydroxy-4-methyl phenyl)
Fig. 13.2 Biosynthesis of carvacrol through the MEP pathway
propan-1-ol, 2-hydroxy methyl-5-(1-methyl ethyl) phenol, 2-(4-hydroxy

methyl-3-hydroxy phenyl) propan-1-ol, 2-(3-hydroxy-4-methyl phenyl) propionic
acid, and 2-hydroxy-4-(1-methyl ethyl) benzoic acid (Austgulen et al. 1987).
Another experiment was done by Michiels et al. (2008), utilizing gastric fermen-
tation simulation technique on pig. Carvacrol was not degraded in jejunum simu-
lations; rather 29% degradation occurs in the cecum. When piglets were
administered with a dose of 13 mg/kg body weight, it was completely absorbed in
the stomach and the proximal section of the small intestine where it showed a
half-life of 2.05 h in the whole digestive tract (Michiels et al. 2008).
Fig. 13.3 Metabolic pathway of carvacrol
13.7 Acute Toxicity of Carvacrol
Data on carvacrol toxicology is limited. Various toxicology studies have been done
in different animal models. Andre et al. (2016) investigated the acute toxicity study
in carvacrol administered through esophageal gavage route in Swiss albino mice.
The result revealed that depression occurs within 10 min of carvacrol administra-
tion, coma, and death of animals within 1 h to 3 days. Administration of the
essential oil carvacrol in rabbits, rats, and dogs exhibited the non-toxic nature,
although the detailed information was not documented (Budavari 1989; Livingston
1921; Andersen 2006; Ghorani et al. 2019). But some reports showed the toxic
behavior of the test drug carvacrol in Osborne-Mendel rats and mice, where
depression, ataxia, decreased spontaneous motor activity, somnolence, coma, and
death were observed (Jenner et al. 1964; Viana et al. 1981) Detail reports of animal
acute toxicity studies have been enlisted in Table 13.1. However, the likely toxicity
impacts of carvacrol on humans have not been reported till date.
Table 13.1 Toxicological study of cavacrol

Species Toxicity study LD50/Doses Toxic signs and death Reference
time (DT)
Osborne-Mendel Acute Oral 810 mg/kg Depression, coma; DT: Jenner
rats Toxicity body weight 1hr-3 days et al.
(1964)
Rabbits Acute Oral 100 mg/kg No detail information Budavari
Toxicity body weight (1989)
Swiss albino Acute toxicity 919 mg/kg Low toxicity, other Andre et al.
mice through body weight effects not specified (2016)
esophageal
gavage route
Rats Acute toxicity >0.5 g/ Toxicity not specified; Andersen,
kg < 5 g/kg DT: 24 h at 5 g/kg dose (2006)
body weight
Rabbits Acute toxicity Dose: 0.5 g/ Toxicity not specified Livingston
kg-2 g/kg DT:18 h to 35 days (1921)
body weight
Dog Acute Parental 0.31 g/kg Not specified Ghorani
Toxicity body weight et al.
(Intravenously) (2019)
Mice Acute Parental 73.3 mg/kg Ataxia, decreased Viana et al.
Toxicity body weight spontaneous (1981)
(Intraperitoneally) motoractivity,
somnolence,death;
DT-5 days
13.8 Antioxidant Activity
Antioxidants are scavenging the ROS, including free radicals, and thereby protecting
cells from cellular stress. They also slow down the prostaglandin synthesis, stimulate
the drug-metabolizing enzymes, and exhibit other biological activities such as DNA
defense, enzyme-induced hepatotoxicity, and cancer imitation inhibition. The
explanation of antioxidant activity was the availability of hydroxyl group
(OH) linked to the aromatic ring (Aeschbach et al. 1994). During the carvacrol
molecule’s reaction with free radicals, it donates hydrogen atoms to an unpaired
electron and generates another radical that is produced by a molecule resonance
structure. Carvacrol can interfere with the cell phospholipid membrane or
low-density lipoprotein and minimize the synthesis of lipid peroxidation and nitric
oxide, contributing to oxidative degradation of cell membranes (Karimian et al.
2011; Teissedre and Waterhouse 2000). Nitric oxide is created by the spontaneous
decomposition of sodium nitroprusside, which has been effectively scavenged by
carvacrol (Aeschbach et al. 1994). Differences in vitro and in vivo studies have
confirmed that carvacrol has strong antioxidant properties. As an antioxidant, car-
vacrol can lower stress by enhancing the antioxidant activity of enzymes like
superoxide dismutases (SOD), catalase (CAT), and glutathione peroxidase
(GPx), and remove free radicals like peroxide, H2O2, superoxide, and nitric oxide
(Samarghandian et al. 2016). In diabetic rats, carvacrol reduced oxidative stress
through the improvement of the activity of antioxidant enzymes (Deng et al. 2013).
In vitro analysis showed that the amount of intracellular ROS production of mouse
V79 fibroblast cells depends on the dose-dependent manner of carvacrol. The pro-
duction of ROS decreases with the increasing concentration of carvacrol (Ündeğer
et al. 2009a). Some anti-oxidant activities are also performed on in vivo studies.
Hydrogen peroxide resistance–induced damage to DNA in hepatic and testicular
tissue was gradually decreasing in rats when carvacrol was administered in drinking
water (at 30 and 60 mg/kg for 7 days or 15 and 30 mg/kg for 14 days) (Slamenova
et al. 2013). Other studies have also shown that carvacrol has a protective role
against lipid peroxidation and induces enhanced endogenous antioxidant defensive
mechanisms in hepatocellular carcinogenesis caused by N-nitrosodiethylamine and
antioxidant function against hepatotoxicity in rats. Carvacrol has enhanced the
reduced function of antioxidant enzymes and improved the lipid peroxidation in
ethanol-exposed rat hippocampus (Wang et al. 2017; Mehrjerdi et al. 2020).
13.9 Antimicrobial Effect
The efficacy of the carvacrol depends on their phenolic group as well as hydroxyl
group which is attached with the phenolic structure and increased membrane per-
meability by working on the micro-organism at low concentration. The inhibitory
activity of phenols may be clarified by associations with micro-organisms’ cell
membrane and is also associated with the compounds’ hydrophobicity. Most
hydrophobic molecules are usually harmful and the cytoplasmic membrane is often
the main source of harmful activity. Besides, lipophilic products showed a strong
affinity for cell membranes, and their insertions induced changes in the physico-
chemical properties of membranes and increased depletion of intercellular ATP
(Ultee et al. 2002; COX et al. 2007). The interactions between antimicrobial
compounds and cell membranes are defined to affect both the lipid order and the
stability of the bilayer, resulting in a decrease in membrane integrity and an increase
in the passive flux of protons through the membrane. This effect is documented
particularly for compounds with a log P greater than 3. By consensus, the most
effective antimicrobial compound was carvacrol, which has a log P of 3.52 (Ben
Arfa et al. 2006). Carvacrol, by partitioning the phospholipid fatty acid chains,
created ion channels across the membrane, eventually allowing ions to exit cyto-
plasm (Ultee et al. 2002). Carvacrol has showed bacteriostatic and bactericidal
activity on food pathogens such as Vibrio cholerae, Campylobacter jejuni, E. Coli,
Listeria monocytogenes, S. aureus, Staphylococcus epidermidis, Lactobacillus
sakei, P. aeruginosa, Pseudomonas putida, Streptococcus mutans, and Bacillus
subtilis (Friedman et al. 2002; Lambert et al. 2001; Mathela et al. n.d.;
Rattanachaikunsopon and Phumkhachorn 2010; Ravishankar et al. 2010). Also, the
presence of a free hydroxyl carvacrol group and its influence on the delocalized
electron system by growing the gradient across the cytoplasmic membrane is

essential for its antibacterial action (Knowles and Roller 2001). Carvacrol not only
inhibited bacterial cell growth but also induces cell death through increased
depolarization of cell membrane which leads to elevated rapid oxidizing burst in
E. coli. Cellular DNA, protein, and K+ were released from cells, which have been
triggered by carvacrol. On treatment with carvacrol, there is a decrease in the
protein expression levels of inflammation, such as TNF-a and COX-2 (Khan et al.
2020)]. Phosphate ion leakage took place in Staphylococcus aureus and
P. aeruginosa cells, which were treated with carvacrol containing oregano essen-
tial oil (Friedman et al. 2004; Lambert et al. 2001). Carvacrol also showed anti-
fungal activity that may act through the membrane and cell wall disruption. An
analysis of the related performance of carvacrol on different micro-organism strains
was presented in Table 13.2.
Table 13.2 Antimicrobial and antifungal activity of carvacrol using different techniques
Sl no Organism Antimicrobial activity Reference
1 Aeromonas salmonicida subsp. Minimum Inhibitory Hayatgheib
Salmonicida Concentration (MIC)-344 lg/ml et al. 2020
ATCC 14174 MIC-688 lg/ml
CAE 235 MIC-344 lg/ml
CAE 452 MIC-344 lg/ml
CAE 258
2 Staphylococcus aureus ATCC MIC- 12.5% v/v (Marino
6538 MIC- 25% v/v et al. 2020)
Staphylococcus epidermidis MIC- 25% v/v
ATCC 34984 MIC- 12.5% v/v
Listeria monocytogenes ATCC MIC- 25% v/v
13932
Bacillus subtilis ATCC 6633
Pseudomonas aeruginosa
ATCC9027
3. Salmonella enterica At 1% concentration, no survival Ravishankar
of the microorginism et al. (2010)
4 Pseudomonas fluorescens MIC-1 g/l Ben Arfa
Escherichia coli MIC-0.25 g/l et al. (2006)
Staphylococcus aureus MIC-0.25 g/l
Bacillus subtilis MIC-0.25 g/l
Lactobacillus plantarum MIC-˃3 g/l
Saccharomyces cerevisiae MIC-0.25 g/l
5 Escherichia coli MIC-0.225 µL/ml Burt, (2004)
Salmonella typhimurium MIC-0.225 µL/ml
Staphylococcus aureus MIC-0.175– 0.450 µL/ml
Listeria monocytogenes MIC-0.375–5 µL/ml
7 Malassezia furfur Disk diffusion assay: 15 mm (Adam et al.
Trichophyton rubrum Complete inhibition 1998)
Trichosporon beigelii Complete inhibition
(continued)

Sl no Organism Antimicrobial activity Reference
8 Bottytis cinerea MIC100 µg/mL Tsao and
Monllinla fmcticokz MIC100 µg/mL Zhou, (2000)
9 Aspergillus niger PTCC 5154 MIC-50 µg/mL,MIF -75 µg/mL Abbaszadeh
Aspergillus fumigatus PTCC MIC-100 µg/mL,MIF -125 µg/ et al. (2014)
5009 mL
Aspergillus flavus PTCC 5004 MIC-100 µg/mL,MIF -125 µg/
Aspergillus ochraceus PTCC mL
5017 MIC-100 µg/mL,MIF -125 µg/
Alternaria alternate PTCC mL
5224 MIC-350 µg/mL,MIF -400 µg/
Botrytis cinerea ATCC 12481 mL
Cladosporium spp. PTCC MIC-300 µg/mL,MIF -350 µg/
5202, Penicillium citrinum mL
PTCC 5304 MIC-100 µg/mL,MIF -125 µg/
Penicillium chrysogenum mL MIC-150 µg/mL,MIF -
PTCC 5271 175 µg/mL
Fusarium oxysporum PTCC MIC-125 µg/mL,MIF -150 µg/
5115 mL
Rhizopus oryzae PTCC 5174 MIC-125 µg/mL,MIF -150 µg/
mL
MIC-200 µg/mL,MIF -250 µg/
mL
10 Candida albicans # Colony forming units Chami et al.
(CFU) 94.46% (2004)
11 Staphylococcus MIC- 146–292 µg/ml Sim et al.
pseudintermedius MIC- 585–1120 µg/ml (2019)
Pseudomonas aeruginosa MIC- 146–292 µg/ml
Proteus mirabilis MIC- 585 µg/ml
Malassezia pachydermatis
12 Vibrio parahemolyticus, MIC-0.5 mg/ml (Fang et al.
Shewanella putrefaciens, MIC-0.5 mg/ml 2019)
Staphylococcus aureus MIC-0.125 mg/ml
Pseudomonas fluorescens MIC-0.5 mg/ml
13 Bacillus subtilis MIC-0.125% v/v Chraibi et al.
2020
14 Escherichia coli MIC-150 µg/ml Khan et al.
Minimum bactericidal 2020
concentration (MBC)-300 µg/ml
13.10 Anticancer Effect of Carvacrol and the Related

Mechanism of Actions
Carvacrol extracted from various medicinal plants showed potent cytotoxic activ-
ities against various carcinoma cell lines by inducing apoptosis through the influ-
ence of numerous proteins associated with the apoptotic pathway, MAPK pathway,
and PI3K/Akt pathway as shown in Table 13.3. It inhibited the proliferation of two
Table 13.3 Pharmacological mechanisms of carvacrol involved in anticancer activities
448
S. Cell lines Effect and mechanism IC50 Reference

No. (µM/l)/dose
1 Human colon cancer cell lines: #Proliferation and migration, cell cycle arrest at HCT116: 544.4 Fan et al. (2015)
HCT116, LoVo G2/M phase, #Bcl-2, "Bax, #MMP-2, #MMP-9, LoVo: 530.2
"apoptosis, " MAPK, #p-ERK, "p-JNKand
"PI3K/Akt signaling pathway, #p-Akt.
2 Ovarian epithelial adenocarcinoma Induction of cytotoxicity, "apoptosis, 322.50 Elbe et al. (2020)
cell line (SKOV-3)
3 Human colorectal carcinoma: antiproliferative effect HCT-116: 92 Pakdemirli et al.
HCT-116 HT-29: 42 (2020)
Human colorectal carcinoma: HT-29
4 Prostate cancer cell line:PC3 Antiproliferative effect, reduce cell viability 25-200 lg/ml dose Trindade et al.
(2019)
5 Choriocarcinoma cell line: (JAR and Antiproliferative effect, "apoptosis, cell cycle Up to 300 lM doses Lim et al. (2019)
JEG3 cells) arrest at sub G1 phase, "Bax, "Bak, "cytochrome
c, ┴MMP, #cytosolic calcium level, " MAPK, #
p-ERK1/2, "PI3K/Akt signaling pathway,
"p-JNK, phosphor-P38, and P90RSK proteins,
#phosphorylation of PDK1, AKT, P70S6K, and
GSK3b
6 Human gastric adenocarcinoma cell Dose-dependent cytotoxicity, "apoptosis, WS-1: 138.1 ± 8.7 lM Günes-Bayir et al.
line AGS cells and normal human genotoxicity and ROS generation, "Bax, AGS:82.57 ± 5.5 lM (2018a)
fibroblast WA-1, invivo analysis in "caspase-3, "caspase-9, # Bcl-2,
Wister rats
7 Human cervical cancer HeLa cells "Cytotoxicity, "apoptosis, " MAPK, HeLa:556 ± 39 lM Potočnjak et al.
"phosphor-ERK1/2, "caspase-9, "PARP (2018)
cleavage, #Cyclin D1, "p21, autophagy, #p-Akt,
#phospho-mTOR
(continued)
A. Mondal et al.
13

No. (µM/l)/dose
8 Human prostate cancer DU145 Antiproliferative agent, "apoptosis, "caspase-3, # DU145:84.39 lM Khan et al. (2017)
DWm, cell cycle arrest at G0/G1 phase
9 Human gastric adenocarcinoma AGS ┴proliferation, "apoptosis,, #GSH levels,, "Bax, AGS:82.57 ± 5.58 lM Günes-Bayir et al.
cells "caspase-3, "caspase-9, # Bcl-2, (2017)
10 Prostate cancer cell lines DU145 Antiproliferative effect, #MMP-2, #p-Akt, and DU145: 498.3 ± 12.2 Luo et al. (2016)
Prostate cancer cell lines: PC3 #p-ERK1/2, ┴migration, ┴invasion PC3: 430.6 ± 21.9
11 Human T lymphocyte Cells: Jurkat Inflection of T cell activity by # IL-2, # IFN-c, 50 lg/ml (Dose) Gholijani et al.
"AP-1 and "NFAT2 (2015)
12 Human acute promyelocytic leukemia Induced apoptosis, "caspase-3, "Bax, # Bcl-2, # HL-60: 100 lM Bhakkiyalakshmi
cell line (HL-60), and (immortalized DWm Jurkat: 50 lM et al. (2016)
human T cell lymphocyte cell line
(Jurkat)
13 Human oral squamous cell carcinoma: ┴Proliferation, "apoptosis, cell cycle arrest at 0-80 lM (Doses) Dai et al. (2016)
Tca-8113 G1/S phase, #cyclin D1, #CDK4, "Bax/Bcl-2
ratio
14 Human hepatomacell lines: HepG2 ┴Proliferation, "apoptosis, cell cycle arrest at HepG2: 425 lM Melušová et al.
Carvacrol (Origanum vulgare): Therapeutic Properties …
G1/S phase (2014)

15 Murine melanoma cell line:B16(F10) ┴tumer growth B16F10: 120 ± 15 lM He et al. (1997)
16 N2a neuroblastoma cells "cytotoxicity, ┴proliferation, anticancer activity >200 mg Aydin et al. (2014)
17 MDA-MB 231 ┴proliferation, induced morphological changes, MDA-MB 231: 100 Arunasree (2010)
"apoptosis
18 Human hepatocellular carcinoma cell Induced anti-proliferation and "apoptosis, HepG2: 0.4 mM/ml Yin et al. (2012)
line HepG2 "caspase-3, # Bcl-2, "cleavage of PARP, #
p-ERK1/2, "phosphorylation of p38
19 Human non-small cell lung cancer Anticancerogenic activity 100–1000 lM Koparal and
cell line: A549 Zeytinoglu (2003)
449
(continued)
450

No. (µM/l)/dose
20 Human cervical cancer cell lines, "Cytotoxicity, "apoptosis, "DNA fragmentation HeLa : 50 ± 5.95 mg/l Mehdi et al. (2011
HeLa and SiHa SiHa: 50 ± 3.89 mg/l
21 Human HepG2 cells Carvacrol protected cells against DNA-damaging 200 lM dose Slamenova et al.
effects of H2O2 was unambiguous (2013)
22 Human breast cancer cell line: MCF-7 " Cytotoxicity, "apoptosis, "caspase-3, MCF-7:244.7 ± 0.71 lM Al-Fatlawi and
"caspase-9, "caspase-6, "p53, "Bax Ahmad (2014)
23 Pparental and drug-resistant human " Cytotoxicity and " membrane and DNA H1299: 380 and 244 lM Ozkan and Erdogan
lung cancer cell lines (H1299) damage (2012)
24 Human colon carcinoma cell line "Oxidative stress at higher concentration Caco-2: 1832 ± 0.11 lM Llana-Ruiz-Cabello
Caco-2 et al. (2015)
25 V79 Chinese hamster lung fibroblast DNA damage at higher concentration Not specified Ündeğer et al.
cells (2009)
A. Mondal et al.
human colon cancer cells, such as HCT116 and LoVo, by inducing apoptosis and
triggering cell cycle arrest at the G2/M phase. It reduced the expression levels of
matrix metalloprotease-2 (MMP-2), MMP-9, and cyclin B1. It also downregulated
the expression levels of Bcl-2 and upregulated the expression levels of Bax. Apart
from this, it decreased the expression levels of phosphorylated protein kinase B
(p-Akt) associated with the activation of the PI3K/Akt signaling pathway. In
association with the activation of the MAPK signaling pathway, there is a decrease
in the expression levels of phosphorylated extracellular signal-regulated kinases
(p-ERK) and an increase in the expression levels of phosphorylated c-Jun
N-terminal kinase (p-JNK) (Fan et al. 2015). The mitochondrial membrane
potential was impaired by carvacrol treatment in choriocarcinoma cell lines, such as
JAR and JEG3 cells. The antiproliferative activity was associated with the induction
of apoptosis and cell cycle arrest at the sub G1 phase. There was a decrease in
cytosolic calcium level in JAR and JEG3 cells. The MAPK and PI3K–AKT sig-
naling pathway was affected similarly in choriocarcinoma cells via regulation of the
phosphorylation of various proteins involved in the pathway (Lim et al. 2019).
Carvacrol inhibited the proliferation of human gastric adenocarcinoma cell line
(AGS cells) and normal human fibroblast (WA-1) by the apoptotic pathway in a
dose-dependent manner but the cytotoxicity towards cancer cells was more in
comparison to normal cells (Günes-Bayir et al. 2018a, b). Apart from the apoptotic
pathway, through autophagy mechanism, the carvacrol exhibited cytotoxic activity
against human cervical cancer cells (HeLa). The mTOR, phospho‐mTOR, and
phospho‐Akt protein expression levels were reduced in cancer treated cells
(Potočnjak et al. 2018). It exhibited its antiproliferative activity against prostate
cancer DU145 cells by inducing apoptosis and activation of caspase 3. Its induced
cell cycle arrest at the G0/G1 phase and loss of mitochondrial membrane potential
occurred in the cancer cells (Khan et al. 2017). Similar observation was reported by
Bhakkiyalakshmi et al. (2016) in which carvacrol showed cytotoxic activity against
human acute promyelocytic leukemia cell line (HL-60), and (immortalized human
T cell lymphocyte cell line (Jurkat). Carvacrol inhibited the PI3K/Akt and MAPK
signaling pathways in prostate cancer cell lines DU145 and PC3, by preventing the
proliferation, migration, and invasion of the cancer cells. It reduced the MMP-2,
p-Akt, and p-ERK1/2 protein expression levels in the prostate cancer cells (Luo
et al. 2016). Carvacrol reduced the production of interleukin-2 (IL-2) and interferon
(IFN-c) in the human Jurkat T cells through the inhibition of activator protein (AP)-
1 and nuclear factors of activated T cells (NFAT2) (Gholijani et al. 2015). It
inhibited the proliferation of human oral squamous cell carcinoma (Tca-8113) as
well as metastasis and invasion of the cells by inducing apoptosis. But it triggers a
cell cycle arrest at the G1/S stage. It reduces the expression level of cyclin D1 and
cyclin-dependent kinase (CDK4) (Dai et al. 2016). A similar mechanism of action
of carvacrol was reported in human hepatoma cells (HepG2) (Melušová et al.
2014). It reduced the tumor growth of murine melanoma cells B16(F10) (He et al.
1997) and displayed potent cytotoxicity at a higher dose against neuroblastoma
cells (N2a cells) (Aydin et al. 2014). Mitogen-activated protein kinase pathway is
altered by carvacrol on treating the HepG2 cell line apart from the apoptotic
pathway (Yin et al. 2012). So overall carvacrol demonstrated potent anticancer

activity against a variety of carcinoma cell lines by inhibiting the proliferation of the
cells by apoptotic pathway triggered by activation of caspases, increased DNA
fragmentation (Koparal and Zeytinoglu 2003; Mehdi et al. 2011; Al-Fatlawi and
Ahmad 2014; Ozkan and Erdogan 2012). It also exhibited protective activity
against H2O2 induced DNA damage (Slamenova et al. 2013; Llana-Ruiz-Cabello
et al. 2015; Ündeğer et al. 2009). The efficacy of carvacrol can further be improved
by preparing a carvacrol b-cyclodextrin inclusion complex, which exhibited higher
antiproliferative activity against prostate cancer cells (PC3), in comparison to free
carvacrol (Trindade et al. 2019).
13.11 Carvacrol Derivatives with Pharmacological

Activities
The structure-activity relationships of carvacrol indicated that the presence of OH

group and delocalized electrons cloud were responsible for the pharmacological
activities of carvacrol (1) (Fig. 13.4) (Ultee et al. 2002). Removal or replacement of
the hydroxyl group with methyl ether produces lesser active compound p-cymene
(2) and methyl ether derivative of carvacrol (3) due to the lower tendency to donate
protons than the hydroxyl group (Ben Arfa et al. 2006; Marinelli et al. 2018).
Carvacryl acetate (4) is an analog of carvacrol synthesized by refluxing carvacrol,
sodium acetate, and acetic anhydride. It was then subsequently purified and char-
acterized [Ben Arfa et al. 2006]. Carvacrol was 28 times more potent than its
structural isomer (positional isomer) thymol (5) (2-isopropyl-5-methyl phenol)
against gram-positive and gram-negative bacteria like Mycobacterium tuberculosis,
Escherichia coli, Staphylococcus aureus, and Candida albicans. Menthol (6)
(2-isopropyl-5-methyl cyclohexanol), eugenol (7) (4-allyl-2-methoxyphenyl),
vanillin (8) (4-hydroxy-3-methoxy benzaldehyde) and 3-isopropyl-5-methyl phenol
(9) were equivalent potent as compared to thymol. Replacement of isopropyl group
of menthol with vinyl group produced neo isopulegol derivative (10) [2-methyl-5-
(prop-1-en-2-yl) cyclohexanol], which was twice more potent antimycobacterial
agent than menthol. 4-chloro-5-isopropyl-2-methyl phenol (11) is a 4-substituted
chloro derivative of carvacrol synthesized from carvacrol exhibited similar
antimycobacterial activity as that of 2-methyl-5-(prop-1-en-2-yl) cyclohexanol (10)
(Alokam et al. 2013).
The aim of the SAR study is to develop carvacrol derivatives with potent bio-
logical activity. Patil et al. (2010) suggested carvacryl derivatives were obtained by
the attachments of acetanilide or a-chloro acetanilide group with carvacrol via ether
bonds. Comparative antimicrobial studies between carvacryl ether derivatives and
carvacrol against Bacillus subtilis, Escherichia coli, Proteus vulgaris, and
Staphylococcus aureus. The phenyl derivative of carvacryl ether (12) provided a
little antimicrobial activity against Bacillus subtulis, and inactive against other
Fig. 13.4 Chemical structures of some carvacrol derivatives (1-33)
bacterial species. The phenyl m-nitro substituted derivative (13) showed a mild
antimicrobial activity against Escherichia coli and remained inactive against other
experimental bacteria. The m-chloro aryl (14) and m, p-dichloro aryl (15) deriva-
tives showed the most potent activity against all bacteria except Escherichia coli;
whereas, the naphthyl-substituted derivative of carvacryl ether (16) exhibited more
potent antibacterial activity than carvacrol against Proteus vulgaris, and Bacillus
subtilis. The parent compound carvacrol also exerted no activity against

Escherichia coli (Patil et al. 2010). The enzymatic conversions (glycosylation) of
carvacrol produced b-glucosides (B7) and b-gentiobiosides (B8) derivatives ame-
liorated the physicochemical properties and the pharmacokinetic profile of car-
vacrol (Shimoda et al. 2006). The flavor, fragrance, and antimicrobial properties
were improved by introducing hydroxymethyl and allyl groups in carvacrol (19-22)
(Lupo et al. 2000) but the bioactivity against all the tested Gram-negative bacteria
was decreased and antimicrobial activity on Gram-positive bacteria strains were
increased in respect to carvacrol. Ortho allyl carvacrol derivative showed the most
significant activity against Propionibacterium acnes but resistant against
Staphylococcus aureus. Introduction of saturated and unsaturated aliphatic side
chains and aromatic units produced ethers (23-28) and esters (29-32) derivatives
that possessed more potent antimicrobial response than carvacrol against Bacillus
megaterium, Bradyrhizobium japonicum, Bacillus polymyxa, and Bacillus substilits
(Nikumbh et al. 2003). Among ethers, the carboxymethyl derivative (28) and
among the esters, the phenylacetate derivative (30) were the most active antibac-
terial compounds. Development of 4-hydroxy-2-isopropyl benzene sulfonic acid
(31), potassium 4-hydroxy-2-isopropyl benzene sulfonate (32), and
5-isopropyl-2-methyl phenyl dodecanoate (33) derivatives of carvacrol (Fig. 13.4)
were synthesized with better physicochemical profiles and antimicrobial activity
(Bassanetti et al. 2017). The parent drug and synthesized compounds were tested
against Clostridium perfringens, Salmonella typhimurium, Salmonella enteritidis,
and Escherichia coli strains. These three analogs along with carvacrol exhibited
potent cytotoxicity against human colorectal carcinoma cell lines (HT-29) by
inhibiting the proliferation of the cancer cells (Bassanetti et al. 2017). Cacciatore
et al. (2015) synthesized a series of alkyl cysteine carvacrol derivatives by using the
co-drug strategy. The hydroxyl group of carvacrol was conjugated with the car-
boxyl portion of sulfur-containing amino acids via an ester bond. The in vitro
studies of all the derivatives showed low water solubility, good stability in an acidic
environment, in human and rat plasma, and a good permeability verified by
PAMPA-GI (parallel artificial gastrointestinal membrane permeability assay). (R)-
5-isopropyl-2-methyl phenyl 2-acetamido-3-(allylthio) propanoate (34) was found
to be the most active antibacterial compound (Fig. 13.5). The carvacrol sulfon-
amide derivatives (35-43) (Fig. 13.5) were synthesized by inserting selected amines
at carvacrol aromatic ring previously treated with chlorosulfonic acid (De Oliveira
et al. 2016). The obtained sulfonamides were superior to carvacrol against selected
Staphylococcus aureus strains and able to reduce bacterial resistance. P-toluene
(35), p-fluorobenzene (37), and 4-amino-N-(thiazol- 2-yl) benzene sulfonamide
(42) groups containing carvacrol derivatives revealed antimicrobial activity at lower
concentration with lower MIC values (from 3.90 to 62.50 ppm) among the tested
compounds. The compound 4-hydroxy-2-isopropyl-5-methyl-N-(p-tolyl) benzene
sulfonamide (35) had a synergistic effect with erythromycin, and compound N-
(4-fluorophenyl)-4-hydroxy-2-isopropyl-5-methyl benzene sulfonamide (37)
showed a synergistic response when incorporated with ampicillin and tetracycline,
respectively (Marinelli et al. 2018).
Fig. 13.5 Chemical structures of some carvacrol derivatives (34-43)
More scientific evaluation of the carvacrol derivatives was required before using
in agriculture, food, and medicine as most of the reported documents revealed
in vitro results.
13.12 Conclusion and Future Perspectives
Cancer is a dangerous health risk that is present worldwide. The morbidity and the
mortality rates connected with cancer are alarming, despite the existence of multiple
treatment modalities for patients suffering from cancer. Natural substances are
important components that can be used for the discovery and the progression of
new anticancer medications. Since natural components have no or negligible
adverse effects, the utilization of these substances to develop novel anticancer
agents provide a promising choice for chemoprevention and novel cancer treatment.
The current therapeutic intervention for the management of cancer poses several
limitations due to the use of mono-targeted synthetic agents, elevated cost, low
effectiveness, and dangerous adverse actions. Consequently, it is necessary to
develop novel and innovative medications for the prevention and treatment of
cancer. Carvacrol is known to prevent the development of many cancers as well as
suppress the growth of cancer cells.
Various signaling pathways were implicated in the proliferation, differentiation,
apoptosis evasion, and survival of neoplastic cells. So these pathway activation
results in cancer-promoting mechanisms. This review indicates the capacity of
phytochemical carvacrol to act as multi-targeted agents to impede cancer cell
growth. Carvacrol’s cytotoxic actions toward specific cancer by inhibition of cell
proliferation, and its antimitotic behavior induced apoptosis and inhibition of
movement, invasion, or metastasis of cancer cells.
Further research studies need to be directed in vivo to disclose the long-term
impacts of carvacrol usage, the cause of the various pathway inhibition, the impact
of carvacrol to prevent cancer in high-risk populations, and its effects when used in
combination with existing chemotherapy and when used in conjunction with
diversified phytoconstituents. An impressive amount of research findings presented
here establishes the promise of this phytochemical as anticancer agents and the
necessity for the implementation of this phytochemical into human clinical trials.
Chemopreventive carvacrol responsible for the inhibition of various pathways
has been discussed throughout this article. Thus, a coordinated effort to discover
new targets to boost the capability to restore the normal signaling process will result
in the future chemopreventive and anticancer drugs. The findings of the studies
presented in this review may enable researchers to create novel and effective cancer
prevention and therapeutic approaches.
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Chapter 14
Pharmaceutical Application
of Bio-actives from Alstonia Genus:
Current Findings and Future Directions
Atish T. Paul, Ginson George, Nisha Yadav, Arjun Jeswani,

and Prashant S. Auti
Abstract Genus Alstonia is comprised of around 155 species throughout the

world. Phytochemical screening of Alstonia genus has demonstrated the presence of
diverse phytochemicals that include at least 800 distinct metabolites. The main
classes of metabolites are alkaloids, iridoids, flavonoids, fatty acids, etc. Secondary
metabolites from this genus also bring positive results when employed as anti-
cancer, anti-spasmodic, antitussive, antiarthritic, antioxidant, etc. Alstonia boonei is
listed as an antimalarial drug in African pharmacopoeia. In China, a formulation
consists of A. scholaris leaves—Dengtaiye tablets are used for the treatment of
cough and fever symptoms. Recently, A. scholaris leaves-derived indole alkaloids
have been registered as an investigational new botanical drug (No. 2011L01436)
and China Food and Drug Administration (CFDA) has approved its phase I/II
clinical trials for the treatment of respiratory diseases. Pharmacokinetic and safety
analysis of this botanical drug in healthy human subjects have revealed a safe
profile under the dose regimen experiment. Apart from that, strictamine, an indole
alkaloid isolated from A. scholaris exhibited a similar in vitro antiviral activity to
that of acyclovir. The present chapter is mainly focused on the critical analysis of
various secondary metabolites isolated from Alstonia (approximately 15 species).
Apart from that, pharmacological, toxicological and intellectual property rights
studies have also been included.

Keywords Genus alstonia Secondary metabolites Pharmacological activities
Pharmacokinetic studies IPR
A. T. Paul (&) G. George N. Yadav A. Jeswani P. S. Auti

Laboratory of Natural Product Chemistry, Department of Pharmacy, Birla Institute of
Technology and Science Pilani (BITS Pilani), Pilani Campus, Pilani 333031, Rajasthan, India
e-mail: atish.paul@pilani.bits-pilani.ac.in
https://doi.org/10.1007/978-3-030-54027-2_14
464 A. T. Paul et al.
14.1 Introduction
Alstonia is a widespread genus of trees and shrubs in the family of Apocynaceae.

This genus is mainly distributed in Central America, Himalayas, China, tropical
Africa, Malesia, Philippine, and Australia. In 1810, Robert Brown (1685–1760)
described the genus in honor of Charles Alston, a renowned botanist at the
University of Edinburgh (1740–1760). A. scholaris, A. spectabilis, A. venenata, and
A. costata were the four species referred by Robert Brown in the Alstonia genus
(Sidiyasa 1998). According to the World Checklist of Selected Plant Families
(WCSP), approximately 155 species (49 accepted species name) were identified in
the Alstonia genus (Govaerts and Leeuwenberg 2016). As per The Plant List, 114
scientific plant species rank for the genus Alstonia, wherein 43 were accepted as a
species name (The Plant List 2013). Alstonia genus is documented to possess
various health benefits for traditional uses. An ancient Ayurvedic textbook
‘Bhavaprakasha’ has been well documented the therapeutic significance of A.
scholaris. According to that saptaparna (vernacular name of A. scholaris) can cure
ulcer, mitigate vaata and kapha, heal various skin diseases including leprosy,
anti-parasitic activity, anti-asthmatic activities, etc. Generally, the Alstonia genus is
traditionally used for liver and intestinal troubles, heart diseases, respiratory-related
symptoms, various skin diseases, fever, and vulnerary conditions (Khyade et al.
2014). In African Pharmacopoeia, A.booeni is listed as an antimalarial drug (Olajide
et al. 2000). Along with the development of the modern scientific era, investigation
of various secondary metabolites from this genus has gained more attention for a
diversified pharmacological activity. Among these endeavors, numerous classes of
phytochemicals such as alkaloids, iridoids, coumarins, flavonoids, simple pheno-
lics, steroids, and fatty acids have been identified. In addition to that various
pharmacological activities of these phytochemicals were also explored. In some of
the cases, identified pharmacological activities were further provided a full proof
basis for their traditional uses. In China, the monoterpenoid indole alkaloidal
component from the A. scholaris leaves was formulated as a capsule and currently,
it has completed the phase I clinical trial for the respiratory-related ailments (Li
et al. 2019). The obtained promising results provided additional value to the tra-
ditional uses of these plants for respiratory problems.
The present chapter is mainly focused on the critical analysis of various species of
Alstonia (approximately 32 species). A comprehensive literature analysis of Alstonia
genus showed the presence of approximately 800 compounds embracing alkaloids,
iridoids, flavonoids, fatty acids, etc., and it exhibited a multifarious range of phar-
macological activity such as anti-cancer, anti-spasmodic, antitussive, antiarthritic,
and antioxidant. In the present chapter, pharmacological activities of the isolated
secondary metabolites are discussed. Further, a higher number of patents have been
filed for variety of applications for Alstonia that includes pharmaceutical applications
also. In many patents, Alstonia is a single component in the polyherbal formulation.
The Alstonia containing polyherbal formulation is beyond the scope of the book
chapter. Hence, in this chapter, we have selected the patents, wherein the Alstonia
14 Pharmaceutical Application of Bio-actives … 465
Number of PubMed publications on Alstonia

90
Number of publications
80
70
60
50
40
30
20
10
0
1942-1980 1981-2000 2001-2005 2006-2010 2011-2015 2016-2020
Years
Fig. 14.1 The number of Alstonia-related publications (PubMed)
Number of Google patents on Alstonia

300
250
Number of patents
200
150
100
50
0
1990-1994 1995-1999 2000-2004 2005-2009 2010-2014 2015-2019
Years
Fig. 14.2 The number of Alstonia-related patents (Google patents)
genus has been claimed for the significant contribution in pharmacological activities.
The increase in the amount of the publication and patents (Figs. 14.1 and 14.2) in the
last few years evinced that the scientific attention toward the Alstonia genus is
increasing in a vast manner. Hence, the present book chapter will help to obtain a
better understanding of the pharmaceutical application of Alstonia-derived
phytochemicals.
This chapter mainly deals with the following species namely A. scholaris, A.
macrophylla, A. boonei, A. angustifolia, A. venenata, A. yunnanesis. A. spatulata,
A. rupestris, A. rostrata, A. pneumatophora, A. penangiana, A. mairei, A. congensis,
A. angustiloba, A. actinophylla (Table 14.1). Other species of Alstonia genus such as

A. boulindaensis, A. constricta, A. coriaceae, A. deplanchi, A. glaucescenes, A.
lanceolate, A. lanciolifera, A. lenormandii, A. mulleriana, A. odontophora, A. plu-
mose, A. quaternata, A. sphaerocapitata, A. undalufolia, A. undulata, A. villosa, A.
vitensis have been reported for the isolation of various phytochemicals but lacks the
evaluation of the pharmacological potencies. Hence, these species are beyond the
scope of this book chapter and have been avoided for further discussions.
14.2 Botanical Description
See Table 14.1.
14.3 Phytochemistry and Pharmacological Activities
14.3.1 Phytochemistry and Pharmacological Activities of

A. Scholaris
14.3.1.1 Anti-inflammatory and Analgesic Activities
Traditional Chinese medicines have highlighted the importance of the aqueous

extract of A. scholaris leaves for relieving tracheitis and cold symptoms. Based on
these reports, numerous attempts have been tried to identify the anti-inflammatory
and analgesic effects of A. scholaris. Although the majority of the reports are
mainly briefed about the use of the whole extract, a few groups tried to identify the
effect of A. scholaris-derived phytochemical in exhibiting desired pharmacological
activities.
Cai et al. isolated scholarisine I and II (1 and 2, Fig. 14.3) from the alkaloidal
fraction of A. scholaris leaves and evaluated its in vitro anti-inflammatory activity
(Cai et al. 2010a). 1 exhibited a non-selective inhibition toward cyclooxygenase
(COX), while 2 resulted in selective inhibition of COX-II with a significant
5-lipoxygenase (5-LOX) inhibitory activity (Table 14.2).
Shang et al. reported the in vitro anti-inflammatory activities (COX-I, COX-II,
and 5-LOX inhibition) of 29 alkaloids (3–31, Fig. 14.4) from the ethanolic extract
of A. scholaris leaves (Shang et al. 2010). Alkaloids fraction resulted in a moderate
anti-inflammatory potential (54–64% inhibition). Among the screened compounds,
16-Formyl-5a-methoxystrictamine (12), picralinal (19), and tubotaiwine (29) ex-
hibited a promising anti-inflammatory activity by selectively inhibiting COX-II and
5-LOX (>90 and 70% inhibition, respectively). As represented in Table 14.3,
Creabanine (6), isogentialutine (14), scholaricine (24), and strictamine (27) showed
the anti-inflammatory activity by selectively inhibiting COX-II enzyme (>90%
Table 14.1 Botanical description of various Alstonia species
14
Species Bark and trunk Leaves Flowers or inflorescences Fruits Seed

A. scholaris Greyish-brown, Simple, whorled with 4–7 Flowers are greenish-white, Linear, 20–50 cm long Oblong, hairs at
(Jøker 2000; lenticellate unequal leaves that are bisexual, compact, paniculate dehiscent, follicles both ends
Khyade et al. narrow elliptic-oblanceolate cyme, pubescent (two-lobed)
2014)
A. actinophylla Greyish bark, Whorls of 3–7, elliptic or Loosely cymose inflorescence, Glabrous, a pair of Oblong, long
(Flora Malesiana copious, glabrous sublanceolate leaf-blades, glabrous corolla and follicles, follicles and about 8– ciliated
DataPortal; branchlets prominulous transverse slender pedicels 20 cm long
Monachino veins
1949)
A. angustifolia Bark smooth, grey 3-whorled, stalked leaves Terminal inflorescence Follicles are 50–70 cm Oblong,
(Monachino or brown, flaky or have leathery leaf-blades long pointed at one
1949) fissured that are narrowly end, and 1 cm
lance-shaped long
A. angustiloba Dark grey, rough, Elliptic leaf-blades, broad, Puberulent inflorescences, ovate Dehiscent, develop as a Numerous dark
Pharmaceutical Application of Bio-actives …
(Monachino fissured, and and acuminate at the apex to lanceolate calyx tubes, and pair of pods and brown, oblong
1949; NParks peeling off in oblong-ligulate corolla lobes cylindrical follicles.
Flora & Fauna rectangular flakes
Web 2019)
A. boonei Buttresses Whorls at nodes, Flowers are white with long Pair of slender follicle Seeds bearing a
(Monachino deep-fluted high oblanceolate, apex rounded pedicels, tomentose follicles with brown floss at either tuft of silky
1949; Adotey and narrow to acuminate end
et al. 2012)
A. congensis Brown, smooth/ Whorls of 4–6, simple and Puberulent or glabrous Glabrous, a pair linear Brown, oblong,
(Monachino rough, lenticellate entire, leaf-blades are inflorescence, glabrous ovary and follicles 20–45 cm long and flattened
1949; Lemmens acuminate at the apex follicles
2005)
(continued)
467
468
Species Bark and trunk Leaves Flowers or inflorescences Fruits Seed

A. macrophylla Smooth, blackish Whorls of 3–4, verticilliate, Pubescent, slender pedicels, and Glabrous, linear, and 20– Oblong, small
(Khyade et al. brown bark, simple, penni-veined, and distinct ovaries 50 cm long hairy seeds
2014) straight trunk, and a glabrous above
narrow crown
A. mairei Glabrous, Whorls of 3–5, oblanceolate Glabrous, not ciliate, cymes Linear and 5–10 cm long Oblong, cilia at
(Monachino lenticellate and glabrous longer than leaves, small stigma and follicles are distinct seed apex
1949) branchlets apiculi
A. penangiana Grey or pale brown Whorls of 3–4, glabrous, Many-flowered, glabrous, and A pair of follicles, Dark brown,
(Sidiyasa 1998) colored smooth obovate pubescent glabrous striate narrowly ovate
barks or elliptic with
long cilia
A. The smooth bark of Leaves are simple, sessile or Cymose terminal inflorescences The fruits are dehiscent Glabrous,
pneumatophora greyish white color sub-sessile, coriaceous, carrying numerous flowers with follicles produced in pair, blackish brown,
(Monachino and milky sap obovate to spatulate with funnel-shaped corolla of containing many oblong oblong, and
1949; Ferry rounded apex yellowish-white color seeds round ended
2016)
A. rostrata Pale or Whorls of 3–4, glabrous, White colored many flowers, Widest solitary follicles Numerous,
(Monachino yellowish-brown oblong or elliptic, undulate glabrous or puberulous, (more than 10 mm), oblong, or
1949) bark without and/or sinuate leaf margins syncarpous ovary single follicle, glabrous elliptical with
buttresses, granular long cilia
A. venenata Greyish-brown Whorls of 3–7, elliptic Ciliate calyx lobes, slender stipes, Fruits are fusiform with Smooth and flat
(Monachino bark and bright leaf-blades, broad lateral short and blunt stigma apiculi, five petals, stalked and with tufts
1949) yellow hard woody nerves sub-umbellar cyme beaked follicles, tapering
root both ends
A. yunnanensis Shrubs or small Whorls of 3–6, glabrous; Many flowers, cymes puberulent, A pair of distinct Oblong or
(Monachino trees with smooth narrowly elliptic or obovate and slender pedicels follicles, glabrous, lacks elliptic, round
1949) or ringed branches stipes ended, and
rough surfaces
A. T. Paul et al.
Fig. 14.3 Chemical

structures of scholarisine I O O O O
and II (1 and 2)
N N
OH
Scholarisine I (1) Scholarisine II (2)
Table 14.2 Anti-inflammatory activities of scholarisine I and II (1 and 2)

Compound (at COX-I inhibition COX-II inhibition 5-LOX inhibition
100 µM) (%) (%) (%)
1 83.3 95.0 4.8
2 53.2 96.7 80.6
SN-560 61.3 – –
NS-398 – 97.1 –
Zileuton – – 83.1
inhibition). Corypalmine (5), echitamine (9), and 5-methoxylstrictamine (16) did

not exhibit anti-inflammatory properties (inactive). Remaining compounds exhib-
ited a poor to moderate inhibition toward the screened enzymes (10–70%
inhibition).
Along with the in vitro anti-inflammatory activity, in vivo anti-inflammatory and
analgesic effects of a group of alkaloids (picrinine-20, 24 and vallesamine-30) along
with the alkaloids fraction were evaluated in ICR mice. To observe the peripheral
and central analgesic effects individually, acetic acid-induced writhing test and hot
plate test were used, respectively. Formalin test was used for understanding both
central and peripheral analgesic effects. In acetic acid (0.6% acetic acid at 0.1 mL/
10 g body weight, i.p.)-induced writhing response test, the alkaloids fraction
(100 mg/kg) exhibited lesser writhing (20.0) than the remaining fractions, while
aspirin resulted in 9.3 writhing responses. Effects of three alkaloids, such as 20
(10 mg/kg), 24 (5 mg/kg), and 30 (8 mg/kg) resulted in the decreased number of
writhing response in the ICR rats (20, i.g. and i.p.—21.7 and 16.8; 24 i.g. and i.p.—
18.7 and 14.9; 30 i.g. and i.p.—23 and 14.7; aspirin—200 mg/kg, i.g—16.0). In the
hot plate latent pain response test, the screened alkaloids did not increase the
latency time that highlighted the absence of central analgesic effects of these
alkaloids. In formalin test, hydrochloric morphine (10 mg/kg, i.p.) resulted in an
87.4% inhibition ratio in the licking time (s), while the screened alkaloids exhibited
a poor inhibition ratio. However, in the next phase, significant nociception ratios
were obtained (24–5 mg/kg, i.p.—62.7%; aspirin—200 mg/kg, i.g.—67.6%). All
these results indicated that the screened alkaloids exhibited an analgesic activity
through peripheral mechanisms.
O O
O
CH2OH O O O
COOCH3
N N OH
N N O N
H O
O O
Akuammidine (3) Cantleyine (4) Corypalmine (5) Crebanine (6)
OH
COOCH3 H
O O HO
O N H
O N OH
N H
O N
H N
O N O OH HO H COOCH3
Dicentrine (7) Discretamine (8) Echitamine (9) 19-Epi-scholarisine (10)
O-
O O OH
COOCH3 N+
AcO O
O
N
N N
N HO H O OH
N O N
OH
3-Epi-dihydrocorymine 16-Formyl-5 - 12-Hydroxy-echitamidine Isogentialutine (14)

17-acetate (11) methoxystrictamine (12) Nb-oxide (13)
O
O COOCH3 N
O HO
N
N
N N O
N O
N OH
N
O
O O OH
Leuconoxine (15) 5-Methoxylstrictamine (16) N4-Demethylechitamine (17) Nareline (18)
H
N
HO N NO H3COOC
H3COOC
N
H
O
O H
N O OCH3
O N N
O H N
O O H
O
O
O
Picralinal (19) Picrinine (20) Scholarisine B (21) Scholarisine E (22)
O O
O O O
N
O HO N N
OH
O N
N O
N N N
HO O O
O O
O
Salutaridine (23) Scholaricine (24) Scholarisine G (25) Stephanine (26) Strictamine (27)
N O N O
N O
H OH
O H
N O
H
O N N
H O
O N
OGlu O
N
Strictosamine (28) Tubotaiwine (29) Vallesamine (30) Vallesiachotamine (31)
Fig. 14.4 Chemical structures of various alkaloids from A. scholaris

Table 14.3 Anti-inflammatory activities of various alkaloids from A. scholaris

Compound (at 100 µM) COX-I COX-II 5-LOX
Inhibition (%) Inhibition (%) Inhibition (%)
Alkaloids fractiona 54.3 63.3 57.3
6 53.3 90.7 38.4
12 45.8 95.7 79.9
14 43.1 95.9 <0
19 44.7 96.4 79.5
24 44.7 92.0 31.0
25 38.5 91.1 57.3
27 47.0 95.6 <0
29 60.8 90.4 69.5
SC-560 61.3 – –
NS-398 – 97.1 –
Zileuton – – 83.1
a
At 300 µg/mL
Further, anti-inflammatory activities of these alkaloids (20, 24, and 30) were
screened by xylene-induced ear edema and the carrageenan-induced air pouch
model. In xylene-induced ear edema, early-stage inflammation resulted in edema-
tisation of the ear. Aspirin resulted in 45.7% inhibition in the edematisation. 20 at
10 mg/kg, i.g. and i.p. resulted in 41.9 and 41.5% inhibition. Although the i.g.
administration of 24 (5 mg/kg) and 30 (8 mg/kg) did not result in good inhibition, i.
p. administration exhibited significant inhibition on the edematisation (40.3 and
42.0%, respectively). In the carrageenan-induced air pouch model, various bio-
chemical parameters raised from inflammatory responses were measured.
Prostaglandin E2 (PGE2), superoxide dismutase (SOD), nitric oxide (NO), and
malondialdehyde (MDA) were measured as the biochemical markers for the
inflammation. The screened alkaloids lowered the MDA levels in serum, reduced
NO production, and increased levels of SOD in exudate. The results indicated that
these alkaloids inhibited the lipid peroxidation and served as a free radical scav-
enging property by the antioxidant enzymes enhancement. Further, a decreased
level of PGE2 in exudates suggested the ability of these alkaloids to interfere with
the COX pathways of arachidonic acid metabolism.
Sultana et al. isolated ten triterpenoid derivatives (32–41) from the ethanolic
extract of A. scholaris aerial parts (Fig. 14.5) and evaluated the anti-inflammatory
potential of newly isolated phytochemical, nighascholarene (34), ursane type
triterpenoid methyl ester (100 mg/kg), by carrageenan-induced paw edema test in
Wistar rats (Sultana et al. 2019). Compound 34 resulted in 44% inhibition in the
paw edema, while indomethacin resulted in 85% inhibition (positive control).
HO
HO HO
HO
OH CH2OH
OH
HO
HO O O O
HO H
HO
HOH2C O O O
O
Lanosta-5-ene,24-ethyl-3-O- Lanosta-5-ene,24-ethyl-3-O- -D- 12-Ursene-2,3,18,19-tetrol,28-acetate
-D-glucopyranoside (32) glucopyranoside ester (33) (nighascholarene) (34)
H
OH O
H
OCH3
O
HO
HO
O
Alstoprenyol (35) 3 -Hydroxy-28-b-acetoxy-5-olea triterpene (36) -Amyrin acetate (37)
H
OH
O H
HO O
H H
O HO
HO
-Amyrin (38) Lupeol acetate (39) 3 -Hydroxy-24-nor-urs-4,12,28- Ursolic acid (41)

triene triterpene (40)
Fig. 14.5 Chemical structures of various triterpenes from A. scholaris (32–41)
14.3.1.2 Cytotoxic Activities
Jagetia et al. evaluated the cytotoxic potential of echitamine chloride (42, Fig. 14.6)
in cervical carcinoma (HeLa), human liver cancer (HepG2), human myeloid leu-
kemia (HL-60), human oral epidermoid cancer (KB) and human breast cancer
(MCF-7) cell lines (in vitro) and in mice bearing Ehrlich ascites carcinoma
(EAC) (Jagetia et al. 2005). Dose-dependent cytotoxicity was observed in all the
cell lines. Among the various cell lines, KB cells were prone more to cytotoxicity
(Table 14.4). To identify the optimum doses in in vivo conditions, a varying
concentration of 42 (1, 2, 4, 8,12,16 mg/kg) was screened. A 120 days survival was
monitored. A dose-dependent increase in the mean survival time (MST) and the
percentage increase in median life span (% IMLS) were observed. The best
anti-tumor result was observed for 8 and 12 mg/kg of 42, wherein the 1.3-fold MST
increased. Various biochemical parameters (glutathione reduced—GSH, MDA
levels) were also evaluated. A time depended reduction in the GSH content was
observed with 42 (16 mg/kg) till 3 h (2.2 µmol/107 cells), further an elevation was
observed till 12 h (2.4 µmol/107 cells) after the post-treatment. In contrary, MDA
levels were increased till 6 h (4.7 nmol/107 cells) and reverted to the normal
condition after 12 h of pre-treatment (4.2 nmol/107 cells). From this study, it is
evident that an increased lipid peroxidation was observed with the treatment of 42,
while GSH concentration was reduced in the tumor cells.
Fig. 14.6 Chemical structure COOCH3

of echitamine chloride (42) HO
N Cl
N
H
OH
Echitamine Chloride (42)
Table 14.4 Cytotoxicity Cell lines IC50 of 42 (µg/mL)

effects of echitamine chloride
(42) against various cell lines KB 10.5
HeLa 30
HepG2 50
HL-60 51
MCF-7 52
O
O O
O
O O
HN O
O HN O
HN
OH
OH OH N
HN HN
Alstoniascholarine L (43) Alstoniascholarine M (44) Alstoniascholarine N (45)
O O
N N N
H H H
OH HO N OH MeO N OH
HO N H H
H H H H
COOMe COOMe COOMe
Alstoniascholarine O (46) Alstoniascholarine P (47) Alstoniascholarine Q (48)
Fig. 14.7 Chemical structures of various alkaloids from A. scholaris (43–48)
Qin et al. isolated Alstoniascholarines L–Q (43–48, Fig. 14.7) from the
methanolic extract of A. scholaris inadequately dried leaves (Qin et al. 2015b). The
isolated new alkaloids were screened for cytotoxicity assay in various cell lines
(human colon adenocarcinoma (SW-480), human hepatocarcinoma (SMMC-7721),
HL-60, MCF-7, and human lung epithelial (A-549), by using cisplatin and pacli-
taxel as a positive control. Further, neurotrophic activities were evaluated in PC12
cells. Unfortunately, the alkaloids neither showed cytotoxic activity nor exhibited
neurite outgrowth-promoting activity.
Fig. 14.8 Chemical structure O HO OH

of echitamidine-N-oxide-19-
N H
O-b-D-glucopyranoside (49) H O OH
O
OH
N H
H
O O
Echitamidine-N-oxide-19-O- -D-glucopyranoside (49)
Table 14.5 Cytotoxicity Cell lines Cell viability (%) at 100 µg/mL
effects of echitamidine-N-
oxide-19-O-b-D- HeLa 28.77
glucopyranoside (49) against HepG2 36.67
various cell lines KB 29.16
MCF-7 32.67
U373MGa 29.08
a
U373MG—Human glioblastoma astrocytoma cell line
D. Subba Reddy isolated echitamidine-N-oxide-19-O-b-D-glucopyranoside (49,

Fig. 14.8) from the methanolic extract of A. scholaris stem bark and evaluated the
in vitro cytotoxicity in various cell lines (Subba Reddy 2016, 2017). Compound 49
caused concentration-dependent cytotoxicity and maximum effect was observed at
100 µg/mL. Further, it exhibited a moderate potential to KB, MCF-7, and U373MG
cell lines (Table 14.5). In another study, the effect of pre-treatment of 49 in the
radio sensitizing capacity of KB cells was evaluated. Compound 49 (20 µg/mL)
pre-treated KB cells with c-radiation reduces the clonogenicity of the cells than the
individual treatment.
In another study, Wang et al. isolated 13 phytochemicals (38, 41, 50–60,
Fig. 14.9) from the hexane fraction of A. scholaris leaves and evaluated the
anti-proliferation (Wang et al. 2016) effects against non-small-cell lung carcinoma
cells (NSCLC) (Wang et al. 2017). Among the isolated compounds (eight triter-
penoids (38, 41, 50–55) and five sterols (56–60), terpenoids exhibited a higher
potential than the sterols. In the case of isolated sterols (Table 14.6), b -sitosterol
(58) resulted in a 20% reduction in the cell viability, while the remaining sterols did
not inhibit the viability of A-549 cells. In the case of triterpenoids, ursolic acid
(41) and betulinic acid (52) demonstrated a strong anti-NSCLC activity. 41 was
reported for the activation of adenosine monophosphate (AMP)-activated protein
kinase and inhibition of mammalian target of rapamycin (mTOR) pathways that
control protein synthesis. Compound 52 induces the apoptosis in cancer cells via
triggering the mitochondrial pathways. Remaining terpenoids exhibited moderate to
poor anti-proliferation effects against NSCLC.
Kuok et al. isolated Melosline A (61), Melosline B (62), 1-[2-[2-(carbox-
ymethyl) indole-3-yl] ethyl]-3-ethylpyridinium hydroxide inner salt (63) from the
OH
R
OH HO
O
HO HO
HO HO
Oleanolic acid (50) Betulin (51); R = CH2OH 2 ,3 ,28-Lup-20(29)-ene-triol (53) Lupeol (54)
Betulinic acid (52); R = COOH
HO
HO HO
-Amyrin (55) Poriferasterol (56) Epicampesterol (57)
O HO
OH OH
OH
HO
-Sitosterol (58) 6 -Hydroxy-4-stigmasten-3-one (59) Ergosta-7,22-diene-3 ,5 ,6 -triol (60)
Fig. 14.9 Chemical structures of various triterpenoids and steroids from A. scholaris (50–60)
Table 14.6 Cytotoxicity Compound IC50 values (µM) against A-549 NSCLC cells
effects of various triterpenoids
from A. scholaris against 41 39.8
NSCLC 52 40.1
53 172.6
51 240.5
50 >400
54 >400
55 >400
38 >400
leaves and twigs of A. scholaris (Fig. 14.10) and evaluated their cytotoxicity effects
against MCF-7 cell lines (Kuok et al. 2017). 61 exhibited moderate cytotoxicity
(IC50 > 39.8 µM), while the remaining compounds, 62 and 63 did not exhibit any
cytotoxic activity (IC50 > 50 µM).
Wang et al. identified the glioma stem cells (GSCs) inhibiting scholarisine Q
(64) and scholarisine R (65) (nor-monoterpenoid alkaloids, Fig. 14.11) from the
methanolic extract of A. scholaris fruits (Wang et al. 2018). As represented in
Table 14.7, 64 exhibited a significant effect to that of taxol. Further cell prolifer-
ation assay of the tested compounds indicated the inhibition of GSCs proliferation.
HO
O N
H HN O
N
HN
N N+ O
O
Melosline A (61) Melosline B (62) 1-[2-[2-(carboxymethyl)indole-3-yl]ethyl]-3-
ethylpyridinium hydroxide (63)
Fig. 14.11 Chemical O

structures of Scholarisine Q
O O
and R (64 and 65)
O O
H N N
HN O
O H
HO
O
HN
H
Scholarisine Q (64) Scholarisine R (65)
Table 14.7 Cytotoxic effects Compound cell lines IC50 values (µg/mL)
of Scholarisine Q, R (64 and
64 65 Taxol
65), taxol against GSCs
GSC-3# 17.9 21.7 13.6
GSC-12# 15.5 19.9 10.7
GSC-18# 20.6 20.6 8.9
Moreover, these compounds induced the GSCs apoptosis by increasing the tumor
necrosis factor-alpha (TNF-a) expression and cleavage of caspase-3.
Wei et al. reported a novel bis-indole scaffold containing Alstoniasidines A and
B (66 and 67, Fig. 14.12) from the methanolic extract of A. scholaris leaves and
evaluated its selective anti-tumor effects in GSCs (Wei et al. 2018). At 25 µM
concentration, both the tested compounds inhibited the growth of GSCs (GSC-12#
and GSC-18#). Cell viability assay indicated that the compounds exhibited sig-
nificant cytotoxicity against GSCs (Table 14.8). The isolated compounds were
more selective toward the cancer cell types (in normal cell lines IC50 > 90 µM). In
order to identify the apoptotic mechanism, several apoptotic regulators were mea-
sured by using real-time polymerase chain reaction (RT-PCR) assay. A significant
increase in the Interleukin 1 (IL-1) and TNF-a level (extrinsic pathway regulators)
was observed while calpain-1, caspase-12 (endoplasmic reticulum stress apoptotic
regulators), Bcl-2, Bax, Bid, Apaf-1 (intrinsic pathway regulators) did not exhibit
any changes. Further, they suggested that the anti-tumor effects mainly occurred
due to the activation of the extrinsic apoptotic pathways.
HN HN
H H
N N
O O
O O O
O O O O
HO O HO
HO OH HO OH
MeOOC MeOOC
O O
N N
N O N O
H H
Alstoniasidine A (66) Alstoniasidine B (67)
Fig. 14.12 Chemical structures of alstoniasidine A and B (66 and 67)
Table 14.8 Cytotoxic effects Compound IC50 values (µg/mL)

of alstoniasidine A, B (66 and Cell lines 66 67 Taxol
67) and taxol against GSC
cells GSC-12# 30.9 20.1 12.5
GSC-18# 19.2 16.1 10.4
Fig. 14.13 Chemical

structure of alstobrogaline MeOOC
(68) H
N
N N O
H
Alstobrogaline (68)
Krishnan et al. isolated alstobrogaline (68, Fig. 14.13) from A. scholaris leaves
and evaluated for the in vitro cytotoxicity in various human breast cancer cell lines
(Krishnan et al. 2019). 68 exhibited significant activity in human breast adeno-
carcinoma (DA-MB-231) and MCF-7 cells (IC50 = 25.3 and 24.1 µM, respec-
tively), while a poor activity was observed in MDA-MB-468, human breast cancer
(SK-BR-3), and T47D cells (IC50 > 30 µM).
14.3.1.3 Antibacterial and Antifungal Effects
Maurya et al. isolated loganetin (69, Fig. 14.14), an iridoid from ethyl acetate
extract of A. scholaris stem barks by fast centrifugal partition chromatography
(FCPC) and evaluated for its antibacterial potential in nalidixic acid-resistant with
MTCC No. 1652 (NAREC), nalidixic acid sensitive strain CA8000 (NASEC), and
Escherichia coli strains (Maurya et al. 2014). Individually, 69 exhibited poor
activity against the selected strains (Minimum Inhibitory concentration-MIC
value-500 µg/mL). However, a combination of 69 (10 µg/mL) with nalidixic
acid exhibited a potential antibacterial activity. Individually nalidixic acid exhibited
a MIC of 6.3 and 100 µg/mL for NAREC and NASEC, while in combination, it
exhibited a MIC of 1.6 and 12.5 µg/mL with a 4- and 8-fold dose reduction of
nalidixic acid concentration, respectively.
Liu et al. isolated 16 compounds (3, 16, 18–19, 24, 27, 30–31, 70–77,
Fig. 14.15) from the methanolic extract of A. scholaris leaves (Liu et al. 2015) and
evaluated the antibacterial effects against Staphylococcus aureus (ATCC 25922),
Pseudomonas aeruginosa (ATCC 27853), Enterococcus faecalis (ATCC 10541),
O O
OH
O
OH
Loganetin (69)
Fig. 14.14 Chemical structure of loganetin (69)
O O O O
H H
N N
O HN OH HN OH N
H O O
HO N O
H N
H N OO
O O
Normavacurine-21-one (70) 5-Hydroxy-19, 20-E- 5-Hydroxy-19, 20-Z- Strictamine N4 oxide (73)

alschomine (71) alschomine (72)
O OH OH
N N
OH O
O H
N O
H
N
N N O
HO H O O H N
N+ O O
O
O-
Vallesamine N4-oxide (74) 19-Epischolaricine (75) 12-Methoxyechitamidine (76) Isovallesiachotamine (77)

Fig. 14.16 Chemical OH O

structure of OH
3,5,7-Trihydroxyflavone (78)
HO O
3, 5, 7-Trihydroxyflavone
(78)
E. coli (ATCC 11775), and Klebsiella pneumoniae (ATCC 13883). Cefotaxime

was used as a positive control. Normavacurine-21-one (70), 27 and vallesamine N4-
oxide (74) exhibited significant antibacterial activity against E. faecalis (MIC
values—0.8 for each compound; cefotaxime MIC—0.2 µg/mL), 74, 18 and
5-hydroxy-19, 20-Z-alschomine (72), and against P. aeruginosa (MIC values—0.
8 each, cefotaxime MIC—0.78 µg/mL), while 18 showed moderate activity (MIC
value of 1.6 µg/mL) against K. pneumoniae (cefotaxime MIC—0.8 µg/mL). The
remaining compounds exhibited moderate to poor antibacterial effects (6.25–
100 µg/mL).
M. Abinaya et al. isolated 3,5,7-Trihydroxyflavone (78, Fig. 14.16) from the
methanolic extract of A. scholaris leaves and evaluated the biofilm formation
inhibition and quorum sensing activity against P. aeruginosa (Abinaya and
Gayathri 2019). Among the various concentrations of 78, 6.2 and 12.5 µg/mL
concentration resulted in 50 and 55% of the reduction, while 1 mg/mL solution
resulted in an 85% reduction in the biofilm formation. Further, the effects of various
pathogens (S. aureus, E. coli, P. vulgaris, B. subtilis, and P. aeruginosa) in the
pyocyanin production from P. aeruginosa were analyzed by a well diffusion
method. The pyocyanin production was more sensitive to S. aureus and B. subtilis
than the remaining species (zone of inhibition of 18.3 and 16.5 mm at 300 µg/mL).
Qin et al. isolated 11 new alkaloids (Alstoniascholarines A-K (79–89,
Fig. 14.17) along with polyneuridinic acid from the aqueous fraction of A. scholaris
leaves (Qin et al. 2015a). Antibacterial effects of these alkaloids were screened in
various bacterial strains, namely as S. aureus (ATCC 25922), P. aeruginosa
(ATCC27853), E. faecalis (ATCC 10541), E. coli (ATCC 8739), P. smaitii (ATCC
29916), and K. pneumoniae (ATCC 13883). Gentamycin was used as a positive
control. Antifungal activity was evaluated in fungal strains, namely as
Epidermophyton floccosum (CBS 566.94), Microsporum gypseum (CBS118893),
and Trichophytom mentagrophytes (ATCC 4439). Griseofulvin was used as a
positive control. In the antibacterial study, Alstoniascholarine F (84) and
Alstoniascholarine J (88) exhibited a potential activity toward P. aeruginosa
(MIC = 3.1 µg/mL each) while gentamycin exhibited a MIC of 0.78 µg/mL.
Further, Alstoniascholarine C (81), 84, and 88 exhibited a potential activity toward
P. smaitii (MIC = 3.1, 6.3, and 1.6 µg/mL, respectively) while gentamycin
exhibited a MIC of 0.20 µg/mL. In the remaining antibacterial strains, the isolated
alkaloids exhibited poor activity (>25 µg/mL). Alstoniascholarine D (82),
Alstoniascholarine G (85), 88 exhibited moderate activity against E. floccosum
O
N N N
HN HN HN
HOOC HOOC OOC
H H H
OH OH OH
Alstoniascholarine A (79) Alstoniascholarine B (80) Alstoniascholarine C (81)
N
O N H
H H
N OH
H N R N
OOC H H
H OEt
N O COOH
HOOC
Alstoniascholarine F (84); R = H
Alstoniascholarine D (82) Alstoniascholarine E (83) Alstoniascholarine G (85); R = OH
Alstoniascholarine H (86); R = OMe
O
N N N
H H H H
H OH H
MeO N OH MeO N H N OH
H H H H
H H
COOH COO COO
Alstoniascholarine I (87) Alstoniascholarine J (88) Alstoniascholarine K (89)
Fig. 14.17 Chemical structures of Alstoniascholarines A-K (79–89)
O
O
HO
3-Hydroxy-11-ursen-28,13-olide (90)
Fig. 14.18 Chemical structure of 3-hydroxy-11-ursen-28,13-olide (90)
(MIC = 31.6 µg/mL each). The remaining antifungal strains were not susceptible to
the reported alkaloids (MIC > 62.5 µg/mL). The MIC values of griseofulvin were
in the range of 3.9–7.8 µg/mL.
Wang et al. isolated various pentacyclic triterpenoids (Fig. 14.18) from the
methanolic extract of A. scholaris leaves and evaluated its antibacterial efficacy
(Wang et al. 2016) against Bacillus cereus (ATCC 9139), E. faecalis (ATCC
29212), Listeria monocytogenes (ATCC 7644), methicillin-sensitive S. aureus
(MSSA, ATCC 29213), methicillin-resistant S. aureus (MRSA, ATCC 43300),
Table 14.9 Antibacterial Compound Minimum Inhibitory concentrations (µg/mL)

effects of various triterpenoids
E. faecalis L. monocytogenes B. cereus
from A. scholaris
50 4 8 16
41 1 2 8
Ampicillin 2 1 128
Tetracycline 4 2 4
E. coli (ATCC 35150), P. aeruginosa (ATCC 27853), Salmonella enterica (ATCC

13311). All the isolated compounds (3-hydroxy-11-ursen-28,13-olide (90), 41, 50–
52 and 54) did not exhibit any antibacterial effects in gram-negative bacteria, while
in gram-positive bacteria, 50 and 41 exhibited a moderate potential (Table 14.9).
Further, a synergistic antibacterial effect was observed against B. cereus and S.
aureus by 41 in combination with ampicillin and tetracycline.
14.3.1.4 Nuclear Factor-jB (NF-jB) Inhibitory Activity and b2

Adrenoreceptor (AR) Activation
Hou et al. identified NF-jB inhibitors and b2-AR agonists from an alkaloidal
extract of A. scholaris via microfractionation bioactivity-based ultra-performance
liquid chromatography/mass spectrometry (Hou et al. 2012b). In the study, cyto-
toxicity of the compounds was determined via the quantities of lactate dehydro-
genase (LDH). For the identification of NF-jB inhibition and b2 AR activation,
luciferase reporter assay was used. Preliminary UPLC-MS analysis identified 17
peaks for NF-jB inhibition, while 11 peaks were identified for b2 AR agonist
activity. According to the relative percent content, nine alkaloids, namely, 3, 18, 19,
20, 24, 27, 19(Z)-vallesamine (91), (Z)-alstoscholarine (92), and (E)-alstoscholarine
(93), were screened for individual screening (Fig. 14.19). Compared to control,
these compounds didn’t exhibit a significant level of LDH in the serum (10 or
100 µmol/L). The obtained results indicated that these compounds did not show
any kind of toxicity to the cells. Further, NF-jB inhibition activity of alkaloids in
O
O
O
O
N
N
N
N N
H H N
COOCH3 O H
HO O
19(Z)-Vallesamine (91) (Z)-alstoscholarine (92) (E)-alstoscholarine (93)

Fig. 14.20 Chemical N

structure of 19,20-(E)-
Vallesamine (94)
N
H
HO COOCH3
19,20-(E)-vallesamine (94)
the non-toxic concentrations was determined via luciferase reporter assay in TNF-a
induced BEAS-2B cells (5 ng/mL) as compared to dexamethasone (positive con-
trol). Although all the tested compounds displayed a good inhibition on TNF-a
induced NF-jB production, 24, 92, and 93 showed significant effects at even low
concentrations (RFU ratio < 10 at 100 µm/L). Apart from that the cytokine release
test (IL-6 and IL-8 expression) was also performed in TNF-a induced BEAS-2B
cells (5 ng/mL). Cells treated with 3, 19, 20, 27, 92, and 93 only resulted in
decreased expression of IL-6 (<500 pg/mL), while all the alkaloids demonstrated
suppression of IL-8 release (<500 pg/mL). Dexamethasone (10 µmol/L) inhibited
IL-6 and IL-8 (200 and 300 pg/mL, respectively).
While in another study, Hou et al. identified several new b2 adrenergic receptor
agonists (3, 13, 20, 24, 27, 92–94) from an alkaloidal extract of A. scholaris leaves via
bioactivity-based LC/MS analysis (Hou et al. 2012a). b2 AR agonist activities were
confirmed by performing in vivo relaxant tests on guinea pig tracheal muscles.
Compounds 3, 13, 20, 24, 92, 93, and 19,20-(E)-vallesamine (94, Fig. 14.20) were
identified as the initial hit for b2 AR activation. Further, chromatographic separation
resulted in the pure form of these phytochemicals (90–95% purity) and b2 AR
activities of these components (0.5 µg/mL), as well as the positive control (salbuta-
mol, 0.01 µmol/L), were determined. Salbutamol exhibited the highest activity (RFU
ratio-2.5). Compared with the control, 3, 92–94 exhibited a significant activity (RFU
ratio < 2.0) while 20 and 24 exhibited a poor b2 agonist activity (RFU ratio < 1). To
confirm the in vitro activity, spasmolytic activity tests were performed by using
isolated guinea pig trachea (in vivo) and the obtained activity at 5 µg/mL follows—3
(EC50 = 243.9 µmol/L), 92 (EC50 = 137.5 µmol/L), 93 (EC50 = 74.8 µmol/L), and
salbutamol (EC50 = 285.7 µmol/L). Remaining alkaloids exhibited a poor spas-
molytic activity (EC50 < 70 µmol/L). The comparable spasmolytic activity of 3 and
92 highlights the probable role of these alkaloids as b2 AR agonists.
Yang et al. isolated various monoterpenoid indole alkaloids (3, 16, 18–20, 24,
30, 75, 95) from the ethanolic extract of A. scholaris leaves and determined its
NF-jB inhibitory activity by using NF-jB luciferase assay in HepG2-NFjB-Luc
cells (Yang et al. 2018). Ammonium pyrrolidine dithiocarbamate (PDTC) was used
as a positive control. Scholarisine S (95, Fig. 14.21), 3, 19, 20, and 75 exhibited
significant NF-jB inhibitory activity (relative NF-jB luciferase activity < 0.8 folds
at 25 µM). The remaining compounds exhibited more than 0.8 folds relative
luciferase activity at 25 µM. Further, 19, 20, and 75 inhibited TNF-a induced
NF-jB activations.
Fig. 14.21 Chemical H

structure of Scholarisine S MeOOC
H
(95)
H
N N
O
HO
Scholarisine-S (95)
Fig. 14.22 Chemical COO

structure of
17-nor-excelsinidine (96) N
N
H
17-nor-excelsinidine (96)
14.3.1.5 Antiviral Activity
Zhang et al. isolated 17-nor-excelsinidine (96, Fig. 14.22) and 27 from the
methanolic extract of A. scholaris leaves and evaluated its anti-HSV and
anti-adenovirus (ADV) activities (Zhang et al. 2014b). HSV-transfected Vero cell
line was used for the evaluation of anti-HSV activity while adenovirus-transfected
Hep-2 cell line was used for anti-adenovirus activity. Compound 27 exhibited a
higher activity (CC50-5.0 and 3.3; EC50-0.36 and 0.28 µg/mL, respectively, for HSV
and ADV) than the 96 (CC50-6.9 and 3.3; EC50-1.09 and 0.94 µg/mL, respectively,
for HSV and ADV). Acyclovir (positive control) exhibited CC50-302.0 and 302.6;
EC50-0.38 and 1.9 µg/mL, respectively, for HSV and ADV. Although 27 exhibited a
higher activity than the positive control, the latter is having higher selectivity
(Acyclovir—790.8 and 153.0; 27−13.9 and 11.8 for HSV and ADV).
Nguyen et al. identified four cystine knot a-amylase inhibitors (Alstotides 1–4)
from the ethanolic extract of A. scholaris leaves (Nguyen et al. 2015). These are the
types of peptides rich in cysteine and proline molecules with high resistance to heat
and protease enzymes. Further, in cytotoxicity assays (Vero cells), Alstotide-1
(AS1) and Alstotide-3 (AS3) did not exhibit a significant activity till 100 µM.
Hence, by taking the concentration range of 0–100 µM, antiviral activities of
alstotides were evaluated in IBV, DENV2, and RSV A. IBV is a c-coronavirus and
the causative agent of infectious bronchitis. AS1 and AS3 exhibited inhibition of
plaque formation in a dose-dependent manner. Furthermore, AS1 exhibited a
moderate inhibition toward DENV2 (EC50 = approx. 90 µM). However, it didn’t
exhibit any activity toward the RSV A up to 100 µM concentration.
14.3.1.6 Antiallergic and Antitussive Effects
Shang et al. evaluated antitussive, anti-asthmatic, and expectorant activities of

various fractions/phytochemicals (20, 24, and 30) from A. scholaris leaves (Shang
et al. 2010). The antitussive activity was evaluated in various animal models
(Ammonia or sulfur dioxide-induced mice coughing and citric acid-induced guinea
pigs coughing) while histamine-induced bronchoconstriction in guinea pig was
used for the evaluation of the anti-asthmatic activity. Codeine phosphate was used
as a positive control in the antitussive screening, while aminophylline is in bron-
choconstriction and ammonium chloride is used in case of expectorant activity. In
ammonia liquor-induced cough in mice, among the various fractions evaluated, the
alkaloid rich fraction exhibited a significant reduction in the coughing frequency
(30.7 and 39.3% for 50 and 100 mg/kg of alkaloid rich fraction), while these
activities were comparatively lesser than the codeine phosphate (55.4% at 30 mg/
kg). In sulfur dioxide-induced cough in mice, alkaloid rich fraction (100 mg/kg)
exhibited a 62.2% increment in the cough latent period, whereas codeine phosphate
(30 mg/kg) resulted in 83.2%. A similar kind of results was obtained in citric
acid-induced cough in guinea pigs wherein a 153.8 and 419.0% increment in the
latent period of cough with the alkaloidal fraction (100 mg/kg), and codeine
phosphate (30 mg/kg) groups, with a cough inhibition frequency of 62.4, and
91.9%, respectively. In the anti-asthmatic effects in guinea pigs, alkaloid rich
fraction (79.9% at 200 mg/kg) exhibited an increased tumble and delitescence of
convulsion with that of aminophylline group (90.1% at 100 mg/kg). In expectorant
effect analysis, ammonium chloride (1500 mg/kg) and the alkaloids fraction (at 60
and 120 mg/kg) could markedly enhance tracheal phenol red output (112.7 and
152.1, 138.6% increase, respectively).
The preliminary screening highlighted the potential role of alkaloid rich fraction
in exhibiting antitussive and anti-asthmatic effects. Hence, the study was further
proceeded with the alkaloids fraction and main alkaloids (20, 24, and 30). In
ammonia-induced cough, i.g. administration of 20 (10 mg/kg) and 24 (5 mg/kg)
resulted in 38.9 and 34.1% increment in the latent period of cough in mice, and
these are comparable with the codeine phosphate (30 mg/kg, i.g., resulted in 47.6%
increment). The same trend was observed within the case of anti-asthmatic effect
screening. At a dose of 10 mg/kg, the 20 group increased the tumble and delites-
cence of convulsion of guinea pigs by 63.0% while aminophylline group resulted in
96.3% of the parameters.
Qin et al. evaluated the antitussive effects of 43–48 (Qin et al. 2015b). In
ammonia-induced cough (mice), the total alkaloids from the inadequately dried
leaves exhibited a lesser % inhibition (38.0 and 27.2% at 20 and 10 mg/kg) than the
dried leaves (58.0 and 45.6% at 20 and 10 mg/kg). Codeine phosphate (30 mg/kg)
was used as a positive control that resulted in 77.6% inhibition.
Zhao et al. identified various alkaloids (20, 24, 75, and 30) from the ethanolic
extract of A. scholaris leaves and evaluated the effects in airways allergic conditions
(Zhao et al. 2017). Ovalbumin-induced airways allergic inflammatory models in SD
rats were used in the study, and dexamethasone (2 mg/kg, once daily) was used as a
positive control. Various parameters such as cellular infiltration, bronchoalveolar

lavage fluid (BALF), IL-4, and IL-10 expression in BALF were analyzed. Along
with the various doses of total alkaloids, the following doses of the phytochemicals
were also screened. Intra-gastric administration of the following doses of alkaloids
were performed once a daily [20 (5 mg/kg); 24 (3 mg/kg); 30 (3 mg/kg); 75
(3 mg/kg)]. The alkaloidal treated group resulted in a decreased amount of total
leukocytes and eosinophils % in BALF. All the screened alkaloids exhibited a
potential activity (WBC content—<0.5 109/mL; eosinophil %—4%). In the
disease control group, IgE and eotaxin in serum were expressed at a level of 2.5 µg/
mL and 6 pg/mL. However, treatment of the alkaloids resulted in a decreased
amount of IgE and eotaxin (<2.0 µg/mL and 4 pg/mL, respectively). Furthermore,
the treatment of alkaloids also resulted in the reduction of inflammatory markers
such as IL-4 and IL-10 in BALF.
In another study, Yun-Li Zhao et al. evaluated the effect of 20, 24, 30, and 75 in
post-infectious cough in mice (Zhao et al. 2018). The animal model was stan-
dardized by instilling lipopolysaccharide (LPS—at 80 µg/50 µL/mouse) at tracheal
region followed by 30 min exposure of cigarette smoke till 30 days. After the
administration of alkaloids (above-mentioned dose, except 73 (i.g.), 1 mg/kg), the
symptoms of cough in mice were attenuated. Total white blood cells (WBC),
neutrophils (NEU) amounts in BALF and MDA, C-reactive protein (CRP), IL-6 in
serum were significantly reduced.
14.3.1.7 Metabolic Enzymes (a-Glucoside, a-Amylase, and Pancreatic

Lipase) Inhibitory Effects
NJong-Anurakkun et al. reported the a-glucoside inhibitory potential of A. schol-

aris leaves-derived phytochemicals (97–99, Fig. 14.23) Jong et al. (2007).
Quercetin 3-O-b-D-xylopyranosyl(1″ ! 2″)-b-D galactopyranoside (97), (+)-lyo-
niresinol 3-O-b-D-glucopyranoside (98), and (−)-lyoniresinol 3-O-b-D-glucopyr-
anoside (99) were isolated from the rat intestine acetone powder. The screened
compounds exhibited a moderate potential toward a-glucoside (Table 14.10).
Nguyen et al. studied the a-amylase inhibitory effect of AS1 to AS3 on
a-amylase from human salivary, T. molitor larvae (TMA), and fungus (Aspergillus
oryzae). AS1 to AS3 exhibited a significant activity toward the a-amylase of TMA
(1.9–5.2 µM). However, it did not exhibit any activity toward human and fungal
a-amylases up to 100 µM concentration (Nguyen et al. 2015).
George et al. evaluated the pancreatic lipase inhibitory potential of A. scholaris
stem barks (George et al. 2019). Bioassay-guided fractionation of the methanolic
extract resulted in the identification of echitamine (9) as a potential pancreatic lipase
inhibitory leads (IC50 = 10.92 µM).
OH
HO HO
O OH O
OH O
O OH
HO O O OH
HO OH HO OH
OH O
O O OH O O OH
O OH O
O O OH O
O O OH
HO OH OH
OH OH
OH
Quercetin 3-O- -D- (+)-Lyoniresinol 3-O- -D- (-)-Lyoniresinol 3-O- -D-
xylopyranosyl(1”’ 2”)- -D glucopyranoside (98) glucopyranoside (99)
galactopyranoside (97)
Fig. 14.23 Chemical structures of various lignans from A. scholaris (97–99)
Table 14.10 a-glucoside Compound IC50 values(mM)

inhibitory potential of various
Sucrase Maltase
lignans from A. scholaris
(97–99) 97 17.2 1.96
98 1.95 1.43
99 >10 >10
14.3.1.8 Anti-tubercular Activities
Macabeo et al. isolated various alkaloids (94, 100–104, Fig. 14.24) from the
methanolic extract of A. scholaris leaves. Further, the anti-tubercular activity of the
isolated compounds was evaluated by microplate alamar blue assay (MABA) in
Mycobacterium tuberculosis H37Rv by using rifampicin as a positive control
(Macabeo et al. 2005, 2008). Among the various compounds screened, 20S-
tubotaiwine (103) only exhibited a potential anti-tubercular activity (MIC—
>100 µg/mL). The remaining compounds exhibited a poor activity toward the
M. tuberculosis (Table 14.11).
14.3.1.9 Antimalarial Activities
Salim et al. isolated seven indole alkaloids (49, 105–110, Fig. 14.25) from the
ethanolic extract of A. scholaris bark (Salim et al. 2004). Isolated compounds are
49, Akuammiginone (105), echitaminic acid (106), echitamidine-N-oxide (107), Nb-
demethyl alstogustine N-oxide (108), akuammicine N-oxide (109), and Nb-deme-
thyl alstogustine (110). Further, in vitro antimalarial activity was evaluated by
hypoxanthine incorporation techniques. Compounds 109 and 110 had an IC50
values of 63.2 and 6.75 µg/mL, respectively, against Plasmodium falciparum (K1,
multidrug-resistance (MDR) strain).
O O
N O O
O H O H
N N
H
N
H N N
OH O
(+)-manilamine (100) N4-methyl angustilobine B (101) Angustilobine B N4-oxide (102)
N
N
H
NH O
N O
H O O
O
20(S)-tubotaiwine (103) 6,7-secoangustilobine (104)
Table 14.11 Anti-tubercular Compound MIC (µg/mL)

activities of various alkaloids
from A. scholaris 94 >128
100 >128
102 >128
101 >128
103 >100
104 >128
Rifampicin 98% inhibition at 0.125 µg/mL
O-
O OH OH
O HN N+
HO
O
N+ N+
N
OH N OH H
O H O
O
Akuammiginone (105) Echitaminic acid (106) Echitamidine N-oxide (107)
O- OH
O N
OH N+
N+
N N
N H O
O -
O
- O O
O
Nb-Demethylalstogustine N-oxide (108) Akuammicine N-oxide (109) Nb-Demethylalstogustine (110)

HO
HO
Cycloeucalenol (111) Cycloartanol (112)
Fig. 14.26 Chemical structures of cycloeucalenol and cycloartenol (111 and 112)
14.3.1.10 Hypoglycemic Activities
Ragasa et al. reported the isolation of a mixture of cycloeucalenol, cycloartanol

(111, 112, Fig. 14.26), 39, 51, and 54 from dichloromethane extract of A. scholaris
leaves (Ragasa et al. 2015). Further, the isolated mixture was screened for hypo-
glycemic assay in male albino mice (Mus musculus L.) at various concentration (25,
50, and 100 mg/kg) by using glimepiride solosa (16.7 µg/kg) as a positive control.
Supplementation of 25 mg/kg resulted in a high impact on the hypoglycemic
properties. In 0.5 h, the mixture exhibited a 52.61% blood glucose reduction while
the glimepiride resulted in 39.07% glucose reduction.
14.3.1.11 Hepatomodular Effects
Singh et al. isolated 38 from the ethanolic extract of A. scholaris stem bark and
evaluated the hepatomodular effect against CCl4-induced hepatic oxidative stress in
Wistar albino rats (Singh et al. 2015). CCl4 (0.2 mL/kg, twice a week, i.p.) was
administered for the induction of hepato-oxidative stress. Concurrently, 38 (20 mg/
kg/day, oral) was administered for 30 days. Various biomarkers such as c-glutamyl
transpeptidase (GGT), aspartate, and alanine transaminases (AST, ALT) were
evaluated. CCl4 produced a significant hepatic oxidative stress, while the treatment
of 38 resulted in the recovery of the oxidative stress.
14.3.1.12 Anticataract Activity
Soni et al. isolated a novel isoflavonoid (113, Fig. 14.27) from the ethanolic extract
of A. scholaris stem bark and evaluated its anticataract activity in goat lens (in vitro)
and fructose-induced experimental cataract (in vivo) condition (Soni et al. 2019). In
the in vitro experiments, cataract was induced in goat lenses by incubating with a
Fig. 14.27 Chemical HO

structure of chromone O OH
analogue from A. scholaris
(113)
OH O
O
6-(2,4-dihydroxyphenyl)-4-hydroxy-2-prop-1-en-2-yl-2,3-
dihydrofuro [3, 2-g] chromen-5-one (113)
higher concentration of glucose (55 mmol/L). Compound 113 (50 µg/mL) resulted
in a slight degree of opacity as compared to the toxic control group. During the
in vivo experiments (albino male Sprague–Dawley rats), animals were administered
with a fructose solution. The high fructose-rich diet will lead to a series of mech-
anisms in the SD rats. Fructose will lead to the elevation in the blood pressure and
blood glucose levels that in turn results in metabolic alterations. Apart from that
fructose will also upregulate the angiotensin-II and increase the sympathetic ner-
vous systems that results in hypertensive conditions.
Further, the accumulation of high fructose increases the lenticular opacity,
subsequently leading to increased osmotic pressure and oxidative stress in the lens
results in cataractogenesis. In the case of systolic and diastolic blood pressure,
dose-dependent activity was observed with the administration of 113. Moreover,
blood glucose levels were reduced. A significant reduction in the lens opacity was
observed with 113 treatment for 8 weeks. Further, various biochemical parameters
were restored.
14.3.1.13 Anti-fertility Effects
Gupta et al. isolated 37 from the ethanolic extract of A. scholaris stem bark and
evaluated the anti-fertility effects (10 mg/rat/day for 60 days) in male albino rats
(Gupta et al. 2008). The administration of the 37 did not exhibit a significant weight
loss. However, a decreased weight in the reproductive organs (testes, seminal
vesicle, ventral prostate, and epididymides) was observed. Apart from that sperm
density and motility were also reduced. The seminiferous tubular diameter, Sertoli
cells cross-sectional surface area, and counts were also decreased significantly.

of A. Macrophylla
14.3.2.1 Cytotoxicity Effects
Keawpradub et al. screened the cytotoxicity effects of various alkaloids (114–116,

Fig. 14.28) isolated from the methanolic extract of A. macrophylla root bark
N O O
O
N N
N O
O O
O N
N N O
O
N
O-Acetyl-macralstonine (114) Macrocarpamine (115)
N O
N
H H
H
NO O
N
O
N N
O O
N
OH
H3COOC O N O
H
OH O
H
Villalstonine (116) Vinblastine Sulphate (117)
Fig. 14.28 Chemical structures of various alkaloids from A. macrophylla (114–116)
(Keawpradub et al. 1998). Among the tested alkaloids, only O-Acetyl macral-
stonine (114) displayed selectivity for cancer cell lines (Table 14.12).
In another report, Keawpradub et al. evaluated the cytotoxicity of 14 indole
alkaloids (114–116,118–128, Fig. 14.29) from the methanolic extract of A.
macrophylla root bark in MOR-P, COR-L23 cell lines using SRB assay
(Keawpradub and PJ Houghton 1997). Except, macralstonine (124) and villal-
stonine Nb-oxide (127) (89.4 and 62.2 µM), all the tested indole alkaloids exhibited
a strong cytotoxicity activity in MOR-P (2.3–19.6 µM) than the monomeric indoles
(61.4 to >100 µM), while the same trend was observed in COR-L23 cell lines
(2.92–20.2 µM for bisindole alkaloids; 57.2 to >100 µM for monomeric indoles).
Bisindole alkaloids such as 114, 115, and 116 exhibited the potent activity (6.3, 4.6,
and 2.3 µM in MOR-P cell line; 4.1, 5.3, and 2.9 µM in COR-L23 cell lines,
respectively). Further, cytotoxicity effects of these alkaloids were evaluated in
various cell lines such as StM 1a, Caki-2, MCF-7, and LS174T. These alkaloids
exhibited an IC50 in the range of 2–7 µM. However, the results from the normal cell
lines (breast fibroblasts) exhibited IC50 values of 8.1–21.9 µM highlighting that
these alkaloids lack the selectivity toward the cancer cell cytotoxicity.
Changwichit et al. isolated secoiridoid (Fig. 14.30) from the methanolic extract
of A. macrophylla stems and evaluated its cytotoxicity effects (Changwichit et al.
2011). At a concentration of 10 µg/mL of naresuanoside (129) resulted in a 22%
inhibition in the growth of human androgen-sensitive prostate cancer cell (LNCaP),
Table 14.12 Cytotoxic Compound IC50 values (µM for the compounds and nM
effects of various alkaloids Cell lines for vinblastine)
from A. macrophylla and
114 115 116 Vinblastine sulfate
vinblastine sulfate against
various cell lines MOR-P 6.3 4.6 2.3 3.1
COR-L23 4.1 5.3 2.9 0.9
BF 21.9 8.1 8.5 >100
StMl la 3.3 2.9 2.4 1.7
Caki-2 4.6 7.3 2.9 1.9
MCF-7 2.4 1.9 3.4 0.5
Ls 174T 2.4 1.8 1.9 0.8
MOR-P-adenocarcinoma, COR-L23 human lung cancer cell
lines; BF-normal human breast fibroblast cell line; StMl
la-melanoma; Caki-2-renal cell carcinoma; Ls174T-colon
adenocarcinoma
O N
N N
O OH H H H
N O
N N
H
N N OH
O
OH
Talcarpine (118) Pleiocarpamine (119) Alstoumerine (120) 20-Epi-antirhine (121)
O O
N
N O R N
O N
R N
N
O
O
Alstonerine (122), R = H Macralstonine (124); R = OH

Alstophylline (123), R = OMe O-Methyl macralstonine (125); R = OMe
N O
N O
O O
H H N
N OH
N
N
O O
N N N N
OH
N N
H3COOC O
O H
OO H
O
Alstomacrophylline (126) Villalstonine Nb-oxide (127) Alstomacroline (128)

Fig. 14.30 Chemical O O O

structure of naresuanoside HO OH H
(129) O
O
HO O
H
O O
O OH
O
O
HO HO
O
Naresuanoside (129)
while in a concentration range of 0.1 ng/mL–10 µg/mL did not exhibit any kind of
inhibition in the cell growth of human foreskin fibroblast cells (HF cells, normal
cells).
Lim et al. isolated 17 alkaloids (130–146) from the ethanolic extract of A.
macrophylla stem bark and leaf (Fig. 14.31). Further, they evaluated the MDR
reversing capacity in KB/S (vincristine-sensitive) and KB/VJ300
(vincristine-sensitive and vincristine-resistant KB cell lines) (Lim et al. 2014).
All the compounds exhibited poor cytotoxicity in the cell lines (IC50 > 25 µg/mL).
However, 11-methoxyvincorine (141), 11-demethoxyquaternine (144), 19,20-Z-
affinisine (136) were reported to reverse MDR in vincristine-resistant KB (VJ300)
cells (IC50 = 4.60, 6.60 and 7.47 µg/mL, respectively).
14.3.2.2 Antiplasmodial Activity
Keawpradub et al. isolated 13 alkaloids (114–116, 118–128) from the methanolic

extract of A. macrophylla leaves, stem bark, root bark and evaluated the antiplas-
modial activity against the K1 strain of P. falciparum by using chloroquine
diphosphate as a positive control (Keawpradub et al. 1999). Bisindole alkaloids
such as 114, 115, 116, and 128 (IC50 values = 0.53, 0.36, 0.27, and 1.12 µM)
exhibited a comparable activity with the chloroquine diphosphate (IC50
value = 0.20 µM). Further, the effects of these alkaloids were evaluated in
chloroquine-resistant (K1) and chloroquine-sensitive strains (T9–96) of
P. falciparum. These bisindoles were found to be significantly less active against
T9–96 (0.94–39 µM; chloroquine diphosphate—0.02 µM). In K1 cells, they
exhibited a significant activity (0.36–1.12 µM; chloroquine diphosphate—
0.20 µM).
Hirasawa et al. isolated alstiphyllanines A-D (147–150, Fig. 14.32) from the
methanolic extract of A. macrophylla leaves and evaluated the antiplasmodial
activity (against P. falciparum, 3D7) (Hirasawa et al. 2009). Screened alkaloids
exhibited a moderate in vitro antiplasmodial activity (IC50 of 6.85, 0.34, 6.20, and
2.75 µg/mL, respectively, for 147–150). In vitro cytotoxicity evaluation resulted in
H H
H H O
H N
H N
H O
N HO N O
N N H HO
N N O H
H H O
H H O O H
O
Alstofolinine A (130) 20,21-Dihydroalstonerine (131) Macrocarpine (132) Macrodasine H (133)
OMe OMe H
CH2OH
HO
O OH H
N N N
N N N
HN HN H
O
O O
HO HO H O
Alstonoxine C (134) Alstonoxine D (135) 19,20-Z-affinisine (136) 2(R)-3-Hydroxycathafoline (137)
R1 R COOMe
COOMe H
H
2
R
O
N H N N
N N
N
O
R O
H H
2(S) cathafoline (138); R = H 10-Demethoxyvincorine (140); R1 = R2 = H 10-demethoxyvincorine-N4-oxide (142); R = H

2(S)-10-methoxycathafoline (139); R = OMe 11-methoxyvincorine (141); R1 = R2 = OMe Vincorine-N4-oxide (143); R = OMe
N O
N
MeO COOMe
H O
O O
N O
O O
N N N
H O
MeO
O
H HO MeO
the conclusion that these compounds are not cytotoxic in nature (IC50 > 25 µg/mL).
148–150 comprised of picraline-type skeletons (arise through a corynanthe-type
skeleton) while 147 consists of an ajmaline-type skeleton.
Cheenpracha et al. isolated 11 alkaloids (118, 151–160) from the methanolic
extract of A. macrophylla bark (Fig. 14.33) and evaluated the antiplasmodial
activity (P. falciparum K1, MDR) and cytotoxicity activities in KB cell lines
(Cheenpracha et al. 2013). Alstonisine (158) exhibited moderate antiplasmodial
activity (IC50 of 7.6 µM), while dihydroartemisinin was used as a positive control
(IC50 = 1.41 nM). Remaining screened compounds did not exhibit any antiplas-
modial activity. In cytotoxicity assays, all the screened compounds were not
cytotoxic to the KB cell line (IC50 = >100 µM). Doxorubicin (IC50 = 0.55 µM)
and ellipticine (IC50 = 4.87 µM) were used as reference cytotoxic substance and
positive control.
O O
N+ O O
O O
N O O
O N O N
-
O
N+ O O O
O O N O
O O
O O
O
Alstiphyllanine B (148); R = H
Alstiphyllanine A (147) Alstiphyllanine C (149); R = OMe Alstiphyllanine D (150)
Fig. 14.32 Chemical structures of alstiphyllanine A-D from A. macrophylla (147–150)
O
O HO O
O-
N+
N O N OH OH
N O O
O O N
Alstoniaphylline A (151) Alstoniaphylline B (152) Angustimaline (153) Alstoniaphylline C (154)
N N
O
HO
O N N
N O
N
O O
O
Alstonerinal (155) Alstophyllal (156) Macrocarpine B (157)
O O
O N
N
HN N
HN
O N O O
O O O O
Alstonisine (158) Nb-Demethylalstophylline Oxindole (159) 10-Methoxy cathafoline (160)
14.3.2.3 Vasorelaxant Activities
Hirasawa et al. evaluated the vasorelaxant effects of various A. macrophylla

leaves-derived alkaloids (147–150) (Hirasawa et al. 2009). The screened molecules
exhibited a slow relaxation activity against phenyl epinephrine (3 10−7 M)-
induced thoracic rat aortic rings with endothelium. Compound 147 exhibited a 70%
vasodilation, while 35, 40, and 42% vasorelaxation was observed by the treatment
of 148–150.
O N O
R1
N O
2 R4 O
R N O
O
R3 N
O OH
Alstiphyllanine I (161); R1 = H, R2 = R3 = OMe, R4 = OCOCH3
O
Alstiphyllanine J (162); R1 = R2 = R3 = OMe, R4 = OCOCH3
Alstiphyllanine K (163); R1 = R2 = R3 = H, R4 = OCOCH3
Alstiphyllanine M (167)
Alstiphyllanine L (164); R1 = H, R2 = R3 = OMe, R4 = OH
Alstiphyllanine N (165); R1 = R2 = R3 = H, R4 = OH
Alstiphyllanine O (166); R1 = R2 = R3 = OMe, R4 = OH
N
N N
N
O- N
N+ O
O O
O
O O O
HO HO
O
Alstiphyllanine H (168) Vincamedine (169) Vincamajine (170)
MeO N
N
O MeO O
MeO
O MeO O N
N
MeO
O O O
O
Vincamajine-17-O-3',4',5'-trimethoxybenzoate (171) Vincamajine-17-O-veratrate (172)
Arai et al. reported the ex vivo (rat aorta) vasorelaxant activities of A. macro-
phylla-derived alkaloids (161–172, Fig. 14.34) (Arai et al. 2012). Various mech-
anisms were proposed for the vasoconstriction. Administration of 0.3 µM of
phenylephrine to the thoracic aortic rings with endothelium resulted in vasocon-
striction. Vincamedine (169) resulted in a potential activity within 5–15 min, while
alstiphyllanine H (168) exhibited poor activity. Further, to understand the
involvement of endothelial cells, vasorelaxant activity was tested using
endothelium-denuded aorta (-EC rings). In the presence of a nitric oxide synthase
(NOS) inhibitor, NG-monomethyl-L-arginine (L-NMMA, 100 µM), the relaxation
was attenuated in -EC rings, clearly indicating that the vasorelaxation was partially
mediated by the NO release from endothelial cells. Further, it was confirmed that
the vasorelaxant effect of 169 did not involve a K+ channel.
14.3.2.4 Neuroleptic Activity
Chatterjee and Dey evaluated the neuroleptic activity of 116 (from A. macrophylla)
by calculating the brain serotonin level in mice (Chatterjee and Dey 1964).
Intraperitoneal administration of 116 (20 mg/kg) has shown an effect in the central
nervous system. Brain serotonin content was identified as 0.64 and 1.64 µg/g after
15, 30 min of villalstonine administration.
14.3.2.5 Contraceptive Effects
Chattopadhyay et al. screened the effects of methanolic extract and its subsequent
n-butanol fractions of A. macrophylla leaves on the forward motility (FM) of
mammalian (goat and human) spermatozoa (Chattopadhyay et al. 2005). n-butanol
fractions exhibited a higher potential than the mother methanolic extract. Further,
fractionation resulted in three fractions, such as 58 (fraction A), 41 (fraction B) and
b-sitosterol glucoside (173, Fig. 14.35) and a mixture of minor compounds (frac-
tion C). Although a semi-purified fraction was utilized for the pharmacological
screening, the majority of activity raised was attributed to the presence of 41 only.
A dose-dependent activity was exhibited by 41. In spectrophotometric motility
assay of goat cauda sperm, control cells showed 40% vigorous FM. However, a
reduced sperm motility was observed with the treatment of 41 at 100 and 200 µg/
mL concentrations (25 and 5%, respectively). In human sperm, 41 (100 µg/mL)
caused 90% inhibition on the FM. From these results, it is clear that 41 exhibited a
higher inhibitory activity toward the human spermatozoa FM than goat cauda
sperm.
14.3.2.6 Sodium-Glucose Cotransporters (SGLT) Inhibiting Potential
Arai et al. isolated 20 alkaloids (19, 20, 122, 147–150, 168–172, 174–181) from the
methanolic extract of A. macrophylla leaves (Fig. 14.36) and evaluated the inhi-
bitory potential of sodium-glucose cotransporters (Arai et al. 2010). The in vitro
Fig. 14.35 Chemical

structure of b-sitosterol
glucoside (173)
OH
HO OH
HO
O O
-Sitosterol glucoside (173)
O
H O
N O N
N O O N
O
O O O N
N O
H O O O O HO
O O
O O
O O
Alstiphyllanine E (174) Alstiphyllanine F (175) Alstiphyllanine G (176)
R'
NO N
10-Methoxy-N(1)-methylburnamine-
R1O O 17-O-veratrate (177); R1 = OMe, R2 = H, R' = Me
O O Burnamine-17-O-30,40,50-trimethoxybenzoate (178);
R1 = H = R' = H, R2 = OMe
R2 OMe
OMe
N H O
O H
N HO N
O N
NH
O H O
O HO
O N O
O
O
Quaternine (179) O-Deacetylpicraline (180) Alstonal (181)
Table 14.13 SGLT Compound % inhibitiona

inhibitory activities of various
SGLT1 SGLT2
alkaloids from A.
macrophylla 150 89.9 101.4
174 60.3 85.9
175 65.2 103.8
177 95.8 102.6
178 53.0 87.3
a
At 50 µM
SGLT inhibitory potential was assessed by monitoring inhibition of uptake of

methyl-a-D-glucopyranoside in cultured cells expressing SGLT1 or SGLT2. As
represented in Table 14.13, 10-methoxy-N1-methylburnamine-17-O-veratrate
(177) resulted in a significant SGLT inhibition, followed by 150 and 175 (95.8,
89.9, and 65.2 for SGLT1 inhibition; 102.6, 101.4, and 103.8 for SGLT2,
respectively). The remaining compounds exhibited a moderate to poor SGLT
inhibition activity (0–30% inhibition).
OH
OH
HO
O
OH O OH
OH O O
H
OH OH
OMe O HO O
O HO O
OH OH
OH
HO O OH O OH O O
OMe
HO
OH
HO OH
OH
OH O OH
Tricin-4'-O- -L-arabinoside (182) Vitexin (183) Myricetin-3'-rhamnoside-
3-O-galactoside (184)
Fig. 14.37 Chemical structures of flavonoid analogues from A. macrophylla (182–184)
14.3.2.7 Antifungal and Antibacterial Effects
Parveen et al. isolated three flavonoids (182–184, Fig. 14.37) from the methanolic
extract of A. macrophylla leaves and evaluated the antifungal and antibacterial
effects of triacin-4′-O-b-L-arabinoside (182) by using agar well diffusion methods
(Parveen et al. 2010). Antibacterial effects were screened in S. aureus (IAO-SA-22)
and E. coli (K-12) by using chloramphenicol as a positive control. Antifungal
activities were performed in S. typhimurium (MTCC-98) and C. albicans
(IAO-109) by using nystatin as a positive control. The compound exhibited a
maximum zone of inhibition with S. typhimurium (20 mm), E. coli (16 mm), fol-
lowed by S. aureus and C. albicans (13 and 12 mm, respectively).
14.3.2.8 Cholinesterase Inhibition Activity
Changwichit et al. isolated 129 from the methanolic extract of A. macrophylla

stems and evaluated the cholinesterase inhibition activity. Compound 129 exhibited
a mild acetylcholinesterase (AChE) inhibitory activity on electric eel AChE
(IC50 = 64.02 µM), human recombinant AChE (IC50 = 88.93 µM), and horse
BChE (IC50 = 110.25 µM). Galantamine was used as a positive control that
exhibited an IC50 of 0.15, 0.10 and 1.30 in electric eel AChE, human recombinant
AChE, and horse BChE, respectively (Changwichit et al. 2011).

of A. Angustifolia
14.3.3.1 Anti-protozoal and Antiplasmodial Activities
Wright et al. isolated ten alkaloids (115, 119, 122–124, 170, 185–188) from the
methanolic extract of A. angustifolia roots (Fig. 14.38) and evaluated the
N
H
H
N N O
H
N N
N O O O
H O
N N R1
H H
N
O N N
O H R2
H3CO2C H
Villastonine (185) Macralstonine acetate Norfluorocurarine (187);

(186) R1 = H, R2 = CHO
11-Methoxyakuammicine (188);
R1 = OMe, R2 = COOCH3
Fig. 14.38 Chemical structures of various alkaloids reported from A. angustifolia (185–188)
anti-protozoal (Entamoeba histolytica) and antiplasmodial (P. falciparum) activities

(Wright et al. 1992). Emetine hydrochloride and chloroquine diphosphate were
used as positive controls for anti-protozoal and antiplasmodial activities, respec-
tively. Among the screened alkaloids, 115, villastonine (185), and macralstonine
acetate (186) exhibited a significant anti-protozoal activity (ED50 = 8.12, 11.8, and
15.51 µM, respectively). Compound 124 and 170 were found to be inactive in the
anti-protozoal screening. Emetine hydrochloride exhibited an ED50 of 2.04 µM. In
the case of anti-spasmodial activity, chloroquine diphosphate exhibited an ED50 of
0.168 µM. Compound 185 exhibited potential antiplasmodial activity
(ED50 = 2.92 µM), followed by 186 and 115 (ED50 = 3.43 and 9.36 µM, respec-
tively). Compound 124 was found to be inactive against the P. falciparum. The
remaining compounds exhibited ED50 values in the range of 20.50–138 µM.
14.3.3.2 Cytotoxicity Studies
Tan et al. isolated 20 alkaloids (189–208, Fig. 14.39) from the ethanolic extract of A.
angustifolia barks, leaves and evaluated the reversing ability toward the MDR in
vincristine-resistant KB cells (Tan et al. 2014). The screened alkaloids did not
exhibit any kind of cytotoxicity against the vincristine-sensitive and
vincristine-resistant (KB/VJ300) cells (IC50 > 25 µg/mL). However, in the presence
of 0.12 µM vincristine, 122 exhibited a strong activity in reversing MDR in
drug-resistant KB/VJ300 cells (IC50 = 10 µM). Apart from that alkaloids alstolac-
tone A (191), O-acetyl talpinine (195), and 119 showed moderate to weak activity in
the presence of 0.12 µM wherein, IC50 values range between 40 and 60 µM.
Pan et al. isolated ten alkaloids (116, 122, 132, 155, and 209–214) from the
methanolic extract of A. angustifolia stem bark (Pan et al. 2014) and screened the
cytotoxic effects against HT-29 human colon cancer cell line (Fig. 14.40).
Paclitaxel (positive control) exhibited an ED50 value of 0.006 µM, while the vil-
lalstonidine E (214), 116 and 122 exhibited an ED50 values of 6.5, 8.0, and 8.6 µM,
respectively. The remaining compounds were considered as inactive in nature
(>20 µM).
O
N O
N
O
N
O N O HN O O N N
N O
O O O
O
Alstofonidine (189) Alstofolinine B (190) Alstolactone A (191) Macrogentine A (192)
N N
O
N
H OH
N H O N
H NH
H HN
H O O O N
HO O
HO
HO OH
Isoalstonoxine B (193) Alstonoxine E (194) O-Acetyltalpinine (195) N4-Methyl-19-epitalpinine (196)
H R'
N
H O H
OH H HO R N
N O
N N
N H H O
O H H CHO
Macrocarpine E (199); R = Me, R' = H

7(S)-Talpinine oxindole (197) 19-Epi talcarpine (198) Macrocarpine F (200); R = H, R' = Me
N H
N H
O N
HO
R1 N R2 N O
N
O O
O
Macrocarpine G (201); R1 = R2 = H
Macrocarpine H (202); R1 = Me, R2 = OMe 1
N -Demethylalstonerine (203) 1
N -demethylalstonerinal (204)
O
O H
O N H
O CH2OH
O N O N
N N N
N H
N
HO OH
HO O HO
7-Hydroxy 6-Oxo Alstoumerine oxindole (207) Normacusine

pleiocarpamine (205) pleiocarpamine (206) B-2(S)-pseudoindoxyl (208)
Fig. 14.39 Chemical structures of various alkaloids from A. angustifolia (189–208)
14.3.3.3 NF-jB (P65) Inhibitory Activity and Antileishmanial Activity
NF-jB (p65) inhibitory activity of the isolated compounds (116, 122, 132, 155, and
209–214) was performed in HeLa cells by using rocaglamide as a positive control
(Pan et al. 2014). Except for N4-methyl talpinine (209; IC50 value = 1.2 µM), the
remaining compounds were identified as inactive to the NF-jB inhibition.
Rocaglamide exhibited an IC50 of 0.08 µM. In the antileishmanial activity
screening, N4-methyl-N4,21-seco talpinine (210), 214, and villalstonine N4-oxide
H H O
N
N N
H CHO N OH
O N+ N H
OH
4
N -Methyltalpinine (209) N4-Methyl-N4,21-secotalpinine (210) Affinisine (211)
O
H
H
H O
N H
R Villalstonine N4-oxide (212); R = O-
N Villalstonidine D (213); R = CH3
N H
Villalstonidine E (214); R = CH2Cl
NH
O OMe
Fig. 14.40 Chemical structures of various alkaloids from A. angustifolia (209–214)
(212) exhibited a moderate activity (IC50 values = 57.8,78.0, and 80.3 µM) while
the remaining compounds exhibited poor IC50 values (>100 µM).

of A. Boonei
14.3.4.1 Behavioral, Acute Toxicity Effects, Diuretic Study,

Cardiovascular Activity Studies, and Neuromuscular Activity
Ojewole reported various pharmacological activities of 9, isolated from the stem

bark of A. boonei. Behavioral and acute toxicity effects (in albino mice), diuretic
study (albino rats), cardiovascular activity studies (normotensive albino rats and
cats), and neuromuscular activity (cats) were performed in whole animals (Ojewole
2008). Effects of 9 in various muscles were screened by using an isolated muscle,
such as isolated atrial muscles and vascular smooth muscles. Guinea pig isolated
spontaneously beating atria, electrically driven left atria represented the atrial
muscles, whereas, rat isolated portal vein that represented the isolated vascular
smooth muscles. The LD50 of 9 was found to be 70.5 mg/kg i.p. and it did not
produce any kind of behavioral changes in rat till 1/7th of LD50 values. Two doses
of 9 (25 and 50 mg/kg/oral) were administered to the albino rats for understanding
the diuretic effects. Hydrochlorothiazide was used as a positive control (157.7% in
urine output at 4 h). At 4 h, 9 exhibited the maximum diuretic effect (87.7% and
118.9% increased urinary output for 25 and 50 mg/kg of 9). A dose-dependent
reduction in systemic arterial blood pressure and heart rate were observed in nor-
motensive albino rats (for blood pressure-8.5–77.2% for 5–180 min; for heart
rate-6–63% at a concentration range of 0.05–10.0 mg/kg i.v of 9) and cats (at a
concentration range of 0.025–10.0 mg/kg i.v of 9). In vivo neuromuscular activity
screening revealed that 9 (5–20 mg/kg, i.p.) depressed (23–81%) the twitches of the
cat anterior soleus or tibialis muscle preparation induced by indirect electrical
stimulation. At a concentration range of 10–100 µg/mL of 9, spontaneously beating
atria isolated from the guinea pig reduced the amplitude and rate of contractions.
However, in the electrically driven left atria, the force of contraction was also
reduced. In the isolated smooth muscles, a depression in the amplitude of the
spontaneous myogenic contractions was observed with the administration of 9 (10–
100 µg/mL).
14.3.4.2 Antiarthritic Effect
Okai and Carroll isolated 39 from the petroleum ether extract of A. boonei root
barks and evaluated its antiarthritic effect in complete Freund’s adjuvant (CFA)-
induced arthritic Wistar rats (Kweifio Okai and Carroll 1993). At the treatment
periods, 66 mg/kg body weight of 39 was administered orally in every 48 h from
32 to 40 days of post-adjuvant. Ankle and paw diameters remained unchanged
among the treatment and control groups. Among the various biochemical param-
eters, alkaline phosphatase (U/L), spleen weight (g%) were altered during the
arthritic conditions (control-265 U/L and 0.19 g%, respectively; arthritic rats-219
U/L and 0.22 g%, respectively). However, treatment with 39 returned the increase
in spleen weight (0.20 g%) and the reduction in serum alkaline phosphatase (272 U/
L).
In another study, Okai et al. investigated the antiarthritic effects of a-amyrin
palmitate (215, Fig. 14.41) in complete Freund’s adjuvant (CFA)-induced arthritic
Wistar rats (Kweifio Okai et al. 1995). However, in the study, acute and chronic
arthritic conditions were identified and evaluated for the effects of 215. During the
acute phase, the ipsilateral ankle swelling was rapidly increased from 60% (11 days
after CFA induction) to 94% (19 days after CFA induction). In the chronic phase,
the percent swelling was similar from 32 to 50 days. Treatment of 215 prevented
the percentage swelling rate by 34% in acute arthritic conditions. Higher blood
granulocyte and serum hyaluronate were observed in the acute conditions of
arthritis with the control groups (1292–9827 106/L and 181–287 µg/L). In
chronic conditions also these biochemical parameters were increased from the
control groups (Blood granulocytes from 668 to 2287 106/L and Serum hya-
luronate from 100 to 277 µg/L). However, 215 treatment results in declined bio-
chemical parameters (For acute condition: blood granulocytes-5681 106/L and
Serum hyaluronate-149 µg/L; for chronic conditions: blood granulocytes—
1813 106/L and Serum hyaluronate—147 µg/L).
-amyrin palmitate (215)
Fig. 14.41 Chemical structure of a-amyrin palmitate (215)
-Amyrin linoleate (216)
Lupeol palmitate (217)
Lupeol linoleate (218)
Fig. 14.42 Chemical structures of various triterpenoids A. boonei (216–218)
14.3.4.3 Serine Protease Inhibitory Potential
Rajic et al. reported the serine protease (trypsin and chymotrypsin) inhibition
potential of triterpenoids (38, 39, 215–218) from the barks and roots of A. boonei
(Fig. 14.42) (Rajic et al. 2000). In the case of trypsin inhibition, lupeol palmitate
(217) exhibited a higher potential (IC50 = 6.0 µM, with a Ki value of 10.4 µM)
than the remaining triterpenoids (IC50 = 10–41 µM). However, for chymotrypsin
inhibition, a-amyrin linoleate (216) exhibited the potential activities of 16 µM with
a Ki value of 27.5 µM. Further, these compounds have inhibited the enzymes
through non-competitive inhibition kinetics.
14.3.4.4 Anti-inflammatory Activity
Okoye et al. isolated 37 and 55 from the methanolic extract of A. boonei stem bark
(Okoye et al. 2014). The anti-inflammatory activity was evaluated by using egg
albumen-induced paw edema and xylene-induced ear edema models in rats. Apart
from this, gastric ulcerogenic, in vivo leukocyte migration, and RBC membrane
stabilization tests were also investigated. Prior (30 min) to the sub-plantar injection
of the egg albumen (0.1 mL), 37 (50 and 100 mg/kg) was administered orally.
Aspirin (100 mg/kg) was used as a positive control. Aspirin recorded significant
reduction in paw edema from the 2nd h, while the screened compound (100 mg/kg)
showed a significant activity from the 5th h. Ulcerogenic effect of 37 (50 and
100 mg/kg) was identified by using an ulcer index value. Indomethacin (40 mg/kg)
was administered as a positive control. Indomethacin evoked significant irritation of
the gastric mucosa compared with the control, while the test compound did not
evoke significant irritation. Heat-induced hemolysis and hypotonicity induced
hemolysis methods were used for the assessment of membrane stabilization
potential of 55 and 37. Diclofenac sodium (100 µg/mL) was used as a positive
control. Screened compounds (50 and 100 µg/mL) exhibited higher inhibition on
heat-induced haemolysis than diclofenac sodium (40% inhibition). Conversely, in
hypotonicity induced haemolysis, diclofenac sodium (50% inhibition) exhibited a
higher potential than the compounds. Further, to evaluate the acute topical
inflammation, the effects of 37 and 55 in xylene-induced ear inflammation were
determined. Compound 37 exhibited a higher % inhibition (>45% at 50 and
100 µg/mL) while 55 exhibited a comparable activity to that of indomethacin (45%
inhibition 100 µg/mL). Furthermore, a significant inhibition of leukocyte migration
and neutrophil infiltration were produced by 37 (100 mg/kg) in comparison with
positive control (indomethacin—50 mg/kg).
14.3.4.5 Antibacterial and Antioxidant Activities
Okoye et al. isolated eight flavonoid glycosides (219–226, Fig. 14.43) from the
methanolic extract of A. boonei leaves and evaluated the antioxidant activity (DPPH
free radical scavenging model) and antimicrobial activity (Agar well diffusion
technique) (Okoye and Okoye 2016). In DPPH scavenging assay, kaempferol
derivatives did not exhibit any kind of activities, while the quercetin derivatives
exhibited dose-dependent activities (IC50) ranging from 36.0 to 66.0 µg/mL.
Quercetin-3-O-[a-L-rhamnopyranosyl (1 ! 4)-b-D-glucopyranoside] 223) and
Quercetin-3-O-robinobioside (220) exhibited a higher DPPH scavenging potential
OH OH
HO OH HO OH OH OH
OH OH HO OH
OH HO OH
HO HO OH
O O O O HO
O O HO
O O O O
HO O OH HO O OH O
HO O OH O
O O HO O OH
O O
O OH O OH
O OH O OH
HO HO
OH OH HO HO
Rutin (219) Quercetin robinobioside (220) Kaempferol-3-O-rutinoside (221) Kaempferol-3-O-robinobioside (222)
OH
HO OH OH
HO OH OH
O O OH
HO HO
HO O O HO HO O OH
OH O OH
HO O
O OH O HO O O OH
HO O OH HO O O OH
O OH O
O HO O OH OH O
O O OH
O OH O OH
O OH HO
HO HO
OH
OH HO OH
Quercetin-3-O-[ -L-rhamnopyranosyl Kaempferol-3-O-[ -L-rhamnopyranosyl Quercetin-3-O-[ -L-rhamnopyranosyl Quercetin-3-O-[ -L-rhamnopyranosyl

(1 4) -Dglucopyranoside] (223) (1 4) -Dglucopyranoside] (224) (1 2) -Dglucopyranoside] (225) (1 2) -Dgalactopyranoside] (226)
Fig. 14.43 Chemical structures of various flavonoid glycosides from A. boonei (219–226)
(IC50 values = 36.0 and 48.0 µg/mL) than the positive control, Vit. C (IC50 of
49.0 µg/mL). Antimicrobial activity was performed against E. coli. Quercetin-3-O-
[a-L-rhamnopyranosyl (1 ! 2)-b-D-glucopyranoside] 225) and Quercetin-3-O-
[a-L-rhamnopyranosyl (1 ! 2)-b-D-glucopyranoside] (226) exhibited a potential
antibacterial activity with a MIC values of 1.77 µg/mL and 1.92 µg/mL,
respectively.
Olaoye S. et al. isolated a cytotoxic indole alkaloid, Alstiboonine (227, Fig. 14.44),
from aqueous methanolic extract of A. boonei stem bark and confirmed its cytotoxic
activities through brine shrimp lethality assay (Balogun et al. 2016). The titled
compound exhibited a potent LD50 value (39.72 µg/mL) and was claimed as a
cytotoxic phytochemical.

of A. Venenata
14.3.5.1 Hypertensive and Neuroleptic Properties
P. K Dey evaluated various pharmacological activities of alstovenine (228,

Fig. 14.45), an alkaloid from A. venenata (Dey 1965). The hypertensive activity of
228 was screened in chloralosed cat, and the desired pharmacological activity was
observed with a concentration of 25–30 µg/kg of body weight. Further, a 100–
Fig. 14.44 Chemical O

structure of alstiboonine (227) HO O
N H
O
N
H
Alstiboonine (227)
Fig. 14.45 Chemical

structure of alstovenine (228) O O
HN
HO
O
N
Alstovenine (228)
200 µg bath of 228 did not exhibit any kind of activity in uterine and intestinal
contractions. Higher nervous activity was screened in the rabbit, by injecting 30 µg/
kg of 228. The treated animal did not exhibit any kind of immediate effect. After
30 min, it became quiet, showed a stupor attitude. Neuroleptic properties of 228
were identified with the help of spontaneous motility tests in mice. Intraperitoneal
administration of 500 µg/kg of the drug resulted in somnolence and a state of
apprehension after 30–40 min.
14.3.5.2 Psychopharmacological Effects
Bhatt acharyaa et al. evaluated the psychopharmacological effects of A. venenata-

derived alkaloids (228, venenatine (229) and echitovenidine (230), Fig. 14.46) by
various methods (Bhattacharya et al. 1975). Compound 228 (1 mg/kg, i.p.) pro-
duced signs of central excitation such as piloerection, irritability, enhanced loco-
motor activity, increased startle response, and exophthalmos. A reduction in motor
N
O
O O
HN O
HO
O N
N H O
O
Venenatine (229) Echitovenidine (230)
Fig. 14.46 Chemical structures of venenatine and echitovenidine (229 and 230)
activity and sedation was resulted by the administration of 229 (50 mg/kg, i.p.).
However, at 100 mg/kg (higher dose), a sedative effect was observed in animals. In
the case of 230, a central stimulation in rats and mice was observed. However, in
the later phase, a central depression had occurred. All the tested compounds
(100 mg/kg, i.p.) potentiated the hexobarbitone hypnosis. Both 228 and 230
antagonized reserpine induced sedation, ptosis at 1 and 2.5 mg/kg, i.p. respectively.
However, 229 (100 mg/kg, i.p.) synergistically enhance the ptosis and sedation.
Further, 228 and 230 (1 and 2.5 mg/kg, i.p., respectively) potentiated the lethal
effects of amphetamine (10 mg/kg, i.p.) in aggregated mice. Furthermore, these
compounds potentiated the behavioral effects of DOPA and 5-HTP. In contrary,
229 did not exhibit any kind of activity. The analgesic activity of a subanalgesic
dose of morphine (2 mg/kg, i.p.) was potentiated by 228 (a latent period of tail-flick
response from 9.2 to 22.3 s.) and 230 (a latent period of tail-flick response from
10.3 to 25.3 s). While 229 had antagonizing effects on the analgesic activity (a
latent period of tail-flick response from 28.6 to 16.2 s.). Diphenylhydantoin
(2.5 mg/kg, i.p.) did not exhibit any anti-convulsant activity. However,
pre-treatment with 228 and 230 (1 and 2.5 mg/kg, i.p., respectively) resulted in
60% of anti-convulsant activity. Compound 229 antagonized the anti-convulsant
activity (effective dose—25 mg/kg, i.p. of diphenylhydantoin exhibited a 100%
inhibition, while pre-treatment resulted in a 40% anti-convulsant activity).
Compounds 228 and 230 potentiated the tryptamine induced convulsions (trypta-
mine control group—10% animals having convulsion, whereas 228 group—70%
animal; 230 group—60% animals). 229 did not exhibit any kind of
tryptamine-related activities. A transient depressor response along with a stimula-
tion of respiration was produced by 228 in anaesthetized dog’s blood pressure,
while 230 exhibited the same kind of activity without respiratory stimulation.
229 produced a moderate depressor response. LD50 of the compounds was found to
be 8.7, 126, and 176 mg/kg, respectively for 228, 230, and 229. These all results
indicated the potential of 228 and 230 as an inhibitor of monoamine oxidase.
14.3.5.3 Antifungal Activities
S. K. Singh et al. evaluated the antifungal activities of D3-Alstovenine (231,

Fig. 14.47), a quaternary alkaloid isolated from the water-soluble base fraction of
the bark of A. venenata (Singh et al. 1999). A series of concentrations (0, 250, 500,
Fig. 14.47 Chemical

structure of D3-Alstovenine O O
HN
(231) H
HO
O
N
H
Cl
-Alstovenine (231)
700, and 1000 mg/L) were evaluated against 17 fungal strains. At 250 mg/L
concentration, spore germination of Cercospora sp. was completely inhibited while
at a concentration of 750 or 1000 mg/L, H. sativum, Helminthosporium maydis and
Erysiphe polygoni were found to be sensitive to 231 (100% inhibition of spore
germination). Alternaria species and Fusarium udum were found to be resistant to
the tested compounds. Further, 231 was active against facultative and biotrophic
pathogens.
In another study, U. P. Singh et al. evaluated the antifungal activity of 229
against ten species of fungi (Singh et al. 2000). Since 229 is insoluble in water, an
acetate form was prepared by dissolving the alkaloid in a molar equivalent of
1 mol/L aqueous acetic acid. A series of concentrations (0.5, 1.0, 1.5, 2.0 mg/L)
were evaluated for antifungal activities. An aqueous solution of 229 as its acetate
showed antifungal activity against all the plant pathogenic and saprophytic fungi. A.
brassicicola and U. cynodontis were most sensitive to 229 (higher inhibitory
potential at 0.5 mg/L concentration). At the higher concentrations (2.0 mg/L),
Fusarium udum, Aspergillus flavus, Alternaria brassicicola, and Ustilago cyn-
odontis showed maximum sensitivity (seed germination is less than 10%). Further,
the effect of 229 on the germination and development of E. pisi conidia on excised
pea leaves were also evaluated. Results indicated that 229 had a marked effect in the
reduction of germination of E. pisi conidia and pre-inoculation treatment with 229
showed greater efficacy than post-inoculation treatment.

of A. Yunnanensis
14.3.6.1 Cytotoxic and Anti-inflammatory Activity
Feng et al. isolated various alkaloids (20, 232–255, Fig. 14.48) from the alkaloid
extract of A. yunnanensis whole plants and cytotoxic and anti-inflammatory
potential were evaluated (Feng et al. 2009). Alstoyunines C (234), E (236), and F
(237) displayed selective inhibition of COX-II (>75%), while NS-398 (positive
control) exhibited a 97.09% COX-II inhibition. All the screened alkaloids exhibited
poor activity against COX-I. In the 5-LOX inhibition assay, 236 exhibited 77.72%
inhibition, while positive control (zileuton) exhibited 83.05% inhibition.
Cytotoxicity evaluations were performed in HL-60, A-549, SMMC-7721, pancre-
atic cancer (PANC-1), and SK-BR-3cells. Except 237 remaining compounds
exhibited a poor activity in all the cell lines (IC50 > 40 µM). 237 exhibited an IC50
of 3.89 µM against HL-60.
In another study, Cao et al. isolated 8 (240, 256–262) monoterpenoid indole
alkaloids (Fig. 14.49) from the ethanolic extract of A. yunnanensis whole plants and
O
H O- O
N OH N
R1 N OH
N N+ -
N+ O
O N+ O - N+ O -
R2 O
O
O
O O
Alstoyunine A (232); R1 = OMe, R2 = OH O
Alstoyunine B (233); R1 = OH, R2 = OMe Alstoyunine C (234) Alstoyunine D (235) Alstoyunine E (236)
O
Cl
N O N O
N N
O N OH
N
O O
N N
O O H O O
O O
O
O
Alstoyunine F (237) Alstoyunine G (238) Alstoyunine H (239) Vinorine (240)
N O N O N
N H
N
N O O
N N
O H H O
O O
O O
Perakine (241) Vellosimine (242) Lochnerinine (243) Tabersonine (244)
O
O
N O
N HO
OH O N
N N
N O R O
N
H N
O H O O
O
O O O
O
O
O 11-Methoxy tabersonine (246); R = H
19-acetoxy-11-methoxytabersonine (247); (-)-Echitoveniline (248) Picraline (249)
Raucaffrinoline (245)
R = OAc
O
O
MeO OMe
O
N O
O H MeO
N O
O
N OH O
N O O N
H O
O
NH
Echitoserpidine (250) Vellosiminol (251) 19(Z)-burnamine-17-O-3',4',5'-trimethoxybenzoate (252)
N OH OH
O O O O
HN HN
HO
N
H O O N N
O
Compactinervine (253) 19-Epi-ajmalicine (254) Alloyohimbine (255)
Fig. 14.48 Chemical structures of various alkaloids from A. yunnanensis (232–255)

O O Cl
O
N N
AcO AcO
O O
OH OH N N
N N
H COOCH3 H COOCH3 N H N H
O CHO CH2OH
11-Hydroxy-6,7-epoxy-8-oxo- 14-Chloro-15-
4 4
vincadifformine (256) hydroxyvincadifformine (257) Perakine N -dioxide (258) Raucaffrinoline N -oxide (259)
O
O AcO AcO
N
O O
N N
O N H N H
N O COOH COOH
H COOCH3
11-Methoxy-6,7-epoxy-8- Vinorine N1,N4-dioxide (261) Vinorine N4-oxide (262)

oxovincadifformine (260)
evaluated its cytotoxic and anti-inflammatory potentials (Cao et al. 2012). For the
cytotoxicity evaluation, following cell lines were used astrocytoma (CCF-STTG1),
glioma (CHG-5, SHG-44, U251), MCF-7 cells, human skin cancer (SK-MEL-2),
and meningioma (BEN-MEN-1). Except for meningioma cell lines, perakine
N4-oxide (258), raucaffrinoline N4-oxide (259), and vinorine N4-oxide (262)
exhibited cytotoxicity against all the tested tumor cell lines (IC50 values ranging
from 9.2 to 65.5 µM). The remaining compounds were identified as non-toxic in
nature (IC50 value > 50 µM). Adriamycin (positive control) exhibited strong
cytotoxicity wherein the IC50 values ranges from 14.1 to 37.6 nM. In in vitro
anti-inflammatory screening, similar results were obtained, wherein 258, 262, and
259 exhibited selective COX-II inhibition (94.77, 94.05, and 88.09%, respectively),
NS-398 was used as a positive control that exhibited 97.13% of COX-II inhibition.
All screened compounds exhibited poor COX-I inhibition. These all results indicate
that N4-oxide is required for cytotoxicity and anti-inflammatory activity.
Li et al. isolated six alkaloids (263–268, Fig. 14.50) from the ethanolic extract of
A. yunnanensis aerial parts and evaluated the in vitro cytotoxicity in eight tumor cell
lines (Li et al. 2017). The selected cell lines are human osteosarcoma (SOSP-9607,
Mg-63, Saos-2, M663), human gastric carcinoma (BGC-823), HepG2, HL-60 and
MCF-7 . Adriamycin was used as a positive control. Among the tested compounds,
alstiyunnanenine D (266), E (267) and alstonia scholaine I (268) exhibited the
potent cytotoxic activity in osteosarcoma cell lines (IC50 = 3.2–5.8 µM). Except
Saos-2 and M663 cell lines, alstiyunnanenine A (263) exhibited poor activity
against all the selected cell lines (IC50 > 35 µM). Alstiyunnanenine B (264) and C
RO COOMe H COOMe
OH
O O
O N N
N
N H N
N OMe H
H H
Alstiyunnanenine A (263) Alstiyunnanenine B (264); Alstiyunnanenine C (265)

R = veratoryl
O O
N N N H
H H
OH OH H H
H H
H H OH
N N N
H COOH H COO O COOH
HO HO H
Alstiyunnanenine D (266) Alstiyunnanenine E (267) Alstoniascholaine I (268)
(265) exhibited moderate cytotoxicity against BGC-823, HepG2, HL-60, and

MCF-7 cells, wherein IC50 values were ranging in 16.9–24.2 µM. Adriamycin IC50
values were observed in the range of 0.01–0.04 µM.

of A. Spatulata
14.3.7.1 Cytotoxicity Activity
Tan et al. isolated 23 (15, 17, 20, 29, 269–287) alkaloids from ethanolic extract of
A. spatulata leaves and stembark (Fig. 14.51) (Tan et al. 2010). Further, they
evaluated the cytotoxicity effects of these alkaloids against KB/S (vincristine-
sensitive) and KB/VJ300 (vincristine-resistant) cell lines. In KB/S cell lines,
15-hydroxyangustilobine A (269) and angustilobine B (281) exhibited a potential
activity (IC50 = 5.26 and 6.99 µg/mL, respectively), while the remaining screened
compounds were found to be inactive (IC50 > 25 µg/mL). 281 exhibited significant
cytotoxicity (IC50 = 8.12 µg/mL). 24 and 269 exhibited moderate cytotoxicity
(IC50 = 13.35 and 14.39 µg/mL, respectively). However, except alstolucine C
(278), nor-6,7-secoangustilobine A (283), and undulifoline (284), all the tested
compounds were found to reverse MDR in vincristine-resistant KB (VJ300) cells
(IC50 = 0.59–19.22 µg/mL).
O
N OH
O O N
H
O H
O
N N H
O N
O N
H N H
O O
N O O
15-Hydroxy angustilobine A (269) 16-Epivincamine (270) 16R,19E-Isositsirikine (271) 20(R)-Tubotaiwine (272)
N
O O O
HN O
N
N O N
O H O O
ON O
O H
H
4,6-Secoangustilobinal A (273) Akuammicine (274) Alstolobine A (275)
O O-
N O
O N
N+
N
R H N
O N R H
O H O
O O
O
Alstolucine B (276); R = H
(-)-Alstolucine C (278) Alstolucine E (279); R = OH
Alstolucine D (277); R = OH
(-)-Alstolucine F (280); R = H
OH
N
O
O O
O
N H
N HN N
O H O H
O O
O
N
Angustilobine B (281) N4-Demethyl-12- Nor-6,7-Seco

methoxyalstogustine (282) angustilobine A (283)
O
H O N N N
N
O
N
HO N
N N HO H
H O O
O O
O O
Undulifoline (284) Vincadifformine (285) Vincamine (286) Vinervine (287)
Fig. 14.51 Chemical structures of various alkaloids from A. spatulata (269–287)


of A. Rupestris
14.3.8.1 Cytotoxicity Activity
Wang et al. isolated 10 alkaloids (288–297) from the ethanolic extract of A.

rupestris leaves (Fig. 14.52) and evaluated its cytotoxicity potentials (Wang et al.
2013). Cytotoxicity effects were evaluated in various cell lines, such as A-549,
HepG2, SMMC-7721, MCF-7, HL-60, BGC-823, and SW-480. Doxorubicin was
used as a positive control. (E)-16-formyl-5a-methoxystrictamine (296), scholarisin
I (288), and scholarisin VI (293) possessed significant cytotoxicity (IC50
values < 30 µM) against all the tested tumor cell lines. Scholarisin IV (291) and
scholarisin V (292) were found to be non-cytotoxic (IC50 > 80 µM). Structural
analysis indicated that these compounds lack a linkage between C-5 and N-4, hence
it is speculated that these bonds are essential for cytotoxicity. The remaining
compounds demonstrated moderate cytotoxicity (IC50 values = 30–80 µM).
Zhang et al. isolated 6 monoterpenoid indole alkaloids (256, 261, 298–301) from
the ethanolic extract of A rupestris aerial parts (Fig. 14.53) and evaluated its cytotoxic
potential (Zhang et al. 2014a). Cytotoxic activity was evaluated in seven head and
neck squamous cell carcinomas (CAL-27, Detroit-562, Hep-2, SCL-1, SCC-PKU,
TCA-83, and UMSCC-1). Doxorubicin was used as positive control (IC50 values in
the range of 14.7–35.4 nM). Among the screened compounds, 11-hydroxy-6,7-
epoxy-8-oxovincadifformine (256) 6,7-epoxy-8-oxo-vincadifformine (298), and
11-acetyl-6,7-epoxy-8-oxo-vincadifformine (299) exhibited significant cytotoxicity
O O
H
O N O O O
N O O
O O O
N
O O
O NH O N OH
R NH
O N
O H N
H
Scholarisin I (288); R = CHO Scholarisin IV (291) Scholarisin V (292) Scholarisin VI (293)
Scholarisin II (289); R = CH2OH
Scholarisin III (290); R = OCOCH3
OH O
O N
O O OH
COOMe
O H N OHC OMe
N O
N N
N
O
O H N OH
O N
Scholarisin VII (294) (3R,5S,7R,15R,16R,19E)- (E)-16-Formyl-5 - 3-Epi-

Scholarisine F (295) methoxystrictamine (296) dihydrocorymine (297)
Fig. 14.52 Chemical structures of various alkaloids from A. rupestris (288–297)

O O Cl
O
N AcO N
O OH
N
R N OH
N H N
H COOCH3 O CHO COOCH3
H
6,7-Epoxy-8-oxo-vincadifformine (298); Perakine N1,N4-dioxide (300) 11-Hydroxy-14-chloro-15-hydroxy
R=H -17 vincadifformine (301)
11-Acetyl-6,7-epoxy-8-oxo-vincadifformine (299);
R = OAc
Fig. 14.53 Chemical structures of various alkaloids from A. rupestris (298–301)
against all tested cell lines with IC50 values less than 20 µM, while remaining com-
pounds were found to be inactive.
14.3.8.2 Antibacterial and Antifungal Activities
Wang et al. further screened antifungal activity of (288–297) against five species of
fungi [Alternaria alternata (TX-8025), Colletotrichum nicotianae (SACC-1922),
Gibberella pulicaris (KZN 4207), Gonatopyricularia amomi (MB-9671),
Phytophthora capsici (KACC-40157)] (Wang et al. 2013). Compounds 288, 289,
290, and 295 exhibited antifungal activity against C. nicotianae and G. pulicaris
wherein MIC values were ranging from 0.69 to 1.7 and 0.64 to 1.55 mM. Nystatin
(positive control) exhibited a MIC value of 0.006 mM.
Zhang et al. further evaluated the in vitro antimicrobial activities (disk diffusion
method) of 256, 261, 298–301 against two species of bacteria [Mycobacterium
tuberculosis (ATCC-25177/H37Ra) and S. aureus (ATCC-25923)] by using rifam-
picin (5 µM/mL) as positive control. Antifungal activity was evaluated against
Alternaria alternata (TX-8025), Colletotrichum nicotianae (SACC-1922),
Gibberella pulicaris (KZN4207), Gonatopyricularia amomi (MB-9671), and
Phytophthora capsica (KACC-40157)] by using nystatin (10 µM/mL) as a positive
control. 298, 256, and 299 exhibited significant activity against A. alternata and
P. capsica with MIC values of 0.66–0.99 mM, 0.87–1.10 mM, and 1.53–1.64 mM,
respectively. Further, a moderate antibacterial activity were observed against
S. aureus (zone of inhibition = 15.72, 16.33 and 14.91 mM). Perakine N1, N4-
dioxide (300) and vinorine N1, N4-dioxide (261) demonstrated strong antibacterial
activities against S. aureus (MIC = 0.49 and 0.83 mM), while 11-hydroxy-14-chloro-
15-hydroxy-vincadifformine (301) had no activity (Zhang et al. 2014a).
14.3.8.3 Anti-inflammatory Activity
Wang et al. screened the in vitro anti-inflammatory properties of A. rupestris-

derived alkaloids (288–297) against COX-I and COX-II (Wang et al. 2013).
Among that 288, 293, and 296 showed selective inhibition of COX-II (96.4, 95.5,
and 92.0%) comparable with the positive control, NS-398 (97.1%). Remaining
compounds exhibited a poor COX-II inhibition (<50% inhibition). The screened
compounds exhibited a poor activity toward COX-I (<45% inhibition).

of A. Rostrata
Cai et al. isolated alstrostine A (302) and B (303) from the methanolic extract of A.
rostrata leaves (Fig. 14.54) and evaluated the cytotoxic effects in cancer cell lines
(MCF-7, SMMC-7721, HL-60, SW-480, and A-549) (Cai et al. 2011). These
compounds did not exhibit any kind of cytotoxic activity (IC50 > 40 µM).
Zhong et al. isolated 21 monoterpenoid indole alkaloids (17, 30, 63, 76, 108,
249, 270, 282, 304–316) from the methanolic extract of A. rostrata trunk
(Fig. 14.55) and evaluated its cytotoxicity and acetylcholinesterase inhibition
activity (Zhong et al. 2017). At 20 µM concentration, screened compounds
exhibited poor cytotoxicity against HeLa, SGC-7901 gastric cancer, and A-549 cell
lines. Further, these compounds did not exhibit acetylcholinesterase inhibition
activity.
In another study, Yuan et al. isolated 6 alkaloids (17, 76, 110, 317–319) from the
methanolic extract of A. rostrata twigs (Fig. 14.56) and evaluated its cytotoxic
effects against five human cancer cell lines, HL-60, SMMC-7721, A-549, MCF-7,
and SW-480 cells (Yuan et al. 2018). However, at a concentration of 40 µM, these
compounds were found to be inactive in nature.
HO HO
N O O N O O
N N
OH
O O
HO HO O
O O
HO O OH O
O O O O O
HO
HO OH O
O OH O
OH HO
HO
Alstrostine A (302) Alstrostine B (303)
Fig. 14.54 Chemical structures of alstrostine A and B (302 and 303)

N N
O N H
O OH
N
N H
O N N
O COOCH3 O H COOCH3 O H
N O COO
Alstrostine G (304) Alstrostine H (305) Alstrostine I (306) Alstrostine J (307)
N OH OH
N N
H
O
N
O O
N
H COOCH3 N N
H O H O O
O O N
Alstrostine K (308) Echitamidine (309)
N4-Demethyl alstogustine (310) 6,7-Seco angustilobine B (311)
O
OH OH
N O
O OH
N O H
O N
O O
N N N
O H O N N
O N H
N OH
H
Eburenine (313) Anthirine (316)
N4-Demethyl-12-methoxy Nb-Methyl-aspidodasycarpine (315)
17-O-Acetyl-Nb-
alstogustine N4 oxide (312)
demethylechitamine (314)
Fig. 14.55 Chemical structures of alkaloids from A. rostrata (304–316)
HOH2C COOMe O
AcOH2C COOMe
N H
OH
N
N
N OH H
OHC N COOMe N OH
OH H
H
Winphylline A (317) Winphylline B (318) 17-O-Acetyl norechitamine (319)
Fig. 14.56 Chemical structures of various alkaloids from A. rostrata (317–319)

of A. Pneumatophora
14.3.10.1 Anti-melanogenic Properties
Koyama et al. isolated various alkaloids (3, 24, 286, 309, 320–327) from the
methanolic extract of A. pneumatophora leaves (Fig. 14.57) and evaluated its
anti-melanogenic properties (Koyama et al. 2010a). A melanin inducer
(3-isobutyl-1-methylxanthine) stimulated B16 melanoma cells were cultured with
various concentration of (6.3–100 µM) isolated alkaloids for four days.
A dose-dependent cell viability (IC50 = 58.3, and 68.9 µM, respectively) and a less
O
N H N N H O
O H O
H H H
H H H
N N N
R H HO H R H
O O O
O O O
Alpneumine A (320); R = OH Alpneumine D (323); R = OH

Alpneumine C (321); R = H Alpneumine B (322) Alpneumine E (324); R = H
N O-
OH N+
O
O H N
N N O
H N
O MeOOC H O
O O
OH
Alpneumine F (325) Alpneumine G (326) Alpneumine H (327)
Fig. 14.57 Chemical structures of various alkaloids from A. pneumatophora (320–327)
than 50% melanin content were observed with the treatment of alpneumine G
(326) and vincamine (286). Melanogenesis was moderately inhibited
(IC50 = 71.4 µM) by alpneumine E (324). Tyrosinase inhibition potential of these
alkaloids wasalso examined in mushroom tyrosinase assay (in vitro). However, it
exhibited a poor activity (IC50 > 100 µM). Furthermore, 326 (50 and 75 µM) in
western blotting analysis resulted in a decreased expression of tyrosinase protein.
14.3.10.2 Nitric Oxide Production Inhibition
Koyama et al. isolated alsmaphorazines A (328) and B (329) from the methanolic
extract of A. pneumatophora leaves (Fig. 14.58) and evaluated the inhibitory
potential on the NO production in LPS-stimulated J774.1 (Koyama et al. 2010b).
Fig. 14.58 Chemical R

structures of alsmaphorazine
A and B (328 and 329)
N
OH O
N O
O
O
Alsmaphorazine A (328); R = OH
Alsmaphorazine B (329); R = H
Compound 328 exhibited a dose-dependent inhibition (IC50 = 49.2 µM), while 329
did not exhibit any inhibition in NO production at 50 µM.

of A. Penangiana
Yeap et al. isolated ten alkaloids (330–339) from the ethanolic extract of A.
penangiana leaves (Fig. 14.59) and assessed their cytotoxic effects against various
cell lines (KB/S: vincristine-sensitive KB; KB/VJ300: vincristine-resistant KB;
PC-3 and LNCaP: MCF-7 and MDA-MB-231: human breast adenocarcinoma;
HT-29 and HCT 116: human colorectal carcinoma; A-549) (Yeap et al. 2018).
Compound 333 and 334 showed pronounced in vitro growth inhibitory activity
against the screened cell lines (IC50 values = 0.3 to 8.3 µM). A potent cytotoxic
effect was observed in HT-29 cell lines (IC50 = 0.7 and 0.3 µM for 333 and 334,
respectively).
H H
O
NMe OH
COMe COOMe
R H H H
H H Me N H H
Me H N
N NH
HN NH O
O
O OH O N HH
O OH H H
H
Alstonisinine A (330); R= CHO Alstonisinine C (332) Angustilongine A (333)
Alstonisinine B (331); R= COMe
H H H H
O O
NMe H NMe
COOMe COOMe H H Me
H
OH H
N H N H N N NCHO
H N OH
CH2OH
O H
N HH O N HH H
H H
Angustilongine B (334) Angustilongine C (335) Alstonoxine F (336)
O O
O HO
O O
O O
O COOMe O COOMe
H H
O Cl N N
NMe OH N N
COOMe H H
H O O
H H CH2Cl
N H N
O N HH O O
O O
H
O O
Angustilongine D (337) Vincamaginine A (338) Vincamaginine B (339)
Fig. 14.59 Chemical structures of various alkaloids from A. penangiana (330–339)


of A. Mairei
Cai et al. isolated 22 indole alkaloids (340–355, 250, 244, 241, 230, 20, 19) from
the ethanolic extract of A. mairei leaves and twigs (Fig. 14.60) and evaluated its
cytotoxic potential of various monoterpenoid from in A-549, PANC-1, HL-60,
SMMC-7721, and SK-BR-3 cells. However, it exhibited poor cytotoxicity
(IC50 > 40.0 µM) (Cai et al. 2010b).
Yan et al. isolated four alkaloids (320, 356–358) from the ethanolic extract of A.
mairei leaves (Fig. 14.61) and evaluated its in vitro cytotoxic activities against four
osteosarcoma cell lines (Saos-2, Mg-63, U2-OS, and SOSP-9607 cells) (Yan et al.
2017). Alstomairine B (357) and alstomairine C (358) exhibited higher cytotoxic
activity (IC50 < 15.0 µM) against all tested tumor cell lines. IC50 of doxorubicin
(positive control) was in the range of 0.02–0.03 µM.

of A. Congensis
14.3.13.1 Antiplasmodial Activity
Kanyanga et al. evaluated the antiplasmodial activity of various phytochemicals (9,

55, 311, 359) from A. congensis root bark (Cimanga et al. 2019). Two strains of
P. falciparum (K1 and NF54 A19A) were used for in vitro screening. Boonein
(359, Fig. 14.62) was inactive against both the strain (IC50 > 64 µM) while 55 and
311 exhibited a moderate activity (IC50 = 20.57 & and 5.05 and 21.26 &
40.70 µM, respectively for K1 and NF54 A19A strains). Quinine was used as a
positive control that exhibited an IC50 of 0.25 and 0.46 µM, respectively, for K1
and NF54 A19A strains. Further, these compounds did not exhibit any kind of
cytotoxicity against MRC-5 cells (CC50 > 64 µM).

of A. Angustiloba
14.3.14.1 Vasorelaxant Activities
Koyama et al. isolated alstilobanines A-E (360–364) from the methanolic extract of
A. angustiloba leaves (Fig. 14.63) and evaluated its vasorelaxant activities in iso-
lated rat aorta (Koyama et al. 2008). Phenylephrine (PE, 3 10−7 M) was
O N
N O
O
N H R
O O
N
N O H O
H O
N O O
H
(14- ,15- )-14,15- Maireine A (341); R = OMe Venalstonine (343)

Epoxyaspidofractinine (340) Maireine B (342); R =H
N N N
O O
O O
R OH O
N N N
H O H O H O
O O O
(-)-Minovincinine (344); R = H Echitovenaldine (347)

(-)-11-Methoxy minovincinine (345); R = OMe (-)-Echitovenine (346)
O
O
N O
N N O
O O
R O O
O
O O
O N O
N O H
N H
H O O O
O O O
Echitoveniline (349); R = H 11-Methoxy echitoserpidine (351)

11-Methoxy echitovenidine (348)
11-Methoxy echitoveniline (350); R = OMe
O H O
N N O N
N
H O
O N
N
O O
N N O O
H H O O O
O O O O
(-)-Lochnericine (353) Rhazimol (355)

(19S)-Mindolinine (352) Deacetyl picraline 3,4,
5-trimethoxybenzoate (354)
Fig. 14.60 Chemical structures of various alkaloids from A. mairei (340–355)
O
N N
H H
H O H O
H H
N N
O H R H
O O
O O
Alstomairine B (357); R = OMe

Alstomairine A (356)
Alstomairine C (358); R = OH
Fig. 14.61 Chemical structures of alstomairine A–C (356–358)

OH
O
Boonein (359)
Fig. 14.62 Chemical structure of boonein (359)
O O
O O
OH O
O O OH
H
N NH N
HO
HN
N+ N
OH -
O OH
Alstilobanine B (361) Angustilobine C (362)

Alstilobanine A (360)
O
O O
H O
NH N O
HN
N+ HO O
-
O
Alstilobanine D (363) Alstilobanine E (364)
Fig. 14.63 Chemical structures of various alkaloids alstilobanines A–E (360–364)
administered for the induction of the contraction and 360 showed more potential
relaxation activity (44.3%), while 364 exhibited the least activity (5%).
14.3.14.2 Cytotoxicity Activities
Ku et al. isolated various alkaloids (4,17, 94, 103, 271, 276, 281, 284, 286, 311,
314, 362, 365–372) from ethanolic extracts of A. angustiloba leaf and stem bark
(Fig. 14.64). Further, they evaluated its cytotoxicity effects in vincristine-sensitive
KB and vincristine-resistant (KB/VJ300) cells (Ku et al. 2011). Angustilobine C
(362) showed moderate cytotoxicity toward vincristine-sensitive KB
(IC50 = 7.76 µg/mL) and KB/VJ300 cells (IC50 = 7.33 µg/mL). Andransinine
(366) did not exhibit appreciable cytotoxicity against both the cell lines
(IC50 > 25 µg/mL), however, it reversed MDR in KB/VJ300 cells (IC50 = 1.61 µg/
mL in the presence of 0.1 µg/mL of vincristine).
O
O O NHO
N O O
OH H O
O N
N
N O
HO O
N
N O N O
Angustilobine A (365) Andransinine (366) O-Acetyl vallesamine (367) 6,7-Seco-19,20-epoxy

angustilobine B (368)
O
O NH
N N H
O
O N
HN O
HO OH
N N N
H O H H
O N MeOOC
H
HO
Condylocarpine (369) Angustiphylline (370) Yunnanensine (371) Venoterpine (372)
Fig. 14.64 Chemical structures of various alkaloids from A. angustiloba (365–372)

of A. Actinophylla
14.3.15.1 Carboxypeptidase U (CPU) Inhibitor Activity
Carroll et al. reported the bioassay-guided isolation for a carboxypeptidase U

(CPU) inhibitors from the aqueous extract of A. actinophylla leaves (Carroll et al.
2005). The study resulted in the identification of actinophyllic acid (373,
Fig. 14.65) as a CPU inhibitor with an IC50 value of 0.84 µM.
14.4 Pharmacokinetics and Metabolite Identification
There are few studies that explore the pharmacokinetic properties of the Alstonia-
derived phytochemicals. These all reported studies are mainly repeated for 20, 24,
30, and 75 (Fig. 14.66).
Cao et al. have characterized the chemical constituents and rat metabolites from
the A. scholaris leaves alkaloid extract (Cao et al. 2016). After the administration of
10 mg/kg b.w per oral of alkaloid fraction, the metabolites were identified by using
an LC/SRM-MS method. Among the 35 metabolites identified, 12 were
picrinine-type 11 scholaricine-type, 9 vallesamine-type, and 3 tubotaiwine-type of
alkaloids. The developed LC/SRM-MS method was able to separate the R and S
confirmation of various components at MS3 spectra (for scholarsine, echitamidine,
etc.). A total of 25 biotransformed metabolites were characterized in the
N
OH
O
N
H
HO O
Actinophyllic Acid (373)
Fig. 14.65 Chemical structure of actinophyllic acid (373)
O OH
H N OH O H N
N
N
O O H
N
N N
O HO H HO H O
O N O
O O
Picrinine (20) Scholaricine (24) Vallesamine (30) 19-Epi scholaricine (75)
Fig. 14.66 Chemical structures of various alkaloids from A. scholaris (20, 24, 30, 75)
experimental animals. Metabolic studies of 24 and 75 highlighted the rapid

absorption of these compounds into circulation. Glucuronidation, sulfation, and N-
oxidation were found to be the metabolic reaction involved in the metabolism of 24,
wherein 4 metabolites were characterized in urine, one metabolite in plasma and
feces was characterized. In case of 30, 6 metabolites were identified in urine, 4 in
plasma, and 4 in fecal samples. Hydroxylation, glucuronidation, N-oxidation, and
demethylation were identified as the metabolic pathways. A high amount of 20 in
feces and lower amounts in plasma and urine highlighted the low absorption of
these compounds. 11 metabolites were identified in urine, 2 in plasma, and 9 in
fecal samples. Compound 20 was mainly metabolized into phase I products.
Further, these results conclude that the absorption of these molecules is directly
related to the polarity of the molecules (Sch/Epi > Val > Pic). Thus, a total of 40
compounds were detected in urine, 33 in plasma, and 38 in feces.
Yun-Li Zhao et al. have studied the pharmacokinetic effects of A. scholaris total
alkaloids (TA) in SD rat plasma by a validated LC-MS/MS method (Zhao et al.
2018). After the oral administration of various doses (10, 25, and 50 mg/kg) of total
alkaloids, the plasma concentration of 4 bioactive alkaloids (24, 75, 30, and 20)
fitted an open two-compartment model. Tmax of 24 and 75 occurred within 10 min
while 30 and 20 occurred in 15 min. These compounds were eliminated from
plasma after 24 h of the administration. A TA dosage proportional increase in the
Cmax and AUClast was obtained for the four indole alkaloids. Further, a short
elimination half-life (T1/2a) was also identified. In the study, 20 and 30 were rapidly
acting while the remaining alkaloids were exhibited faster activity in in vivo con-
ditions. Thus, it has been suggested that three times a day administration of total
alkaloids will produce a best therapeutic effect in humans.
Rui Li et al. registered a mixture of indole alkaloids from the leaf of A. scholaris
as an investigational new botanical drug (No. 2011L01436) for the treatment of
various respiratory-related ailments and was approved for phase I/II clinical trials
by CFDA (Li et al. 2019). A randomized, open-labeled, clinical trial was performed
in healthy Chinese volunteers (40) for evaluating the safety and pharmacokinetics
of capsule of alkaloids from the leaf of A. scholaris (CALAS) at different doses (20,
40, 80, 120 mg per oral). 24, 75, 30, and 20 are the major compounds reported for
the desired pharmacological activities (respiratory diseases). A dose depended
effect were observed in Cmax, AUC 0-t, and AUC0-∞. Among the alkaloids, 30 was
detected at higher concentration in blood (Cmax: 12.909–60.889 ng/ml), followed
by 24 (Cmax: 2.162–21.025 ng/ml), 19-epischoalricine (Cmax: 1.172–9.391 ng/ml)
and least by 20 (Cmax: 1.295–8.674 ng/ml). Further, 30 and 20 had a half-life up to
3–4 h, while 24 and 75 were up to 10 h. 24 exhibited a non-linear nature in a
multi-dose linear relationship, while the remaining alkaloids exhibited a linear
characteristic. CALAS was safe and well-tolerated without any serious adverse
events during the whole trial periods. From these promised results, it can be
deduced that 40–80 mg/time t.i.d capsule is required per day to maintain an
effective blood concentration, and the effectiveness of these results has to be con-
firmed by performing further the phase II clinical trials by using these dosage
regiments.
14.5 Intellectual Property Rights (IPR) Values of Alstonia

Genus
IPR refers to the general term for the assignment of property rights by an inter-
governmental organization in forms of patents, copyrights, and trademarks. These
property rights provide an exclusive right to an inventor or assignee to exercise a
monopoly on the use of the item for a specified period. Many commercial and
academic institutions protect their research information through IPR which makes
these as early sources of cutting-edge research. A huge number (>300) of patents
has been filed that comes under the Alstonia genus. However, the majority of these
patents come with Alstonia as a single component in polyherbal formulations.
These kinds of patents are beyond the scope of this book chapter; hence they were
excluded. In the present chapter, Table 14.14 indicates various patents with
Alstonia as one of the major components. The majority of the patents have to
explore the traditional knowledge of Alstonia genus-related activities. For instance,
a higher number of patents can be observed with respiratory-related ailments and
skin-related problems. Apart from that various formulations were also patented.
Table 14.14 List of patents published on Alstonia genus

S. No. Composition/ Use/Indication Patent ID/Title References
formulation
1 Alstonia-leaf Antiasthmatic-antitussive CN109985078A Zhi and
buccal tablet effect A kind of Alstonia- Xuemei (2019)
leaf buccal tablet
and preparation
method
2 Alstonia-leaf and Enriches the drinking CN101983574A Mingzhi et al.
de-enzyming tea health-care efficacy of Alstonia-leaf tea (2010)
containing bag traditional tea bag and processing
method thereof
3 Alstonia-leaf total phlegm-dispelling CN107693499A Xiaolin et al.
flavones functions and relieve A kind of (2017)
dispersible tablet inflammations such as preparation method
(not less than throat and tonsillotomes. of Alstonia-leaf
62.5 mg/tablet) total flavones
dispersible tablet
4 Extract of Alstonia Treatment of nervous CN107412775A Addington and
species or the disorders such as With the method for Newman
extract of Alzheimer disease, Alstonia species or (2017)
Luckynut Thevetia Huntington chorea, or the extract for
Seed species apoplexy treating nervous
disorders of
Luckynut Thevetia
Seed species
5 A. mairei extract To treat liver, lung and CN101347475B Yalin et al.
colon cancer Use of Alstonia (2008)
mairei extract in
preparing
anti-tumor
medicament
6 Alstonia-leaf For cough-relieving, CN106727787A Yuguo et al.
dispersible tablet eliminating the phlegm, A kind of Alstonia- (2015)
anti-inflammatory, leaf dispersible
spasmolysis, it is tablet and
antipyretic the effect of, preparation method
for chronic bronchitis, etc. thereof
7 Alstonia-leaf Anti-cancer (lung) CN102631396B Yan et al.
traditional Chinese Anti-lung cancer (2012)
herbal composite Alstonia-leaf
traditional Chinese
herbal composite,
method for
preparing same and
application thereof
in preparing
anti-lung cancer
medicine
(continued)

formulation
8 Sugar-free Relief cough, eliminate CN102600220A Jun (2011)
Alstonia-leaf phlegm, and can diminish Sugar-free Alstonia-
granules inflammation leaf granules and
preparation method
thereof
9 Extract of A. Treating cold, fever, and CN101084951B Xiaodong et al.
scholaris respiratory disease Medicine for (2006)
treating diseases
concerned with
respiratory and
preparation method
and application
thereof
10 Mixture of extracts Cough-relieving CN106890209A Guangrong
consists of compound preparation A kind of et al. (2016a)
Alstonia leaf, cough-relieving
Momordica compound
grosvenori, and preparation and
American Ginseng preparation method
thereof
11 Mixture of extract For producing analgesia, CN106853148A Guangrong
consists of A. anti-inflammatory, Compound et al. (2016b)
scholaris leaf eliminating the phlegm, longxuejie
Resina Draconis medicine of the curative preparation and
and borneol effect such as relieving preparation method
asthma thereof
12 A composition For treating a respiratory US20180344792A1 Xiaodong et al.
consists of disease Pharmaceutical (2015)
picrinine, composition for
vallesamine, treating respiratory
scholaricine, and disease
19-epischolaricine
13 A mixture extracts For treating livestock and CN107569538A Min et al.
consists of bird’s respiratory disease A kind of (2017)
Alstonia, pharmaceutical
Astragalus Root, preparation for
Osmanthi preventing and
Fragrantis extract, treating livestock
and and bird’s
sulfadimethoxine respiratory disease
and preparation
method thereof
(continued)

formulation
14 Mixture extract For the treatment of fever US20060141069A1 Pushpangadan
consists of A. and related symptoms Synergistic et al. (2004)
scholaris, Berberis antipyretic
aristata, Tinospora formulation
cordifolia,
Andrographis
paniculata, and
Hedychium
spicatum
15 Effective amount Preventing prostate CN101027070A Hall and
of a mixture of cancer and/or alleviating Flavopereirine and Belensky
flavopereirine and the symptoms of BPH alstonine (2005)
alstonine (Benign Prostatic combinations in the
Hyperplasia) or PIN treatment and
(prostatic intraepithelial prevention of
neoplasia prostate cancer
16 Hydrolipid matrix Vaginal contraceptive IN1128/DEL/2005 Mandal and
consists of leaf and Herbal vaginal Chattopadhyay
bark of A. contraceptive an (2006a)
macrophylla effective birth
control measure
(topical)
17 Echitamidine-N- Anti-cancer and IN201741044816 Subba Reddy
oxide-19-O-b-D- anti-metastatic Anti-cancer and (2017)
glucopyranoside anti-metastatic
isolated from A. activity of EOG
scholaris (echitamidine-N-
oxide-19-O- b-D-
glucopyranoside)
an isolated
compound obtained
from ethanolic
extract of Alstonia
scholaris
18 Ursolic acid from Antibacterial, antifungal, IN1121/DEL/2005 Mandal and
the A. macrophylla anti-inflammatory, and Herbal Chattopadhyay
antihistaminic properties antimicrobial, (2016b)
anti-inflammatory,
and antihistaminic
formulation
(topical/oral)
14.6 Conclusion and Future Perspective
Alstonia genus consists of many related species, and traditional usage of these
species highlighted the potential of such plants in exhibiting various pharmaco-
logical activities. Although many reviews are available on Alstonia species, the
present chapter is mainly focused on the pharmaceutical applications of the sec-
ondary metabolites from Alstonia genus. A preliminary literature review indicated
that more than 800 phytochemicals have been identified/isolated from the Alstonia
species, and these studies were reported mainly from China, India, Malaysia, South
Africa, etc., wherein a strong ethnomedical usage of these species have took place.
Further, in African Pharmacopoeia, A. boonei was listed as an antimalarial drug.
The majority of reported studies is mainly relying on the crude extract; however, the
present chapter dealt with the pharmacological application of the isolated phyto-
chemicals from the Alstonia genus. To the best of our knowledge, around 32
species of Alstonia have been undergone the isolation process. However, 15
species-derived phytochemicals are only evaluated for the various pharmacological
activities. Among the vast isolated/identified phytochemicals, a few numbers (ap-
prox. 400) have been evaluated for a limited number of pharmacological activities.
In that scenario also, all the species-derived phytochemicals did not evaluate all the
reported activities. Especially, the search for potent pharmacologically active
phytochemicals from the A. scholaris is increasing in a dramatic manner, remaining
species are getting a negligible focus for their diverse activity. Evaluation of a
species correlated pharmacological activities might be helpful for the development
of a proper structural activity relationship of these compounds with respect to the
individual pharmacological activity as well as the species. Moreover, it will also
help to explore the structural similarities and their required modification for the
desired pharmacological activity. Monoterpenoid indole alkaloids are the major
components explored from the Alstonia genus and were exhaustively evaluated for
various pharmacological activities such as anti-cancer, antiviral, anti-spasmodic,
antitussive, antiarthritic, and antioxidant. Apart from that it also comprises iridoids,
flavonoids, steroids, fatty acids, etc.
Among the isolated phytochemicals, a varying degree of pharmacological
activities was observed. For instance, 2, 12, and 19 selectively inhibited COX-II.
Further, 67 against GSC-12# and GSC-18# exhibited a significant cytotoxicity.
Apart from that 27 exhibited a good in vitro antiviral activity similar to that of
acyclovir but lacked the selectivity. All these data have suggested that a proper
synthetic modification, either via chemical modification or by selecting the active
pharmacophore, might also result in the development of various novel drugs.
Although Alstonia-derived phytochemicals exhibited numerous activity, the
pharmacokinetic effects of these compounds are unexplored. A few studies are only
reported for the pharmacokinetics effects that mainly include 24, 75, 30, and 20.
The lack of pharmacokinetic studies might be taken care of by the scientific world
and will be helpful for obtaining a further exploration of the Alstonia genus.
In a nutshell, diverse pharmaceutical application of Alstonia genus-derived

phytochemicals indicates that a proper evaluation might result in the development
of various drug/drug candidates for diverse pharmaceutical applications.
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Chapter 15
Role of Natural Bio-active Compounds
as Antidiabetic Agents
Sandra N. Jimenez-Garcia, Lina Garcia-Mier,

Moises A. Vazquez-Cruz, Xochitl S. Ramirez-Gomez,
Abstract Diabetes Mellitus (DM) is a metabolic disease caused by secretion and/

or poor use of insulin, which produces an excess of glucose in the blood. DMs are
responsible for 1.5 million deaths per year and according to the World Health
Organization (OMS) it is estimated that there are 422 million people with DM
worldwide, growing dramatically in low- and middle-income countries. A poor
public healthcare system, a low purchasing power, and a lack of human resources
are just some of the conditions that cause the diabetic population to look for
supplements or adjunctive alternatives for this disease. On the other hand, the use of
plants to cure and/or prevent diseases has prevailed over time. In this sense, there
are various plants in popular use that have been attributed antidiabetic properties
since they contain bioactive compounds of secondary metabolites such as saponins,
polyphenols, terpenes, among others, which act by inhibiting enzymes involved in
the adsorption of carbohydrates, improving secretion and insulin sensitivity,
have antioxidant and hepatoprotective properties, among other mechanisms of
S. N. Jimenez-Garcia X. S. Ramirez-Gomez
División de Ciencias de la Salud e Ingeniería, Universidad de Guanajuato, Ejido de Santa
María del Refugio Celaya, Campus Celaya-Salvatierra, C.A. Enfermedades no Transmisibles,
Av. Ing. Javier Barros Sierra no. 201 Esq, C.P. 38140 Guanajuato, Baja California, Mexico
L. Garcia-Mier
Departamento de Ciencias de la Salud, Universidad del Valle de México, Campus Querétaro,
Blvd, Juriquilla no. 1000 a, Delegación Santa Rosa Jáuregui, C.P. 76230 Santiago de
Querétaro, Querétaro, Mexico
MoisesA. Vazquez-Cruz
Departamento de Investigación y Desarrollo, KOPPERT MEXICO, Circuito el Marques Nte.
82, Parque Industrial El Marqués, C.P. 76246 Santiago de Querétaro, Mexico
RamonG. Guevara-Gonzalez J. F. Garcia-Trejo A. A. Feregrino-Perez (&)
División de Estudios de Posgrado Ingeniería de Biosistemas, Facultad de Ingeniería,
Universidad Autónoma de Querétaro, C.U. Cerro de las Campanas S/N, Colonia Las
Campanas, C.P. 76010 Santiago de Querétaro, Querétaro, Mexico
e-mail: feregrino.angge@hotmail.com
https://doi.org/10.1007/978-3-030-54027-2_15
536 S. N. Jimenez-Garcia et al.
action, thus helping in prevention and/or as adjunctive therapy in the treatment of

blood glucose levels regulation and delaying some of the complications derived of
the DM.
Keywords Diabetes mellitus Bioactive compounds Antidiabetic Antioxidant
15.1 Introduction
Diabetes mellitus (DM) is a serious, chronic, and complex metabolic disorder of

multiple aetiologies with profound consequences, both acute and chronic (Sudha
et al. 2011; Soumya and Srilatha 2011). Also known only as diabetes, DM and its
complications affect people both in the developing and developed countries, leading
to a major socioeconomic challenge. The WHO projects that diabetes will be the
seventh leading cause of death in 2030 (Kumar et al. 2011b). In 2011, the global
prevalence of diabetes for adults of 20–69 years was 7.7%, this was predicted to
rise to 9% by 2030 (Wou et al. 2019). More of 415 million people worldwide was
affected to diabetes in 2017, and this amount may be increased to 642 million by
2040 due to change in lifestyle, eating habits, and growth of the urban area
(Ogurtsova et al. 2017). Diabetes is distinguished by chronic hyperglycemia with
disturbances in the macromolecules’ metabolism as a result of impairments in
insulin secretion, insulin action, or both. There are various types of diabetes of
which type 1 DM (T1DM) and type-2 DM (T2DM). The T1DM is also known as
insulin-dependent diabetes. It is primarily due to pancreatic islet beta-cell
destruction and is characterized by deficient insulin production in the body. On
the other hand, T2DM, also known as non-insulin-dependent diabetes, results from
the body’s inactive use of insulin and hyperglycemia (Baeyens et al. 2018) and
accounts for the vast majority of people with diabetes around the world.
The increase in the morbidity and mortality of diabetes is related to whit
macrovascular (stroke, peripheral vascular disease, and among other) and
microvascular (nephropathy, retinopathy, and neuropathy) complications (Harding
et al. 2019). Therefore, avoiding hyperglycemic states without abusing hypo-
glycemic states in both type 1 and type-2 diabetes helps to retards the development
of microvascular complications, without being a determining factor in this (Baeyens
et al. 2018; Furman et al. 2020). To achieve the regulation of glucose levels, are
administered mainly insulin secretagogues, insulin sensitizers, carbohydrate-
degrading enzyme inhibitors, peptide analogs, dipeptidyl peptidase-4 inhibitors,
and glucagon-like peptide-1, however, they all have limited efficacy and tolerance,
as well as side effects (Rotenstein et al. 2012).
In this sense, medicinal plants have been a good alternative in the search for
compounds that help control glucose levels in both developed and developing
countries. It is considered that more of 1200 plants and various secondary metabolites
such as alkaloids, carotenoids, flavonoids, glycoside molecules, polyphenols, ter-
penoids, and tannins have been used to treat Diabetes (Sachithanandam et al. 2019).
15 Role of Natural Bio-active Compounds … 537
Other experts indicated than only 800 plants are used in the management of Diabetes
and only 30% of plants used in folk medicines for Diabetes have been biologically
assayed (Chinsembu 2019). Therefore, the potential to discover new antidiabetic
drugs from plants is still untapped.
15.2 Mechanisms of Action of Antidiabetic Substances
Hypoglycemic drugs exert antidiabetic effects through different mechanisms,

namely, sulfonylureas (reduce blood sugar, mostly by elevating insulin release from
islets of Langerhans), biguanides (improve the body’s sensitivity towards insulin and
reduce the amount of sugar absorbed by the intestine), a-glucosidase inhibitors
(reduce the absorption rate of carbohydrates in the body), thiazolidinediones (im-
prove muscle and adipose tissue sensitivity to insulin and reduce liver glucose
production. Also augment b-cell function by lowering free fatty acid levels that
ultimately lead to b-cell death), and non-sulfonylureas secretagogues (increase the
secretion of insulin from active b-cells). These drugs alongside with insulin are the
main way for controlling diabetes; however, they have different side effects. The
antidiabetic action of plant materials has been accredited to the mixture of phyto-
chemicals (alkaloids, phenolic acids, flavonoids, glycosides, saponins, polysaccha-
rides, stilbenes, and tannin) or to a single component of plant extracts through
various mechanisms such as regulation of glucose and lipid metabolism, insulin
secretion, stimulating b cells, NF-jB signaling pathway, inhibition of gluconeogenic
enzymes (pyruvate carboxylase, PEP carboxykinase, fructose-1, 6-bisphosphatase,
and glucose 6-phosphatase), and reactive oxygen species (ROS) protective action
(Salehi et al. 2019).
He et al. (2019) summarize naturally antidiabetic drugs and identified their
molecular targets. a-Glucosidase and a-amylase are crucial enzymes that hydrolyze
complex dietary carbohydrates to glucose or monosaccharides in the digestive
system. Glycosidase inhibitors decrease the digestion process and postprandial
blood glucose levels through the blockage of various amylases and glycosidases.
The inactivation of DPP4 prolong the degradation rate of glucagon-like peptide-1
(GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) normalizing
insulin release. GLP-1 and GIP can modulate b-cell differentiation, insulin
biosynthesis and release, gastric emptying, and execute some other functions.
Plasma levels of GLP-1 and GIP promote insulin secretion, reduce glucagon
secretion, and in this way, control blood glucose levels. The stimulation of PPAR c
(a family of nuclear receptors and ligands activated by transcription factors that
modulate adipocyte differentiation, lipid metabolism, inflammatory processes, and
glucose homeostasis) serves as an insulin sensitizer in adipose tissue and muscle
throughout the improvement of glucose and lipid metabolism and inflammatory
processes. The antidiabetic effect of alkaloids glycosides, polyphenols, carotenoids,
terpenoids, flavonoids, anthocyanins, tocopherols, peptidoglycans, steroids, sapo-
nins, xanthones, and polysaccharides may also be related to these other
mechanisms: blockage of PTP1B to enhance leptin and insulin sensitivity to exhibit

synergistic effects on glucose and lipidic metabolism and modulation of GLUT4
translocation via activating PI3K/AKT pathway in the liver and muscle. Finally, the
amelioration of glucose and lipidic metabolism and insulin level through the
functional enzymes, insulin-related oxidant stress, and inflammatory factors are also
implicated in the antidiabetic action of some natural bioactive compounds.
15.3 a-Amilase Inhibitors
Since ancient times, plants have been considered as a source of compounds for the
treatment of various diseases. Currently, given the availability of these compounds,
their low cost, ease of preparation and/or minimal side effects have increased their
use throughout the country world (Sachithanandam et al. 2019), as well as the
interest in discovering new antidiabetic drugs.
Several studies have focused on plants that have been attributed to antidiabetic
activity by inhibiting carbohydrate-degrading enzymes (Trinh et al. 2020) also
called glucosidases. Glucosidases are a group of digestive enzymes that break down
the dietary carbohydrates into simple monosaccharides. One of these glucosidases
is a-amylase. The a-amylase is one of the major secretory products of the pancreas
and salivary glands, catalyze the hydrolysis of starch via a double displacement
mechanism involving the formation and hydrolysis of a covalent-glycosyl enzyme
intermediate by using active site carboxylic acids for it (Sales et al. 2012).
Glucosidases inhibitor enzymes reduce the rate of carbohydrate digestion and delay
the carbohydrate absorption from the digestive tract; therefore, they have the
potential to prevent the development of diabetes by lowering the after-meal glucose
levels (Miao et al. 2015). a-Amylase inhibitors are also called starch blockers since
they prevent or slow down the absorption of starch into the body mainly by
blocking the hydrolysis of 1,4-glycosidic linkages of starch and other oligosac-
charides into maltose, maltotriose, and other simple sugars.
Plants have a wide diversity of compounds including polyphenols than are good
inhibitors of enzymes responsible for carbohydrate digestion. The inhibition of
a-amylase by a polyphenol is highly related to its molecular structure, as the
inhibition results from binding interactions between polyphenols and the enzyme
(Sun et al. 2019). Hydroxyl groups, galloyl substituents, and conjugated systems
are some of the important characteristics of polyphenols for effective inhibition of
the enzyme (Table 15.1).
There are other compounds such as oligosaccharide inhibitors of the trestatin
family, proteinaceous inhibitors isolated from plant tissues. Acarbose is obtained
from this type of compound. Acarbose is a compound used in the clinical treatment
of diabetes mellitus, it is a competitive a-Amylase inhibitor. Terpenoids (oleanane,
ursane, and lupane) have been linked to a-Amylase inhibition although their
mechanism is not yet known (Sales et al. 2012).
Table 15.1 Indicates different examples of polyphenols with inhibitory activity of a-amylase
Compounds Inhibition References
a-amylase
(IC50)
Flavonoids Quercetagetin 10.2 uM Lo Piparo et al.
(2008)
Scutellarein 9.64 uM Lo Piparo et al.
(2008)
Kamferol, Apigenin, Naringenin, 0.5–6.0 mM Barrett et al. (2013)
Daidzein, Catechin
Luteolin 0.01 mg/ml Komaki et al. (2003)
Phenolic Caffeic acid 0.4 mM Narita and Inouye
acids 3.49 mg/ml (2011), Sun et al.
(2016a, b)
Quinic acid 26.5 mM Narita and Inouye
(2011)
Chlorogenic acids (Caffeoylquinic, 0.02– Narita and Inouye
Feruloylquinic, Dicaffeoylquinic, 2.55 mM (2011)
Dihydrocaffeic) 1.96 mg/ml Sun et al. (2016a, b)
Chlorogenic acid
Cinnamic acid >6.0 mM Narita and Inouye
(2011)
Coumaric acid 4.51– Narita and Inouye
4.86 mM (2011)
Ferulic acid 4.27– Narita and Inouye
5.45 mM (2011)
Tannic Tannic acid 0.301 mg/ml Sun et al. (2016a, b)
acid
Galloyl Epigallocatechin 2.07 mg/ml Miao et al. (2015)
moiety Catechin 3.552 mg/ml Sun et al. (2016b)
Epicatechin 9.32 mg/ml Sun et al. (2016b)
Theaflavin 0.412 mg/ml Sun et al. (2016b)
On the other hand, worldwide the use of plants for the treatment of diabetes is
increasing, some of them have been attributed an inhibitory effect on a-Amylase.
Table 15.2 indicates some parts of plants that have been used to regulate glycemic
indexes. According to the review made by Seetaloo et al. (2018), it can be observed
that there is a wide diversity of plants, plant parts, and types of extracts used to
maintain the hypoglycemic state. Additionally, Chinsembu (2019) in his review of
various plants used in the world for the control of diabetes, where alpha-amylase
inhibition is included, concludes that plants are a prelude in a more realistic and
more inclusive to control diabetes a world level.
Table 15.2 Plants whit a-Amylase inhibitory activity

Scientific name Part used % Inhibition References
(IC50)
Amaranthus Leaves 2.4 mg/ml Sudha et al. (2011)
spinosus
Antidrographis Leaves 46.02 ug/ml Rahmatullah et al. (2012), Kumar et al.
lineata Wallich (2011a)
Artocarpus Leaves 70.58 ug/ml Nair et al. (2013), Mahomoodally et al.
heterophyllus (2016)
Bixa orellana Leaves 50 ug/ml Ponnusamy et al. (2011), Ezuruike and
Prieto (2014)
Calendula Leaves 72.60 ug/ml Kumar et al. (2011b), Ezuruike and
officinalis Prieto (2014)
Camellia sinensis Leaves 0.52 to Olennikov and Kashchenko (2014)
1.54 mg/ml
Cichorium intybus Leaves 18.3 mg/ml Dalar and Konczak (2014), Street et al.
(2013)
Cinnamomum Leaves 40 ug/ml Ponnusamy et al. (2011), Telli et al.
verum (2016)
Cinnamomum Bark 40–46 ug/ml Ali-Shtayeh et al. (2012), Salehi et al.
zeylanicum (2013)
Cecropia Leaves 14 ug/ml Andrade-Cetto et al. (2008)
obtusifolia
Cyperus Tuber 5.19 mg/ml Khan and Yadava (2010), Sabiu et al.
esculentus (2017)
Ficus natalensis Leaves 17.85 ug/ml Olaokun et al. (2013), Keter and Mutiso
(2012)
Kandelia candel Bark 206 ug/ml Trinh et al. (2020), Sathe et al. (2014)
Malva neglecta Fruit 15.2 mg/ml (Yaseen et al. 2015; Türker and Dalar
2013)
Malmea depressa Roots 21 ug/ml Andrade-Cetto et al. (2008)
Momordica dioica Fruit 8 ug/ml Yaseen et al. (2015), Rao and Mohan
(2017)
Orthosiphon Leaves 36.70 mg/ml Mohamed et al. (2012), Ching et al.
stamineus (2013), Ameer et al. (2012)
Parkia roxburghii Pod 7.09–37.2 Sheikh et al. (2015)
ug/ml
Phoenix Leaves, stem, 10.43 to Mahomoodally et al. (2016)
dactylifera whole plant 985.45 ug/ml
Pipper betel Leaves 84.63 ug/ml Mahomoodally et al. (2016), Nair et al.
Blanco (2013)
Rubus fruticosus Leave 53.7 ug/ml Salehi et al. (2013), Shaheen et al.
(2017)
Rumex crispus Root 113.3–199.1 Minh et al. (2019)
ug/ml
(continued)

Scientific name Part used % Inhibition References
(IC50)
Senna urattensis Leave 123.95 ug/ml Thilagam et al. (2013)
Stevia rebaudiana Leave 198.40 ug/ml Mootoosamy and Mahomoodally
(2014), Ruiz-Ruiz et al. (2015)
Trigonella Leave 1.92 mg/ml Nickavar and Yousefian (2011)
foenum-graecum
Urtica dioica Leave 1.89 mg/ml Nickavar and Yousefian (2011),
Barkaoui et al. (2017)
Vaccinium Fruit 57 ug/ml Salehi et al. (2013), Nowbandegani
arctostaphylos et al. (2015)
Zea mays Flower 5.89 mg/ml Ezuruike and Prieto (2014), Sabiu et al.
(2017)
15.4 a-Glucosidase Inhibition
Among the therapeutic options in the treatment of type-2 diabetes mellitus (T2DM)
a-glucosidase enzyme inhibition is an effective one. One strategy that has been used
for the treatment of type-2 diabetes is the inhibition of the activity of
alpha-glucosidases using synthetic drugs. a-glucosidase inhibitors are known to
exist for clinical use (e.g.,voglibose, acarbose, and miglitol); nevertheless, these
inhibitors are habitually connected with gastrointestinal side effects as well as long
synthetic routes. For this reason, the development of inhibitors from natural
products could be an alternative option for the management of postprandial
hyperglycemic condition in T2DM (Dhameja and Gupta 2019). Several studies
have been directed to identify a-glucosidases inhibitors from natural sources, and
many candidates have resulted to be secondary metabolites (alkaloids, flavonoids,
phenols, and terpenoids) (Assefa et al. 2020; Choudhury et al. 2018; Di Stefano
et al. 2018).
a-glucosidase, located in the brush border of the small intestine, catalyzes the
liberation of a-glucose from the non-reducing end of the substrate. By the inhibition
of a-glucosidase in the intestine, the process of carbohydrate digestion spreads to
the lower part of small intestine delaying the absorption rate of glucose into the
blood. Several phytochemicals have shown antidiabetic activity by lowering the
glucose absorption throughout the inhibition of hydrolyzing enzymes a-glucosidase
(Ota and Ulrih 2017).
The World Health Organization has listed a plethora of plants for medical
purposes (21,000). According to the review made by Amjad et al. (2019) around
1200 plants are being used worldwide for the control of diabetes mellitus; from this,
only 30% were chemically and pharmacologically investigated. In this review, it
mentions a list of commonly used herbs in India for diabetes treatment. In the same
way, Bagetta et al. (2020) reviewed the bioactive substances contained in some
foods typical of the Mediterranean area (olive oil, onion, liquorice, rosemary,
oregano, hazelnut, pistachio, apple, red wine, hot pepper, Citrus sp. fruits, saffron,
and garlic) focusing on their impact on insulin resistance and T2DM, endothelial
dysfunctions, inflammatory response, oxidative stress, and dyslipidaemic and
hypercholesterolemic effects. A broad range of vegetables displayed differente
ranges of a-glucosidade inhibition activity (pepper, tomato, eggplant, potato, onion,
bitter melon, pumpkin, shallot, red cabbage, kiwi, cinnamon, mustard, Chinese
rhubarb, soybeans, among other). Assefa et al. (2020) addressed the percentage of
a-glucosidade inhibition according to different parts of plants and plant extracts.
Also, it is aborded the chemical compounds implicated in such inhibition such as
luteolin-7-O-glucoside a flavonoid isolated from pepper leaves and lactucaxanthin a
carotenoid extracted from lettuce. Cyanidin-3-rutinoside as well as different glu-
cosides derived from cyanidin has been found to exert a-glucosidase inhibitory
activity. Different anthocyanins are postulated as bioactive components against
diabetes (Gowd et al. 2017). Many a-glucosidase inhibitors belonging to plants
such as flavonoids, alkaloids, terpenoids, anthocyanins, glycosides, phenolic
compounds, and so on are aborded in the review made by Kumar et al. (2011b) with
their inhibitory potency along with IC50 values.
Plants commonly used to help manage blood glucose include bitter melon,
fenugreek gurmar, ivy gourd, nopal, ginseng, Russian tarragon, cinnamon, psyl-
lium, and garlic. The review made by Ota and Ulrih (2017) states that many of them
prevent glucose absorption by inhibiting intestinal a-amylase and a-glucosidase
throughout the action of several flavonoids (e.g., quercetin and kaempferol). In this
same sense is the review by Bi et al. (2017) about the used of spices in the
management of diabetes mellitus. A wide range of plant species from Africa,
Central America, Mexico, South Asia, and Iran implicate in the decrease of blood
sugar levels by inhibiting the enzymes a-amylase and a-glucosidase are summa-
rized by Chinsembu (2019). Here it is stated that the mechanisms of action are
mediated by phytochemical agents such as saponins, polyphenols, ellagitannins,
triterpenes, and elements (Mg, P, Ca, K, Mn, Cu, Zn, S, Cr, Co, Ni, and V).
Sesquiterpene lactones that could be used to treat diabetes because of a-glucosidase
inhibition are summarized by Chen et al. (2019b). Reviews from plants all over the
word state as an antidiabetic mechanisms the inhibition of a-glucosidase
(Samarakoon et al. 2019; Hamza et al. 2019; Rafe 2017; Rezaei et al. 2015;
Esquivel-Gutiérrez et al. 2012; Salehi et al. 2019).
It is well known the richens of secondary metabolites of Moringa oleifera
(quercetin, kaempferol, myricetin, ferulic acid, gallic acid, rutin, caffeic acid, oleic
acid, a-tocopherol, b-sitosterol, stigmasterol, campesterol and Δ5-avenasterol,
among others), hence their implications in ameliorating different health diseases
have also been evaluated. Hexane root extract exhibited the high a-glucosidase
inhibition of 88.045 ± 0.765% at 1.00 mg/ml extract concentration (IC50 value of
0.382 ± 0.006 mg/ml). This value is higher than that of acarbose (Magaji et al.
2020). Due to significant amount of saponins, alkaloids, flavonoids, phenols, ter-
penoids, and steroids found in Carica papaya seeds extracts, inhibitory effects of on
a-glucosidase enzymes have been evaluated. The hexane extract (IC50 = 75.78 mg/
ml) displayed the most potent and prominent effect compared to other extracts
(Agada et al. 2020). In the same sense, hexane extract of the roots of Paramignya
trimera (Rutaceae), a woody shrub endemic from the south of Vietnam, exhibited
a-glucosidase inhibitory activity presumably related to new compound found in
there, comprising two coumarins, pyranocoumarins A and B, and an acridone
alkaloid, paramiacridone (Trinh et al. 2020). Methanolic, aqueous, petroleum, and
hexane extracts from seeds, barks, and leaves of S. mahogany have shown
antidiabetics activities by different mechanisms, among them, inhibition of glu-
cosidase. The compounds implicated in such activity are phenolics (flavonoids
(swietemacrophyllanin, catechin, and epicatechin) and tannins), triterpenoids,
tetranortriterpenoid (limonoid: mahonin, secomahoganin, swietmanins, swiemaho-
gins, swietenine, and swietenolide), saponins, and alkaloids (Ervina 2020).
Alkaloids isolated from the mushroom Hericium erinaceus exhibited IC50 values
for the inhibition of a-glucosidase in the range of 12.7–23.3 lM (Wang et al. 2015).
A vast array of plants displays antidiabetic activity throughout a-glucosidase
inhibition; Table 15.3 shows some plants that have been reported with
a-glucosidase inhibition activity.
Table 15.3 Overview of plants whit a-glucosidase inhibitory activity

Plant/vegetable Part used % Inhibition (IC50) References
specie
Moringa oleifera Root 0.382 ± 0.006 mg/ml Magaji et al.
(2020)
Carica papaya Seeds 75.78 mg/ml Agada et al.
(2020)
Paramignya Root Paramiacridone: 62.5 lM Trinh et al.
trimera Paramicoumarin B: (2020)
65.5 lM
Ziziphus Leaves Nummularine-R: Choudhary
oxyphylla 212.1 ± 1.6 lM, et al. (2011)
Nummularin-C:
215.1 ± 1.2 lM,
Hemsine-A:
394.0 ± 2.4 lM
Curcuma longa Rhizomes 1.32–0.38 lg/ml Lekshmi
et al. (2012)
Callistephus Flower 8.14 lg/ml Zhang et al.
chinensis (2013)
Thymus praecox Aerial parts – Cam et al.
(2019)
Cinnamomum Bark 670 mg/ml Shihabudeen
zeylanicum et al. (2011)
Ficus Bark – Deepa et al.
benghalensis (2018)
Swietenia Seed – Wresdiyati
mahagoni et al. (2015)
(continued)

specie
Clitoria ternatea Petals – Escher et al.
L. (2020)
Phaseolus Fruit 19.52 ± 1.10 lg/ml Tan and
vulgaris L. Chang
(2017)
Glycine max L. Fruit 13.81 ± 0.08 lg/ml Tan and
Chang
(2017)
Red cabbage Leaves 280.03 ± 18.02 lg/ml Tan and
Chang
(2017)
Broccoli Flower 260.05 ± 10.20 lg/ml Tan and
Chang
(2017)
Blueberry Fruit 31.22 ± 1.10 lg/ml Tan and
Chang
(2017)
Blackberry Fruit 42.05 ± 1.81 lg/ml Tan and
Chang
(2017)
Salacia chinensis Heartwood 5.01 ± 1.51 lg/ml Thengyai
et al. (2019)
Vitex glabrata Bark 11.22 ± 1.70 lg/ml Thengyai
et al. (2019)
Senna siamea Buds 14.12 ± 1.59 lg/ml Thengyai
Heartwood et al. (2019)
Terminalia Leaves 15.84 ± 1.34 lg/ml Thengyai
catappa et al. (2019)
Phyllanthus Whole plant 25.11 ± 1.44 lg/ml Thengyai
amarus et al. (2019)
Erythroxylum – – Picot et al.
laurifolium (2014)
Elaeodendron – – Picot et al.
orientale (2014)
Antidesma – – Picot et al.
madagascariensis (2014)
Stillingia lineata – – Picot et al.
(2014)
Polyscias Leaves. 3-O-[- 440.5 ± 12.7 lg/ml Hanh et al.
fruticosa D-glucopyranosyl-(1 ! 4)– (2016)
D-glucuronopyranosyl]
oleanolic acid 28-O–
D-glucopyranosyl
Ester
(continued)

specie
Pheidole Leaves 0.874 mg/dl Kidane et al.
punctulata (2018)
Meriandra Leaves 0.599 mg/dl Kidane et al.
bengalensis (2018)
Agaricus blazei Fruit body 357.23 ± 20.11 Stojkovic
et al. (2019)
Coprinus Fruit body 322.74 ± 1.21 Stojkovic
comatus et al. (2019)
Canna indica L. Rhizome 2.35 lg/ml, 27.1 lg/ml. Ayusman
et al. (2020)
Canarium Bark 18.93 lg/ml Quan et al.
tramdenum (2019)
Vitis aestivalis Peel 0.384 mg/ml Zhang et al.
(2011)
– Not specified
15.5 Activation of Glucose Transporters
Glucose is a key fuel for most cells and a vital substrate for many biochemical
reactions. Since glucose is essential for every cell of the body, so are the glucose
transporters. There are three main of glucose transporters: sodium–glucose linked
transporters (SGLTs), facilitated diffusion glucose transporters (GLUT), this one
can be divided into subclasses (Class I, II, and III), and SWEET (this one was
recently discovered. SWEET1 is the one found in humans and it is suggested to
provide glucose for lactose synthesis in the mammary gland). About GLUT
transporters, Class I facilitative glucose transporters are represented by GLUT1 to
GLUT4. Class II of glucose transporters has four members, namely, GLUT5,
GLUT7, GLUT9, and GLUT11. There are five known Class III glucose facilitative
transporters, namely, GLUT6, GLUT8, GLUT10, GLUT12, and GLUT13 (HMIT).
GLUT isoforms vary in their tissue specificity and affinity for glucose (Navale and
Paranjape 2016; Szablewski 2019).
Peripheral resistance to insulin is a prominent feature of both insulin-dependent
and non-insulin-dependent diabetes. Skeletal muscle is the principal site in charge
of the decreased insulin-induced glucose consumption in diabetic subjects. The
rate-limiting step for glucose utilization in muscle is the glucose transporters. This
cellular process malfunctions in human and animal diabetes. Glucose transport
crosswise the muscle cell plasma membrane is facilitated by glucose transporter
proteins. The isoforms GLUT1 and GLUT4 are expressed in muscle. Insulin
increases glucose transport in muscle by the recruitment of the GLUT4 transporter
(but not GLUT1) from an intracellular pool to the plasma membrane. GLUT4 is
involved in the pathophysiology of T2DM. Defects in GLUT4 expression or
translocation to the peripheral cell plasma membrane in T2DM patients hinders the
entrance of glucose into the cell for energy production. In addition to suitable drugs,
a proper diet and/or exercise can be executed to target the increase in GLUT4
expression. Glucose transporters have major implications for the control of the
delivery of glucose to mammalian cells; for this reason, they will become more and
more prominent for the management as diabetes (Alam et al. 2016).
In the next lines are going to be aborded phytochemicals as source materials for
the modulation of glucose transporters and therefore implicated in the development
of new antidiabetic drugs or therapies for the management of diabetes.
The potential of flavonoids such as anthocyanins and myricetin against diabetes
is primarily through the modulation effects on glucose transporter by enhancing
GLUT2 expression in pancreatic b cells and increasing expression and encouraging
translocation of GLUT4 via PI3K/AKT, CAP/Cb1/TC10, and AMPK pathway
(Hajiaghaalipour et al. 2015). Resveratrol is a compound derived from the
phenylpropanoid pathway found in Japanese knotweed, peanuts, berries, grape
skins, red wine, and roots of rhubarb; studies have reported blood-glucose-lowering
effects of resveratrol in animal models, among the propose mechanisms is the
enhancement of GLUT4 translocation (Ota and Ulrih 2017). Quercetin influences
glucose and lipid metabolism, probably because of the increase of the glucose
uptake by means of mitogen-activated protein kinases (MAPKs) insulin-dependent
mechanism, resulting in the translocation of GLUT4. Rutin also has shown stim-
ulation of glucose transport into muscle through activating the synthesis and
translocation of the transporter GLUT4 (Bagetta et al. 2020). Isoflavones improve
glucose uptake in white adipose tissue by means of increasing the expression of
GLUT4 whereas improves insulin resistance in the skeletal muscles by means of
upregulating GLUT 4 translocation (Duru et al. 2018). Epigallocatechin gallate, a
bioactive polyphenol in green tea, promotes GLUT4 translocation to improve
adipose insulin resistance (Xu et al. 2018), this same mechanism is activated by
ginsenosides (Shao et al. 2019) and by para-nitro derivative of caffeic acid phe-
nethyl ester (Li et al. 2019). Dinda et al. (2020) in their comprehensive review
about role of dietary flavonoids in prevention of obesity and diabetes states that
flavonoids improve GLUT4 expression and translocation to plasma membrane. In
the same sense is the review made by Unuofin and Lebelo (2020). Abscisic acid, a
terpenoid, in in vitro condition has been reported to adjust GLUT4-mediated glu-
cose uptake. Plumbagin, a quinone, contributes to glucose homeostasis through
GLUT4 translocation (Xu et al. 2018). P-coumaric acid has potentially beneficial
effects in improving or treating metabolic disorders by modulating modulates
glucose and lipid metabolism via GLUT2 (Amalan et al. 2016).
Regarding SGLT inhibitors, alkaloid compounds isolated from the leaves of A.
macrophylla have shown good SGLT1 and SGLT2 inhibition. Two stilbene tri-
mers, gneyulin A and B from the dried bark of G. gnemonoides, displayed inhi-
bition for SGLT1 and SGLT2. Also, inhibition for these transporters was found for
Schisandra chinens. This plant contains polyphenols, lignans, and triterpenoids
whose pharmacological effects have been evaluated (Choi 2016). SGLT2 inhibitors
inhibit renal glucose reabsorption and promote glucosuria, thereby leading to
Table 15.4 Overview of plants with antidiabetic effect through effect in glucose transporters
Plant/vegetable specie Effect in glucose References
transporters
Saffron GLUT4 Bagetta et al. (2020)
Onion GLUT4 Akash et al. (2014), Bagetta et al.
(2020)
Opuntia ficus-indica GLUT1 Dinda et al. (2020)
Brachylaena elliptica GLUT2 Sagbo et al. (2018)
Cinnamon GLUT4 Şanlier and Gencer (2020)
Ginger GLUT4 Şanlier and Gencer (2020)
Astragalus GLUT4 Venkatakrishnan et al. (2019)
membranaceus
Gymnema sylvestre GLUT2, GLUT4 Venkatakrishnan et al. (2019)
Fenugreek GLUT4 Venkatakrishnan et al. (2019)
Nigella sativa GLUT4 Benhaddou-Andaloussi et al.
(2011)
Black bean GLUT2, SGLT1 Mojica et al. (2017)
Centratherum GLUT2, GLUT4 Arya et al. (2012)
anthelminticum
Thymus praecox SGLT-1, SGLT-2, Cam et al. (2019)
GLUT2
Curcumin GLUT4 Chauhan et al. (2018)
Acer nikoense SGLT Choi (2016)
Sophora flavescens SGLT Choi (2016)
reductions in plasma glucose concentrations, SGLT1 are similar, but their mode of
action is in the intestines. Phlorizin from the bark of P. communis was the first
compound to be known to exert SGLT1 and SGLT2 inhibition. This flavonoid
along with some other flavonoids and some alkaloids (10-methoxy-N(1)-
methylburnamine-17-O-veratrate and allstiphyllanine D) with SGLT inhibitor
capacity are reviewed by Blaschek (2017). Table 15.4 presents an overview of plant
with antidiabetic effect through the modulation of glucose transporters.
15.6 Activation of Insulin Secretion
DM has become an important health problem with its high morbidity and mortality
since it is considered a chronic-degenerative disease with an increase in its
prevalence. Etiologically, DM suggests that alterations in genetic and environ-
mental factors, as well as increased insulin resistance (Fang et al. 2016). Insulin
resistance in DM is mainly caused by the modification of insulin signaling making
the body insulin sensitive (Wang et al. 2019). The result of these modifications is
the reduction of glucose absorption from myocytes, hepatocytes, adipocytes, and
the elevation of blood glucose levels, that is, the development of insulin resistance
and hyperglycemia (Perry et al. 2014). For many years of research, insulin resis-
tance mechanisms remain a focal point of study and the ability to maintain glycemic
homeostasis improves the progression and severity of diabetes mellitus, for this
reason, drugs that include oral antidiabetic agents are used, insulin and others like
incretin to control blood homeostasis (Chen et al. 2019a). Medicinal plants have
been used for many years to treat various diseases. The use of these medicinal
plants alone or in combination with other medicinal plants is currently known as an
alternative therapy for the treatment of chronic-degenerative diseases such as DM
using phytochemical compounds and the bioactivity of medicinal plants (Engin
et al. 2018). Plants can have many phytochemical compounds with an effect on
various metabolic pathways. On the other hand, the bioactive compounds of
medicinal plants and the antidiabetic, anti-hyperglycemic, and hypoglycemic
potential have been reviewed in various investigations in different models in the
long or short term, as well as these can be used in conjunction with pharmacological
drugs (Martín and Ramos 2017). Table 15.5 shows the compounds with antidia-
betic potential such as bitter gourd, ginger, turmeric, black cumin, cinnamon, garlic,
green tea, among others. The most common compounds found in these medicinal
plants are anthocyanins, quercetin, cinnamaldehyde, curcumin, catechins, S-methyl
cysteine sulfoxide, and oleoresin A among others (Beidokhti and Jäger 2017).
These phytochemical compounds have been shown to improve insulin sensitivity,
stimulate insulin secretion, increase peripheral glucose uptake, inhibit hepatic
glycogenolysis, exert antioxidant effects, inhibit hepatic glycogenolysis, and
potentiate endogenous incretins. Glycogenolysis and the enhancement of endoge-
nous incretins are some pharmacological mechanisms of these herbs. Currently,
there is biological knowledge about the specifications and the process of action in
the treatment of diabetes of medicinal plants. Polyphenols are the compounds that
we find mostly in fruits, vegetables, cereals, and most foods. Polyphenol amounts
are specific to each food. The products with the highest amounts of polyphenols are
spices, dried herbs, cocoa, purple or red berries and seeds, which can even contain
200–300 mg per 100 g. Epicatechin and catechin monomers or oligomers of epi-
catechin and/or catechin (procyanidins) and flavan-3-ols have various beneficial
effects such as possible effects to improve insulin resistance (Ghorbani et al. 2019).
Rowley et al. (2017) demonstrated in a clinical analysis that catechins improve the
cellular redox state. Thus, migration of Nrf2 (Nuclear factor erythroid 2-related
factor-2) to the nucleus is stimulated, key genes for mitochondrial respiration are
regulated, glucose-stimulated insulin secretion is increased, and beta-cell function is
improved as a result (Fu et al. 2016). On the other hand, they also beneficially
modulate the concentrations of transforming growth factor beta1 (TGF-beta1) and
TNF(Tumor necrosis factor-alpha) in blood mononuclear cells, regulate NF-kappaB
(Nuclear factorkappa B), and IL-1beta (interleukin-1-beta), thus considerably
increasing the stimulated Akt phosphorylation (Fu et al. 2016; Matzinger et al.
2018). Insulin resistance is a cause, at least in part, of endothelial dysfunction.
Therefore, flavonoids improve endothelial dysfunction, stimulate the elevation of
NO (Nitric oxide) bioavailability, therefore, protect the vascular endothelium and
decrease risk factors for cardiovascular disease (Bhattacharya et al. 2019). Thus, the
use of in vivo models with rats and different types of extracts, such as Aloe Vera
extracts, reduces blood glucose levels, a-glucosidase activity (Chang et al. 2013). In
other investigations, the use of phenolic extracts of avocado that reduces serum
insulin levels and in a clinical model use of omega fatty acids was able to maintain
glycemic control (Thenmozhi et al. 2012). Also, in an in vivo model using the musa
paradisiaca flowers, and green fruits it was possible to control plasma glucose and
Hba1c (glycosylated hemoglobin) (Dikshit et al. 2012). Also, in a medicinal shrub
berberis integerrima conducted in vivo and clinical studies in which it produced a
decrease in blood glucose and a decrease in serum glucose, as well as Hbca1 levels,
respectively (Moazezi and Qujeq 2014). Likewise, for cinnamon, clinical studies
showed that the polyphenols it contains were applied to women with polycystic
ovary syndrome, and during the period of application there is insulin resistance and
hyperinsulinemia (Singab et al. 2014). Other in vivo studies showed that black tea
and black cumin in water and hexane extracts, respectively, are associated with a
lower risk of DM, showing hypoglycemic, anti-hyperglycemic, and oral antidia-
betic activity in rats, and a sensitizing action in the black tea (Abeywickrama et al.
2011; Ahmad et al. 2013). Based on this, treatment with Nigella sativa (black
cumin extract) caused a decrease in serum glucose, an increase in decreased serum
insulin levels and a partial proliferation/regeneration of pancreatic b cells
(Mohebbati and Abbasnezhad 2020). On the other hand, garlic, ginger, and shallot
were applied in models with diabetic rats, and when isolating in S-alyl cysteine
sulfoxide from garlic it stimulated in vitro insulin secretion of b cells, besides that
the ginger extracts showed increased adipocytes of mouse 3T3-L1 preadipocytes
and thus insulin sensitivity was improved (Jafri et al. 2011; Moradabadi et al.
2013). Besides, the hypoglycemic properties of the shallot were shown by the
quercetin content in the extracts applied in diabetic models producing a hypo-
glycemic effect and also the extract decreased plasma glucose and HbA1c levels
and increased insulin level (Mehdi et al. 2012). There is increasing evidence
showing that flavonoids such as eupatilin, fisetin, genistein, naringenin, proantho-
cyanidins, puerarin, quercetin, and rutin increase plasma insulin tested in vivo
models (Akash et al. 2014; Gowd et al. 2017). The increase in plasma insulin can be
attributed to the preservation of beta-cell survival, and the stimulation of insulin
release. These beneficial effects are mediated by influencing the activation and
amplification pathways of insulin secretion. Thus, the accumulation of bioactive
compounds inhibits the electron transport chain in the mitochondria and alters the
function of the KATP channel (Şanlier and Gencer 2020). Other research indicates
that flavonoids can improve glucose-induced insulin secretion by influencing the
Amplification pathways including PLC/PKC and cAMP/PKA signaling cascades as
demonstrated in the pancreatic islets of rats by applying various doses of routine,
also, genistein, and kaemferol have an effect on cAMP/PKA signaling cascades (Fu
et al. 2016). Chen et al. (2019a) demonstrated that quercetin participates in ERK1/2
phosphorylation by enhancing insulin release.
Table 15.5 Antidiabetic activity of dietary plants in vivo, in vitro and clinical trials
Plant/vegetable Extract/compounds Effects References
specie
Aloe vera Aloeresin A/no Decrease fasting blood Chang et al.
quantity glucose and improve (2013)
insulin levels in plasma
Avocado Monounsaturated Shown significant recovery Thenmozhi et al.
fatty acids of in the level of serum (2012)
avocado/300 mg/kg insulin, glycosylated
hemoglobin and activities
of carbohydrate metabolic
enzymes
Banana (Musa Flower and Reduced significantly Dikshit et al.
paradisiaca lyophilized stem fasting and postprandial (2012)
fruit) juice/50 mg/kg plasma glucose and HbA1c
and increased serum insulin
Berberis Anthocyanin/1 mg, Showed a significant Moazezi and
integerrima twice daily decrease in serum glucose Qujeq (2014)
and HbA1c levels
Bitter melon Triterpene, phenolic The increased glucose Joseph and Jini
(Momordica compounds/10 mg/ utilization by the liver, (2013)
charantia) kg significantly stimulation of insulin
release from pancreatic
b-cell, increasing number
of b-cell, and suppression
of the key gluconeogenic
enzymes
glucose-6-phosphatase and
fructose-1,6-bisphosphatase
Black cumin or Oil/1 ml/kg Significantly reduced the Ahmad et al.
black seed glucose levels (2013)
(Nigella sativa)
Black tea Flavonoids and Hypoglycemic, Abeywickrama
(Camellia caffeine/480 mg/ml anti-hyperglycemic, and et al. (2011)
sinensis L.) antidiabetic actions.
Blueberry Anthocyanin/22.5 g Increased insulin sensitivity Stull et al.
(Vaccinium of powdered dry and decreased glucose (2010)
angustifolium blueberries/twice concentrations
Aiton) daily
Caraway, Flavonoids (rutin, Improving kidney and Shaffie et al.
coriander, and quercitin, and pancreas cell dysfunction (2010)
fennel kaempferol)/
Caraway (10 mg/
kg), coriander
(40 mg/kg), and
fennel (30 mg/kg)
Cinnamon Bark extract A significant decrease in Singab et al.
(cinnamaldehyde)/ blood glucose, (2014)
200 mg/kg a-glycosidase activity, and
increase in serum insulin
levels and HDL-cholesterol
levels
(continued)

specie
Coffee (Coffea Quinides and Improvement in glucose Chang et al.
arabica L.) chlorogenic acids/ tolerance and insulin (2013),
120 mg/kg sensitivity and a lower risk Tunnicliffe and
of DM Shearer (2008)
Garlic (Allium Oil/150 mg/kg Significantly lowered the Moradabadi
sativum L.) levels of blood glucose, and et al. (2013)
improved the levels of
plasma insulin, increased
insulin secretion and
insulin sensitivity
Ginger (Zingiber Gingerol/500 mg/kg Showed significant Jafri et al. (2011)
officinale decreased blood glucose
Roscoe)
Grape (Vitis Condensed tannins Showed significantly Sapwarobol
vinifera L.) and flavonoinds/ reduces the postprandial et al. (2012)
100 mg/kg plasma glucose
Guava (Psidium Leaf Tea/200 mg/kg Showed significantly Deguchi and
guajava L.) reduces the postprandial Miyazaki (2010)
plasma glucose
Jackfruit Condensed tannins Significant decrease in Shahin et al.
(Artocarpus and flavonoinds/ blood glucose (2012)
heterophyllus 400 mg/kg
Lam.)
Loquat Total triterpene acid Caused significant Li et al. (2007)
(Eriobotrya fraction at 300 mg/ hypoglycemic effects on
japonica Lindl.) kg day and Total normal, alloxan and
sesquiterpenes at STZ-induced diabetic mice
30 g/kg day
Lychee (Litchi Oligonol and Attenuated Noh et al. (2011)
chinensis Sonn) polyphenol/10 mg/ diabetes-induced hepatic
kg damage via regulation of
oxidative stress and lipid
metabolism
Mango Aqueous extract of Was effective in Villas Boas et al.
(Mangifera leaves/250, 500 and maintaining the long-term (2020)
indica Linn. 1000 mg/kg hypoglycemic effect, as
(Anacardiaceae)) well as, significantly
increased the sensitivity of
diabetic animals to insulin
and the plasma insulin level
Nopal (Opuntia Aqueous extract Play role in intestinal Leem et al.
ficus-indica var. (water-soluble glucose absorption, glucose (2016)
saboten) dietary fiber, uptake to peripheral tissues,
vitamin c, and and improvement of insulin
polyphenols/ action
flavonoids)/250,
500, and 1000
mg/kg
(continued)

specie
Onion (Allium S-methyl cysteine Significantly controlled the Ogunmodede
cepa L.) sulphoxide/200 blood glucose and altered et al. (2012),
mg/kg the activities of liver Ozougwu (2011)
hexokinase and glucose
6-phosphatase towards
normal
Papaya (Carica Phenolics, and Reduced blood glucose Venkateshwarlu
papaya L.) b-cryptoxanthin/ levels and possessed et al. (2013)
100 mg/kg antidiabetic and
anti-hyperglycemic
activities
Pomegranate Oleanolic, ursolic, Reduced blood glucose Li et al. (2008)
(Punica and gallic acids/ levels, and stimulation,
granatum L.) 300 mg/kg regeneration, and increased
number of b-cell, by
protecting pancreatic tissue
and subsequent release of
insulin
Saffron (Crocus Carotenoids/ significantly decreased Elgazar et al.
sativus L.) 600 mg/kg blood glucose levels and (2013)
increased serum insulin and
improved liver and kidney
functions
Shallot (Allium Flavonoids Lowered the plasma Mehdi et al.
ascalonicum L.) (quercetin)/100 mg/ glucose and HbA1c levels (2012)
kg and enhanced insulin level
Soybean Isoflavones/100 g/ Produced effective Nanri et al.
(Glycine max L.) dia antidiabetic activity and (2010)
blood glucose regulators
Turmeric Curcuminoids, Increased the production of Chang et al.
(Curcuma longa (vanillin, and ferulic insulin and actions likely (2013), Pawar
L.) acid/200 mg/kg via regulation of insulin et al. (2014)
resistance, b-cell function,
and gut absorption
Curcumol/40 mg/kg Is 9.4% more potent than Raafat and Omar
turmeric extract (100 mg/ (2016)
kg) in subchronic
management of diabetes
Watermelon L-citrulline/94.5% Increase serum arginine El-Razek and
(Citrullus watermelon juice concentration and has a Sadeek (2011)
lanatus (Thunb)) significant hypoglycemic,
hypolipidemic effect and
significantly
15.7 Future Perspective and Conclusion
DM is a metabolic syndrome that is difficult to define due to the conditions it has

and the alternatives used to control or decrease it. The use of phytochemical
compounds plays important roles in DM control based on the state of homeostasis
inducing antioxidant enzymes by large amounts of reactive oxidation species by
controlling Nrf2. And this provides us with tools to apply antioxidant therapies to
treat DM. Therefore, precision medicine targeting a certain type of antioxidant in
specific cells in a limited period of time can be helpful in treating the disease.
Lifestyle changes and better control of diet are more effective than medicines to
prevent the disease. On the other hand, clinical trials have positively described
changes in biomarkers in diabetes and other diseases when using polyphenolic
compounds, but there is still a long way to go in establishing the dose and type of
phenolic compound responsible for the preventive potential and/or therapeutic
effects against these chronic diseases. Accordingly, all of the medicinal plants
described in this review suggest that the content of phytochemical compounds they
contain may be used as a strategy to improve glucose/insulin homeostasis through
increased insulin sensitivity, leading to preservation. of function of pancreatic islets
in a prediabetic condition. The low availability of the isolated compounds also leads
us to investigate other alternatives to improve bioavailability such as the use of
microencapsulates.
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EXCLI J 12:956
Chapter 16
An Overview of the Bioactivities
of Gedunin
Yong Sze Ong, Kooi Yeong Khaw, Loh Teng-Hern Tan,

Peng-Nian Yew, Kai-Boon Tan, Wei Hsum Yap, Siah Ying Tang,
Liang Ee Low, Learn-Han Lee, and Bey-Hing Goh
Abstract Gedunin is a naturally occurring pentacyclic triterpenoid secondary

metabolite presents in Meliaceae family. Since the first isolation and characteri-
zation of this compound at 1960s, the chemical synthesis and bioactive properties
of this compound have been continuing researched. The plant has been historically
used as the folk medicine to treat malaria and infectious diseases. Evidence-based
pharmacology study showed that gedunin and its derivatives exerted enormous
potential in the treatment of cancer, neurodegenerative and infectious diseases.
Considering this year marks 60 years since the isolation of gedunin, this review
presents an update of the bioactivity of gedunin and its derivatives with future
prospect of this bioactive compound.
Keywords Gedunin Bioactivities Anti-cancer Triterpenoid Pharmacology
Y. S. Ong K. Y. Khaw K.-B. Tan L. E. Low B.-H. Goh (&)

Biofunctional Molecule Exploratory (BMEX) Research Group, School of Pharmacy,
Monash University Malaysia, Bandar Sunway, 47500 Selangor Darul Ehsan, Malaysia
e-mail: goh.bey.hing@monash.edu
L. T.-H. Tan L.-H. Lee
Novel Bacteria and Drug Discovery (NBDD) Research Group, Microbiome and Bioresource
Research Strength, Jeffrey Cheah School of Medicine and Health Sciences, Monash
University Malaysia, Bandar Sunway, 47500 Selangor Darul Ehsan, Malaysia
P.-N. Yew
Faculty of Applied Sciences (FOAS), Department of Bioscience, Tunku Abdul Rahman
University College (TARUC), Jalan Genting Kelang, 53300 Kuala Lumpur,
Setapak, Malaysia
K.-B. Tan
The University of Edinburgh, Centre for Discovery Brain Sciences, Hugh Robson Building,
15 George Square, Edinburgh EH8 9XD, Scotland
W. H. Yap
School of Biosciences, Taylor’s University, Subang Jaya, 47500 Selangor Darul Ehsan,
Malaysia
https://doi.org/10.1007/978-3-030-54027-2_16
564 Y. S. Ong et al.
16.1 Introduction
Ever since humankind begins, a close connection between humans and mother earth
has been partly attributed to the discovery of cures for illnesses from nature. The
use of medicinal plants to treat various illnesses back then was totally based on
experience, due to the fact that there was no evidence-based knowledge behind the
cause and pathogenesis of illnesses. Over the time, medicinal plants play an
important role in the discovery and development of the drugs due to the advantages
of readily available and cost effectiveness (Atanasov et al. 2015). As a result of
nutritional needs and the adaption to the environmental challenges, plants produce
chemical compounds which then become the valuable source for novel therapeutic
agents (Khaw et al. 2017). Examples of clinically available plant-derived thera-
peutic compounds are vincristine from Catharanthus roseus, capsaicin from
Capsicum spp., paclitaxel from Taxus brevifolia and camptothecin from
Camptotheca acuminate.
Plant-derived compounds have contributed to the drug development as they
served as the lead compounds for some of the chemically-modified therapeutics
such as acetylsalicylic acid from salicylic acid, warfarin from dicoumarol and
topotecan from campthothecin. Public gives attention to the plant-derived com-
pounds as proven by the award of Nobel Prize in Physiology or Medicine in 2015
for the discovery of artemisinin which was isolated from Artemisia annua for the
treatment of parasitic diseases (Efferth et al. 2015). The therapeutic effects of
S. Y. Tang
Chemical Engineering Discipline, School of Engineering, Monash University Malaysia,
Bandar Sunway, Selangor Darul Ehsan, Malaysia
Advanced Engineering Platform, Monash University Malaysia, Bandar Sunway,
Selangor Darul Ehsan, Malaysia
L. E. Low
Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou, Zhejiang 310058, P.R. China
Key Laboratory of Biomedical Engineering of the Ministry of Education,
College of Biomedical Engineering & Instrument Science, Zhejiang University,
Hangzhou, Zhejiang 310027, P.R. China
L.-H. Lee
Health and Well-Being Cluster, Global Asia in the 21st Century (GA21) Platform,
Monash University Malaysia, Bandar Sunway, 47500 Selangor Darul Ehsan, Malaysia
B.-H. Goh
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058,
P.R. China
16 An Overview of the Bioactivities of Gedunin 565
medicinal plants on an array of chronic diseases including cancer, diabetes, and

neurological disorders are still being extensively studied, partly due to the necessity
arisen from decreasing efficacy of the contemporary drugs.
Meliaceae, the fast-growing flowering plant family, consists of medicinal
evergreens members that could withstand dry season in the tropical and subtropical
areas. Neem tree (Azadirachta indica) is one of the most well-known species
among members of the Meliaceae family for its wide health-promoting activities.
The scientific name of Azadirachta indica (Azadi = free, diracht = tree, indica = of
India, in Persian) literally denotes neem as a ‘free tree of India’, describing that the
tree is intrinsically free from insect and illnesses (Kumar and Navaratnam 2013).
Over the centuries, neem has been traditionally used by folklores for various pur-
poses: The aqueous extract of neem leaf is used by Indians and Burmese to treat
malaria (Badam et al. 1987) while the neem leaf is added in toothpaste by German
and Indians for anti-septic purpose (Uko et al. 1995).
Over the decades, much attentions have been paid to discover the bioactive
compounds of the neem tree, in which more than 300 structurally complex
bioactive compounds have been purified and structurally characterized (Subapriya
and Nagini 2005). It has been reported that the vast biological activities of the neem
are contributed to some of the active constituents such as azadirachtin, gedunin,
nimbolinin, nimbin and quercetin (Alzohairy 2016). Over the years, tremendous
amount of attentions has been drawn to one of the bioactive compounds known as
gedunin. This limonoid type of tetranortriterpenoid compound consists of four
trans-fused six-membered rings with an oxirane annelated to the fourth ring. It has
been reported that gedunin possesses a vast array of biological activities such as
anti-cancer, anti-parasitic, anti-inflammatory, antimicrobial and insect growth
inhibitory effects.
In addition to neem, gedunin and its natural occurring derivatives can be purified
from the other Meliacae family including cannonball mangrove (Xylocarpus
granatum), African mahogany (khaya grandifoliola) and Spanish cedar (Cedrela
odorata) (Bickii et al. 2000; Lakshmi et al. 2010; Brandt et al. 2008). The current
review summarizes the recent findings on the bioactivities of gedunin and its
derivatives, and also evaluate the potential and significance of gedunin and its
derivatives as a potential therapeutic agent.
16.2 Bioactivities of Gedunin
16.2.1 Anti-Cancer Properties of Gedunin
More than 60% of the clinically-approved anti-cancer drugs are derived from plants
and microorganisms. Owing to the genetic elasticity of cancer cells and the con-
sequent emergence of chemoresistance, considerable attention has been drawn to
natural bioactive compounds with anti-cancer properties. Gedunin has been studied
extensively for its in vitro anti-cancer properties against a variety of cancers such as
colon (Uddin et al. 2007), ovarian (Johnson et al. 2014; Patwardhan et al. 2013),
mammary (Brandt et al. 2008; Kikuchi et al. 2011), pancreatic (Boopalan et al.
2013; Subramani et al. 2017), lung, stomach cancers, melanoma and leukemic
(Kikuchi et al. 2011) (Table 16.1). The anti-cancer activities of neem compounds
including gedunin in gynecological cancers (breast, cervical and ovarian) was
recently reviewed by Moga et al. (2018). A number of literatures reported gedunin
showed notable anti-cancer mechanisms such as induction of autophagy, apoptosis,
mitotic arrest and inhibition of metastasis (Fig. 16.1). The compound exhibited a
promising in vitro efficacy with IC50 values in a range of 3.22–30 µM; in which the
efficacy was more prominent in breast cancer cell lines (MCF-7 and SkBr3) and
leukemia cell line (HL60).
In 2006, a research group led by Lamb et al. (2006) discovered that the
anti-proliferative property of gedunin was modulated by the 90-kDa heat shock
protein (Hsp90) through connectivity map. Hsp90 is a molecular chaperone that is
responsible for the stability and function of a wide variety of client proteins for cells
growths and survivals. In cancerous cells, these client proteins are frequently
mutated or/and overexpressed, henceforth it is being actively pursued as individual
therapeutic target (Garg et al. 2016). Hsp90 is essential for normal cellular
homeostasis. It is also known to play an important role in several pathological
conditions. Hsp90 and its co-chaperones (which help to regulate the function of the
Hsp90 protein-folding machine) are extensively studied in tumorigenesis. Hsp90
inhibitors bind and inactivate Hsp90 N-terminal ATP-binding site, causing pro-
teasomal degradation of Hsp90-dependent client protein (Patwardhan et al. 2013).
Unlike most of the drug inhibitor of Hsp90, gedunin interacted with Hsp90 without
the involvement of competitive inhibitor of ATP, suggesting that a novel and
unique mechanism of Hsp90 modulation (Hieronymus et al. 2006).
Through Hsp90 inhibition, gedunin has been further investigated for the
downstream mechanisms of action responsible for its anti-cancer effect. Research
revealed that gedunin inhibited cancerous cell proliferation by inducing mitotic
arrest between metaphase and anaphase. The significant overexpressed of check-
point kinase-1 (CHK1) and polo-like kinase-1 (PLK1) in the cancerous cells has led
to the mitotic halt. In a study performed with pancreatic cancer cells, gedunin has
shown to exhibit anti-proliferative effect by suppressing mTOR and 4EBP, which
are the key phosphoproteins responsible for protein translation.
Induction of apoptosis in cancerous cells has been the main strategy in cancer
treatment (Ong et al. 2018). Several studies reported that treatment with gedunin
has induced apoptosis and autophagy in various cancer cells through modulation of
cell death executors such as LC3B, WIPI-1, VMP-1, pJNK, caspases and
PARP. Gedunin demonstrated its potential as effective agent against pancreatic
cancer which currently lacks of effective treatment. In vitro study by Subramani and
colleagues showed that gedunin at 25 µM has induced high percentage of apoptotic
cell death in three pancreatic cancer cell lines HPAC (46.5%), MIAPaCa-2 (37.6%)
and PANC-1 (44.7%), but it was relatively safe against non-cancerous pancreatic
cell (hTERT-HPNE). In addition, gedunin induced apoptosis through intrinsic and
Table 16.1 Bioactivities of gedunin or derivatives tested with respective experimental model
16
Bioactivities Gedunin/Derivatives Experimental model Efficacy References

Anti-cancer Gedunin Caco2 colon cancer cell line 16.8 lMa Uddin et al.
(2007)
MCF-7 breast cancer cell lines 8.84 ± 0.03 lMa Brandt et al.
(2008)
SkBr3 breast cancer cell lines 3.22 ± 0.06 lMa Brandt et al.
(2008)
Pancreatic cancer cell lines Unidentified Boopalan
et al. (2013)
ID8, ID8TaxR, A2870, C30 and CP70 0–30 lMb Johnson
ovarian cancer cell lines et al. (2014)
A2870 and ID8 chemoresistant ovarian 2.5 lM (Synergizes with Johnson
cancer cell lines cisplatin and paclitaxel)b et al. (2014)
Cervical cancer cell line HeLa-PRB and 0–40 lMb Patwardhan
An Overview of the Bioactivities of Gedunin
Breast carcinoma cell lines MDA-MB-231, et al. (2013)

MDA-MB-453, Hs578T, T47D and MCF7
Leukemia cell line HL60 5.9 lMa Kikuchi
et al. (2011)
Lung cancer cell line A549 >20 lMa Kikuchi
et al.(2011)
Stomach cancer cell line AZ521 16.9 lMa Kikuchi
et al. (2011)
Breast cancer cell line SK-BR-3 8.3 lMa Kikuchi
et al. (2011)
Melanoma cell line CRL1579 >20 lMa Kikuchi
et al. (2011)
Caco2 colon cancer cell line Unidentified Uddin et al.
(2007)
567
(continued)
568

Pancrease cell line 5.0 lM
1a-hydroxy-1,2-dihydrogedunin Leukemia cell line HL60 >20 lMa Kikuchi
et al. (2011)
7-deacetylgedunin Lung cancer cell line A549 >20 lMa Kikuchi
et al. (2011)
Stomach cancer cell line AZ521 16.9 lMa Kikuchi
et al. (2011)
Breast cancer cell line SK-BR-3 12 lMa Kikuchi
et al. (2011)
Melanoma cell line CRL1579 >20 lMa Akihisa et al.
(2009),
Kikuchi
et al. (2011)
In vitro epstein-bar virus early antigen 488 mol ratio/32 pmol TPAa Akihisa et al.
(EBV-EA) activation assay (2009)
Leukemia cell line HL60 2.9 lMa Kikuchi
et al. (2011)
7-deacetyl-7-benzoylgedunin Lung cancer cell line A549 >20 lMa Kikuchi
et al. (2011)
Stomach cancer cell line AZ521 >20 lMa Kikuchi
et al. (2011)
Breast cancer cell line SK-BR-3 >20 lMa Kikuchi
et al. (2011)
Melanoma cell line CRL1579 >20 lMa Kikuchi
et al. (2011)
Neuroblastoma cell line SH-SY5Y 38.95 ± 3.225 Lu et al.
(2009)
Y. S. Ong et al.
(continued)
16

Cryoprotective Gedunin Experimental rat (in vivo) 56.86 lg/mLa Lakshmi
et al. (2010)
Primary neuronal and astrocyte cells 0–24 lMb Smirnova
et al. (2011)
Experimental rat (in vivo) 66.54 lg/mLa Lakshmi
et al. (2010)
Photogedunin Primary rat hippocampal neurons 500 nMb Jang et al.
(2010)
Deoxygedunin Neuroblastoma cell line SK-N-SH 24.03c Zhang et al.
(2009)
Neuroblastoma cell line SK-N-SH 12.77c Zhang et al.
(2009)
Deacetylgedunin Neuroblastoma cell line SK-N-SH 6.02c Zhang et al.
(2009)
Deacetoxy-7-oxogedunin In vivo articular inflammation Pretreatment: 0.005–5 mg/ Conte et al.
experimental model kgb (2015)
Post-treatment: 0.05 mg/kgb
Anti-neurological Gedunin Inhibit NF-jB activation 3.5–7.5 lM Tom et al.
(2019)
Neuro-2a cells 20 lM Yang et al.
(2019)
Anti-inflammation Gedunin TPA-induced ear edema inflammation in 0.16d Akihisa et al.
Mice (2009)
Inhibited NLRP3 expression in vivo 0.5 mg/kg Borges et al.
(2017)
(continued)
569
570

+ a
7-deacetylgedunin In vitro parasitized human O erythrocyte 1 lM Khalid et al.
with Plasmodium falciparum (1986)
Salannin Inhibit expression levels of nitric oxide and 30–50 lM Akihisa et al.
7-deacetylgedunin COX proteins (2017)
Anti-parasitic Gedunin In vitro parasitized human O+ erythrocyte 1.25 lg/mLa Bickii et al.
with Plasmodium falciparum (2000)
In vivo Plamodium berghei-infected 50 mg kg−1 day−1b to Omar et al.
experimental model achieve 44.6% parasitaemia (2003b)
clearance
50 mg kg−1 day−1b
synergize with
25 mg kg−1 day−1 dillapiol
to achieve 70% parasitaemia
clearance
experimental model achieve 44% parasitaemia (2003a)
clearance
synergize with
clearance
In vitro parasitized human O+ erythrocyte Babesia bovis: 21.72 lMa Azirwan
with Babesia bovis, Babesia bigemina, Babesia bigemina: et al. (2019)
Babesia caballi and Theleria equi 15.25 lMa
Babesia caballi: 22.1 lMa
Theleria equi: 33.21 lMa
(continued)
Y. S. Ong et al.
16

a
In vitro antimalarial microdilution assay D6: 306 ng/mL Omar et al.
against Plasmodium falciparum W2: 315 ng/mLa (2003a)
chloroquine-sensitive clone W2 and
chloroquine-resistance clone D6
In vitro human erythrocyte infected with D10: 1.66 ± 0.37a Chianese
Plasmodium falciparum W2: 1.31 ± 0.42a et al. (2010)
chloroquine-sensitive clone D10 and
chloroquine-resistance clone W2
In vitro MTT assay for female Brugia 0.24 lMa Misra et al.
malayi adult worm (2011)
In vitro MTT assay for female Brugia 2.03 lMa MacKinnon
malayi microfilaria et al. (1997),
In vivo experimental model −80.0 ± 10.0%b changes in Misra et al.
intra-peritonealy inoculated with Brugia worm recovery (2011)

malayi adult worm
In vitro antimalarial microdilution assay D6: 39 ng/mLa
against Plasmodium falciparum W2: 20 ng/mLa
chloroquine-sensitive clone W2 and
chloroquine-resistance clone D6
In vitro antimalarial microdilution assay D6: >10,000 ng/mLa MacKinnon
against Plasmodium falciparum W2: 840 ng/mLa et al. (1997),
chloroquine-sensitive clone W2 and Chianese
chloroquine-resistance clone D6 et al. (2010)
1,2-Dihydrogegunin In vitro antimalarial microdilution assay D6: 2580 ng/mLa
1,2-Epoxygedunin chloroquine-sensitive clone W2 and D6: 4210 ng/mLa
chloroquine-resistance clone D6 W2: 2440 ng/mLa
In vitro human erythrocyte infected with
1,2-Dihydro-3b-gedunol D6: > 10,000 ng/mLa
571
Plasmodium falciparum
W2: > 10,000 ng/mLa
(continued)
572

chloroquine-sensitive clone D10 and a
7-Ketogedunin D6: 2500 ng/mL
chloroquine-resistance clone W2 W2: 900 ng/mLa
Tetrahydrogedunin D6: 133 ng/mLa
W2: 39 ng/mLa
21-Acetylgedunin D6: 832 ng/mLa
W2: 156 ng/mLa
23-Acetylgedunin D6: 10,000 ng/mLa
W2: 2130 ng/mLa
Hexahydrogedunin D6: 2610 ng/mLa
W2: 1280 ng/mLa
7-Deacetylgedunin D10: 5.14 ± 1.23a
W2: 3.29 ± 0.59a
experimental model achieve 67.5% parasitaemia (2003b)
clearance
synergize with
to achieve 80.7%
parasitaemia clearance
7-Methoxygedunin In vivo Plamodium berghei-infected 50 mg kg−1 day−1b to Omar et al.
experimental model achieve 67% parasitaemia (2003a)
In vitro antimalarial microdilution assay clearance
against Plasmodium falciparum 50 mg kg−1 day−1b
chloroquine-sensitive clone W2 and synergize with
chloroquine-resistance clone D6 25 mg kg−1 day−1 dillapiol
(continued)
Y. S. Ong et al.
16

clearance
D6: 783 ng/mLa Omar et al.
W2: 749 ng/mLa (2003a)
In vitro antimalarial microdilution assay D6: 556 ng/mLa
7-Benzyloxygedunin chloroquine-sensitive clone W2 and D6: 1828 ng/mLa
chloroquine-resistance clone D6 W2: 1998 ng/mLa
In vitro MTT assay for female Brugia
7-Hydroxygedunin 0.21 lg/mLa
malayi adult worm
Photogedunin In vitro MTT assay for female Brugia 2.23 lg/mLa Misra et al.
malayi microfilaria (2011),
In vivo experimental model −70.0 ± 10.0%b changes in Okhale et al.
(2012)
intra-peritoneally inoculated with Brugia worm recovery

malayi adult worm
Minimum inhibitory concentration Bs: NA
(MIC) against: Bacillus subtilis, Sa: NA
Staphylococcus aureus, Klebsiella Kp: NA
pneumonia, Escherichia coli Ec: NA
Antimicrobial Gedunin Minimum inhibitory concentration Bs: NA Okhale et al.
(MIC) against: Bacillus subtilis, Sa: 2000 lg/mLb (2013),
Staphylococcus aureus, Klebsiella Kp: 1000 lg/mLb Cespedes
pneumonia, Escherichia coli Ec: 2000 lg/mLb et al. (2016)
7-deacetoxy-7a-hydroxygedunin Minimum inhibitory concentration Bs: 2000 lg/mLb
potassium salt (MIC) against: Bacillus subtilis, Sa: NAb
Staphylococcus aureus, Klebsiella Kp: 1000 lg/mLb
pneumonia, Escherichia coli Ec: 2000 lg/mLb
7-deacetoxy-7a-hydroxygedunin In vitro bioassay with fall armyworm 39.0 ppmb with 48.3% larvae
573
larvae survival
(continued)
574

Insect growth Gedunin In vitro bioassay with the gram pod borer, H. armigera: 50.8 ppma Koul et al.
inhibitory Helicoverpa armigera (Hubner) and the S. litura: 40.4 ppma (2003)
Asian armyworm larvae, Spodoptera litura
(Fabricius)
In vitro bioassay with Anopheles 0.1 ppmb Nathan et al.
stephensi’s larvae, pupae and adult Larval mortality: 55–67% (2005)
Pupal mortality:
61.6 ± 5.9%
Adult mortality:
61.0 ± 5.9%
In vitro bioassay with fall armyworm 8.0 ppmb with 17% larvae Céspedes
larvae survival et al. (2000)
Photogedunin acetate In vitro bioassay with fall armyworm 10.0 ppmb with 50% larvae Céspedes
larvae survival et al. (2000)
Photogedunin epimeric In vitro bioassay with the gram pod borer, H. armigera: 24.2 ppma Koul et al.
Helicoverpa armigera (Hubner) and the S. litura: 21.5 ppma (2003)
Asian armyworm larvae, Spodoptera litura
(Fabricius)
6b-hydroxygedunin In vitro bioassay with Anopheles 0.1 ppmb Nathan et al.
stephensi’s larvae, pupae and adult Larval mortality: 78–94% (2005)
Pupal mortality:
84.3 ± 7.6%
Adult mortality:
87.5 ± 8.5%
a
50% inhibitory dose; bDose with highest reported efficacy; cHalf maximal effective concentration; dEffective dose for 50% of sample population
Y. S. Ong et al.
Fig. 16.1 Graphical summary of the anti-cancer mechanisms of gedunin, which includes the
degradation of proteasome via binding to p23, activation of autophagy via modulation of PI3K/Akt
pathway, arrest cell cycle by upregulation of CHK1 and PLK1, induction of apoptosis via intrinsic
pathway and inhibition of metastasis via downregulation of FAK, JNK, MMP-2 and MMP-9
extrinsic pathways by modulation of Bax, caspase 3 and PARP in pancreatic cancer

cells PANC-1 (Subramani et al. 2017).
Gedunin showed anti–metastatic effect through inhibition of sonic hedgehog
signaling. In the study performed by Subramani and colleagues, the migratory
characteristics of HPAC, MIAPaCa-2 and PANC-1 were significantly inhibited
(45–86%) by 15 µM of gedunin in a scratch assay (Subramani et al. 2017).
Additionally, 5 µM gedunin efficiently inhibited rhShh–induced invasion of pan-
creatic cancer cells by downregulation of proteins involved in Hedgehog/Gli sig-
naling pathway such as PTCH1, PTCH2, Gli1, SUFU and Shh proteins. Another
study by Li et al. (2018) demonstrated that gedunin inhibited cell migration and
invasion at concentration of 15 µM in glioma primary brain tumor cell U-251 MG
by reducing the expression of FAK, MMP-2, MMP-9 and uPA after 48 h of
incubation. Even though gedunin has been well-studied in vitro, its mechanisms of
action have not been studied extensively under in vivo conditions, leaving a huge
gap towards the realization of its clinical translation as an anti-cancer agent.
Apart from gedunin, some studies have been carried out to investigate the
anti-cancer activities of its derivatives. The isoform, 7-deacetyl-7-benzoylgedunin,
exhibited lower anti-cancer efficacy with higher IC50 (more than 20 µM) as com-
pared to gedunin across the variety of cancerous cell lines. In 2008, Brandt’s
research team has synthesized a series of gedunin derivatives and found out that
none of those were more effective than gedunin against breast cancer cells (MCF-7
and SkBr3) (Brandt et al. 2008). In fact, there is no literature available to explain
why its derivatives cannot outperform gedunin.
A recent study reported that gedunin derivatives,

3-a-acetoxydihydrodeoxygedunin (3-a-DOG) were partial agonist towards adhe-
sion G protein coupled receptors (aGPCR) GPR56/ADGRG1 (Stoveken et al.
2018). aGPCRs is broadly distributed in various tumor cells and has become
prominent as a potential therapeutic target in various neurological diseases and
cancers (Paavola and Hall 2012). The activating mechanism of most aGPCRs have
only been recently characterized and limited modulatory compound are being
defined. Hence, 3-a-DOG and possibly other gedunin derivatives as partial agonist
to aGPCRs could indicate an invaluable area for further research.
16.2.1.1 Combinatorial Treatment Using Gedunin Enhances

Anti-cancer Properties
The complex pathway of tumorigenesis has significant impact on the efficacy of

anti-cancer drugs. Many studies have demonstrated that combinatorial treatments
using multiple agent have proven to improve the clinical therapeutic efficacy of
anti-cancer drugs as compared to single agent regimen. Specifically, gedunin in
neem has been shown to synergize with other anti-cancer drugs apart from its
anti-cancer properties as a single agent. (Sharma et al. 2014; Gupta et al. 2017;
Bodduluru et al. 2014; Kamath et al. 2009).
Combinatory treatment with gedunin and epalrestat has shown to attenuate the
Aldose Reductase (AR) enzyme in the SCC131 oral cancer cell line (Tanagala et al.
2018). Inhibition of diabetic associated enzymes Aldose Reductase (AR) are cor-
related to the inhibition of SCC131 cell proliferation, apoptosis evasion and
angiogenesis through enervating downstream PI3K/Akt/mTOR/ERK/NF-jB sig-
nalling axis. It is reported that the combinatory treatment resulted in diversion of
cell death in favor of apoptosis in the treated cell lines. Further, pro-invasive and
proangiogenic proteins were downregulated in treated groups, hence inhibiting
cancer cell migration. In all events, combined treatment of gedunin and epalrestat
was reported to be more effective than single agents. In a similar study, Kishore and
the team (2016) suggested that the abrogation of AR enzyme, PI3K/Akt and NF-jB
pathways by gedunin has downregulated the expression of miR-21, hypoxia
inducible factor-1 alpha (HIF-1a) and the pro-angiogenic factors vascular
endothelial growth factor in a hamster model of oral carcinogenesis, which inhib-
ited angiogenesis (Kishore et al. 2016).
Interestingly, gedunin acted synergistically with the chemotherapy agents cis-
platin (C30, CP75) and paclitaxel (ID8TaxR) at doses as low as 2.5 lM, which
have no effect on the chemoresistant cancer cell lines when treated alone (Johnson
et al. 2014). In a similar study, the anti-cancer effect of neem-derived gedunin alone
and in combination with cisplatin was also tested against SKOV3, OVCAR4, and
OVCAR8 ovarian cancer cell lines. Gedunin reduced cell proliferation by 20%
when treated alone. When treat synergistically with cisplatin, further 47% decrease
in cell proliferation was reported (Kamath et al. 2009).
In another study, Sharma et al. (2014) proved the synergistic effect sub-lethal
dose of ethanolic neem leaf extract and cisplatin in reducing the viability of breast
(MCF-7) and cervical (HeLa) cancer cells compared to individual compound alone.
It was reported that 1 µM of cisplatin used in combination with 50–100 µg/mL
neem leaf extract resulted in a significant (71–82%) reduction of MCF-7 cells as
compared to the compounds alone (85–93.1%). The calculated Combinational
indices (CI) in the studies were reported to be <1, indicating a synergistic interaction
between the two drugs at their respective treatment dosage (Sharma et al. 2014).
16.2.2 Anti-neurological Disorders and Cryoprotective

Effects of Gedunin
Neurodegeneration is a progressive deterioration of neuronal structures and func-

tions resulting to cognitive disability and dementia primarily affecting aging pop-
ulation (Ramanan and Saykin 2013). Alzheimer’s disease, Parkinson's disease,
Amyotrophic lateral sclerosis and Hungtington disease are the few commonly
known neurodegenerative disorders. These diseases remain uncured despite
extensive basic research and drug discovery to ameliorate the conditions. Some
research showed that gedunin and its derivatives were able to protect the neurons
against b-amyloid (Ab) and other stressors.
It is widely known that the accumulation of Ab oligomers or fibrils contributes
to neurodegenerative pathology in Alzheimer’s disease (Murphy and LeVine 2010).
Gedunin significantly suppressed the cell death in neuronal cells subjected to Ab
cytotoxicity (Lu et al. 2009). Gedunin derivatives, deacetylgedunin,
deacetoxy-7-oxogedunin and deoxygedunin are the HSF1/Hsp70 activators and
thus protecting neuronal cells from cell death induced by MG-132 cytotoxicity
(Zhang et al. 2009). Recent study by Tom and his colleagues demonstrated that
gedunin inhibited Ab1-42-induced NF-jB activation, the expression of nitric oxide
and IL-1b level (Tom et al. 2019).
Brain-derived neurotrophic factors (BDNF) are a family of neurotrophins that
plays an essential role in the maintenance of central nervous system (Thoenen
1991). BDNF binds to transmembrane TrkB receptor and activates downstream
signaling cascades that are responsible for a variety of neuronal protections against
ischemic injury, glutamate toxicity, and programmed cell death (Leeds et al. 2005;
Lindholm et al. 1993; Schabitz et al. 2000). Smirnova et al. (2011) reported that
gedunin was able to rescue neuronal cells from the cell death caused by glutamate
and its analog by acting as a direct activator of NF-E2-related factor 2 (Nrf2), a
transcriptional regulator of cellular detoxification and antioxidant defense.
Deoxygedunin has been reported as an agonist of BDNF by eliciting the strongest
stimulatory effect on TrkB signaling cascades and executing neuronal protection
against cell death induced by oxygen–glucose deprivation (OGD) and glutamate. In
a similar study, deoxygedunin appeared to have anti-depression and learning

enhancement effects (Jang et al. 2010).
Huntington disease is a neurological disease characterized by abnormal move-
ments, cognitive dysfunction, and psychiatric disease (Frank 2014). Till date, there
are no treatment to reverse the progression of this disease. A study by Yang and the
team showed that gedunin disaggregated mutant Huntingtin protein in Neuro-2a
cells, endogenous mutant Huntingtin progression and intranuclear inclusions in
Hungtinton disease patients derived cells (Yang et al. 2019).
16.2.3 Anti-inflammatory Effects of Gedunin
Inflammation is an adaptive immune response that underlies myriads of physio-

logical and pathological conditions (Medzhitov 2008). Dysregulated inflammation
plays a crucial role in the development of many cellular and systemic disorders such
as cardiovascular diseases, cancer, neurodegenerative disorders, metabolic syn-
dromes and autoimmune disease. For this reason, researchers have begun to
understand the underlying mechanism of inflammation in order to control or cure
the aforementioned diseases.
Ferraris et al. (2012) showed that pretreatment of gedunin reduced the level of
chemotactic mediators such as CCL2, CCL3, CCL5, CCL11, IL-5 and the lipid
mediator LTB4 thus impairing the recruitment of eosinophil and T lymphocyte to the
site of inflammation of experimental mice. Subsequent study by Ferraris et al. (2012)
revealed that treatment with gedunin and derivatives in zymosan-induced inflam-
mation experimental model has significantly improved knee edema progression,
reduced neutrophil accumulation and production of inflammation mediators such as
CXCL8/IL-8, IL-1b, IL-6, TNF-a, LBT4 and PGE2 (Conte et al. 2015).
Penido et al. (2005) studied on the anti-inflammatory and analgesic effects of
bioactive compounds from the seeds of Carapa guianensis Aublet, which consist of
gedunin and 6-acetoxygedunin, 7-deacetocy-7-oxogedunin. Orally given tetranor-
triterpenoids (TNTP) appeared to be able to reduce ovalbumin- and zymosan-induced
edema inflammation and protein extravasation to the site of inflammation in mice. It is
noteworthy that such efficacy of TNTP at 25–100 mg/kg was comparable to that of
commercially available anti-inflammatory drugs such as dexamethasone and
promethazine. Besides, the influx of leukocyte and the increase in inflammation
mediators such as TNF-a, IL-1b, CXCL8/IL-8 and prostaglandin E2 induced by
various noxious stimuli were markedly observed after the treatment with TNTP
(Penido et al. 2006, 2005). Gedunin derivatives, 7-deacetylgedunin was reported to
reduce ear edema inflammation promoted by 12-O-tetradecanoylphorbol-13-acetate
(TPA) in experimental mice (Akihisa et al. 2009). A continuous investigation by
Akihisa et al. (2017) showed that Salannin and 7-deacetylgedunin reduced the
expression levels of the inducible nitric oxide synthase and cyclooxygenase proteins
in a dose dependent manner (Akihisa et al. 2017). Another study showed that gedunin
inhibited NLRP3 expression and inpaired production of TNF-a, IL-6 and nitric oxide
in-vivo (Borges et al. 2017).
16.2.4 Anti-parasitic Effects of Gedunin and Derivatives
Malaria is a vector-borne infectious disease caused by Plasmodium spp. parasitic

protozoan that belongs to the Apicomplexa phylum. Malaria is one of the major
public health problems that negatively impact on social and economy. In 2010, 1.2
million people worldwide were dead due to malaria (Murray et al. 2012). The
situation continues to deteriorate due to the fact that no preventive vaccination
against Plasmodium is available. The control of the disease thus relies heavily on
the treatment with antimalarial medications such as quinine, chloroquine,
sulfadoxine-pyrimethamine and amodiaquine over the decades (Sinha et al. 2014).
Due to massive spreads of antimalarial resistance, alternative therapeutic strategies
are urgently needed.
Gedunin was reported to possess antimalarial activities against Plasmodium fal-
ciparum, which accounted for most of the malaria cases worldwide (Khalid et al.
1986). The IC50 of gedunin was about 1 lM upon 48 h of treatment, which was
comparable to the established antimalarial compound quinine. MacKinnon et al.
(1997) tested the antimalarial activities of gedunin and 9 derivatives isolated from
neem tree against P. falciparum. However, only gedunin exhibited the highest anti-
malarial activity with the IC50 of 39 ng/mL against chloroquine-sensitive
P. falciparum clone, which was even higher than chloroquine and quinine
(MacKinnon et al. 1997). On top of that, synergistic study between gedunin and
chloroquine showed that gedunin extracted from the bark and the seed of Khaya
grandifoliola was able to exert synergistic effect when treated together with chloro-
quine (Bickii et al. 2000). Moreover, gedunin and derivative 7-methoxygedunin were
shown to have synergistic effect when acting together with dillapiol, which is a
commonly known antimalarial compound extracted from dill weed, in the mice
infected with Plasmodium berghei (Omar et al. 2003b). When acting alone,
7-methoxygedunin was able to inhibit the parasite level in infected mice by 67% at
50 mg/kg, while 80% of inhibition was achieved when 25 mg/kg dillapiole was
administered together with the same dose of 7-methoxygedunin (OMAR et al. 2003a).
On the other hand, gedunin and photogedunin have been reported for their
antifilarial activity against parasitic filarial nematodes Brugia malayi. In vitro study
showed that gedunin and photogedunin were active against adult worm (IC50 of
0.24 and 0.21 lg/mL) comparable to the antiparasitic drug ivermectin (IC50 of
1.61 lg/mL). In addition, in vivo study showed that gedunin and photogedunin
significantly suppressed the survival of the parasite in jird transplanted model
(Misra et al. 2011). Gedunin inhibited the growth of Babesia bovis, Babesia
bigemina, Babesia caballi and Theleria equi which are the causative agents of
bovine and equine piroplasmosis (Azirwan et al. 2019). Taken together, it is
plausible to mention that gedunin possesses strong antiparasitic activities that is
noteworthy for further investigations. Research by Yerbanga et al. (2014) proved

that the ethanol extract of neem leaves with gedunin as a major component
exhibited in vitro anti-malarial effect through transmission blocking activity in
P. falciparum. Yet, no further study has been done to investigate the underlying
mechanisms of action (Yerbanga et al. 2014).
16.2.5 Antimicrobial Effects of Gedunin and Derivatives
The occurrence of bacterial resistance has increased over the years in several
countries where by common antibiotics such as aminoglycosides, cephalosporins
and penicillins are no longer effective against pathogens (Chaves et al. 2015; Davies
and Davies 2010). Such resistance causes negative impacts on public health with
increasing mortality and morbidity and increase in economic burden with rising
cost for antimicrobial treatment. Therefore, myriads of natural products derived
from animal, plant and microorganism have been deemed as an alternative to
antibiotics.
Okhale et al. (2012) revealed that gedunin was inactive against Bacillus subtilis,
Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae whereas the
semisynthetic derivative 7-deacetoxy-7a-hydroxygedunin potassium salt possessed
greater antimicrobial activity against all tested bacterial species except B. subtilis. The
subsequent study by the group showed that another derivative,
7-deacetoxy-7a-hydroxygedunin exhibited antimicrobial activity against B. subtilis,
K. pneumonia and Escherichia coli but not S. aureus (Okhale et al. 2012, 2013).
Although various studies have been carried out to investigate the bioactivities of
gedunin, the antimicrobial property of gedunin is overlooked by the research
community.
16.2.6 Insect Growth Inhibition Effects of Gedunin

and Derivatives
Due to insect resistance and ecological impacts caused by commercialized insec-

ticides, more efforts have been devoted to investigate the potential of natural
products on the insect’s growth inhibitory effect. One of the functions of the
plant-based phytochemicals as the defense system is to dissuades herbivores and
insecticides from consuming them. Over the years, studies have been carried out to
investigate the inhibitory effect of gedunin and its derivatives against insect growth
(Cespedes et al. 2016). Gedunin and photogedunin from Cedrela dugessi and
Cedrela salvadorensis were tested against the growth larvae of fall armyworm. The
results showed that these compounds were able to inhibit the growth of larvae in a
concentration dependent manner (Céspedes et al. 2000). Another study has reported
the larval growth inhibitory effects of gedunin derivative, 6b-hydroxygedunin,

against the gram pod borer, Helicoverpa armigera (Hubner) and the Asian army-
worm, Spodoptera litura (Fabricius). The 6b-hydroxygedunin inhibited the growth
of the larvae by 50% at a concentration 21.5 lg/mL. Interestingly, the efficacy of
6b-hydroxygedunin was two-fold greater than gedunin. In addition, gedunin and
deacetylgedunin were tested against Anopheles stephensi which is the major vector
of malaria. Nathan et al. (2005) reported that deacetylgedunin possessed high
inhibitory activity against the growth of larvae, pupae and adult as well as the
production of egg of A. stephensi. However, the mechanisms of the insecticidal,
repellent and growth inhibitory of these compounds are not well understood.
16.3 Commercial Potential of Gedunin
Considering the multiple therapeutic effects of gedunin that have been reported in
the literature, it is not surprised that patents have been registered for gedunin and its
derivatives. Thus far, Vinson-Hieronymus et al. (2011) has patented gedunin and its
derivatives (publication number: US 2011/0,263,693 A1), together with celastrol to
inhibit Hsp90. As mentioned earlier, Hsp90 is a multitasking molecule that governs
the maturation of an array of diverse cellular functions. It is also a specific client
protein that acts as signal transducers implicated in the molecular mechanisms of
normal cellular biology, disease and evolutionary processes (Kaplan and Li 2012).
Thus, by inhibiting Hsp90, pathological processes like cancer and inflammation can
thus be suppressed (Kaplan and Li 2012; Chatterjee et al. 2007). On the other hand,
gedunin has been patented as a substrate of polymeric wound care material (pub-
lication number: US 2013/0,209,534 A1) for its antimicrobial properties and the
ability to aid in wound healing. The neuroprotective effects of gedunin and a
number of limonoids have also granted the inventor Steiner and colleagues a patent
(publication number: US 2010/0,056,617 A1) disclosing the method of preparation
and the utilization of these compounds.
16.4 Conclusion and Gaps of Knowledge
The tremendous robust evidences to date have shown that gedunin and its
derivative possess multiple therapeutic effects including anti-cancer,
anti-inflammatory, anti-parasitic, antimicrobial and insect growth inhibition.
However, most of the studies are limited to in vitro phase with or without the
comparison to commercialized drugs. We thus look forward to have extensive
animal model-based evidences in order to understand how gedunin and its
derivatives work in in vivo systems. This will then fill the gaps of the pharmaco-
logical understandings of gedunin and its derivative such as bioavailability, phar-
macokinetics and pharmacodynamics. Nonetheless, toxicology screening is yet to
be carried out to ensure the safety of gedunin and its derivatives, especially for
semisynthetic derivatives of gedunin, which involve inorganic components and
chemicals in the production. To entirely elucidate the molecular mechanism action
of gedunin, more in-depth and holistic studies have yet to be carried out.
Acknowledgements This work is financially supported by Monash Global Asia in the 21st
Century (GA21) research grant (GA-HW-19-L01, GA-HW-19-L06 and GA-HW-19-S02);
University grants from the University of Malaya (PG136-2016A and PG135-2016A), External
Industry Grants from Biotek Abadi Sdn Bhd (vote no. GBA-81811A) and Fundamental Research
Grant Scheme (FRGS/1/2019/WAB09/MUSM/02/1 and FRGS/1/2019/SKK08/MUSM/02/7).
Author Contributions The writing was performed by K-YK, Y-SO, LT-HT, P-NY, K-BT. The
manuscript was further critically reviewed and improved by W-SY, S-YT, K-YK, Y-SO, LT-HT,
L-EL, L-HL and B-HG. While B-HG, LT-HT, L-HL provided vital guidance and insight to the
work. The project was conceptualized by B-HG.
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bbrc.2009.10.079
Chapter 17
Biological Activities of Marine Products
and Nutritional Importance
Dilipkumar Pal and Khushboo Raj
Abstract Bioactive compounds, also known as phyto-nutrients are those com-

pounds which enhance or we can say that promote good health. They are found in a
limited amount in plants, animals, marine, and other natural food sources which
help in the prevention of many diseases e.g. cancers, cardiac disorders, diabetes,
etc. It is well known to us that nearly half of the worldwide biodiversity is con-
stituted by different types of marine species and as a result, oceans, sea are enriched
with valuable natural bioactive compounds such as proteins, peptides, amino acids,
fatty acids, sterols, oligosaccharides, vitamins, and minerals, etc. These agents also
help to enhance the nutritional as well as the therapeutic value of the food products.
Every year nearly thousands of new compounds are isolated from a marine
organism which further help in the discovery of new leads for the development of
new drugs to treat or diagnose human diseases like cancer, viral diseases, inflam-
mation, etc. For example, thyrsiferol, which is isolated from marine red algae
(genus: Laurencia) on the experimental studies shows potent anti-viral and
anti-tumour activities. Another example is marine sponge (genus: Insignia) which is
a good source of terpenoids consisting of tetronic acids which act as anti-
inflammatory agent, analgesic, and antibiotics. There are numerous examples of
such marine products that are enriched with nutritional values and show many
potent biological activities. In this article, we are going to discuss different marine
products one by one with their biological and nutritional importances and also their
role in the development of new drugs in the treatment of various human diseases.
Keywords Bioactive compounds Cancers Cardiac disorders Diabetes

Marine organism Thyrsiferol Anti-viral
D. Pal (&) K. Raj

(A Central University), Bilaspur, Chhattisgarh 49551, India
https://doi.org/10.1007/978-3-030-54027-2_17
588 D. Pal and K. Raj
17.1 Introduction
Ocean or Marine life denotes the life of various organism which inhabits in the
saltwater of the sea or near the coastal subculture and due to its remarkable bio-
diversity, it has become a huge stock of natural resources for many biologically
active compounds (Bellisle et al. 1998). More than 200,000 marine species are
already reported and 2 million are yet to be documented (Fiszman and Salvador
2003). Marine species vary in size including small phytoplankton (0.02 mm) to
large cetaceans e.g. whales, dolphins, blue whale, etc. It also includes various
microorganisms like bacteria, algae, fungi, viruses, etc. (Plaza et al. 2008). Today
marine organism is well known as a potent source of the bioactive compound
because of its complex habitat and exposure to extreme condition. Thus, these
potent bioactive compounds which act as a good source of nutrition also exhibit
potent biological activities. Peptides, polysaccharides which are isolated from
various marine sources produce anticancer, anticoagulant activities. A marine
sponge, (genus: Insignia) for example is a good source of terpenoids and these
terpenoids consists of tetronic acids which act as anti-inflammatory, analgesic and
antibiotic agents (Diplock et al. 1999).
17.2 Marine Organisms: Source of Nutrition
17.2.1 Proteins
Proteins are considered as one of the most essential nutrients for our body system. It
consists of one or more than one long chain of amino acid residues (the building
block of protein) which is coded by the genetic (DNA) code along with a no. of
other amino acids (Kadam and Prabhasankar 2005). There are twenty different
types of amino acids which are classified into two categories (i) EAA (Essential
amino acid) and (ii) NEAA (Non-Essential amino acid). EAAs are nine in numbers
and they cannot be produced by the body itself, thus they are taken from foods or
outer sources. On another hand, NEAAs are eleven in numbers and they can be
produced by the body itself (Amado et al. 2013). In Table 17.1 name of twenty
amino acids with their 3 letters abbreviation is mentioned (Kim et al. 2010).
The primary function of protein includes catalytic reactions, DNA replication,
production of antibodies, etc. (Sheraji et al. 2013). Seafood is well known for its
outstanding source of protein which includes all essential amino acids in an ade-
quate amount for humans (Freitas et al. 2012). On various chemical analysis, it was
found that marine food consists of 9–24% of protein which is well digestible to
human beings. For example, Halibut fish consists of 19% proteins with all EAA in
an adequate amount (Ar-6%, His-1.66%, Cys-6.16%, Trp-1.64%, and Cys 1.45%)
(Damodaran et al. 1997; Lum et al. 2013).
17 Biological Activities of Marine Products … 589
Table 17.1 Name of essential amino acid and non-essential amino acid
S. No. Essential amino acids Non-essential
(eaas) amino acids
(neaas)
1 Histidine (His) Alanine (Ala)
2 Isoleucine (Ile) Arginine (Arg)
3 Leucine (Leu) Asparagines
(Asn)
4 Lysine (Lys) Aspartic acid
(Asp)
5 Methionine (Met) Cysteine (Cys)
6 Phenylalanine (Phe) Glutamic acid
(Glu)
7 Threonine (Thr) Glutamine (Gln)
8 Tryptophan (Trp) Glycine (Gly)
9 Valine (Val) Proline (Pro)
10 Serine (Ser)
11 Tyrosine (Tyr)
Also, the other protein sources are marine mammals, algae, bacteria, etc. Seal
liver is recently documented as an enriching source of protein as same as the
amount of it present in beef liver (Asha et al. 2014). Different species of Whales
reported as an excellent source of protein ranges from (17–24%) (Lordan et al.
2011). Another example of protein source is Laminaria (alga) and their different
species having a total content of 14% of crude protein. The EAA which is found in
Laminaria includes aspartic acid, glutanic acid, glycine, alonine, valive, leucine,
isoleucine, and also some serine, threonine, proline, phenylalanine, lysine and a
small amount of arginine (Rasmussen and Morrissey 2007; Cho et al. 2008).
Spirulina, which is a biomass of cyanobacteria is reported as rich in high protein
content which ranges from 60 to 70% with an ample amount of EAA like leucin
tryptophan, methionine, phenylalanine, lysine, threonine, isoleucine, and valine
(Kim et al. 2010). It also helps in repairing damages, lowering total cholesterol
(CDC), raising good cholesterol (HDC) and acts as an anti-inflammatory agent
(Bocanegra et al. 2009). In Table 17.2 protein percentage in a marine organism is
mentioned.
17.2.2 Lipids and Fatty Acids
Omega-3 or polyunsaturated fatty acids (PUFA) play a vital role to keep the human
body healthy and free from various diseases like CVS, retinal diseases, diabetics,
etc. On various chemical and biological analyses, it is found that a regular con-
sumption of seafood like fishes, crabs, etc. can help to lower the risk of cardiac
590
Table 17.2 Different types of marine species with their protein percentage
Species Protein content (%) Ar (%) His (%) Lys (%) Trp (%) Cys (%) References
Fishes amiuriscatus (catfish) *28 *0.97 Zheng et al. (2013)
Oncorhynchus tschawytscha (salmon) *16 *5.02 *1.41 *6.27 *1.20 *1.27 Jun et al. (2004)
O.nerka (salmon) *11 – – – – *1.25 Rocha et al. (2015)
O.keta (salmon) *5 *5.55 *1.30 *5.69 *1.33 –
O.gorbuscha (salmon) *6 – – – *1.09 *1.15
O.kisutch (salmon) *8 *5.68 *1.87 *6.57 *1.94 *1.39
Germaalalunga (tuna) – – – *1.18 – Kanazawa (2001)
Melanogrammusaeglefinus 18 5.70 1.17 6.41 0.85 1.16
(Haddock)
Algae Spirulina platensis 60 7.3 2.2 4.8 0.3 0.9 Karuppasamy et al. (2013)
Porphyratenra 20 16.4 1.4 4.5 1.3 – Ge et al. (2006)
Ulva 13.6 3.7 0.7 0.0 0.6 –
Sargassum 9–20 4.0 1.9 4.5 1.8 –
Shellfish crabs 17 7.6 1.5 6.4 1.1 Mayer et al. (2010)
Oysters 7 5.7 1.8 5.2 – – Paul et al. (2011)
Clams 9 5.3 1.5 5.4 1.2 –
Shrimp 25 6.6 1.8 8.3 1.2
D. Pal and K. Raj
related disorders (CHD,CAD) and other diseases also because seafood has a very
low content of saturated fatty acid and higher content of omega-3 fatty acids. The
main reason of a high amount of PUFA in fishes is because of their daily con-
sumption of algae where the synthesis of long-chain PUFA takes place (Aneiros
and Garateix 2004).
Eicosapentenoic (EPA) acid and docosahexenoic (DHA) acid are the examples
of Omega-3 fatty acid that is mainly found in the brain, cerebral cortex, skin, retinal
part of the human body system. They can be synthesized in the human body from
their precursor alpha-linolenic acid (ALA), but the rate of conversion is very low
because of the insufficient amount of the enzyme Δ6-desaturase (Dharmaraj et al.
2009). That’s why PUFA is taken in diet from marine food (fish oil, algae oil, etc.)
or other food resources. In Fig. 17.1 the synthesis process of Eicosapentenoic acid
and docosahexenoic acid are illustrated. There are so many examples of marine
sources available which enrich PUFA, EPA, DHA, etc. For example, salmon,
swordfish and halibut are excellent sources of PUFA (EPA, DPA, DHA) with
respective average levels of 1434, 3625, 3358, 2654 mg-PUFAs/100 g fw (Ma
et al. 2008).
Also, C. Vulgaris, the microalgae is enriched with oleic acid, palmitic acid and
linolenic acid. Haematococcus, a green microalgae includes short-chain fatty acids
and exhibits antimicrobial activity (Sijtsma et al. 2004). Figure 17.2 exemplify
various marine sources with the fatty acid profiles (LøvstadHoldt and Kraan 2011).
17.2.3 Sterols
These are another class of lipids that are also found in marine food. On experimental
studies, it has been noticed that the sterols which are extracted from macro and
microalgae, fish and other marine invertebrates (like corals, molluscs, sponges) are
capable of lowering the level of LDC (low- density lipoprotein) and also show
anti-inflammation bioactivity (Wall et al. 2010). Sargasseum ringgoldianum (brown
algae) has been reported to have various types of sterols in which saringosterol is
associated with anti-tubercular activity. Pelvetia siliquosa (marine algae) has also
been reported to have fucosterol and anti-oxidant activity (Pal 2013; Nimse and Pal
2015). Phytosterols (C28 and C29 sterols) are essential precursors of some vitamins.
For instance, ergosterol is a precursor of vitamin D2 and cortisone. Clionasterol
which is found in Spirulina is associated with increasing PAF in vascular endothelial
cells. Fucosterol, which is isolated from P. Siliquosa exhibits potent anti-diabetic
activity at a dose level of 100 and 300 mg/kg. It also decreases the glycogen
degradation of mouse liver by 23–29% (Wang et al. 2006). Various sterols with their
sources are mentioned In Table 17.3 (Özyurt et al. 2013).
C OH
alpha-linolienic acid
O
(ALA) 18:3
delta6- desaturase
(FADS2)
O2 + NADH+ H+
(2)H2O +NAD+
C OH
stearidonic acid 18:4
malonyl-CoA O
ER-mediated elongation
ELOVL2, ELVOL5
CoASH
Eicosatetraenoic acid 20:4

delta6- desaturase
(FADS1) O2 + NADH+ H+
(2)H2O +NAD+
OH
Eicosapentenoic acid ( EPA) O
omega-3 PUFA 20:5
ELOVL2,ELOVL5
Docospentaenoic acid (DPA)

22:5
ELOVL2,ELOVL5
Tetracosahexaemic acid 24:6 Tetracospentaenoic acid 24:5
TRANSLOCATION
Tetracosahexaenoic acid 24:6
beta- oxidation,
AcylCoA oxidase
Thiolases
PEROXISOMES Bif unctional protein HSD 1784
C O
OH
Docosahexenoic acid 22:6
Fig. 17.1 Synthesis process of eicosapentenoic acid and docosahexenoic acid

50
45
40
% Of total faƩy acids
35
30
25 Salmon salar L.
20 Laminaria sp.
15 Thysanoessa raschii
10
5 Proteomonas sulcata
0 Teleaulax acuta
Storeatula major
A
ID
ID
ID
ID
ID
EP
DH
AC
AC
AC
AC
AC
C
IC
IC
C
EI
NI
RI
LE
IT
OL
U
O
LM
O
LA
ID
LIN
PA
CH
A-
AR
PH
AL
VARIOUS FATTY ACID
Fig. 17.2 Marine sources with fatty acid profile
Table 17.3 Structures of sterols with their sources and uses

Sterols Structure Sources Use
Ergostérol Chlamydomonas In breast cancer
reinhardtii treatment
H
H H
OH
Clionasterol Spirulina PAF activity
H
H
OH
Fucosterol Turbinariaornata Anti-cancer

activity
H
H
H H
OH
(continued)

Sterols Structure Sources Use
Saringasterol OH Lessonia nigrescenes (anti-tubercular
activity)
OH
Chondrillasterol Brown algae Anti-bacterial

(scenedesmusobliquus) activity
H
H
OH
17.2.4 Carbohydrates
17.2.4.1 Polysaccharides
Polysaccharides (glycan) are a long chain of polymeric carbohydrates composed of

monosaccharide (simple sugar) linked by glycosidic bonds. They are found in high
content in marine seaweed and algae. Some of the examples of polysaccharides
include agar, alginate, carvageen, isolated from macroalgae whereas other
polysaccharides like chitin, chitosan, isolated from cuticles of various crustaceans,
crabs and shrimps (Spolaore et al. 2006). Red algae-like gelidium, arcicilaria,
hypnea, and gigartina are the main source of agar and they are used extensively in
food, pharmaceutical, cosmetic, paper and textiles industries for various purposes
like food gums, emulsifying agent, etc. (Prasad et al. 2006 and Calder 2009).
Various polysaccharides are not digestible for human GIT because of the absence of
suitable degradation enzymes and thus they are regarded as dietary fibre. The total
dietary fiber content of seaweeds (25–75% of the dry weight of marine algae) is
higher than the fiber content of most fruits and vegetables. Dietary fibres are cat-
egorized into two classes, one is soluble and another is insoluble. Marine algae are a
reservoir of different types of carbohydrates. For example, Phaeophyta (brown
algae) includes alginates, fucans, laminarans, etc. and all these are soluble fibres.
Other examples are xylans, floridean starch, sulphated galactans and mannans, ionic
polysaccharides which are obtained from Rhodophyta (red algae) and green algae
(Lloret 2010) respectively.
It is well proven now that human consumption of algal fibre is very beneficial
and can resolve many health-related problems with its many biological activities
like antitumor, anticoagulant, antiviral, and many others.
Fucidan which is a sulfated polysaccharide and made up of primarily L-fucose
can be found from the various sources of brown seaweeds like Saccharina
japonica, Fucus vesiculosus, Undaria pinnatifida, and Hizikia fusiformis and also
from marine invertebrates such as sea cucumber. Experimental analysis shows that,
fucoidans can be widely used in cosmetic products. When it is used topically it
forms a protective layer, enhances skin hydration and also helps in prevention and
treatment of skin photo aging. (Zheng et al. 2013 and Charissoux et al. 2013).
Carrageenan is also an example of sulfated polysaccharides which is found in
Rhodophyta (red algae). It is composed of D-galactose units. Carrageenan is used in
cosmetic industries as skin lotions, toothpaste binders, shaving foams, etc. It also
exhibits antioxidant, antiaging, and anti-cancer properties (Li et al. 2008).
Also, laminaran is an example of marine polysaccharides that produces anti-
coagulant activity when structural modification like sulfatin, reduction, or oxidation
occurs (Kanekiyo et al. 2007; Heo et al. 2002; Je et al. 2014).
17.2.4.2 Oligosaccharides
They are another class of carbohydrates that contains 3–10 sugar units. Marine
oligosaccharides are produced either in algae by natural process or by hydrolysis of
derived polysaccharides.
Oligosaccharides derived from seaweed have been proven as a potent antifungal,
antibacterial precursor. They also show defence action by enhancing pathogens
protection. In addition marine algae also includes oligosaccharides that are used as
prebiotic as egxylo/func-oligosaccharides and non-digestible oligomers (cannot
pass through GIT) so they can be used to enhance the growth of beneficial probiotic
bacteria (Rodríguez-Meizoso et al. 2010; Le et al. 2009). Bifidobacteria is an
example of probiotics which live in intestines and stomach and helps in digestion
and also provide protection from pathogens. K-Carrageenan isolated from
Kappaphycus alvarezii is very much beneficial for the prevention of colon car-
cinogenesis and also exerts cholesterol-lowering, anticoagulant, immunomodula-
tory, antiviral and antioxidant properties. On the other hand, alginate
oligosaccharides exerts antioxidant activities along with inhibitory action on
neuro-inflammation and enhances the microglial phagocytosis, thus promises a
potent nutraceutical agent against neurodegenerative diseases e.g. Alzheimer’s
disease, Parkinson’s diseases etc. Marine oligosaccharides have also been used in
food products, cosmetics, biomedicine etc. (Zaporozhets et al. 2014).
17.2.5 Antioxidants
Today food industry has been more focused towards the development of antioxi-
dants from the natural sources as it is a safer option in comparison to many syn-
thetic commercial antioxidants. Antioxidants are those agents which prevents the
oxidation of lipids due to which there is no spoilage of food. Lipid oxidation is
caused by reactive oxygen species (ROS) e.g. hydrogen peroxide, free radicals, etc.
and its leads to the deterioration of nutritional values of lipid content of food. Thus
to reduce lipid peroxidation. Antioxidants like propyl gallate, butylated hydroxy-
toluene, butylated hydroxytoluene, tert-butylhydroquinone, etc. are used
(Wijesekara et al. 2011; Pal and Nandi 2005; Soni et al. 2008) to prevent oxidation.
The main problem with synthetic antioxidants is that they are not safe: may include
serious side effects. That’s why researchers are now more focused on naturally
derived anti-oxidants with proper utilisations. Apart from deterioration of food
products, these ROS are also responsible for causing various diseases such as
cancer, neurodegenerative disease, inflammatory diseases, etc. (Rinaudo et al. 2006,
Pal 2013; Nimse and Pal 2015).
The reaction of ROS with biomolecules like proteins, membrane lipids and DNA
result in cellular or tissue level injuries. Equilibrium between endogenous
anti-oxidant systems and oxidant formation protect cellular biomolecules, however
a disturbance in this balance can lead to oxidative stress. Therefore, anti-oxidants
play a vital role in maintaining the cellular redox state and protecting the body
against damage caused by ROS (Mata et al. 2010, Pal 2013; Nimse and Pal 2015)
(Fig. 17.3).
HO
OH
OH
OH
OH
HO
OH
HO OH
OH
HO OH
P h lo ro tan n in s
Fig. 17.3 Structure of phlorotannins

17.2.5.1 Polyphenolic Compounds
Marine polyphenolic compounds have been extracted from micro and macroalgae
and they are characterised in ten different categories according to their structures.
These ten classes include- phenolic acid, hydroxycinnamic acids, simple phenols,
coumarines, xanthones, naphthoquinones, flavonoids, stibenes, anthraquinones and
lignins (Fig. 17.4) (Vo et al. 2011).
Phlorotannins that is one of the classes of polyphenol compounds produced by
brown algae families as a secondary metabolite is very effective in bacterial
infection. It also shows UV- protective and anti-proliferative effects (Fig. 17.3)
(Patel and Goyal 2011).
17.2.5.2 Photosynthetic Pigments
These are the pigments that capture solar energy and use it for photosynthesis. In
marine life generally micro and microalgae, plant, fungus have these types of
photosynthetic pigment. Carotenoids and chlorophyll are such examples of pho-
tosynthetic pigment that are present in macroalgae (Ishihara et al. 2010).
Carotenoids pigment exhibit anti-oxidant property and also act as pro-vitamin A,
which is further converted into vitamin A. Carotenoids also provide profusion
against cancer, cardio-vascular disease and AMD (age-related macular degenera-
tion) (Mussatto and Mancilha 2007).
Beta-carotene and astaxanthins, found in microalgae along with carotenoids also
act as a antioxidants, anti-cancer agents and helps in treatment of arthritis disease
(Bin et al. 2013).
17.2.6 Vitamins and Minerals
They are considered as necessary nutrients which performs many vital roles to keep
human body healthy. They provide strength to bones, boost the immune system,
heal wounds and also repair cellular damages. Seafoods are considered as a good
source of vitamins and minerals. Palmarialongat, a macroalgae is an excellent
source of vit. B12 and green seaweeds like Laminaria digitata, U. Pinnatifida is a
great source of Vit. C and E. Also U. Pinnatifida and sargassum sp. contain an
adequate amount of major minerals like Na, Ca and Mg as well as trace minerals
(Fe, Zn, Mn and Cu) (Kuroiwa et al. 2009). Vitamin D is very important for the
growth and development of the bones. It makes the bone stronger and prevents
various bone related diseases like rickets (in infants and children) and Osteoporosis
(in adults). Thus fatty fishes like salmon, pilchard etc. are good source of vit.
D (Ngo et al. 2010). In Table 17.4, examples of different microalgae with different
number of vitamins are mentioned. Figure 17.5 describes mineral content of dif-
ferent algae (Shahidi and Zhong 2010; Miyashita et al. 2011).
O OH
R1 O
OH R2
OH
R3
p h en o lic acid
R4
h y d ro x y cin n am ic acid s O
OH
x an th o n es
p h en o ls
O O O
co u m arin es
O f lav o n e
n ap h th o q u in o n es
O O
E -stilb en e
O
an th raq u in o n es
Fig. 17.4 Structure of various polyphenolic derivatives
17.3 Marine Sources for Pharmacological Effect
17.3.1 Anti-cancer
Cancer is a frightful human disease and has become a major burden to human
worldwide. According to a recent report in 2015, 90.5 million people have been
suffering from cancer and about 14.1 million new cases occurring a year with 8.8
million death. According to an estimate of 2021, 21 million new cases of cancer are
17
Table 17.4 Different microalgae with different amount of vitamins

Microalga
(mg/kg)
Vitamins Spirulina Chlorella Senedesmusquadricauda T. suecica L galbana D. tertiolecta C. stigmatophora
(mg/kg) platensis pyrenoidosa
A 840 480 554 263 850 76 500 82 500 49,380
Thiamin (B1) 44 10 11.5 32.3 14.0 29.0 14.6
Riboflavin (Bz) 37 36 27 19.1 30.0 31.2 19.6
Biological Activities of Marine Products …
Pyridoxin (B6) 3 23 – 2.8 1.8 2.2 1.9

Pantothenic acid – – – 37.7 9.1 13.2 21.4
(B5)
Biotin (H) (B8) 0.3 0.15 – 0.8 1.0 0.9 1.1
Ascorbic acid 80 396 191.0 I 19.0 163.2 100.2
(C)
Tocopherol (E) 120 421.8 58.2 116.3 669.0
599
1200
1000
800
mg/100g
600
400
200
0
Ca Cu Mg Mn K Na Fe Zn
Chlorella vulgaris (Microalga) Hypnea valenƟae ( Seaweed)

Ompok bimaculatus Labeo pangusia
Fig. 17.5 Mineral content of different algae
expected with 13 million deaths of cancer patients (Miyashita 2013). However, to

fight with this dreadful disease many chemotherapeutic drugs are currently avail-
able in the market but due to their serious side effects they become risky to use for
the cancer patients. Thus, today researches are more focused on natural products
and their derivatives to treat the cancer patients and 60% of drugs from natural
origin have already been documented for cancer treatment (Saha and Pal 2016;
Saha et al. 2017; Sannigrahi et al. 2012). As we know oceans cover more than 70%
of earth’s surface and an exultingly marine source has emerged as a new hope for
cancer treatment. The chemical adaptations generally take the form of so-called
“secondary metabolites” and involve such well-known chemical classes as ter-
penoids, alkaloids, polypeptides, peptides, shikimic acid derivatives, sugars, ster-
oids and multitude of mixed biogenesis metabolites (Prokschet et al. 2002). Marine
bioactive compounds are self-sufficient to treat cancer by imitating insensitivity to
anti-growth signals evasions of apoptosis, limitless replication, presenting metas-
tasis and tissue invasion.
There are many examples of marine compounds like Parmariapatmate (sea-
weed) is effective against cancer disease. It inhibits cancer cell proliferation. It has
been already discussed that seaweed is enriched with protein polysaccharides
PUFA, vitamins, and minerals. It is also used for different pharmaceutical purposes,
especially for cancer treatment (Anitha et al. 2010). Among the three types of
seaweeds, phaeophyta (brown seed) has the highest phytochemical content such as
terpenes, carotenoids and phenolic compounds. These phytochemicals exhibit a
potent anticancer activity.
Since past decades marine sponges have been considered as a huge source of
bioactive natural chemical substances. Poriferas is the oldest one with excellent
living fossils and approx. 8000 documented species and perhaps twice as many
un-described species. Sponges are capable of surviving in every type of marine
environment; either it is polar seas or temperate and tropical waters. They vary in
shape, size, and colours. From many years marine compounds derived from
sponges are under doing preclinical and clinical trials for anticancer activity and the
compounds are discodermolide, hemiasterlins A and B, modified halichondrin B,
KRN-70000, Alipkinidine (alkaloid), fascaphysins (alkaloid), isohomohalichondrin
B, Halichondrin B, Laulimalide/Fijianolide, 5-methoxyamphimedine (alkaloid) and
Variolin (alkaloid) respectively. In recent years, 43 new marine originated
anti-angiogenic agents have been investigated with unknown molecular mecha-
nisms (Halim et al. 2011; Migliore and Coppede 2009).
Ara-C (1-beta-D-Arabnofuranosylcytosine or Cytarabine) is the first documented
marine derived anti-cancer agent. It is a nucleoside derivative of spongothymidine
and spongouridine, isolated from Tectibethrya crypta. Thyrsiferol, which is isolated
from marine red algae (genus: Laurencia), on the experimental studies shows a
potent anti-viral and anti-tumour activity. Renieramycins is isolated from a marine
sponge Reniera sp. which persuades apoptosis by following p53—a dependent
pathway and enable to inhibit progression and metastasis of lung cancer.
Monanchocidin is a polycyclic guanidine alkaloid isolated from Monanchora pul-
chra (a marine sponge) that encourages cell death in human monocytic leukemia,
human cervical cancer and mouse epidermal cells (Perdicaris et al. 2013).
Smenospongine, a sesquiterpene aminoquinone, extracted from the sponge
Smenospongia sp. is associated with anti-proliferetive and antiangiogenic activities.
Spongistatin 1, a macrocyclic lactone polyether, isolated from the marine
sponge Spongia sp. is associated with the inhibition of mitosis process, inclusion of
microtubule assembly and induction of cytotoxicity in cancer cells. In addition,
scientists purify a lectin from the marine sponge Cinachyrellaapion (CaL) and find
that it exerts hemolytic, cytotoxic and anti-proliferative activities. A marine ses-
tertespene, isolated from the sponge Hyrtios sp. acts as an anticancer agent by
inhibiting chronic myclogenousrecekmia cells by regulating cell cycle, apoptosis,
mitogen activated protein kinase (MAPKs) pathway and nuclear factor kappa B
(NF-Ke) signalling cascade (Thakur and Müller 2004).
Marine algae include phytoplankton, seaweeds, sea anemones, etc. Seaweeds are
further categorized into four classes: (i) Chlorophyta (green algae) (ii) Phaeophyta
(brown algae) (iii) Rhodophyta (red algae) and (iv) Cyanobacteria (blue-green
algae). There are 6000 species of rhodophyta, 2000 species of phaeophyta and 1200
species of chlorophyta. Seaweeds are used in a variety of fields such as food
industry, fertilizer industry, pharmaceutical industry, cosmetic industry, etc.
Seaweeds have been proved as promising sources of bioactive compounds that
exert many biological activities including anti-cancer action. In many works of
literature, it has mentioned that daily intake of dietary carotenoids lowers the risk of
cancer. Dimethylsulfonioacetate, derived from Chlorophyta can bring improvement
in cancer cells and neural degeneration caused by brain cancer (Solanki et al. 2008;
Nguemfo et al. 2004; Zubia et al. 2007).
Microbes are the single-celled marine phytoplankton which constitutes 90% of

the ocean’s total biomass. They are tiny microscopic organisms. Bacteria, fungi,
and plankton along with the viruses originate from them. They are in billion
inhabiting in seawater. Many marine microbes constitute biochemical diversity
along with a reservoir of novel drugs. They are considered as essential sources for
drug discovery and development. Marine bacteria are associated with the devel-
opment of novel drugs like antibiotics and other pharmaceuticals. Meroterpenoids
are the class of secondary metabolites in which the terpenoid moieties are linked to
molecules from different biosynthetic pathways (Rahman and McFadden 2011).
They include quinones with prenylated naphthoquinones and reduced hydro-
quinone analogues and are mainly derived from marine fungi and actinomycetes.
Meroterpenoids exert anti-cancer activity. Polyketide synthases belong to a class of
enzymes associated with the biosynthesis of secondary metabolites and show potent
anti-cancer activity. Actinomycetes come under the systematic groups of secondary
metabolite producers. They exert various biological activities including anti-cancer
property (Huheihel et al. 2002).
Arisostatin A and arisostatin B, glycosylated polyketides are isolated from
the Micromonospora sp.. They come under the class of tetrocarcin–type cytotoxic
compounds and especially Arisostatin A exhibit potent anti-cancer activity because
of its cytotoxic effect on human cancer cells. It also activates caspase 3, a key
effector protease responsible for apoptosis induction (Wang et al. 2013).
Toluquinol, isolated from marine fungus interferes with one of the factors
causing cancer by impairing the unlimited replicative potential, characteristic fea-
tures of tumour cells. It suppresses the proliferation of the promyelocytic leukemia
cell line, fibrosarcoma cell, and colon adenocarcinoma cell. Diketopiperazines,
isolated from marine-derived fungi, have gained a lot of attention from the
researchers’ attention because of their varying chemical structure and biological
activities. Halimide ((-)-phenylahistin), originated from Aspergillus ustus, is a
fungal prenylated Diketopiperazines showed anticancer activity by arresting the cell
cycle of cancer (P388) in its G2/M phase (Schumacher et al. 2010).
Tunicates or urochordates, comes under subphylum Tunicata or Urochordata.
They have been shown as a primitive model organism to study immunodefense
since the innate immune system has been hypothesized as an important functional
component that may partially explain the lack of metastatic tumors in invertebrates
(Janakiram et al. 2010). Marine-derived compounds such as didemnin B, Aplidine,
and ecteinascidin have reached clinical trials for antitumor activity. Didemnin B,
isolated from Trididemnum solidum, is a cyclic depsipeptide and today it is rec-
ognized as a first marine-originated compound to enter phases I and II clinical trials.
The phase II studies suggest the complete or partial remissions with non-Hodgkins
lymphoma, but due to cardiotoxicity as a side effect, scientists chose didemnin B for
further study. The closely related dehydrodidemnin B (DDB, Aplidine) is isolated
from Aplidium albicans in 1988. Spectroscopic studies assign a structural formula
in which a pyruvyl group in dehydrodidemnin B is replaced by the lactyl group in
Didemnin B and syntheses of dehydrodidemnin B have been achieved. It is also
found that aplidine is more active than Didemnin B and lacks Didemnin B’s car-
diotoxicity (Rabelo et al. 2012).
Ecteinascidins, which are considered as the second family of tunicates, origi-
nated from the extracts of Ecteinascidia turbinate. They exert anti-cancer activity,
this property of ecteinascidins is first described in 1969 (Menna et al. 2013). Apart
from sponge, algae, tunicate, microbes’ other marine organisms like sea cucumber,
sear hare, molluscs and bryozoans also have an anticancer function including
microtubule-interfering action, DNA-interaction, phosphatase inhibitions, etc.
(Smithet al. 2010). Alkaloids pyridoacridines isolated from various marine sources
have been reported to possess significant cytotoxicity against cultured cells, and the
family as a whole seems to be of great interest as a source of new lead structures for
the development of a future generation of therapeutic agents. Sea cucumbers are
those marine animals that are considered as an essential human food resource, and
their extracts have been used for OTC dietary health supplements. Triterpene
glycosides from sea cucumbers show a wide range of biological effects, such as
antifungal, antitumor, hemolytic, cytostatic, pro-apoptotic, and immunomodulatory
activities (Thiansilakul et al. 2007). Frondoside A and Cucumariosides exhibit
anti-cancer effects on both in vitro and in vivo models. The dolastatins are extracted
from the Indian Ocean sea hare, Dolabella auricularia. Subsequently, several
dolastatins and related molecules are isolated from filamentous marine cyanobac-
teria, which are the natural diet of the sea hares. The dolastatins are the most active
molecules in inhibiting cancer cell growth (Yam et al. 2001).
17.3.2 Anti-Cardiovascular Effect
Omega-3 or (PUFA) is very much beneficial to our health. It lowers the risk of
cardiovascular disease. Many studies and reports suggest that fish consumption has
a direct relation in reducing the risk of congenital heart defect, myocardial
infarction, etc. Many research reports suggest that omega-3-fatty acids help to lower
the blood pressure (systolic and diastolic) in people with hypertension problems.
Also, it suggests that fish oil supplements are very much beneficial for arrhythmia
patients (Xia et al. 2011).
A study on Mediterranean people suggests that people who intake a high amount
of marine food have a low risk of congenial heart disease because Omega-3-fatty
acids reduce the risk factors associated with triglyceride concentrations, blood
pressure, platelet aggregation and heart arrhythmias. Also the report shows that on
daily consumptions of fish can lower the severe symptoms of depressions in adults
and asthma and respiratory disorders in children. Also, a protein that is derived
from marine (macroalgae) is better ACF-1 inhibition because of fewer side effects
and also, they act as a potent drug beneficial for hypertension patients (Mozaffarian
and Wu 2011).
17.3.3 Anti-coagulant Activity
Literature survey suggest that marine organisms also exert anti-coagulant activity.
They show anti-coagulant effect either by inhibiting thrombin or by activating
anti-thrombin (III) or by increasing the clotting time both in the intrinsic and
extrinsic pathways (Mauro et al. 2012). In a recent study, marine-derived sulphated
glycans have emerged with a potent coagulant and thrombosis property. These
glycans are categorized into two class (i) GAGs such as fucosylated chondroitin
sulfates (isolated from sea cucumber) and (ii) GAG mimetics like sulphated
galactans and sulphated fucans. Moreover, sulphate content plays a key role in
anti-coagulant activity because the presence and amount of distribution of sulphate
decide the process of coagulation or platelet aggregation (Fan et al. 2011).
However, in some cases of fucoidan and fucans, the outcome of anti-coagulant
activity is related to some factors such as (i) the content of sulfate or disulfate or
fucose (ii) the higher molecular weight that usually induced a stronger anticoagulant
activity and (iii) the molecule presents a linear backbone. Laminaran is also an
example of a marine polysaccharide which after structural modification like sul-
fation, reduction, or oxidation exerts anticoagulant activity (Mauro et al. 2012).
Heparin is a sulfated polysaccharide that is mainly present in mammalian tissues,
one of the most common anti-coagulant drugs that has been used for the last
15 years as a commercial anti-coagulant in thromboembolic disorders. However, it
shows several side effects like thrombocytopenia and is not able to inhibit thrombin
bound to fibrin, and shows deficiencies and unwanted bleeding. More than 300
marine organisms have been documented as heparin alternatives and around 301
new anti-thrombotic and anti-coagulant derivatives have been documented as well.
These molecules vary from polysaccharides to protein structures and are originated
from a variety of marine sources (Sharon and Lis 2004).
In vivo studies suggest that S-galactofucan, isolated from Spatoglossum schroederi
(brown seaweed) exerts potent antithrombotic activity. Spirulan extracted
from Arthrospira platensis interferes with the blood coagulation-fibrinolytic system
and exhibited anti-thrombogenic properties. The degree of sulfation of chitosan is an
important point. Highly sulfated chitosans induce an increase of thrombin, activated
partial thromboplastin time and thrombin time (Desai 2004).
17.3.4 Anti-obesity
Obesity is a complex disease that includes the deposition of excessive fat in the
body’s tissue. It is considered as chronic metabolic disorders that occur due to an
imbalance between energy intake and energy outlay. Obesity is a matter of concern
because it can lead to many health-related problems such as B.P., cardiac related
disorders, sleep apnoea, type-2 diabetes mellitus, high cholesterol levels; etc.
Obesity also harms people on the social platform. Sometimes obese people may
face multiple forms of prejudice and discrimination due to their over-weight, thus
obesity has become a medical as well as a social problem. Many studies showed
that fish oil is capable to reduce body weight (Mauro et al. 2012). A study on 324
men and women aged between 20 and 40 years from different countries (Spain,
Iceland, and Ireland) is conducted. They are given sunflower oil and fish oil as their
diet. Various measurements are noted down which include: total cholesterol,
high-density lipoprotein, low-density lipoprotein, cholesterol, triacylglycerol, and
anthropometric measurements. It is found that the weight loss diet with fish oil has
high triacylglycerol, low total cholesterol, and high-density lipoprotein. Other
studies are also done by comparing the body mass index between fish consuming
people and meat consuming people. The study is conducted on men and women
aged 20–97 years. A study report shows that people consuming fish has a lower
body mass index in comparison to people consuming meat (Maeda et al. 2009).
Eicosapentenoic (EPA) acid and docosahexenoic (DHA) acid also play a major
role to prevent obesity. They mainly inhibit lipid synthesis enzymes like fatty acid
synthase and stearoyl-CoA desaturase-1. EPA and DHA prevent the entry of fatty
acid to adipocytes for lipogenesis (Aminin et al. 2010). Polyunsaturated fatty acids
are also very effective against obesity as they suppress the factors which are
involved in adipocyte differentiation and fat deposition. Animal studies show that if
EPA and DHA intake increases then, it can protect the body from obesity, and can
also reduce fat when they are already obese. A study on mice is performed in which
they are received sunflower and fish oil diet. In this study, plasma lipid level,
hepatic triglycerides, cholesterol, hepatic mRNA of expression of lipogenic and
lipidolytic genes are measured. The results which is found suggest that a group of
mice who are fed with fish oil has lower body weight in comparison with those who
are fed with sun flower oil (Imhoff et al. 2011).
Marine algae also exert anti-obesity activity e.g. U. pinnatifida, Hijikia fusi-
formis, and Sargassum fulvellum, which are part of the Asian diet but are also
consumed in many other places (Venegas-Calerón et al. 2010). Fucoxanthin has a
unique structure involving an allene bond and a-monoepoxide which is associated
with anti-obesity activity as well as anti-diabetic activity. Also a nutrigenomic
report suggest that fucoxanthin induces uncoupling protein 1 (UCP1) expression in
mitochondria of white adipose tissue resulting in oxidation of fatty acids and heat
generation in white adipose tissue. Uncoupling protein 1 is known as co-factor of
anti-obesity enzyme because its expression can result in the total organism’s energy
expenditure and its dysfunction can cause obesity. Fucoxanthin induces a clear
UCP1 signal and its mRNA is detected using Western and Northern blot analyses of
abdominal WAT). Further experiments with obese rats and mice show that the
fucoxanthin-fed groups always has improved insulin resistance and decreased blood
glucose In another study, purified Fx and macroalgae lipids containing fucoxanthin
are compared, in which the latter induces a higher expression of UCP1 when
Fucoxanthin is present with the other components, especially lipids. Furthermore, a
synergistic action of omega‐3 fatty acids is noticed on the anti‐obesity effect of
fucoxanthin (Tsukui et al. 2007).
Fucoidan, as we all know, can reduce lipid accumulation by stimulating lipolysis

which decreases weight gain. Researchers have studied the lipolitic activity of
fucoidan by examining the protein level of the hormone‐sensitive lipase (HSL) and
the phosphorylated HSL (p-HSL) using Western blots. HSL is one of the most
important targets of lipolytic regulation (Menna et al. 2013). The subsequent
phosphorylation and activation of HSL result in an increase in the hydrolysis of
stored triacylglycerols into mono‐acylglycerols and free fatty acids. The level of
HSL and p‐HST has increased compared to the controls after fucoidan treatment of
differentiated 3T3‐L1 adipocytes (Venegas-Calerón et al. 2010).
17.3.5 Bone Growth and Healing
Osteoporosis is the most common bone disease in which the density of bone decrease.
It mainly occurs when the body reabsorbs more bone tissue and produces less to
replace it. According to IOF (International Osteoporosis foundation) every 1 in 3
women over age 50 will experience osteoporotic fractures, as will 1 in 5 men aged
over 50. Overall, 61% osteoporotic fractures occur in women with a female to male
ratio of 1:6. Marine organisms have been proved itself as a potent source of osteo-
genic bioactive. Fucoidan isolated from Apostichopus japonicas (a sea cucumber)
exerts anti-resorptive and osteogenic activity (Granito et al. 2017; Al et al. 2013).
Kim et al. (2014) demonstrate the osteogenic effect with brown algal extracts
added to bone marrow macrophage cultures. This includes inhibition of
RANKL-dependent MAPKs and downregulation of c-Fos and NFATc1 transcrip-
tion factors. Other examples of algal extract which show osteogenic activity
is Sargassum horneri (brown algae) which stimulate osteoblast genesis and inhibit
osteoclast genesis in vitro in preosteoblastic and monocytic cell lines. Similarly
in vitro and in vivo work is performed on rat femoral tissue to study the ability of S.
horneri extracts and it is found that it increases the bone calcium content and
inhibits bone resorption.
Nacre which is generally available in powder form and also known as the mother
of pearl has now become the body of research. It is a lustrous aragonitic inner layer
that is found in molluscan shells and belongs to mussels and abalone taxa (Singh
et al. 2011). Similar to the bone, nacre also consists of both inorganic and organic
contents with an organic shell matrix comprises of proteins and other nutrition. It is
used as a template for calcium carbonate mineralisation. Various studies on nacre
have been conducted since the early 1990s, with initial in vitro work showing its
capacity to stimulate the mineralization of human osteoblasts and the ability of
nacre to aid bone reconstruction in human maxillary defects. Here, nacre powder is
mixed with the blood of patients and injected into the defect site of eight
middle-aged female patients. The results show no evidence of toxic effect and
demonstrate enhanced mineralisation and good bio-dissolution of nacre within the
area of injection (Akakabe et al. 2014). The significance of this discovery is not
realised until a subsequent commentary is published, emphasising the remarkable
ability of a raw and unrefined natural product to promote bone growth. Since this
early work, there has been a surge of research effort, including in vitro and in vivo
studies, as well as those specifically focusing on the proteins and mechanisms
involved in enhancing cellular activity, making nacre an excellent case study of
bioactive research (Kose et al. 2016).
A good example of the in vitro work which is conducted with used water soluble
matrix (WSM) that is extracted from the oyster Pinctada fucata. This study
demonstrates both the ability of nacre to enhance osteoblast differentiation (in-
creased Col-I, osteocalcin, and ALP expression) and its ability to scavenge free
radicals, suggesting an antioxidant potential that may also support bone regenera-
tion. WSM has also been shown to increase bone mineral density (BMD) in an
ovariectomized mouse model of osteoporosis, in part attributed to increased Runx2
and Fos-related antigen-1 expression as a result of JNK pathway stimulation in
osteoblasts. Furthermore, the extract suppresses actin ring formation and
RANKL-induced upregulation of c-Fos and NFATc1 in osteoclasts. Other in vivo
work shows nacre implanted into rat femurs supported new bone formation,
implant/bone fusion, and increased expression of numerous markers indicative of
increased BMU action (Uchiyama et al. 2003).
Aquamin derived from Lithothamnioncorallioides (red alage) includes calcium,
magnesium and approx 72 other trace minerals. L. corallioides is famous for its
uniqueness that comes under the few algal species who produces a calcareous
skeleton. L. corallioides generally found on muddy/sandy substrates (at less than
20 m in depth) with assemblage of unattached algae (Yamaguchi and Matsumoto
2012).
Other examples include Haliotis discus, which is associated with osteoblastic
activity. Zoanthus sp. Includes Norzoanthamine, an alkaloid, responsible for anabolic
effects on bones and increases the formation of a collagen-hydroxyapatite composite.
Another natural marine compound with osteogenic potential is Phorbaketal A, derived
from the marine sponge Phorbas sp. This bioactive is shown to stimulate osteoblast
differentiation in mesenchymal stem cells, predominantly through activation of the
extracellular signal-regulated kinase (ERK) pathway (Kose et al. 2016).
17.3.6 Anti-inflammatory Activities
Marine food or seafood also exerts anti-inflammatory effects because of the inhi-
bition of certain inflammation mediators like cytokines, prostaglandins, leuko-
trienes, etc. by polyunsaturated fatty acids (PUFAs) especially omega-3. Many
studies show that enhancing the ratio of omega-3 to omega-6 fatty acids (which
have pro-inflammatory and immunoactivity properties) in diet, can result in a
decrease in inflammation because eicosanoids derived from omega-3 have
anti-inflammatory activity (Sonani et al. 2014). Many works of literature and
experimental studies suggest that daily intake of fish or fish oil is very much
effective on inflammation. It is also found that fish oil can be taken as an alternative
for NSAID (Non-steroidal anti-inflammatory drugs) to avoid the side effects of

NSAIDs like gastric ulcer, bleeding, etc. (Ellis 2001).
Marine seaweeds also exert anti-inflammatory activities. For example, fucox-
anthin derived from brown algae helps to decrease the production of prostaglandin
(E2) (PGE2) and inhibits COX-2 protein expressions. The sulfoglycolipidic fraction
(SF) derived from Porphyridium cruentum includes a high concentration of pal-
mitic acids (26.1%), archidonic acid (6.8%), EPA (16.6%) and omega -9 fatty acids
(10.5%) and thus it exhibits anti-inflammatory activity (Park et al. 2011). In
addition, heterofucan isolated from brown algae Dictyota menstrualis inhibits the
leukocyte migration and reduces the level of pro-inflammatory cytokines.
Moreover, Fucoidan isolated from seaweed E. Cava decreases COX-2, nitric acid,
and prostaglandins (Gunnarsdottir et al. 2008).
17.3.7 Neuroprotective Agents
Neurodegenerative diseases refer to the death of a certain part of the brain due to
some illness which generally occurs in the older age group. These are generally
incurable which results in progressively degeneration of the nervous system (CNS
as well as ANS) and neurons Ferrari et al. (2008), Gordaliza (2010). The most
common neurodegenerative diseases are Parkinson’s disease and Alzheimer's dis-
ease. Many synthetic compounds are available in the market for the treatment of
neurodegenerative diseases but they all involve certain side effects such as anxiety,
nervousness, drowsiness (Pal et al. 2008, 2009; Pal and Mazumder 2014; Gupta
et al. 2003), mouth dryness or tiredness, etc. (Shibata et al. 2008; Davies-Coleman
2012). For this reason, many scientists and researchers have focused on the
development of novel naturally derived drugs with low side effects.
There are various causes for neurodegenerative disease and oxidative stress is
one of them. Oxidative stress occurs due to an imbalance between pro-oxidant and
anti-oxidant homeostasis that further results in the occurrence of toxic reactive
oxygen species. Our CNS is very much sensitive to oxidative stress and that results
in lipid peroxidation, DNA, and protein damage and ultimately becomes the reason
for neuronal death. Thus, antioxidants become a major saviour for the prevention of
neurodegenerative diseases. Lim et al. demonstrate that Neorhodomela aculeate,
which is also known as Rhodomela confervoides, is able to scavenge DPPH with an
IC50 = 90 lg/mL It at a concentration of 20 lg/mL completely suppresses H2O2
induced lipid peroxidation in rat brain homogenate (Maeda et al. 2007). In addition,
lowering the risk of neurodegenerative disease and high DHA blood levels is
interlinked. Experimental studies on animal models suggest that DHA depletion can
result in amyloid protein accumulation and hyperphosphorylation of tau and can
become the reason for Alzheimer's disease (Santiago-Santos et al. 2004). Many
works of literature and reports suggest that an adequate intake of fish and marine
algae can help to maintain the DHA blood level and can help in the prevention of
Alzheimer's disease. Houghton et al. Suggest in his report that
Crinum jagus and Crinum glaucum, two Nigerian Crinum species consist of cho-
linesterase (ChE) inhibitory activity that is very much beneficial for the treatment of
Alzheimer disease. There are many examples of algal species that are endowed with
cholinesterase (ChE) inhibitory activity and can be used for the treatment of neu-
rodegenerative diseases for e.g. (Dalisay DS et al. 2009) Dictyotahumifusa,
Hypneavalentiae, Padina gymnospora, Ulva reticulate, etc.
17.4 Summary
In Table 17.5 various marine sources along with their bioactive ingredients and
biological activity is mentioned (Ustyuzhanina et al. 2013; Theodore and
Kristinsson 2007; VonPost-Skagegard et al. 2006).
Table 17.5 A summary of marine sources with their bioactive ingredients and biological activities
Sources Bioactive ingredients Biological activities
Fish oil Omega-3 fatty acid Antitumoral and
Antimetastasis activities
Ascidian Omega-3 PUFA Anticancer
Crustaceans (shrimp, crab, chitin and chitosan Anticancer, Antimicrobial
crayfish) Anti‐inflammatory
Hypocholesterolemic activities
Marine sponge Monanchora Polycyclic guanidine Anticancer activities
pulchra alkaloid
Sponge Smenospongiasp. Smenospongine, a Antiproliferetive and
sesquiterpene Antiangiogenic activities
aminoquinone
Micromonosporasp. arisostatin A and Cytotoxic effects
arisostatin B
Aspergillus ustus Halimide ((-)- Anticancer effects
phenylahistin)
Cladophora rupestris Crude extract Osteogenic effects
Laurencia undulata Floridoside Osteogenic effects
Haliotis laevigata Perlucin protein Osteogenic effects
Lithothamnioncorallioides Aquamin Osteogenic effects
Nannochloropsisoculata Peptide Osteogenic activity
Symploca sp. Largazole Osteogenic effects
(depsipeptide)
Phorbas sp. Phorbaketal A Neuroprotective activity
B. triquetrum ferulic acid Neuroprotective activity
L. japonica fucoidan Neuroprotective actvity
Eisenia bicyclis Phlorotannins Neuroprotective activity
Ecklonia stolonifera Sterols, phlorotannins AChE, Neuroprotective
actvity
(continued)

Sources Bioactive ingredients Biological activities
Nemalionhelminthoides Mannans Antiviral immunomodulatory
actvity
Codium fragile Galactan Antiviral immunostimulating
activity
Arthrospira platensis Spirulan Antiviral anticoagulant effects
Cuttlefish (Sepiellamaindroni, Polysaccharide Antimutagenic effects
Euprymnaberryi) Antimicrobial effects
Krill (Euphausiasuperba) Omega‐3 PUFA (DHA Anti-cardiovascular diseases
and EPA effects
Cholesterol reducing effects
Bonito Omega‐3 PUFA (DHA Anti-cardiovascular effects
and EPA)
Herring Omega‐3 PUFA (DHA Anticardiovascular effects
and EPA) Anti‐obesity actvity
Antitumor effects
Cuttelfish Peptides Antihypertensive activity
Antioxidant actvity
Chrysophrys (pagrus) major Chrysophsins Antimicrobial actvity
Microalgae Chlorella Astaxanthin AGE formation inhibition
zofingiensis
Microalgae Chlorella – Antioxidant potential,
pyrenoidosa a-amylase and a-glucosidase
inhibition
Fungus Cosmospora sp. Aquastatin A PTP1B inhibition
Actinomycetes Streptomyces sp. Pyrostatins A and B N-acetyl-glucosaminidase
inhibition
17.5 Conclusion
Marine sources are reservoir of essential bioactive ingredients which are beneficial
for human body system. They can be used in wide ranges of fields such as, cos-
metics, pharmaceuticals, food industry, etc. Marine foods have ability to prevent
and cure many diseases like CVS disorders, bone related disease, cancer, etc. The
marine organisms offer an enormous resource for drug discovery and development,
and are considered as the largest remaining reservoir for natural molecules. They
may be used as functional ingredients in the food industry. In this regards, efforts
should be made to create awareness about the beneficial effects of marine food since
their consumption and utilisation in day to day life will help prevention of many
chronic diseases.
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Chapter 18
Cardiac Tissue Engineering: A Role
for Natural Biomaterials
Pallavi Pushp and Mukesh Kumar Gupta
Abstract Cardiovascular diseases (CVDs) are the major cause of death all over the
world, being responsible for 7.4 million deaths in a year. In the past two decades,
advances in biomaterials, stem cell biology, and engineering have allowed the
generation of tissue constructs that can imitate the complex structure of the heart
and, upon transplantation into animal models, improved the cardiac function.
Consequently, stem cell-based cardiac tissue engineering (CTE) has emerged as a
potential therapeutic strategy for the treatment of CVDs. The choice of appropriate
biomaterials that can provide differentiating and paracrine milieu to mimic the
extracellular matrix (ECM) is the key to the proper functioning of cardiac tissue
construct. Plant- or animal-derived natural polymers such as alginate, chitosan,
collagen, fibrin, gelatin, and glycosaminoglycan are biocompatible and facilitate
cell adhesion, proliferation, and differentiation for development of cardiac tissue
constructs. On the other hand, natural biomaterials such as collagen, fibronectin,
laminin, nephronectin, etc. are often used to biofunctionalize the synthetic
biomaterials-derived scaffolds to enhance cell adhesion and cell-to-cell communi-
cation. This book chapter discusses various approaches for CTE and the role of
different natural biomaterials and their importance in the biofunctionalization of
synthetic biomaterials for developing cardiac tissue constructs.

Keywords Bioactive polymers Biomaterials Cardiac tissue engineering

Hybrid polymers Scaffolds Stem cells
P. Pushp
Department of Biotechnology, Institute of Engineering and Technology (IET),
Bundelkhand University, Jhansi, Uttar Pradesh 284 128, India
P. Pushp M. K. Gupta (&)
Department of Biotechnology and Medical Engineering,
National Institute of Technology, Rourkela, Odisha 769 008, India
e-mail: guptam@nitrkl.ac.in
https://doi.org/10.1007/978-3-030-54027-2_18
618 P. Pushp and M. K. Gupta
18.1 Introduction
Cardiovascular diseases (CVDs) such as myocardial infarction (MI), atherosclero-

sis, cardiomyopathies, heart valve diseases, and long-QT syndromes (LQTS) are the
major causes of death worldwide. They are generally associated with apoptotic and
necrotic changes in the cardiac tissue and loss of functional cardiomyocytes (CMs).
Since the self-renewal or regenerative potential of CMs is extremely limited, the
lost CMs are challenging to be compensated by therapeutic approaches. Thus,
despite significant research in diagnosis and treatment, CVDs remain major ther-
apeutic challenges in the medical field. In recent years, cardiac tissue engineering
(CTE) has been emerging as a potential therapeutic strategy for the treatment of
CVDs. These CTE approaches involve isolation and expansion of autologous
patient cells, including stem cells such as mesenchymal stem cells (MSCs) or
induced pluripotent stem cells (iPSCs) and their differentiation into cardiac tissue
on an appropriate extracellular matrix (ECM) for in vitro incubation followed by
in vivo transplantation (Fig. 18.1).
In initial studies, ‘cell-based therapy’ by cardiomyoplasty were attempted
wherein embryonic, fetal and neonatal CMs, skeletal myoblasts, bone marrow stem
cells, or resident cardiac progenitors were directly injected into the injured heart.
Several clinical trials on cardiomyoplasty reported functional improvements in
CVDs but the functional benefits were transient. The cardiomyoplasty procedure
also suffer from poor availability of cells, low cell retention and poor survival of
Fig. 18.1 Approaches for cardiac tissue engineering

18 Cardiac Tissue Engineering: A Role for Natural Biomaterials 619
implanted cells, loss of intracellular communication between CMs and ECM, acute
inflammation and injury caused by injection procedure and blockage of the
microcirculatory network by the injected cells. Some studies even reported negative
effect of cardiomyoplasty, resulting in arrhythmias due to inappropriate electrical
integration. Thus, in vitro developed cardiac tissue constructs, seeded with stem
cells or their progenitors, were explored as alternative options. Since then, the field
of CTE exponential advanced with advancements in the design and development of
ECM-mimicking scaffolds, efficient perfusion bioreactors and, newer sources of
stem cells, and advanced analytical techniques. This book chapter summarises
various approaches for CTE and the role of natural biomaterials in fabricating the
scaffolds for seeding of stem cells.
18.2 Approaches for CTE Using Natural Biomaterials,

Cell-Sheet and Decellularized Tissues
In ‘classical’ CTE, cardiac tissue construct is created by seeding desired cells into
biomaterial-derived scaffolds. The scaffolds are initially fabricated, without cells,
using natural or synthetic or hybrid biomaterials and, the cells are then seeded into
the scaffold. The scaffolds are expected to allow neovascularisation, neuronal
innervations and electromechanical cell coupling with host myocardium following
their transplantation into the host. In order to achieve these characteristics, several
modifications such as incorporation of growth factors and pro-angiogenic factors,
co-culturing of CMs with other cell types etc. are often required to further improve
the survival of the tissue construct (Fig. 18.1). The scaffolds are traditionally fab-
ricated using natural biomaterials such as alginate, chitosan, collagen, fibrin, gelatin,
and glycosaminoglycan, fibronectin, laminin, nephronectin. Many natural polymers
have low elastic modulus and poor electrical conductivity required for the CTE and
necessitates doping with synthetic biomaterials or nanomaterials such as carbon
nanofibers to develop a conductive scaffold (Martins et al. 2014). Natural bioma-
terials such as gelatine, collagen, fibronectin, laminin, stromal-derived factor-1,
nephronectin, and bioactive glass etc. are also used to bio-functionalize the synthetic
biomaterials-derived scaffolds to enhance cell adhesion, cell-to-cell communication
and cellular interaction with scaffold (Oliver et al. 2019; Yang et al. 2018).
In a newer development, engineered heart tissue (EHT) were created by seeding
CMs into collagen I and Matrigel™ (an extracellular matrix compound derived
from Engelbreth-Holm-Swarm mouse sarcoma) matrix followed by mechanical
stretching into circular form (Yildirim et al. 2007). Guo et al. generated embryonic
stem cell (ESC)-derived myocardium in mouse wherein CMs were seeded into
circular moulds with collagen I and Matrigel™ to produce EHT, which were
subsequently subjected to unidirectional cyclic stretch at 10% strain and 2 Hz (Guo
et al. 2006). Transplantation of the EHT improved ventricular function in MI model
but formed transmural thrombus in a heterotropic heart transplant model
(Zimmermann et al. 2006).
Scaffold-free cell sheet/cell patch engineering is a yet another attractive approach

for producing multi-layered cardiac patch. This technique was first developed in
Japan by Kushida and co-workers. In this technology, a temperature-responsive
culture surface is first fabricated by layering a temperature-responsive polymer (e.g.
poly (N-isopropylacrylamide)) onto polystyrene petri plates. Monolayers of CMs
are then grown on such cell culture plates and, upon confluency, are recovered as an
intact cell sheet by lowering the temperature to 20 °C. Scaffold-free multi-layered
cell sheets are then generated as 3D tissue by stacking of these monolayer sheets
using a silicon mould (Haraguchi et al. 2014). Scaffold-free vascularized CMs
patches have also been developed by Stevens et al. (2009) by utilizing a rotating
orbital shaker. The 3D myocardial tissue, developed from these stacked CMs
sheets, resembled native heart tissue and showed synchronous beats (Haraguchi
et al. 2011). In a clinical trial conducted in Japan, transplantation of autologous
myoblast cell sheets was shown to improve the cardiac dysfunction in heart failure
cases (Matsuura et al. 2014). However, the stacked cell sheets are generally limited
in thickness up to 3 layers (*80 µm) due to the lack of vascularization and oxygen
transport to the centre of the tissue. Furthermore, cell sheet tissue lack native
orientation and spatial placement of cardiac cells. The later can however, be
overcome by photolithography-based microtexturing of culture dishes before
growing the monolayers (Isenberg et al. 2008).
Decellularized whole heart tissue has also been used as scaffold for CTE.
Cadaveric hearts can be decellularized by using detergents to obtain whole heart
ECM scaffolds with well preserved natural 3D structure of ECM, vascular archi-
tecture, mechanical anisotropy, competent acellular valves and intact chamber
geometry. The decellularized whole heart can then be re-seeded with CMs and
endothelial cells (ECs) and, cultured in a perfusion bioreactor to generate a spon-
taneously beating ‘whole heart’, which can be used as allograft or xenograft (Ozlu
et al. 2019). Such strategy has been successful in both human and animal. It is also
possible to develop personalized bioartificial hear by seeding of patient’s own cells
(Kang et al. 2020). Unfortunately, re-seeding of cells into the decelluralized heart in
a uniform manner is quite difficult and requires specialized multi-stimulation
bioreactor to provide coordinated mechanical and electrical stimulation and nutrient
supply (Wang et al. 2013a). To circumvent non-uniform re-seeding some studies
have suggested the sectioning of myocardium into 2 mm thick portions before
decellularization and using them as cardiac patches (Wang et al. 2010). In other
studies, decellularized matrix of heart or other organ has been incorporated into
scaffolds for CTE.
18.3 Natural Biomaterials Used in ‘Classical’ CTE
Cells within a tissue are surrounded by ECMs that are composed of biomolecules
and fibers, synthesized by cells themselves, blood vessels and innervating neurons.
The ECMs provide micro-niche whose physico-chemical microenvironment allows
the cells to interact through cell signalling molecules and perform their physio-
logical function. Thus, a tissue engineered cardiac construct must be designed to the
mimic ECM of the heart. Ideally, a scaffold for human cardiac tissue must have a
thickness of *0.5 cm with adequate porosity for oxygen transport and to allow a
cell density of *105 cells/cm3 of the construct. The construct should also have the
ability to generate a force of 2–4 mN/mm2 during contraction and to support
electrical signals propagation at *25 cm/s. It should also allow neovascularisation,
neuronal innervations and electromechanical cell coupling with the host myo-
cardium upon transplantation into the host. An ideal biomaterial must be biocom-
patible and bio-mimetic yet biodegraddable in concomitant with the development of
new ECM after transplantation. It should also be amenable to laboratory procedures
for fabricating the scaffolds of desired physical, chemical and biological properties
and should be scalable of large-scale clinical applications. Some of the commer-
cially available biomaterials for CTE are shown in Table 18.1.
Scaffolds for CTE have been developed using plant- or animal-derived natural
polymers such as alginate (Choe et al. 2019), chitosan (He et al. 2018), collagen
(Hosoyama et al. 2018), fibrin (Bagheri-Hosseinabadi et al. 2018), gelatin (Majidi
et al. 2018), hyaluronic acid (HA) (Hadisi et al. 2020), Poly (3-hydroxyoctanoate)
(Bagdadi et al. 2016) and glycosaminoglycan (Flanagan et al. 2006). Natural
polymer-derived scaffolds are generally biocompatible and biodegradable and
provide better cellular ECM-like environments to facilitate cell adhesion, prolif-
eration and differentiation (Yi et al. 2017). Some polymers may be easily degraded
by body’s natural enzymes or have relatively weak mechanical properties, which
can be controlled by combing with synthetic polymers. The rate of degradation can
also be controlled by chemical cross-linking (Wu et al. 2014) of the biomaterials.
Some natural polymers have also been shown to have immunogenicity or may act
as a potential reservoir of infectious agents (Jawad et al. 2008). The later can
however, be controlled by use of genetically engineered and ‘immunologically
virgin’ animals reared under gnotobiotic conditions (Hwang et al. 2015).
18.3.1 Collagen I
Collage I is a predominant ECM protein of heart and is FDA approved for thera-
peutic use in skin and bone. It contains peptide sequences that interact with inte-
grins to promote cell attachment. It can also modify the substrate stiffness and
thereby alter the cellular functions such as growth, proliferation and differentiation.
Thus, it has been used extensively in CTE as hydrogel, sponge-like scaffold or as
coating material for enhancing hydrophilicity of synthetic biomaterials. Electrospun
of sub-micron diameter fibrils of collagen has also been possible, which could be
rapidly and densely infiltrated by the cells and helped in the formation of functional
blood vessels (Telemeco et al. 2005). Hamdi et al. (2009) observed significant
increase in angiogenesis at the site of MI upon transplantation of myoblast-seeded
collagen scaffold. Similar results were also observed with bone marrow-derived
622
Table 18.1 Commercially available biomaterials for cardiac tissue engineering

Sl. No Product Company Material Processing Application
1 Restore ® DePuy Small Intestine Biological Soft tissue
Orthopaedics, Inc Submucosa
2 PEEK-OPTIMA® Invibio Polyether Polymer Heart valve
ether ketone
3 ENDURAGen® Stryker Collagen Polymer Vascularization
4 SurgiMend® TEI Biosciences Acellular Collagen Biological Soft tissue and
vascularization
5 Cymetra® LifeCell Tissue Biological Revascularization
and cell
population
6 Ultramax® Atrium Medical Polyethylene Polymer Vascular grafts
Corporation terephthalate
7 CardioPass® CardioTech Polyurethane Polymer Synthetic
coronary artery
8 HYAFF-11® Fidia Advanced Hyaluronic acid Biological Myocytes
Biopolymer
P. Pushp and M. K. Gupta
CD133+ cells loaded on collagen patch (Joshi et al. 2018). Zhang et al. (2013)
microfabricated chitosan-collagen scaffold with micropores (200 µm) by using an
array of parallel channel designed for CTE. The parallel channels enhanced oxygen
transport, established cell to cell contact and improved cell alignment and elon-
gation. Another way to improve number of stem cell growth on collagen matrix is
to conjugate it with anti-Sca-1 monoclonal antibody (Shi et al. 2011).
Collagen has also been used in combination with other natural polymers such as
glycosaminoglycan, gelatin or Matrigel™ to have a synergistic effect (Kleinman
and Martin 2005). Transplantation of MSCs in collagen-glycosaminoglycan com-
posite to the infarct region induced neovascularisation at the site of the infarct and
increased MSCs in both the scaffold and the heart wall (Qu et al. 2018). The
structural integrity and bio-stability of collagen can be improved by cross-linking of
with gluteraldehyde (GA), dimethylaminopropyl-ethlcabodiimide (EDC) (Ahn
et al. 2013) glyoxal, b-glycerophosphate (Wang et al. 2013b), malic acid deriva-
tives (Saito et al. 2008), dendrimers (Duan and Sheardown 2005), diamine and
diaminohexane (McKegney et al. 2001), formaldehyde, genipin, and carbodiimide
hydrochloride (EDAC) (Madhavan et al. 2010).
18.3.2 Gelatin
Gelatin is generally formed by thermal denaturation or partial hydrolysis of

animal-derived collagen I. Thus, it has been used in CTE to promote cellular
attachment and their integration into the infracted heart. It can also be blended with
other natural polymers such as gelatine. A blend of alginate/gelatin in 20:80 has
been successfully used for fabrication of scaffolds for CTE (Rosellini et al. 2009).
In another study, Ravichandran et al. (2013a) have electrospun nanofibrous tissue
construct by using a blend of gelatin, hemoglobin and fibrinogen and seeding them
with 5-azacytidine-treated mesenchymal stem cells (MSCs). The developed cardiac
tissue construct was shown to provide superior biological and functional effects and
high oxygen carrying potential for CTE. Combination of chitosan-agarose-gelatin
(Bhat and Kumar 2012) and fibrinogen-gelatin (Balasubramanian et al. 2013)
composite nanofibers was also shown to enhance the proliferation of CMs and
expression of functional cardiac proteins such as a-actinin, troponin I, connexin-43
and myosin heavy chain.
The gelatine has also been combined with synthetic biomaterials for growing
aligned CM with improved contractility. Composite of gelatine with polyglycerol
sebacate (PGS) could be used to develop an hybrid scaffold which allowed aligned
cell proliferation and resulted into well-defined anisotropy with enhanced con-
tractibility of the CMs (Kharaziha et al. 2013). Aligned patterned 3D bioprinted
gelatin hydrogel with synchronized beating of CMs, differentiated from MSCs, has
also been reported (Tijore et al. 2018).
18.3.3 Fibrin
Fibrin is a naturally occurring blood clotting protein and is FDA approved for use as
a surgical sealant. The fibrin network has a nanometric fibrous structure, which upon
treatment with thrombin can create fibrin gel. Upon introduction into the heart, the
fibrin gels can get replaced by natural ECM and therefore, it serve as a temporary
scaffold in CTE (Grassl et al. 2003). Fibrin glue (Christman et al. 2004), and
hydrogel scaffolds (Kaiser et al. 2019) have been explored as possible cardiac grafts.
Thomson et al. described a high-density microtemplated fibrin scaffold seeded with a
tri-cell mixture of CM, Epithelial cells (ECs), and fibroblasts to mimic native cardiac
tissue in structure and cellular composition (Thomson et al. 2013).
18.3.4 Alginate
Alginate of algal origin has also been used as natural biomaterials for CTE. Sodium
alginate has been extensively used as hydrogel for encapsulation of cells and
delivery of growth factors for CTE (Sun et al. 2010). The physico-chemical
properties such as gelation time, uniformity, mechanical strength, tensile properties,
and mechanics of alginate gel formation can be controlled or altered by using
different cation sources such as CaCl2, CaSO4, and CaCO3 (Kuo and Ma 2001),
temperature (Drury et al. 2004) and molecular weight distribution (Augst et al.
2006). Alginates containing higher amount of glucuronic acid (G-type) formed
stiffer gels due to the presence of diaxial links and had higher elastic and Young's
moduli than mannuronic acid (M-type) alginate gels and were more adaptable for
cardiac implantation (Ceccaldi et al. 2012). Fetal CMs, seeded onto alginate
hydrogels, stimulated neovascularization and healing of the infracted myocardium
in a rat model of MI (Leor et al. 2000). In addition to hydrogel system, alginate has
also been used to develop fibrous scaffold by electrospinning and porous scaffold
by freeze-drying (Lee et al. 2009). Macroporous scaffolds with interconnected pores
showed efficient cell seeding and maintained high metabolic activity of the cardiac
cells (Dar et al. 2002).
In earlier studies, it was difficult to electrospun the alginates as continuous and
uniform nanofibers due to lack of chain entanglements caused by rigid and
extended chain conformation. Electrospinning of alginates could successfully be
done by using glycerol as a co-solvent with water in different volume ratio (Nie
et al. 2008) and combining with water–soluble synthetic polymers such as poly-
ethylene oxide (PEO) and poly (vinyl alcohol) (PVA). Alginate/PEO nanofibers
showed good uniformity, integrity, cell adherence, cell viability and cellular
compatibility (Narayan and Miqin 2007). The cell adherence of alginate scaffolds
could be increased by incorporation of natural peptides such as fibronectin, laminin,
nephronectin or immobilization of RGD (arginine-glycine-aspartic acid) peptides
(Shachar et al. 2011).
Several researchers have also used alginate to create hybrid biomaterials for
CTE. Bai et al. (2011) made alginate/collagen composite microbeads for EHT. The
3D porous composite scaffold formed from different ratio of alginate and chitosan
by freeze drying method showed significant increase in ejection fraction, improved
neo-vascularisation, attenuated fibrosis and proliferation of CMs (Ceccaldi et al.
2013). Mollar et al. developed hydrogel composites from methacrylated hyaluronic
acid, alginate and gelatin (Moller et al. 2011). The composite of alginate/elastin and
PEG showed large pore size (approx 60–75 µm) as compared to alginate and elastin
composite at 20 °C (Chandy et al. 2003). The larger pore size membranes retained
less water content and high degradation profile and hence, were suitable for CTE.
Sapir et al. also created a functional cardiac patch from rat cardiac cells seeded on
macroporous alginate scaffold incorporated with magnetically responsive
nanoparticles (MNPs) (Sapir et al. 2014b).
18.3.5 Chitosan
Chitosan, a natural cationic polysaccharide and linear polymer obtained from chitin
through deacetylation process and linked via b, 1–4 linkage, has also been found
suitable as for CTE (Hussain et al. 2013). The low mechanical strength of chitosan
scaffolds can be overcome by formation of composite materials and creation of
nanofibers (Albanna et al. 2012). Chitosan hydrogel, incorporated with glutathione,
suppressed reactive oxygen species (ROS) and showed high survival of CMs (Li
et al. 2013). Liu et al. (2012) reported that chitosan hydrogel can scavenge ROS
molecules and improve the environment for cell engraftment, survival and homing
for ischemic heart.
Chitosan is occasionally used as hybrid biomaterials along with other natural or
synthetic biomaterials. For example, copolymer of chitosan and poly(e-caprolactone)
(PCL) provided better cell adhesion and mechanical strength for development of
cardiac tissue construct than Chitosan or PCL alone (Chatzinikolaidou et al. 2014).
The multilayer scaffold prepared from combination of chitosan, gelatin and PCL was
also found to be suitable for CTE (Pok et al. 2013). Similarly, micro-patterned aligned
collagen-chitosan hydrogel enhanced the success rate of beating CMs, oxygen
transport and provided better cell connections with construct (Zhang et al. 2013). Baei
et al. (2016) developed a thermo-sensitive conductive hydrogel with a highly porous
network of interconnected pores by incorporating nanoscale electro-conductive gold
nanoparticles into chitosan hydrogels.
18.3.6 Fibroin/Silk Fibroin
Fiboin present as natural form in silkworms, moths, spiders, silverfish, beetles etc.
is also a good source of natural biomaterial for CTE. Silk from mulberry Bombyx
mori as well as non-mulberry Antheraea mylitta has been used for CTE (Patra et al.
2012). It is composed of heavy and light chain of core proteins called fibroins,
which is coated by another protein called sericin—a glue, which help to bind silk
fibroins together due to its sticky nature. It is a natural biopolymer with excellent
mechanical strength, extensibility, elasticity and strain hardening. The strength-to-
density ratio of silk is much higher than that of steel. The mechanical properties of
silk, which include extreme elasticity (5–17 GPa), toughness, tensile strength (500–
900 MPa), and compression resistance, depends on the hydrophobic region com-
posed of repetitive amino acid sequences. Silk also has small diameter and occur as
aligned fibers which increases the surface area to volume ratio. Properties such as
slow degradation, high tensile strength, good oxygen and water permeability sup-
port better cell adhesion and growth, which made it one of the demanding
biopolymer for CTE. In fact, scaffolds prepared from silk fibroin not only induced
alignment of the cells but also helped in synthesis of titin, a protein critical to
sarcomere assembly that provide elasticity to the muscles (Cutts et al. 2015).
The main limitation in the use of silk is its hypersensitivity reactions and adverse
immunological effects due to non-mammalian origin. The immune response can be
improved by removing sericin through a process called degumming (Altman et al.
2003). After degumming, the silk used to dissolve in appropriate solvent such as
formic acid, calcium nitrate, calcium chloride, ionic liquids, hexafluoroisopropanol,
lithium bromide (Kluge et al. 2008) to form scaffold. Silk fibrous has been used as
films, hydrogel, 3-D macro or micro capsules, porous scaffolds, and electro-spun
and wet-spun fibers. The porous silk-based matrix can be formed by salt leaching,
gas foaming and freeze drying whereas fibrous scaffolds can be formed by elec-
trospinning or microfluidic approaches. The porosity and mechanical properties of
matrix formed can be controlled by concentration of silk fibroin and particle size of
salt. Several authors have reported efficient seeding of CM into silk fibroin scaffolds
to form contractile patches (Patra et al. 2012). Rahimi et al. developed a
thermo-sensitive conductive hydrogel with a highly porous network of intercon-
nected pores by incorporating nanoscale electro-conductive gold nanoparticles
(Au-NPs) into chitosan hydrogels (Rahimi et al. 2014). Silk fibroin exhibited
similar properties of cell attachment as fibronectin, a component of the natural
matrix for CM, probably due to its RGD domains.
Composite of silk fibroin with chitosan or hyaluronic acid has also been used to
generate cardiac patch from seeded MSCs, which promoted cell growth and car-
diomyogenic differentiation (Yang et al. 2010) and, upon transplantation into rat
heart model of MI, improved cardiac repair (Chi et al. 2013). Silk fibroin has also
been used as a coating material for surface modification of synthetic biopolymers.
Liang et al. immobilized silk fibroin on poly (ethylene terephthalate) (PET) film via
plasma pre-treatment followed by dip coating, which gave high surface roughness
and promoted MSCs culture (Liang et al. 2013).
18.3.7 Decellularized ECM
Decellularized ECM of heart (Singelyn et al. 2009), and other organs such as small
intestinal submucosa (SIS) (Crapo and Wang 2010), and urinary bladder (Robinson
et al. 2005) has also been incorporated into scaffolds for CTE. Decellularization of
tissues by detergents such as SDS can remove cells without affecting the bio-
chemical composition and mechanical properties of the ECM in terms of elasticity,
strength and durability (Eitan et al. 2010). The viscoelasticity and strength of the
decelluralized cardiac ECM was reported to be similar to those of native tissue,
although its elasticity and apparent viscosity are higher (Bronshtein et al. 2013).
Seeding and culturing of decelluralized cardiac tissue with MSCs partially restored
the mechanical properties lost after decellularization (Bronshtein et al. 2013). Thus,
decellularized ECM of heart are being extensively explored as a natural biomaterial
for CTE and variable factors such efficient methods of ECM removal, homoge-
neous re-cellularization strategies and tailoring of ECM for enhancing bioactivity
(Kc et al. 2019).
Decellularized ECM has also been used in combination with other natural and
synthetic biomaterials. Stoppel et al. developed a silk-based scaffold containing
decellularized ECM of heart (Stoppel et al. 2015). These silk-ECM composite
scaffolds had tunable architectures, degradation rates, and mechanical properties
and their subcutaneous implantation in rats resulted in remarkable endogenous
cell infiltration and vascularization after 4 weeks in vivo. In vitro, silk-cECM
scaffolds maintained both the atrial CMs and the human ESC-derived CMs (Stoppel
et al. 2015).
Interestingly, xenogenic ECMs were also shown to be beneficial in promoting
human CM proliferation (Sarig et al. 2015). Crapo and Wang (2010) combined rat
CMs with pig SIS gel and cultured on porous elastomeric PCS scaffolds. They
observed that cardiac tissue engineered from pig SIS gel had a more physiological
rate of contraction than those produced on Matrigel™. However, the composition
of ECM varies from batch to batch and chances of immunogenicity and zoonosis
with xenogeneic sources of ECM cannot be ruled out.
18.3.8 Other Natural Biomaterials
Other natural biomaterials such as glycosaminoglycans, hyaluronic acid, Poly

(3-hydroxyoctanoate) etc. have also been used in CTE. Glycosaminoglycans are the
most abundant polysaccharide present in the body in the form of long unbranched
disaccharide units, where units contain either of two such as N-acetylgalactosamine
or N-acetylglucosamine and a uronic acid such as glucuronate or iduronate.
Hyaluronic acid is a macromolecule made of linear glycosaminoglycans and has been
reported to improve neovascularization of tissues and normalize the left ventricular
function in infracted rat hearts (Ventura et al. 2007). Poly (3-hydroxyoctanoate), Poly
(3-hydroxyoctanoate) is a medium chain length polyhydroxyalkanoate produced by

bacterial fermentation. Poly (3-hydroxyoctanoate) polymer was found to support cell
viability, proliferation and adhesion and resembled cardiac tissues in terms of their
mechanical properties (Bagdadi et al. 2016). In another study, Mussel-inspired
conductive Ti2C cryogel was shown to promote the functional maturation of CMs
(Ye et al. 2020). The cryogel was developed as 3D vessels-shape framework and
when seeded with CMs, they remarkable aligned sarcomere with primitive interca-
lated disc between the CMs and showed synchronous beating. Transplanted of the
Ti2C cryogel into MI rat model, could improve cardiac function (Ye et al. 2020).
More recently, Walker et al. (2019) engineered an adhesive and electrocon-
ductive cardiac patch by electrospinning of gelatin methacryloyl (GelMA) and
conjugation of a choline-based bio-ionic liquid (Bio-IL). These patches strongly
adhered to the myocardium by forming ionic bonds and did not require suturing.
Culture of primary CMs and fibroblasts could form contractile tissue constructs and
established gap junction with superior mechanical and electroconductivity. List of
natural biopolymers and other commercially available biomaterials used in CTE is
shown in Tables 18.1 and 18.2.
Table 18.2 Overview of natural biomaterials used for cardiac tissue engineering
Polymer Co-polymer/ Fabrication Cell type References
Crosslinking method seeded
Collagen Chitosan, Electrospinning MSCs, Shi et al. (2011),
Glycosaminoglycan, CD133+ Telemeco et al.
gelatin or Matrigel/ cells (2005), Xiang et al.
GA, EDC or EDAC (2006)
Gelatin Alginate, Electrospinning MSCs Balasubramanian
Chitosan-agarose, et al. (2013), Bhat
Fibrinogen, PGS and Kumar (2012),
Ravichandran et al.
(2013a)
Fibrin Thrombin Hydrogel CMs, Black et al. (2009),
EC, Thomson et al.
fibroblast (2013)
Alginate PEO, PVA, Elastin, Electrospinning, Fetal Moller et al. (2011),
and PEG Hydrogel CMs Nie et al. (2008)
Chitosan PCL, Gelatin Hydrogel CMs Chatzinikolaidou
et al. (2014), Chiu
et al. (2012)
Fibroin Chitosan/HA Films, Hydrogel, MSCs, Rahimi et al. (2014),
CMs Yang et al. (2009)
Decellularized Silk Porous aligned MSCs, Bronshtein et al.
ECM scaffold CMs (2013), Stoppel et al.
(2015)
18.4 Enhancing the Elasticity and Electrical Conductivity

of Natural Biomaterials for CTE
Polymeric scaffolds for CTE must exhibit elasticity and high electrical conductivity
to mimic the flexibility, electrical conductance, and contractility of native cardiac
tissues (Ye et al. 2020). Many of biomaterials currently used in CTE lack of
electrical conductivity and appropriate mechanical properties. This can be over-
come by use of hybrid biomaterials combining two or more natural and synthetic
biomaterials or combining with conductive nanomaterials. For examples, natural
polymers such as chitosan have low elastic modulus and poor electrical conduc-
tivity, which could be improved by doping with carbon nanofibers to develop a
highly conductive composite scaffold (Martins et al. 2014). Chitosan/carbon
composite scaffolds had an elastic modulus of 28.1 ± 3.3 kPa, similar to that
measured for rat myocardium, and excellent electrical properties, with a conduc-
tivity of 0.25 ± 0.09 S/m (Martins et al. 2014). Similarly, incorporation of carbon
nanohorns into collagen was reported to promote the proliferation of rat CMs and
inhibit the proliferation of cardiac fibroblasts (Wu et al. 2016).
Metallic nanoparticles such as Au-NPs have also been combined with natural
polymers to develop thermosensitive and/or electro-conductive scaffolds (Min et al.
2018). Baei et al. (2016) developed a thermosensitive conductive hydrogel with a
highly porous network of interconnected pores by incorporating nanoscale
electro-conductive Au-NPs into chitosan hydrogels. AuNP-collagen matrix with
localized nanoscale stiffness in the substrate activated the b1-integrin signalling,
which mediates the activation of integrin-linked kinase and its downstream signal
kinase for cardiac cell differentiation (Li et al. 2016). Sapir et al. (2014a) created a
functional cardiac patch by incorporating magnetically responsive nanoparticles
into macroporous alginate scaffold and applying external magnetic stimulation. In
recent years, incorporation of reduced graphene oxide (rGO) into biomaterials has
also attracted great attention. Shin et al. engineered cardiac constructs using
rGO-incorporated gelatin methacryloyl (GelMA) hybrid hydrogels (Shin et al.
2016). The incorporation of rGO into the GelMA matrix significantly enhanced
electrical conductivity and mechanical properties of material and exhibited better
viability, proliferation, and maturation of CMs compared to those cultured on
GelMA hydrogels. Similarly, Jiang et al. (2019) developed electroconductive
porous scaffold of chitosan for CTE by blending with GO. Incorporation of GO into
chitosan resulted in an electro-conductivity of 0.134 S/m, which is in the range of
conductivity required for native heart tissue. Combination of natural biomaterials
with synthetic biomaterials has also been tried. Bhaarathy et al. used copolymer of
poly (l-lactic acid)-co-poly (e-caprolactone) (PLACL), silk fibroin and Aloe Vera
for fabricating biocomposite nanofibrous scaffolds for CTE (Bhaarathy et al. 2014).
18.5 Enhancing the Mechanical Properties of Natural

Polymers for CTE
While natural biomaterials such as gelatine, chitosan etc. provide good

hydrophilicity to support cell attachment, they are difficult to handle during surgical
reconstruction of heart defects. It can be overcome by use of sandwiched scaffold
wherein biomaterial of high tensile strength such as PCL is sandwiched in natural
biomaterials such as gelatine-chitosan (Pok et al. 2013). The PCL core could
provide surgical handling, suturability and high initial tensile strength, while the
gelatin-chitosan scaffold allowed for cell attachment, with pore size and mechanical
properties conducive to CMs migration and function. Similarly, Ravichandran et al.
fabricated PGS/fibrinogen, core/shell fibers with core as elastomeric PGS that
provided suitable mechanical properties comparable to that of native tissue and
shell as fibrinogen to promote cell-biomaterial interactions (Ravichandran et al.
2013b). The PCL has high mechanical strength with slow degradation rate, there-
fore incorporation of hybrid composite with polydioxanone fibers by electrospin-
ning improves mechanical properties compared to PCL alone for vascular smooth
muscles generation (Pan et al. 2017). Hence, hybrid materials with tunable
mechanical properties have attracted researcher’s attention in the field of tissue
engineering. The hybrid material of polylactide with aniline tetramer shows
excellent ductility and enhanced modulus for the differentiation of myoblast to
myotubes (Xie et al. 2015). In addition, crosslinking of hybrid materials with
genipin represented lower elastic modulus suitable for soft tissue engineering
(Garnica-Palafox and Sanchez-Arevalo 2016). The first reported hybrid scaffolds
with the help of 3-D stereolithographic printing of chitosan and polyethylene glycol
diacrylate were found to have *400 kPa elastic modulus with interconnected pores
suitable for the repair of soft tissues (Morris et al. 2017).
18.6 Natural Biomaterials as Coating Materials
Natural biomaterials such as gelatine, collagen, Matrigel™, fibronectin, laminin,

bioactive glass etc. have also been used commonly to bio-functionalize the synthetic
biomaterials-derived scaffolds to enhance cell adhesion, cell-to-cell communication
and cellular interaction with scaffold (Oliver et al. 2019). Synthetic RGD has also
been used to mimic biochemical signals for cell attachment (Lambshead et al. 2018).
Most cell types attach to the biomaterials in an RGD domain-dependent manner and
therefore, synthetic RGD peptides were effecting interaction of CMs, osteocytes and
other cells with biomaterials (Hoyos-Nogues et al. 2019). CMs are also responsive to
synthetic fragments of fibronectin, vitronectin, and stromal-derived factor-1 (Liao
et al. 2018). Thus, inclusions of these synthetic sequences into biomaterial formu-
lation are expected to improve cell adhesion and play an important role in cell
retention during dynamic cell seeding of scaffolds.
18.7 Types of Scaffolds for CTE from Natural

Biomaterials
Generally, scaffolds are designed to be fibrous or porous in nature. Fibrous scaffolds

mimic the nanoscale structure of the native ECM and their topology and porosity
can be designed to modify the cell behaviour. They contain a large
surface-to-volume ratio, high permeability and interconnecting pore structure,
which allow efficient infiltration and proliferation of cells. On the other hand,
porous scaffolds support host cell infiltration and vascularization, and provide a
large surface area for cell adhesion, differentiation and proliferation, thus making
them advantageous for growing functional 3D tissues.
18.7.1 Fibrous Scaffolds
Fibrous scaffolds are generally created by electrospinning of polymer solutions. The

process of electrospinning involves dispensing of a biopolymer out of a syringe
needle and its exposure through a charged voltage gradient between the needle and
a collector. The diameter, density, and alignment of electrospun fibers can be
controlled by optimizing the viscosity and flow-rate of the polymer solution, the
voltage gradient, and the topography of the collector (Zhou et al. 2019). Natural and
hybrid biomaterials have been used for electrospinning (Pushp et al. 2017). In
addition, composite materials comprised of layers of different polymers such as
PLGA/gelatine can also be created by electrospinning for developing cardiac pat-
ches (Prabhakaran et al. 2011). Bio-functionalized fibrous scaffolds can also be
electrospun by adding functional materials to the polymers before or after elec-
trospinning. Kai et al. incorporated Polypyrrole (PPy) to PCL/gelatine solution
before electrospinning to impart electroconductivity to the nanofibers.
Various modifications for creating specialized nanofibrous scaffolds have also
been reported. Ravichandran et al. (2013b) fabricated core/shell fibers by co-axial
electrospinning, with PGS as core material and fibrinogen as shell material. Among
various modifications, the creation of aligned nanofibrous scaffolds is considered as
the most important for CTE as they structurally mimic oriented ECM of the heart
(Parrag et al. 2012). Gelatin-coated aligned PCL nanofibrous scaffold greatly pro-
moted cell attachment and alignment because of biological components and ordered
topography of scaffolds (Kai et al. 2011). In our study, we observed that PCL
aligned nanofibers, coated with collagen I, promoted cell attachment and prolifer-
ation of MSCs cells. The MSCs attached, penetrated, and spread well on the surface
of aligned nanofibrous scaffold than random nanofiber (Pushp et al. 2019). Similar
results were also observed with iPSCs (Fig. 18.2). However, the biggest limitation
of electrospinning is the inability to form thick scaffolds due to low fiber packaging
density and entrapment of cells (Venkataraman et al. 2018).
Fig. 18.2 Growth of human induced pluripotent stem cells (iPSCs) on Matrigel coated (A) plates
and aligned PCL nanofibers (B)
18.7.2 Porous Scaffolds/sponges
Porous scaffolds or sponges can provide true 3D scaffolds with desired thickness.
Highly porous scaffolds with interconnected pores can be created by lyophilizing
the biomaterial solution (Petrauskaite et al. 2016), salt extraction (Caspi et al. 2007),
selective laser sintering (Mota et al. 2015) or microfabrication (Engelmayr et al.
2008). Porous scaffolds for cardiac grafts have been most commonly created using
collagen (Copes et al. 2019). Collagen composite with fibrin resulted into com-
pressive stiffness of 18 and 21 kPa comparable to native myocardium for
iPSCs-derived CMs whereas the beating CMs were appeared only on pure collagen
groups (Kaiser et al. 2019). A major limitation of porous scaffolds is difficulty in
uniform seeding of cells into the scaffold (Alaribe et al. 2016) during the cell
culture. Cell seeding generally results in higher cell densities towards the surface
and lower towards core of the scaffolds (Nikolova and Chavali 2019).
18.7.3 Hydrogels
In another approach, cells can be suspended into non-porous, non-fibrous hydro-

gels, which are then cross-linked for complete encapsulation of the cells into
construct. Hydrogels are cross-linked networks of polymers with high water content
between polymer chains. They not only acts as scaffolds to provide physical support
but also permit the suspended cells to migrate and assemble into contractile tissues
to form spheroid-like micro-tissues (Fang and Eglen 2017). Hydrogels have been
created using natural biomaterials such as fibrin (Myu Mai Ja et al. 2018), collagen
(Hasan et al. 2015), and chitosan (Lu et al. 2009) etc. Composite hydrogels have
also been created by combining natural and synthetic biomaterials such as
PEGylated fibrin (Zhang et al. 2008). Cross-linking of polymers for hydrogel
formation can be done by various methods such as temperature modulation,

enzymatic activation, alteration of pH, or ionic cross-linkage. Unlike porous or
fibrous scaffolds, the shape, stiffness and thickness of cell-encapsulated hydrogels
can be easily controlled and therefore, they can be directly injected into the heart
tissue in vivo as in situ construct (Ye and Black 2011). A major limitation in the use
of hydrogels for CTE is its inherent heterogeneous nature which can influence
mechanical properties of the construct and may result in graft failure due to
improper mechanical and electrical integration into recipient myocardium.
18.7.4 Bio-Fabricated/Micro-Fabricated Scaffolds
In recent years, need for microfabrication of scaffolds has been felt to mimic the
natural structure of the heart. Mammalian heart has unique and intricate structural
organization such as parallel aligned myocytes and myofibers which varies along
the depth of the ventricular wall. These topological cues and structural anisotropy of
heart are important for CTE and therefore, strategies such as nanopatterning of
biomaterials, stamping of ECM proteins on scaffolds, 3D tissue printing,
textile-templated scaffolds etc. have been attempted as novel means to mimic native
ECM and enhance functionality of cardiac tissue construct (Zhang et al. 2019).
Thomson et al. microtemplated fibrin scaffolds with uniform architecture of parallel
microchannels (60 lm), surrounded by an interconnected microporous network of
pores (27 lm) and mechanical stiffness (70–90 kPa) comparable to native cardiac
tissues (Thomson et al. 2013). Tri-culture of CMs, EC and fibroblasts on
microtemplated fibrin scaffolds mimicked native cardiac tissue in structure and
cellular composition.
Natural biomaterials have also been used as bioink to develop scaffolds of
pre-defined and organized architecture by 3D tissue printing or prototyping. The 3D
bioprinting allows the deposition of cells, biomaterials and signalling molecules
into precisely organized geometrics and therefore, helps in biofabrication of tissue
constructs similar to those found in the native heart (Alonzo et al. 2019). A number
of bioprinting technology including extrusion, inkjet, laser-assisted and stere-
olithography with natural as well as synthetic bioinks have been developed and
applied to the CTE. Gaetani et al. (2012) successfully used tissue printing tech-
nology to fabricate scaffold for culturing human cardiac progenitor cells. In yet
another methodology, using knitted conventional textiles made of cotton or
polyester yarns as template targets. Hong et al. (2020) used digital lighting pro-
cessing printer for 3D printing of silk fibroin natural polymer with
gycidyl-methacrylate as bioink. Silk fibroin was also combined with Collagen I for
3D printing and development of cardiac patches after seeding with MSC
(Sanz-Fraile et al. 2020). Mixture of different cells with hydrogels have been
printed for functional vascularized patches with the help 3D printing that meets
immunological and anatomical properties of native heart tissue (Noor et al. 2019).
18.8 Conclusion and Future Direction
Physico-chemical properties such as biodegradability, biocompatibility, porosity,

cell adhesion property, cytocompactibility etc. make natural biomaterial an excel-
lent choice for fabrication of scaffolds for CTE. However, natural biomaterials may
require to be tuned up for mechanical integrity and electrical coupling for mitigating
native cardiac tissue. Such tuning requires generation of hybrid or composite bio-
materials with nanomaterials or synthetic polymers. Conversely, natural polymers
are commonly used for biofunctionalization of synthetic biomaterials and
improving their hydrophilicity, cell adhesion property and biocompatibility. Newer
scaffold fabrication techniques such as 3D printing, prototyping and microfabri-
cation technology are also being developed which allows generation of aligned and
anisotropic heart tissue.
Since the first description of engineered heart tissue nearly two decades ago,
various research groups have also developed methods of cultivating contractile 3D
cardiac grafts. These cardiac grafts displayed functional and morphological prop-
erties of native tissue and upon grafting, they integrated into native tissue to
improve the cardiac function. However, despite progressive improvements, several
critical challenges such as identification of the best cell source and biofabrication
technique for generation of 3D vascularised tissue constructs exist. Furthermore,
automation and standardization are also needed for translation of the technology
from laboratory animals to human.
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Chapter 19
The Importance of Natural Products
in Cosmetics
Nagarjuna Reddy Desam and Abdul Jabbar Al-Rajab
Abstract People from worldwide have been using plant-based substances (Natural
Products) to enhance the appearance since the existence of mankind. In the ancient
Egypt, around 3000 BC, there is evidence of using cosmetics, and their usages have
been a necessary part in our everyday life in all cultures. Initially, natural products
have been used for beauty products; occasionally augment with paints and dyes.
Natural products have approached back with present trend cosmetic products which
are mainly derived from plant sources. Since from longer time, plant products
(Natural Products) are source of food and medicines. A broad range of natural
products is used in cosmetics preparations, skin care such as treatment of dryness,
treatment of eczema and acne, as well as antioxidant, anti-inflammatory, anti-aging,
hair care products such as hair growth imputes, hair color, scalp complaints like
dandruff, and skin protection, and also toiletry preparations. Essential oils are major
source of plants; essential oils have been used in preparation of perfumes, hair care
substances, emollient of the skin. For example, natural products have been used in
cosmetic industry avoiding side effects with traditional preparations for herbal
beauty such as Emblica officinalis (Amla), Acacica concinna (Shikakai), and
Callicarpa macrophylla (Priyangu) have been used strongly in skin care and hair
care. Moreover, Indian women are still using natural products such as Pterocarpus
santalinus L. and Curcuma longa (skin care), Lawsonia inermis L. (hair color), and
natural oils such as coconut, olive, shea butter, jojoba, and essential oils in perfumes
for their bodies. The present book chapter represents the importance of natural
products in cosmetics.
Keywords Natural products Herbs Cosmetics Essential oils
N. R. Desam (&)
Anantha Lakshmi Institute of Technology & Sciences, Anantapur,
Andhra Pradesh 515001, India
e-mail: dndnrchem@gmail.com
A. J. Al-Rajab
Etcetera Publications, Chesterville, ON K0C 10, Canada
https://doi.org/10.1007/978-3-030-54027-2_19
644 N. R. Desam and A. J. Al-Rajab
19.1 Introduction
Since the ancient times, humans from all advancements have been using infinite
substance as a source to improve their beauty, looking younger, enhance their
sexiness, and normally protect their health (Freitas et al. 2015). The substances used
commonly nowadays for these momentums are generally known as cosmetics and
cosmeceuticals (Freitas et al. 2015). Normally, cosmetics do not consist of phar-
macologically active substances, at the minimum level, and do not demonstrate the
amount of benefit of skin scientifically (Freitas et al. 2015). Moreover, in com-
parison with the classic cosmetics, they are safe, universally available from
supermarkets, medical shops, beauty salons, and online traders. Classic cosmetic
products include maquillage, toothpastes, conditioners and shampoos, hair colors,
nail enamel, perfumes, and antiperspirant (Joshi and Pawar 2015; Wanjari and
Waghmare 2015).
Cosmeceuticals, also known as cosmedics or cosmedicals, are skin care sub-
stances with active natural products with the therapeutic or drugs like interest
includes cosmetic property (Joshi and Pawar 2015; Wanjari and Waghmare 2015).
The skin care substances methodological demonstrate measures that productive
affects the skin and are issued by the functional substances (Joshi and Pawar 2015;
Wanjari and Waghmare 2015). Cosmetics could summon constructional adjust-
ments in pores and skin and beneficial outcomes in skin situations including
blackheads, pimples, hyperpigmentation, and rosacea (Joshi and Pawar 2015;
Wanjari and Waghmare 2015). Although cosmeceuticals are not considered as
medicines or pharmaceutical substances, so they don’t require any prescription to
omit, and they could be used frequently without risk and major side effects (Joshi
and Pawar 2015; Wanjari and Waghmare 2015). Cosmeceuticals of herbs and
artificial substances and their derivatives include antioxidants, antidandruff, vita-
mins, shampoos, antifungal compound, sunscreen lotions for sun protection factor,
anti-acne, anti-wrinkle, anti-aging, toothpastes, and deodorants that contain
antiperspirants (Freitas et al. 2015; Joshi and Pawar 2015; Wanjari and Waghmare
2015). Natural products such as haldi, chandan, manjistha, yastimadhu, khas, and
nagkheshara are used to gleam complexion; while arusa, amala bavchi, guduchi,
and chakmarad are mentioned as kustaharan (Kuno and Matsumoto 2004).
Moreover, natural products such as haridra, khadira, vidyanga, amalaki, abhaya,
jatisaptaparna, karacira of various promises from Knahshthag and Mahakashiya are
mentioned productively for skin disorder. Charak and Sages stated natural products
are used to remove toxins from the body, clear the entangled that conduct grow on
the skin and also secured from kushtha and boils (Kuno and Matsumoto 2004). For
example, natural products such as Eladi Gana contain ela, kusstha, tagar, jatamani,
tvak, dhmamaka, potra harenuka, shutki, stuuneyaka, choraka, guggol sarjarasa,
agaru, devedaru, and padmakesher.
As per 1989 safety regulations, consumer substance or substance deliberate
application should be used on the outermost surface of human body which includes
lips, nails, hair system, epidermis, and external medicine, as well as denticle,
19 The Importance of Natural Products in Cosmetics 645
mucous membrane for wash, fragrances used for protecting from bad smell for the
determination of treating and stopping disease (Zuorro and Lavecchia 2010).
Novel natural active chemical constituents are derived from the plant kingdom,
earth, and sea. Approved active chemical constituents include vitamins, food fibers,
minerals, enzymes, hormones, antioxidants, multitude of naturals, and Chinese
herbs. Historically, natural products have been used commercially, and till now,
several new natural products consist of natural oil and herbs that are available in the
market. In cosmetics, plants are the major source and base. Furthermore, natural
products are used as drugs in pharmaceutical industry, but they are not acceptable to
be used as cosmetics products. According to the cosmetic safety regulatory act in
1989, Atropa belladonna and Digitalis purpurea plant materials were forbidden
(Zuorro and Lavecchia 2010). Natural product complete extracts and specific
extracts have been used in cosmetics. Total natural products and specific extracts
are employed which are the major cause of determination of their specific activities.
Some selective natural products were applied in different zone of use, for example,
Glycyrrhiza glabra for skin annoyance; Ginkgo biloba as antioxident; Berberis
vulgaris as skin glowing (Zuorro and Lavecchia 2010). Natural products are major
source to make soaps such as S. officinalis (soapwort) in Europe; Y. glauca (yucca)
in southern USA; S. indica as soapnut in India; Phytolacca dodecandra L., and
Quillaja saponaria L., in Africa and America, respectively (Zuorro and Lavecchia
2010). This is extremely interesting to know how plants are differing from other
plants used which differ from tree bark to berries. The present chapter represents the
importance of natural products in cosmetics and cosmeceuticals.
19.2 Background
In Africa Homo sapiens, over 100,000 years ago, African Middle Stone Age dis-
covered the red bister including crayons are the application of cosmetic body
(Murube 2013). An additional proof in ancient Egypt, about 10,000 BC, humans
used scented oil, lotions to clean, skin soothe, and mask their body odor (Murube
2013). Then, about 3,000 BC, the use of cosmetics such as hair and skin care
products became more common in Egypt and also expanded to large parts of Asia
and Africa (Hetta 2016a, b; Murube 2013). Historically, women and men were
applied kohl—as eye makeup which also protects the eyes from dry winds and sun
radiation (Hetta 2016a, b; Murube 2013). Castor oil skin lotions and creams were
prepared and used for the skin protection. Also, extracts from red algae for abrade
lipsticks; sometimes from fish scales to obtain the glittering substance and crunch
on Glycyrrhiza glabra root sticks to improve their breath. Silent basic natural active
ingredients such as almond oil, sesame oil, lily, rosemary, peppermint, rose, aloe,
lavender, chamomile marjoram, and thyme oils were used in perfumes (Hetta
2016a, b; Murube 2013).
Around 100 AD, Ancient Rome traditionally used makeup and beauty products.
It was their trend and faith that became a necessary part of their daily life
(Blanco-Dávila 2000). Beauty products like body lotions, eyeshadow, liners, talcum
powders, nail products, perfumes, and toothpaste products were used by women in
Roman daily and also beauty masks to make their faces glow, while Roman men
were used it as hair dye. Particularly, women’s social status, attractiveness, wealth,
clothes, makeup embellish in. In 215 BC, Lex Oppia established a law extravagance
to control gorgeous and luxurious; women could purchase and wear (Hsieh et al.
2016; Naidoo et al. 2016; Stutesman 2016; Watson 2012). After six years, this law
was reversed; but people from Ancient Rome took cosmetics to new heights and
limits. Now, in Milan, there are the largest beauty empires in the world.
Prior to twentieth century, about the year 1910, cosmetics were used more in
fashion in Europe and USA. In 1910, in Paris, they introduced color
makeup. Hence, in 1920s, film industry introduced in Hollywood had an advanced
influence on cosmetics (Jones 2011). Theda Bara was a great movie star of the
silent movie period, and her makeup artist has been developing the cosmetics
production (Jones 2011). In 1920s, the cosmetic substances were highly demanded
to inculcate consciousness in American women. The conduct among others to the
wing variety which was identified by fearless dark eyes, red nail polish, red lipstick,
eyebrow pencil, mascaras artificial hair color, brownish sunscreens (Murnen and
Seabrook 2012; Schlessinger 2007). In the entire world, cosmetics were shortsupply
between 1939 and 1945 during the Second World War. During this war, important
part owed to the fact that alcoholic and petroleum products are the best active
ingredients of several cosmetic products.
The late twentieth century, between 1960 and 1970s, women in the western
world used the cosmetic products desert to cheapen women to sex objects (Oréal
2015). In 1970s, it was a trend to develop the natural looking products.
Coincidently different lipstick colors (like green, red lilac, silver, and pink),
non-allergenic cosmetics makeup powders with longer staying have been been
developed (Oréal 2015), and also males started to use cosmetics to improve facial
features (Ribeiro et al. 2015). They have been developed the most popular cos-
metics such as mask for dark circles, age spots, and blemishes on the skin and large
pores on the skin (Ribeiro et al. 2015).
Cosmetics and cosmeceuticals industries have a large market worldwide which
was estimated over 200 billion US dollars in the year 2015 (Chaudhri and Jain
2009; Ribeiro et al. 2015). This might be qualified to increase in world economy,
changing the way of life, increasing utilization and personal skin and hair care of
new commercial naturally developed substances. Worldwide in the main retailers,
36.9% of share is from Asia and Pacific regions, and share from America, Europe,
Africa, and Middile East holds 30.8%, 29.5%, and 2.9%, respectively (Chaudhri
and Jain 2009). In global markets, accounted skin, hair, makeup, fragrances, and
hygiene products are 36.3%, 22.9%, 18.2%, 12%, and 10.5%, respectively. These
cosmetics and beauty products are usually purchased from online stores, retail
shops, supermarkets, drug stores, departmental stores, and brand outlets (Chaudhri
and Jain 2009).
19.3 From Past to Future
A modern development of cosmetics and cosmeceuticals industry is the natural

beauty products (Atanasov et al. 2015). Since ancient times, extracts from various
parts of aromatic and medicinal plants are introduced in cosmetics and cosme-
ceutical products (Barbulova et al. 2015; Kapoor 2005). Around early 1900s,
naturally a derived product adequately increases in cosmetics and cosmeceuticals
industry (Jones 2011; Kapoor 2005). Currently, organic substances or products are
more on demand throughout the world as the utilizing of natural products at low
risk of undesirable effects (Carola et al. 2012).
19.4 Source of Natural Products
Natural products are generally acquired from aromatic and medicinal plants, mix-
ture of volatile and nonvolatile chemical constituents with strong odor (Bakkali
et al. 2008). Natural products are considered as one of the most control plant
products in agriculture, as they exhibit antiviral, antibacterial, anti-cancer, antiox-
idant, antidiabetic, insect repellent, antifungal, anti-inflammatory, and cosmetic
properties (Reddy 2019; Said et al. 2016; Swamy et al. 2016). For example, from
willow tree bark, Digitalis lanata flowers and opium medicinal products derived as
aspirin, digoxin, and morphine, respectively, and also extracted from T. indica and
S. alata areal parts and seeds contains polyphenols, flavonoids, and hemicellulose
xyloglucan that were used for skin protection from sun radiation. Leaf extracts from
T. serrulata (Vahl) have been used for treatment for hair growth and hair loss
(Freitas et al. 2015). Essential oils extracted from L. aestuans are used as facial
toners, lotions, lip balms, ointments, creams, scrubs, massage oils, masks, and
shampoos for antidandruff and also used for treatment for eczema, acne, chicken
fox, insect bites and scarring from burns (Reddy 2019). M. olefira leaf and seed oil
extracts contain considerable amount of b-carotenes, vitamins A, C, & E, and
polyphenols using anti-inflammatory and antioxidant activities also have been used
for body oils, scrubs, lotions, balms, creams, hair care, moistures sun protection,
and perfumes (General Bureau of statistics 2014). Some of these plants were used
for traditional use because monoterpenes, sesquiterpenes and triterpenoids and
phenolic compounds are present. For example, P. amboinics consist of mono, di,
and sesquiterpenes which are used for antioxidant, anti-inflammatory, antimicro-
bial, skin cleansers, anti-wrinkle, anti-skin cleansers, anti-aging night creams, and
cosmetics also used for itchy skin and insect bite (Freitas et al. 2015; Kleynhans
et al. 2017). A. indica L. oils extracted from fruits and seeds are used for preparing
soaps, creams, balms, shampoos, nail polishes and toothpastes (Mans et al. 2017;
Nagarjuna et al. 2017). These essential oils could be used as perfumes, flavoring
agents, fragrances for different cosmetics and cosmeceuticals (Swamy et al. 2016).
19.5 Extraction and Isolation of Natural Products

or Essential Oils
Natural products or volatile oils extracted by using different methods could be

applied for essential oil extraction, such as steam distillation, hydro distillation,
solvent extraction, or continuous extraction such as Soxhlet extraction, liquid–solid
extraction, liquid carbon dioxide, microwave extraction. Generally, for essential oil
extraction hydrodistillation or steam distillation quintessential is used, resulting in
less molecular weight, mixture of chemical constitutes is obtained. For example,
Lamiaceae and Citrus families have used steam and hydrodistillation. For extraction
of natural products, Soxhlet extraction and liquid–solid extraction quintessential are
used, resulting in high molecular weight of the compound.
All these natural products and essential oils are mixtures with the other chemical
constituents from the natural sources; the products must be isolated and purified.
Isolated purified compounds are acquired in milligrams to grams. After extraction,
natural products or essential oils are isolated using these methods by adsorption,
precipitation, chromatography, and crystallization methods, etc. After isolation
product needs to study chemical and physical properties, chemical structure elu-
cidation, and structural activity of relationship of the product.
19.6 Plants-Derived Cosmetics and Cosmeceuticals
A rich and diverse traditional medicinal system has been employed for traditional
medicine using different variety of plants worldwide. The uses of these plants in the
world thoroughly have been addressed by Raghoenandan (Hindustani), van’t
Klooster (Maroons), van Adel and Ruysschaert (Creoles), and Tjong Ayong
(Javanese) (Raghoenandan 1994; Tjong Ayong 1989; Van Andel and Ruysschaert
2011; Van’t Klooster et al. 2016). Around 789 medicinal plants have been pub-
lished in the Surinamese, among 10% (seventy two) of plants were used for the
purpose of cosmetics and cosmeceuticals. Among seventy-two plants, three quarter
(fifty eight) is used cosmeceuticals for treating scars, warts, pimple, pigment skin,
scaly skin, skin vexation, inflammation, pustules, and boils as well as skin fungi and
skin parasites. These properties indicated plants have significant cosmaceuticals and
medicinal properties. More than twenty-five plant families are belonging to the
cosmeceuticals, which are recommended for treating the skin-related problems.
More than 20% of medicinal plants have been used for cosmetics and cosmeceu-
ticals which include perfumes, skin care, hair care, refreshment and steam bath and
some of the cosmetics and cosmeceutical plants as shown in Table 19.1.
Table 19.1 Plants used for cosmetics and cosmeceuticals

Plant Scientific name Condition
P. guajava L. Body odor
T. serrulata (Vahl.) Genital steam baths
E. hieracifolia (L.), S. alata (L.) Flaky skin
M. maripa L., M. citrifolia L., B. excels L. Hair care
M. indica L., M. micrantha, S. trilobata, U.camphorata, M. Local skin lesions (e.g., boils
charantia L., S. alata L., A. indica, A. Juss, C. odorata L., and pustules)
C. citrates, Citrus sp., Cecropia sp.
B. orellana L., Z. mays L. Makeup
Sida rhombifolia L. Cedrela odorata L. Pigment spots
A. vera L., C. odorata L., A. galanga L. Scars
E. foetidum L., C. nucifera L., E. oleracea, M. maripa, M. Skin care
citrifolia L., L. aestuans L.
A. sativum L., L. purpureus L., S. alata L., S. reticulate L. Skin fungi
P. stellis, L. aestuans L. Skin rejuvenation
P. amboinicus Lour. G. barbadense L., Orchid spp. Skin refreshment
Amaranthus sp., C. nucifera L., C. odorata L., E. prostrata Pimples
L., M. micrantha Kunth, R. fruticosa L., U. camphorata L.,
O. cochinellifera Steud., V. guianensis, C. sativus L.,
J. curcas L., R. communis L., C. guyanensis Desf., S. alata
L., T. indica L., B. guianensis Aubl, C. alatus Aubl, C.
ramosa Aubl, M. glabra L.
A. moschatus Medik., A.purpurascens Aubl, A. indica, A.
Juss, C. odorata L., A. bilimbi L., P. coccinea Aubl., O.
sativa L., Q.amara L., C. latifolium Lam., S.leucocarpon
Dual., S. stramoniifolium Jacq., W. indica L., L. rosea, A.
galanga L.
A. curassavica L., P. acuminata Ait., C. difussa Burm.f. Warts
B. pinnatum (Lam), G. barbadense L. Warts
E. prostrata L., M. micrantha Kunth., U.camphorata L., C. Skin irritation (e.g., rash,
cujete L., B. orellana L., T. ulei Vaupel., C. difussa Burm.f., dermatitis, and eczema)
C. urens L., M. guianensis Aubl., S. alata L., L. nepetifolia
L., O. tenuiflorum L., A. moschatus Medik., A. indica, A.
Juss, C. guianensis Aubl., C. odorata L., F. schumacheri,
P. marginatum Jacq.
U. camphorata L., C. guianensis Aubl. Skin parasites
19.6.1 Manufacture of Plant-Derived Cosmetics

and Cosmeceuticals
Based on the earlier abundant plant, raw material could be processed into cos-
metics, cosmeceuticals, and medicinal preparations. As a result in the worldwide
increasing number of entrepreneurs such as individuals, small, medium, and large
scale gross on medicinal plants sector. Hence, either collecting or cultivating the
raw materials homeless they act as retailers or mediator or supplier for processing or
preparation of cosmetics or cosmeceuticals intermediates or final products (Playfair
et al. 2011). For example, in Suriname Skin Glanz cosmetics Odany Jewa, and Jomi
cosmetics are specialized in skin care-related cosmetics and cosmeceuticals from
start. These include scrubs, face washes, liver spots, eye bags, day and night
creams, ointments, removal of impurities on the skin and facial cleaners, etc. (Grant
2017). However, while using the cosmetics/cosmeceutical materials caution and
possible side effects such as photosensitivity and skin allergy are perfectly men-
tioned in the user instructions (Grant 2017).
Cosmetics and cosmeceuticals are usually produced from different fresh plant
parts such as leaves, fruits, barks, seeds as well as unfinished substances such as
waxes and vegetable oils from plants as shown in Table 19.2. The acquire plant raw
materials are from growers, collectors, and vendors who mainly operate in different
places in the world. Hence, plants of these parts grow easily and persistently
encountered in the wild. L. aestuans is an exception West Indian wood nettles
relatively rare (Grant 2017). Extraction of other important constitutes such as oils
are complicated process from these plants such as C. nucifera (coconut tree), C.
guyanensis, V. paradoxa (shea butter), A. chinensis (jojoba oil), and C, guianensis.
Table 19.2 Plants used for preparation of cosmetics and cosmeceuticals

Family Plant Scientific name Use of cosmetics/
cosmeceuticals
Asphodelaceae A. vera (L.) Skin care products
Arecaceae C. nucifera L., E. oleracea Mart., M. Skin, Hair, and Eye care
maripa products
Asteraceae E. prostata L. Skin care products
Crassulaceae B. pinnatum Skin care products
Cucurbitaceae C. sativus L. Skin care products
Commelinaceae T. serrulata L. Hair care products
Fabaceae C. guyanensis Eye and hair care products
S. alata Deep skin cleaners
T. indica L. Skin care products
Lecythidaceae B. excelsa Hair and skin care products
Lamiaceae L. nepetifolia Eye care products
P. amboinicus Skin care products
Moringaceae M. oleifera Skin care products
Malvaceae H. sabdariffa Skin and hair care products
Meliaceae C. guianensis Eye, Hair and Skin care
products
Orchidaceae Orchid spp. Skin care products
Poaceae C. citratus Skin care products
Urticaceae L. aestuans Skin care products
Rubiaceae M. citrifolia Hair and skin care products
19.6.2 Substances of Plant-Based Cosmetics

and Cosmeceuticals
The most frequently used plants in cosmetic and cosmeceuticals are shown in
Table 19.2. There are some plant leaves especially for facial washes such as A.
indica, A. vera, M. oleifera, C. citrates, and Y. indica. C. citrates and P. amboinicus
leaves are used for facial milks. The leaves and fruit juices of these important plants
such as E. oleracea, C. sativus, M. citrifolia, A. indica, C. nucifera, T. indica, and
H. subdariffa L. are used to synthesis day and night skin lotions. To remove dead
skin cells, stimulate blood circulation and facial scrubs E. oleracea fruit granules
are used mainly and also for deep skin cleaning E. prostate, S. alata, and T. indica
essential components of leaf juices are used. M. pleifera and L. aestuans leaves
extracts contain active ingredients for treating eczema, chapped lips, insect bites,
and acne, also leaves extracts of T. serrulata are used for treating hair growth.
Particularly most of the above-mentioned plant extracts are used for skin care
creams, lotions, and scrubs. Oils extracted from leaves, fruits, and bark from these
plants B. excels (Brazil oil), C. nucifera (coconut oil), C. guianensis (karapa oil), M.
maripa (maripa oil), and C. guyanensis (hoped oil from bark) are used for treating
hair growth and hair care products. A. indica, B. pinnatum, and L. nepetifolia leaves
extracts are used eye masks, ointments, and treating eye bags and acne. Significant
unfinished products such as shea butter, grapeseed oil, jojoba oil, coconut oils are
used for preparing hair care, skin care, soaps, moisturizers and sunscreens, hair
conditioners, anti-wrinkle formulations, hair conditioners, ointments, baby oils,
skin inflammation, and juices extracted from A. indica and P. amboinicus are
commonly used for preparations of facial masks.
Some of the above indicated medicinal plants used for cosmetics and cosme-
ceuticals and their plants phytochemical composition are widely presented in
Table 19.3. Few of the plants are summarized below, for example,
Aloe vera (L) Burm.f.
This plant is mainly used in traditional medicinal treatment of microbial infections,
skin conditions, constipation, and diabetic mellitus (Manvitha and Bidya 2014;
Mahor and Ali 2016). This indicates that leaves of A. vera extracts contain sig-
nificant therapeutic agents such as polymannans, lectins, anthraquinones, and
acetylated mannans (Hamman 2008; Moghaddasi and Verma 2011); that’s why the
leaf gels are included in soft drinks and dilatory additives for digestion (Eshun and
He 2004; Qadir 2009). It is moreover used in sunburns, ointments, shampoos,
conditioners, skin lotions, sunscreens, facial tissues, soaps, makeup, moisturizers,
and shaving creams which are used for the main applications for smoothening and
moisturizing effects of A. vera leaf gel (Eshun and He 2004; Hamman 2008;
Manvitha and Bidya 2014; Mahor and Ali 2016; Moghaddasi and Verma 2011;
Periasamy et al. 2014; Qadir 2009).
Table 19.3 Plants and its main parts used for preparation of cosmetics and cosmeceuticals and its
presumed active chemical constituents
Plant Scientific Main plant part’s Active chemical constituent
name used
A. vera L. Gel from fresh Polysaccharides
leaves
A. indica, A. Juss Fresh leaves and Limonoids
seed
B. excelsa Fresh leaves Glycosides, saponins
C. guianensis Seeds Limonoids, fatty acids
C. nucifera L Fruits and seed Phenolic compounds, vitamin E, terpenes,
saponins
C. guyanensis Trunk Terpenes
C. sativus L. Fruits Water, antioxidants
C. citratus Leaves Essential oils
E. prostata L. Leaves Coumestans, glycosides, amyrins
E. oleracea Mart Fruits, seeds Anthocyanins
H. sabdariffa L. Fresh calyces Anthocyanins, vitamin E, flavonoids
L. aestuans L. Leaves Essential oils
L. nepetifolia L. Leaves Flavonoids, terpenes, essential oils, coumarins
M. maripa Seeds Vitamins A and E, fatty acids
M. citrifolia L. Fruits and seeds Flavonoids, fatty acids
M. olifeira Leaves Ben oil
Orchid spp. Seed Essential oils
P.boinicus Leaves Essential oils
S. alata L. Leaves Phenolic compounds, essential oils
T. indica L. Leaves and seeds Polyphenols, flavonoids, xyloglucan, fatty
acids
Azadirachta indica A. juss.

In India, A. indica leaves, fruits, seeds, bark, and root extracts were used against
different types of diseases in Ayurveda over two millennia such as diabetic mellitus,
high blood pressure, fever, cold respiratory conditions, and cosmetics. Especially,
oils extracted from seeds and fruits have been used for traditional medicine against
redness, inflammation of the skin and acne. These results indicate that A. indica
leaves extract contains nimbinin and Azadirachtin phytochemicals, which are useful
for antimicrobial and antiparasitic activities. Neem oil contains oleic acid, palmitic
acid, linoleic acid, and stearic acid phytochemicals. Hence, it could be used in
synthesis of different cosmetics like nail polishes, toothpastes, shampoos, soaps,
and balms (Djibril et al. 2015; Galeane et al. 2017; Hashmat et al. 2012;
Mak-Mensah and Firempong 2011).
Bertholletia excelsa Humb. & Bonpl.

B. excelsa is a native of Amazon forest, essential seeds of the Brazilian nut.
These seeds are more abundant of vitamins, digestible minerals, staple diet, and
dietary fiber. Brazilian nuts are commercially collected and mixed with different
nuts. Oils extracted from these nuts consist of more than 75% of unsaturated fatty
acids, mainly selenium, linoleic acid, oleic acid, phytosterols, polyphenolic
acids, and vitamin E, particularly polyphenols and vitamin E show antioxidant
properties. Hence, these phytochemicals could be used mainly in lotions, creams,
shampoos, hair care products, aging skin, flaky skin, skin inflammation, and
acne. Hence, the fatty acids are the applications of moisturizing effects
(Chunhieng et al. 2004; Chunhieng et al. 2008; John and Shahidi 2010;
Kluczkovski et al. 2015; Yang 2009).
Bryophyllum pinnatum (Lam) Oken.
Since ancient times, mother-of-thousands B. pinnatum (Fig. 19.1) is especially used
for the sores, boils, wounds, treat for burns, boils, as well as it has antiseptic,
amphiphatic, antimicrobial, anti-inflammatory and conventional astringent proper-
ties. Leaves of B. pinnatum extracts consist of mainly fumaric acid and saponins
(from various parts of plant), which is more essential for cosmetics and cosme-
ceuticals substances for skin regeneration formulas, hair growth syrups, facial
creams, creams for wrinkles, treatment for acne, and anti-aging lotions (Afzal et al.
2012; Akpuaka and Ezem 2011; Amenta et al. 2000; Dey et al. 2012; El-Abdellaoui
et al. 2010; Kaur et al. 2014; Nagaratna and Hegde 2015).
Fig. 19.1 The

mother-of-thousands
Bryophyllum pinnatum
(Lam) Oken. (Crassulaceae)
Carapa guianensis Audl.

The bark and seed oil extracts of C. guianensis are composed of alkaloids and
terpenoids, respectively, which is used for antipyretic and antiallergic, antiparasitic,
antimicrobial, anti-inflammatory and also having anti-wound healing medication
purpose, respectively. Leaf and fruit extracts were traditional medicine for treatment
of itching and intestinal worms. The crab or carap oil is composed of many fatty
acids such as oleic acid, linolic acid, and palmitic acid which is used for preparation
of skin and hair care products (Campos et al. 2007; Cabral et al. 2013; Henriques
and Penido 2014; Letawe et al. 1998; Miranda Júnior et al. 2012; Nayak et al. 2010;
Pereira et al. 2014).
Cocos nucifera L.
Parts of C. nucifera plant are used for the traditional medicine, for example, in
Brazil, Haiti, Papua New Guinea it used for treatment of arthritis from the husk
fiber, root extracts are used to treat diarrhea, amenorrhea, and stomach pins,
respectively. As per Indonesian and Fijan beliefs, coconut oil prevents hair loss.
These are causes for the presence of tannins, phenols, flavonoids, triterpenes,
steroids, saponins, alkaloids, and condensed tannins as well as fatty acids and
vitamin E in different parts of plant. For these causes, coconut oil is base essential
element for hair conditioners, shampoos, shower gel, sunscreens, moisturizers,
body butters, nourishing, and emollients, skin infections, anti-aging, prevents dry
skin and anti-redness (Da Fonseca et al. 2014; Esquenazi et al. 2002; Gopala et al.
2010; Holdsworth 1992; Lima et al. 2015; Sachs et al. 2002; Weniger et al. 1986).
Copaifera guyanensis Desf.
Copaiba oils extracted from C. guyanensis (Fig. 19.2) tree trunk and bark have been
many medicinal and cosmetic properties, were already known to American Indians.
The trunk of C. guyanensis is used for injured animals to heal their wound and also
it could be used for the treatment of skin diseases, to stimulate wound healing,
infections, inflammations, and malignancies. Copaiba oils consist of biologically
active chemical constituents such as diterpenens and sesquiterpenes, kaurenoic acid,
copalic acid and caryophyllene. Because of these, they exhibit antibacterial, anti-
eczema, anti-inflammatory, antibacterial, antifungal (especially S. aureus) and
antioxidant properties. Hence, copaiba oils are significantly used in cosmetic
industry in scars, stretch masks, shampoos, capillary lotions, soaps, bathing foams,
and anti-acne (Da Silva et al. 2012; Leandro et al. 2012; Lucas et al. 2017; Veiga
et al. 2007).
Cucumis sativus L.
The plant of C. sativus is composed of more than 90% of water; also contains
vitamin B, C, & K and b-carotene phenols and flavonoids phytochemicals. C.
sativus fruits, fruit juices, fruit water, and fruit extracts are essential to be used in
cosmetic formulation, the water considerably is essential benefit for the skin such as
skin conditioning, eye and facial makeup, neck and face products, bath foams,
Fig. 19.2 Harvesting copaiba

oil from the trunk of
Copaifera guyanensis Desf.
(Fabaceae)
soaps, skin hydrating products, cleaning products, detergents, facial peel products,
skin rejuvenation products, body and hand lotions and hair care products (Akhtar
et al. 2011; Ibrahim et al. 2010; Kumar et al. 2010; Mukherjee et al. 2013; Murad
and Nyc 2016; Rajasree et al. 2016).
Cymbopogon citrates (Dc) stapf.
The essential oils and phytochemicals are extracted from the leaves of C. citates
(lemongrass), extensively used for the antimicrobial, antipyretics, diuretics, insect
repellents, anti-inflammatory, antiprotozoals, antidyspeptics, spasmolytics,
antibacterial, antifungal, antidiarrheal, antioxidants, antipyretics, cytoprotective.
Oils are extracted from the leaves, phytochemical is composed of flavonoids,
phenolic compounds, and essential oil is composed of citral, geraniol, citronella,
citronellol, and myrcene. Essential oils are nice smelling, resulting in it could be
used as fragrances, perfumes, creams, detergents, and creams. Preparation of
ionones for cosmetics and perfumes citral is the main starting molecule. Essential
oils are mainly used for the synthesis of skin care products, like creams, lotions,
facial cleansers (Ekpenyong et al. 2014, 2015; Ganjewala 2009; Maheshwari et al.
2014; Mosquera et al. 2016; Olorunnisola et al. 2014; Pandey 2017; Shah et al.
2011).
Eclipta prostate (L.) L.

Extracts from different parts of E. prostate are used for respiratory disorders,
gastrointestinal complaints, fever, skin problems, microbial, parasitic infections,
spleen enlargement, liver ailments, as well as hair greying and hair loss. Ethonoilc
and petroleum ether extracts of E. prostrate leaves are used for hair oils in
Ayurveda and hair growth respectively. Extracts are composed of various phyto-
chemicals such as oleanane type glycosides (eclalbasaponins) and triterpenes
(oleanolic acid, ursolic acid, & amyrins). These compounds show potential
antibacterial, antieczema, anti-inflammatory, and antioxidant properties. Hence,
because of these reasons oils and extracts of E. prostrara are used for skin nour-
ishing and skin anti-aging agents, as well as hair and hair growth (Baldi et al. 2011;
Chan et al. 2014; Datta et al. 2009; Kaur and Chandola 2010; Kumar et al. 2005;
Roy et al. 2008; Saraswat et al. 2015; Uddin et al. 2010).
Euterpe oleracea Mart.
Various parts of E. oleracea extracts are used worldwide especially in Brazil
processed into pulp, which is used for food products, juice in beverages, smoothies,
and dietary supplements. Extracts of E. oleracea pulps are mainly composed of
polyphenolic anthocyanin, palmitic acid, and oleic acids. Resultant extract shows
potential antioxidant properties. Phytochemicals such as anthocyanins and phenolic
compounds are significantly used for the anti-aging lotions, skin regenerating
creams, treatment for skin damage, sunscreens, and anti-inflammatory products as
well as fatty acids are used as cosmetics as hair conditioners, skin moisturizers,
soaps, and shampoos (Bobbio et al. 2000; Daher et al. 2014; De Santana et al. 2017;
Hogan et al. 2010; Kang et al. 2010; Portinho et al. 2012; Schauss et al. 2006;
Yamaguchi et al. 2015).
Hibiscus sabdariffa L.
H. sabdariffa L. (Fig. 19.3) is extracted from calyces used in traditional folk
medicine system for a wide range of kidney disease, cough and bronchitis, gas-
trointestinal problems microbial infections and cosmetics. These applications
indicate that the plant extracts contain vitamin E, flavonoids, phenolic substances as
well as antioxidant anthocyanins, antibacterial and anti-inflammatory effects.
Hence, H. sabdaritha L extracts are considerably used for preparations of hair and
skin care products as well as flowers of H.sabdaritha L. crude polysaccharides acts
as stimulatory effects (Abou et al. 2011; Brunold et al. 2004; Da-Costa-Rocha et al.
2014; Ismail et al. 2008; Liu et al. 2002; Mohamed et al. 2007; Mounnissamy et al.
2002).
Laportea aestuans (L.) Chew.
Extracts from L. aestuans are potentially used for traditional medicine system
which includes eye infections, parasitic infections, and microbial as well as gon-
orrhea and syphilis and also exhibit antioxidant and antimicrobial activity. Essential
oils extracts from the leaves of L. aestuans are composed of methyl salicylate and
Fig. 19.3 The roselle Hibiscus sabdariffa L. (Malvaceae)
chrysene -2-ol derivatives. Hence, essential oils are used for face masks, massage
oils, lotions, creams, scrubs, facial tones, antidandruff shampoos and conditioners,
lip blames as well as used for treatment for insect bites, chicken fox, acne, eczema,
and blemishes (Chukwuma et al. 2015; Essiett et al. 2011; Lans 2007; Okereke
et al. 2017; Oloyede 2016; Oloyede and Ayanbadejo 2014).
Lenotis nepetifolia (L.)
L. nepetifolia (Fig. 19.4) plants parts (sepals, leaves, flowers, and roots) are used in
traditional medicinal system. Hence, extracts are composed of active ingredients
such as terpenes, terpenoids, quinines, alkaloids, Saponins, and coumarins as well
as essential oils. Because of these active chemical constituents, they exhibit
antibacterial, antioxidant, insecticidal, radical scavenging anti-inflammatory, and
cosmetic properties. L. neperifolia has been used for skin allergies, calm agitation,
counteract muscle spasms, treatment for bronchial asthma, heal burns, pain, fever,
cold, anti-malaria, and arthritis as well as it used for cosmetics such as skin
regenerating agents, skin rashes, skin infections, and skin rejuvenating. Essential
oils from this plant shown pleasant fragrance, it could be used for preparation of
perfumes, because of the diterpenens and coumarins (Imran et al. 2012; Niteshwar
and Kumari 2012; Oyedeji and Afolayan 2005; Oyedeji et al. 1999; Pedro et al.
1991; Udaya et al. 2013).
Fig. 19.4 The lion’s ear

Leonotis nepetifolia (L) R. Br.
(Lamiaceae)
Maximiliana marpia (Correa) Drude.

M. maripa edible oils are extracted from fruit, seeds, and pulp, composed of fatty
acids, phytosterols, tocotrienols, glycolipids, tocopherols, and a-carotene. These
chemical constituents exhibit antimicrobial, antioxidant, and moistening properties.
For these reasons, M. maripa edible oils are used traditionally in skin ageing, skin
care products, smoothen scared skin, massage oil, moisturizers, rejuvenation, soaps,
shampoos, treating acne, and hair conditioners (Balslev et al. 2008; Bereau et al.
2001; Dos Santos et al. 2015a, b; Dos Santos et al. 2015a, b; Fernández et al. 2016;
Pereira et al. 2013).
Morinda citrifolia L.
The extracts from M. citrifolia are potentially used for the treatment of wounds,
sprains, sores and diabetic mellitus, AIDS, high blood pressure, malignant neo-
plasm’s and also used in skin injury, infection by ultraviolet radiation, and seed oil
is used as anti-inflammatory for acne. Hence, these extracts are used in preparation
of skin and hair care cosmetics and cosmeceuticals like shampoos and conditioners,
body and foot lotions, deodorants, ointments, hand and facial soaps, eye creams,
face masks, moisturizers, day and night creams as well as for treatment for acne.
(Assi et al. 2017; Kakad et al. 2015; Krishnaiah et al. 2012; Levand and Larson
1979; Palu et al. 2012; Potterat and Hamburger 2007; West and Sabin 2012).
Moringa oleifera Lam.
M. oleifera leaf and seed oils are used in traditional medicinal system, M. oleifera
seed oil is known as ben oil, ben oil is composed of fatty acids mainly behenic acid.
Because of this active substance, it could be used for moisturizers, massage oils,
and aromatherapy. Leaf extracts of M. oleifera are composed of significant amount
of vitamins A, C, & E, polyphenols and b-carotene which might own antioxidant
and anti-inflammatory properties. Hence, for these active ingredients of leaf and
seed oil extracts might be used in sunscreens, scrubs, body oils, creams, lotions,
balms, hair care products, moisturizers as well as seed oils are used in preparation
of perfumes. M. olifeira extracts relieve spasms, cardiac stimulant, diabetes mel-
litus, cardiac stimulant, antimicrobial, antiparasitic, and other conditions could be
used (Ali et al. 2013; Ashraf and Gilani 2007; Kale and Megha 2011; Ogbunugafor
et al. 2011; Ojiako and Okeke 2013; Taher et al. 2017; Warra 2012; Warra 2014).
Orchidaceae.
Generally, Orchidaceae (Fig. 19.5) plants and its parts show potentially various
traditional medicinal systems. Especially these family plants are used in Chinese
traditional medicine for treatment of cancer, diabetes mellitus, hypertension, and
urinary tract infections. For example, in this family, V. planifolia is used for the
preparation of perfumes, deodorants, and aromatherapy products. Some of these
family plant extracts are used in cosmetics and cosmeceuticals. Some of them are
composed of flavonoids and phenolic compound, exhibit anti-inflammatory,
Fig. 19.5 The tiger orchid

Oncidium jonesianum
(Orchidaceae)
anti-aging, and antioxidant property. Some plants from this family are water bound
or hydrophilic chemical constituents. These constituents increase corneum hydra-
tion and have been used for skin moisturizers and emollients. Flowers of this family
are the source of perfumes, fragrances, skin care products, hair care products, and
bathing products, as well as flower bouquets, are useful to identity scent and
perfumes (Bulpitt et al. 2007; Hadi et al. 2015; Hossain 2011;Menon and Nayeem
2013; Minh et al. 2016; Paul et al. 2013; Ribeiro et al. 2015; Sadler et al. 2011).
Plectranthus amboinicus Lour.
Extracts from P. amboinicus have been used for the treatment of fever, infections,
and genitourinary disorders, gastrointestinal and respiratory diseases. Essential oils
extracted from this species are used for scent as well as in the treatment of skin
diseases like wounds, burns, sores, and also used for insect bites, allergies,
antiseptic dressing for wounds and parasitic infections. Plant extracts are composed
of phenolic compounds, monoterpenes, sesquiterpenes, and diterpenens. These
active constituents show significant antioxidant, anti-inflammatory, and antimi-
crobial properties. These properties indicate that essential oils could be used in
soaps, skin cleansers, anti-wrinkle, anti-ageing, day and night creams, ointments,
skin itchy, moisturizers and skin rejuvenating (Bhatt and Negi 2012; Chifundera
2001; Erny et al. 2014; Harsha et al. 2003; Lukhoba et al. 2006; Rabe and Van
Staden 1998; Roshan et al. 2010).
Senna alata (L.) Roxb.
S. alata L. (Fig. 19.6) extracts of natural products and essential oils from various
parts such as roots, bark, fruits, seeds, and leaves are composed of bioactive sub-
stances such as phenolic substances, alkaloids, terpenes, anthraquinones, tannins,
and phytosterols. Few of these chemical constituents exhibit laxative and purgative
properties. Essential oils are used for treatment of ringworms infections, fungal
infections, as well as scabies. Bioactive chemical substances from this plant show
antifungal, antioxidant, and anti-inflammatory activity. Essential oils reduce skin
damage from ultraviolet irradiation. Hence, S. alata leaf extracts are considered as
cosmetics. Leaf extracts are used in skin care products like skin-repairing agents,
anti-ageing, sunscreens, and soaps (Adelowo and Oladeji 2017; Ehiowemwenguan
et al. 2014; Meenupriya et al. 2014; Moriyama et al. 2003; Oladeji et al. 2016;
Oresajo et al. 2010; Sule et al. 2011).
Tamaringus Indica L.
Preparation of T. indica extracts is composed of flavonoids, polyphenols, and
linoleic acid and oleic acid. These phytochemicals exhibit antioxidant, antibacterial,
antifungal, antiviral, antiparasitic, and anti-inflammatory properties. Hence, plant
extracts could be used in traditional medicinal system like malaria, fever, parasitic
infections, and respiratory problems. Flavonoids and polyphenols are present in T.
indica leaves extract, so it exhibits wound healing properties and hemicelluloses
Fig. 19.6 The candle bush

Senna alata (L.) Roxb.
(Fabaceae)
xyloglucan substances extracted from seeds of T. indica used for skin damage from
UV radiation. Hence, extracts from T. indica are used for preparation of skin care
products such as moisturizers, face masks, skin rashes, anti-aging night creams,
ointments, sunscreens, soaps, body lotions facemasks, facial toners and lip balms
(Al-Fatimi et al. 2007; Attah et al. 2015; Escalona-Arranz et al. 2010; Havinga et al.
2010; Kuru 2014; Luzia and Jorge 2011; Mesfin et al. 2012; Naik et al. 2017;
Strickland et al. 2004).
Tripogandra serrulata (vahl) Handlos.
T. serruulata extracts are prepared from various parts of the plant; it is native of
Caribbean and Southern American countries. These extracts are traditional medi-
cine used for kidney disorders, uterus cleaning, treating traumas, wounds, and
oviducts. Moreover, leaves are used to treat hair growth and hair loss treatment. For
these reasons, plant extracts are used for the preparation of hair care products.
Despite, the shortage of comprehensive data on the phytochemical information
about the plant extracts (Caballero-George and Gupta 2011; De Filipps et al. 2017;
Funasaki et al. 2016; May 1982; Pereira and Bartolo 2016; Valadeau et al. 2010;
Sedoc 1992; Venugopalan et al. 2011).
19.7 Applications of Natural Products in Cosmetics
Some of the natural products from the plants have been used for skin and hair
problems. For instance, different parts of these plants Rubia cordifolia, Linn.
Callicarpa macrophylla Vahl, Acacia concinna DC, emblica officinalis Gaertn, and
Curcuma longa have been used directly for face and hair problems. Ethnic and
community group’s historically natural products have been used for the treatment of
different hair conditions and skin diseases. Natural products intimate potential
properties like antioxidants, antimicrobial, anti-inflammatory, antimelanogenesis,
antihyluronidase, and antityrosinase in cosmetics. Some of natural products have
been used in different applications shown below.
19.7.1 Natural Products as Skin Care Agents
Dry skin treatment.

Stratum corneum binding of water indissoluble could be compromised and inef-
fective. For this reason, it is beneficial to decrease the transepidermal water loss by
applying occlusive films. Hence, there is no cause why mineral oil or petrolatum
should not be used. Hence, vegetable oils have more benefits for dry skin treatment.
Castor oil formed from (Ricinum communis) castor seeds, consists of more than
50% of fixed oil, more viscous, pure when colorless and has slight odor. Castor oil
is composed of ricinoleic acid and unsaturated fatty acids. Hence, castor oil is used
to skin smoothing, protect skin from harsh climate as well as used to prepare soaps.
Ricinolic acid and its analogs are used to improve the skin moistening and
smoothing qualities as well as enhance the skin conditions such as acne and rough
skin. Hydrogenated castor oil or its esters oils has been used for solubilizers for
toiletry as well as skin and hair care cosmetic formulations and are useful condition
and cleaning of the skin and hair (Sato 2002). Cocoa butter is used for the
smoothing of sunburn and windburn. Cocoa butter is obtained from T. cacao.
Cocoa butter composed of triglycerides consists of palmitic acid, stearic acid, and
oleic acid as well as monounsaturated fatty acid. Hence, it is widely used in
moisturizer and also used in cosmetic preparations and it has reported as potential
antioxidant (Sato 2002). Sunflower oil is composed of polyunsaturated fats,
triglycerides, or linoleic acid as well as essential fatty acids. These constituents are
used for maintaining good skin. Linoleic acid reduces the water loss transepidermal
and eliminates the scaly lesions normally with patients. Hydrated sunflower oil and
oleic acid contents are used in natural and functional raw materials in cosmetic
preparations. Olive oil is composed of triglycerides, tocopherols, squalene, car-
otenoids, sterols, chlorophylla, polyphenols, squalene, volatile oils, flavored com-
pounds, and fatty acids. These constituents are useful for treatment of dry skin, as
well as used in lip balm, soap, hand lotions, shampoos and conditioners and
massage oils. Extracts of leaves and fruits of olive tree show anti-inflammatory and
oxygen scavenging effects (Tehara and Hachimaki 2002). Olive oil shows potential
free radical scavenging effects, due to rich in polyphenols; it is applied for contact
dermatitis, skin damage, atopic dermatitis, eczema, xerosis, seborrhea, psoriasis,
rosacea thermal and radiation burns as well as other skin aging and inflammations.
Eczema.
Eczema is a skin condition specified by scaling, itching, redness, and swelling. For
treating eczema, turmeric has been used potentially. C. longa L; turmeric is pro-
cessed rhizome portion, which is used for traditional medicine. It is usually boiled
yellow powder. Curcumin is a major chemical component in turmeric, which has
potential biological activities such as anti-HIV, antiparasitic, antibacterial, anti-
carcinogenic, antioxidant, anti-inflammatory as well as wound healing powder,
applied to septic and aseptic wounds and inhibition of lipid peroxidation. Curcumin
is also used to check and prevention or treatment for psoriasis, skin damage from
sun radiation, wounds, burns, acne, and premature aging (Phan et al. 2001).
Acne, spots, and pimples.
Skin condition damage causes whiteheads, blackheads, inflammation, sweat glands,
and hair follicles. Some natural product extracts are traditionally used for the
treatment of acne, spots, and pimples. Extracts from A. vulgaris, A. absinthum, and
A. campestris are used for rapid healing wounds, skin ulcers as well as eczema,
herpes, and purulent scabies (Aniya et al. 2000). Extracts from O. gratissimum and
O. basilicum essential oils are used for treatment of acne, pimples and spots as well
as it shows antibacterial treatment for acne, antiseptic and antimicrobial activities
(Orafidiya et al. 2002). P. sativum extracts are used for the treatment of acne, due to
peas composed of fats, salts, proteins, lecithins, and carbohydrates. For example,
crushed peas are used for face masks, acne, and wrinkled skin (Orafidiya et al.
2002). C. pepo (pumpkin) extracts are composed of fatty acids; it has been used for
traditional medicine, and it shows potential anti-inflammatory properties due to
fatty acids are mainly composed of palmitic, stearic, and oleic acid. The roots,
leaves, and seeds of these extracts are used for pimples, blackheads, sores, and
herpes lesion (Orafidiya et al. 2002). A. cepa (red onion) has been used for tradi-
tional medicinal system, it could be used externally for boils, blackheads, and
abscesses to draw out of the infection as well as reduce the inflammation and
improve the healing. Red onions are composed of high amount of flavonoids;
hence, it shows potential anti-inflammatory and antiallergic properties as well as
onion juice shows antimicrobial and antifungal effects. In Africa, onion juice is
applied for scalds, infection, and burns, especially in East Africa onion skin has
been used for body sores and facial (Aburjai and Natsheh 2003).
Anti-aging skin treatment.
Human Skin aging caused by UV radiation from the sun is one major environ-
mental factor. Skin aging is due to exceeded degenerative and regenerative changes
and epidermises are analyzed by wrinkling and thinning together in the aspect of
Table 19.4 Plants used in skin cosmetics and toiletries as cosmeceuticals

Plant Scientific Active chemical constituent Cosmetic use
name
A. Catechu Catechin Antioxidant
A. Vera Aloin Antidermatitis
A. Recutita Chammomile Antiphologistic
A. Sativum Alliin and Allicin Antioxidant
B. Seeds Rutin Anti-wrinkle
C. Sativus Crocetin Protective
C. Longa Curcumin Antibacterial
C. Asiatica Centella Skin Firming/Conditioning
C. Limonus Hesperedin Fungal Infection of Skin
G. Glabra Glycyrrhizin Skin Whitner
G. Tea Chammomile Photoprotective
C. Murula Lupenol Anti-aging
R. Officinalis Rosemary Anti-aging
E. Officinale Ascorbic Acid, Tannins Protective
G. Biloba Ginki Skin Tonic
P. Corlifolia Psorolin Skin Staining & Pigmenting Agen
T. Viridis Gallic Acid, Catechin and Rutin Antioxidant
V. Vinifera Carotene Eczema
D. Carota Beta Corotene UV Protection
L. Esculantum Tamotine and Tamotidine Potent Bacteriostatic
H. Virginiana Gallic Acid Cooling Agent
lines, groove, crack, and wrinkle, especially in lines of facial expressions. This is
the reason for rapidly evident morphological alterations. There are many so-called
anti-aging that is traditionally applied material nothing more than moisturizers.
Ginseng (P. ginseng) is a traditional medicinal system for treatment of anti-ageing,
for more than 2000 years in Korea. Compared to other countries, Korean Ginseng
is chemically and physically different. Extract of this plant is more active to
enhance the skin metabolism, provide soften and moisture as well as enhance the
skin whiteness and reduce keratinization. The anti-aging effect leads due to the
increase of blood circulation and increase of skin nutrition and cell proliferation
(Aburjai and Natsheh 2003). Moreover, natural extracts or oils are a major source of
phytosterols, and tocopherol chemical constituents, which has been used for
antioxidant and bioactivity skin formulations. Generally, wheat germ oil, corn oil,
and seaweed extracts help to maintain skin elasticity and moisture. The details of
plants and their benefits in cosmetic benefits are shown in (Table 19.4).
Free-radical scavenging effects.
Natural product extracts are major source of plants, hence plant extracts shows
potential free-radical activity, due to polyphenols its derivatives, tannins, and
flavonoids (Ashawat et al. 2000; Pietta 2000). C. sinesis yields Black and Green
Tea. Black and green tea acquire leaves fermentation and after harvest leaves are
immediately steamed and dried, respectively. Tea is composed of more than 500
chemical constituents such as amino acids, tannins, flavonoids, vitamins, caffeine
and polysaccharides, which are similarly found in lemons. Black and Green tea
contain almost same amount of vitamin B6, vitamin E, and vitamin K, but 90% of
vitamin C destroyed during the fermentation process. Flavonoids which is present
in tea proven potential properties such as antibacterial, antiviral, antioxidant,
anti-inflammatory, antiallergic and while tannins show antiseptic and antioxidant
properties. Root extracts from tea are composed of Saponins; it shows potential
anti-inflammatory and antioxidant properties. Green tea consists of polyphenols
which are major active constituents; catechins are major important polyphenols,
while flavonoids such as phenolic acids and flavones. Recent days Green tea is now
subjected key attention, it is proven as potential antioxidant property for its capa-
bility to repair UV photo-damage and phototoxicity. Green tea and its extracts are
used for dry skin treatment and thus it shows potential anti-inflammatory and
anticarcinogenic effects of skin disorders (Katiyar and Elmets 2001). While com-
position of polyphenols is less abundance in black tea when compared with green
tea; but, it is still considered as a good source of antioxidant properties. Applying
black and green tea oral extracts decrease the photochemical damages to the skin
(Katiyar and Elmets 2001). Green and black tea extracts are remaining essential in
preventing the early sign of UV radiation phototoxic effects (Katiyar and Elmets
2001). V. vinifera L. (grape seed) is composed of different types of polyphenolic
proanthocyanidins. These polyphenols show potential antioxidant activity when
compared with Vitamin C and Vitamin E and it shows tyrosinase-inhibiting
activity, which has shown potential skin-lightening and anti-aging cosmetics (Lee
et al. 2001).
Anti-inflammatory effects.
In many diseases, inflammation is usual response. It’s bearing and controlling in the
treatment of these pathologies. There are many natural products that show
anti-inflammatory properties. T. pretense L. (Red clover) shows potential
anti-inflammation for multiple skin conditions like acne, rash, psoriasis, and
eczema. Red clover consists of isoflavones, which is used for UV radiation pro-
tection, reduces the inflammatory aedema reaction hypersensitivity induces simu-
lated UV radiation. M. recutia L. (German chamomile) and A. nobilis Linn. (Roman
chamomile) extracts similar chemical constitutes with almost same ratio. Extracts
from these plants traditionally are used in the form lotions, ointments, and
inhalations. Essential oil extracts and its isolated substances from these plants are
used for treatment and prevention of different skin disorders. Chamomile essential
oil extracts consist of flavonoids like apigenin and glycosides, which shows
potential antipruritic, anti-inflammatory, and antierythema effect (Aburjai and
Natsheh 2003). Extracts from chamomile consist of chamazulene and bisabolol
shows anti-inflammatory activity. T. foenum (Fenugreek) is fragrant herb, whose
seed is used for traditional medicinal system for all ages. Europe, Greek, Romans,
Egyptians, and among othera are used for medicinal and delicious purpose. Seeds
of this extract show potential antioxidant and anti-inflammation and emollient
properties. Extracts are traditionally used for treatment of skin inflammations,
mouth ulcers, and chapped lips. Worldwide G. glabra L. (Licorice root) extracts
used in traditional medicinal system. Root extracts of these plants composed of 5–
10% of glycyrrhizin, a sweet taste and in general, it is less soluble in blood com-
pared with Saponins. Hence, Licorice root extracts consist of glycyrrhetic acid,
which shows potential anti-inflammatory effects as well as used for skin problems
like irritations and acne (Aburjai and Natsheh 2003).
Miscellaneous.
Cucumber (C. sativa Linn.) is palliative, which cools and heals the irritated skin by
sun radiation or cutaneous eruption. Cucumber and lemon extracts are used in
cosmetics and cosmedicals products that are used for the treatment of hyperpig-
mentation. Both extracts are not interfering with each other and provide skin
lighting. Many scientific reports indicate the presence of antioxidant enzymes and
superoxide’s activity with cucumber fruit and highest in the skin (Aburjai and
Natsheh 2003).
Enumeration.
E. officinalis Gaertn; commonly known as Amla, dry fruits of this plant are used for
traditional medicinal system and cosmetics. Fruit extracts are composed of
phyllembic acid as well as isolated other chemical constituents such as ellagic acid,
corilagin, terchebin, trigalloylglucose from fruits. These extracts are used for
treatment of cooling, diuretic, laxative, and it is rich source of vitamin C. Vitamin C
is five times rich when compared with orange juice; for this reason, it is used for
hair dyes. The fruit powder and extracts are used as hair care products such as
shampoos and used as medicine for hair roots. Rubia coedifolia Linn. its common
name Manjit, from this plant dry roots and stems are used for traditional medicine,
especially used in cosmetics. Roots and stem extracts are composed of purpurin,
munjistin, Alzarin, and glucosides as well as anthraquinone derivatives. These
chemical constituents are used for antiseptic, antidysenteric deobstruent and root
ionic. Decoctions of leaves and stems extracts are used for vermifuge. Especially
extracts from stem are used for rhinosinal infections due to the presence of septilin
drug as well as roots extracts are used as coloring medicinal oils. Extract could be
applied directly in skins care such as removal of dark spots on the face, treatment
for acne, skin regeneration, and anti-ageing.
Acacia concunna DC. Is commonly known as Ritha and Shikakai in India. The
extracts from this plant are used especially as hair care products; treatment for hair
growth, hair splitting, dandruff, and hair fall. It is used to keep original hair color.
These applications due to plant extracts are composed of saponin, mixture of
acacinin-A and B as well as carbohydrates are composed of fructose, glucose, and
xylose. Sometimes seed extracts could be used for fish poison.
According to the ethnic or community groups understanding, the
above-mentioned plants are successfully used for skin and face problems such as
dark shadows, wrinkles on the face, acne, and pimples. Skikakai and amla could be
used for the hair problems such as hair color, scalp care, hair falling, and dandruff.
Particularly, Indians used cosmetics and cosmedicals for skin and hair care as
shown in (Table 19.5). Besides some other medicinal plants having soap properties
which can be used for skin and hair care products are shown in (Table 19.6)
(Sharma et al. 2003).
19.7.2 Natural Products as Hair Care Agents
Natural products are used for stimulating hair growth, hair color, and dyes as well
as scalp problems like dandruff.
Hair growth stimulants.
Nowadays, natural products and its derivatives are used for hair growth and hair
tonic products and precaution of alopecia, due to increasing blood circulation,
Table 19.5 Plants used for cosmetics for skin and hair care
Plant Scientific Family Part Cosmetic use
name used
A. concinna DC Mimosaceae Pods Soaps, hair splitting, hair falling, and
dandruff
A. barbadensis Liliaceae Leaves Hair falling, dandruff, and sun burn
A. racemous Liliaceae Roots Used in cure of wrinkle on face
A. indica Meliaceae whole Skin, hair, and scalp care
plant
B. orellana Bixaceae Seed Seeds used for color, mascaras, &
pulp lipcare
C. macrophyllaia Verbenaceae Fruits The fruits are blended in creams to treat
acne
C. longa Zingiberaceae Rhizome Improves the color of the skin
C. amada Zingiberaceae Rhizome A good face pack
E. prostrate Asteraceae Whole Used in keep the hairs in original color
plant
E. officinalis Euphorbiaceae Fruits Commonly used in hair care
A. moschatus Malvaceae Seeds Used to provide musk like fragrance to
cosmetics
H. abelmoschus Malvaceae Seeds Used to provide musk like fragrance to
cosmetics
L. inermis Lythraceae Leaves Used to color for hairs
O. dillenii Cactaceae Fruits Used in lipcare
R. cardifolia Rubiaceae Whole Application on skin and lip care and
plant treat for acne and pimples
Table 19.6 Selected plants Plant Scientific Family Common name

in India used in soaps as name
cosmetics
A. concinna DC Mimosaceae Shikakai
A. millefolium Linn Asteraceae Gandana
A. nobilis Linn Asteraceae Roman
Chamomile
L. inermis Linn Lythraceae Henna
M. chamomilla Linn Asteraceae Babuna
M. fragrans Houtt Myristicaceae Jayaphal
N. jatamansi DC Valerianaceae Jatamansi
P. emblica Linn Euphorbiaceae Amla
S. mukorossi Gaertn Sapindaceae Reetha (soap nut)
S. saponaria Lour Sapindaceae Reetha (soap nut)
S. trifoliatus Linn Sapindaceae Reetha (soap nut)
V. officianalis Linn Valerianaceae Tagger
activation of dermal papilla, increased nutrition through increased blood flow and
antitestosterone action is not yet clear (Mans and Grant 2017). Grape seeds and G.
biloba leaf extracts are composed of proanthocyanidins. This has promoted pro-
liferation of apoptosis hair follicle cells. Thus, these extracts are suggesting
potential hair tonic (Aburjai and Natsheh 2003). But other plants claimed potential
hair growth such as aloe, henna, rosemary, and sage. But it required further clinical
trials to use traditional medicine. A. barbadensis and A. vera gels are used tradi-
tionally for hair growth (alopecia) and hair loss. The plant extracts consists of
aloenin that is a major chemical constituent, which has major responsible for hair
growth without any skin irritation and side effects (Aburjai and Natsheh 2003).
Henna (L. alba L.) has been used for hair dyes. Since ancient time, Egyptians are
used to hair loss. More than 500 species of sage is used for traditional medicine as
general medicine. Generally, it is used for healing purpose. S. officinalis L. is also
known as garden sage. The extracts of these sages are used as lotions to improve the
skin and hair growth as well as conditioner. Combination of sage and rosemary
used to maintain the dark wavy hair and stimulate hair growth. S. officinalis L active
chemical constituents such as tannins, Saponins as well as camphor and borneol are
accountable for the effect on hair (Aburjai and Natsheh 2003). Rosemary (R.
officinalis Linn), is a known aromatic traditional medicinal plant. It is used in folk
medicine to stimulate hair growth. It is delicious, medicinal, and cosmetic prop-
erties. Rosemary consists of coffeic acid and its derivatives like rosmarinic acid
which shows potential antioxidant property.
Dandruff treatment.
In the recent years, dandruff became a major problem due to fungal infections,
microbial, and environmental conditions. The dandruff has been knowledgeable,
because of scaling and flaking of the scalp. Dandruff sufferers get damaged scalp as
well as decreased lipid level, ceramides, cholesterol and fatty acids. Hence, epi-
dermal water level reduces on the scalp which causes dandruff (Harding et al.
2002). Rosemary and sage extracts are potentially used for dandruff. The extracts
are used for the treatment of greasy hair; hair loss, and skin as well as extract sage
massaged on the scalp could control dandruff, loss of hair, or hair falling. Thyme
essential oils also used to inhibit the dandruff, scalp rub to avert hair loss as well as
rosemary and thyme encourage hair health. Garlic extracts are used traditional
medicine for dandruff and as a vegetable, it includes potential medicinal properties
such as antifungal, antibacterial, antioxidant, antiseptic, tonic, and anti-
inflammatory (Agiga and Seki 2000). Garlic could not be used directly; it causes
burning sensation, contact dermatitis, and allergic reactions for some people.
Walnut leaves extracts are used in external applications for hair loss, itching,
eczema, acne, peeling, and dandruff as well as skin disorder treatment such as
itching, emollient, abrasions, frostbite, treatment for sun burns, and nappy rashes as
well as dandruff and scalp problems (Aburjai and Natsheh 2003).
Hair coloring.
For hair coloring, vegetable oils are usually recommended, because of low aller-
genic power. For hair color, natural products do not have great advance, sinceplant
extract dyes or natural dyes are unstable in solution form, liable to oxidation, pH
color shift, fading and discoloration and single dye may not give right color for hair.
But only walnut and henna are suitable for hair color by mixing of leaves of other
plants (Aburjai and Natsheh 2003). L. inermis is known as henna. It is used for hair
color, feet, and hand color as well as used for certain skin disorders. Henna consists
of lawsone chemical constituent, which is responsible for developing red color in
henna. The chemical constituent is isolated from leaves as brown powder. Lawsone
has strong binding capacity to the hair, due to the chemical reaction between thiol
groups with keratin. German chamomile leaves extracts consist of apigenin flavo-
noids, which could be used for dull golden yellow color as well as flowers extracts
are used for hair rinse. Turmeric also is used for hair color to convert yellow to deep
orange color which is due to Curcumin pigment present, which is responsible for
yellow color of herb. H. sabdariffa L. extracts show red color due to extracts
composed of red color anthocyanidins such as delphinidin could be used. But red
color depends on pH of the solution.
19.7.3 Essential Oils Used as Cosmetics
Essential oils are the complex and composition of mixtures. Aromatic and
medicinal plants and its oils have been used in perfumes, cosmetics, medicines, and
culinary applications. Essential oils could be used for bath and skin massage, as
scent (inhaled directly or diffused). Since ancient time, essential oils are used for
pain relief, tension alleviation, skin care, fatigue, produce sense of relaxation and
revitalize the entire body which is potential benefit for cosmetics. Essential oils and
Table 19.7 Plant essential oils are used in cosmetics

Plant Family Essential oils extracted from parts
A. squarrosus L. Gramineae Roots
V. zizanioides L. Gramineae Roots
C. aurantiacum L. Rutaceae Flowers
E. dives Schauer Myrtaceae Leaves
J. officinale Linn Oleaceae Flowers
J. virginiana Linn Pinaceae Wood
M. piperita Linn Labiateae Aerial parts
M. champaca Linn Magnoliaceae Flowers
M. elenhi Linn Sapotaceace Flowers
P. fascicularis Lamk. Syn Pandanaceae Male inflorescence
R. damascene Mill Rosaceae Flowers
S. album Linn Santalaceae Wood
S. lappa Clarke Compositae Roots
S. robusta Roth Dipterocarpaceae Stem
S. aromaticum Linn Myrtaceae Flower bud
V. odorata Linn Violaceae Flowers
its derivatives are used for skin emollient, hair conditioners, pleasant aroma, and
skin elasticity. Essential oils are composed of terpenoids, phenylpropanoids, and
fatty acids. Terpenoids belong to mainly mono, di, and sesquiterpenes. These lar-
gest terpenoids more than 30,000 groups are synthesized by isopentenyl diphos-
phate and other chemical constituents are present such as small chain alcohols and
aldehydes, it is formed by conversion of fatty acids and phospholipids. Essential
oils show different medicinal and cosmetic properties (Reddy 2019) as well as some
essential oils producing plants are used in cosmetics shown (Table 19.7).
Cosmetic benefits.
Essential oils could be used in perfumes, skin lotions, hair care products and
pleasant smell and glow. Essential oils show potential antibacterial activity, hence it
could be used in preservative system in cosmetics. The essential oil efficiency
depends on both concentration as well as the microbial strain. Essential oils could
be used in cosmetics, for instance, menthol, mint, camphor, and eucalyptus oils as
cooling agents, as well as menthol analogs such as methoxypropanediol, menthyl
hydroxybutyrate, menthyl glucoside, menthoxy furan, and menthy lactate as a
refreshing the skin (Aburjai and Natsheh 2003).
Perfumery.
Perfume word is derived from Latin. Essential oils are major applications for the
perfumes. In ancient times, perfumes are obtained by simple method to produce
perfume, soak flower petals in fat known as pomade (Barel et al. 2001). Plant flower
fragrance has rapidly converted volatile constituents into high impact commercial
commodity. Cosmetics, perfumes, and therapeutic fragrances are extracted from

flowers of some aroma plants such as narcissus, tuberose, gardenia, and rose.
Perfumes are mainly used in two purposes, one it could be used as cosmetics and
toiletries precuts such as hair products, personal care products, bath products,
deodorants, and fine fragrance and other types used as household products include
laundry products, surface cleaners, room fresheners, washing liquids, and disin-
fections. Essential oils are major ingredients for preparation of perfumes. Essential
oils are composed of hundreds of suitable ingredients, which are added to fragrance
composition to improve the smell. Still successful fragrances contain significant
quantities of naturally occurring essential oils (Barel et al. 2001).
Hair care.
Essential oils are used for hair care products to remove negative smell from perm
lotion; it helps for hair conditioning and shampoos, improvement of hair texture and
also longer lasting pleasant smell. Rosemary and chamomile essential oils are added
straight to mild shampoo. Rosemary oil can help hair condition and potential hair
growth as well as tea tree could help controlling dandruff. Lavender could be used
to repel lice and fleas. Essential oils mixed with hair care products; it would be
improved shine and conditioning effects.
Aromatic skin care.
Skin problems are generally the surface manifestation of deeper conditions such as
environmental pollution, toxins from blood, hormonal imbalance, nervous and
emotional difficulties. Essential oils are potential use for such problems. Essential
oils are soluble in oils and alcohols and important scent to water. Hence, essential
oils provide ideal chemical constituents for cosmetics and specific disease. Essential
oils are made up of very small molecules, and interact through the skin, it means of
lipophilic fractions reacting with the lipid parts of the cell membrane. For instance,
citrus oils such as lemons, grapefruits, oranges, and tangerines are composed of
mixture of alcohol, acids, esters, carbohydrates, ketones, and aldehydes. These
chemical constituents are rich in fragrance and flavor. Hence, essential oils are used
to important taste to different products, aroma, and taste. Essential oils are used
personal perfumes widely, where they are used pure and frequently combined with
synthetic substances. Lavender oils distillated from mainly Lavandula have been
used therapeutically and cosmetic agents. These oils are extremely used for the
preparation of soaps, perfumes, and skin lotions. Lavender oils show potential
antibacterial, antioxidant, antifungal properties. Essential oils from black cumin are
composed of thymoquinone, carvacrol, 4-terpineol, and t-anethole shows antioxi-
dant, antibacterial, antifungal and anticarcinogenic, analgesic and anti-inflammatory
properties. Hence, these oils could be used for skin care (Aburjai and Natsheh 2003).
19.8 New Trends in Cosmetics (Plant Origin

of By-Products)
In recent days, fruits, vegetables, and foods (agronomical) industries increase the
amount of waste, which produce different types of expendable by-products; rich in
valuable components such as pharmaceuticals, food, and cosmetics. There are many
cosmetic active constituents which are extracted from meat, fish, and dairy prod-
ucts. Furthermore, in agronomical disposable wastes are used in cosmetic field.
Hence, these products are commercially less expensive, bio-feasible, more effective,
and environmental friendly. In consequences to plant-derived extracts from wastes
are adopted in cosmetic industry. Moreover, the generated wastes or products from
organic farming are absolutely more valuable source and safe to use in cosmetics.
Since decades, fruits and vegetables and agricultural products are considerable more
significant in our everyday diet and their indubitable nutritional values have been
comprehensively studied (Barbulova et al. 2014). Since decades, human kinds used
fruits and vegetables for fragrance, perfumes, flavoring, preservatives, cosmetics,
and pharmaceuticals. Vegetables, fruits, dairy, and agricultural products have
enormous health benefits such as heart diseases, heart stokes, respiratory disorders
as well as variety of cancers. Fruits and vegetables are composed of flavonoids
exhibit that shows potential antioxidant activity; grapes and apples present active
substances such as flavonoids, catechins, epicatechins, and procyanidins, which are
used for heart diseases; some vegetables such as red tomatoes and orange are
composed of carotenoids, b-carotenes is present, which are effective for neutralizing
the free radicals; in cherries, red grapes, and berries anthocyanidins are present,
which are used for brain function; citrus fruits are more dominant for flavonoids.
Because of the above reasons, fruits and vegetable consumptions considerably
increased in last few decades and also the amount of wastes and residues increased
substantially. Without observing waste during food processing, every year,
agro-food industries generate 800,000 tons wastes around worldwide (Ayala-Zavala
et al. 2010; Tuck et al. 2012).
Food and agriculture industries are generating 10–60% (Table 19.8) of solid
waste and raw materials. It was demonstrated that in few cases main products are
less valuable than waste products (Liu et al. 2012). It was indicated in percentages,
which is preferred to call by-products. Industry by-products are capable to be
recycled as valuable products. These by-products are potentially made by fruits,
skin, stems, seeds, leaves unusable pulp, and wastewaters generally through way. In
few cases, wastes represent more than 40% of total plant food (like mango, papaya,
pineapple, citrus fruits, artichoke, and asparagus) (Liu et al. 2012). Food
by-products know to minerals, sugars, and organic acids, bioactive constituents as
flavonoids, carotenoids, and polyphenols parallel to their essential counterparts.
Because of this phytochemical composition, finding natural chemical constituents
are as an alternate for synthetic substances. The by-products in industrial fields are
used in food, pharmaceutics, nutraceutics, and cosmetics.
Table 19.8 Percentages of by-products generated from fruits and vegetables processing industries
Plant Edible part (%) % of by-products
Agave 60 40 (rind and pith)
Apple 89 11 (pulp and seed core)
Artichoke 40 60 (outer bracts, receptacles, and stems)
Asparagus 50–60 40–50 (spear)
Banana 70 30 (peel)
Cactus cladodes 80 20 (spines, glochids, and peel)
Carrot 60–70 30–40 (pomace)
Mandarin 84 16 (peels)
Mango 58 42 (seeds, peels, unusable pulp)
Citrus fruits 44 66 (peel)
Papaya 53 47 (seeds, peels, unusable pulp)
Passion fruit 25 75 (rind and seeds)
Pineapple 48 52 (core, peels, top, pulp)
Potato 60–85 15–40 (peel)
Tomato 93–97 3–7 (peel and seeds)
19.8.1 By-Products from Citrus Fruits
Rutaceae (citrus family plants) also known as agrumes are one of the largest fruit
crops worldwide. Citrus fruits are rich in vitamin C (citric acid) and vitamin B (like
thiamin, niacin, pyridoxine, riboflavin, pantothemic acid, and folate), as well as
flavonoids, limonoids, and carotenoids chemical constituents are present. These
ingredients are the major source of dietary fibers, lowered circulating cholesterol,
and gastrointestinal diseases. Citrus fruits contribute to human diet and exhibit good
antioxidant properties. From citrus family, some of them are eaten fresh such as
grapes, oranges, and tangerins, as well as others around 85% of industrial processed
consumption such as lemon and orange juices. Phytochemicals of these natural
sources are used for human color for skin, hair color, and eyes, as well as skin
protection from ultraviolet radiation. Ethanol extracts waste by-products from the
citrus family shown potential antimicrobial, antioxidant, and anti-inflammatory
properties, because these extracts are major source of essential oils and flavonoids.
Essential oils are used particularly as preservatives against spoilage, pharmaceuti-
cals, and cosmetics. For example, C. unshiu peel essential oils composed of
limonene (80.5%), c-terpinene (6.80%), and cymene (4.02%) are major com-
pounds. These essential oils exhibit potential antibacterial activity, anti-
inflammatory, antimicrobial, and antioxidant properties. Essential oils and
extracts of citrus family by-products are used as hair and skin care products.
19.8.2 By-Products from Tomato and Olive
Tomato and olive are the most important food products worldwide. Tomato and
olive oil food processed products are mainly located in the Mediterranean areas and
shipped worldwide.
The annual production of tomato in the world is around 160 million tons, about
40 million tons of which are processed such as tomato peel, tomato paste, unpeeled,
and chopped tomatoes (Tomato Processing industries 2014). High amount of
important tomato by-products (peels, pulps, and seed) generated due to tomato
processed industrially are rich in lycopene which has antioxidant properties, and
used for disease prevention. Tomatoes are highly demanded for food, pharma-
ceuticals, and cosmetic industries. Lycopene is extracted from peel approximately
five times more than pulp. Extraction of lycopene commercially is expensive due to
its difficult extraction process. Tomatoes by-products’ high moisture content and
sensitivity to microbial spoilage make the storage and processing of this material
problematic (Papaioannou and Karabelas 2012). Hence, lycopene ensuring to be
used in food and cosmetic industry (Papaioannou and Karabelas 2012).
Mediterranean diet olive oil has most important to improve health status in terms
of heart disease. It is already know that olive oils are principle ingredients which are
used to variety of cosmetic products such as face and body creams, soaps, lotions,
shampoos, body massage oil, and hair oils. Olive oils by-products are composed of
phenolic compounds and monounsaturated fatty acids. Olive oil and by-products
show potential biological activity in epithelial, endothelial, platelets, neurons,
neoplastic cells, and immune system. By-products are composed of phenolic
compounds such as hydroxytyrosol, oleuropein, and other derivates show in vivo
and in vitro antioxidant property. A lot of research efforts were published especially
about oleuropein and its pharmacological activities which include
anti-inflammatory, anti-atherogenic, anti-cancer, antimicrobial, antiviral, antioxi-
dant, hypoglycemic, and hypolipidemic effect (Omar 2010). Olive oil and its
by-products already known to skin and hair care products.
19.8.3 By-Products Processing from Coffee
Coffee is among the most crops generating large amount of by-products in the
coffee processing industry. Globally, coffee (C. arabica and C. robusta) is usually
cultivated in South Asia, India, Latin America, and Africa. By-products of coffee
are produced from the coffee pulp and husk processing, it has less applications such
as prepare compost, fertilizer, and livestock feed (Murthy and Naidu 2012). While
collection and selection of coffee beans some of coffee berries are unused green
beans, they have been mechanically damaged and sufficiently ripe and big to pass
the next processing step. The green beans have valuable antioxidants, which not yet
been broken by the roasting process (Murthy and Naidu 2012).
Otherwise, the unroasted beans are extracted with water, carbon dioxide, and
ethanol; these extracts are rich in bioactive antioxidant constituents. The removal of
ethanol from the extract results in jelly like extract which is used to improve the
skin tone, natural skin cell renewal, reinforced the epidermal barrier, and decreasing
inflammatory process.
19.9 Future Prospects and Conclusions
This chapter presents the selected plants extracts and their by-products used in the
preparation of cosmetic products worldwide. Globally, more than 5,000 plants are
used in cosmetics and cosmeceuticals. Currently to develop cosmetics and cos-
meceuticals, natural products are untapped reservoir.
This clench identifying and developing many plants-derived natural beauty
products. For occasion B. orellana L (Fig. 19.7) seed extracts are used for
orange-red wax or lipstick plant, Amazon base native people has been used for
body and facial embellishment, as well as used in lipsticks, powders, nail polishes,
eye shadows, and cream blushes. Baobab (Adansonia digitatal L) is generally
known as monkey tree or upside-down tree. It is native of Madagascar and
southwest of Africa. This plant seed extracts have been used for normal and dry
skin anti-irritating, antioxidant, and anti-sensitizing as well as hair and nail con-
ditioning purpose (Hetta 2016a, b). Mafura (Trichilia emetica L.) common name
Natal mahogany is growing in Zimbabwe, Sudan, South Africa, and Uganda. Its
plant extracts show potential antioxidant, and anti-inflammatory effects, preparing
Fig. 19.7 Seeds of the

annatto Bixa orellana L.
(Bixaceae)
natural soaps, making candles, as well as used for hair and skin care products
traditionally (Hetta 2016a, b). Ximenia american L. (common name Sour plum), it
has been distributed in Africa. Extracts of this plant are used in traditional medicinal
system like skin problems, angina, fever, toothache, and others, it is also used
traditionally in cosmetics such as lipsticks, lubricant, soaps, vegetable butters,
emollient, conditioner, body massage oil, hair oil and conditioner and skin softener
(Hetta 2016a, b). The early shoots of C. aurantium L. (sour orange) chewed for
fresh breath in elderly age women’s in Creole as well as essential oils from this
plant might be used in soaps, skin care products, deodorants, and mouthwashes.
Essential oils from seeds of D. odorata are extensively used in perfumes industry
due to major amounts of coumarins with pleasant odors and also used in preparation
of skin and hair care products.
Moreover, an important pathology to take advantage of these opportunities to
develop active chemical constituents, mechanism and its characterization must be
necessary for cosmetics and cosmaseuticals. For example, A. vera is used for
wound healing, but it needs high level of supporting evidence and its promoting
activity, A. vera analog products are used as topical agents for treatment of skin
lesion (Pereira and Bártolo 2016). E. olearacea products are used worldwide in
most foods, cosmetics, and cosmeceuticals; but, health and scientific evidence are
still insufficient (Yamaguchi et al. 2015). M. citrifolia fruits are used in general
health disease, power and energy drinks, fruit juices as well as leaves formulated as
capsules or pills high in demand. But most of assert of noni plant products are not
carried by hard scientific evidence (Assi et al. 2017).
Over few decades, enormous understanding of cosmetic skin formulations and
skin biology was developed. Different types of commercial cosmetics are available
for skin care products such as skin whitening, skin protection lotions, skin creams,
and anti-ageing. Several natural products are serving cosmetic industry in addition
to their medicinal benefits to the skin. More research efforts are required for
delivery of natural products cosmaceuticals ingredients and report of their activities
in cosmetic field could lead the development in the next decade.
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Chapter 20
Encapsulation of Bioactive Compound
and Its Therapeutic Potential
Lalduhsanga Pachuau, Laldinchhana, Probin Kumar Roy,

James H. Zothantluanga, Supratim Ray, and Sanjib Das
Abstract Micro- or nanoencapsulation has become one of the most attractive

approaches to enhance the stability and bioavailability of bioactive compounds
isolated from natural products. In addition, such encapsulation also provides an
opportunity to modulate the release of bioactive compounds by using functional
polymers that can be fine-tuned according to the need of the body. Over the last
10 years, increasing research works have been dedicated toward the encapsulation
of bioactive compounds for various purposes. Numerous techniques, embracing
micro- and nanoplatforms including spray drying, freeze drying, micro- and mul-
tiple emulsification, electrospinning, and coacervation, have been utilized to
achieve the encapsulations. Such encapsulations have been found to improve the
physicochemical properties of the bioactive compound, provide stability and
enhanced bioavailability, controlled release of the compound, enhancing bioactiv-
ity, and masking of flavor or taste, along with several other benefits. Wide ranges of
materials including lipids, synthetic, and natural polymers have been utilized, and
the type and amount of the wall formers have been found to influence the perfor-
mance and functionality of these preparations.
Keywords Encapsulation Bioavailability Bioactive compounds Hydrogels

Electrodynamic processes
L. Pachuau (&) S. Ray S. Das

Department of Pharmaceutical Sciences, Assam University, Silchar 788011, India
e-mail: aduhapc@gmail.com
Laldinchhana P. K. Roy
Department of Pharmacy, Regional Institute of Paramedical and Nursing Sciences,
Aizawl 796017, India
J. H. Zothantluanga
Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh 786004, India
https://doi.org/10.1007/978-3-030-54027-2_20
688 L. Pachuau et al.
20.1 Introduction
Phytochemicals in plants that produce therapeutic activities are collectively known

as bioactive compounds. These bioactive compounds are predominantly plant
secondary metabolites and nutrients such as vitamins and minerals that may induce
pharmacological activity at high doses are usually excluded from the definition of
bioactive compounds (Bernhoft 2010). Abundant bioactive phytochemicals are
found in vegetables, fruits, seeds, nuts, legumes, leaves, and other parts of plants
(Al Juhaimi et al. 2018; Pachuau et al. 2019; Sagar et al. 2018; Septembre-
Malaterre et al. 2018; Xiao and Bai 2019). Phenolic compounds such as flavonoids,
courmarins, phenolic acids, xanthones, and ellagitannins along with alkaloids,
phytosterols, carotenoids, anthocyanins, and tocopherols are some of the commonly
found bioactive compounds from plant sources that are responsible for their
bioactivities (Fig. 20.1) (Altemini et al. 2017; Da Silva et al. 2016; Xiao and Bai
2019). In addition to their bioactivities, phytochemicals such as phenolics in veg-
etables and fruits may also contribute to their stability against oxidation, taste,
color, flavor, and odor (Naczk and Shahidi 2006).
Studies have reported myriads of pharmacological activities including antimi-
crobial, anti-hyperglycemic, anti-inflammatory, antioxidant, anti-proliferative, and
anti-diabetic effects of bioactive phytochemicals isolated and characterized from
various plant species (Martins et al. 2016; Ramesh Kumar et al. 2018;
Rodriguez-Garcia et al. 2017). Diverse preparations made from these wide varieties
of plant species have been used in the treatment of different diseases since ancient
times as documented in Eber’s Papyrus, Traditional Chinese Medicines (TCM), and
Ayurvedic formulations (Atanasov et al. 2015; Khan et al. 2019; Yang and Yue
2012). These traditional practices still remain valuable sources of information for
modern drug discovery and development programs. In fact, data from Food and
Fig. 20.1 Structures of some phytochemicals

20 Encapsulation of Bioactive Compound and Its Therapeutic Potential 689
Drug Administration (FDA) revealed that about 40% of the approved molecules are
either natural compounds or inspired by them and, from these, 74% are indicated
for anticancer therapy (Seca and Pinto 2018). Still, it has been reported that about
95% of the world’s biodiversity are yet to be systemically investigated for their
pharmacological activity and only 15% have been evaluated phytochemically
(Atanasov et al. 2015; David et al. 2015). The current global scenario and the urgent
need for effective therapy to treat chronic as well as infectious diseases including
cancer and HIV implied that natural products will continue to play a pivotal role in
drug discovery and development processes (Cragg and Newman 2013).
Although natural products are considered to be safe and their bioactive phyto-
chemicals exhibit excellent pharmacological activities in vitro, they often failed to
translate this into clinical or therapeutic effects in vivo. The major problems
associated with bioactive phytochemicals include their low oral absorption and
rapid systemic clearance which ultimately lowers their bioavailability and thera-
peutic efficacy in vivo (Kumar et al. 2010; Rein et al. 2012). For instance, oral
administration of curcumin at 500 mg/kg in Sprague-Dawley rats produced maxi-
mum plasma concentration (Cmax) of 0.06 ± 0.01 µg/ml after 41.7 ± 5.4 min
(Yang et al. 2007), while in healthy human volunteers, curcumin was detected in
the serum only when it was administered orally at a dose higher than 8 g (Lao et al.
2006). Similarly, other potent bioactive compounds such as quercetin (Almeida
et al. 2018; Kasikci and Bagdatlioglu 2016), paclitaxel (Malingre et al. 2001;
Tiwari and Amiji 2006), and ellagic acid (Bala et al. 2006; Lei et al. 2003) also
produced extremely low oral bioavailability eventually resulting in suboptimal oral
therapy in vivo. Predominant factors that led to the poor oral absorption of these
natural bioactive compounds are their eminently low aqueous solubility, poor
dispersibility, and bioaccessibility which are the prerequisites for absorption
through the gastrointestinal (GI) tract. For example, the maximum solubility of
curcumin in aqueous buffer pH 5.0 is only 11 ng/ml (Ma et al. 2019) and the
aqueous solubility of ellagic acid is about 9.7 µg/ml (Alfei et al. 2019). In addition,
instability of various bioactive compounds on exposure to environmental factors
such as pH, enzymes, light, heat, oxygen, and moisture may also contribute to their
degradation in vitro and in vivo (Bohn et al. 2015; Islam Shishir et al. 2018).
Moreover, biological factors including permeability of the intestinal epithelium, the
presence of efflux transporters such as P-glycoprotein and the induction or inhi-
bition of metabolizing enzymes may also serve as barriers to oral bioavailability of
several bioactive compounds (Chu et al. 2008; Ma et al. 2019; Xie et al. 2011).
Overcoming these challenges has become one of the most important focuses of the
current research on improving the oral bioavailability of bioactive compounds.
Techniques such as micronization (Aguiar et al. 2018) or nanonization (Aditya et al.
2019; Borhan et al. 2014) to reduce the particle size, co-administration of phyto-
chemicals with bio-enhancers such as piperine (Gorgani et al. 2017), micro- and
nanoencapsulation with different types of coating materials (Dias et al. 2017;
Ezhilarasi et al. 2013) are some of the approaches designed to achieve the oral
bioavailability enhancement of bioactive compounds.
The process of applying relatively thin coatings around a substance which may
be solids, liquids, or even gases is called encapsulation. Based on the size of the
coated particles, the process may be microencapsulation or nanoencapsulation.
Encapsulation separates the encapsulated materials also known as the core materials
from the environment with the help of a coating material, usually a polymer. Thus,
encapsulation provides protection of the core material and it may also add certain
functionalities such as color for identification, masking of taste, or sustaining the
release of the core materials. As a result, micro- or nanoencapsulation has become
an indispensable technology in various industries including pharmaceuticals, cos-
metics, nutraceuticals, and agriculture.
Encapsulation is one of the techniques that can be applied to enhance oral
bioavailability, solubility, dispersibility, and stability of bioactive compounds
(Beevers and Huang 2011; Ezhilarasi et al. 2013; Gomez-Estaca et al. 2015; Li
et al. 2015; Shaikh et al. 2009). Numerous methods have been applied in the
encapsulation of various natural bioactive compounds utilizing wide range of
coating materials such as lipids, natural, and synthetic polymers (Anirudhan and
Binusree 2016; Islam Shishir et al. 2018; Oehlke et al. 2017; Pinho et al. 2013;
Shao et al. 2011; Sharma et al. 2007; Young et al. 2005). The selection of the
coating materials is critical as the size, shape, and stability of the final encapsulated
product as well as the release characteristics of the core material is largely deter-
mined by the type and amount of the coating materials used (Dias et al. 2017).
20.2 Rationale for Encapsulation of Bioactive Compounds
Encapsulation of natural bioactive compounds into various nano- or

micro-platforms has the potential to resolve several drawbacks that are inherent to
these phytochemicals. This may range from improving their physicochemical
properties such as solubility and stability, to their pharmacokinetics such as
absorption and the overall bioavailability. Some of the advantages offered by
encapsulation of bioactive compounds can be summarized as follows:
(a) Enhanced bioavailability and therapeutic activity: Improvement in
bioavailability of poorly absorbable phytochemicals such as polyphenols has been
reported after their encapsulation into various systems such as solid lipid
nanoparticles (SLN), micelles, liposomes, and others (Aqil et al. 2013; Murugan
et al. 2009; Puligundla et al. 2017; Xie et al. 2011; Yang et al. 2008). This
enhancement in bioavailability can be manyfold, as at least ninefold increase in oral
bioavailability had been reported for poly(lactic-co-glycolic acid) (PLGA)
nanoencapsulation of curcumin (Shaikh et al. 2009), while 942.53% enhancement
in relative bioavailability was also reported for curcumin encapsulated in SLNs (Ji
et al. 2014).
(b) Stability: Most of the bioactive compounds from plants are susceptible to
light, heat, and pH changes developing unpleasant flavor or colors (Alborzi et al.
2012; Fang and Bhandari 2010; Prakash et al. 2018; Rezaei et al. 2019; Soukoulis
and Bohn 2018). Encapsulation can prevent in vitro or in vivo degradation of

bioactive compounds against these factors.
(c) Taste masking: Food or therapeutic applications of several phytochemicals
have been limited by their unpleasant flavor, astringency, and bitter taste (De Souza
et al. 2020). Encapsulation can eliminate this problem as the bioactive compound is
not in contact with the taste buds located in the oral cavity. For instance,
microencapsulation of quercetin with carnauba wax, shellac, or zein was able to
reduce or mask the bitter taste of the compound (Khor et al. 2017), while the flavor
of turmeric extract was effectively masked by brown rice flour and
b-cyclodextrin-based microcapsules (Laokuldilok et al. 2016).
(d) Improvement of physicochemical properties: Micro- or nanoencapsulation
offers the opportunity to improve the overall physicochemical properties such as
morphology and wettability, presenting bioactive compounds into spherical, uni-
form size, and free-flowing powders which also facilitate their processing during
manufacturing (Bertoni et al. 2019; Dima et al. 2016; Lu et al. 2011). Such
improvement in powder flow property reduces the possibility of variation in the
quality of the end products (Guajardo-Flores et al. 2015). Dissolution rate of poorly
aqueous-soluble phytochemicals such as naringin and quercetin has also been
reported to be enhanced by 20–55% (Pai et al. 2015) and 100 times (Barras et al.
2009), respectively, probably due to the dispersion of the bioactive compound in
amorphous form within the matrix.
(e) Controlled or targeted release of bioactive compounds: Encapsulation
enables controlled and targeted release of the encapsulated bioactive molecules,
protecting against degradation and first-pass metabolism while enhancing their
bioactivity following oral administration (Goiun 2004; Moreno et al. 2018; Sun
et al. 2015; Yao et al. 2015) (Table 20.1).
20.3 Encapsulation of Bioactive Compounds
20.3.1 Microencapsulation
Microencapsulation is one of the most common methods employed to provide wide

range of functionalities including protection of bioactive compounds against
environmental factors and to enhance their bioavailability (Table 20.2). It is also
possible to encapsulate bioactive oils such as essential oils and is a means of
converting these oils into free-flowing powders which facilitate its handling, sta-
bility, and bioactivities. Due to their small and uniform particle size, microcapsules
are distributed homogenously along the GI tract which promote absorption of their
encapsulated compounds and enhance oral bioavailability (Lengyel et al. 2019).
Various methods such as spray drying (Boonchu and Utama-ang 2015), spray
congealing (Tomsik et al. 2019), coacervation phase separation (Silva et al. 2011;
Jain et al. 2015), solvent evaporation (Paulo and Santos 2018; Sawale et al. 2017),
Table 20.1 List of some bioactive compounds with nanoencapsulation technology for improved
characteristics
Bioactive Encapsulation type/ Improvement after encapsulation References
compound techniques
Eugenol Oil-in-water Thermal stability Woranuch
emulsion and ionic and Yoksan
gelation (2013)
Curcumin Nano-precipitation Solubility and antioxidant activity Dutta et al.
(2018)
Nanoemulsion Solubility and stability Ahmed
et al. (2012)
Liposomes Increased bioavailability compared Takahashi
to suspension et al. (2009)
Caffeic acid Inclusion complex Enhanced bioactivity Pinho et al.
by b-cyclodextrin (2015)
Liposomes Enhanced antioxidant properties Katuwavila
et al. (2016)
Chlorogenic Nanoparticles by Sustained release property, retained Nallamuthu
acid ionic gelation antioxidant activity, and enhanced et al. (2015)
techniques bioavailability
Liposomes Enhanced oral bioavailability, Feng et al.
increased antioxidant properties (2016)
Coumaric Nano-sized Increased stability and Huang et al.
acid multi-phase bioaccessibility, sustained release (2019)
emulsion
Resveratrol Nanoemulsion by Solubility, stability, and Sessa et al.
high-pressure bioavailability (2014)
homogenization
Quercetin Solid lipid Increased bioavailability Li et al.
nanoparticle (2009)
Microemulsion Increased bioavailability Sun et al.
(2010)
Nanoliposomes by Enhanced bioactivity, enhanced Rodriguez
film-sonication release et al. (2019)
Capsaicin Nanoemulsion Increased bioavailability Choi et al.
(2013)
Retinoic acid Solvent evaporation Controlled release Ezpeleta
et al. (1996)
Ellagic acid Emulsion– Sustained release, enhanced Mady and
diffusion– bioavailability Shaker
evaporation (2017)
Sesamol Phosphatidyl Enhanced bioavailability and Yashaswini
choline micelles bioactivity et al. (2017)
d-limonene High-pressure Enhanced antimicrobial activity Donsi et al.
homogenization (2011)
Carvacrol Liposome/lipid film Sustained released, increased Engel et al.
hydration technique activity (2017)
(continued)

Bioactive Encapsulation type/ Improvement after encapsulation References
compound techniques
Thymol Liposome/lipid film Sustained released, increased Engel et al.
hydration technique activity (2017)
Naringenin Liposome/thin film Enhanced solubility and Wang et al.
hydration bioavailability (2017)
Table 20.2 Reasons for microencapsulation of bioactive compounds

Reasons
To minimize bitterness and astringency of bioactive compounds
Encapsulation of bioactive fixed and essential oils
Controlling of organoleptic properties such as color, flavor, and odor
Protection against degradation due to oxidation, pH, enzymatic reactions, etc.
Controlled release and improved therapeutic activity of bioactive compounds
Ease of administration
Improvement in physicochemical properties
Enhance dissolution rate
and ionic gelation (Borgogna et al. 2010) have been utilized for encapsulating
bioactive compounds. Wide range of wall-forming materials from natural and
synthetic sources have been used in microencapsulation of bioactive compounds
and the nature and quantity of these wall formers determined, to a large extent, the
quality and functionality of the resultant microcapsules.
In an ideal delivery system, the bioactive compounds should not be allowed to
affect the color or flavor or interact with the sensory organs and also the particles are
presented small enough not to interfere with the texture (Champagne and Fustier
2007). Microencapsulation is a technology to achieve this objective in effective
delivery of bioactive compounds. An extract of Gentiana lutea root containing
bitter secoiridoids was coated with ethylcellulose–stearate system into microcap-
sules and was found to mask the bitterness in the mouth and the release of the
bioactive compound in the GI effectively reduces the daily energy intake in human
subjects (Mennella et al. 2016). Not only with the bitter taste alone, natural com-
pounds often come with strong odor or flavor, leaving behind a sensation of
astringency. Apart from enhancing its stability, the strong flavor and astringency of
cinnamon extract, a rich source of proanthocyanidins, were successfully concealed
by microcapsulation with gelatin/gum arabic and gelatin/j-carrageenan systems
(De Souza et al. 2020).
Spray drying is one of the most common methods employed for microencap-
sulation of bioactive compounds. Spray drying was employed to effectively
encapsulate both the seed oils and peel extract of pomegranate to provide protection
of the phenolics and fatty acid contents which are otherwise highly sensitive to
environmental factors during their storage (Bustamante et al. 2017). Procyanidins

extracted from grape seed oil was also encapsulated by spray-drying method using
gum arabic and maltodextrin as coating materials and in this manner, the stability
and shelf-life of the product were enhanced (Zhang et al. 2007). When freeze drying
was compared against spray drying for microencapsulation of bioactive compounds
extracted from Hibiscus Calyces using various encapsulating materials, encapsu-
lation of anthocyanins was higher with freeze drying using gum arabic and yields
higher antioxidant activity, while in terms of physical properties, better results were
obtained with spray-drying method (Piovesana and Norena 2018).
Another improvement that microcapsules offered is the prospect of enhancing
the solubility, and hence the oral bioavailability of bioactive compounds.
Spray-congealing microencapsulation of wild garlic (Allium ursinum L.) extract
was found to enhance the aqueous solubility more than 18 times of the pure extract,
and in addition, there was only a minor decrease in the content of the bioactive
compounds, allicin, and S-methyl methanethiosulfonate over 3-month period of
storage (Tomsik et al. 2019). Microencapsulation also provides the opportunity for
sustained or controlled release of bioactive compounds for prolonged therapeutic
activity (Jain et al. 2015; Saifullah et al. 2019).
20.3.2 Nano-based Encapsulation Platforms for Bioactive

Compounds
Various nanotechnology-based platforms have been developed to overcome the low

oral bioavailability, systemic toxicity, or stability problems facing bioactive com-
pounds. Enhancing oral bioavailability has been one of the major objectives of
these nanomedicinal preparations. The main focus of such enhancement schemes
encompasses the three oral bioavailability requirement steps like increasing the
aqueous solubility of bioactive compounds, enhancing the lipid partitioning or
permeability and reduction of first-pass metabolism (Pachuau 2019). Studies have
shown that encapsulation of bioactive phytochemicals in various nanoparticulate
systems was successful in achieving these objectives by controlling their release,
facilitating their systemic absorption, bypassing pre-systemic metabolism, and
enhancing the cellular uptake while also providing their stability (Ahmed et al.
2012; Aqil et al. 2013; Mukherjee et al. 2015).
20.3.2.1 Polymeric Nanocapsules
Nanocapsules differ from microcapsules only in their size; however, reduction of

particle size to nanometer range leads to remarkable advancement in their
physicochemical properties. Nanoencapsulation enhances dissolution of the drug,
membrane permeability stabilization of labile drugs, and controlled release of the
encapsulated drug (Pandey et al. 2005). As a result, polymeric nanocapsules

facilitate both oral and parenteral administration of bioactive compounds. The type
and amount of the wall formers in nanocapsules have been shown to influence the
size, surface properties, stability, drug-loading capacity, aqueous solubility, and the
release profile of the encapsulated compounds (Dos Santosa et al. 2016). Therefore,
selection of appropriate wall-forming material is crucial to impart the desired
functionality to the nanocapsules. Biocompatible and biodegradable poly(lactic
acid) (PLA), poly(glycolic acid) (PGA), and their copolymer poly
(lactide-coglycolide) (PLGA) are among the most commonly used polymers for
nanoencapsulation (Liu and Feng 2015). Encapsulation of ellagic acid in PLGA
nanoparticles was found to increase the intestinal permeability from 66% in
aqueous suspension of ellagic acid from to 87% in nanocapsules while also
exhibiting its antioxidant effects (Bala et al. 2006). PLGA-based silymarin
nanocapsule was also shown to sustain the release, improve bioavailability, and
exhibit preferential toxicity toward prostate cancer cells, thus enhancing its overall
therapeutic efficacy (Snima et al. 2014). Nanoencapsulation of curcumin with
PLGA also delayed the progression of diabetic cataract in animal model (Grama
et al. 2013) (Table 20.3).
Poorly aqueous-soluble anticancer phytochemical paclitaxel, isolated from
Taxus brevifolia, which also suffers from P-glycoprotein-mediated efflux and
metabolism by cytochrome P450 metabolic enzymes (Singla et al. 2002), had been
alleviated through nanoencapsulation. The improvement in permeability of
nanoencapsulated paclitaxel across Caco-2 monolayers was found to be 6.7–7.4
times of the lone paclitaxel and the plasma concentration time curve (AUC) and
Cmax following oral administration also increased up to 5.7 times and 7.3 times,
respectively (Iqbal et al. 2011).
Coating with mucoadhesive polymers is another means to improve oral
bioavailability of bioactive compounds due to their ability to bring prolonged and
intimate contact with GI tract surfaces resulting in better absorption. Chitosan has
often been used to coat the polymeric nanocapsules due to its ability to adhere to the
negatively charged mucosal surface, thus improving absorption and hence thera-
peutic activity (Chuah et al. 2014; Ensign et al. 2012; Mazzarino et al. 2012).
Capsaicin is a natural bioactive compound isolated from peppers with pungent
odor and quick degradation and nanoencapsulation with natural wall formers
gelatin and acacia was able to mask the pungency and instability of capsaicin
(Jincheng et al. 2010; Wang et al. 2008). pH-dependent release and thermal sta-
bilization of bioactive compounds have also been achieved using natural-based
encapsulating materials (Akbari and Wu 2016; Pinheiro et al. 2015).
20.3.2.2 Solid Lipid Nanoparticles (SLN) and Nanostructured Lipid

Carriers (NLC)
Lipid-based nanocarriers like SLNs have become effective alternative delivery

systems to conventional colloidal carriers like emulsions, liposomes, and polymeric
Table 20.3 Various methods for microencapsulation of bioactive compounds
696
Methods Bioactive compounds Coating materials Particle Percent Purpose

size encapsulation
(µm) (%)
Spray drying (Mennella et al. Gentiana lutea root extract Ethylcellulose and stearate – – Bitter-taste
2016) (secoiridoids) masking
Spray-drying (Boonchu and Grape (Vitis vinifera L.) pomace Maltodextrin and – – Masking of
Utama-ang 2015) extract Carboxymethyl-cellulose bitterness and
astringency
Spray drying (Bustamante Pomegranate (Punica granatum Capsul C (modified starch) 4.34– 35.1–92.7 Stabilization
et al. 2017) var. Wonderful) peel extract and 4.67
seed oil
Spray drying (Zhang et al. Grape seed extract (Procyanidins) Gum arabic and 5–30 88.84 Stabilization
2007) maltodextrin
Spray drying and freeze Bioactive compounds from Partially hydrolyzed guar 5.43– 59.84–74.28 Improvement of
drying (Piovesana and Hibiscus calyces extract gum, poly-dextrose, gum 143.08 physical properties
Norena 2018) arabic
Spray drying (Romo-Hualde Bioactive compounds from red Gum arabic 5.46 63.60–77.10 Stabilization
et al. 2012) pepper (Capsicum annum L.)
Spraydrying (Saenz et al. Bioactive compounds from cactus Maltodextrin and inulin – 39.41–99.49 Stabilization
2009) pear (Opuntia ficus-indica)
Double emulsion solvent Caffeic acid Ethylcellulose 1.4–298 65.9–92.3 Controlled release
evaporation (Paulo and cosmetic
Santos 2018) formulations
Spray congealing (Tomsik Wild garlic (Allium ursinum L.) Gelucire 50/13 (Stearoyl 100– 99 Stability and oral
et al. 2019) extract polyoxyl-32 glycerides) 200 bioavailability
Complex coacervation (Jain b-Carotene Whey protein isolates and 1–500 70 Controlled release
et al. 2015) gum acacia
L. Pachuau et al.
micelles (Lin et al. 2017). In SLNs, the bioactive compounds are part of the solid
lipid core matrix (Fig. 20.2) which is stabilized by a surfactant or a mixture of
surfactants (Weiss et al. 2008). They provide a safe means of effective delivery
system for poorly aqueous-soluble bioactive phytochemicals, enhancing their sta-
bility while also promoting their permeability and bioavailability. They originate
from an O/W-type emulsion where the liquid oil (lipid) is replaced by solid oil that
remains solid at body temperature (Akhavan et al. 2018). SLNs are biocompatible,
considerably stable with good encapsulation efficiency, and can prolong the release
of the encapsulated compounds (Akhavan et al. 2018; Weiss et al. 2008). The
surface of SLN can also be functionalized with agents to facilitate their uptake and
targeting the release of the drug to the desired site of action (Ganesan et al. 2018).
In addition, SLN can also carry both lipophilic and hydrophilic drugs and can be
sterilized and produced in large scale (Liu and Feng 2015). Nanostructured lipid
carriers (NLC) are new generations of SLNs developed in the 1990s which
incorporate a mixture of solid and liquid lipids to overcome the limitation of SLNs
in effective drug delivery (Akhavan et al. 2018). NLCs improve the bioactive
retention capacity of these nanostructures and facilitate controlled release of the
bioactive compounds.
Stabilization to oxidation offered by SLNs seemed to depend on the physico-
chemical properties of the encapsulated bioactives. Within the SLN, hydrophobic
and high melting bioactive compounds like b-carotene are reported to be located
within the inner matrix and kept at the a-subcell of the crystal structure thereby
protecting it from oxidation, while less hydrophobic compounds like vitamin A
arranged on the surfaces of the SLN leading to rapid oxidation (Salminen et al.
2016). However, between the SLNs and NLCs, NLCs were reported to provide
better stability to b-carotene than the SLNs (Qian et al. 2013). The tendency of
droplets aggregation and to coalescence still exists in SLNs which have the capacity
to expel the encapsulated b-carotene to the particle exterior on storage, making it
sensitive to degradation than when it is encapsulated in NLCs.
Fig. 20.2 Structures of solid

lipid nanoparticles (SLNs)
(Reproduced with permission
from Lin et al. 2017,
Copyright © Food and Drug
Administration, Taiwan.
Published by Elsevier Taiwan
LLC)
Flavonoids like quercetin and resveratrol suffer from low aqueous solubility,
rapid metabolism, and photosensitivity which limited their oral bioavailability when
taken orally (Li et al. 2009; Pandita et al. 2014). Formulation of quercetin into SLN
remarkably enhances its gastrointestinal absorption and pharmacokinetic study in
animal model demonstrated that the relative bioavailability of the SLN formulation
against the quercetin suspension 571.4% indicating phenomenal increase in its oral
bioavailability (Li et al. 2009). Stearic acid-based SLN of resveratrol was also
reported to increase the oral bioavailability of the bioactive compound by eightfolds
when compared to its suspension. Similarly, oral bioavailability of curcumin was
also increased in a dose-dependent manner when formulated into soy lecithin-based
SLN in animals. Improvement in oral bioavailability of curcumin-loaded SLN
against solubilized curcumin rat plasma was reported to be 155 times at the dose of
1 mg/kg, 59 times at 12.5 mg/kg, 32 times at 25 mg/kg, and 39 times at the dose of
50 mg/kg (Kakkar et al. 2011). Bioavailability enhancement through SLN formu-
lation has also been reported for other bioactive phytochemicals like an alkaloid
vinpocetine which exhibit poor aqueous solubility and extensive first-pass meta-
bolism (Luo et al. 2006; Medina 2010). Oral bioavailability from SLN vinpocetine
formulation was found to be 4.16–4.17 times that of the suspension (Luo et al.
2006).
Recently, SLN-based sclareol was prepared following hot homogenization
technique and evaluated its anti-proliferative activity in vitro against A549 human
lung epithelial cancer cells which showed similar cytotoxicity to the plain sclareol
but long-term stability and sustained release of sclareol were obtained with the SLN
formulation (Hamishehkar et al. 2018).
NLC-based formulations also exhibit promising results in enhancing bioavail-
ability of bioactive compounds. Silymarin was encapsulated in NLC-based for-
mulation using glycerol distearates, oleic acids, lecithin, and Tween-80, which
increases the oral bioavailability of silymarin against solid dispersion pellets and
commercially available silymarin preparation, Legalon®, was 2.54- and 3.10-folds,
respectively (Shangguan et al. 2014). However, when NLC was compared against
microemulsion in its capacity to enhance oral bioavailability of luteolin in animal
model, microemulsion fared better than the NLCs (Liu et al. 2014). The relative
bioavailability of NLC-based and microemulsion-based luteolin formulations to the
luteolin suspension were found to be 515.06% and 885.46%, respectively.
20.3.2.3 Liposomes and Phytosomes
Both liposomes and phytosomes are also lipid-based formulations suitable to

facilitate the delivery of poorly aqueous-soluble phytochemicals. Liposomes are
spherical vesicles (15–100 nm) consisting of phospholipid with a liquid core and
variable layers, where the hydrophobic tails of the phospholipids face each other,
while the hydrophilic heads are projected toward the inner aqueous core or the
outside boundary of the liposome (Fig. 20.3) (Bonechi et al. 2019; Emami et al.
2018). They resemble biological membrane and can accommodate both hydrophilic
Fig. 20.3 Major difference between liposome and phytosome® (Reproduced with permission
from Selmaty et al. 2010, Copyright © Elsevier Science BV 2009)
and hydrophobic drugs within their layers which make them unique carrier for drug
delivery. In spite of their advantages, successful delivery of liposomes through oral
route has always been a challenge as the question remains whether they would
remain stable along the GI tract conditions (Park et al. 2011). However, the
effective delivery of phytochemicals through oral route has been demonstrated in
animal models (Yi et al. 2013). Flammulina velutipes sterols was orally delivered
through liposomes in Kunming mice and relative bioavailability of ergosterol and
22,23- dihydroergosterol at 162.9 and 244.2%, respectively, was obtained. The
sterols, however, was rapidly eliminated within 4 h. A faster and better absorption
of curcumin was also reported when it was encapsulated in leicithin-based liposome

exhibiting improved oral bioavailability in animal model (Takahashi et al. 2009).
Formulating the Curcuma longa (Ukon) extract (LUE) into liposomes was found to
deliver better protection against carbon tetrachloride-induced liver injury as com-
pared to the uncapsulated extract (Takahashi et al. 2008). Liposomes are also
adaptable to functionalization with targeting ligands to improve their effectiveness
in cancer therapy. Kappaphycus alvarezii extract containing multi-bioactive com-
pounds was encapsulated into PEGylated-liposome and then functionalized with
folic acid to target folate positive breast cancer cells (MCF-7) (Baskararaj et al.
2020). The constructed liposome was highly stable in the physiological buffers with
steady drug release and also exhibit cytotoxicity toward MCF-7 in a
concentration-dependent manner.
Liposomal formulation also contributes to the improvement in physicochemical
properties of natural bioactive compounds such as solubility and stability. Chemical
stability of resveratrol was enhanced by liposomes and along with 3-oxo-C12-
homoserine lactone, about 70% inhibition of tumor growth in murine tumor model
was reported when they are administered intravenously (Coimbra et al. 2011).
Encapsulation of enriched-phenolic fraction of the extract from Pistacia vera L
into nanoliposome exhibits multiple bioactivity including antioxidant, anti-
inflammatory, and anti-melanogenic activities making it a promising cosmeceuti-
cal preparation for skin pigmentation disorders (Oskoueian et al. 2020). Promotion
of stability and enhancement in bioactivity of quercetin (Hao et al. 2017) and bitter
gourd (Momordica charantia) (Erami et al. 2019) were also reported with their
nanoliposomal formulations.
Proliposome systems were also developed with an objective of enhancing the
stability and oral bioavailability of the encapsulated drugs. Compared to liposomes,
proliposomes are highly stable and obtained in dry, free-flowing particles which
form liposomal suspension immediately on contact with water (Yan-yu et al. 2006).
Presenting the whole system in the form of solid makes proliposome highly stable
while the intrinsic pharmacological properties remain intact.
Phytosomes are complex of phospholipid with herbal extracts developed to
enhance the oral bioavailability of natural bioactive compounds. It is a patented
technology developed in Italy around 1989 and several phytosome products are
already available in the market (Pachuau 2019). The complexation of phos-
phatidylcholine (phospholipid) with polyphenols in phytosomes led to the forma-
tion of intermolecular bond between these two and the amphiphilic nature of the
phosphatidylcholine promotes the miscibility of the complex with aqueous and lipid
solvents which guide the polyphenol to permeate the lipid-based GI tract lumen
(Kidd 2009). Delivery of polyphenols in the form of phytosomes has the capacity to
enhance the oral bioavailability of polyphenols at least by 2–6 times (Ajazuddin and
Saraf 2010; Kidd 2009). The ratio of the bioactive compounds to the phos-
phatidylcholine has been considered to be important factor in phytosome formu-
lations. This bioactive compound:phospholipid ratio may range from 0.5:1 to 3:1
and the optimum ratio may depend on the kind of phytochemicals being complexed
(Pachuau 2019). Frequently, a combination of bioactive phytochemicals has been
incorporated into phytosomes to attain their synergistic effects (Rathee and Kamboj
2018; Sharma and Sahu 2016).
20.3.2.4 Self-emulsifying Systems
Both micro- and nano-self-emulsifying systems are capable of enhancing oral

bioavailability of poorly aqueous-soluble drugs by enhancing their solubility and
dissolution rate. They are composed of a lipid, surfactant, bioactive compound, and
a co-surfactant, and following oral administration, they swiftly form microemul-
sions with particle size less than 100 nm within the GI fluid bringing the drug into
solution for enhanced absorption (Cui et al. 2009; Dwivedi et al. 2014). Micro- and
nano-self-emulsifying systems have been demonstrated to enhance the oral
bioavailability of various phytochemicals including curcumin (Cui et al. 2009),
arteether (Dwivedi et al. 2014), silymarin (Li et al. 2010; Wu et al. 2006), curcumin,
and thymoquinone (Alwadei et al. 2019), naringenin (Khan et al. 2015) as well as
plant extracts (Arun and Maneesh 2016).
20.3.2.5 Niosomes
Niosomes are self-assembled non-ionic surfactant vesicles analogous to phospho-

lipid vesicles of liposomes where hydrophilic drugs are encapsulated and
hydrophobic drugs are partitioned into the hydrophobic tails of the non-ionic sur-
factant (Hu and Rhodes 1999; Uchegbu and Vyas 1998). Niosomes are more stable
than liposomes and are more economical to prepare from a wide range of surfac-
tants which make them an attractive alternative to liposomes (Song et al. 2015).
Niosomal preparations are reportedly initiated from the cosmetic industry in the
1970s and gradually expanded toward drug delivery applications including phy-
tochemicals (Muzzalupo and Mazzotta 2019). Niosomes of poorly soluble
D-limonene were reported to prolong the release of D-limonene and its cytotoxicity
toward HepG2, Macf-7, and A549 cancer cells was significantly enhanced
(Hajizadeh et al. 2019). Niosome also facilitates the solubilization of Lawsone and
compared to free Lawsone solution, the antitumor activity of Lawsone noisome
against MCF-7 breast cancer cells was increased significantly (Barani et al. 2018).
Studies have shown that niosomes are also effective carrier for the delivery of
bioactive phytochemicals across the skin. Compared to their solutions, more effi-
cient delivery of ellagic acid across the human epidermis and dermis was observed
when it was formulated as niosomes and this penetration of the skin was reported to
be dependent on the vesicle size (Junyaprasert et al. 2012).
Niosomes may suffer from the issue of aggregation, fusion, and leakage of the
encapsulated bioactive compounds. To alleviate this problem, proniosomes have
been prepared which are dry and free-flowing type that spontaneously form nio-
somes on gentle agitation with hot water. Proniosomes were demonstrated to
increase the oral bioavailability of poorly soluble alkaloid, vinpocetine by about 4-

to 4.9-folds (Song et al. 2015).
20.3.2.6 Hydrogels
Hydrogels are hydrophilic three-dimensional polymer networks which can take up

large amount of water. They are biodegradable and the three-dimensional network
of the hydrogel disintegrates into non-toxic materials with excellent biocompati-
bility (Akhtar et al. 2016). Hydrogels are prepared from wide ranges of natural and
synthetic polymers and can be presented in the various physical forms such as
beads, microparticles, nanoparticles, and films and they have become one of the
promising encapsulating systems to provide protection to sensitive phytochemicals
and for their controlled delivery (Abaee et al. 2017; Gomez-Mascaraque et al.
2016a). The advantages hydrogels offer in encapsulation of bioactive compounds
include the following:
(i) Enhancing stability and bioacessibility of sensitive, poorly aqueous-soluble
bioactive molecules (Gomez-Mascaraque et al. 2016a; Han et al. 2020;
Zhang et al. 2016).
(ii) pH-dependent and controlled release of bioactive compounds (Bourbon et al.
2016; Lopez Cordoba et al. 2013; Rutz et al. 2013).
(iii) Efficient encapsulation of both hydrophilic and hydrophobic bioactive
compounds (Bourbon et al. 2016).
(iv) Enhancing bioactivity (Chan et al. 2010).
(v) Improving bioavailability (McClements 2017).
(vi) High drug-loading capacity due to their large spacing within the polymeric
network (Teng et al. 2015).
Various hybrid hydrogel systems such as emulsion hydrogels, organogels and
bigels have also been evaluated for encapsulation of bioactive compounds.
Encapsulation of bioactive compounds within these networks can improve their
stability, solubility and dispersibility thereby improving their overall bioavailability
(Mao et al. 2019). Suitable functionalities such as responsiveness to pH, temper-
ature, enzymes etc. can also be imparted to these hybrid hydrogels to provide the
desired release characteristics of the formulation (McClements 2017). In emulsion
(O/W) gel systems, the continuous aqueous phase gets solidified or gelled to pro-
vide structural integrity and flexibility to the whole system. As a result, it has wide
application in functional food industry. Bioactive compounds such as curcumin
(Geremias-Andrade et al. 2017), quercetin (Chen et al. 2018), a-tocopherol (Freire
et al. 2018) have been delivered through emulsion gel systems with improved
stability, bioaccessibility and controlled release. In addition, the structural state of
emulsion hydrogels also allows them to be used as an efficient fat-replacement in
various food products without affecting their sensory characteristics thereby pro-
moting health benefits to consumers (Pintado et al. 2015, 2016).
20.3.2.7 Electrodynamic Processes
In recent years, electrodynamic encapsulation processes such as electrospraying and

electrospinning have received increasing attention as an alternative to the more
established and commonly employed techniques such as microencapsulation
(Alehosseini et al. 2018; Jacobsen et al. 2018). During electrospraying, the high
voltage electrostatic force converts the drug-loaded liquid solutions into fine dro-
plets and gets evaporated during their flight toward the ground electrode
(Alehosseini et al. 2018). A high degree of molecular interaction in the sprayed
liquid produces electrospun fibers, while lower degree of interaction results in
electrosprayed particles (Fig. 20.4). In conventional spray-drying process, drugs or
bioactive compounds are exposed to high temperature in the range of 170–220 °C
to dry and encapsulate the material and this condition of drying may degrade
thermolabile drug substances (Jacobsen et al. 2018). However, in electrospraying,
exposure to such high temperature has been eliminated which makes it more
conducive for encapsulation of heat-sensitive compounds. Electrodynamic encap-
sulation of bioactive compounds also offers the following advantages (Drosou et al.
2017; Ghorani and Tucker 2015; Wen et al. 2017a; Wen et al. 2017b):
(i) It is a relatively simple, cost-effective, and flexible method for fabrication of
nanomaterials.
(ii) It has the potential to scale up to large-scale manufacturing.
(iii) The method is adaptable to wide range of encapsulating materials.
Fig. 20.4 Left: Schematic of physical representation at the molecular level of entanglement
regimes for dilute and concentrated polymer concentration. Middle: Schematic diagram of a basic
electrospinning (jet formation) and electrospraying (liquid-droplet atomization) processes. Right:
Examples of SEM image of microspheres (electrospraying) and nanofibers (Electrospinning)
(Reproduced with permission from Ghorani and Tucker 2015, Copyright © Elsevier Science BV
2015)
(iv) Enhanced stability of bioactive compounds.

(v) High surface-to-volume ratio of the nanoscale materials results in better
bioavailability.
(vi) Both hydrophilic and hydrophobic bioactive compounds can be
encapsulated.
Electrospraying was employed to successfully encapsulate b-carotene in its
solubilized form to enhance its stability and also to improve its bioavailability
(Basar et al. 2020). When exposed under UV light, the free-b-carotene degrades
within 180 min while only 20% degradation was found with the encapsulated
b-carotene. Wide ranges of polymers are adaptable to electrodynamic processing
including polysaccharides cellulose and its derivatives, guar gum, pectin, starch,
chitosan, alginate, etc. (Wen et al. 2017b). Viscosity, surface tension, and electrical
conductivity of the polymeric solution were found to influence the morphology of
the electrosprayed capsules or fibers (Gomez-Mascaraque et al. 2016b).
20.3.2.8 Solid Dispersion and Micelles
Solid dispersion technique is one of the most commonly employed methods to

improve the solubility and bioavailability of poorly aqueous-soluble drugs.
Crystalline drugs often exhibit poor solubility in aqueous-based solvents as the
lattice energy must be overcome to bring them into solution (Li et al. 2013).
Formulation into amorphous solid dispersion helps solubilization of such crystalline
drugs as the encapsulating polymers in the solid dispersions trapped them in the
amorphous state which facilitates their dissolution once they come in contact with
the aqueous solvents. Various cellulose derivatives have been reported to exhibit
strong enhancement of curcumin dissolution while also protecting from chemical
degradation and affecting pH-dependent release (Li et al. 2013). Various formu-
lations of curcumin such as nanocrystal solid dispersion prepared with hydrox-
ypropyl cellulose SL (CSD-Cur), amorphous solid dispersion with hydroxypropyl
methylcellulose acetate succinate (ASD-Cur), and nanoemulsion (NE-Cur) were
prepared and the increase in oral bioavailability compared to the unformulated
curcumin was reported to be 12-folds for ASD-Cur, 16-folds for CSD-Cur and,
9-folds for NE-Cur (Onoue et al. 2010).
Self-assembled colloidal dispersion of amphiphilic surfactants, also known as
micelles, has been another important technique to enhance the solubility of
hydrophobic drugs. Certain amphiphilic polymers are also known to form micelles
and are known as polymeric micelles (Husseini and Pitt 2008). Polymeric micelles
have been shown to enhance the solubility and bioactivity of bioactive compounds.
When Pluronic P123- and Solutol HS15-based polymeric micelles were prepared
loaded with naringenin, sustained release of naringerin and significant enhancement
in its cytotoxicity against cancer cell line were observed (Zhai et al. 2013). Oral
bioavailability of paclitaxel in glycyrrhizic acid micelle was also found to enhance
sixfolds as compared to Taxol (Yang et al. 2015).
20.4 Conclusion
Enhancement in stability and bioavailability of phytochemicals has been one of the

most important challenges in their effective utilization. Thousands of bioactive
compounds have been isolated and characterized over the years; however, their
therapeutic effectiveness in vivo is often limited by their poor physicochemical
properties and modest oral absorption. Several studies have demonstrated that
encapsulation into various micro-and nanoplatforms is capable of alleviating such
obstacle. Microencapsulation techniques have been proven and commonly
employed method for such enhancement. Novel techniques like hydrogels and their
variants such as emulsion gels, organo gels, and bigels along with electrodynamic
processes like electrospraying and electrospinning techniques are certainly
promising approaches to facilitate the effective delivery of these bioactive
compounds.
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Chapter 21
Tannins and Polyphenols Extracted
from Natural Plants and Their Versatile
Application
Suvadeep Mal and Dilipkumar Pal
Abstract From the beginning of lives on earth, nature is contributing different

products to the system constantly and endlessly. Plants synthesize a large number of
organic compounds, which are commonly known as primary and secondary
metabolites with various applications. Tannins are one of the secondary metabolites
solely obtained from the natural or plant sources where it present in the woods,
barks, leaves, fruits, cell sap or in vacuoles. Chemically, they are polyphenolic
colloidal solutions with complex astringent properties and it has the ability to tan or
convert the skin of animals into leather. Depending on the complexity of chemical
nature, tannins are classified into two types i.e., hydrolysable tannins and con-
densed tannins. More than 8000 different tannins of free or bound forms have been
detected which can be used in various sector. Despite of its astringent property,
tannins and polyphenols can show their identity with different applications with
properties like anti-oxidant, anti-inflammatory, anti-microbial, anti-aging, stom-
achic, cardio-tonic, diuretics, laxatives, hypoglycemic, anti-corrosive or in pho-
tography, food, neutraceuticals or cosmeceuticals. In this review, we discuss about
different tannins and polyphenols obtained from different sources, their types, about
important chemicals and their remarkable applications in different fields of the
system.

Keywords Secondary metabolite Colloidal solution Tannins Hydrolysable

tannin Condensed tannin Astringent Polyphenols Laxative Cardio-tonic

Anti-oxidants Anti-inflammatory Anti-microbial Diuretics Neutraceuticals
Cosmeceuticals
S. Mal
Haldia Institute of Pharmacy, Kshudiram Nagar, West Bengal, Haldia-721657, India
D. Pal (&)
https://doi.org/10.1007/978-3-030-54027-2_21
716 S. Mal and D. Pal
21.1 Introduction
Natural products are the chemicals and compounds that are given to us by nature.
The significance of the nature and the natural products are well understood when we
come to the ecosystem. For the thousands of years, plants are defending and
reigning rank holder which works day and night in their own body for giving us
remarkable natural products. The various and different parts of a plant can be a
good source for natural compounds. Plants metabolize different organic compounds
in their body by complex mechanism comprises physical and chemical episodes of
photosynthesis, respiration, degradation. There are two types of metabolism occurs
in plant system, like primary and secondary metabolism. Primary metabolism yields
primary metabolites which are essential for the growth, development, functioning
and the survival of the plant whereas, secondary metabolites play a direct or indirect
role in plant’s growth and they are not required for a plant to survive. The sec-
ondary metabolites of a plant play a key role in plant’s defence system, fighting
herbivores and pathogens. Also they are found useful in other important functions
like giving plant or plant portions a color, maintaining and signaling the primary
metabolic pathways etc. (Mazid et al. 2011). Some of the much known important
secondary metabolites are terpenes, phenolic metabolites, glycosides and alkaloids.
Tannins and polyphenols are one of the most widely used secondary metabolites
obtained from different species of higher altitude plants. It is found from different
parts of myrobalan, nutgall, chestnut, rhubarb, bahera, arjuna, Indian goose berry,
ashoka bark, black and pale catechu, pterocarpus etc. and they are extracted by
maceration. Chemically, they are the mixture of complex organic substances in
which polyphenols are present. Tannins are basically phenylpropanoids, condensed
to lengthy polymers with molecular weight ranging from 300 to 3000 (Khanbabaee
and Van Ree 2001). They are non-nitrogenous and not crystalline in nature, they do
possess a bitter taste and they give positive results with Goldbeater’s skin test
(Nierenstein 1932). Tannins are classified into two types generally, hydrolysable
tannins and condensed tannins. Hydrolysable tannins can be hydrolysed easily by
enzymes or different acids into gallic acid or ellagic acid, which further process
pyrogallol, glucogallin, etc., where condensed tannins do not respond to hydrolysis
and being a flavone derivative, they can be related to flavonoid dyes and pigments or
phlobatannins or proanthocyanidins (Schofield et al. 2001). It is having astringent
property and it helps in precipitation of different proteins, gelatins, glycosides etc.
from any solution. The word ‘tannin’ helps to give a simple knowledge about its
property to tan, i.e. ability to convert leather from skin surface (Sieniawska and Baj
2017). Because of this property, tannins and tannin containing drugs can be used in
burn treatment. Apart from the astringent property, tannins do possess versatile
applications like medicinal uses, industrial uses and biological applications. Tannins
are used to treat different diseases and biological conditions like fever, diarrhea,
diabetes, eye infections, gall bladder stone, intestinal disorder, constipation etc.
(Sieniawska and Baj 2017). Different tannins and tannic acid derivatives obtained
from different parts of plants like roots, barks, stems, leaves, galls, fruits, herbs are
21 Tannins and Polyphenols Extracted from Natural Plants … 717
used as anti-viral, anti-oxidant, anti-inflammatory, anti-microbial, anti-aging,

stomachic, cardio-tonic, diuretics, laxatives, anti-hyperglycemic, anti-cancer,
anti-coagulant etc. (Smeriglio et al. 2017). Despite of all these beneficial effects on
body, tannins also cause side effects like loss of appetite, sickness, nausea, stomach
irritation, over urination, or sometimes more serious side effects like hepatotoxicity,
sore and throat cancer, and heart failure (Kumar and Singh 1984).
21.2 Tannin Occurrence: Plants Containing Tannins
Tannin, being a secondary metabolite, can be found in almost all higher plants.
Tannins are found commonly in angiosperms and gymnosperms. Among the plants,
tannins are distributed through the different parts of plants like, fruits, roots, barks,
woods, leaves, seeds and plant galls. In major cases, tannins are found normally in
the plants belonging to different families like combretaceae, fagaceae, euphor-
biaceae, leguminosae, rubiaceae, anacardiaceae, aceraceae, actinidiaceae, bixaceae,
burseraceae, dipterocarpaceae, ericaceae, myricaceae among dicot plants and
najadaceae, typhaceae among monocot plants. Condensed tannins are most abun-
dant polyphenols and it is found in almost all families of plants. Tannins are often
found in the xylem and secondary phloem or in the layer between the epidermis and
cortex (Ashok and Upadhyaya 2012). The production of condensed tannins
occurred inside tannosome, a chlorophyllus organelle, enclosed within tannoplast,
attached on the cytosolic face of the endoplasmic reticulum (Brillouet et al. 2013).
After synthesis, tannins are stored in vacuoles or in the surface wax of the plants
giving protection to the plants from the outer environmental predators. While
staying in the vacuoles, tannins present in the active form but it does not show any
of its activeness i.e., it does not interfere with plant metabolism or proteins until cell
breakdown or cell death occurs and it is released (Cannas 2008). Tannin can also be
accumulated in the vacuole of tannin cells, which are the idioblasts of parenchyma
cells (Kanzaki et al. 2001). The presence of tannin can be seen generally in the outer
part of the bud and in case of leaf tissues, the most common part where tannin is
obtained is in the upper epidermis. They protect the leaf tissues against predators as
it is distributed in leaf tissues also. In root tissues the most common region is the
hypodermis, where they act as a chemical barrier of pathogens to penetration and
colonization in roots. In seeds, they are located in between outer integument and
aleuronic layer, where they exhibit allopathic and bactericidal properties (Ashok
and Upadhyaya 2012). The different tannins obtained from different plant sources
with their pharmacological indications are given in Table 21.1.
Table 21.1 Tannin containing plants, their sources and pharmacological indications
718
Source Plant Tannins obtained Indications References

parts
Acacia catechu Bark Catechin, epicatechin, Cough, diarrhoea, protection against skin ulcer Shen et al. (2006)
(L.) catechutannic acid,
Family: epicatechin-3-O-gallate,
Fabaceae and epigallocatechin-3-
O-gallate
Acacia nilotica Pod Gallocatechin-gallate Diarrhoea, diabetes, sore gums, and skin infection (arim and Azlan
(L.) methyl gallate, catechin, (2012)
Family: epicatechin, catechin gallate,
Fabaceae galloylglucose
Agrimonia Herb Catechin; procyanidin Eye infections, diarrhoea and disorders of gall Granica et al.
eupatoria (L.) B3, agrimoniin bladder, liver, and kidneys (2013)
Family:
Rosaceae
Camellia Leaves Catechin, epicatechin Aiding digestion, blood purification, boosting immune Shoji and
sinensis gallate, epigallocatechin system, heart disorder, antiviral, hyperglycemia Nakashima
(L.) gallate, epigallocatechin (2006)
Family:
Theaceae
Diospyros kaki Fruit Proanthocyanidin Antiseptic, hypercholesterolamia, cardiac disorder Zhang et al.
Family: catechin, gallocatechin, (2011)
Ebenaceae catechin-3-O-gallate,
gallocatechin-3-Ogallate
Emblica Fruits Phyllemblin Laxative, diuretic Variya et al.
officinalis (2016)
Family:
Euphorbiaceae
(continued)
S. Mal and D. Pal
21

parts
Geranium Leaves Geraniin Intestinal disorders Okuda and Ito
Thunbergii (2011)
Family:
Geraniaceae
Geum japonicum Herb O-galloyl-betaglucoside, Diuretic, anti-coagulant Xie et al. (2007),
(L.) pedunculagin, Dong et al. (1998)
Family: tellimagrandin II, 2,6-di-
Rosaceae O-galloyl-D-glucose,
casuariin, and 5-
desgalloylstachyurin
Hamamelis Bark and Hamamelitannin, Swellings, wound healing, burns therapy, Hartisch and
virginiana (L.) leaves pentagalloylglucose, anti-inflammatory, and anti-tumors Kolodziej (1996)
Family: derivatives of
Hamamelidaceae epicatechin-(4B-8)-
catechins,
proanthocyanidins
Krameria Root Tannic acid, Dysentery, menorrhagia, (Singh)
Tannins and Polyphenols Extracted from Natural Plants …
trianda (L.) rhataniatannic acid, incontinence of urine, haematuria, sore throat

Family: crameric acid,
Krameriaceae phlobaphene,
phloroglucin, proanthocyanidins
Mallotus Bark 3,4,11-Tri-Ogalloylbergenin Gastritis, gastric ulcer, diarrhoea, and constipation Okada et al.
japonicas (1973)
(L.)
Family:
Euphorbiaceae
(continued)
719
720

parts
Mouriri pusa Leaves Catechins and Gastritis and ulcers Vasconcelos et al.
Family: condensed tannins (2010)
Melastomataceae
Phyllanthus Leaves, Geraniin, Chronic wounds, skin eruptions, anti-tumor Agyare et al.
Muellerianus Stem, corilagin, furosin (2011)
Family: bark
Phyllanthaceae
Potentilla erecta Roots Pentadigalloylglucose, Inflammations, wounds, bleeding, dysentery, diarrhoea, Tomczyk and
Family: pedunculagin, bowel disease, microbial infections, cancer, antiseptic for Latté (2009)
Rosaceae agrimoniin, the mouth and throat
epigallocatechin,
catechins, proanthocyanidins
Potentilla Aerial Agrimoniin, Diarrhoea, bleeding, influenza, parotitis, lymphadenitis, Okuda et al.
kleiniana parts potentillin hepatitis, scare, numbness of limbs, dysmenorrhea, ulcer (1984)
Family:
Rosaceae
Pterocarpus Whole Kinotannic acid, k-pyrocatechin Anti-hyperglycemic, anti-hyperlipidemic, astringent Dhanabal et al.
marsupium plant (2006)
Family:
Leguminosae
Quercus Gall Tannic acid Astringent, inflammation, local anesthetic, bacterial, Ikram and
infectoria (Turkish fungal and viral infections Nowshad (1977)
(Oak) gall)
Family:
Fagaceae
(continued)
S. Mal and D. Pal
21

parts
Quercus robur Bark Grandinin, castalagin, Diarrhoea, oral mucosa or skin inflammation, Mämmelä et al.
(L.) glucogallin hemorrhoids (2000)
Family:
Fagaceae
Rhus chinensis Gall Chinese gallotannin Antiseptic, burn therapy, Djakpo and Yao
(Mill.) (Chinese traumatic injuries, (2010)
Family: gall) haemorrhoids, and ulcers
Anacardiaceae in the mouth
Sanguisorba Root Sanguiin H-6 Dysentery with blood, Tsukahara et al.
officinalis (L.) nosebleeds, burns (2001)
Family:
Rosaceae
Saraca indica Bark Catechol Oxytocic, larvicidal activity Mathew et al.
Family: (2009)
Leguminosae
Syzygium cumini Bark Corilagin and related Sore throat, bronchitis, Lanitis et al.
ellagitannins asthma, dysentery and ulcers (2012)
Tannins and Polyphenols Extracted from Natural Plants …
Terminalia Stem b-sitosterol Cardioprotection, angina pectoris, hypercholesterolemia Bharani et al.

arjuna bark treatment (2002)
(Arjuna)
Family:
Combretaceae
Terminalia Dried Gallic acid, chebulagic acid Anti-diabetic and anti-oxidant Hazra et al.
belerica mature (2010)
(Bahera) fruits
Family:
Combretaceae
(continued)
721
722

parts
Terminalia Dried 2,4-Chebulyl-b-Dglucopyranose, Cold-related coughs, ulcer, purgative Pfundstein et al.
chebula mature chebulinic acid, (2010)
(Myrobalan) fruit punicalagin, terflavin A,
Family: terchebin, tannic acid
Combretaceae
Uncaria Leave, Catechutannic acid, catechin Anti-oxidant, anthelmintic Amir et al. (2012)
gambier young
Family: shoot
Rubiaceae
S. Mal and D. Pal
21.3 Classification of Tannins
Chemically, tannins are high molecular weight compounds, which are soluble in
water and can be broadly classified in various types according to their complexity of
their chemical nature or according to the structural moieties or according to the
behavior on dry distillation or on the action of enzyme tannase. Firstly, on the basis
of the chemical nature or better to say, according to the functionality, tannins are
classified as “true tannins’ and “pseudo tannins”. In case of this first divisional
group, true tannins are those, which is having high molecular weight and give
positive result in Goldbeater’s skin test (Rangari 2008), so they can be used as
tanning agents. Whereas, the latter is of low molecular weight, do not respond to the
Goldbeater’s skin test i.e. they cannot be used as the tanning agents. The basic
nucleus of true tannins and pseudo tannins are also in contrast like, true tannins are
made of gallic acid or egallic acid derivatives but the basic nucleus of pseudo
tannins are chlorogenic acid or catechins (Shah 2009). Catechins, and chlorogenic
acid from cocoa and Nux vomica respectively are two common examples of
pseudotannins (Ashok and Upadhyaya 2012). True tannins are further classified as
hydrolysable tannins, condensed tannins and complex tannins, according to their
behavior in acid hydrolysis. Hydrolysable tannins are those, which upon hydrolysis
by acids like HCl or H2SO4, yield gallic acid or ellagic acid. Since, it is a pyrogallol
type of tannin, further dry distillation of gallic acid and ellagic acid gives pyrogallol
(Ramakrishnan and Krishnan 1994). On the other hand, condensed tannins are
resistant to hydrolysis by acids. Instead of hydrolysis, they depolymerize or
decompose into red or brown colored pigment called phlobaphene (Foo and
Karchesy 1989). So, the hydrolysable tannins comprise gallotannins and hexahy-
droxydiphenic acid made polyesters, whereas condensed tannins contain favan-3-ol
nuclei (proanthocyanidins) (De Bruyne et al. 1999). Complex tannins are mixtures
of both, hydrolysable and condensed tannins. But according to modern studies,
structural characteristics leads to categorise tannins into four major groups i.e.
gallotannins, ellagitannins, complex tannins and condensed tannins (Khanbabaee
and Van Ree 2001).
21.3.1 Gallotannins
Gallotannins are one of the compounds, belonging to the subclass of hydrolysable

tannins because it can be hydrolysed by mineral acids and yield gallic acid. It is a
polymer, obtained from the condensation of the carboxy group (–COOH) of gallic
acid and its polymeric derivatives with the hydroxyl groups (–OH) of a carbohy-
drate (mainly glucose). In case of partial substitution with galloyl unit (1), the
remaining –OH groups of polyol can make substitutions with various other residues
or remain un-substituted (Mueller-Harvey 2001).
The gallotannins 2,3,4,6-tetra-O-galloyl-D-glucopyranose (TGG) (2) and

1,2,3,4,6-penta-O-galloyl-b-D-glucopyranose (b-PGG) (3), found in many plant
families, are the major intermediates for the biosynthesis of hydrolysable tannins
(Gross 1993) (Hagenah and Gross 1993). Most of the galloyl units at the anomeric
centre of their D-glucosyl unit have the b configuration at the anomeric centre. But
in some cases like 1,4-di-O-galloyl-a-D-glucopyranose (4), the anomeric centre of
the D-glucopyranose has the a configuration (Khanbabaee and van Ree 2001).
Examples of some gallotannins are: tannic acid (Chinese gallotannin) (5), Turkish
gallotannin (6), acertannin (7), hamamelitannin (8) etc. (Fig. 21.1).
Tannic acid is a gallotannin with chemical formula given as C76H52O46. It
contains decagalloyl glucose (5) moiety and it is a commercially available tannin.
Tannic acid is found in the nutgalls formed by insects on twigs of certain oak trees
(Quercus infectoria and other Quercus species). While Chinese gallotannin contains
decagalloylglucose residue, Turkish gallotannins tends to have penta-, hexa-, or
heptagalloylglucose moiety.
21.3.2 Ellagitannins
Ellagitannins contains at least two galloyl units coupled to each other by C–C
bonds, but they do not form any catechin units. Ellagitannins are formed by the
first-stage biogenetic oxidative coupling of at least two galloyl units, yielding an
axially chiral hexahydroxydiphenoyl moiety or HHDP unit (9) (Khanbabaee and
Van Ree 2001). Remarkably, all ellagitannins, with HHDP units bound to the
polyol unit (basically, D-glucopyranose) in 2,3- or 4,6- or 1,6-positions, always
exert the (S)-configuration, while 2,4- or 3,6-coupled HHDP units show the (R)-
configuration generally (Feldman and Ensel 1994). Ellagitannins differ from gal-
lotannins, in their galloyl groups linkage. Ellagitannins link their galloyl units
through C–C bonds, whereas gallotannins follows depside bond linkage (Radebe
et al. 2013). Some of the ellagitannins are chebulinic acid (10), corilagin (11),
geraniin (12), tellimagrandin II (13), casuarinin (14), potentillin (15) etc.
(Fig. 21.2).
21.3.3 Condensed Tannins
These tannins are derivatives of flavonoid (16), catechins (17), flavan-3–4-diol.

They do not contain sugar residues. Monomeric catechins and leukoanthocyanidins
(flavan-3–4-diol) (18) don’t have any tanning properties, but when they oligomerize
under acidic condition or enzymatic action, they can produce significant tanning
action (Khanbabaee and van Ree 2001) (Ferreira and Slade 2002). Condensed
tannins are the oligomeric and polymeric proanthocyanidins formed via linking
catechins one another in C-4 to C-6 or C-8 positions.
Proanthocyanidins (19) can be classified according to variation in hydroxylation

pattern into several subgroups like propelargonidins (3,4ʹ,5,7–OH) (20), procyanidins
(3,3ʹ,4ʹ,5,7–OH) (21), prodelphinidins (3,3ʹ,4ʹ,5,5ʹ,7-OH) (22), proguibourtinidins
(3,4ʹ,7-OH) (23), profisetinidins (3,3ʹ,4ʹ,7-OH) (24), prorobinetinidins
(3,3ʹ,4ʹ,5ʹ,7-OH) (25), proteracacidins (4ʹ,7,8-OH) (26), promelacacidins
(3ʹ,4ʹ,7,8-OH) (27), pro-apigeninidins (4ʹ,5,7-OH), and proluteolinidins
(3ʹ,4ʹ5,7-OH) (Fig. 21.3) (Khanbabaee and Van Ree 2001).
21.3.4 Complex Tannins
Complex tannins are formed when, a hydrolysable unit binds with a gallotannins or
an ellagitannins through a glycoside linkage. An example of complex tannin is
acutissimin A (27), which is isolated from the bark Korean chestnut (Castanea
HO OH
HO OH
O OH OH
OH
O HO
O OH
O HO O O
OH O OH
OH HO O O OH
O OH HO O
HO O
O
HO O O
HO
1 OH O O OH O
OH
HO OH
OH OH
HO HO
HO HO OH
OH
HO 2 3
HO OH
O OH HO OH
O
OH
OH HO
OH
O
OH O HO OH O
O
HO O
O O
OH HO HO O
O OH
O O O
HO OH O HO
O O O OH HO
O O HO O O
HO OH
HO O OH HO
O O O OH
HO HO O
O O
HO OH
HO
O O 4
HO
O OH O
O OH O
O OH
HO
OH
HO HO O OH
OH HO
HO OH
O
HO
5 O
HO HO HO OH
O OH
OH
HO OH 6 OH
OH
OH O OH
O
O O
HO O O
OH
O O
HO OH
OH
OH
OH 8
7
Fig. 21.1 Structures of some gallotannins

HO
HO OH HO OH
O
O O OH HO
HO OH HO
HO O
O
HO OH
HO OH
O O O
HO O
OH O OH
HO OH HO OH O
O O O O
9 O O O O
O
OH
HO
HO OH OH
HO 11
HO OH HO OH OH
O O OH
HO
OH
HO OH OH
10
OH
HO OH
O O
O OO
OH HO OH
O O
OH O
HO O
O O O OH
HO
O O
O O
HO
O HO OH O OH
O
OH HO O
HO OH
O 13 OH
12 OH
OH
HO
HO OH
OH OH
HO O
HO O
O OH
O
HO
HO
O O OH O
HO O OH
O HO
O O O
O OH O O
HO O
O HO O
OH
O
O HO OH
HO
HO
OH O OH OH
OH
HO
HO OH
14
OH
15
Fig. 21.2 Structures of some ellagitannins
crenata) (Okuda and Ito 2011). This is a flavo-galloyl unit bound glucosidically to
C-1, with an additional three hydrolysable ester bonds to a D-glucose-derived
open-chain polyol. More example of complex tannin is epigallocatechin gallate
(28), which is widely present in tea leafs and responsible for variety of medicinal
activities or camelliatannin A (29) (Fig. 21.4) obtained from Camellia japonica.
21.4 Biosynthetic Pathways of Tannins
The biosynthesis of tannins follow two different synthetic pathways for hydro-
lysable tannins and condensed tannins. The biosynthesis of hydrolysable tannins
comprises via the key intermediate formation of 1,2,3,4,6-pentagalloyl glucose
(Hagenah and Gross 1993). The starting material or the precursor is quinic acid.
Gallotannins or depsidic metabolites can be formed by the galloylation of the
OH OH
OH OH OH
O HO O O OH
OH HO O
O
OH OH
HO OH
16 OH
17 OH
O
18 OH
OH
HO OH HO 19
HO OH OH
O OH
HO HO O
OH OH
OH OH HO O
O O
OH OH
OH OH OH OH OH
O
21 OH
OH HO O HO O
HO OH
OH
HO OH OH
20 OH
OH 23
22
OH OH (Prodelphinidin B1)
OH OH
HO O HO O
OH OH
HO O
OH OH
25
24 26
Fig. 21.3 Structures and basic units of condensed tannins
HO HO
OH OH OH
HO HO OH HO
O HO
OH O
OH
HO O OH
OH HO O
HO
OH O
O OH O
O HO O OH
HO
OH
HO O O O O
O HO O
OH HO
HO OH
O OH O
O
HO HO OH
HO O
HO O OH
HO OH HO OH
O
HO OH
OH OH 29
O
27
OH
28 OH
Fig. 21.4 Structures of some complex tannins
intermediates and ellagitannins are formed due to the oxidation leading to C–C
linkage.
21.4.1 Biosynthesis of Hydrolysable Tannins
Hydrolysable tannins biosynthesis starts from gallic acid synthesis from quinic acid.
Quinic acid comes from the one of the most important intermediate in glycolysis in
plant, phosphoenolpyruvate. Phosphoenolpyruvate via phosphoenolpyruvate
translocator (PEPTr), forms quinic acid. Under the catalytic action of gallate
1-b-glycosyl transferase enzyme, and via the formation of UDP from UDP-a-glucose,
gallic acid yields 1-O-galloyl-b-D-glucose. From 1-O-galloyl-b-D-glucose, one
molecule of D-glucopyranose is cleaved consecutively in each step and it results in
galloylation to form 1,6-digalloyl-b-D-glucose, 1,2,6-trigalloyl-b-D-glucose,
1,2,3,6-tetragalloyl-b-D-glucose and finally 1,2,3,4,6-pentagalloyl glucose under
the catalysis of enzymes like b-glucogallin-dikisgalloyl glucose-O-galloyl trans-
ferase, b-glucogallin-trikisgalloyl glucose-O-galloyl transferase, b-glucogallin-
tetrakisgalloyl glucose-O-galloyl transferase respectively. 1,2,3,4,6-pentagalloyl-
b-D-glucopyranose or simply 1,2,3,4,6-pentagalloyl glucose is the key intermediate
found in many plant families. After the intermediate 1,2,3,4,6-pentagalloyl glucose
formation, the galloylation of pentagalloylglucose continues and ester linkage
between two galloyl moieties forms gallotannins or depsidic metabolites. The enzy-
matic catalysis of 1,2,3,4,6-pentagalloyl glucose by different galloyltransferase
enzymes forms different linkages to form different gallotannins (Niemetz and Gross
et al. 2005).
After the formation of intermediate 1,2,3,4,6-pentagalloyl glucose, oxidation
takes place by the action of different oxido-reductase enzymes, leading to C–C
linkage formation between suitably orientated galloyl residues of glucogalloyl
molecules and it results in hexahydroxydiphenoyl (HHDP) units formation which is
the core moiety of ellagitannins (Grundhöfer et al. 2001). The biosynthetic pathway
of hydrolysable tannin and complex tannin is given in Fig. 21.5.
21.4.2 Biosynthesis of Condensed and Complex Tannins
The phosphoenolpyruvate obtained from glycolysis in plants, forms quinic acid by

the phosphoenolpyruvate translocator (PEPTr). Quinic acid in the next step, yields
shikimic acid and chorismate. Shikimic acid is found to be the main precursor of
different metabolite formation in plants. In the next step, chorismate gives pheny-
lalanine when asparagine (Asn) forms aspartate (Asp) side by side. After this, the
enzyme called phenylalanine ammonia lyase (PAL) helps to convert phenylalanine
into cinnamic acid or cinnamate. Hydroxyl group insertion occurs to the para
position of cinnamic acid by the help of the enzyme called cinnamic acid
hydroxylase (CAH) to form 4-coumaroyl-CoA. P-coumaroyl-CoA gives chalcone,
when it is condensed with malonyl CoA. The enzymes involved in this step are
4-coumaroyl-CoA ligase (4CL) and chalcone synthase (CHS). Naringenin chalcone
isomerizes under the enzymatic condition produced by chalcone isomerase (CHI) to
form naringenin, which is a flavanone. Naringenin then forms a dihydroflavonol
called dihydrokaempferol via the attachment of a hydroxyl group in 3rd position by
flavanone 3-hydroxylase (F3H). In the next step, another hydroxyl group insertion
occurs in dihydrokaempferol and gives dihydroquercetin by flavonoid 3′-hydro-
xylase (F3′H). Further, reduction takes place by dihydroflavonol reductase
(DFR) which results in the formation of leucocyanidin. Leucocyanidin cleaves the
OH
O OH
O OH UDP OH
O HO OH
UDP- -glucose
HO HO O
OH
P OH PEPTr
HO O O
O gallate 1- -glycosyltransferase HO
CH2 HO OH
HO OH
OH
Phosphoenolpyruvate OH
OH
Quinic acid 1-O-galloyl- -D-glucose
Gallic acid
-glucogallin-O-
galloyl transferase
D-glucopyranose
OH
HO OH OH
OH
OH HO OH
OH
-glucogallin-trikisgalloyl O -glucogallin-dikisgalloyl OH
O O OH
glucose-O-galloyl transferase glucose-O-galloyl transferase
O O O O
O O O OH
OH O O O
1-O-galloyl- - HO O
D-glucose OH
OH D-glucopyranose 1-O-galloyl- - HO OH
OH D-glucose OH
1,2,6-trigalloyl- -D-glucose 1,6-digalloyl- -D-glucose
D-glucopyranose
OH
HO OH
OH OH
HO OH OH OH
OH -glucogallin-tetrakisgalloyl
O
glucose-O-galloyl transferase O O OH
O O O O
OH O O OH O
HO O OH
O O 1-O-galloyl- - O O
D-glucopyranose
O OH D-glucose HO O O
HO O O O OH
O OH OH
OH HO
OH OH
ues HO OH
1,2,3,6-tetragalloyl- -D-glucose sid
l re OH
l l oy les 1,2,3,4,6-pentagalloyl glucose
ga ecu
een mol
etw yl
g e b gallo
a o Galloylation
ink luc
C l of g
HO
OH C- OH
HO OH
OH OH
OH
HO HO OH
OH
OH O
O O O OH
HO O O HO
O OH
OH O O O
O
O O O OH
OH HO
HO O O O O
O O O O O
O O OH
HO O OH
OH
HO
HO Ellagitannin OH
HO OH
OH
Gallotannin
Fig. 21.5 Biosynthetic pathway of gallotannin and ellagitannin
hydroxyl group in 4th position by the help of leucocyanidin deoxygenase (LDOX)

to produce cyanidin or anthocyanidin, which gives (–)-epicatechin by the reduction
of ring double bond. Leucocyanidin upon the enzymatic action of leucoantho-
cyanidin 4-reductase (LAR), gives (+)-catechin. Leucocyanidin polymerises in the
presence of anthocyanidin synthase (ANS) to produce dimer and trimer procyanidin
via C4–C6 or C4–C8 linkage. Procyanidin polymer finally gives condensed tannin
by different C–C linkage formation. Epicatechin units can go through glycosylation
to produce complex tannins (Harding et al. 2014). The biosynthetic pathway of
condensed tannin and complex tannin is given in Fig. 21.6.
O OH
O O OH
O HO Phosphoenolpyruvate
OH
HO
O OH
P OH PEPTr O
HO
O HO OH HO OH OH O
Phosphoenolpyruvate OH
OH Chorismic acid
H2O
Quinic acid
Shikimic acid
Asn
Asp
O O O
SCoA CAH OH PAL OH

NH2
HO
4-coumaroyl-CoA Cinnamic acid Phenylalanine
SCoA
HOOC
O CHS OH
Malonyl CoA
HO OH OH
HO O
CHI
OH O OH O
Chalcone Naringenin
OH F3H
OH OH
HO O HO O
F3'H
OH
OH
OH O
OH O
Dihydroquercetin Dihydrokaempferol
OH
OH
DFR OH
OH
OH
OH
HO O
HO O
LDOX HO O LAR
OH
OH OH
OH
OH
Anthocyanidin OH OH (+)-Catechin
Leucocyanidin
OH
ANR
OH
OH HO O
OH
Glycosylation OH
OH
HO O OH
OH
HO O
OH OH
OH OH
HO
(-)-Epicatechin OH OH
O OH
HO HO O
HO O
O OR OH OH
HO O
OH
O OH
O
HO RO Procyanidin oligomer
OR
R= HO OH OH
Complex tannin Condensed tannin
Fig. 21.6 Biosynthetic pathway of condensed tannins and complex tannins

21.5 Extraction Process of Tannins
As tannins show its solubility in water, alcohols, acetone, ethyl acetate etc., the
solvents which are commonly used to extract different tannins (better say hydro-
lysable tannin) from natural sources are: aqueous solutions of methanol, ethanol, or
acetone and also ethyl acetate. Nonpolar organic solvents like benzene, n-hexane,
petroleum ether, chloroform, dichloromethane etc. are usually used in pre-treatment
of sample for the purpose of removing lipids and chlorophyll. These non-polar
solvents with low extraction strengths are also beneficial in preventing enzymatic
reactions of tannins (Arapitsas 2012). In case of handling tannins with low
molecular weight or in the processing of matrices containing large amounts of
enzymes (i.e., bark or fruit), extraction is done with methanol treatment, while the
use of acetone is preferentially done with tannins of higher mol. Wt. But the
temperature should be taken into account as higher temperatures for extended times
results in hydrolysis of the galloyl moiety and it may force ellagitannins releasing
ellagic acid (Okuda et al. 1989). In addition, extraction with alcohols like ethanol
and methanol may produce ethyl or methyl esters of gallic acid, respectively
(Mueller-Harvey 2001) (Okuda et al. 1989). Not only the extraction solvents, but
also the other factors like temperature, pH, solvent/material ratio etc. contributes in
the extraction procedure. Some of the extraction process applied in the extraction of
tannins from different plants are shortly described below:
1. Classic extraction is done via boiling the solvent. The optimal conditions for
extracting Assam green tea polyphenols lies under maintaining sample and water
ratio (1:20), with a pH of 4–5, utilizing fresh green tea leaves (Wati et al. 2009).
2. Pressurized liquid extraction is another way of extracting tannins, treating
with the solvent at an elevated temperature (usually between 50 and 200 °C) and
under high pressure (between 10 to 15 Mpa). Herein, the best recoveries were
obtained by way of employing this technique for releasing catechin and epi-
catechin from grape seeds, as well as from non-fermented tea leaves,
medium-fermented tea leaves, and fermented tea leaves. Such release comes
about within 10 min (Piñeiro et al. 2004).
3. Supercritical carbon dioxide extraction is applicable using supercritical car-
bon dioxide (SC–CO2) with critical point at 31.1 °C and under a pressure of
73.8 bar. But while performing this technique, one condition should be taken
care of. Normally, carbon dioxide (CO2) is not a suitable solvent for extracting
polyphenols because of its nonpolar nature, hence, to overcome this problem, a
polar organic solvent such as a solution of ethanol and water, can be used during
extraction or before the extraction (Lang and Wai 2001). Ethanol is used in this
technique, as a co-solvent, which was successfully utilized in the extraction of
green tea catechins (Gadkari et al. 2015).
4. Microwave-assisted extraction is done with solvent that is directly heated by
microwaves. The optimal performance of tea polyphenols extraction was
obtained under a microwave intensity of 600 W, a microwave radiation time of
3 min, and a one-time microwave radiate action, with tea/water ratio of 1:20 (Li
and Jiang 2010).
5. Ultrasound/sonication-assisted extraction method demonstrates excellent
solvent penetration into cellular materials. In this process, optimal extraction
conditions for several hydrolysable tannins came about by way of treatment
with 19.7% ethanol, for 26.4 min, at 24 °C (Lee et al. 2013). Of note, for
deriving catechins from commercial Chinese tea samples, the novel dynamic
ultrasound-assisted extraction method was found to be more effective than static
ultrasound-assisted extraction (Gu et al. 2007).
6. Extraction by lyophilisation can be done by lyophilizing sample and macer-
ation by acetone and water in 4:1 ratio overnight. After that, extracted sample is
shaken on planar shaker for several hours (3 h.) and it is then centrifuged to get
the supernatant, which is concentrated and frozen. Then the frozen material is
taken and extracted again with methanol (Suvanto et al. 2017).
21.6 Physical and Chemical Properties of Tannins
• These are high molecular weight phenolic compounds. These are complex
organic, non-nitrogenous and non-crystalline substances.
• Physically, tannins have a colour of reddish brown and dark brown, it possess a
state of non-crystalline matter and a taste with bitterness and puckering (Jaiswal
et al. 2018). A tannin can also possess light yellow or white amorphous or shiny
powder like appearance (Li et al. 2013). Tannins are soluble in water, acetone,
glycerol, dilute alkalies and alcohols; sparingly soluble in ethyl acetate, chlo-
roform and insoluble in benzene. Gallotannins are rapidly soluble but ellagi-
tannins are slowly soluble in water in comparison to gallotannin (Mingshu et al.
2006). They are practically insoluble in ether, carbon-disulphide and benzene
(Maximilian Nierenstein and Skene 1934).
• Tannins show its individual characteristics when they are treated with mineral
acids. Hydrolysable tannin is hydrolyzed into gallic acid or ellagic acid when it
gets a treatment with different mineral acids like HCl or H2SO4. Upon dry
distillation, hydrolysable tannin results in the production of pyrogallol and
phenolic compounds. Condensed tannins, upon acid treatment in elevated
temperature, it polymerizes and is converted into a red insoluble complexes,
called phlobaphenes (Pizzi 2019).
• Precipitation and astringency: Tannins form insoluble cross-linked complexes
by interacting with protein molecules. They combine with skin and hide to form
leather through a process of crosslinking the skin collagen (Khanbabaee and
Van Ree 2001) and with gelatin and isinglass to form an insoluble compound.
They combine with alkaloids to form tannates, most of which are insoluble in
water. Tannins have the ability to precipitate different compounds like gelatin,
alkaloid, protein, glycoside, skin hide and heavy metal.
• Reaction with salts or bases: Tannins yield purple, violet or black precipitates
with iron compounds. They are precipitated by number of metallic salts notably
potassium dichromate, and lead acetate and sub acetate. Tannins are precipitated
by copper acetate solution. Tannins when mixed with potassium ferricyanide in
the presence of ammonia, it produce deep red colour (Wei and Xingjin 1989).
• When it is boiled with acetic anhydride, it is converted into a crystalline acetyl
derivative, which melts at 137 °C (Ramakrishnan and Krishnan 1994).
• Carcinogenicity: Prolonged use of tannins can cause cancer. An evidence about
the carcinogenic effect of Areca catechu was observed by typical use of it that
resulted in oral and esophageal cancer (Hernandez et al. 2017).
• Anti-oxidizing properties: Tannins can show their anti-oxidant property because
of the large number of the hydroxyl (OH) groups present in it (Bagyalakshmi
et al. 2019).
21.7 Chemical Tests of Tannins
The presence and the amount of tannins can be clarified with the help of different
chemical tests of tannins. It can be divided into two groups, like, qualitative tests
and quantitative test. By qualitative test we can understand about the presence of
different tannins and by the quantitative tests, the amount of the tannin present in
the sample can be determined.
21.7.1 Qualitative Tests
1. Goldbeater's skin test: This is the major qualitative test for the confirmation of
true tannins based on the astringent property of tannins. Goldbeater’s shin is the
untanned hide (membrane) prepared from the intestine of ox. The gold-beater’s
skin test is originally described by Atkinson and Hazleton (Honorá Price 1924).
The first stage of performing this test is to make the Gold-beater’s skin to soak
in 2% HCl and make the skin hide permeable for tannin. After that, the skin is
rinsed with distilled water and is dipped in the tannin solution for 5 min. After
this step, the skin is washed with water and is treated with 1% ferrous sulphate
solution. The brown or black colouration of skin gives the confirmation about
the presence of tannins. Pseudo tannins don’t give positive result to this test
(Baig and Anand).
2. Water test: Tannins create a light yellow to dark brown discoloration in the
water. A simple test for tannins involves filling a clear glass with water and
letting it sit overnight. If the colour settles to the bottom of the glass, the
discoloration is most likely caused by iron and/or manganese and not tannins,
but if the intensity of the colour remains intact, it is most likely caused by
tannins (Franzmann et al. 2001).
3. Ferric chloride test: In two millilitres (2 mL) of the hot aqueous solution of
tannin extract, few drops of 10% Ferric chloride solution is added. The
occurrence of blackish blue colour showed the presence of gallotannins and a
green-blackish colour indicated presence of catechol tannins (Auwal et al.
2014) and also the occurrence of greenish brown colour indicates the presence
of condensed tannins (Pizzolato 1977).
4. Alkaline reagent test: To 1 ml of the plant extract, a few drops of 10% Sodium
hydroxide (NaOH) solution was added. An instant appearance of yellow to red
precipitate indicates the presence of tannins (Bharudin et al. 2013).
5. Phenazone test: In a 5 ml aqueous extract of tannin, sodium acid phosphate (0.5
gm.) is added and the mixture is heated, cooled and filtered. A solution of 2%
phenazone is added to the filtrate. A bulky coloured precipitate is formed
(Alotaibi 2019).
6. Match stick test (Catechin test): Match stick test is specially done for detection
of catechins. The principle of this qualitative test based upon simple reaction of
catechins, which can produce phloroglucinol in the presence of acid. A match
stick (which contains lignin) is dipped in aqueous extract of tannin (catechins),
dried and moistened with concentrated hydrochloric acid and is warmed in the
flame. Catechins in the presence of HCl produces phloroglucinol which stains
the lignin (present in match stick) produces pink or red colour (Thiruchenduran
et al. 2017).
7. Gambir-Fluorescein test: It is often performed to identify the pale catechu. The
alcoholic extract of pale catechu is mixed with sodium hydroxide solution, and
petroleum ether. After the mixing, the whole mixture is shaken and kept aside
for few minutes. The petroleum ether layer show green fluorescence due to the
presence of fluorescein present in pale catechu. Black catechu gives negative
result in this test (Ray et al. 2006).
8. Bromine water test: In an aqueous extract of condensed tannins, the addition of
acetic acid followed by bromine water gives a result of buff coloured precip-
itate. Hydrolysable tannin doesn’t response to this test (El Sissi and El
Sherbeiny 1967).
9. Lead sub-acetate test: In the ethanolic extract of tannin, addition of acetic acid
and lead sub-acetate solution results in gelatinous cream coloured precipitate
(Ukoha et al. 2011).
10. Chlorogenic acid test: When an extract of chlorogenic acid containing sample is
treated with aqueous ammonia. A green colour is formed on exposure to air
(Ashok and Upadhyaya 2012).
11. Vanillin-hydrochloric acid test: This test is performed by taking alcoholic
extract of sample, vanillin alcohol (vanillin 1 gm, alcohol 10 ml) and con-
centrated hydrochloric acid in a ratio of 1:10:10. The presence of tannin is
confirmed by a pink or red colour formation due to phloroglucinol (Rangra
et al. 2019).
12. Phlobatannin test: The occurrence of phlobatannin can be confirmed by the

deposition of red precipitate when the aqueous solution of tannin is mixed with
5 ml of 1% hydrochloric acid (Ezeonu and Ejikeme 2016).
21.7.2 Quantitative Tests
1. Hide-powder method: The aqueous solution of tannin is mixed with previously

dried chromated hide powder in vacuum for 24 h. over CaCl2 and the mixture is
stirred for 1 h. at ambient temperature. The suspension forms and it is filtered
through sintered glass filter. The weight gain of the hide-powder after filtration
is expressed as a percentage of the weight of the starting material is referred to
the percentage of tannin in the sample (Guangcheng et al. 1991).
2. Stiasny's method: In the aqueous solution of tannin, 1 ml of 10 (M) HCl and 2 ml
of 37% formaldehyde are added. The reaction mixture is heated over reflux for
30 min and it is then filtered through sintered glass. The precipitate obtained, is
washed with hot water for several times and dried over CaCl2 bed. The per-
centage yield of tannin is calculated with the starting material (Zhang et al. 2008).
3. Folin–Ciocalteau spectrophotometric method: The Folin-Ciocalteu reagent
(FCR) or Folin-Denis reagent, is named after Otto Folin, Vintilă Ciocâlteu,
and Willey Glover Denis. This reagent is prepared by dissolving 10 g of sodium
tungstate and 2.5 g of sodium molybdate in 70 ml of water, followed by the
addition of 5 ml of 85% phosphoric acid and 10 ml of concentrated
hydrochloric acid and 15 g of lithium sulphate, 5 ml of water, and 1 drop of
bromine (Peterson 1979). Polyphenols in plant extracts react with the reagent to
form a phosphotungstic-phosphomolybdenum complex, which gives blue
fluorescence under UV visible spectrophotometry (Schofield et al. 2001). The
maximum absorption of the chromophore depends on the alkaline solution and
the concentration of phenolic compounds. The maximum absorption is generally
obtained in a wave length of 760 nm (Blainski et al. 2013).
4. Turbidimetric method: This method is based upon the simple precipitation
reaction between Cu2+ from copper acetate and tannin at 4.5 pH and the
absorbance is being monitored at around 470 nm (McDonald et al. 1996). The
precipitate is formed due to the presence of functional groups such as
ortho-dihydroxyphenyl and carboxyl groups in tannins, which act as mono or
bidentate ligands and forms chelates (Lima et al. 2012).
5. Ferrous tartrate method (Colorimetric method): This method is based on the
formation, in a buffered medium (pH 6.8) of a complex between ferrous tartrate
and tannins in the tea, which absorbs light at around 560 nm (Iwasa and Torii
1962). The ferrous tartrate method is more selective than the Folin–Ciocalteau
method, and is unaffected by the co-existence of reducing agents, such as
ascorbic acid (Y T Hung et al. 2010).
6. Iodometric method: This method follows simple titration. Due to the phenolic
nature of tannins, they can consume iodine from any alkaline medium. The
titration is done by taking standard sodium thiosulphate solution and using
starch as indicator, and the excess consumed iodine determined which is then
used to determine total phenolic contains in the extract (Al-Alimi et al. 2017).
7. Agglutination method: Erythrocytes have a tendency of agglutination when it
comes in contact with tannins but the RBCs do not take part in haemolysis. The
agglutination occurs because of the formation of sticky membrane on their
surface. The end point is reached when all RBCs present, forms precipitation i.e.
solution remains colourless on shaking. The quantity of precipitated RBC
determines the amount of tannin present in sample (Pirofsky et al. 1962).
21.8 Activities of Tannins
Lots of research provide the knowledge about tannins that they are used biologi-
cally or can be used in different therapeutic purposes, in manufacturing purposes
etc. From the discovery of this secondary metabolites, tannins are extensively
studied and the outcome of those research shows tannin’s ability to perform in a
versatile way which can show its different activities like anti-oxidant,
anti-inflammatory, anti-microbial, anti-aging, stomachic, cardio-tonic, diuretics,
laxatives, hypoglycemic, anti-corrosive property etc. or it can be used in photog-
raphy, food, neutraceuticals or cosmeceuticals and so in different sectors. It is
believed that tannins show its applications by two mechanistic way.
1. As un-absorbable, which is usually the complexes with binding properties which
may exert local actions like antioxidant, radical scavenging, antimicrobial,
antiviral, anti-mutagenic, and anti-nutrient effects, or
2. As absorbable, which is having a benefit of possessing lower molecular weight
so that they are easily absorbed, and produce systemic effects in various organs
(Serrano et al. 2009).
21.8.1 Anti-oxidant Properties
Among all of the in-vitro studies, the most extensively studied property is its
anti-oxidant property. Tannins may show the anti-oxidant property because of its
several hydroxyl (OH) groups present in the phenolic moiety. Tannins do have an
ability of scavenging free radicals and inhibiting lipid peroxidation depending on
their structure and degree of polymerization (Tian et al. 2012). The antioxidant
activities of tannins are generally calculated by DPPH radical scavenging method.
The absorbance values of the compounds which changes the colour from violet to
yellow were measured at 517 nm. The percentage inhibition of DPPH free radical
scavenging activity was calculated using the following equation:
%Inhibition ¼ ½ðADPPH ASample Þ=ADPPH 100%
Where, ADPPH = Absorbance of DPPH, ASample = Absorbance of sample (Bora

et al. 2019; Pal 2013; Nimse and Pal 2015).
Different hydrolysable tannins like geraniin, pentagalloyl glucose, chebulinic
acid, geraniin isoterchebin, mallotusinic acid, pedunculagin, pedunculagin, tel-
limagrandins I and II exert their anti-oxidant property by lipid peroxidation pre-
vention interfering with xanthin-xanthin oxidase complex, adenine diphosphate and
ascorbic acid or with DPPH (Okuda 2005). Different types of condensed tannins
such as procyanidins B1 and B3 (Ariga et al. 1988) and kaki tannin also helps to
inhibit H2O2 or Fe2+ mediated lipid peroxidation (Jerez et al. 2007). Another study
using DPPH assay and oxygen radical absorbance capacity (ORAC) assays, showed
that Bengal coffee extract is having more potency towards anti-oxidant property
than Arabic coffee (Patay et al. 2016). Arsenic mediated liver injury and
obese-diabetic rat models are protected by administering tannin rich cocoa extract
for 4 weeks (Chandranayagam et al. 2013; Jalil et al. 2008). Different anti-oxidant
properties of tannins is listed below in Table 21.2.
Table 21.2 Anti-oxidant property of different tannins

Tannin Characteristics Assay
type
Geraniin, Pentagalloyl Lipid peroxidation, interferes with xanthin-xanthin In-vitro
glucose oxidase complex, adenine diphosphate and ascorbic acid Assay
Chebulinic acid, and scavenging effects on DPPH free radical
geraniin
isoterchebin,
mallotusinic acid,
pedunculagin,
pentagalloylglucose,
tellimagrandins I and
II
Pedunculagin, Lipid peroxidation in rat liver mitochondria stimulated
epigallocatechin by adenine di-phosphate and ascorbic acid
gallate
Kaki-tannin Significant inhibition of the auto-peroxidation of lipids
caused with H2O2 or Fe2+/
Ascorbic acid
Tannin-rich cocoa Arsenic mediated liver injury protection In-vivo
Cocoa extracts Anti-oxidant effects in the obese-diabetic rats
Table 21.3 Anti-microbial property of different tannins

type
Gallotannin Inhibits glucan synthesis. Activity seen In-vivo
on S. Mutans, S. Salivarius, and A.
Viscosus
Catechins and epigallocatechin gallate Potency against H. Pylori, E. coli In-vitro
Tellimagrandin I, rugosin B, corilagin Reduce the MIC of antibiotics in the
and theasinensin A, procyanidin B3 MRSA treatment
and B4
Catechin Inhibition towards C. histolyticum
21.8.2 Anti-microbial Properties
The anti-microbial properties of tannins also have been a field of interest for dif-
ferent researchers. Tannins can show their anti-microbial activity in direct way or
indirect way. The direct way comprises of the action of tannin in microbial
metabolism by affecting oxidative phosphorylation, whereas by inhibiting extra-
cellular microbial enzymes and depriving the substrate required for microbial
growth, tannins exert their indirect way of anti-microbial action (Cardona et al.
2013; Mohanta et al. 2007; Gurjar and Pal 2020; Howell 2001). Gallotannins can
exert bacteriocidal activity on Streptococcus mutans, S. Salivarius, and
Actinomyces viscosus by inhibiting the synthesis of water insoluble glucan
(Wu-Yuan et al. 1988). Different complex tannins like catechins and epigallocat-
echin gallate showed in-vitro anti-bacterial potency against Helicobacterium pylori,
Escherichia coli (Díaz-Gómez et al. 2014) (Díaz-Gómez et al. 2013). Different
hydrolysable tannins like Tellimagrandin I, rugosin B, corilagin and theasinensin A
(complex tannin) and procyanidin B3 and B4 can remarkably reduce the MIC
(minimum inhibitory concentration) of antibiotics like oxacillin, penicillin G,
aminoglycosides and ampicillin in the treatment of methicillin resistant
Staphylococcus aureus (MRSA) (Hatano et al. 2005). Catechins are shown to have
inhibitory property towards Clostridium histolyticum (Cardona et al. 2013). The
potency of different tannins obtained from different sources are listed in Table 21.3.
21.9 Anti-viral Properties
Tannins show anti-viral activity via the inhibition of virus absorption (Fukuchi et al.
1989). Different hydrolysable tannins like chebulagic acid and punicalagin can be
effective against hepatitis C, dengue, measles and bronchitis (Lin et al. 2013).
Potency against Herpes simplex can be exerted by some hydrolysable tannin or
galloylated condensed tannins by inhibiting its absorption (Fukuchi et al. 1989;
Table 21.4 Anti-viral property exhibited from different tannins

type
Chebulagic acid and Effective against human cytomegalovirus, hepatitis c In-vitro
punicalagin virus, dengue virus, measles virus, and respiratory
syncytial virus
Hydrolysable tannin Potency against Herpes simplex by inhibiting its
absorption
Casuarinin Potency against Herpes simplex by inhibiting its
attachment and penetration
Oenothein B, Activity against Human Immuno deficiency Virus
Coriariin A, and (HIV)
Agrimoniin
Takechi et al. 1985) or by some ellagitanninns like casuarinin, through the inhi-
bition of the attachment and penetration power of the virus (Cheng et al. 2002).
Dimeric ellagitannins like oenothein B, coriariin A, and agrimoniin showed activity
against HIV (Cheng et al. 2002). The anti-viral properties are listed below
(Table 21.4).
21.9.1 Cardioprotective Activity
The mechanism by which tannins show their cardiac activity is believed to be via
the inhibition of enzymatic degradation of elastin, induced stabilization of peri-
cardial tissue, stabilization of endothelial dependant vaso-relaxation (Flesch et al.
1998), depressing cardiac papillary muscle contraction (Lee et al. 2010) and the
reduction of the calcification of the glutaraldehyde-fixed aortic wall (Sieniawska
and Baj 2017). Condensed tannins can also give cardioprotective effect against
myocardial injury in rat models (Karthikeyan et al. 2007). Hydrolysable tannins
give protection to the heart and show anti-ischemic activity by TNF-a inhibition,
scavenging free radicals and reactive oxygen species and endothelial nitric oxide
synthase stimulation (Beretta et al. 2009). Purified hydrolysable tannins obtained
from Geum japonicum relaxes phenylephrine mediated vaso-contraction by nitric
oxide (NO)-cGMP pathway and gives hypotensive effects (Xie et al. 2007). The
galloyl moiety of gallic acids, digallic acid and 1-desgalloyl regusin F is responsible
for the inhibitory effect on propranolol induced negative inotropism (H Lee et al.
2010). Condensed tannins tend to form endothelium derived relaxing factor which
relaxes noradrenaline mediated contracted pulmonary artery (Russell and Rohrbach
1989). Proanthocyanins exert long lasting anti-hypertensive effects by regulating
nitric oxide mediated endothelium related factors (Magos et al. 2008) and give
cardio-protection against myocardial infarction induced by isoproterenol
(Karthikeyan et al. 2007). Tannic acid relaxes pre-contraction of human coronary
artery in both endothelium dependant and non-dependant manner, by interfering or
Table 21.5 Cardioprotective, cardiotonic and cardio-active properties of tannins

type
Tannin from red and Prevention of elastin degredation, stabilizes tissues on In-vitro
white wine heart, endotheial dependant vaso-relaxation
Hydrolysable tannin Action on cardiac papillary muscle, anti-ischemic
activity by TNF-a inhibition, scavenging free radicals
and reactive oxygen species and endothelial nitric oxide
synthase stimulation
Digallic acid and Inhibition o negative inotropic effect
1-desgalloyl regusin F
Condensed tannins Relaxes pulmonary artery, myocardial injury relief
Proanthocyanin Fights against myocardial infarction and interferes with
cGMP level
increasing vascular cyclic guanosin monophosphate (cGMP) levels (Flesch et al.

1998). The cardioactive effects of tannins are listed in Table 21.5.
21.9.2 Anti-histaminic Property
Depending upon the number and composition of the galloyl groups, HHDP groups
and phenolic moiety present, hydrolysable tannins like geraniin, agrimoniin,
euphorbin C, oenothein B can significantly inhibit jO2 induced histamine release
(Kanoh et al. 2000; Gurjar and Pal 2018, 2020).
21.9.3 Cytotoxic and Anticancer Activity
Procyanidins and various monomeric flavanols can exert their cytotoxic activity by
the presence of the galloyl moiety present in the 3′ position of C ring (digalloyl
procyanidin) (Actis-Goretta et al. 2008). Among different hydrolysable tannins,
geraniin and corilagin inhibits TNF-a release (Okuda 2005) whereas hydrolysable
tannins obtained from Eugenia jambolana like, vescalagin, acutissimin A/B, epia-
cutissimin A/B, grandinin/roburin E, hexagalloyl glucose and heptagalloyl glucose
etc. inhibits a-amylase which helps in cancer growth (Tong et al. 2014). Different
complex tannins like catechins also exhibit cytotoxic activity by the previous
mechanism or interfering with cellular signalling pathways (Miao et al. 2014).
Proanthocyanidins like Procyanidin B1 and B2 inhibit cell proliferation (Actis-Goretta
et al. 2008), effectively suppress the epidermal growth factor receptor phosphoryla-
tion, inhibiting the growth of human colon carcinoma cell line (Serrano et al. 2009).
Some in-vivo activities are also reported. Inhibition of skin-tumor promotion can be
seen by different tannins like 1,2,3,4,6-penta-O-galloyl-b-D-glucose, tenophyllanin
A and alienanin B and C, epigallocatechin gallate in the rat models which had been
pre-treated with different tumour initiators like 7,12-dimethylbenzo[a]anthracene
(DMBA), tetradecanoylphorbol-13-acetate, teleocidin (Okuda 2005). Apple and red
wine proanthocyanidins inhibited tumor promotion with azoxymethane induced
colon carcinomas in rat models (Serrano et al. 2009). Procyanidins hexamer from
cocoa and Japanese quince exerted anti-cancer activity arrests G2/M cell cycle col-
orectal cancer cells, which can possibly be mediated by the Akt pathway (Choy et al.
2016) and showed pro‐apoptotic effects on caco‐2 colon cancer cells respectively
(Gorlach et al. 2011; Saha and Pal 2016; Sannigrahi et al. 2012; Saha et al. 2017; Rani
et al. 2016; Pal et al. 2012).
Ellagic acid inhibits the growth of MCF-7 breast cancer cells, by G0/G1 cell
cycle arrest (Chen et al. 2015). Another research briefly explains about androgen
independent prostate cancer cells suppression by cell invasion and motility or the
down-regulation of MMPs by ellagitannins (Pitchakarn et al. 2013) and at higher
dose, ellagic acid treatment was found to be effective to induce growth inhibition
and caspase-dependent apoptosis in PC3 prostate cancer cells in a dose
approachable manner (Malik et al. 2011).
Gallic acid from blackberry, raspberry, walnuts, chocolate, wine, green tea and
vinegar may inhibits the migration of AGS gastric cancer cells possibly by
up-regulating RhoB as well as down-regulating AKT/small GTPase signalling
pathways and regulating NF-jB activity (Ho et al. 2013). It also showed
ROS-dependent pro-apoptotic effects in different cell lines, such as HCT-15 colon
cancer cells (L H Russell et al. 2012) and LNCaP prostate cancer cells
(Subramanian et al. 2016). Gallic acid treatment can selectively inhibited growth of
liver cancer cells through the mitochondria-mediated apoptotic pathways (Sun et al.
2016). However, gallic acid suppressed the invasion and migration of PC3 prostate
cancer cells via the down-regulation of MMP-2 and MMP-9 (Liu et al. 2011) or
decreased cell proliferation, invasion and angiogenesis of HeLa and HTB-35 cer-
vical cancer cell lines (Zhao and Hu 2013).
Among anthocyanins, delphinidin exhibits strong anticancer activities by
inducing apoptosis and cell cycle arrest in several types of cancer. The cytotoxic
effect of delphidine is believed to be due to the suppression of the nuclear factor
kappa B (NF-jB) pathway (Bin Hafeez et al. 2008). Another research study
proves that peonidin-3-glucoside treatment pointedly down-regulates the matrix
metalloproteinase (MMP) and prevents lung cancer cells invasion and metastasis
(Liu et al. 2013).
A study showed that epigallocatechin gallate treatment suppressed
nicotine-induced migration and invasion of lung cancer cell lines in-vitro as well as
in-vivo through inhibition of angiogenesis and epithelial-mesenchymal transition
(EMT) (Shi et al. 2015). Another study found that epigallocatechin gallate inhibits
survivin (a potent anti-apoptotic protein) and induces apoptosis (Onoda et al. 2011) or
prevents b-catenin/Wnt oncogenic signalling pathway to several gastric cancer cell
lines (Tanaka et al. 2011). Another study on colon cancer suggested that the Akt,
extracellular signal-related kinase (ERK 1 or 2) and alternative p38MAPK signaling
pathways were involved in the chemopreventive effects of epigallocatechin gallate
Table 21.6 In-vitro and in-vivo activities of tannins against cancer

type
Geraniin and corilagin Inhibits TNF-a In vitro
Hydrolysable tannins from Eugenia Inhibits a-amylase
jambolana, catechin
Procyanidin B1 and B2 Epidermal growth factor receptor
phosphorylation, inhibiting the growth of
human colon carcinoma cell line
Gallic acid from Blackberry, RhoB regulation and AKT/GTPase
raspberry, walnuts, chocolate, wine, signalling down-regulation
green tea and vinegar
Delphinidin Prevents NF-jB pathway
Peonidin-3-glucoside Prevents the invasion and metastasis of
lung cancer cells
Epigallocatechin gallate Inhibits nicotine-induced lung cancer,
inhibits surviving, interferes with Wnt
pathway in gastric cancer, Akt, ERK and
p38MAPK signaling pathways in colon
cancer
Tenophyllanin A and alienanin B and Skin tumour promotion by DMBA, In vivo
C, Epigallocatechin gallate tetradecanoylphorbol-13-acetate,
teleocidin is inhibited
Proanthocyanidins from Apple and Azoxymethane induced colon carcinoma
red wine prevention
Procyanidins hexamer from cocoa and Arrests G2/M cell cycle colorectal cancer
Japanese quince cells via Akt pathway and interferes with
Caco‐2 colon cancer cell
Ellagic acid G0/G1 cell cycle arrest in breast cancer
cell, androgen independent prostate
cancer suppression, inhibition of
MMP-9, caspase-dependent apoptosis of
prostate cancer cell line
Gallo tannin ROS-dependent pro-apoptotic effects on
colon and prostate cancer cells
Gallic acid down-regulation of MMP-2 and MMP-9
in prostate and cervical cancer cell line
Epigallocatechin gallate Prevents angiogenesis and EMT, inhibits
androgen action in prostate cancer and
estrogen action in breast cancer
Anthocyanidins Inhibits tumor growth in lung cancer cell
line, suppressing the growth of
HER2-positive tumor
(Cerezo-Guisado et al. 2015). Anti-cancer activity on hormonal regulation is also

shown by epigallocatechin as it suppresses estrogen (estradiol, E2) induced breast
cancer cell proliferation (Tu et al. 2011) or it antagonizes androgen, leading to sup-
pression of prostate cancer growth both in vitro and in vivo (Siddiqui et al. 2011).
Different anti-tumor and anti-cancer activity of annis is as follow listed in Table 21.6.
21.9.4 Anti-diabetic Property
Tannins possess potential anti-diabetic action by several mechanisms (Karan et al.

2013). Firstly, tannins do possess the ability to lower blood glucose levels by
delaying intestinal glucose absorption or through the inhibition of a-amylase and as
well as by exerting a-glucosidase activity. It has been suggested that the interaction
of tannins with human a-amylase depends on the possessed free hydroxyl group
that are able to participate in hydrogen bonding (Kandra et al. 2004) and the
inhibition of intestinal a-glucosidase activity is given by proanthocyanidin content
(McDougall et al. 2005). Tannins can also work on insulin and insulin receptor cells
by inducing insulin-like effect on insulin sensitive tissues or by delaying the onset
of insulin-dependent diabetes mellitus by regulating the antioxidant environment of
pancreatic b-cells (Serrano et al. 2009). Procyanidin found in grape seed can show
insulinomimetic action resulting hypoglycaemia (Pinent et al. 2004). 1,2,3,4,6-
penta-o-galloyl-b-D-glucopyranose binds to insulin receptor, stimulates glucose
transport in adipocytes and maintains blood glucose levels in body (Li et al. 2005).
In-vivo activities have been reported in defining diabetes prevention by bark extract
of Syzygium cumini. Tannins from Syzygium cumini have been reported to increase
plasma insulin and C-peptide levels with reduction of blood glucose levels of both
normal and diabetic rats after 45 days oral administration (Saravanan and
Leelavinothan 2006). Tannins with anti-diabetic activity are listed below
(Table 21.7).
Table 21.7 Anti-diabetic property of different tannins

type
Proanthocyanidin Inhibits of intestinal a- In-vitro
glucosidase activity
Hydrolysable tannin Controls anti-oxidant environment
of pancreatic b-cells
Procyanidin Insulinomimetic action is shown
1,2,3,4,6-penta-o-galloyl-b-D-glucopyranose Stimulates glucose transport in
adipocytes
Tannins from Syzygium cumini Up-regulates concentration of In-vivo
plasma insulin and C-peptide
levels
Table 21.8 Tannins in obesity prevention

type
Epigallocatechin Inhibits lipid accumulation In-vitro
gallate
Complex tannin G0/G1 phase or G2/M phase cell cycle arrest in adipocyte
differentiation
Tannin from Adipocyte apoptosis via caspase 3 activation and ERK and
green tea Cdk2 pathway regulation
21.9.5 Anti-obesity Action
It is believed that polyphenol-rich diets may relate to anti-obesity action as tannins

possess the ability to interact with adipose tissues like pre-adipocytes, adipose stem
cells, and immune cells directly or indirectly (Sieniawska and Baj 2017)
(Table 21.8). It can inhibit lipid accumulation (Moon et al. 2007) and adipogenesis,
or can induce apoptosis of adipocytes (Lin et al. 2005). Different complex tannins
like (+)/(-) epigallocatechin galllate, epigallocatechin or epicatechin arrest cell cycle
of adipocyte cells in adipocyte differentiation at G0/G1 phase or G2/M phase and
induce apoptosis in mature adipocytes or murine preadipocytes (Wang et al.
2014). It also activates caspase 3 and follows ERK and Cdk2 pathway which is
responsible for adipocyte cell apoptosis (Wu et al. 2005; Hung et al. 2005).
21.9.6 Anti-inflammatory Action
Several laboratory researches confirm that tannins can inhibit hyaluronidase

enzyme and elastase enzyme which is connected with the inflammation factors
(Piwowarski et al. 2011). Studies on tannins obtained from Terminalia chebula
(pentagalloylglucose and trigalloyl glucose) or grape seed proanthocyanidins like
geraniin, corilagin, and furosin showed good wound healing property (Zhang et al.
2005). Tannic acid crosslinks fibrous collagen and prevents collagen matrix
degradation which inhibits MMP (matrix metalloprotease) activity in-vitro (Zhang
et al. 2009). Corilagin showed its potency against lung inflammation by reducing
bleomycin-induced lung fibrosis number in apoptotic lung cells and prevented
membrane breakdown of lung epithelial cells in-vivo (Wang et al. 2014; Rani et al.
2014). The anti-inflammatory action of tannins are listed in Table 21.9.
Table 21.9 Anti-inflammatory property of different tannins

type
Tannin from Terminalia Keratinocyte and fibroblast proliferation mediated In-vitro
chebula wound healing property
Grape seed Amelioration of mitochondrial oxidative stress
proanthocyanidins
Tannic acid Crosslinks fibrous collagen and prevents collagen
matrix degradation
Corilagin Prevents membrane breakdown of lung epithelial cell In-vivo
and fibrosis
21.9.7 Anti-aging Properties
The extract of Boerhaavia diffusa can be served as diuretics, anti-convulsants,

opthalmic and anti-aging products (Rajpoot and Mishra 2011). The gall of
Terminalia chebula was tested for inhibitory activity against tyrosinase as well as
the proliferative and MMP-2 inhibition activity on early aging human skin
fibroblasts in order to evaluate their in vitro anti-aging activity and it resulted in a
potent tannin extract that could be used as anti-aging drug (Manosroi et al. 2010).
Phyllanthus emblica extracted tannins showed marked anti-aging and skin pro-
tection activity (Chaudhuri et al. 2007). The tannins, catechins, epigallocatechin
gallate, naringenin obtained from Cochlospermum vitifolium also possess anti-aging
potency due to its ability to inhibit collagenase, elastase, hyaluronidase enzymes
(German-Baez et al. 2017).
21.9.8 Other Therapeutic Activities of Tannins
Tannins from Mouriri pusa showed a good in-vivo anti-ulcer activity by giving
cyto-protective effects against gastric ulcers in rats (Vasconcelos et al. 2010).
Kaki-tannin is seen to have the ability in preventing the rise of total plasma
cholesterol, non-HDL cholesterol, triglycerides in type 2 diabetic mice fed high fat
diet. It also induces the genes which can metabolize cholesterol (Matsumoto and
Yokoyama 2012). Induction of immune response by stimulating interleukin 1
(IL-1) can be seen in case of different ellagitannin oligomers such as oenothein A
and B, camellin B, woodfordin C, D, E and F (Okuda and Ito 2011).
Some in-vivo and in-vitro assays had been done to check its effectiveness against
alzheimer’s disease and epilepsy (convulsion) and tannic acid was seen to inhibit
b-secretase activity via interfering with amyloid beta production and also it pre-
vented cognitive impairment (Pal et al. 2008, 2009; Pal and Mazumder 2014; Gupta
et al. 2003; Pal and Nandi 2005; Mori et al. 2012). The hydrolysable tannins
obtained from Terminalia chebula also can act as anti-alzheimer by inhibiting

acetylcholinesterase and NMDA receptor (Afshari et al. 2016).
In-vivo anti-diarrhoeal activity was evaluated of tannin (procyanidin) obtained
from the bark of Sclerocarya birrea as it inhibits intestinal transit rather than to
inhibition of net secretion of fluid and electrolytes provoked by the laxative agents
(Galvez et al. 1991). The gallotannin-enriched extract isolated from Galla Rhois
(GEGR) gave laxative effects due to its ability to recover the colon structure, to
increase the production of mucin, and downstream signaling pathway of muscarinic
acetylcholine receptors via G-protein coupled receptor (Kim et al. 2016). Laxative
effect can also be seen by tannin extract of Mareya micrantha, Aloe ferox, Urginea
indica, Fumaria parviflora, Phyllanthus emblica etc. (Hwang 2018).
21.10 Tannins in Industry
Tannins or tannic acid are found to be in charge of the application in ink manu-
facture, dye industry, plastic resin industry, water purification process, manufac-
turing of adhesives, surface coatings, and cosmetics etc. (Ramakrishnan and
Krishnan 1994). Tannins are mainly considered in processing leather from animal
skin in industry. They bind to the protein collagen present in the skin and make
crosslinking among them to produce leather, thus preventing the disintegration and
decompose of the fibres. A variety of tanning agents are used for tanning but the
three main types of tanning are vegetable tanning, chromium III and synthetic
tanning (Falcão and Araújo 2018; Covington 1997). The leather possess the
properties of better resistance, elasticity and softness due to its better durability. In
this way, the leather becomes an extremely flexible material that can be used for
different applications from bags to shoes, from belts to jewellery, but even for
leather sofas and armchairs, cell phone and tablet cases, clothing and outerwear.
21.11 Tannins in Cosmeceuticals
For the effectiveness of cosmeceuticals, the topically applied cosmetic products have
to overcome stratum corneum barrier and penetrate into the skin epidermis and
dermis layer after the release of active ingredients to give their activities. This whole
process solely depends upon different properties such as molecular weight,
lipophilicity, vehicle formulation etc. (Löf et al. 2011). It was reported that
polyphenols can show slight rheological properties as well as their stability, par-
ticularly, the decrease of viscosity, surface active properties (Di Mambro and
Fonseca 2005). For example, catechin can decrease the surface tension by emerging
itself at the air/water interface. In the o/w emulsion, different tannin units like gallic
acid, catechin etc. decreases the surface tension, which results in alteration of droplet
sizes of emulsions in some cases and thus improving the dispersion state of emulsion
(Di Mattia et al. 2010). Despite of the ability of tannins to work on the air–water
interface, tannins can provide the faster release of active ingredients. The smallest
and the most hydrophilic protocatechuic acid exhibited the highest permeation rates,
followed by catechins and epigallocatechin gallate (Dal Belo et al. 2009).
Due to their antioxidant properties, polyphenols can improve the oxidative
stability and storage stability of emulsions (Di Mattia et al. 2010). In case of tannins
with anti-ageing property, the tannin should release from the formulation and work
on the dermis and epidermis of the skin (Zillich et al. 2015). Tannins from
Phyllanthus emblica can be used in formulation of cosmetics as it possess good skin
protection activity and the skin lightening lotion with 2% extract showed remark-
able in-vivo activity by inhibiting the conversion of dihydroxy indole and dihy-
droxy indol carboxylic acid to melanin (Chaudhuri et al. 2007). Hydrophilic tannic
acid and lipophilic ellagic acid had been tasted for skin protection and it gave
anti-aging property by protecting dermal elastin from enzymatic degradation and its
deposition into fibroblast (Jimenez et al. 2006).
Different hydrolysable tannins and catechins obtained from chestnut can be used
as adhesives, cosmeceuticals, and food processors (Aires et al. 2016). Another
research study gave a knowledge about the collagen stabilization ability of veg-
etable tannin. It was found that pre-treated collagen fibres with acrylic polymer and
with tannin extract of Acacia mollissima exhibited an increase in hydrothermal
stability by 25 °C (Madhan et al. 2002).
21.12 Tannins in Neutraceuticals
Tannins can be found in some food bearings in an extensible amount and by taking
those in a definite amount can be beneficial for our health. Camellia sinensis (tea
plant) contain high tannin proportion (Ashok and Upadhyaya 2012) and the tannins
found in tea can provide health benefits beyond satisfying traditional nutritional
requirement. The tannins obtained from tea can be effective in conditions like
hypertension (Hara and Suzuki 1989), diabetes (Tong et al. 2014), and obese (Ra
2002). Chewing gum, containing tea polyphenols has been claimed to be effective
against influenza, and said to inhibit dissemination of the virus (Hara and
Nakayama 2001). High amounts of tannins (mainly condensed tannins and pro-
cyanidins) can be found in wines (more in red wines) and beers. Procyanidins was
shown to improve pathological oxidative state in a diabetic condition (Kumari and
Jain 2012) or asthma (Schmitz 2001). Pomegranate seed extract associated with
ellagitannins can be effective against atherosclerosis. De-caffeinated coffee extract
and chlorogenic acid supplement can make a person to lose weight. The stability of
flora in the intestine and the anti-fungal activity can be monitored with the help of
tannins or condensed tannins extract from acerin fruit, canaigre root or Swedish
birch bark (Sieniawska and Baj 2017).
21.13 Conclusion
Tannins possess an important place in medicinal chemistry, phytochemistry, as well

as in the industrial or manufacturing field. For the many past years, this secondary
metabolite has been extensively studied and for their application in different sectors,
tannin holds a favourite and remarkable place for the researchers in the way of
phytochemical researches. Many more years to come, many more researches to be
done and hopefully more applicability of tannins in more different fields yet to be
discovered. Giving a little contribution, this book chapter describes about different
types of tannins, their occurrence, extraction processes and about their versatile
applications and importance in the field of science.
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Chapter 22
Piperine: Sources, Properties,
Applications, and Biotechnological
Production
Neetu Sachan, Dilipkumar Pal, and Phool Chandra
Abstract From ancient times, phytopharmaceuticals have played an important role

in the management of human health. Piperine, an alkaloid with the piperidine
nucleus was discovered and isolated by Hans Christian Ørsted, from the fruits of
Piper nigrum. Piperine forms is slightly water soluble and forms monoclinic nee-
dles and possess a strong pungent taste. Piperine contains plentiful established
health effects and beneficial therapeutic properties. Cells and enzymes are key
elements in biotechnological processes to carry out a wide variety of very specific
reactions under judicious conditions to produce piperine and their products.
Piperine also serves as bio-enhancers in conjunction with drugs to stimulate drug
molecules’ activity across different routes by improving the drug's bioavailability
across the membrane, raising the drug's effect across conformational interaction,
and working as a drug receptor. In recent years, there has been significant interest in
the use of piperine to treat many illnesses, its health-beneficial effects, and its work
as bio-enhancers. Due to their biological activity, piperine has the potential to be
used in health and medicine.

Keywords Piperine Alkaloid Piper nigrum Bio-enhancers Therapeutics
Phytopharmaceuticals
N. Sachan (&) P. Chandra

School of Pharmaceutical Sciences, IFTM University, Lodhipur Rajput, Delhi Road (NH-24),
Moradabad 244 102, Uttar Pradesh, India
e-mail: neetuphool@gmail.com
D. Pal
Institute of Pharmaceutical Sciences, Guru Ghasidas Viswavidyalaya, Bilaspur-495 009,
Koni, Chhattisgarh, India
https://doi.org/10.1007/978-3-030-54027-2_22
760 N. Sachan et al.
22.1 Introduction
Piperine contains numerous pharmacological activities and many health benefits,

particularly against chronic conditions such as insulin resistance reduction,
anti-inflammatory activity, and correction in hepatic steatosis (Derosa et al. 2016).
Piperine was first isolated in 1820 by the Danish chemist Hans Christian Orstedt; it
occurs as a yellow crystalline solid (MW = 285.33 g mol−1, m.p. = 128–130 ˚C)
poorly soluble in water and presents weak base properties (Chavarria et al. 2016;
Koleva et al. 2012).
22.2 Biosynthesis of Piperine
Piperine is a secondary metabolite biosynthetically derived from L-lysine and a

cinnamoyl-CoA precursor. Decarboxylation of L-lysine by lysine decarboxylase
(LDC) yields cadaverine, which undergoes oxidative deamination by copper amine
oxidase (CuAO,) originating 5- aminopentanal. This compound is rapidly cyclized
into D1-piperidine Schiff base and subsequently reduced to form piperidine. In
parallel, piperonyl-CoA is generated from a cinnamoyl –CoA precursor; this pre-
cursor undergoes chain elongation with malonylCoA in a Claisen-like reaction,
generating a keto-ester that is reduced by NADPH and then dehydrated to afford
piperonyl-CoA. The piperine unit reacts with piperonyl-CoA affording piperine
(Fig. 22.1).
22.3 Extraction Techniques
The extraction of piperine from the plants can be made with different techniques
and that are present in Fig. 22.2 (Raman and Gaikar 2002; Rathod 2014).
22.4 Effect on Heart
Piperine produced both positive chronotropic and inotropic responses in the isolated
rat atria but not in the ventricular muscles. A tachyphylaxis to piperine occurred
rapidly depending on the dose of preincubation. It has been revealed that piperine
diminishes the level of substance P in the rat spinal cord, possibly as a result of an
extensive discharge of this neuropeptide. In the heart, it has been shown that
capsaicin releases CGRP from NANC nerves and the released CGRP shows pos-
itive chronotropic and inotropic effects (Franco-Cereceda and Lundberg 1985;
Franco-Cereceda et al. 1988; Miyauchi et al. 1987, 1988). Therefore, the lack of
22 Piperine: Sources, Properties, Applications … 761
O
O SCoA O
O H2N Lysine
Malonyl-CoA OH
NH2
O O
LDC
O SCoA
O NADPH
H2N NH2 Cadaverine
OH O
O CuAO
SCoA
O
O NH2 5-Aminopentanal
H2O O
O SCoA
O
N Piperideine
Piperonyl-CoA
NH LDC: Lysine decarboxylase

O CuAO: copper amine oxidase
O NADPH: nicotinamide adenine
N dinucleotide phosphate
O CoA: coenzyme A
Piperine
Fig. 22.1 Biosynthesis of piperine
effects of capsaicin after preincubation with piperine suggests that endogenous

CGRP might be depleted during preincubation with piperine. This is supported by
the immunohistochemical study, where a marked reduction of CGRP-I was found
after the incubation of the tissue with piperine. It has been demonstrated that CGRP
produces positive motropic responses and an increase in the formation of cyclic
AMP in the atria but not in ventricles of rats and also of guinea pigs. In the present
study, CGRP also did not show positive inotropic effects on the ventricular muscle.
However, these results are not in accordance with the demonstration that
CGRP-binding sites are present in both atrial and ventricular muscle membranes
(Sigrist et al. 1986). They showed that the number of receptors is highest in the
right atrium followed by the left atrium, and then the right and left ventricles. The
exact reasons for the difference in the effects of CGRP between the atria and
• Extraction time: 18
minute; Extraction yield • Extraction time: 2
(w/w): 0.58% minute; Extraction yield
• Benefits: Short running (w/w): 94%
Ultrasound
time, higher extractive Microwave • Benefits: Selective,
assisted yield, controllable short running time, high
extraction parameters assisted extraction
• Disadvantage: Small
extraction • Disadvantage: More
particle size, more filtration steps, time
filtration steps consuming during
cooling
• Extraction time: 12 ± 1
h; Extraction yield
Double (w/w): 3.90% ± 0.10%
bypass • Benefits: Easily
Soxhlet operate, simple
apparatus • Disadvantage: Long
extraction time, solvent
consuming
• Extraction time: 2-5 h;

Extraction yield (w/w):
6.7%-7.6%
• Extraction time: 30 Supercritical • Benefits: Efficient,
Ionic min; Extraction yield fluid
selective, clean, fast
liquid (w/w): 3.57% extraction
• Disadvantage: High
ultrasound • Benefits: Environment cost, less pressure
assisted friendly, Short resistant
extraction extraction run, high
efficiency
Fig. 22.2 Different extraction techniques used to extract piperine
ventricles have yet to be determined. The supply of CGRP-containing nerves was

considerably poor in the ventricles compared to those in the atria, indicating that the
amount of releasable CGRP is small in the ventricles. Even if CGRP is released
from sparse CGRP-containing nerves, it does not exert positive inotropic actions on
the ventricles. This may be a reason why both piperine and capsaicin failed to
produce positive inotropic responses in the ventricles (Miyauchi et al. 1988).
22.5 Effect on Pentobarbitone Sleeping Time
Piperine potentiated pentobarbitone sleeping time in dose-dependant manner, with

peak effect at 30 min. Blood and brain pentobarbitone levels were higher in
piperine-treated animals. Piperine treatment in rats, treated chronically with phe-
nobarbitone, significantly potentiated pentobarbitone sleeping time, as compared to
the controls. There was no alteration in barbital sodium sleeping time. It is possible
that piperine inhibits liver microsomal enzyme system, and thereby potentiates the
pentobarbitone sleeping time (Gupta et al. 2003; Mujumdar et al. 1990; Pal and
Nandi 2005; Pal and Mazumder 2014; Sachan et al. 2015; Pal et al. 2009).
22.6 Bioavailability of Drugs
Piperine, an essential constituent of black and long peppers, has been mentioned to
boost the bioavailability of drugs. The goal of the present studies was to understand
piperine ’s interaction with intracellular drug biotransforming processes in vitro and
in vivo liver tissue. During in vitro findings in rat filtrate, piperine suppressed
removal of the hydroxy group from aryl hydrocarbon, N-methyl group from
ethylmorphine, O-ethyl group from 7-ethoxycoumarin, and 3-hydroxy-benzo(a)
pyrene glucuronidation in graded order. Piperine suppression of certain conse-
quences from 3-methylcholanthrene- and phenobarbital-treated rats in post mito-
chondrial filtrate was identical to those controls. Piperine suppression of
arylhydrocarbon hydroxylase from rats treated with 3-methylcholanthrene was
similar to 7,8-benzoflavone. Piperine has induced noncompetitive suppression of
hepatic hydroxylase from non-treated and 3-methylcholanthrene-treated animals
with a Ki of thirty microM below the significant Km of arylhydrocarbon hydrox-
ylase detected at controls. Additionally, the kinetic studies of ethylmorphine-
N-demethylase suppression from monitoring rat hepatic microsomes showed non-
competitive suppression with significant 0.8 mM Km and thirty-five microM Ki.
Such studies have shown that piperine is a non-specific drug metabolic inhibitor
that displays no distinction between the various cytochrome P-450. Oral piperine
administration in rats significantly suppressed the functions of the hepatic arylhy-
drocarbon hydroxylation and UDP-glucuronyltransferase. Absolute arylhydrocar-
bon hydroxylation inhibition detected within 1 h was restored to normal range
within 6 h. Piperine pretreatment extended in time of sleeping and paralysis in
animals at 50% of the SKF-525A dose. These verdicts confirm that piperine as
effective drug metabolism inhibitor (Atal et al. 1985).
22.7 Effect on Enzymes
In vitro and in vivo modulation of drug-metabolizing enzymes by piperine was

investigated in microsomes of rats and guinea pigs. In vitro piperine caused
concentration-related inhibition (50% at 100 microM) of arylhydrocarbon
hydroxylase (AHH) and 7-ethoxycourmarin deethylase (7ECDE) activities, which
were comparable in control and 3-methylcholanthrene (3MC) treated rats. In guinea
pig microsomes, however, piperine caused strong inhibition at lower concentrations
(35% at 10 microM) and relatively much lesser inhibition with further increase in
piperine concentrations. A Dixon plot of the kinetic data of both AHH and 7ECDE
indicated noncompetitive inhibition with a Ki of approx. 100 microM. In vivo,
piperine given at a dose of 25 mg/kg body wt to rats caused a maximal inhibition at
1 h of both the enzymes, while only AHH returned to normal value within 4 h.
Similarly, upon daily treatment of piperine (15 mg/kg body wt) to rats for 7 days,
7ECDE was consistently inhibited, while AHH showed faster recovery. Piperine
thus appeared to cause differential inhibition of two forms of cytochrome P450, and
thus would accordingly affect the steady-state level of those drugs metabolized by
these pulmonary forms of cytochromes P450 (Reen and Singh 1991).
22.8 Effects on Antioxidant Pathways in Tissues

from Diabetic Rats
Excessive oxidative stress has been implicated in the pathology and complications
of diabetes mellitus (Matough et al. 2012), as well as other conditions including
cancer, multiple sclerosis, muscular dystrophy, emphysema, Parkinson’s disease,
Alzheimer’s disease, and the aging process (Kehrer and Klotz 2015).
Hyperglycemia generates abnormally high levels of free radicals by a mechanism
involving autoxidation of glucose, followed by oxidative degeneration and protein
glycation (Hunt et al. 1988). In addition, nonenzymatic glycosylation of those
enzymes that normally detoxify free radical species may exacerbate oxidative stress
in diabetes. Thus, clinical complications in diabetes and other oxidative
stress-related diseases may be due partially to the inability of key antioxidant
enzymes to function at normal levels (Karan et al. 2012; Pal et al. 2008; Pal 2013;
Rauscher et al. 2000).
Complications of diabetes mellitus, which include atherosclerosis, ischemic heart
disease, fatty liver, retinopathy, cataract, nephropathy, and neuropathy, are associ-
ated with oxidative modifications of various tissue components (Matough et al.
2012; Oberley 1988; Wolff 1993). In diabetes, cell death resulting from inflamma-
tion or oxidative damage may release products of protein catabolism, including
transition metals. These may serve to perpetuate the generation of reactive oxygen
species such as hydroxyl and superoxide radicals. The effects of diabetes on
antioxidant defense, consistent with those seen by other researchers, include alter-
ations in detoxifying enzyme activity in every tissue studied, as well as increases in
hepatic lipid peroxidation and decreases in the hepatic concentration of GSH, an
endogenous free radical scavenger. In diabetic rats, piperine treatment resulted in the
reversal of diabetic effects on cardiac lipid peroxidation (the 33% reduction by
diabetes was returned to untreated normal values after piperine), on cardiac glu-
tathione reductase activity (the 50% increase by diabetes was reduced to 25% above
untreated normal values after piperine), on renal glutathione peroxidase activity
(145% increase by diabetes was reversed to 52% above normal after piperine), and
on superoxide dismutase activity in kidney (the 90% increase by diabetes was
somewhat decreased after piperine). Likewise, piperine reversed the
diabetes-induced changes in the concentration of GSSG in brain (the 54% increase
by diabetes was decreased almost to untreated normal values after piperine). Piperine
treatment appeared to have no effect on hepatic TBA and GSH concentrations or
catalase or glutathione peroxidase activities or on cardiac catalase or glutathione
peroxidase activities that were altered by diabetes. The 19% increase in glutathione
peroxidase activity in the brain of diabetic rats after piperine and the 35% decrease in
concentration of hepatic GSSG in piperine-treated diabetic rats as compared to
untreated diabetic rats appear unrelated to the effects of diabetes. In the study, the
lipophilic piperine was more likely to interact with membranes, influencing lipid
peroxidation and GSH transport. Superoxide dismutase and catalase, on the other
hand, are part of the defense system in the hydrophilic compartment of the cell, and
were less likely to be modulated by piperine treatment.
22.9 Effect on the CNS
After exposure to piperine for 96 h, the concentration toxicity relationships of

piperine on hippocampal neurons and astrocytes displayed marked differences in
terms of cellular vulnerability to piperine. Cultured astrocytes showed only a
moderate increase in LDH release without any significant change in morphology at
100 piperine. On the contrary, cultured hippocampal neurons showed marked
injuries both biochemically and morphologically. In the time course study, piperine
induced delayed neuronal injury in which significant damage was apparent after
48 h and increased gradually up to 96 h of incubation, whereas the same concen-
trations did not affect astrocytes at any of the times studied (Unchern et al. 1997).
Their data suggest that piperine is selectively cytotoxic to cultured neurons
compared with cultured astrocytes. It was notable that MTT reduction of
piperine-treated astrocytes increased at all concentrations used in spite of the
unchanged protein content. Mitochondrial dehydrogenases in viable cells are
mainly responsible for MTT reduction activity. These findings imply that the
apparent increase in MTT reduction in piperine-treated astrocytes may be due to
enhanced mitochondrial enzymatic activity rather than increased cell proliferation.
In addition, the fact that astrocyte cultures were grown to confluence before being
used in the experiment does not support piperine-induced astrocyte proliferation.
The relationship between increased mitochondrial function and the tolerance of
astrocytes to piperine toxicity is still unknown.
Several lines of evidence indicate that astrocytes possess more defences, par-
ticularly the glutathione system, against free radicals than neurons. In neurons, the
level of glutathione is much lower than in astroglial cells (Bolaños et al. 1995;
Makar et al. 1994). This is likely because the activity of c-glutamyl cysteine
synthetase, a key enzyme in glutathione synthesis, is approximately eightfold
higher in astrocytes compared with neurons. Therefore, cultured neurons seem to be
more vulnerable to reactive compounds such as peroxynitrite (ONOO–) than cul-
tured astroglial cells. In this connection, it is possible that the differences in sen-
sitivity of cultured neurons and cultured astrocytes to piperine may be explained by
their different cellular content of glutathione. Piperine neurotoxicity was prevented
by free radical scavengers thereby suggesting the involvement of lipid peroxidation
and/or free radicals in its toxic action. The reason for the failure of SOD and
catalase to protect hippocampal neurons may be because these agents cannot cross
the cell membrane, whereas the effective agents, DO-tocopherol and trolox, are
expected to cross the cell membrane readily. These results suggest that intracellular
target(s) may be involved in the cytotoxicity of piperine. The protective emcacy of a
lipoxygenase inhibitor against piperine-induced neurotoxicity suggests that the
lipoxygenase pathway may play a role in piperine-induced free radical generation.
In addition to the harmful effects of the free radicals formed, lipoxygenase
metabolites by themselves may provoke various kinds of cell damage (Ochi et al.
1992; Sakagami et al. 1989).
It had been reported that SOD and catalase, but not fail to protect cultured
pulmonary artery (Kachel et al. 1990) endothelial cells against amidarone-induced
injury. However, SOD and catalase prevent the generation of reactive oxygen
species and glutamate-induced neurotoxicity in cultured cerebellar granule neurons
(Gunasekar et al. 1995). This discrepancy may be due to the different sites of free
radical generation in these insulting conditions. SOD and catalase form the defense
system in the hydrophilic compartment of the cell whereas u-tocopherol is the key
membrane-bound antioxidant (Cotgreave et al. 1988). In this connection, it is also
significant that piperine, due to its high lipophilicity, may directly interact with
membrane and may facilitate lipid peroxidation resulting in neuronal injury.
Although the piperine molecule contains unsaturated double-bonds in the
side-chain which may suggest antioxidant properties, recent studies indicate that
piperine contains, if any, very (Joe and Lokesh 1994; Reddy and Lokesh 1992)
weak antioxidant and free radical scavenging activity. Indeed, piperine in a con-
centration range similar to that used in our study was shown to increase lipid
peroxidation (Johri et al. 1992) in isolated rat jejunum epithelial cells.
22.10 Effect on Acute Kidney Injury
Acute kidney injury (AKI) is a serious complication that may be observed in up to

5% of the hospitalized patients (Basile and Yoder 2014). A chief cause of Acute
renal injury is renal ischemia–reperfusion (IR). Renal injuries caused by ischemia–
reperfusion include oxidative stress, inflammation, and damages of tubular
epithelium and vascular endothelium (Abuelo 2007). After ischemia–reperfusion,
inflammation initiates as a consequence of cell injury, subsequent molecular
products which trigger parenchymal cells of kidney and dendritic cells (DCs),
demanding the exudation of chemokines (Chen and Nuñez 2010; Kurts et al. 2013;
Rabb et al. 2016). Renal ischemia primes to leukocyte permeation and tissue injury
progress through upregulating the adhesive molecules (including ICAM-1) in
vascular endothelium, and creating cytokines (proinflammatory), viz., interleukin-6
(IL-6), interleukin-1 (IL-1), and TNF-a in kidney (Kurts et al. 2013; Thurman
2007), Moreover, renal ischemia–reperfusion increases the amalgamation of indu-
cible nitric oxide synthase (iNOS), enhancing the production of NO which bind
with reactive oxygen species (ROS) to form peroxynitrite (ONOO), prevents
endothelial nitric oxide synthase (eNOS), diminishes NO production in
endothelium, and consequences in vasoconstriction (Chatterjee et al. 2003;

Goligorsky et al. 2002; Ling et al. 1999). They reported that piperine gavage shields
kidney from 30-min ischemia and 24-h reperfusion influenced injuries in rat.
Piperine was ingested by gavage with 97% of absorption without any modification.
In the research, renal ischemia–reperfusion increased plasma creatinine and
urea-nitrogen concentrations. Given the contrary relationship between plasma cre-
atinine concentration and glomerular filtration rate (GFR), it is likely that the rise in
the plasma creatinine and urea-nitrogen concentrations is because of the diminution
in GFR. It has been shown that different factors are at play in reducing GFR
following IR, among which, mention can be made of the back-leak of filtered
substances (due to damaged tubular epithelial cells), activation of tubuloglomerular
feedback because of increased NaCl (Mohammadi et al. 2019).
22.11 Anticonvulsant Mechanisms of Piperine
Experimental findings suggest that piperine considerably slowed down the onset of
seizures in the PTZ-induced seizure test, telling a potential involvement of
GABA A receptors. In addition, prolonged onset of seizures in picrotoxin and
strychnine-initiated piperine-treated seizure models, although deprived of upsetting
mortality, also indicates the involvement of GABAergic and glycinergic signaling
paths, possibly with a widely recognized downstream pathway. Certainly, piper-
ine’s acute effect on cortical and hippocampal GABA estimate the prevalence that
piperine intervention has increased GABA levels in these areas and fits into the PTZ
experiment with its anticonvulsant influence. Earlier works indicate the involve-
ment of GABA transmission in animal models with anticonvulsant responses of
piperine (Zaugg et al. 2010). In addition, da Cruz et al. study (2013) encourage our
results because it advocates that piperine enhances basal GABA and glycine
amounts in the brain (da Cruz et al. 2013). From the other hand, serotonergic
modulation in MES induced convulsions could be a potential mechanism for the
protective role (Browning et al. 1983). In fact, our acute study indicated that
piperine facilitated the release of serotonin in cortex and hippocampus (Li et al.
2007) which may result in increased seizure threshold and this facilitatory effect on
serotonin release (Pei 1983). Interestingly, piperine has provided effective protec-
tion in seizures provoked by BAYK-8644 (a glutamate receptor agonist dependent
on L-type voltage) (Gasior et al. 1995), in line with the PASS prediction that
piperine may affect L-type channels. Piperine modulatory impacts of the Ca2+
channel have been reported earlier in in vitro studies utilizing rat hippocampal
neurons (Fu et al. 2010). In patch clamp tests, piperine merely poorly blocked
L-type channels. Piperine may reduce the inclination of BAYK-8644 to L-type
calcium channels allosterically, thus compensating for the difference between both
the in vitro and in vivo effects (Bukhari et al. 2013; D'Hooge et al. 1996; Mishra
et al. 2015). The dose-dependent anticonvulsant impact of piperine in the convul-
sions induced by MES mentioned the idea of piperine inhibiting sodium channel
activity. In fact, this possibility had been predicted by in-silico PASS prediction,
and our in vitro findings show that piperine reduces the peak current of the Na C
isoform Nav1.4 canal.
We note also that Nav1.4 converts the muscle tissue isoform of sodium channels,
but many of the antagonistic locations are preserved in the sodium channel family
as well as we thus anticipate that other types of sodium channels, including neu-
ronal ones, would also be similarly blocked (Fozzard et al. 2005). Therefore, it is
possible that piperine in animal models evaluated by inhibiting sodium channel
interaction may reflect its anti-convulsive attributes. We further found that the
TRPV1 receptor has also, in recent times, reported to play a role in the epilepto-
genesis method. TRPV1 is a broad—spectrum, high Ca2+ permeability cation
channel (Kauer and Gibson 2009), and this specific receptors usually promotes the
release of glutamate by boosting the excitability of neurons and synaptic
C-terminals (Chávez et al. 2010; Gibson et al. 2008; Schöbel et al. 2012; Zsombok
et al. 2011). Piperine has been shown to inhibit TRPV1 receptors, and this activity
may thus also contribute to the anticonvulsant action of this compound (Chen et al.
2013; McNamara et al. 2005), perhaps in conjunction with sodium channel block.
22.12 Immunomodulatory and Antitumor Activity
The dosage of piperine was selected on the basis of cytotoxicity. 1.14 mg/dose/
animal for piperine is the lowest concentration with maximum activity.
Administration of piperine showed increased number of total WBC count. This
indicates piperine can stimulate the hemopoietic system. The differential count
shows the drug did not alter the ratio of different WBC types. Bone marrow fills in
as the significant wellspring of all blood cells, including lymphocytes.
Administration of piperine demonstrated an expansion in bone marrow cellularity
and a-esterase positive cells showing its impact on stem cell multiplication.
Piperine was found to expand the coursing immunizer titer and counter-acting agent
framing cells demonstrating its stimulatory impact on the humoral arm of the
immune system. Administration of piperine could likewise altogether repress the
development of strong tumor incited by DLA cells and ascites tumor instigated by
EAC cells. Immunomodulators may enact cytotoxic effector cells, for example,
cytotoxic T lymphocytes, natural killer (NK) lymphocytes, macrophages, and ini-
tiated neutrophils (Fidler 1985, 1988). Utilization of chemotherapy in addition to
target-explicit immunomodulators hold a sensible guarantee for clinical utility in
future (Sunila and Kuttan 2004). Immunomodulatory activity of piperine may be
due to the combined action of humoral and cell-mediated immune responses.
Hence, the results indicated that piperine could act as a non-toxic immunomodu-
lator which possesses antitumor property also (Sunila and Kuttan 2004).
22.13 Larvicidal Effects
The group of scientist studied the consequences of piperine against Anopheles

larvae. Larvae were given to eat mixtures of typical larval diet and piperine in
changed amounts. Mortality was documented 48 h after the test was placed to the
larval containers. Piperine mixtures produced high number of deaths in the
Anopheles gambiae. The Anopheles funestus were noticeably less penetrating to
piperine which may reproduce a noticeable change in the nourishing ways of this
species to Gambiae complex or a change in nutrition breakdown by alterations in
breeding habitation flanked by species. They inferred that Insecticide safe and
vulnerable by species demonstrated defenseless to piperine (Samuel et al. 2016).
22.14 Inhibits B Lymphocyte Activation and Effector

Functions
B cells play a vital role in humoral immune responses that guard against microbial
pathogens are yet also connected with the pathogenesis of certain autoimmune and
allergic disorders (Gadermaier et al. 2014; Khan et al. 2013). Keeping this in mind a
group of scientists started to keep on searching and identifying a novel pharma-
cological agent that is able to modulate B cell activation and effector functions. The
inhibitory effect of the test compound was devoid of TRPV1 as piperine inhibited
the multiplication of B cells from TRPV1-deficient mice. In addition, piperine
repressed B cell production of interleukin (IL)-6 and IL-10 cytokines, as well as
IgM, IgG2b, and IgG3 immunoglobulins (Soutar et al. 2017).
22.15 The Anti-tumor Effectiveness and Mechanisms

Accompanied with the Combination
of Docetaxel-Piperine
Malignant growth of Prostate is the utmost widely recognized non-cutaneous car-

cinoma in men. Metastatic castrate-resistant prostate cancer (mCRPC) can be
treated by docetaxel a chemotherapeutic agent but soon it develops resistance
(Guirgis 2015), and that can be overcome by simultaneous treatment with docetaxel
and piperine. These effects can be due to the suppression response of piperine to
CYPs and P-gp intervention along with alterations in gene expression linked to
tumorigenesis and cellular responses (Li et al. 2018).
22.16 Allergic Encephalomyelitis
Multiple sclerosis (MS), an inflammatory T cell facilitated autoimmune compli-

cation of CNS (Ridderstad Wollberg et al. 2014). In clinical MS patients and
Experimental Allergic Encephalomyelitis (EAE) preclinical model, T leukocytes
transmigrate from the bloodstream into the CNS to attack myelin sheath encircling
nerve fibers (Zozulya and Wiendl 2008). Autoreactive CD4+ and CD8 + T cells
were considered as the key driver events in EAE models, indicating important
immunopathologic features of MS (Friese and Fugger 2005; Lassmann and Bradl
2017). In highly proliferative cells, such as activated T lymphocytes, increased de
novo pyrimidine biosynthesis by Human Dihydroorotate dehydrogenase can enable
their superior growth capacity (Quéméneur et al. 2003), thereby inhibiting this
enzyme serve the purpose. The scientists’ result showed that piperine is a potent,
direct DHODH inhibitor with an IC50 value of 0.88 ± 0.04 lM. Meanwhile,
piperine effectively reduced T cell proliferation through DHODH inhibition in vitro,
suggesting a potential role of piperine in immune function modulating.
Furthermore, piperine treatment significantly reduced immune cell infiltration in
central nervous tissue and resultant remission of EAE symptoms. The 13.2–15.8 h
terminal half-life (t1/2) for elimination of piperine (Ren and Zuo 2019) is largely
shorter than A771726. Since DHODH is a main functional target of EAE, we
corroborated the efficacy of piperine in MS model by inhibiting DHODH activity,
although it remains to be investigated in the future whether other proteins were
involved in the therapeutic effect of piperine in EAE (Liu et al. 2020).
22.17 Memory Enhancer and Restoration of Myelin

Damage
Piperine enhanced remembrance damage in lysolecithin-stimulated demyelination

model by mitigating astrocytes initiation, iNOS, and provocative cytokines
appearance level by enhancing total antioxidant capability in demyelination situa-
tion. Also, Piperine amplified the level of IL10, Foxp3, and antioxidant gene
markers. Piperine upgraded the myelin restoration progression by intensifying the
amount of MBP and BDNF (Roshanbakhsh et al. 2020).
22.18 Effect on Carbamazepine Metabolism
Carbamazepine (CBZ) is a drug of choice to treat different type of seizures. It works

for seizure control in the dose range of 4–12 lg/ml in human plasma. Increase in
concentration may cause side effects such as diplopia, nystagmus, and aplastic
anemia (Bialer et al. 1998). Carbamazepine-10, 11-epoxide (CBZE), a metabolic
product of Carbamazepine in liver by CYP3A4 with lesser concern of CYP2C8

(Kerr et al. 1994) and contributes to seizure control and toxicity (Tomson et al.
1990). The impact of piperine on the CBZ metabolic pathways was verified by
nurturing 50 lM CBZ with piperine in animal or human hepatic microsome nurture
arrangement at various concentration levels. The intensity of CBZ metabolism was
measured by the amount of CBZE formation. CBZE formation was shown to have
decreased after adding piperine in both the rat and the human nurture arrangement,
suggesting that piperine could supress both animal and human CBZ metabolism.
The measured piperine inhibition IC50 on CBZ breakdown in hepatic microsomes,
as shown in Fig. 22.3, was 9.20 ± 1.36 lM and 10.00 ± 1.22 lM individually for
alive primates and human. The related IC50 values of piperine between alive pri-
mates and human hepatic microsomes direct that alive primates hepatic microsome
can be exercised to predict such a suppressive activity by piperine against mam-
malian CBZ metabolism (Ren et al. 2019b).
Fig. 22.3 Sigmoidal dose–

response curves of piperine on
CBZ metabolism in rat
(A) and human (B) liver
microsome incubation
system. Each point represents
mean ± S.D. in triplicate
experiments. With copyright
permission (Ren et al. 2019b)
Fig. 22.4 Representative plots for irreversible inhibition of CBZ metabolism by piperine in rat
(Upper) and human (Bottom) microsome incubation system. A CBZ metabolic activity remained
after preincubation with different concentration of piperine versus preincubation time plot.
B Observed inactivation rates (kobs) versus piperine concentration plot for inactivation kinetic
parameter calculation (kinact and KI). Each point represents mean ± S.D. in triplicate
experiments. With copyright permission (Ren et al. 2019b)
Meanwhile, noncompetitive repression and time-dependent repression estab-

lished comparable kinetic variations in the demonstrative graphs, the time-dependent
repression power was also examined by preincubation of hepatic microsome with
piperine for dissimilar time and then verified the residual metabolic pursuit on CBZ
breakdown (Fig. 22.4) (Ren et al. 2019b).
The consequence of piperine on microsomal action was explored, additionally,
using the hepatic microsome from animals ingested with 3.5 mg/kg and 35 mg/kg
piperine for 14 days. The metabolic events were examined by CBZE creation using
CBZ as enquiry substratum. Associated with regulator group, a substantial inhi-
bition of CBZE formation was found in higher amount of piperine ingested group
with comparative CBZE creation of 67.0 ± 18.8% against control, indicating
suppression of CBZ metabolic action by chronic piperine administration (Ren et al.
2019b). Piperine’s effect on mRNA and protein appearance of genes controlling
CBZ breakdown was further examined by means of hepatic illustrations from
animals ingested with manifold piperine dose (Fig. 22.5). Against the control
Fig. 22.5 Comparison of liver mRNA expression levels of genes regulating CBZ metabolism
from rats after 14 days treatment of 3.5 mg/kg or 35 mg/kg piperine (LD PIP or HD PIP) or
vehicle (Control). (*p < 0.05, compared with control group). Each point represents mean ± S.D
(n = 6). With copyright permission (Ren et al. 2019b)
group, significant decreases were found in the expression level of rCyp3a2 and
rCAR mRNA in the elevated dose piperine treatment group (Ren et al. 2019b).
Finally, Ren et al. (2019a) concluded that the study demonstrated time-
dependent repression of carbamazepine metabolism through piperine as a CBZ and
piperine interference effect. Sustained use of high-dose piperine could not only
suppress the metabolic response of the CBZ but also decrease the level of rCyp3a2
mRNA and protein creation and rCAR mRNA illustration. Simultaneous ingestion
of CBZ with piperine, particularly after extended usage at a high-dose level,
therefore requires further attention.
22.19 Bioenhancer Effects
The piperine enhances the bioavailability of different drugs explored in Fig. 22.6
(Atal et al. 1985; Balakrishnan et al. 2001; Bano et al. 1987; Dama et al. 2008;
Gupta et al. 1998; Janakiraman and Manavalan 2008; Karan et al. 1988; Kasibhatta
and Naidu 2007; Mujumdar et al. 1990; Pooja et al. 2007; Singh et al. 2010, 2005).
22.20 Ayurvedic Formulations
The piperine is used in the ayurvedic formulations and they are presented in
Fig. 22.7 (Gupta and Jain 2011a, b, c; Patel et al. 2012; Rode et al. 2013; Rout et al.
2007; Sarkar et al. 2011; Shailajan et al. 2011; Singh et al. 2011; Vyas et al. 2011).
Metronidaole (Nitroimidazole antibiotic) Rifampicin (Bactericidal antibiotic)
Diclofenac Sodium (NSAID's) Isoniazid (Antibacterial agent)
Pentazocine (Opioid analgesic) Pefloxacin (Antibacterial)
Nimusulide (NSAID's) Tetracyclines (Broad-spectrum antibiotic)
Phenytoin (Antiepileptic) Sulfadiazine (Sulphonamide)
Pentobarbitone (Short-acting barbiturate) Oxytetracyclines (Broad-spectrum antibiotic)
Propranolol (β-adrenoceptor blocker) Ampicillin ( -lactam antibiotic )
Theophylline (Bronchodilators) Norfloxacin (Antibacterial agent)
Carbamazepine (Anticonvulsant) Nevirapine (NNRTI)
Piperine
Fig. 22.6 Bioenhancer effects of piperine on various drugs
ChitrakadiVati
Trikatu Churna
Kaphaketu rasa
Pippli Churna
Marichyadivati
Ayurvedic
formulation
of Piperine
Sitopaladichurna
LasunadiVati
Eladi Gutika
Ajmodadichurna
Triphalaguggulu
Fig. 22.7 Marketed ayurvedic formulations made up of piperine

22.21 Death of Cerebellar Granule Neurons Induced

by Piperine is Distinct from that Induced by Low
Potassium Medium
In this study, piperine-induced cell death in cultured cerebellar granule neurons was
compared with that induced by K+ withdrawal. Exposures to piperine resulted in
concentration-related neuronal death. In contrast to low K+-induced granule neu-
ronal death, piperine-induced death was unaffected by inhibitors of protein syn-
thesis and endonuclease activity. It is well-known that death of cerebellar granule
neurons induced by K+ withdrawal displays the requisite morphological and bio-
chemical characteristics of apoptosis including the dependence on de novo protein
synthesis and endonuclease activation (Unchern et al. 1998). Therefore, our data
suggest an involvement of non-apoptotic mechanism in piperine-induced granule
neuronal death. It was notable that a decrease in cellular MTT reduction preceded
the onset of neuronal death induced by both piperine and low K+ exposures. This
indicated that compromised mitochondrial function may be, at least partly,
responsible for the observed neuronal death. It was evident that neurons rapidly lost
their mitochondrial membrane potential, energy charge, and subsequently the
ability to metabolize MTT before proceeding to necrosis. Due to its high
lipophilicity and unsaturated double bonds, it is conceivable that piperine may
directly interact with neuronal membranes and initiate lipid peroxidation. This
speculation is in accordance with an observation that piperine (25–100 µM)
increased lipid peroxidation in isolated epithelial cells of rat jejunum (Johri et al.
1992). The consequences of piperine-induced lipid peroxidation are the disruption
of neuronal membrane integrity and the liberation of cytotoxic reactive oxygen
species (ROS). ROS have been implicated as mediators of both glutamate-induced
cell death (Gunasekar et al. 1995) and low K+ -induce apoptosis of cerebellar
granule neurons (Schulz et al. 1996). However, we did not observe any protective
effects of lipophilic free radical scavengers against low K+ -induced granule neu-
ronal death. This ineffectiveness may be due to the fact that major intracellular sites
of oxygen free radical generation in apoptosis include mitochondria, endoplasmic
reticulum, and perhaps nuclear membranes (Korsmeyer et al. 1995); whereas the
free radical scavengers used in our study exert their effects mainly on cell mem-
branes. Schulz et al. (1996) observed that vitamin E (>2 mM) partially protected
cerebellar granule neurons against apoptosis induced by 24 h K+ withdrawal
(Schulz et al. 1996). At very high concentrations, the protective effect of vitamin E
may be related to its stabilizing effect on the cell membrane rather than its
antioxidant or free radical scavenging property (Diplock 1982).
22.22 KV Channel as Therapeutic Target for Prostate

Cancer Treatment
The concept of KV channel as therapeutic target for prostate cancer treatment attracts
increasing interest, but the lack of effective and selective KV channel modulators has
hindered the progression of treatment strategy (Schönherr 2005). Exploring the
functional properties of ion channels in cancer progression and metastatic behavior
is emerging as a novel approach for the development of effective anticancer treat-
ment. Potassium (K+) channels in the plasma membrane of tumor cells contribute to
a wide range of cellular processes including cell cycle progression, cell proliferation,
and apoptosis (Ouadid-Ahidouch and Ahidouch 2008). In particular, K+ channels
play a crucial role in cell proliferation, as its activation is a prime factor for cell cycle
progression through early G1 phase of the cell cycle (Wonderlin and Strobl 1996).
Hence, the blockage of K+ channel activity has shown to inhibit cell proliferation in
several cancer cell lines including prostate (Ouadid-Ahidouch and Ahidouch 2013).
In prostate cancer cells, KV channel is quite prominently expressed and involved in
cell proliferation (Prevarskaya et al. 2007). Blockade of KV channel by K+ channel
blocker 4-aminopyridine (4-AP) inhibited cell growth in both androgen-sensitive
(AT-2) and androgen-insensitive (MAT-LyLu) rat prostate cancer cell lines (Fraser
et al. 2000). Similarly, the channel blockers, dequalinium, amiodarone, and
glibenclamide have shown to induce apoptosis in PC-3 cells (Abdul and Hoosein
2002). Characterization of Kv channel in LNCaP and PC-3 Cells is presented in
Fig. 22.8, Concentration-dependent effect of piperine on IK in LNCaP cells
Fig. 22.9 and Concentration-dependent effect of piperine on IK in PC-3 cells
Fig. 22.10 (George et al. 2019; Rauscher et al. 2000).
22.23 Piperine Impairs the Migration and T

Cell-Activating Function of Dendritic Cells
Group of scientist attribute piperine-mediated inhibition of DC migration to

impairment of the chemokine receptor switch from CCR5 to CCR7 since CCL21
causes DC migration via stimulation of CCR7 (Yoshida et al. 1998). Reduced
in vivo migration of piperine-treated DCs was likely also caused by reduced
expression of CCR7. In addition, piperine-treated DCs exhibited decreased
LPS-induced expression of CD40 and MHC II molecules, as well as reduced
synthesis of the pro-inflammatory cytokines, TNF-a, IL-6, MCP-1. Furthermore,
exposure to piperine caused a significant decrease in MHC II high-expressing DC,
which is indicative of a reduction in highly mature DCs (Lutz et al. 1999). Taken
together, these findings suggest that exposure to piperine caused DCs to retain an
immature or semi-mature DC phenotype in spite of TLR4 stimulation with LPS.
Interestingly, piperine-treated DCs have increased phagocytic activity (Bae et al.
2012), which is also consistent with an immature DC phenotype. Our findings are in
Fig. 22.8 Characterization of K V channel in LNCaP and PC-3 Cells. Typical recordings of the
whole cell outward K + current were elicited by voltage command pulses of increasing step pulses
from −140 mV to +70 mV in 10 mV increments. (A and C) Representative current traces of I
recorded in the absence and presence of 10 mM TEA in LNCaP and PC-3 cells. (B and D) The I-V
relationship in the absence and presence of TEA in LNCaP and PC-3 cells. (E and
G) Representative tail current traces recorded in LNCaP and PC-3 cells. (F and H) Tail
current–voltage relationship of LNCaP and PC-3 cells. Data are plotted as mean ± SEM (n > 7).
With copyright permission (George et al. 2019)
general agreement with two previous studies that showed reduced expression of
maturation markers and proinflammatory cytokines following DC exposure to
piperine (Bae et al. 2012); however, unlike these earlier reports, we observed
reduced synthesis of IL-6, but no effect on CD86 expression by piperine-treated
DCs. Reduced production of proinflammatory cytokines has also been reported in
piperine-treated B16-F10 melanoma cells, adipocytes, and macrophages (Pradeep
and Kuttan 2003, 2004; Woo et al. 2007) indicating that this effect is not cell
type-specific. Moreover, normal expression of ICAM-1, MAC-1, and LFA-1
adhesion molecules by piperine-treated DCs shows selective inhibition of DC
surface marker expression, which may reflect targeting of specific signal trans-
duction pathways. Indeed, piperine is a known inhibitor of extracellular
signal-regulated and c-Jun N-terminal kinases that are activated in mouse DCs as a
result of LPS stimulation (Bae et al. 2012).
Piperine-mediated inhibition of DC maturation marker expression induced by
LPS, CpG, and poly I:C indicates that the inhibitory effect of piperine on DC mat-
uration caused by pattern recognition receptor stimulation was not restricted to the
Fig. 22.9 Concentration-dependent effect of piperine on IK in LNCaP cells. A Representative

current traces of I recorded in the absence and presence of piperine at different concentrations (0.1,
25, 50 and 100 µM). B The mean I-V of IK in the absence and presence of piperine at different
concentrations. Data are plotted as mean ± SEM (n > 7). With copyright permission (George
et al. 2019)
LPS/TLR4 axis. Furthermore, this finding suggests that piperine modulates signaling
pathways associated with the TLR adaptor molecule Toll/IL-1R domain-containing
adaptor inducing interferon (TRIF) and the TLR adaptor protein MyD88, since TLR3
agonists such as poly I:C act via TRIF, TLR9 agonists such as CpG act via MyD88,
and TLR4 agonists such as LPS activate both TRIF and MyD88 signaling pathways
(Trinchieri and Sher 2007). It, therefore, seems likely that piperine acts downstream
of TRIF and MyD88 by targeting NF-jB, which stimulates the production of genes
participating in the inflammatory response (Gasparini and Feldmann 2012). Other
investigators have also reported piperine-mediated suppression of the NF-jB route.
For instance, nuclear aggregation of the NF-jB subunits p65, p50, and cRel is
repressed in piperine-medicated B16-F10 melanoma cells (Pradeep and Kuttan
2004), while piperine interferes with NF-jB transcriptional activation in HT-1080
fibrosarcoma cells (Hwang et al. 2011). Piperine also inhibits the degradation of IjBa
Fig. 22.10 Concentration-dependent effect of piperine on IK in PC-3 cells. A Representative

current traces of IK recorded in the absence and presence of piperine at different concentrations
(0.1, 25, 50 and 100 lM). B The mean I-V of IK in the absence and presence of piperine at
different concentration. Data are plotted as mean ± SEM (n > 7). With copyright permission
(George et al. 2019)
in TNFa-stimulated endothelial cells and PMA-stimulated HT-1080 cells (Hwang

et al. 2011; Kumar et al. 2007). However, piperine does not affect the IL-1b-induced
transcriptional activity of NF-jB in synoviocytes (Bang et al. 2009).
Piperine-mediated inhibition of the NF-jB pathway, therefore, appears to be cell
type-specific. We report here that piperine-treated DCs had decreased nuclear levels
of the NFjB subunit RelB, which is normally abundant in the nuclei of DCs matured
by stimulation with pathogen-associated molecular pattern molecules (Neumann
et al. 2000). Since RelB is critical for DC maturation and antigen-presenting function
(Zanetti et al. 2003) and NF-jB controls CCR7 transcript (Sánchez-Sánchez et al.
2006), and reduced RelB expression in the cores of piperine pickled DCs shows
partially for suppressive response for piperine in DC development, immigration, and
T cell excitation detected in the experiment. Though, piperine suppresses
mitogen-activated protein kinase signaling in numerous cells (Bang et al. 2009;
Hwang et al. 2011), including DCs (Bae et al. 2012). We suggest that
piperine-mediated inhibition of mitogen-activated protein kinases also contributes to

the failure of DCs to migrate in response to CCL21 since CCR7-induced chemotaxis
of DCs involves the activation of extracellular signal-regulated kinase and p38
mitogen-activated protein kinase (Riol-Blanco et al. 2005). Antigen-specific stimu-
lation of OVA323–339-specific CD4+ T-cells with piperine-treated DCs resulted in
decreased production of IL-2 and IFN-c, indicating that T helper cells were directed
away from the Th1 phenotype that is typically induced by DCs differentiated with
granulocyte–macrophage colony-stimulating factor (Eksioglu et al. 2007). In con-
trast, there was no reduction in CD4+ T-cell production of IL-4 and IL-17, suggesting
that T helper cells had not been alternatively directed to a Th2 or Th17 phenotype.
A similar decrease in CD4+ T-cell synthesis of IFN-c with no change in IL-4 pro-
duction occurs when CD4+ T-cells are stimulated with DCs that were treated with
dexamethasone prior to LPS maturation (Matyszak et al. 2000). Interestingly, mul-
tiple restipulation of CD4+ T-cells with dexamethasone-treated DCs resulted in the
selective induction of T regulatory cells. Although we did not determine whether
piperine-treated DCs also induced T regulatory cells, such an outcome seems unlikely
given that T regulatory cells require IL-2 for their development and function (Burchill
et al. 2007; de la Rosa et al. 2004). We also observed that the in vivo proliferation of
OVA323–339-specific CD4+ T-cells was virtually ablated when piperine-treated
DCs were used as antigen-presenting cells. This profound inhibitory effect on
CD4+ T-cell activation was likely due to reduced migration of piperine-treated DCs
to lymph nodes and impaired interactions with CD4+ T-cells as a result of decreased
DC expression of CD40 and MHC II molecules (Rodgers et al. 2016).
22.24 Piperine-Laden Nanoparticles with Increased

Dissolving and Improved Bioavailability
for Controlling Epilepsy
The reduction of size is a regular exercise to enhance the drug solubility by raising
the surface area to mass quotient, altering particle curvature that institute defects
into crystal lattice (Williams et al. 2013). The solubility and dissolution of piperine
have been improved by several formulation techniques, such as solid dispersion
with hot melt extrusion technology, Tween 80 coated solid lipid nanoparticles and
microemulsion with self-emulsifying drug delivery system (Ashour et al. 2016;
Elnaggar et al. 2015; Shao et al. 2015). However, none of these formulations have
been verified to confirm its anti-epileptic effect. Although nanoprecipitation method
has been used to develop a piperine and curcumin co-loaded nanoparticle prepa-
ration before, the feasibility for nanoprecipitation with piperine only and its sub-
sequent effect for epilepsy control have never been attempted (Moorthi et al. 2012).
In the study, they developed a nanosized formulation of piperine by nanoprecipi-
tation method to enhance its solubility, dissolution, and improve systemic exposure
of piperine after oral administration, which is a noninvasive route that is suitable for
long-term use during epilepsy control. The anti-epileptic effects of piperine

nanoparticles were further evaluated in acute seizure modes in mice (Pal et al.
2009).
The nanosized piperine was articulated by employing nanoprecipitation method
(Ren et al. 2019a) and they observed the characteristics of nanoparticles under TEM
with high spacial resolution. As shown in Fig. 22.11, piperine nanoparticles appeared
as spherical black spots with a smooth surface. Smaller particle size (around 67 nm)
was found for piperine nanoparticles under TEM observation compared with DLS,
which may be attributed to the shrinkage of nanoparticles upon drying process during
TEM preparation. Thus, the outcome of the particle size from DLS would be reflected
as a more precise result for piperine nanoparticles particle size.
The release pattern of piperine nanoparticles was constructed to demonstrate its
dissolution pattern and compared with that of unformulated piperine (Fig. 22.12).
They studied the anti-epileptic effect of piperine nanoparticles with a mouse
model with PTZ-induced acute seizures. Characteristic behavioral patterns such as
myoclonic twitch and tonic limb extension were observed after i.p. injection of PTZ
at 80 mg/kg in Kunming mice, indicating the successful development of seizure
model in mice (Raol and Brooks-Kayal 2012). As shown in Fig. 22.13, unformu-
lated piperine at 15 mg/kg failed to prevent the PTZ-induced seizure since there
was no significant difference in average seizure frequency or latency to first seizure
compared with the PTZ group. In contrast, no seizure was found during the 30 min
observation for piperine nanosuspensions at same dose level (15 mg/kg), which
was significantly different from PTZ group for seizure frequency and latency to first
seizure (p < 0.01). After lowering the dose of piperine nanoparticles to 7.5 mg/kg,
effective anti-seizure effect was still demonstrated with reduced seizure frequency
Fig. 22.11 Micrograph of

piperine nanoparticles
revealed by transmission
electron microscopy (TEM).
With copyright permission
(Ren et al. 2019a)
Fig. 22.12 In vitro release

profiles of piperine
nanoparticles and
unformulated piperine in
phosphate buffer at 37 °C for
24 h. Each point represents
mean ± S.D (n = 3). With
copyright permission (Ren
et al. 2019a)
and delayed onset of seizure compared with PTZ group (p < 0.05). Therefore, they
concluded that the nanosuspensions preparation could significantly improve the
anti-epileptic effect piperine (Ren et al. 2019a).
22.25 In Vitro Cytotoxic and In Silico Activity
The obtained MTT values showed that piperine has a cytotoxic effect because the
IC50 was recorded as 61.94 ± 0.054 µg/ml (Nimse and Pal 2015; Paarakh et al.
2015; Sannigrahi et al. 2012; Saha and Pal 2016). The tyrosine kinase receptors
have multidomain extracellular Ligands for specific Ligand, a signal pass trans-
membrane hydrophobic helix and tyrosine kinase domain. The receptor tyrosine
kinases are not only cell surfaces transmembrane receptors, but are also enzymes
having kinase activity (Bari et al. 2012). Angiogenesis in cancer is a significant
stage in which new capillaries form to supply a vasculature for nutrient supply and
waste material removal. Thus, the kinase inhibitor is a new cancer treatment as such
an anti-angiogenic agent. The trend nowadays is to develop herbal drugs and drug
candidates as inhibitors.
Low molecular weight entities originated in the extracellular part of the receptor
impede tyrosine kinase phosphorylation block signaling (Manley et al. 2002). Since
the type I receptor tyrosine kinase is a major regulator of several distinct and diverse
cellular pathways. They took Piperine and docked to get the superior conformer.
The docking demonstrates that piperine has a −7.6 kJ mol−1 binding energy with
two hydrogen bonds formed. Molecular docking with EGFR tyrosine kinase
domain revealed that piperine has suppressive capability and thereby has interac-
tions in active pockets one or the other amino acids. The topology of the active
position of EGFR tyrosine kinase was similar in all synthesized molecules, which is
Fig. 22.13 The seizure frequency (A) and latency to first seizure (B) of Kunming mice within
30 min after inducing acute seizure by PTZ (80 mg/kg, i.p.) in absence or presence of oral
administration of unformulated piperine at 15 mg/kg or piperine nanoparticles at 7.5 or 15 mg/kg
45 min before PTZ injection. Each point represents mean ± S.D (n = 6, *p < 0.05, **p < 0.01
compared with PTZ group). With copyright permission (Ren et al. 2019a)
lined by interacting amino acids as predicted from the ligplot. In in vitro experi-
ments, the molecule emerged to be active against the cell line used in inhibiting the
cell growth (Paarakh et al. 2015).
22.26 Conclusion
Piperine is a versatile chemical entity and it possesses different anticancer properties

as well it has the capability to enhance the bioavailability of important drugs either
in its form or in a modified dosage form. Further activity is required to understand
its utility in other important metabolic pathways.
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Chapter 23
Protein and Enzymes Isolated
from Plant Sources and Their Utilization
in Pharmaceutical Field
Om Prakash Panda, Sitansu Sekhar Nanda, Dong Kee Yi,

Dilipkumar Pal, and Souvik Mukherjee
Abstract For transporting out original BDY functions, minerals, ViT, carbohy-
drates, pRt and fibres necessitated, received from animal or PlT sources or both. pRt
reckoned as important compounds among all nutrients for the HuM BDY because
they facilitated in cells to build up and tissues repair in the BDY. The BDY used pRt
for energy production in the shortage of carbohydrates and fats, is essential for the
BuLd of MusL mass. An active Ezm is extracted from any living organism. Sources of
Ezm are fungi, yeast, bacteria, animals and PlT. A very much larger number of Ezm is
found its use in diagnosis and chemical analysis. Non-microbial sources provided a
larger proportion of enzyme. Ezm prevailed from PlT sources are bromelain,
actinidin, ficin, a-amylase-amylase, papain, LipOXse. Application of Ezm finds its
way in industries for food and beverage processing, animal feed, detergents
biosensors, Pharmaceuticals, wastewater treatment and recent biofuels.

Keywords Protein Nutrient building muscle mass Enzyme Chemical

analysis Chemical diagnosis Bromelanin Ficin Lipooxygenase
Abbreviations
ALBM Albumins
Ama Amino acid
BDY Body
BuLd Building
CoNjT Conjugated
DIse Diseases
Ezm Enzymes
O. P. Panda
Centurion University of Technology and Management, Balasore, Odisha, India
S. S. Nanda (&) D. K. Yi
Department of Chemistry, Myongji University, Yongin, South Korea
e-mail: nandasitansusekhar@gmail.com
D. Pal S. Mukherjee
Department of Pharmaceutical Sciences, Guru Ghasidas Viswavidyalaya (A Central
University), Bilaspur, Chhattisgarh 495 009, India
https://doi.org/10.1007/978-3-030-54027-2_23
794 O. P. Panda et al.
FbRs Fibrous
HIStd Histidine
H uM Human
ISLe Isoleucine
Le Leucine
LipOXse Lipooxygenase
MusL Muscles
PEpt Peptide
PlT Plants
pR t Proteins
Reat Reactions
SeD Seeds
SysT System
VaL Valine
ViT Vitamin
23.1 Introduction
Proteins (pRt) are the most abundant organic molecules of the living system (SysT).
The name protein is derived from the Greek word “proteios” meaning holding the
first place. In 1838 the Dutch chemist Mulder used the term pRt to denote the high
molecular weight nitrogen-rich and most abundant substances present in animals
and plants (PlT). The body (BDY) regularly needs nutrients for carrying out original
BDY functions, among these, pRt are the most important compounds because they
aid in building (BuLd) cells and tissues for a human(HuM) BDY. A high pRt richen
diet is needed for BuLd BDY or muscles (MusL). If the BDY deficiencies in fat and
carbohydrates, it creates energy production by using pRt as they are requisite for
MusL mass BuLd MusL. It reduces the long polymer chain of pRt from the amino acid
(Ama) monomer. The Ama monomers is coupled with the peptide (PEpt) bonds
between the amino and carboxyl groups. It is applied in pRt in industry and in the
HuM BDY. So, an effort produced to review on enzymes and pRt and their phar-
maceutical application.
23.1.1 Classification of pRt
Based on the structure, composition, shape and solubility of Ama, pRt are assorted
into following classes: simple, fibrous (FbRs), conjugated (CoNjT) and derived pRt.
Simple pRt are two types such as (a) Globular pRt (Albumins (ALBM), Globulins,
Glutelins, Histones, Globins, Protamines, and Prolamins etc.), (b) FbRs pRt
(Collagens, Elastin, and Keratins etc.). The example of CoNjT pRt is Nucleo, Glyco,
23 Protein and Enzymes Isolated from Plant Sources … 795
Muco, Lipo, Phospho, and Metallo pRt etc. However Derived pRt are two types such
as Primary pRt (Coagulated pRt, Proteans, and Meta pRt etc.), and Secondary pRt
(Proteoses, Peptones, Poly PEpt, PEpt etc.). The simple pRt only comprise of Ama and
are further classified in to: Globular pRt: These pRt are oval or spherical shaped,
soluble in water or in other solvents and digestible (Table 23.1). FbRs pRt: These are
Fiber like shape, water- insoluble and cannot be digested. ALBM ous or sclero pRt
establish the major group of FbRs pRt. (Table 23.2) CoNjT pRt: It makes these pRt up
of Ama and non- pRt moiety which is also known as a prosthetic or CoNjT group
(Table 23.3). Derived pRt: These pRt are derived from simple and CoNjT pRt by
denaturation or degradation process. These are further divided into primary derived
pRt. It may also know as first hydrolyzed products of pRt. For example: Coagulated,
Proteans, Meta pRt. Secondary Derived pRt: These pRt are the degraded products of
pRt or the progressive hydrolytic products of pRt undergoing hydrolysis. Examples
include proteoses, peptones, poly PEpt and PEpt (Fig. 23.1).
Table 23.1 Examples of Globular pRt

pRt Properties Examples
ALBMs Coagulates on heating and soluble in water forming Serum ALBM,
dilute salt solutions Ov ALBM (egg),
Lact ALBM (milk)
Globulins Soluble in neutral and dilute salt solutions Serum globulins,
Vitelline (egg yolk)
Glutelins Soluble in dilute acids & alkalis and are mostly found Oryzenin (rice),
in PlT Glutelin (wheat)
Prolamins Soluble in 70% alcohol Zein (maize),
Gliadin (wheat)
Histones Soluble in dilute acids & water but insoluble in dilute Thymus histones
ammonium hydroxide Histones of codfish
sperm
Globins Behaves like histones but is not basic and is not Thymus histones,
precipitated by ammonium hydroxide Histones of codfish
sperm
Protamines Strongly basic and resemble histones but smaller and Sperm pRt
soluble in ammonium hydroxide
Table 23.2 Examples of FbRs pRt

Examples Properties
Collagens Connective tissue pRt but do not have tryptophan (TrypN). On boiling with dilute
acids or water, yield gelatin which is soluble and digestible
Elastin pRt of Elastic tissues such as arteries and tendons
Keratins Present in an exoskeletal structure like hairs, horns, nails, etc.
Table 23.3 Examples of CoNjT pRt

pRt Properties Examples
Nucleo Here prosthetic group is nucleic acid, either Nucleohistones,
pRt RNA or DNA nucleoprotamines
Glyco Here prosthetic group is carbohydrate Mucin (saliva), ovomucoid (egg
pRt white)
Lipo pRt Prosthetic group is lipid Serum lipo pRt, membrane lipo
pRt
Phospho Prosthetic group is phosphoric acid Vitelline (egg yolk), Casein
pRt (milk)
Chromo It colors prosthetic group substance Hemoglobin, cytochromes
pRt
Metallo pRt contain metal ions such as Co, Cu, Mg, Carbonic anhydrase (Zn),
pRt Fe, Zn Ceruloplasmin (Cu)
Fig. 23.1 Structure of various proteins
23.1.2 Elemental Composition of Proteins
pRt are predominantly formed by five major elements in following proportion

Hydrogen: 6–7.3%,
Carbon: 50–55%, Nitrogen: 13–19%, Oxygen: 19–24%, Sulfur: 0–4%. Essential
Ama are the chief constituents of pRt found in naturally occurring organic com-
pounds containing Ama and carboxyl groups, and necessary for and animal growth
and nutrition, Hence the source of essential Ama is rich food consumption as a HuM
BDY does not produce them. pRt or poly PEpt are the substance made by Ama which
are joined by PEpt bonds (Millward 1999). It highlights the roles of the various Ama
in Table 23.4.
Table 23.4 Ama and their biological role

Ama Structure Role
ISLe O Forms hemoglobin; prevents MusL
wasting in debilitated individuals
OH
NH2
e
L O Promotes healing of skin and broken
bones; reduces MusL pRt breakdown
OH
NH2
a
V L O Influences brain uptake of other
neurotransmitter precursors (TrypN,
phenylalanine and tyrosine)
OH
NH2
td
HIS O Produces red and white blood cells;
used in the treatment of anemia
N
OH
HN NH2
Lysine O Inhibits viruses; used in the
treatment of herpes simplex; Lysine
H2N and vitamin (ViT)-c together form L-
OH carnitine: a biochemical that enables
MusL tissue to use oxygen more
NH2 efficiently, delaying fatigue
Methionine O Increases the antioxidant levels

(glutathione) and reduces blood
S cholesterol level`
OH
NH2
Phenylalanine O Produces collagen, it acts as
precursor of tyrosine; enhances
learning, memory, mood and
OH alertness
NH2
(continued)

Ama Structure Role
Threonine OH O Prevents fatty build up in the liver; it
is also amino detoxifier
OH
NH2
TrypN H Prevents fatty build up in the liver, it
N acts as precursor of key
neurotransmitter serotonin; which
exerts a calming effect
NH2
OH
O
23.1.3 Classification Ama
They are classified on the basis of carbon chains present, nutritional requirement
and polarity. Based on carbon chain present Ama are of three types: Aliphatic Ama:
These are further classified in to Neutral (Monoamine-Monocarboxylic Acid) for
example, Glycine, Alanine, Serine, Threonine, Valine (VaL), Leucine (Le) and
Isoleucine (ISLe). Acidic (Monoamine-Dicarboxylic Acids) for example, Aspartic
acid, Asparagine, Glutamic acid and Glutamine. Basic: for example, Arginine,
Lysine and Hydroxyl sine. Sulphur containing Ama for Example, Cysteine, Cystine
(di cysteine) and Methionine, Aromatic Ama for example, Phenylalanine, Tyrosine
and Thyroxine. Heterocyclic Ama: for example, Proline, hydroxy Proline, TrypN,
Histidine (HIStd). Based on nutritional requirements amino Ama are two types:
Essential Ama: they are essentials for the body and these Ama are also indispensable
Ama. They are not synthesized in the BDY and are obtained from dietary sources.
Examples; VaL ISLe, TrypN, Methionine, Le, Phenylalanine, Threonine and lysine.
Exceptionally HIStd and Arginine are indicated as semi-essential Ama as a little
amount of them are synthesized in the BDY. Deficiency of these Ama may give
adverse effects like retarded growth, weak immunity, early ageing, etc.
Non-Essential Ama: These Ama are also termed as dispensable Ama and are syn-
thesized in the BDY. Examples; Glycine, Serine, Threonine, Glutamate, Cysteine,
Glutamine, Proline, etc. (Table 23.5) (Fink et al.2012).
Table 23.5 Essential and Essential Non-essential

non-essential Ama
VaL Glycine
ISLe Tyrosine
TrypN Proline
Methionine Cysteine
Arginine Aspartic acid
Le Alanine
Phenylalanine Serine
Threonine Hydroxyproline
Lysine Cystine
HIStd Glutamic acid
23.1.4 Properties of pRt
23.1.4.1 Solubility
The solubility properties of pRt is like as follows: Forms Colloidal solutions in

water (because of its enormous size), Solubility depends on electrostatic charges:
identity net charge depends on number, pH of solvent, location of Ama and pH of
solvent. It depends upon isoelectric point (range 5–8.5): Isoelectric point depends
on seven-charge Ama, example aspartate (b-carboxyl group), glutamate (d-carboxyl
group), cysteine (thiol group), HIStd (imidazole side chains), tyrosine (phenol
group), arginine (guanidinium group) and lysine (ammonium group).
23.1.4.2 Molecular Weight
pRt molecular weight variation depends on the number of Ama residues. Each Ama
contributes 110 value increases in a pRt′ molecular weight e.g., Myoglobin—1700;
Insulin—5700; Hemoglobin—64,450. pRt shape varies as globular (insulin), FbRs or
elongated (fibrinogen), oval (ALBM). For identification of Ama present in pRt the
below given in Table 23.6 is recommended;
23.1.4.3 Chemical Nature of pRt
Most of the pRt fold up to form a unique 3-D structure and the shape in order to
which a pRt naturally folds is its native conformation. Depending upon the chemical
properties of Ama, most of the pRt fold up accordingly while others fold up in to
their native states with the help of molecular chaperones. Four original structure of
pRt have been introduced by the biochemists: (A) Primary, (B) Secondary,
(C) Tertiary & (D) Quaternary.
Table 23.6 Color Reat of pRt

Reat Observations Specific group/Ama
at
Biuret Re Violet or purple color Two PEpt linkages
Ninhydrin Reat Blue color a-Ama
Xanthoproteic Reat Orange color Aromatic Ama
Millions Reat Red color Phenolic group
Hopkins-Cole Violet ring Indole ring
Sakaguchi Intense red color Guanidino group
Nitroprusside – Sulfhydryl groups (Cys)
Sulfur test Black ppt Sulfhydryl groups (Cys)
Pauly’s test – Imidazole ring (His)
Folin–Coicalteau’s test – Phenolic groups
Primary Structure
In 1953 Frederick Sanger determined the complete sequence of Ama of Insulin by

studying the primary structure of pRt, this sequence can be determined. Actually,
each pRt has a unique sequence of Ama which is determined by the genes contained
in DNA. The primary structure of a pRt is largely responsible for its function (Kyte
and Doolittle 1982). The Ama composition of a pRt determines its physical and
chemical properties. The primary structures formed during the translation (a process
of pRt biosynthesis) are held together by covalent PEpt bonds. Depending on the
nature of the free group on each terminal of the PEpt chain, each extremely is
referred as the carboxyl terminus (C-terminus) and the amino terminus
(N-terminus). Numbering of residues always starts at the N-terminal end (–NH2
group) from where the amino group is involved in a PEpt bond. The primary
structure of pRt depends on the gene corresponding to the pRt through transcription
mRNA is synthesized by a particular sequence of nucleotides. The mRNA is further
read by the ribosome in a process known as translation. The sequence of pRt is
specific for a pRt which defines the function and structure of that pRt. For the
determination of Ama sequence in a pRt, they commonly use methods like Edman
degradation or tandem mass spectrometry (Bender and Smith 1997).
Secondary Structure
It refers to the conformation of poly PEpt chain by twisting or folding to a secondary

structure. Two types of secondary structures are mainly identified i.e., a helix, & b
sheet.
(a) a helix
Pauling and Corey propose this in 1951, which is regarded as one milestone in the
biochemistry research. In this structure, the poly PEpt chains fold around the long
axis in such a way that the -NH group of each Ama bind to the –CO group of the
fourth residue by a hydrogen bond, throughout the chain These hydrogen bonds are
parallel to the long axis with the side chains protruding outward in a manner that
each turn of a helix comprises 3.6 Ama residues. Each Ama residue is present at a
distance of 0.15 nm from one another along the axis. Example of a helical pRt
structure is hair pRt i.e., Keratin (Ikai 1980).
(b) b sheet
The b sheet also known as b -Pleated sheet is a common motif of regular secondary
structure of pRt. Beta sheet consists of beta strands connected laterally by at least
two or three backbone hydrogen bonds forming a twisted, pleated sheet. A beta
strand is a stretch of poly PEpt chain typically 3–10 Ama long with backbone in an
extended conformation. They have implicated the supramolecular association of
beta sheets in formation of the pRt aggregates and fibrils observed in many HuM
diseases (DIse), notably the amyloidosis such as Alzheimer’s DIse′.
Tertiary Structure
Tertiary structure represents the folding of pRt that also controls the basic func-
tioning of pRt. Stabilization of tertiary structure is achieved by non-local interac-
tions, through salt bridges, hydrogen bonds, di sulphide bonds and
post-translational modifications. Tertiary structure involves three-dimensional
folding of the chain; stabilized by the bonding between the distant parts of the
sequence. The long poly PEpt chains are tightly folded in to a compact form because
of the following interactions of -R groups of side chains of Ama.
Hydrogen Bonding
Along with the hydrogen bonding between PEpt bonds that give rise to the sec-
ondary structure of pRt, the hydrogen linkage may also be present between the Ama
side chains.
Disulphide Bonding
The disulphide bonds cross-link with the poly PEpt chain. The pRt folds to bring two
cysteine residues together thus the two -SH side chains oxidize and form a covalent
disulphide (S–S) linkage (Mc Ardle et al. 2001).
Electrostatic/Ionic Bonding
The forces of attraction can stabilize the tertiary structure of pRt between Ama side
chains of opposite charge. For example; an electrostatic bond is present between –
NH3+ side chains of Leu and –COO side chains of Asp.
Hydrophobic Bonding
Sometimes pRt folds so that the hydrophobic side chains of Ama (e.g.; Gly, Ala, Val,
Leu, lie, Pro, Met, Phe and Trp) are suppressed within the pRt, where they can
interact to form hydrophobic linkage and thus stabilizes the structure of pRt.
23.1.4.4 Quaternary Structure
Quaternary structure arises when two or more pRt molecules interact with each other
and form a large assembly or complex of pRt. Often, pRt comprise multiple poly PEpt
chains and are mostly referred as pRt subunits in this context. These pRt subunits
may have different (as in a heterodimer) or same (as in a homodimer) poly PEpt
chains. Thus, the quaternary structure is the interaction and arrangement of these pRt
with each other to form a larger aggregate pRt complex. The pRt with two or more
poly PEpt linked with non-covalent interactions are quaternary pRt, e.g.;
Hemoglobin (a2b2) (Fig. 23.2).
23.2 pRt Sources: Animals and PlT
Meat, milk products, milk, egg, fish, and poultry are rich sources of pRt containing
Ama with a balanced level. Legumes, PlT food items, and nuts are also a source of
the same. PlT (vegetable) pRt and animal pRt are differentiated as: When animals’ pRt
consumed in enormous amounts, it leads to high risks of DIse like high blood
pressure and heart DIse because of high fat content in animals’ pRt. Animal pRt
hollered complete pRt because they balance it in a combination of all Ama; hence,
PlT (vegetable) pRt is incomplete pRt; an exception is soya bean pRt. pRt can find
from vegetables, legumes and fruits whereas vegetable and fruits have less content
of pRt than legumes. It distributes pRt in every part of the plants. PlT are essential to
isolate pRt and useful for pharmaceutical industry (Tables 23.7 and 23.8).
Fig. 23.2 Structural idea of

protein
Table 23.7 Parts of PlT as a source of pRt

pRt source as part of Examples
part of PlT
Legume Garbanzo beans, kidney beans, lentils, lima beans, navy beans,
soybeans, split peas
Grain Barley, brown rice, buckwheat, millet, oatmeal, quinoa, rye, wheat
germ, wheat, wild rice
Vegetable Barley, brown rice, buckwheat, millet, oatmeal, quinoa, rye, wheat
germ, wheat, wild rice
Fruit Apple, banana, cantaloupe, grape, grapefruit, honeydew, melon,
orange, papaya, peach, pear, pineapple, strawberry, yangerine,
watermelon
Nuts and seeds (SeD) Almonds, cashews, filberts, hemp SeD, pea nuts, pumpkin, sesame,
sunflower SeD, walnuts (black)
23.3 Some Important pRt, Their Characteristics and Uses
23.3.1 Soybean
Soy pRt obtained from the PlT species Glycine max, family Fabaceae. It composed
of conglycinin (140–170 kDa with glycosylated three subunits)–globular pRt and
Glycinin (340–375 kDa with six AB subunits comprising a basic [B] poly PEpt and
an acidic [A] linked via disulfide bonds). Based on the molecular weight and
sedimentation coefficient, it separates into fractions, 2S, 7S, 11S or 15S. The 7S
globulin and 11S globulin. With other film forming coating combination, glycinin
is known as the emulsifier, gelling agent, and foaming agent. B-conglycinin gets
denatured at a temperature of 70–80 degree centigrade and also less stable than
glycinin. The pRt which are found from Soybean are called Soy pRt are used to
replace the animal pRt in an individual diet. As discussed above the soybean is a
legume that contains no cholesterol and is low in saturated fat. Soybeans are the
Table 23.8 PlT and their pRt

PlT (Scientific Part of pRt Pharmaceutical/Medicinal uses
name) PlT
Soybean S eD B-Conglycinin Potential diagnostic marker for severe
(Glycine max) (7Sglobulin) allergic Reat to soy
Pea (Plsum S eD Glycinin Emulsifying and surfactant properties,
sativum) (11sglobulin) a RNA N-glycosidase activity to release
and b-pisavins an Endo’s fragment
Pea (Pisum S eD Plsumin, Antifungal, micro particle preparation
sativum var Legumin, Vicilin,
macrocarpon) ALBM
Peanuts (Arachis S eD Hypogin Antifungal
hypogaea)
Rice (Oryza S eD Glutenin, Microencapsulation
sativa) Globulin, ALBM,
Prolamin
Quinoa S eD ALBM, Globulins Microencapsulation
(Chenopodium
quinoa)
Peach (Prunus Fruit Thaumatin like Protection against chilling injury in
persica) pRt peach fruit
Almonds S eD Amadin Essential Ama
(Prunus dulcis)
Spinach Leaves Biotinyl pRt ViT
(Spinacia
Oleracea)
Wheat (Triticum S eD Gluten Microencapsulation
aestivium)
only vegetable food that contains all eight essential Ama. These are also excellent
sources of fiber, iron, calcium, zinc and B ViT.
23.3.2 Wheat
Based on solubility, the wheat pRt fractions are classified as, albumins (water sol-
uble), globulins (dilute salt solutions soluble), gliadins (soluble in 70–90% ethanol,
glutenin (insoluble under all the previously mentioned conditions, comprise 34% of
total pRt) and comprise 47% of the total pRt). Gliadin (40 kDa) have four distinct
fractions, a single chain PEpt, and containing intermolecular disulfide bonds. These
play an important role in strength, film formation, strength and elasticity. A mixture
of pRt, Glutenin, has a molecular weight distribution between 100 and 1000 kDa.
The strength of the pRt matrix determined by the disulfide bonds present in gliadin
and glutenin (Fig. 23.3).
23.3.3 Corn Zein
A class of prolamin pRt, Zein, found in maize (corn). It is a powder from corn gluten
meal, one of the best PlT pRt. Pure Zein is clear, tasteless, water-insoluble, odorless,
hard, and edible. High percentages of non-polar Ama, like leucine (20%), glutamine
(26%), proline (10%) and also basic and acidic Ama in low proportions contained by
Zein. Two major fractions of Zein are a-Zein, soluble in 95% ethanol and b-Zein
soluble in 60% ethanol. Commercially, it is used for coating of tablets and in
biodegradable packaging.
Fig. 23.3 Composition of wheat protein

23.3.4 Pea pRt
It is food with a neutral taste that is used in diary alternative such as cheeses and
yogurt. They extract it from the yellow pea, Pisum sativum and has a typical legume
Ama profile. Pea pRt includes four major classes of pRt such as globulin, albumin,
prolamin and glutelin. Mainly globulin (65–80%) and some fractions of albumins,
prolamins and Glutelins are extracted from pea SeD. Three different pRt - legumin,
vicilin and convicilin are comprising by globulin. The 11S globulin fraction with a
molar mass between 350–400 kDa is represented by legumin, while the 7S globulin
fraction with a molar mass of 150 kDa is represented by vicilin and convicilin.
Pea pRt can be found in energy bars, meal-replacement shakes and vegetarian
burgers, etc.
23.3.5 Rice pRt
Rice is a vegetarian pRt isolate that is an alternative to the more common soy and
whey pRt isolates. They can treat Brown rice with enzymes (Ezm) that will cause
carbohydrates to separate from pRt. The resulting powder is then sometimes added
to smoothies or flavored or health shakes. Most other forms of pRt powder, Rice pRt
powder, has a more distinct taste. Pea pRt is high in lysine, low in cysteine and
methionine. Thus, the combination of pea pRt and rice offer a superior Ama, com-
parable to egg pRt or diary, but without the intestinal issues or potential for allergies
that some users have with these pRt. Compared with rice bran, the pRt content in rice
grains is slightly lower, varying from 6 to 15%, prepared by alkali extraction,
followed by subcritical water treatment and by isoelectric precipitation. After
sequential extraction, it has received the following distribution. About 6% albumin,
15% globulin, 3% prolamin and majorly 75% glutenin Albumin from egg white
have foaming properties, found similar also from rice pRt; the emulsifying capac-
ities of albumin from bovine serum (BSA) are significantly higher than those of rice
pRt; isoelectric point at pH 4, minimum pRt solubility occurred whereas maximum
at pH 10; main Ama content of rice pRt is like that of soy and casein pRt; and
denaturation temperature of the rice pRt isolate is about 83.4 °C.
23.3.6 Sunflower pRt
Sunflower oil cakes are the source for major constituents of pRt. A high quantity of
pRt, defatted sunflower flour, contains around 27% in dry weight. The hulled SeD
contains about 20–40% crude pRt. Four fractions of pRt are present in the sunflower
pRt: Albumins, 17–23% of total pRt; major Globulins, 55–60%; and two minor
fractions, prolamins and glutelin, comprising 1–4% and 11–17% of the total pRt
fractions, respectively. It shows two major fractions: 2S albumins and 11S glob-
ulins (also named helianthinin). Helianthinin, a globular oligomeric pRt with a
molecular weight of 300–350 kDa and this pRt mainly exists in the 11S hexametric
form.
23.4 Isolation of pRt
They can use selective precipitation of pRt as: Fractionate a subset of pRt from a pRt
solution. Bulk method used major recovery of the pRt from a crude lysate and
recover a single pRt of interest from a purification step.
23.4.1 Selective Precipitation Methods
23.4.1.1 Salting Out
They apply ammonium sulfate to form co precipitation with pRt because the satu-
ration concentration provides high molarity. It causes precipitation of most pRt, and
does not have a large heat of solution. So, the generated heat gets easily dissipated;
a saturated solution (4.40 M at 20 °C) of pRt has a density of 1.235 g/cm3, does not
interfere with the precipitated pRt sedimentation by centrifugation. It also has
bacteriostatic properties and its concentrated solutions are protecting most pRt from
denaturation in solution state.
23.4.2 Isoionic Precipitation
23.4.2.1 Column Method
pRt are less soluble and showing more precipitation when they are isoionic. The pRt
are their least hydrated conformation (a phenomenon that closely associated to the
condition of pRt at their isoelectric point) when they are isoionic salt- free state. Tan
ford proposes the procedure applicable for this method. To determine the solubility
of many pRt, it applies two important parameters:( a) Low salt concentration (0 to
0.1 to 0.2 M salt) (b) Solution pH regarding each pRt Isoionic point. Column
method can determine by adjusting the pRt to their Isoionic pH to pRt that remain
soluble at their Isoionic point to strip away all salts from pRt the mixed-bed
deionization methods.
23.4.2.2 Dialysis Method
For rendering pRt become salt free (Isoionic), it is one of the oldest methods,
however, two problems arise often with conventional dialysis: Because of presence
of appreciable amount of pRt, the salt diffuses outward and swelling of the dialysis
bag occurs, these total things for osmotic effects. It is uncertain where the Isoionic
point is? If it is workable to deionize by dialysis against buffer. Automatically
adjusts a pRt precisely to its Isoionic pH without prior knowledge of the same by
resin deionization method, so this method also known as Dintzis method. It traps
the counter ions exchange between salt ions and pRt through the membrane and
outside in the exchanger resins. The precipitated pRt in the dialysis tubing is
recovered by centrifugation of the bag’s contents when the exchange is completed.
The dialysis technique is time consuming method as compare to flow-through
column method.
23.4.3 Reversed-Phase High-Performance Liquid

Chromatography (RP-HPLC)
The PEpt and pRt obtained from synthetically or biologically are used for both
preparations and analytical applications can isolate by a versatile technique known
as RP-HPLC. Separation of molecule from the mobile phase to immobilized
hydrophobic ligands attached to the stationary phase is the function of this tech-
nique. The stationary phase containing n-alkyl silica whereas acetonitrile containing
an ionic modifier trifluoroacetic (TFA) used in a gradient analysis of RP-HPLC.
Pharmaceutically important pRt like; PEpt, globular pRt, and pRt having molecular
weight 10,000 undergo purification by this technique. Because of hydrophobicity of
n-alkyl silica and acidic buffering SysT supports results into loss of biological
activity of larger poly PEpt. This technique is very limited in large scale of
utilisation.
23.4.4 Mass Spectrometry of pRt
Electrospray and matrix-assisted laser desorption ionization (MALDI) techniques

are used because it is too difficult to ionize and transfer of large and polar bio-
molecules in to the gas phase. The major areas where the Mass spectrometry in
proteomics are used (a) Identification of pRt, either in large scale proteomic ones or
in classical biochemical projects (b) Characterization of macromolecule, recombi-
nant pRt, and quality control of pRt in biotechnology (c) It applies for detection of
molecular weight of the pRt.
23.5 Application of pRt
They use pRt as albumin-based nanoparticles, hydrogels, film coater, micro parti-
cles, as beads, as composites and also as pRt-based nano carriers in drug and gene
delivery SysT. Some examples are nanoparticles SysT for encapsulation and con-
trolled delivery of bioactive compounds. When pRt used as hydrogels, because of
pRt-micelle structure, solubility of curcumin is enhanced, acts as a nano vehicle in
the food industry. It also acts as vehicles for bioactive like milk pRt. Plsumin pRt
obtained from Pisum sativum acts as novel antifungal. Casein-derived four main
bioactive PEpt act on immune, cardiovascular, nervous SysT, and nutrition SysT so
applied as a source of bioactive PEpt. It uses vegetable pRt in microencapsulation.
23.6 Introduction of Ezm
It defines a catalyst as a substance that increases the velocity or rate of a chemical

Reat without itself undergoing any changes in the overall process. Ezm are the
biocatalyst or pRt that catalyze the chemical Reat. Synthesize Ezm, pRt in nature
(except RNA acting as ribozyme), colloidal and thermo labile in properties and also
specific in their action (Mauguet et al. 2002). French scientist Louis Pasteur in the
year of 1850s states that fermentation of sugar in to alcohol by the yeast is catalyzed
by ferments. As per suggestion, they cannot separate ferments from the structure of
living yeast cells, this hypothesis of a scientist is called vitalism (Cho et al. 2004). It
continues for a decade, later in the year 1897, Eduard Buchner finds that yeast
extracts can ferment sugar solution in to alcohol and he also proves that chemicals
which continues to function when is removed from the cells facilitates that fer-
mentation (Renkema and Vliet 2002). Finally, Frederick gives name such chemical
molecules Ezm. James Sumner in 1926 isolates and crystallizes Ezm urease and he
finds that crystals of urease are formed only of pRt, thus he postulates that chem-
ically all Ezm are pRt. Because of the lack of other examples, this discovery remains
controversial for years. This hypothesis is widely accepted, when John Northrop
and Moses Kunitz crystallize pepsin, trypsin, and other digestive Ezm and chemi-
cally find them in pRt. Over the last decades of the twentieth century, enormous
advances have been made like isolation purification, and structure elucidation of
several Ezm have been done (Shukla and Cheryan 2002).
23.6.1 Nomenclature and Classification of Ezm
All most all Ezm contain a pRt backbone. In some Ezm pRt are the major component
in their structure whereas some Ezm also have pRt and additional non-pRt moieties,
which may or may not take part in the catalytic activity of the Ezm. We commonly
encounter carbohydrates groups which are covalently attached to structural features,

which have no catalytic activity but they support Ezm in stability and solubility
(Edsall and Wyman 1958). Other factors which are associated in Ezm activity are
cofactors metal ions and low molecular weight organic molecules i.e. co Ezm. These
may be loosely or tightly bound by monovalent or covalent forces, contributing to
both Ezm activity and stability (Morr and Ha 1993). Ezm are named by adding the
suffix “-ase” to substrate e.g.; DNA polymerase Ezm catalyzes polymerization of
nucleotides to form DNA (Gonzalez-Perez and Vereijken 2007). Urease Ezm cat-
alyzes urea hydrolysis, etc. Based on functions some Ezm are also named e.g. pepsin
a digestive Ezm, derived from Greek word Pepsis’ means digestion; lysozyme
responsible for cell wall lysis of bacteria (Ordonez et al. 2001). It derives some Ezm
names from their source from where they are obtained, e.g. trypsin is produced by
pancreatic tissue after rubbing it with glycerin hence is named after Greek word
“tryein” meaning “to wear down”. As the time passes away, several new Ezm are
discovered, some Ezm have multiple names. To overcome these problems, they are
classified Ezm based on the Reat they catalyze after the international agreement
(Chandi and Sogi 2007). So, it classifies Ezm according to the report of a
Nomenclature committee appointed by the International Union of Biochemistry. As
a result, all Ezm get a formal E.C. (Ezm Commission) number and some have their
trivial names (Hamada 2000). The Ezm commission (EC) numbers divide Ezm in
order to six major groups according to the Reat catalyzed (Table 23.9).
23.6.2 Chemical Nature and Properties of Ezm
As discussed above, all Ezm are pRt, and even it is found that it also acts on some
RNA molecules. Each Ezm has its own specific structure and specific conformation,
which are essential things for catalytic activity (Garfin 2003). Holo Ezm which
comprises of Apo Ezm (the pRt part) and a co Ezm (non- pRt organic part) is the
functional unit of the Ezm. The non-pRt moiety, which is covalently bound with the
Apo Ezm termed as prosthetic group, is the integral part of Ezm structure. Dialysis
can separate the co Ezm from the Ezm, whereas they cannot separate the prosthetic
group (Aguilar 2004). If Ezm is made up of a single poly PEpt, it is referred as
monomeric Ezm, if over one poly PEpt chain present, known as oligomeric Ezm, e.g.
lactate dehydrogenase. Multi Ezm complexes possessing Ezm have specific sites to
catalyze uncommon Reat in a sequence. Some process can regulate Ezm activity such
as phosphorylation, glycosylation. These processes alter the structure of a pRt found
in the Ezm. It requires Ezm in very minute quantity (Aguilar and Hearn 1996). It
does not absorb them in the overall Ezm Reat, rather is received whole after com-
pletion of Reat. Ezm do not alter the equilibrium constant (K) of a Reat but only
increases the velocity of Reat.
Table 23.9 International classification of Ezm

S. No. Classes Type of Reat catalysed
1 Oxidoreductases Transfer of electrons (Hydride ions or H atom)
2 Transferases Group transfer Reat
3 Hydrolases Hydrolysis Reat (Transfer of functional groups of water)
4 Lyases Addition of groups to double bonds, formation of double bonds
by removal of groups
5 Isomerases Transfer of groups within molecules to yield isomeric forms
6 Ligases Formation of C–C, C–S, C–O, and C–N bonds by condensation
Reat coupled to ATP cleavage
23.6.3 Mechanism of Ezm Action
Ezm are the powerful catalyst; the nature of catalysis taking place in the biological
SysT is like that of non-biological catalysis. In any Reat the reactants have to be a
triggered state or transition state. We know the energy required by the reactants to
carry out Reat as activation energy (Aguilar and Hearn 1996). When the reactant is
heated then attain the energy. The catalyst reduces the activation energy (Ezm in the
biological SysT) and this causes the Reat to proceed at a lower temperature. As we
have discussed that Ezm do not alter the equilibrium constant, it just enhances the
rate of Reat. The Ezm lowers energy barrier of reactants, making the Reat go faster.
The Ezm reduce the activation energy of the reactants in such a way that all the
biological SysT occur at BDY temperature (below 40 °C) (Mant and Hodges 1996).
23.6.4 Important Industrial Ezm and Their Sources
It may extract Ezm those biologically active from any living organism. It uses a wide
range of sources for commercial Ezm production from spinach to snake venom.
More than a hundred Ezm being used industrially, it may prevail Ezm from PlT,
animals, bacteria, fungi or yeast sources (Aguilar 2004). A very much larger
number of Ezm find use in chemical analysis and chemical diagnosis (Lin and
Karger 1990). They prefer microbes to PlT and animals as a source of Ezm. The
details are remarked in the Tables 23.10, 23.11, 23.12, 23.13 and 23.14.
Table 23.10 List of Ezm Ezm EC Source Industrial

received from animal source number use
Catalase 1.11.1.6 Liver Food
Chymotrypsin 3.4.21.1 Pancreas Leather
Lipase 3.4.23.4 Pancreas Food
Rennet 3.4.23.4 Abomasum Cheese
Trypsin 3.4.21.4 Pancreas Leather
Table 23.11 List of Ezm received from PlT source

Ezm EC number Source Industrial use
Actinidin 3.4.22.14 Kiwi fruit Food
a-Amylase 3.2.1.1 Malted barley Brewing
b-Amylase 3.2.1.2 Malted barley Brewing
Bromelain 3.4.22.4 Pineapple latex Brewing
b-Glucanase 3.2.1.6 Malted barley Brewing
Ficin 3.4.22.3 Fig latex Food
Lipoxygenase 1.13.11.12 Soybeans Food
Papain 3.4.22.2 Pawpaw latex Meat
Table 23.12 List of Ezm received from bacterial source

Ezm EC number Source Industrial use
a-Amylase 3.2.1.1 Bacillus Starch
b-Amylase 3.2.1.2 Bacillus Starch
Asparaginase 3.5.1.1 Escherichia coli Health
Glucose isomerase 5.3.1.5 Bacillus Fructose syrup
Penicillin amidase 3.5.1.11 Bacillus Pharmaceuticals
Protease 3.4.21.14 Bacillus Detergent
Pullulanase 3.2.1.41 Klebsiella Starch
Table 23.13 List of Ezm Ezm EC Source Industrial use

received from fungal source number
a-Amylase 3.2.1.1 Aspergillus Baking
Amino 3.5.1.14 Aspergillus Pharmaceutical
acylase
Glucoamylase 3.2.1.3 Aspergillus Starch
Catalase 1.11.1.6 Aspergillus Food
Cellulase 3.2.1.4 Trichoderma Waste
Dextranase 3.2.1.11 Penicillium Food
Glucose 1.1.3.4 Aspergillus Food
oxidase
Lactase 3.2.1.23 Aspergillus Diary
Lipase 3.1.1.3 Rhizopus Food
Rennet 3.4.23.6 Mucor Cheese
miehei
Pectinase 3.2.1.15 Aspergillus Drinks
Pectin lyase 4.2.2.10 Aspergillus Drinks
Protease 3.4.23.6 Aspergillus Baking
Raffinase 3.2.1.22 Mortierella Food
Table 23.14 List of Ezm Ezm EC Source Industrial use

received from yeast source number
Invertase 3.2.1.26 Saccharomyces Confectionery
Lactase 3.2.1.23 Kluvveromyces Dairy
Lipase 3.1.1.3 Candida Food
Raffinase 3.2.1.22 Saccharomyces Food
23.6.5 Ezm Derived from PlT Sources
23.6.5.1 Actinidin
Kiwi fruits are the source of actinidin Ezm, which is a proteolytic Ezm. Actinidin
plays a vital role in aiding the digestive process (Mann et al. 2001). Kiwi fruits
rather than actinidin also contain other Ezm which have no functions or little
functions (Yamada et al.2008). There is also a wide range of Ezm involved in the
ripening of kiwi fruit, particularly Ezm involved in polysaccharide and oligosac-
charide metabolism and in the development of flavor and aroma compounds
(Zengion and Yarnell 2011). Some Ezm influence flavor, texture and nutritional
values, during storage, processing and preparation of kiwifruit (Caballero et al.
2003).
23.6.5.2 a-Amylase
The major source of alpha-amylase (a-Amylase) is malted barley. It is an

oligosaccharide endoglycosidase having the character to cleave an internal glyco-
sidic bond within a poly or oligosaccharide. It requires calcium for producing
activity along with certain anions like chloride, phosphate and others (Ott and Lu
1991). a-Amylase can be produced from certain tissues, the forms found in serum
are most often from the pancreas and salivary glands. a-Amylase can be found in a
variety of BDY fluids and some Ezm are also found in urine in healthy individuals.
The chief purpose in testing amylase, especially when the symptoms are present, is
to diagnose pancreatitis and other primary and secondary pancreatic pathologies
(Moridani and Bromberg 2003). We can make this more specific by testing amylase
iso Ezm specific to pancreas. It also plays a vital role in diagnosing cancers other
than pancreatic cancer like myeloma, ovarian cancer, etc. (Moriyama 2008).
23.6.5.3 b-Amylase
The key source of beta-Amylase (b -Amylase) is also malted barley. It is exo Ezm
that able to cleave alpha (1, 4) linkage from the non-reducing end of the polymeric
chains and release maltose, the disaccharide (Pal et al. 2020). It acts alone and can
degrade amylose completely to maltose. This cannot attack the alpha (1, 6) linkages
or alpha (1, 4) linkages close to the alpha (1, 6) links. b-Amylase is made up in
endosperm in barley and so they do not synthesize it during germination
(Calvo-Villas et al. 2007).
23.6.5.4 Bromelain
The fundamental source of this Ezm is pineapple, it is a mixture of Ezm and has
proteolytic activity (Pal et al. 2019a, b.b). The major function of bromelain is that it
stimulates fibrinolysis by increasing plasmin and also prevent kinin production by
inhibiting platelet aggregation because its mechanism of action is
anti-inflammatory. So, it is used to treat a variety of pain and inflammation (Pal
et al. 2020). When it is applied to reduce pain, it must be dispensed away from food
because it acts as a digestive Ezm if consumed with food. It has also wound healing
activity and a useful tool to shorten healing time post surgically and to reduce levels
of edema, pain and ecchymoses (Mir et al. 2019).
23.6.5.5 b–Glucanase
It is derived from Malted barley. It is also produced by Bacillus amyloliquefaciens.

Fungal b-Glucans are also made by fungi of the Aspergillus group. It is also formed
as a side activity pectinase preparation (Nayak et al. 2019). The structure of PlT
glucans differs from that of the fungal glucan: the former being composed of beta
(1, 3)–(1, 4) structure which are hydrolyzed by the beta (1, 4) glucanase and the
latter the fungal glucans presenting a beta (1, 3) backbone being hydrolyzed by the
beta (1, 3) glucanase. In wine making, specific glucanase have been developed to
hydrolyze Botrytis and yeast glucans in order to improve clarification and filter-
ability of wines (Nayak et al. 2020).
23.6.5.6 Ficin
It is derived from figs latex and a family of proteases known as the cysteine endo
peptidases, it is mainly found in alcoholic beverages and acts as chill proofing agent
for beer, meat tenderizer, dough conditioner, rennet substitute, processing aid for
precooked cereals. It is one of the most commonly used for differentiating a good
deal of blood group antigens: e.g. destroy M, N, S, Duffy a & Duffy b and enhance
some other antigens (Gurjar and Pal 2020).
23.6.5.7 Lipooxygenase (LipOXse)
In the year of 1932, Andre et al. find that the bean flavor in soybeans is mainly
caused by lipoxygenase (LOX) and in 1947, Theorell et al. first extract LipOXse
crystals from soybeans. The pRt content in soybean is about 40% and in mature SeD,
LipOXse accounts for 1–2% of total pRt content (Pal et al. 2020). As compared to
other PlT the LipOXse activity is higher in soybeans. Algae, baker’s yeast, fungi and
cyanogen bacteria also contain LipOXse. The soybean SeD have three iso Ezm types
i.e. LOX-1, LOX-2, LOX-3 and soybean leaves (young), flowers and immature
pods have LOX-7 AND LOX-8 isozymes. LOX is an Oxido reductase, a non-heme
iron-containing pRt that specifically catalyzes polyunsaturated fatty acids with a
cis-1, 4-pentadiene producing a hydrogen peroxide derivative having a CoNjT
double bond by intermolecular oxygenation. Though the active site of LipOXse is not
fully understood but active site group may contain iron, aromatic Ama and
methionine residue (Pal et al. 2019a; b).
23.6.5.8 Papain
It is derived from the roots, leaves, latex, and fruits of the PlT Carica papaya
(Papaya). It catalyzes the breakdown of pRt by hydrolysis (Pal et al. 2019a, b). It is
useful for the analysis of pRt, in tenderizing meat, in clarifying beer. It is also used
in toothpastes and cosmetics and also preparation of various remedies for indi-
gestion, ulcers, fever and swelling (Saha and Pal 2020). Papain can trigger allergic
Reat in susceptible individuals. Skin Reat may occur following contact with fresh
latex from papaya. Hypersensitivity Reat may be especially pronounced in persons
allergic to latex (Sachan et al. 2019).
23.7 Pharmaceutical Applications
Many Ezm find many applications in pharmaceutical industries and various other
industries. It uses Ezm for production of glucose syrups, crystalline glucose, high
fructose corn syrups, maltose syrups, and food, detergent, paper and textile
industries (Saha et al. 2017). It may be used in detergent industry as additives to
remove starch-based stains. It uses textile industry amylases for warp sizing of
textile fibers. In paper industry, it uses them for the reduction of starch viscosity for
coating of paper. Ezm like proteoses, lipases or xylanases have a significant con-
tribution in food industry. Bromelain is used in treatment of inflammation of soft
tissues and edema because of surgery and injury. Papain is used in clarification of
beverages and meat tenderizer; it also find its applications in cheese manufacture as
a substitute of renin (Nayak et al. 2018). Papain is used as an anti-inflammatory
agent; it has shown relieving symptoms of episiotomy. It uses Ezm like pancreatic as
a digestive aid for converting starch in to dextrin and sugar (Soni et al.2017). Renin
is used to coagulate milk and hence making the milk easily digestible for weak
patients. Ezm is applied as a contact lens cleaner (Pal et al. 2019a, b). It is used to
manipulate DNA in genetic engineering and detect the amount of glucose present in
blood. It uses supplement for Ezm deficiencies. Prolactin Ezm is used to treat lactose
intolerance (Nayak et al. 2016). Collagenase is used for skin ulcers. Asparaginase is
used to treat leukemia.
23.8 Conclusion
This book chapter highlights the pharmaceutical importance of pRt and Ezm isolated
from the PlT sources along with their classifications, structure and strategies used
for isolation of PlT pRt. It may be concluded that PlT pRt and Ezm are very much
useful in the pharmaceutical field. The present review would help the scientific
community to think about further modifications or development towards phyto-
chemical screening and isolation of pRt and Ezm prevailing in PlT sources. The
researcher and academicians will also be benefited towards achieving fundamental
knowledges regarding enzymes and proteins.
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Chapter 24
Advances and Perspectives
of Gamma-Aminobutyric Acid
as a Bioactive Compound in Food
Priti Jain and Mangesh S. Ghodke
Abstract GABA is a novel potent bioactive amino acid, non-proteinaceous in

nature and comprising of four carbon units. Its role as an inhibitory neurotransmitter
of the neuronal cortex is well known and it had proven its effects on central nervous
system. Recent studies reveal that this neurotransmitter is also able to act as a
hypotensive agent, anti-diabetic and as tranquilizer. Besides this, GABA is also a
bioactive compound of food, pharmaceutical, and feed industries. It is present in
beans, pulses, milk, green, black, and oolong tea, as well as in fermented foods
including kefir, yogurt, and tempeh and other dairy products. It is normally syn-
thesized in the plant from Glutamate and nowadays various advanced strategies are
being used for enhanced production of GABA. The reason for this may be
accounted for use of GABA as a supplement to treat high blood pressure, stress, and
anxiety, and sleep, as well as to stimulate the body's natural growth hormone, often
by athletes. This chapter would focus on the production of GABA, its effects on
humans as neurotransmitter along with the physiological effects being studied
recently. The chapter will also cover various types of food containing GABA and
later it deals with the strategies being adopted for large scale production of GABA.

Keywords Gamma-amino butyric acid Glutamate GAD Lactobacilli Lactic

acid bacteria Hypertension Neurodegeneration Obesity Anti-diabetic
24.1 Introduction
The human nervous system comprises of more than 40 neurotransmitters. Few have
excitatory roles while few have an inhibitory role. Out of them one of the important
neurotransmitter is Gamma aminobutyric acid. Gamma aminobutyric acid com-
monly known as GABA is an amino acid, non-proteinaceous in nature and com-
prising of four carbon atoms. It is predominating inhibitory neurotransmitter of the
P. Jain (&) M. S. Ghodke

R C Patel Institute of Pharmaceutical Education and Research, Shirpur 425405, India
e-mail: pmj.grv@gmail.com
https://doi.org/10.1007/978-3-030-54027-2_24
820 P. Jain and M. S. Ghodke
Central nervous system. This amino acid is widely distributed not only in our
physiological system but is also present in plants, animals, and micro-organisms
where its role in Krebs cycle is known in plants and microbes while in animals as a
neural signal neurotransmitter. Its wide applications in pharmaceutical, medical,
nutraceuticals and recently for manufacturing of nylon and other compounds make
this simple amino acid “GABA” very important (Dhakal et al. 2012; Dai-Hung and
Sang 2019).
GABA binds to GABA receptors which are sub-classified as GABA-A and
GABA-B. Another minor class GABA-C has also been reported and it has a major
role in retinal signal processing. GABA is biosynthesized from L-glutamic acid by
the decarboxylation reaction catalyzed by Glutamate decarboxylase (GAD, EC
4.1.1.15) and pyridoxal phosphate (PLP) that acts as a cofactor. GABA after
biosynthesis gets packaged into synaptic vesicles by VGAT (vesicular GABA
transporter) (Fig. 24.1), later released in the synaptic cleft and then binds to the
GABA receptors on postsynaptic region which is either GABA-A or GABA-B.
Though the main concern of this chapter is not to deal with GABA receptors, we are
presenting in very brief these receptors for reader's concerns. Both receptors vary in
their physiological and pharmacological properties. GABA-A receptors are ligand
gated chloride channel receptors located post-synaptically while GABA-B is
GPCR’s located pre as well as post-synaptic. On one hand, GABA-A receptors are
involved in mediating cardiovascular, anti-anxiety and anti-convulsive activities
while on the other hand, GABA–B receptors are mainly indulged in depression and
analgesic effects (Matsumoto 1989; Diana et al. 2014; Richard and Timothy 1999).
Fig. 24.1 Biosynthesis of GABA and its fate in Glia

24 Advances and Perspectives of Gamma-Aminobutyric Acid … 821
Alongside the binding to GABA receptors, some GABA undergoes reuptake and
some reach the glia with the help of GABA transporters. GABA which reaches
presynapse may be reused but those GABA entities that move to glia are metab-
olized to form succinic semialdehyde using GABA-Transaminase (GABA-T or EC
2.6.1.19) (Seigel et al. 1999; Rashmi et al. 2018).
24.1.1 Why is GABA Important?
GABA is considered to be an important inhibitory neurotransmitter because of the

pivotal role it plays in mammalian CNS. It has vital roles to play in neuronal
development, synaptic activities, counteracting depression and sleep disorders
(Ziskind-Conhaim 1998; Wong et al. 2003). Besides documented shreds of evi-
dence for its activities on CNS, its innervation to other peripheral systems is also
considered crucial. GABA has proven to be effective anti-hypertensive, anti-cancer,
anti-oxidant, anti-obesity, anti-diabetic, tranquilizer, and many other effects are well
documented (Sheng et al. 2012; Teresa 2013; Injae et al. 2019; Shimada et al.
2009). Hence, the pharmaceutical applications of GABA give it an edge over other
neurotransmitters and its abundance in a variety of food products make it usable by
humans for health benefits. The enzyme GAD that catalyzes the conversion of
glutamate to GABA is found in many bacterial strains like Escherchia, Aspergillus,
Lactic acid bacteria, Neurospora etc. (Tavakoli et al. 2015; Diana et al. 2014). This
enzyme is also present in various plants such as soyabean, tomatoes, tea, germi-
nated brown rice and also in few insects like flies, housefly and cockroaches (Diana
et al. 2014; Nikmaram et al. 2017). Scientists have been struggling hard to utilize
these microorganisms for GABA production through various technologies. The
levels of GABA may vary from trace quantity to micromolar range depending on
various factors. Despite of these low quantities present in plants, the pharmaceutical
and food industries and recently other manufacturing industries are paying strong
attention in increasing the concentration by various means. Several such techniques
include anoxia, enzymatic treatment, germination, old conventional methods,
microbial fermentation, use of excess glutamic acid and many more (Yongqi et al.
2018; Benincasa and Falcinelli 2019; Li et al. 2010a, b).
Very recent reports suggest that GABA is not only important from pharma-
ceutical and nutraceutical front but it is also important in industrial biotechnology
due to its need as a precursor for production of polymer called “Nylon 4” (Park
et al. 2013).
24.1.2 Alternative Synthetic Methods of GABA
Since biosynthesis of GABA does not always provide sufficient levels, it is

important to synthesize it by alternative methods. Hence, various synthetic methods
are used for amplifying the GABA production. Few such synthetic methods are
given below:
Usually, GABA is synthesized using 4-bromobutyric acid ester with pthalimide
or ammonia as nitrogen source. Zhinyong et al. reported the synthesis from
c-butyrolactone with thionyl chloride in the presence of methanol which needs to be
aminated with ammonium hydroxide as the Nitrogen source. (Scheme 24.1)
(Zhiyong et al. 2013; Mohamed et al. 2019).
Another simple method was reported Nudelman et al. They reacted 2-bromo
propionic acid with radio labeled cyano compound, which on reduction with
platinum dioxide gave desired product (Scheme 24.2) (Song et al. 2014; Nudelman
et al. 2008).
Hua and Cai filed a patent exploring simple synthesis for GABA. They used
glutamic acid and converted it to glutaric anhydride. It was then treated with
aqueous ammonia solution to produce imide which was then oxidized with sodium
hypochlorite to produce the desired product. (Scheme 24.3) (Hua and Cai 1995;
Mohamed et al. 2019).
Though many synthetic schemes have been proposed for GABA synthesis but
none is suitable for large scale synthesis because of the corrosive nature of the
chemicals and health-related hazards. Therefore, it is always preferred to use “green
synthesis”. Since the green synthetic approaches for GABA mainly use microor-
ganisms, these methods are cumbersome because of the low levels present in
Scheme 24.1 Synthesis of GABA using Gamma butyrolactone
Scheme 24.2 Synthesis of GABA using 2-bromopropionic acid
Scheme 24.3 Synthesis of GABA using Glutamic acid

microbes but are efficient and safer than normal chemical synthetic methods. The
greener synthesis will be discussed later in this chapter.
GABA, owing to its large profile of pharmacological activities and beneficial
effects in humans, is regarded as a “BIOACTIVE COMPOUND”. In this chapter,
we will, therefore, discuss in detail the Pharmaceutical applications of GABA, its
implication in various diseases, GABA as the bioactive compound in food and
microbes and finally, we would discuss the advances in techniques being adopted
for increased production of GABA.
24.2 Pharmaceutical Properties of GABA
24.2.1 Anti-Hypertensive Effect of GABA
In recent years because of drastic changes in eating habits and decreased physical
activity most of the people are associated with increased metabolic syndrome. The
metabolic syndrome is a collection of interrelated risk factors that seem to promote
atherosclerotic cardiovascular disease. The most commonly known metabolic risk
factors are atherogenic dyslipidemia, elevated blood pressure, and elevated plasma
glucose (Fig. 24.2) (Akama et al. 2009; Ebizuka et al. 2009).
GABA has been reported to reduce blood pressure in experimental animals and
humans. In a study reported by Morio Shimada et al., the anti-hypertensive effect of
GABA-rich Chlorella was given by oral administration for 12 weeks in subjects
Fig. 24.2 Different pharmaceutical roles of GABA

with high-normal blood pressure (130–139 mmHg for systolic blood

pressure-SBP), 85–89 mmHg for diastolic blood pressure (DBP), and borderline
hypertension (140–159 mmHg for SBP and 90–99 mmHg for DBP) in a
placebo-controlled, double-blind study. In the GABA group, SBP significantly
decreased from week 4 to week 12, and also on week 16 of follow-up compared to
the baseline level (p < 0.05 or p < 0.01). Systolic blood pressure levels were sig-
nificantly lower on weeks 8, 10, and 12 than those in the placebo group. Systolic
blood pressure in the placebo group did not significantly change from the baseline
throughout the study period. Diastolic blood pressure in the placebo group was not
significant to the baseline throughout the study period. Though, mechanism for
hypotensive action of GABA has not been fully explained; it is reasonable to
consider that the anti-hypertensive effect of GABA is attributable to its vasodilator
action or the inhibition of peripheral sympathetic nerve or a decrease in total
vascular resistance, because, GABA poorly passes the blood–brain barrier due to its
low lipid solubility. It is known that Angiotensin converting enzyme (ACE) plays
essential role in regulating blood pressure. Several food supplements containing
GABA are found to elicit antihypertensive effect by inhibiting ACE. Eg.
Lactococcus lactis DIBCA2 and Lactobacillus plantarum PU11 fermented milk,
soybean fermented by GABA enriched dairy products, GABA enriched rice grains,
white rice, Chingshey purple sweet potato-fermented milk by lactic acid bacteria,
GABA rich tomatoes, bread have also been reported to reduce BP in animal studies
(Kawakami et al. 2018a, b; Nishimura et al. 2016; Tsai et al. 2013; Lin et al. 2012;
Suwanmanon and Hsieh 2014; Aoki et al. 2003).
24.2.2 GABA as Neuroprotective Compound

and for Neurological Disorders
Injuries and damage caused to nervous system leads to increased inflammatory

responses which lead to release of inflammatory mediators. Several reactive oxygen
species, cytokines, leukotrienes, nitric oxides are released which trigger neurode-
generation and may serve as a responsible factor for diseases like Alzheimer's,
Parkinson's and sclerosis (Cho et al. 2007).
Many studies have been performed to find newer GABA containing food that
may be used to treat neurodegeneration or reduce neurodegenerative effects. It is
proven that GABA agonist protects neurons from inflammation and injury. The
most common symptom observed in Parkinson's disease (PD) are visual halluci-
nations, which increases as the disease advances, and may range from simple
flashes of light or color to complex hallucinations. It is also reported that GABA
levels are reduced in dementia with lewy bodies. This may be attributed to poor
visual input, disrupted connectivity with other visual areas, reduced GABAergic
inhibition (Li et al. 2016).
Scientists harnessed magnetic resonance spectroscopy to govern GABA levels in

occipital lobe and used 36 subjects with Parkinson’s, 19 with and 17 without
complex visual hallucinations, together with 20 healthy controls without halluci-
nations. Through this study they found lower GABA + /creatine in PD with visual
hallucinations (0.091 ± 0.010) versus those without (0.101 ± 0.010) and controls
(0.099 ± 0.010) (F2.49 = 4.5; p = 0.016).
It was observed that there were widespread reductions in white matter integrity in
the visual hallucinations group but was insignificant after controlling the cognition.
Since GABA is reported to be involved in neurodegeneration, the GABA enriched
food may directly or indirectly be beneficial in controlling the neurodegeneration,
mainly by protecting through the toxins or injury (Li et al. 2016; Okada et al. 2000).
Besides Parkinson’s, GABA also has shown to improve the cognitive abilities
and suppress neurodegeneration. Eg. GABA enriched rice germ, Gaba-rich
Monascus-fermented food, Gaba-enriched fermented Laminaria japonica was
examined for the treatment of anxiety, sleeplessness, and depression, cognition
(Yamatsu et al. 2016; Reid et al. 2018).
24.2.3 GABA as Anti-obesity Agent
Reports suggest that the worldwide prevalence of obesity is high and constantly
increasing. Hence, there is an urgent need for search of newer therapies to treat
obesity-related pathologies (NCD Risk Factor Collaboration (NCD-RisC), 2017).
Brown adipose tissues (BAT) were characterized as a thermogenic organ and as per
the reports adult humans also possess functional BAT, and studies have shown that
it is a metabolically active organ with the potential to regulate systemic metabolism.
The metabolomic analyses reveal that GABA levels are increased in the inter-
scapular BAT of mice with dietary obesity.
The study results reveal that constitutive activation of GABA/GABA-BR1
signaling causes BAT dysfunction and systemic metabolic derangement in condi-
tions of obesity. The results also provide a substance that, in healthy volunteers,
GABA-BR1 is significantly increased in individuals with low UCP1 expression.
Obesity is linked to functional decline in BAT levels and metabolic stress reduces
Ucp1 expression. It implicates that GABA-BR1 levels are related inversely to
UCP1 in humans (Ippei 2018).
24.2.4 Antimutagenic and Antimicrobial Activities

of c-Aminobutyric Acid
The most widely appreciated and consumed beverage in vast quantities worldwide
is Tea. GABA tea is a special kind of tea enriched with GABA. Alternative cycles
of anaerobic and aerobic conditions help to accumulate higher GABA levels.

GABA is proposed to act as a natural relaxant and anti-anxiety compound, and its
administration could concurrently enhance immunity under stress conditions.
Jeng-Leun Mau et al. reported a study using GABA tea powder as test materials.
Ethanolic extract was prepared using the optimal extraction conditions (50%
aqueous ethanol and 75 °C). The extraction yield determined was 32.21 ± 0.68%,
and phenol content was 492.08 ± 3.16 mg/g. The results presented that 50%
ethanolic extract of GABA tea was nontoxic and non-mutagenic towards
Salmonella typhimurium (TA98 and TA 100). This extract of TA 98 and TA 100
was highly anti-mutagenic as experimental microbes in the presence of metabolic
activators. In addition, the growth of V. parahaemolyticus, S. aureus, and B. cereus
was inhibited by the addition of tea extract (Mau et al. 2012).
Further studies revealed that Gaba tea extract exhibited inhibitory activity
against Vibrio parahaemolyticus, Staphylococcus aureus, Bacillus cereus,
Salmonella typhimurium, and Escherichia coli (Mau et al. 2012).
24.2.5 GABA as Anti-stress Compound
Stress is defined as a disruption of the body’s homeostasis and their activation is

essential for our survival because it helps us to cope with varying internal and
external factors. Tea (Camellia sinensis) is reported to be the most widely con-
sumed beverage worldwide and it is also reported to reduce physiological stress,
anxiety, and induce relaxation. A popular intervention in complementary medicine
to remove chronic stress involves dietary food supplemented with GABA which is
said to exert the effect peripherally, by acting on the autonomic nervous system
ganglia, and central processes. One such commercially available product is
GABA-enriched oolong tea (Hinton et al. 2019).
Jelinek et al. reported a study to measure the effects of GABA-fortified tea
consumption on heart rate variability (HRV) and stress in 30 participants using a
pre-post cohort study design. ECG recordings, frequency domain parameters
including total power, high and low-frequency power and heart rate were deter-
mined. The control group comprised of subjects with non–fortified tea consump-
tion. Two-way ANOVA was performed and it was concluded that oolong tea
consumption led to a significant decrease in the immediate stress. The authors
concluded that autonomic imbalance and HRV in people with acute stress is sig-
nificantly reduced following a cup of GABA fortified oolong tea and highlighted
the complex interaction between autonomic nervous system function and mood
(Zhenxing et al. 2014). A study carried in 2009 showed that chocolate containing
GABA was successful in reducing the stress condition.
24.2.6 Gamma-Aminobutyric Acid in Thyroid Dysfunction
Obesity is a worldwide epidemic and a key factor for the development of metabolic
syndrome and type 2 diabetes (TD2). Nowadays, high energy diet is an important
cause of obesity occurrence for which the endocrine mechanism remains unclear.
Recently, many surveys have shown that obesity is closely associated with
hypothyroidism (Zhenxing et al. 2014).
Le et al. performed a study where 20-week high-fat diet-fed (HFD) mice were
utilized to study redox status and thyroid functions of diet-induced obesity
(DIO) and DIO-resistant (DIOR) mice in their 1st study. The study also aimed at
determining whether anti-obesity activity of GABA was related to antioxidant effect
and if it improved thyroid function by using GABA in drinking water (0.2, 0.12 and
0.06%). It was observed that in DIO mice, TSH (thyroid stimulating hormone)
levels increased, free thyroid hormone decreased. On the other hand, DIO-R mice
showed normal TSH levels, increased THs and its functions. Down-regulated
thyroid and THs functions in DIO mice may be accounted for obesity. GABA could
prevent obesity by ameliorating oxidative stress and HFD-disrupted functions of
thyroid and THs (Zhenxing et al. 2014).
24.2.7 GABA as Renoprotective
Chronic renal failure (CRF) is characterized as a pathophysiological condition of

the kidney due to the permanent loss of nephrons, leading to the development of
glomerular and tubular lesions. The kidney also contains significant levels of
GABA, and specific binding sites for GABA have been confirmed in the kidney.
Patients with end-stage renal disease and dialysis encephalopathy showed reduction
in GABA levels in many areas of their brains, especially in the cerebral cortex and
thalamus (Sumiyo et al. 2006). A study by Sasaki et al., used a remnant kidney
model with 5/6 nephrectomized rats to analyze the protective effect of
c-aminobutyric acid against chronic renal failure (CRF). Nephrectomy causes renal
dysfunctioning, which was evaluated via several parameters like serum urea
nitrogen, creatinine levels and creatinine clearance. However, GABA administra-
tion ameliorated renal dysfunction, and a longer administration period of GABA
increased its protective effect. In addition, nephrectomized control rats showed an
elevation in the fractional excretion of sodium (FENa) with an increase in urinary
sodium, while GABA led to a significant decline in FENa. Rats administered with
GABA showed improvement in marked levels associated with CRF caused due to
nephrectomy. This might be a proof that GABA inhibits disease progression and
has a protective role against CRF. The protective role may also be accounted for
improvement in the serum lipid profile, with reduced triglyceride and total
cholesterol levels (Kim et al. 2004).
Furthermore, nephrectomy also caused renal oxidative stress with a decrease in

the activity of antioxidative enzymes and elevation of lipid peroxidation. The
administration of GABA attenuated oxidative stress induced by nephrectomy
through an increase in superoxide dismutase and catalase, and decrease in lipid
peroxidation. The histopathological lesions, including glomerular, tubular and
interstitial lesions, under nephrectomy were also improved by GABA with the
inhibition of fibronectin expression. This study revealed that GABA regulated
blood pressure and lipid profile and hence, attenuated renal dysfunction, and it also
ameliorated the oxidative stress induced by nephrectomy, suggesting the promising
potential of GABA in protecting against renal failure progression. In addition to
this, it is documented that oral administration of GABA and its supplements
improved the other comorbidities associated with renal failure (Kim et al. 2004;
Talebi et al. 2016).
24.3 GABA as Bioactive Compound in Food
As discussed in the previous section, GABA is produced from glutamic acid by

enzymatic action of GAD. This reaction occurs in anaerobic conditions at pH below
5. GAD is, therefore, an important enzyme found in many strains of bacteria and
fungi (Lactic acid bacteria, Escherichia, Aspergillus, Streptococcus, Neurospora,
Lactobacillus, Streptomyces, Monascus, Enterococcus, Rhizopus, Haematococcus),
plants (tomato, soybean, tea, mulberry leaves, petunia, pulses, germinated rice,
pulses, brown rice), dairy products and human brain. Besides bacteria, algae are
also a good source of GABA. Section 24.2 discussed the importance of GABA in
various physiological functions and this is the reason why the search and devel-
opment of food containing GABA are given high priority. Using GABA enriched
food products represent a natural and economic way to get rid of many ailments.
Table 24.1 Few GABA containing plants

Botanical Name Common name Amount of GABA Part of plant
Zinziber officinale Ginger 0.0114% Rhizome
Solanum torvum Potato 0.0119% Plant
Ananas comosos Pineapple 124 ppm Fruit
Arctium lappa Beggars button 25 ppm Root
Hypericum perforatum Goat weed 700 ppm Plant
Lycopersicum esculentum Tomato 220–480 Fruit
Phoenix dactylifera Date palm 2660–3370 Fruit
Pisum sativum shoot 153 Green pea
Rehmannia gltinosa Root 4000–31,000 Chinese fox-glove
Urtica diocia Root 250 Common nettle
https://www.naturalmedicinefacts.info/chemical-detected/12318-1.html
Chemical modifications in existing food products is a method that may be used to

enhance the GABA levels and use them as nutraceuticals (Table 24.1).
24.3.1 Microorganisms as Sources of GABA/GAD
Microorganisms like bacteria, fungi, algae, molds, yeast isolated from different
foodstuff are a big pool of GABA and GAD. The first instance of GABA is known
to be isolated from yeast extracts which were acid-treated and later it was also
isolated from red yeast named Rhodotorula glutinis, Neurospora crassa spore
germination, Aspergillus nidulans and A. niger. In case of bacteria, the specific
class called Lactic acid bacteria (LAB) are major group for GABA production.
From amongst the several, strains of Lactobacillus and Lactococcus have been
isolated from variety of fermented foodstuff like Kimchi, paocai, raspberry juice,
etc. The best known GABA producing strains are Lactobacillus paracasei PF6,
Lactobacillus delbrueckii subsp. Bulgaricus PR1, Lactobacillus lactis PU1, and
Lactobacillus brevis PM17. It has also been detected in red yeast, Rhodotorula
glutinis (Kawakami et al. 2018a, b). GABA pool was also seen in Neurospora
crassa spore germination during early phases (Krishnaswamy and Giri 1953;
Schmit and Brody 1975). Besides this, fungi like Aspergillus nidulans and A. niger
contain GABA (Kubicek et al. 1979). As previously stated, the biosynthetic
methods of GABA production are beneficial due to simple and mild reactions, high
efficiency, simple workup, high yield, and environmental compatibility. Therefore,
isolated enzymes like GAD are used for GABA production using different strate-
gies. The other method is to perform microbial fermentation of low-cost feedstock.
This is called a “natural method of production” and is preferred more than chemical
methods. Many factors govern the amount of GABA produced which in turn
depends upon the characteristics of GAD. These are discussed in Sect. 24.4.
24.3.2 Plants as a Source of GABA and GABA Enriched

Food
GABA is found naturally in small quantities in many plant sources. The first source
where GABA was found is potato tubers. Besides this, natural GABA is found in
many fruits, vegetables, and cereals. Vegetables such as spinach, tomatoes, cab-
bage, broccoli, ginger, asparagus, peas, mushrooms, etc. and fruits like Pineapple,
apples, grapes, sweet potato, chestnuts contain GABA. Besides fruits and vegeta-
bles, GABA is also found in a number of cereals and pulses as such or they are
enriched by fermentation using different LAB. Many cereals, legumes contain
GABA producing bacterial strains which are further utilized to generate GABA
enriched breads and sourdoughs. Sourdough breads are better quality breads than
normal because of the effect of Lactobacillus and other LAB which enrich them
better than baker’s yeast. Lactobacillus plantarum C48 and Lactococcus lactis
subsp. lactis PU1 have been used for fermentation of sourdough of many cereals,
pseudocereals and flour obtained from legumes (Coda et al. 2010). Various flours
that have been GABA enriched are amaranth, chickpea, buckwheat, quinoa.
Bathura sourdough bread has been an example of achieving highest levels of
GABA enrichment i.e. 226.22 mg/100 gm (Bhanwar et al. 2013). Oats when fer-
mented with Aspergillus oryzae has very high level of GABA i.e. 435.2 microg/g)
(Briguglio et al. 2018; Shengbao et al. 2014).
Fermentation is not the only process to improve the GABA content. Alteration
of physical conditions needed for plant growth are also important for improving the
GABA content in plants. Changing the germination conditions, pH, stimuli and
other controlled condition help to improve the GABA contents in many cereals. e.g.
42.9 mg/100 g GABA levels are reported in foxtail millet (Bai and Fan 2008) and
14.3 mg/100 g in germinated waxy hull-less barley (Hyun et al. 2009). The most
important and commonest cereal to note is RICE. Exceptional attention has been
paid to increase GABA content in different varieties of rice. The highest GABA
content is found in germinated brown rice as compared to white rice (10 times less)
and brown rice (2 times less) (Patil and Khan 2011). Other cereals rich in GABA
are sprouts of brown rice, barley, oats, millets, corn and beans.
Pulses, also known as leguminous crops are also an excellent source of GABA
because faba beans and mung beans contain high levels of glutamic acid and
applying environmental stress further enhances the levels. Abscisic acid acts as a
stress hormone and under hypoxic-NaCl conditions, it also increases GABA levels
in beans. Azduki beans, wultari beans also possess high GABA levels after proper
treatment. Azduki beans are reported to reduce the risk of cardiac disease and
acetaminophen-induced liver damage. Kidney beans when treated with glutamic
acid during solid state or liquid state fermentation with Baccilus subtilis and
Lactobacillus plantarum respectively, resulted in high GABA levels (Limón et al.
2014).
Overall, pulses may be categorized in 11 groups: dry beans, dry broad beans, dry
peas, chickpeas, lentils, lupins, pigeon peas, black-eyed peas, bambara groundnut,
vetch, other pulses. All are associated with health benefits and have a little amount
of GABA which may be enriched on treatment (Nikmaram et al. 2017).
24.3.3 Dairy Products and Beverages as GABA Sources
Dairy products like cheese, yogurt, milk are GABA enriched by LAB.
Lactobacillus lactis is used for GABA enrichment in cheese (manages hyperten-
sion), fermented goats milk with LAB has very high GABA amount (28 mg/kg). L.
plantarum fermented skim milk is reported to reduce systolic and diastolic blood
pressure in rats. Many varieties of cheese also have natural presence of GABA with
cheese strain like ULAAC-A23 and ULAAC-H13 having highest content (Lacroix
et al. 2013). Chen et al. evaluated anti-diabetic effects in GABA enriched yogurt
and reported it to lower glucose levels and raise serum insulin concentration.
Yogurt-sake, an alcoholic fermented beverage had high levels of GABA than the
total amount observed in yogurt and sake alone. Streptococcus thermophilus Hp
was responsible for high levels while Streptococcus thermophilus Lp produced low
levels (Ohmori et al. 2018).
Many beverages like white tea, fermented juices, alcoholic beverages also
contain GABA and these are being demonstrated for beneficial effects mainly in
treatment of hypertension. Maintaining anaerobic conditions have been reported to
enhance the GABA in tea leaves by 8.9 folds. A variety of Gaba enriched tea is
known as Gabaron tea which contains greater than 150 mg GABA per 100 gm.
This helps to reduce blood pressure and improves sleep disorders. Few such
commercialized compounds are oolong tea, GABA black tea and GABA green tea
(Lacroix et al. 2013).
24.3.4 Marine Sources of GABA
Marine sources have phenomenal biodiversity and this makes them very useful as a
source of healthy food. Marine organisms are also a proven source of GABA and
GAD. Many Irish marine cyanobacteria are reported to be GABA producers. Few
such examples are Calothrix contarenii SABC022701, Chlorogloea microcystoides
SABC022904, Phormidium africanum SABC010301, P. angustissimum
SABC022612 and P. laminosum SABC022613 possessing 99*102–7284 102
nmol g−1 on dry-weight biomass (Shiels et al. 2019). Marine Pseudomonas is
another example to possess this metabolite. pH, stress, osmotic pressure are known
to enhance GABA production in marine and freshwater cyanobacteria. Since
cyanobacteria are microphototrophs, they have become choice for the development
of various bioactive metabolites. Synechocystis sp. PCC6803 has been character-
ized for improved GAD activity and in another case, double engineering was
performed (GADox/DKgd) to improve GABA production (Kanwal and
Incharoensakdi 2019).
As discussed earlier, GABA gets metabolized by GABA shunt pathway which is
important to maintain carbon and nitrogen balance intracellularly. This pathway is
also an important metabolic pathway found in cyanobacteria.
Masuda et al. have demonstrated the presence of GABA in unicellular marine
fungi (Masuda et al. 2008). Microalgae also possess GABA which acts as neuro-
transmitter, anti-oxidant, and anti-inflammatory (Gupta and Dhan 2018).
Halotolerant cyanobacterium Aphanothece halophytica were treated in stress con-
ditions and the normal levels of GABA increased to double than initial amounts
along with enhanced GAD activity. These results also have proven successful in
Arthrospira platensis (Boonburapong and Incharoensakdi 2016). Red Microalgae
Rhodosorus marinus also contains GABA and GABA-Alanine which have proven
to exhibit neuro-soothing effect and regulate skin sensitization by decreasing
TRPV1 over-expression in normal human astrocytes under PMA-induced inflam-

matory conditions (Scandolera et al. 2018). One of the Pseudomonas species of
marine origin also produces GABA (Mountfort and Pybus 1992).
24.4 Techniques for GABA Enrichment and Advances

in GABA Production
Recent consumer awareness and hence need for adopting healthier products with
added advantages, called as “Nutraceuticals” have resulted in the market growth of
such bioactive or functional components. The natural abundance of bioactive
compounds like GABA is usually not very high and does not meet the
ever-increasing market needs. Hence, scientists work untiringly to develop newer
methods and techniques to enhance the concentration of bioactive compounds by
various methods.
Similarly, reports reveal many novel techniques used for optimization and
improvement of GABA content in plants, microbes, and dairy products. (Details on
these techniques are discussed in this section below).
24.4.1 GABA Production by LAB
Microorganisms contain low GABA content which makes it utmost difficult to

extract. Also, the chemical synthetic methods as described previously are hazardous
and difficult to adopt for large scale production. In this case, scientists have dis-
covered many species of Lactic acid bacteria (LAB) that can produce GAD or
produce high levels of GABA. LAB are gram-positive bacteria found ubiquitously
in vegetables, fermented food and LAB are residents and normal flora present in
human gastrointestinal tract. Hence, these are safe for human use and are widely
used by the food industry. These are used as cell factories for GABA production.
LAB generally includes strains of Lactococci and Lactobacilli. Many LAB species
like Lactobacillus brevis PM17, Lactobacillus plantarum C48, Lactobacillus
paracasei PF6, Lactobacillus delbrueckii subsp. bulgaricus PR1 and Lactococcus
lactis PU1 were isolated from cheese and these have been used for GABA pro-
duction (15–63 mg/Kg) (Diana et al. 2014). It has been reported that GABA pro-
duced by LAB possesses higher biological activities compared to natural and
synthetically produced form. Besides these, Lb. buchneri, Lb. helveticus and
Streptococcus salivarius subsp. Thermophilus are also GABA producing and the
highest levels have been determined in Lb. brevis i.e. 345.83 mM.
Zhong et al. have reported the use of fermented mulberry fruits to isolate
Lactobacillus pentosus SS6. This was then used as a starter culture for mulberry
leaves fermentation and incubated at 300C for 6 h. 10% saccharose, 6% peptone,
1.6% K2HPO4 and 1% L-sodium glutamate addition with above incubation also
helped to increase the GABA levels. Mulberry leaves have multiple pharmaceuti-
cals like anti-diabetic, anti-bacterial, anti-inflammatory, etc. Therefore, many efforts
have been needed to increase GABA content in these leaves (Zhong et al. 2019).
Lb. plantarum DSM19463 has been used to ferment grapes which on GABA
enrichment are used as anti-hypertensive and dermatological protecting agents.
Pediococcus acidilactici, P. pentosaceus, E. durans, E. faecalis, E. faecium and
Leuconostocs (L.) are other GABA producing bacteria.
Yogurt, cheese, kimchi sourdough, paocai are few GABA enriched food which
have been fermented with LAB. Except for Lb. brevis CGMCC 1306 (fresh milk
without pasteurization), most other strains may be obtained from sources given
below (Table 24.2).
24.4.2 GABA Production by Other Microorganisms
Though maximum GABA production relies on LAB, there are other microbes that
also produce GABA. Aspergillus niger, Aspergillus nidulans, Neurospora crassa
are few filamentous fungi that have GABA pool. Monascus purpureus is reported
to increase GABA levels in rice (Jannoey et al. 2010) and Rhizopus microspores
increases its level in fermented soybeans.
24.4.3 Factors Affecting GABA Levels
GABA producing ability of each LAB strain is different and it can be modulated by
optimizing the culture conditions and medium composition. Stress conditions (bi-
otic or abiotic) like drought, wounds, salt level, infection, hypoxia, germination,
soaking are few factors that contribute to enhanced GABA levels. Other con-
tributing factors are pH, temperature, carbon source, nitrogen source, glutamate
concentration, PLP. All these parameters are further dependant on GAD properties
which is essential for glutamate decarboxylation (Li and Cao 2010). The optimal
pH needed for GAD activity is specifically governed by the strain of the organism.
e.g. E.coli needs pH of 3.8, N. crassa needs 5.0 and L. brevis needs 4.2.
24.4.3.1 Effect of pH
It is observed that the conversion of glutamate to GABA by GAD is stoichiometric

and it eventually leads to increased pH of the cytosol and neighboring environment.
This increase in pH may further hamper the GABA biosynthesis and hence it needs
to be maintained to acidic pH. H2SO4 is used in fermentation broth to maintain the
acidic pH using Lb. brevis for GABA production. Similarly, maintaining pH 5.0 for
Table 24.2 List of GABA producing strains and their isolation sources
LAB strain Isolation sources Culture GABA levels References
medium
Lactobacillus Korean Sodium 15 mg/Kg Siragusa et al.
brevisPM17 fermented acetate (2007)
vegetable buffer
kimchi, Chinese
traditional
paocai, fresh
milk, alcohol
distillery lees
and black
raspberry juice
Lb. delbrueckii Cheese, Sodium 63, 16, Siragusa et al.
subsp. bulgaricus, Japanese acetate 99.9 mg/Kg (2007)
Lb. plantarum and fermented fish buffer respectively
Lb. paracasei
Lb. paracasei PF6, Pecorino di Sodium 100 mg/Kg Siragusa et al.
Lb. delbrueckii Filiano, acetate (2007)
subsp. bulgaricus Pecorino del buffer
PR1, L. lactis PU1 Reatino,
and Lb. brevis Pecorino
PM17 Umbro, and
Pecorino
Marchigiano
cheeses,
respectively,
Lb. brevis Fresh milk GYP 4599.2 mg/l Huang et al.
CGMCC 1306 without (2007)
pasteurization
Lactobacillus Paocai Nutrient 19.3 mg/L Li et al. (2010a, b)
brevis NCL912 medium,
MSG
Lb paracasei PF6 Indian cheese Sodium 100 mg/L Siragusa et al.
acetate (2007)
buffer,
MSg
L. lactis ssp. lactis cheese starters Wheat 258.71 mg/ Rizzello et al.
01–4, 01–7, 53–1, flour kg (2008), Dhakal
and 53–7 et al. (2012)
Lb helveticus Koumiss Skim 165.11 mg/l Sun et al. (2009)
milk
Lb. paracasei Fermented MRS 31,145.3 mg/ Komatsuzakiet al.
NFRI 7415 crucians Broth l (2005)
fermentation conditions of Lb. paracasei NFRI 7415 produced 302 mM GABA

from 500 mM glutamate and fermentation at pH 4.5 for Streptococcus salivarius
subsp. Thermophilus Y2 also enhanced the production. These examples imply that
optimum pH needed for GABA production is highly species-dependent.
Maintaining the pH towards acidic conditions is also important because increase in
the pH shall increase the activity of GABA degrading or metabolizing enzymes like
GABA transaminase to form succinic semi-aldehyde. This enzyme is highly active
at pH 8.5 in Pseudomonas aeruginosa and transaminases GABA. Succinic
semi-aldehyde dehydrogenase also has been reported to be active at pH 8.4 when
isolated from Saccharomyeces.
24.4.3.2 Effect of Temperature and Time
Incubation temperature during fermentation is also an important factor that governs

GABA production. It affects the thermodynamic equilibrium, biocatalyst activity
and stability. It is observed that the growth of Lb. brevis NCL912 increases up to
temperature of 35 °C and then on the further rise, growth declines. The usual
temperature needed by most strains for high GABA production ranges between 25
and 405 °C. Further, the time for which the incubation is allowed is also a deter-
mining factor. Table 24.3 represents some cases revealing the importance of time,
temperature and pH. The time needed for fermentation is equally important as pH
and temperature. Lb. plantarum DSM19463 and Lb. paracasei NFRI 7415 pro-
duced high levels of GABA (4.83 mM and 60 mM) when fermented for 72 hr and
144 h respectively. In addition, the time at which the additives like GABA sub-
strate, MSG (monosodium glutamate), PLP are added also make a big difference in
the yield. Adding PLP at 0, 24 and 48 h produced 6272, 6570 and 7333 mg/l
GABA respectively after 72 h (Dhakal et al. 2012).
24.4.3.3 Effect of Additives
GABA levels are also influenced by nutrient composition and media additives; PLP
and glutamate being the major additives needed during fermentation while carbon
and nitrogen source being the other additives. 1.25% glucose is found to be the best
carbon source chosen from different pool of carbohydrates like glucose, galactose,
fructose, ribose, xylose, arabinose, melibiose, etc. and 0.5% urea as the nitrogen
source in general. Glutamate is an important additive which generally increases
GABA production. This has been observed in Lb. paracasei NFRI 7415 (500 mM
glutamate for 144 h yielded 161 mM GABA) and Lb. brevis but no effect was
observed in the case of S. salivarius. Besides glutamate, PLP used as a cofactor can
also be given due consideration as it enhances GAD activity. S. salivarius
subsp. Thermophilus Y2, Lb. paracasei NFRI 74,150 and Lb. plantrum
C48demonstrated GABA levels of 7333 mg/l, 200 mM and 504 mg/kg respec-
tively on PLP addition. Sulfate ion also in some cases play a beneficial role by
Table 24.3 Various conditions adapted to increase GABA levels

Strain Optimum Amount of Optimum Amount of
pH GABA temp GABA
produced at produced at
given pH given temp
Lb. brevis 5.0 Higher than NC –
normal
Lb. paracasei NFRI 7415 5.0 302 mM NC –
Streptococcus salivarius 4.5 – NC –
subsp. thermophilus Y2
Lb. plantarum 6.0 59micromole/ NC –
DSM19463 h
Lb. paracasei NFRI 7415 5.0 210 mM NC –
S. salivarius 4.5 7984 mg/l NC –
subsp. thermophilus
Lactobacillus, Lb. paracasei 4.6–5.7 289 mg/kg - NC –
PF6, PF8, PF13, Lb. 391 mg/kg
plantarum PF14, Lb.
sp. strain PF7 and E. durans
PF15
Lb. paracasei 5 210 Mm NC –
Lb. lactis 7.5–8 7.2 g/l NC –
Lb. brevis NCL912 NC – 35C –
Lb. plantarum DSM19463 NC – 30–35 59microM/h
Lb. brevis GAD NC – 30 –-
Lb. brevis CGMCC 1306 NC – 37 –-
Lb. brevis GABA 3.5 – 30 27.6 mg/ml
Lb. buchneri in MRS broth NC – 30 –-
medium
Immobilized whole cells of NC – 40 92%
Lb. brevis
Lc. Lactis NC – 33 310 mg/ml
34 439 mg/ml
Lb. paracasei NFRI 7415 NC – 37 302 mM
Black raspberry juice 4 – 25 25.4 mg/ml
fermented with Lb. brevis 5.5 37 on 15th day
GABA 100 3.5 30 26.5 mg/ml
on 15th day
Highest level
on 12th day
NC indicates the normal conditions used
increasing GAD activity by hydrophobic interactions. Many other substrates like

sourdough, tomo koji, erythritiol, skim milk, iso-malto oligosaccharide, pectin are
also being utilized for increasing GABA production (Dhakal et al. 2012; Yang et al.
2008; Li et al. 2010a, b).
24.4.4 Advances in GABA Production Techniques
24.4.4.1 Immobilized Cell Technology
As discussed previously in GABA biosynthesis, GAD plays the vital role of

decarboxylation of glutamate to synthesize GABA. GAD is also important to
maintain the intracellular pH and for oxidative stress tolerance. This enzyme is
present in prokaryotes and eukaryotes but those which are obtained from microbial
sources are considered prime important for large scale synthesis of GABA. Most of
the GAD obtained from bacterial sources is active only at acidic pH which limits
their application. Hence, newer technologies of molecular engineering have been
adopted to make them activated towards near-neutral pH. To ensue this, the tech-
niques used are immobilized cell technology and gradient controlling fermentation.
Immobilized cell technique leads to the reduction of non-productive growth
phase which in turn increases the cell density of immobilized cells and hence
enhances the overall product yield. This technique also protects the cells from stress
conditions like pH, temperature, salts, and self-destruction (Ying 2007).
Immobilization is considered to be an economical and powerful strategy for GABA
production. E coli GAD has been immobilized by his-tag method which resulted in
223.8 g/l GABA in 100 min and 58% activity could be retained even after ten
cycles of consecutive uses (Lee et al. 2013). Choi et al. immobilized Lb. brevis
GABA 057 to enhance GABA yield as a result of increased glutamate from 2 to
12%. Moreover, these immobilized cells can be used for four such cycles (Choi
et al. 2006).
24.4.4.2 Gradient–Controlling Fermentation
It is to be noted that high cell densities are required for high GABA yields and all
the bacterial strains do not possess the required amount of cell density. For such
cases, gradient-controlling fermentation is used. Such an experiment was designed
by Yang et al (2008) where they used temperature control and double stage pH and
succeeded in achieving high GABA levels from S. salivarius subsp. thermophilus
Y2 (Li and Cao 2010).
24.4.4.3 Molecularly Engineered GAD
It is notable that most GAD’s act in acidic pH. Hence, they are being molecularly
engineered to extend active pH ranges. Knowledge of crystal structure of various
GAD has been helpful to understand the mechanism and active site responsible for
the activity. The method has been successfully applied to GAD isoforms present in
E coli. Its active site was determined which lies in PLP-binding domain and
comprises of Lys276, Glu89, His275, Leu306, and His465 as important amino
acids of binding cleft. Exploiting this information, site-specific mutation was per-
formed and resultant mutants had activity in expanded ph ranges (Pennacchietti
et al. 2009; Thu et al. 2013; Jun et al. 2014). GAD enzymes for L. brevis and B.
megaterium have been molecularly engineered. The crystal structure of these two is
not yet known but taking the clues from structural sequence alignment, the engi-
neering was performed. In the case of L. brevis CGMCC 1306, C-terminal mutant
was designed and it was found to have an activity to near neutral pH values. For B.
megaterium, E294R and H467A, two mutants were designed which succeeded in
displaying enhanced catalytic activity at pH 5.0 (Xu and Wei 2017).
In some studies the genes that encode GAD have also been overexpressed either
homologously or heterologously in various bacterial strains like E coli,
Corynebacterium glutamicum, Lactobacillus sakei, Lactobacillus plantarum,
Bifidobacterium longum. In the case of C glutamicum, the GABA production
increased more than double by co-expressing two genes gadB1 nad gadB2,
obtained from Lb brevis Lb85 (Cui et al. 2020).
24.4.4.4 Coculturing GABA Producing Strains
Co-culturing techniques have been in use for long to enhance the production using
more than one strain for culture. This technique has also been used for enhancing
GABA production. One such study for GABA has been reported by Barrett et al.
where they used human-derived strains. Strains of lactobacilli and bifidobacteria
were cultured in MRS broth supplemented with 005% (w/v) L-cysteine-
hydrochloride (mMRS) under anaerobic conditions at 37 °C. Strains were then
subcultured in mMRS broth for 16–24 h prior to inoculation. The study aimed at
assessing the ability of human intestinally derived strains of Lactobacillus and
Bifidobacterium to produce GABA. From a total of 91 intestinally derived bacterias
assessed, one Lb strain and four Bifidobacterium strains produced GABA.
Lactobacillus brevis DPC6108 was found to be the most efficient onverting up to
100% of MSG to GABA. The addition of Lb brevis DPC6108 to a faeces-based
fermentation significantly improved GABA concentration, evidencing that this
biosynthesis could occur in vivo. The result of this study shows that the production
of GABA by bifidobacteria exhibited considerable interspecies variation.
Lactobacillus brevis and Bifidobacterium dentium proved to most efficient GABA
producing strains among the range of microorganisms tested. The addition of Lact.
brevis DPC6108 to the culturable gut microbiota increased the GABA concentra-
tion in fermented faecal slurry at physiological pH (Barrett et al. 2012).
24.4.4.5 Other Techniques
Various other techniques have also been used to improve the GABA content. Some
of them are Batch fermentation, Sourdough fermentation and improving variety of
medium culture. Many studies have been reported for improving the culture media
and conditions and one such study was done by Alejandra et al. They used wild
GABA-producing LAB isolated from artisanal Mexican cheese and evaluated the
conditions needed for fermentation in milk. The experiments were performed dif-
ferent conditions and additive concentrations [using two inoculum concentrations
(107 and 109 CFU/mL), two incubation temperatures (30 and 37 °C), three gluta-
mate concentrations (1, 3, and 5 g/L), and three pyridoxal 5′-phosphate
(PLP) concentrations (0, 100, and 200 lM)] to determine appropriate conditions to
enhance the GABA. Results revealed that from a total of 94 LAB strains, fermented
milk with two Lb lactis strains (L-571 or L-572) presented the highest GABA
production (Santos-Espinosa et al. 2020). Batch fermentation has been performed on
fungal strains Basidiomycetes and Ascomycetes (Wan et al. 2019) and sourdough
fermentation has been utilized on lactic acid bacteria strains, isolated from Andean
amaranth (A) and Real Hornillos quinoa (Qr) sourdoughs (Villegas et al. 2016).
24.5 Conclusion
The ever increasing search for safe and healthy food products, rose the interest of
researchers and customers in GABA enriched products. Literature reveals that
GABA, an important inhibitory neurotransmitter plays an important functional role
in various diseases and pathologies. The pharmaceutical industries in association
with the food industry are therefore focused on the search of novel methods for
enhancing the quantity of GABA in food products and also in microorganisms and
to inculcate it as a bioactive molecule to control many diseases. There are many
roadblocks and challenges that need to be considered because most of the studies
have been done on animals to date and their efficacy in humans is yet to be
established.
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Chapter 25
Medicinal Attribution of Ginsenoside:
A Huge Source of Plant Bioactive
Compound
Dilipkumar Pal, Souvik Mukherjee, Satish Balasaheb Nimse,

and K. K. Chandra
Abstract Ginsenosides (GND) are chemically triterpenoid saponin in nature.

According to the presence of aglycones, dammarane and oleanane are the two types
of GND. These are mostly observed in species of Panax. The researchers have
discovered over one hundred fifty substances from stocks, grasses, shoots, florets,
drupes from the ginseng plant. GND and their derivatives are the main chemical
constituents of the ginseng plant. Recently, GND are gaining increasing interests
among natural product scientists. GND have many significant pharmacological
activities, including anti-oxidation, mmunomodulation, and preventive actions in
cancer, inflammation, stress, and hypertension, etc. The metabolism of GND
involves two significant metabolic reactions, including acid hydrolysis and
hydrolytic reactions oriented from bacterial origins. After metabolism, GND are
transformed into a more active GND derivatives. The utilization and changes of
unblemished GND, which appears to assume a significant job for their potential
wellbeing impacts, are discussed in this chapter.
Keywords Triterpene Ginseng Saponin Gut flora Biosynthesis

Metabolism
List of Abbreviations
Bsyt Biosynthesis
CNr Cancer
CeL Cell
D. Pal (&) S. Mukherjee

S. B. Nimse
Institute of Applied Chemistry and Department of Chemistry, Hallym University, Chuncheon
200-702, Korea
K. K. Chandra
Department of Forestry, Wildlife and Environmental Science, Guru Ghasidas Viswavidyalaya
https://doi.org/10.1007/978-3-030-54027-2_25
846 D. Pal et al.
CyTk Cytokines
CAsp Caspase
ChLn Cholinergic
ytx
C Cytotoxicity
DEaS Disease
G nS Ginseng
GND Ginsenosides
I nF l Inflammatory
ImM Immuno
LyMT Lymphocytes
LS Lanosterol
MLgT Malignant
MLg Malignancy
MaTs Metastasis
NePL Neoplasm
NEur Neuro
PtW Pathway
PLt Plant
PrLf Proliferative
PHSP Phosphate
PNX Panax
SpN Saponin
SQL Squalene
TrId Triterpenoid
TUm Triterpenoid
25.1 Introduction
Ginseng (GnS) is a significant restorative plant (PLt) having a place in family

Araliaceae. Ginsenosides (GND) and Gintonin are the primary concoction con-
stituents of GnS. As indicated by the nearness of synthetic constituents and different
land inception, there are four kinds of GnS seen in different nations of the world, for
example, South China, American, Vietnam GnS. It is the root of Panax (PNX) family
(Bilia and Bergonzi 2019). The base of GnS has been utilized on antiquated occa-
sions where it gives protection from stress, infection, and fatigue. There are a variety
of GnS based products in the market that are used to advance personal satisfaction.
The GnS products contain a variety of active constituents, including GND (Fig. 25.1),
polyacetylenes, polyphenolic mixes, and acidic polysaccharides. The GND deriva-
tives are steroidal nature and are sometimes called as triterpene saponin (SpN),
which is a particular form of oleanane families . GND is made from the cytoplasm
and plastid region of the GnS PLt. The oral route is the most favorable way for GnS
administration. It is metabolized by gut flora. Configuration of those compound
25 Medicinal Attribution of Ginsenoside: A Huge Source … 847

ginsenoside
shows that it contains four rings with steroidal moieties (Nimse and Pal 2015). In
proto PNX adiols, sugar gatherings are found at the three-position within the carbon
frame, whereas carbohydrate congregations append at the six positions of the same
frame. GND from the oleanane family are pentacyclic containing a five-membered
ring carbon skeleton (Xue et al. 2019).
25.2 Biosynthesis of GND
Biosynthesis (Bsyt) of GND can be divided into four steps, which are shown in
Fig. 25.2. Firstly 2, 3-oxidosqualene (SQL) is produced from acetyl-CoA. Then it is
cyclized and modified with the help of the glycosylation process. There two car-
bocations (CT) have existed, namely 3-isopentyl salt and dimethylallyl radical salt,
which is measured as the primary precursor for synthesis (Tang et al. 2019).
Isopentenyl Di-phosphate (PHSP) pathway (IPP) is originated from the radical acetyl
CoA of Mevalonic acid pathway (PtW). Di PHSP Mevalonic acid PtW is generated
from phosphoglyceraldehyde. Here condensation additionally occurs. As a result,
geranyl salt is created (Zhang et al. 2019). Geranyl Pyro PHSP e is condensed with
IPP, fashioned farnesyl salt. However, the Farnesyl Pyro PHSP skeleton comprising
848 D. Pal et al.
Fig. 25.2 Biosynthesis of ginsenoside
of thirty carbon structures is referred to as SQL (Zhang et al. 2019). Cyclization of 2,

3-oxido compound is the second step (Rahimi et al. 2019). Oxido SQL is transferred
into cyclic derivatives by the assistance of protonation and ring gap feature. During
this method, a stereospecific reaction has additionally happened. Here c–c-c
[triterpenoid (TrId) SpN] or c-b-c [steroid] conformation is discovered and produces
a tetracyclic prostosteryl as result lanosterol (LS) is produced. (Senbagalakshmi
et al. 2019). More LS carbocation is created as a Cyclo fused ring, as found in

cycloartenol. Finally, LS has converted again into LS. When de-methylation occurs,
it produces steroid alcohol. Ultimately, p450, amyrin, dammarenediol CT are
changed into GND via hydroxylation and glycosylation method (Hong et al. 2019).
25.3 Biotransformation of GND
Therapeutic herb items or dietary enhancements are regularly taken orally,

including those based on GnS (Kim et al. 2019a). At the point when most thera-
peutic herbs/dietary enhancements are taken orally, then their constituents will be
contacted with stomachic liquids (stomach acids), and the small floral proteins
within the organ are well maintained in the alimentary tract (Liu et al. 2019). There
is a bit of the mixture at the starting point within the medicinal herb items/dietary
elements, which are used to different alimentary tract, which may be GD secondary
metabolites. The metabolic destiny of the segments of dietary enhancements con-
sequently may be beneficial to a superior comprehension of their organic movement
and also the medical specialty activities of individual elements (Hu et al. 2019).
Moreover, it has been planned that the small floral metabolic movement is influ-
enced by intake programmed modification and physiological components, rather
than by varieties within the microscopic organisms. It will be important in asso-
ciation to the medical specialty activities and impacts of bioactive constituents (Dou
et al. 2019). The metabolism and retention of GND are contemplated seriously. Thus
clarification is needed for the medical specialty activities of GND and their con-
stituents to the clinical adequacy of GND. For many examinations that are embraced
to clarify the destiny of GND through the alimentary tract utilizing acids, chemicals,
and human enteral microscopic organisms, clearly, a massive piece of the perfect
GND is used/changed to GND with all the additional upgrading natural impacts
contrasted distinguishable in plasma. For GND of the PPD-type, for instance, Rb1,
Rb2, and Rc, it has been exhibited in each in vitro and in vivo examinations (He
et al. 2019). Then again, it has been incontestable that the GND Rb1, Rb2, and Rc
may be decayed to GND Rg3 (GRg3) by mellow corrosive treatment, for instance,
abdomen acids, in spite of the very fact that a few of examinations have shown that
PPD GND are scarcely disintegrated within the abdomen. In any case, if GND Rg3 is
delivered within the abdomen, it is transformed to GND Rh2 or 20(S)-PPD by
human enteral microorganisms as exhibited in some in vitro studies (Fu 2019).
When the pharmacokinetic profile of GND is concerned in rodents, it is found that
once an intra-gastric organization of 10 mg/kg GND Rg3 make a plasma grouping in
around forty hours and likewise a 100 mg/ml of GND Rh2 or 20(S)-PPD form the
same separately in four hours. GND Rh2 and 20(S)-PPD has been significantly
changed physiological response than the unflawed GND Rb1, Rb2 and Rc. GND Rh2
and 20(S)-PPD have, as an example, of additional cytotoxicity(Cytx) against neo-
plasm(NePL) cell(CeL) lines and also the unflawed GND (Kim et al. 2019b). For GND
of the PPT-type, for instance, Rg1 and Re, a couple of investigators have exhibited
850 D. Pal et al.
that these mixtures are processed to GND Rh1 and GND F1 and then at long last to
20(S)-PPT following numerous conditions suggesting thereby a stepwise cleavage
of the sugar moieties. GND Rg1 on the oral organization may be transformed into
GND Rh1 and hydrous subsidiaries of Rh1 in the abdomen. It is the fact that all GND
Rg1 is not hydrolyzed within the abdomen, and unflawed GND Rg1 might hit the
interior organ, wherever enteral microbes use it to GND F1 and 20(S)-PPT GND Re.
Then again, it can be hydrolyzed by stomachic liquids to form GND Rg2, which is
then modified within the system to GND Rh1 by the disposal of rhamnose through
enteral microorganisms. Unflawed GND Re might likewise hit the interior organ
wherever it tends to be used by enteral microbes to GND F1 and 20(S)-PPT through
GD Rg1. Apart from enteral microscopic organisms, a couple of nourishment
microorganisms have incontestable conditions to deliver specific sorts of GND,
together with those created by enteral human microbes. This demonstrates that it
would be doable to create up a selected bioconversion procedure to induce
expressly structured useful things by the acceptable mixture of GND substrate and
specific microorganism chemicals from sustenance microorganisms (Darsandhari
et al. 2019). Pharmacokinetic examines have exhibited that GND, once taken orally,
may be distinguished in plasma. Pee tests as unflawed GND or glycosylated cor-
ruption things, the first debasement things recognized in urine, and plasma tests on
oral admission of PPD and PPT GND are considered as the monoglycosylated GND
compound K, GND Rh1 and GND F1. Deglycosylase GND are usually additional
promptly eaten into the cardiovascular system considered as more dynamic mix-
tures than the relating unrotten GND. The bioavailability of perfect GND is poorly
contrasted, as also found in the case of de-glycosylated GND (Yang et al. 2019).
Thusly, the direct physiological impact of unflawed GND in vivo will consequently
be talked regarding and want examinations. Compound K and GND F1 are usually
distinguished in plasma from seven h once the admission of GnS and in pee from
twelve h after the intake, whereas GND Rh1 is recognizable from one h in plasma
and three h in pee once oral. The pharmacodynamics of compound K for which
once endovenous organization is established to mice have become incontestable
than compound K for the foremost half discharged in biliary tract. In any case, some
compound K are esterified with unsaturated fats at C-3 of the aglycone moiety or
C10 of the aldohexose moiety within the liver. Thusly, the esterified sorts of
compound K are collected longer within the liver than compound K itself. Further,
it has been incontestable that esterified compound K inhibits growth more than
compound K in vivo (Zuo et al. 2019). These outcomes propose that liver chemicals
may be involved within the digestion of GND and in the development of dynamic
standards of GND in the body, which is shown in Fig. 25.3 (Lee et al. 2019).
Fig. 25.3 Biotransformation process of ginsenoside

852 D. Pal et al.
25.4 Medicinal and Nutraceutical Applications
Pharmacological impacts of GnS are exhibited within the focal sensory system
(CNS). Besides, broad diagnosis and epidemiologic examinations have shown that
GnS and GnS things have potential cancer (CNr) preventive impacts even as con-
sequences for hyperglycemia. The dynamic segments in GnS comprise primarily of
polysaccharides, polyacetylenes, and GND, of that the GND are viewed because of
the real dynamic standards of GnS. The GND have exhibited a capability to focus on
varied types of tissues, making a range of medicine reactions. Since GND might
produce impacts that do not seem to be constant as one another and single GND and
in addition, their used things might begin varied activities in a very similar tissue,
the overall medicine of GnS things is quite unpredictable (Nguyen and Nguyen
2019). Within the concomitant, the fascinating medicinal impact of GND are
obtained, and hence their potential successfulness with advancing impacts are
talked concerning with forwarding (Karra et al. 2019).
25.4.1 Anti-carcinogenic Effects
GND are applied to evaluate their anti-carcinogenic impacts in vitro and in vivo
using varied approaches. Several numbers of them show a direct Cytx and devel-
opment restrictive impacts against tumor(TUm) CeL, whereas others are looked as if
they would repress metastasis (MaTs) and TUm development (Wang et al. 2018).
Results from epidemiologic and companion contemplate with white and red GnS
have clearly shown that they need nonorganic specific preventive impact against
malignant (MLgT) growth. This impact is perhaps about to be due owing to their
substance of chemicals, specifically GND (Santangelo et al. 2018).
25.4.2 Cytotoxic and Anti TUm Activity
The Cytx and antiproliferative (PrLf) impacts of GD toward human and creature
MLgT growth CeL lines are shown in numerous examinations. In Associate in the
nursing examination, Wang et al. (2007) undertake to assess the toxicity of ten GND
(20(S)-PPD, 5, 12, 14, 15, 44, 84, 88, 91 and 124), detached from the product of P.
GnS, toward a couple of human MLgT growth CeL lines, together with bosom
unwellness CeL lines (e.g., MCF-7 CeL), respiratory organ MLgT growth CeL lines
(e.g., H838 CeL) and prostate unwellness CeL lines (e.g., LNCaP and PC3 CeL).
Amongst the GND tried, GND 20(S)-PPD, Rh2 (15), and GND 20(R)-25-OH PPD
indicate significant movement of all told CeL lines and are significantly the most
effective inhibitors of malignancy (MLg) CeL development and enlargement. For 20
(R)-25-OH PPD, the IC50 esteems for many CeL lines are within the scope of 10–60
metric linear unit, which is at any rate two-fold less than for any of the various GND
tried. Each 20(S)-PPD and 20(R)-25-OH PPD distends CeL death (apoptosis) and
CeL cycle movement during a portion of the subordinate method, whereas these
impacts are less articulated for GND Rh2. It is eminent that 20(R)-25-OH PPD has
an additional grounded impact than GND 20(S)-Rg3 (14) on CeL development
restraint. It has IC50 qualities, which is 5-to 15-overlap less than for GND Rg3, a
compound antecedently being showcased for treatment. Moreover, GND Rb1 (5),
Rd (12), and Rg3 have much zero impact on CeL development and enlargement.
The impact on CeL multiplication of GND Rh2 is furthermore discovered to be of the
same size because the aglycones 20(S)-PPD and 20(S)-PPT, through the restrictive
impact of GND 20(S)-Rh1 is ten times less. Moreover, the distance of sugars in PPD
and PPT aglycone structures seems to decrease the strength to actuate caspase
(CAsp)-mediated CeL death as PPD and PPT are found to instigate CAsp-mediated
CeL death to the next degree than GD Rh2, though Rh1 failed to incite CAsp-
mediated CeL death. This shows that matters of sugar moieties at C-3 or C-6 may
boot assume employment within the anti-TUm impact of GND. It projects the
anti-PrLf impacts of GND. Here, completely different bioactive compounds destitute
GD 65 on the capability of GD to collaborate with CeL layer capacities based on
their hydrophobic nature. This is often likewise as per the structure–action rela-
tionship on the anti-PrLf impacts of GND and, therefore, the upgraded action wat-
ched for unsaturated fat conjugate GND (Bilia and Bergonzi 2019). GND of the 20
(S)-PPD family is the best-contemplated gathering of GND with relevancy anti-TUm
impact of that GND Rh2, and it could be a standout amongst alternative
thought-about GND (Rani et al. 2014). GND Rh2 has been looked as if it would stifle
enlargement in numerous human unwellness CeL, together with bosom, colorectal,
prostate, hepatic, intestinal, melanoma, and creature CeL lines (Li et al. 2019). The
anti-PrLf impact of Rh2 provides off a sway of being connected to its capability to
actuate CAsp-mediated CeL death further as by capturing CeL cycle movement. As
an example, Rh2 has been accounted for to actuate CAsp-3 enzyme, a stimulating
organic compound engaged with CAsp-mediated CeL death and to capture CeL cycle
movement at the G1 section of MCF-7 human bosom MLg CeL, SK-HEP-1 MLgT
hepatoma CeL (Rani et al. 2016), and B16-BL6 MLgT melanoma CeL. Rh2 also can
restrain TUm development in vivo of clean mice bearing human female internal
reproductive organ unwellness CeL (Lee et al. 2018). The anti-PrLf impacts toward
MLg CeL of alternative PPD GND, as an example, Rg3, Rg5 (72), Rs3 (18) and Rs 4
to boot seem to be as a result of their capability to instigate CAsp-mediated CeL
death and to irritate standard CeL cycle occasions. It is a very fact that the anti-PrLf
impacts of GND (Ryoo et al. 2019), together with PPD and PPT, toward urinary
organ proximal tube-shaped structure CeL can be as a result of a change of c-fos and
c-jun quality articulation (Medina-Franco 2019).
854 D. Pal et al.
25.4.3 Inhibition of TUm CeL Invasion and MaTs
The counteractive action of MLg MaTs is critical, and therefore there should be an
improvement in the guess of CNr patients. The first trademark venture of MLg MaTs
is the NePL CeL intrusion of encompassing tissues and vasculature. Kitagawa
Associate in nursing partners designed up an intrusion model for evaluating NePL
CeL attack capability in vitro (Pal and Saha 2019). During this model, NePL CeL are
seeded on a vital refined monolayer of host CeL, for instance, mesothelium or
epithelial tissue CeL. The NePL CeL infiltrate the monolayer and develop and
structure NePL CeL states beneath the monolayer. The limit of the entrance of NePL
CeL in vitro relates well thereupon of in vivo implantation into guinea pigs.
Afterward, the in vitro model permits concentrating on the impacts of drugs on NePL
CeL attack. By utilizing this in vitro model, over ten GD are tried for the hindrance
of NePL CeL attack and MaTs. GD 20(R)-Rg3 (42) has been ascertained to be
associate in nursing intense matter of attack of a couple of NePL CeL as well as
haptonema (MM1), skin CNr (B16FE7), human tiny respiratory organ MLgT neo-
plastic disease (DEaS) (OC10), and human exocrine gland glandular CNr (PSN-1)
CeL Whereas GD Rb2 (7), 20(R)-Rg2 (111), and 20(S)-Rg3 (14) have simply
indicated lowest repressing action on NePL CeL intrusion. Neither GD Rc (10), Re
(84), Rh1 (91), Rh2 (15), nor 20(R)-Rh1 (112) were found to possess any impact
within the model. As shown by Azuma and Mochizuki (1994) and Mochizuki et al.
(1995), the enantiomers 20(S)- and 20(R)-Rg3 appear to possess an impact on NePL
MaTs development as exhibited in vitro on 2 extremely MLgT TUm CeL, B16-BL6
skin CNr and colon 26-M3. MLgT neoplastic DEaS, and in vivo by NePL immu-
nization of B16-BL6 skin CNr in mice. In any case, the impacts of 20(S)- and 20(R)-
Rg3 against pneumonic MaTs in vitro and in vivo appear, by all accounts, to look as
one thing else, with 20(S)-Rg3 demonstrating the weakest impact in vivo and
therefore the most grounded impact in vitro contrasted and 20(R)-Rg3.
25.4.4 Inhibition of TUm Angiogenesis
Angiogenesis is a physiological process involving the growth of new blood vessels

from preexisting vessels and is considered a normal process in growth and devel-
opment, as well as in wound healing. However, this is also a fundamental step in
the transition of TUm from a dormant state to a state where the TUm CeL proliferate
(MLgT state). Inhibition of angiogenesis, therefore, prevents TUm growth, prolifer-
ation, and secondary MaTs and is essential for the prevention and treatment of CNr (
Folkman et al. 1995). Only a few studies on the angio-suppressive effects of GD
have been performed, and they mainly concern the GND Rb2 (7) and 20(R)-Rg3
(42). Sato et al. (1994) study the effect of GND Rb2 on angiogenesis and MaTs
produced by B16-BL6 melanoma CeL in syngeneic mice. Intravenous administra-
tion of GND Rb2 on day 1, 3, or 7 after TUm inoculation results in a remarkable
reduction in the number of vessels oriented toward the TUm mass, but do not cause
significant inhibition of TUm growth. The angio-suppressive effect is
dose-dependent in the ranges of 10–50 mg/mouse.
In contrast, intratumorally or oral administration of GND Rb2 causes a marked
inhibition of both neovascularization and TUm growth. GND Rb2 does not affect the
growth of rat lung endothelial CeL. However, it inhibits in a dose-dependent fashion
the invasion of rat lung endothelial CeL into the reconstituted basement membrane
(Matrigel), which is considered to be an essential event in TUm neovascularization.
Multiple administrations of GD Rb2 after the i.v. inoculation of B16-BL6 mela-
noma CeL results in significant inhibition of lung MaTs as compared with that of the
untreated control. The results suggest that the inhibition of TUm-associated angio-
genesis by GND Rb2 may partly contribute to the inhibition of lung TUm MaTs. Yue
et al. (2006) examine the ability of GD 20(R)-Rg3 to interfere with the various steps
of TUm angiogenesis. GD 20(R)-Rg3 is, for example, found to inhibit the prolif-
eration of human umbilical vein endothelial CeL (HUVEC) with an IC50 value of
10 nM. GD 20(R)-Rg3 also dose-dependently suppresses the capillary tube for-
mation of HUVEC on the Matrigel from 1 to 1000 nM in the presence or absence of
20 ng/ml vascular endothelial growth factor (VEGF). The TUm angio-suppressive
effects and the inhibiting effect of MaTs of GD Rb2 and 20(R)-Rg3 are probably
related to their inhibitive effect on the release of VEGF from TUm CeL.
25.4.5 Immunomodulatory Effects
The Immuno (ImM) modulatory activities of GND square measure are closely
associated with their anti-carcinogenic, anti-inflammatory (InFl) and anti-allergic
activities. The immune responses square measure is controlled by T helper (Th) CeL
and may broadly categorize into cellular mediated responses (CeL-mediated
immunity) mediated by Th1 CeL, macrophages, and protein (antibody-mediated
immunity) responses directed by Th2 CeL. The CeL square measure is concerned
with activation and directional different immune CeL like Cytx.
T CeL and natural killer (NK) CeL, and thence square measure is significantly
necessary within the system. The event and differentiation of Th CeL square sure
strictly regulated by antigen-presenting nerve fiber CeL (DCs). DCs that generate
Th1 responses could also be achieved to forestall or treat pathological conditions
that square measure caused by infections and MLgT disorders via secretion of sort
one cytokines(CyTk) like interferon-g (IFN-g) and interleukin-2 (IL-2) to facilitate
T-CeL-mediated toxicity. In distinction, DCs that generate Th2 responses could also
be wont to forestall or treat conditions within which Th1 responses square measure
disturbed, for instance, contact allergic reaction and response disorders, by secretion
of the sort a pair of CyTk, like IL-4 and IL-10, to assist B CeL to secrete protecting
antibodies (Takei et al. 2004). Therefore, any compound capable of modulating or
operating particularly phagocyte activation by making assembly of small and
enormous lymphocytes (LyMT) (e.g., NK, T, and B CeL) becomes very important
856 D. Pal et al.
within the interference and treatment of TUm, infectious agents, and chronic InFl
DEaS (e.g. Autoimmune disorder, asthma, and atherosclerosis). It is renowned that
numerous GnS species have different ImM modulatory activities in which the most
active elements is square measure GND. Yu et al. (2005) investigate numerous
PPT-type GND isolated from P. GnS leaves (20(S)-PPT, PNX atriol (20(S)-PT), F1
(80), Re (84), Rg1 (88), Rh1 (92), and a pair of 0(R)-Rh1 (112)) for his ability to
modulate sort one differentially and sort 2 CyTk productions from murine spleno-
cytes. GD F1 and Rg1 are found to influence a pair of CyTk production through
regulation of the expression. For instance, in IL-4, GD Rh1 and 20(R)-Rh1 influ-
ence one CyTk production by regulation of the assembly of IL-12 and thereby
influence the expression of IFN-g and T-bet. The latter being a particular Th1
transcriptional subject, is thought to initiate the development of Th1 and inhibit
differentiation of Th2. The results clearly show that PPT-type GND have different
ImM modulatory effects together with each immune-stimulatory and ImM logical
disorder effects. This can be additionally in accordance with a study of Cho et al.
(2002). World Health Organization finds that the GND Rb1 (5), Rb2 (7), Re (84),
and Rg1 modulate WBC proliferation elicited by T LyMT mitogens [e.g., con-
canavalin A] and therefore the lymph CeL agent, lipopolysaccharide GND sixty-nine
(LPS), yet acts as protein IL-2, a potent trigger of WBC proliferation. GND Rb1 and
Re considerably increase Con A-induced WBC proliferation, whereas Rg1 does not
affect the proliferation. On the opposite hand, Rb2 powerfully blocks the
mitogen-induced WBC proliferation with IC50 values around twenty-one. This
clearly shows that GND Rb2 may be a potent ImM logical disorder agent. GND Rb2
and Rb1 have no restrictive effects on the proliferation of IL-2- aroused CD8þ T
CeL, whereas Re and Rg1 show robust restrictive effects with IC50 values of 57 and
64.7 mM, severally. These results clearly indicate that GND could modulate WBC
proliferation. GND of P. noto GnS and P. GnS, like Rb1, Rb2, and Rg1 have
additionally shown to powerfully suppress the assembly of TNF-a in macrophages
treated with LPS.
25.4.6 Anti-inflammatory Activity
Furthermore, these GND also seem to suppress the production of other InFl CyTk,
such as IL-6 and IL-1b, and hence demonstrate that widely distributed GND pos-
sesses anti-InFl and ImM suppressive properties in vitro. The activation of macro-
phages and hence the production of various types of LyMT has been shown to be
essential for the prevention and treatment of TUm and infectious DEaS. GND Rg1 has
been reported to have mainly ImM modulatory effects that increase both humoral
and CeL-mediated immunities by enhancing the activity of Th CeL and NK CeL
responsive to given antigens. Furthermore, it has been reported that maturation of
DCs is promoted by metabolized GND such as compound K. The anti-InFl and
anti-allergic properties of GND are more or less directly linked to their
immune-stimulatory and anti-carcinogenic effects as well as in DEaS where InFl
conditions play a significant role such as in atherosclerosis and neuro (NEur)

degenerative DEaS. Allergic DEaS of type 1, such as asthma, allergic rhinitis, atopic
dermatitis, and food allergy, afflicts up to 20% of the human population in many
countries. Allergen reactivity in these allergic DEaS is based on ImM globin E (IgE)-
mediated pharmacological processes in a variety of CeL populations, in particular
basophils and mast CeL. Degranulation of basophils and mast CeL with antigen
cross-linked IgE releases histamine, prostaglandins, leukotrienes, and CyTk affecting
macrophages, LyMT, eosinophils, and neutrophils, causing tissue injuries and InFl
DEaS. CyTk and/or bacterial LPS induce nitric oxide synthase and cyclooxygenase-2
expression in, for example, macrophages and hence the production of nitric oxide
and prostaglandins, respectively. Sustained production of NO and PGs has been
implicated in the pathogenesis of InFl DEaS and CNr (Zhang et al. 2019). Several
GND have shown to reduce the expression of iNOS and COX-2 and to inhibit the
production of NO and PGs in macrophages as well as the inhibition of nuclear
factor (NF)-kB transcription factor, which regulates iNOS and COX-2 gene
expression. GND Rh1 (92) and Rh2 (15) and GND 20(S)-PPT, a metabolite of, for
example, Rh1 or Rg1 and compound K, a metabolite of, for example, GND Rb1,
have been reported to inhibit the production of NO and PGE2 and to inhibit the
activation of NF-kB, in LPS-stimulated murine macrophages (RAW 264.7 CeL)
(Kwon et al. 2018). The inhibition of NF-kB and COX-2 expression has also been
demonstrated for compound K in mouse ear edema induced by the prototype TUm
promoter 12-O-tetradecanylphorbol-13-acetate (Moon et al. 2018). The results
suggest that these GND can inhibit NO and PGs production by regulation of the
signal transduction related to the activation of NF-kB. The anti-InFl effects of GND
have also been demonstrated in microglial CeL, which are resident macrophages of
the CNS. It is found that the PPDs GND Rb2, Rd, and the PPTs GND Rg1, Re are
able to inhibit LPS-induced NO formation and TNF-a production due to the inhi-
bition of NF-kB in N9 microglial CeL. Thus, these GND may be used in the pre-
vention or treatment of InFl DEaS, such as allergic inflammation and NEur logical
DEaS (e.g., Alzheimer’s and Parkinson’s DEaS) as well as CNr. The anti-allergic
effect of GND has been studied in vitro and in vivo on rodent peritoneal mast CeL
and on IgE-induced passive cutaneous anaphylaxis (PCA), the latter being a model
for study of type 1 sensitivity reactions. GD Rb1, Rc, Rd, F2, and Rh1 have been
shown to inhibit histamine and/or leukotriene release from peritoneal mast CeL,
whereas GD Rh1, Rh2, and compound K have been shown to be potent inhibitors
of the PCA reaction in rodents (Chen et al. 2019). The inhibitory activity of Rh1,
Rh2, and compound K on the PCA reaction is found to be more potent than the
commercial anti-allergic drug disodium cromoglycate. These GND furthermore
show a membrane-stabilizing effect, and it has been suggested that this
membrane-stabilizing effect, which may prevent membrane perturbations, is the
leading cause of their anti-allergic activity.
858 D. Pal et al.
25.4.7 Antistress Activity
Antistress impact of GND complete SpN, GND Rg3, and Rb1 toward immobilization
stress has likewise been exhibited by researches on the cerebrum level of endoge-
nous polyamines, which are fundamental for CeL development, multiplication,
recovery, separation of the mind and outstanding pressure boosts markers. In this
examination, it is discovered that GND Rg3 and Rb1 hinder the action of the catalyst
ornithine decarboxylase, associated with the digestion and catabolism of polyamines
and constricting the degrees of the polyamine putrescine. Along these lines, GND
Rg3 and Rb1 may assume a NEur protective job in the immobilization-focused on the
mind (Tam et al. 2018). Impacts on the CNS by various GND species have been
appeared to have both stimulatory and inhibitory consequences and may adjust NEur
transmission. GND, and specifically GND Rb1, Rg1, and Re appear to assume an
outstanding job in these impacts (Zheng et al. 2018).
25.4.8 Memory, Learning, and NEur Protection
Focal cholinergic (ChLn) frameworks have been embroiled in intercession learning

and memory forms. Since scopolamine is a ChLn receptor opponent, the exhibition
debilitated by scopolamine may bring about the brokenness of focal ChLn compo-
nents and subsequently may bring about memory shortages. Results from creature
studies have demonstrated that GND Rb1, Rg1, and Re can forestall
(scopolamine-incited memory shortages). These ameliorative impacts of Rg1 and
Re have been demonstrated to be firmly identified with an expansion of choline
acetyltransferase movement in the average septum of youthful and mature rodents
(Majid 2019). GDRb1 and Rg1 have likewise demonstrated to be prepared to do in
part, turning around scopolamine-instigated amnesia by improving ChLn movement
and using halfway NEur trophic and NEur protective impacts.
Furthermore, it has been shown that GND Rb1 expands the take-up of ChLn
system in focal ChLn nerve ending and encourages the arrival of acetylcholine from
hippocampal cuts. These outcomes obviously recommend that GND may encourage
learning and improving the fundamental synaptic transmission just as nerve
development (Metwaly et al. 2019). GND additionally appears to have a NEur
protective impact where nerve development likewise assumes a significant job. GND
on in vitro investigations appeared to build survival of refined NEur nal CeL and
upgrade the outgrowth of neuritis. For instance, GND Rb1 has appeared to build the
neurite outgrowth of refined cerebral cortex neurons and animate neurite outgrowth
of PC12 CeL without nerve development factor. The capacity of GND to recover
NEur nal systems has additionally been exhibited in SK-N-SH CeL for PPD-type
SpN. For example, GND & noto GND R4 & Fa, while PPT, ocotillol, and oleanolic
corrosive sort SpN had no impact on neurite outgrowth (Wu et al. 2019). This
plainly demonstrates that some GND can broaden axons and dendrites in neurons
that may make up and fix harmed organizers, as found in the dementia brain
(Szczuka et al. 2019).
25.4.9 Anti-diabetic Activity
It has been demonstrated that the anti-diabetic GD 77 impacts of GDRb1 and 20(S)-
PPT are likely identified with their capacity to enact peroxisome PPAR gamma
(PPARG). PPARG is an individual component obtained from the atomic receptor of
ligand-initiated translation factors that direct the declaration of critical qualities
engaged with lipid and glucose digestion and adipocyte separation (Wang et al.
2019). PPARG is fundamentally communicated in fat tissue, and the enactment of
PPARG improves the capacity of adipocytes to store lipids, in this way lessening
lipotoxicity in muscle and liver. The qualities communicated by the initiation of
PPARG depend to a great extent on the sort of actuating ligand present as they
select an alternate arrangement of cofactors (Park et al. 2019). Consequently, the
transcriptional reaction of the PPARG results in cofactors that lead to expanded
lipid stockpiling and diminished vitality use. For example, transcriptional factor-2
or enlistment of cofactors lead to expanding insulin-animated glucose take-up and
positive guideline of glucose digestion and vitality consumption (e.g., the steroid
receptor coactivator-1).The enactment of PPARG causes body-wide lipid reparti-
tioning by expanding the triglyceride content in fat tissue and bringing down free
unsaturated fats triglycerides available for use. Liver and muscle, along these lines,
improve insulin sensitives (Barman et al. 2019). A few examinations have shown
that the hypoglycemic impact of GnS items depends both on the GND content and
the 78 large P. Christensen profile, plainly shows that the anti-diabetic impact of
GnS relies upon the convergence of single GD and hence, it is inferred that not all
GD has anti-diabetic impacts (Truong et al. 2019).
25.5 Conclusions
GND is particularly TrId interestingly present in PNX species. In light of the

restorative significance of GD, examinations on their compound structure, thera-
peutic exercises, Bsyt proteins & generation improvement have gained much con-
sideration. The Bsyt PtW of GnS SpN imparts normal and expanded chemicals to
those of TrId in natural botanical systems. Until this point in time, there are more
than 150 known diverse GND with different numbers, linkage positions, and sorts of
the sugar moiety, and a large portion of them are oleanonic in nature. Current proof
recommends that quality development coming out from genome and additionally
quality duplication pursued by quality obsession through the affirmative determi-
nation of sub-and neo-functionalized homologs, may give the basic hereditary base
860 D. Pal et al.
to GND Bsyt and PLt adjustment and species radiation. The mechanism(s) driving the
development of GND, just as the occasions hidden the expansion of GND in PNX
species, still cannot seem to be explained. Another zone needing further research
identifies the fundamental blend of the different GnS SpN inside explicit tissues. GnS
SpN are assumed to go as resistance atoms in PLt pressure and pathogen associa-
tions. The pharmacological viability of GND depends on their auxiliary premise,
particularly their hydroxyl gatherings and sugar moieties, associating with layer
lipids. Later on, more understanding is found in the basic restorative impacts of
GND. For example, crosstalk with hormone flagging PtW will enable auxiliary
alterations to accomplish improved beneficial exercises and capacities. The tedious
and work serious development of GnS in the field has driven bioengineering
approaches. For example, the culture of tissues and CeL compound elicitation
during creation, transgenic PLt, and designed yeast frameworks are used to improve
GND generation. Especially, transgenic PLt over-communicating qualities associated
with GND amalgamation, for example, HMGR1, SS, CYPs, and DDS have huge
expanded GND yields. As of late, built yeast CeL communicating GND delivering
catalysts, bring about the creation of PPD, PPT and oleanolic corrosive just as
compound K. These achievements give a modest and proficient mechanical stage
for the production of GND for clinical applications. Distinguishing proof of extra
practical catalysts for biosynthesizing GND will prompt more methodologies for
proficient and huge scale generation of GND variations.
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Advanced Structured Materials

More information about this series at http://www.springer.com/series/8611

Bioactive Natural Products

ISSN 1869-8433 ISSN 1869-8441 (electronic)

Prof. (Dr.) Upal Kanti Mazumder

camptothecin as anticancer drug have been presented in Chap. 9. Chapter 10

Bilaspur, India Dr. Dilipkumar Pal

A thorough review of the natural bioactive compounds collected from plants,

1 Elicitor Signal Transduction Leading to the Production of Plant

2.5 Delivery Technology for Bioactive Natural Products . . . . . . . . 76

4.6 Role of Phytochemicals in Cancer Cure Via Autophagy

6.3.3 Allium Sativum (Family: Amaryllidaceae) . . . . . . . . . 237

8 Capillary Electrophoresis: A New Evolutionary Platform of Plant

10.4 Phenolic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340

11.3.3 Colon Drug Delivery . . . . . . . . . . . . . . . . . . . . . . . 389

13 Carvacrol (Origanum vulgare): Therapeutic Properties

14.3.10 Phytochemistry and Pharmacological Activities

16.2.5 Antimicrobial Effects of Gedunin and Derivatives . . . 580

18.4 Enhancing the Elasticity and Electrical Conductivity

20.3 Encapsulation of Bioactive Compounds . . . . . . . . . . . . . . . . . 691

22 Piperine: Sources, Properties, Applications, and Biotechnological

23.1.3 Classiﬁcation Ama . . . . . . . . . . . . . . . . . . . . . . . . . . 798

24.3 GABA as Bioactive Compound in Food . . . . . . . . . . . . . . . . . 828

Dr. Dilipkumar Pal, (born in West Bengal, India)

reviewer and editorial board member of 30 and 29

Dr. Amit Kumar Nayak (M.Pharm., Ph.D.) is cur-

Supriyo Saha and Dilipkumar Pal

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2021 1

1.2 Types of Plant Metabolites

Characteristics of secondary metabolites:

vi. Bioconversions: Biotransformation process using microorganisms was

1.3 Elicitor and Its Type

Fig. 1.1 Elicitor and its types

1.4 Application of Elicitors on the Production

1.4.1 Production of Secondary Metabolites Using Abiotic

1.4.1.1 Effect of Arachidonic and Jasmonic Acid Elicitors

1.4.1.2 Effect Abiotic Elicitors on Secondary Metabolites of Thyme,

1.4.1.3 Effect of Abiotic Elicitor on Triterpenoid Accumulation

1.4.1.4 Effect of Abiotic Elicitor on Afﬁnin Contents in Heliopsis

1.4.1.5 Effects of Abiotic Elicitor on Hypocrellin a Content in Shiraia

1.4.1.6 Effects of Abiotic Elicitor on Steviosides Production in Stevia

1.4.1.7 Effect of Light as Abiotic Elicitor on the Secondary Metabolite

1.4.1.8 Effect of Gamma Radiation on Secondary Metabolites

1.4.1.9 Effects of Abiotic Elicitors on Secondary Metabolites of Vitis

1.4.1.10 Effect of Salicylic Acid on Alkaloid Biosynthesis in Marine

Hadizadeh et al. experimentally proved the importance of salicylic acid as abiotic

1.4.1.11 Effect of Abiotic Elicitor on Growth of Secondary Metabolites

1.4.1.12 Effect of Abiotic Elicitors on Steviol and Adventitious Root

1.4.1.13 Effect of Ultrasound on Secondary Metabolite Accumulation

Lu et al. experimentally proved the importance of high intensity ultrasound on

ultrasound treatment after 48 h. Maximum phenylalanine ammonia-lyase activity

1.4.1.14 Effect of Abiotic Elicitors on Metabolite Accumulation

1.4.1.15 Effect of MJ Abiotic Elicitor on Anthraquinone Production

1.4.1.16 Effect of Polyunsaturated Fatty Acids on Gymnemic Acid

Praveen et al. scientiﬁcally proved the importance of polyunsaturated fatty acids

1.4.1.17 Effect of Magnesium Oxide Nanoparticle on Secondary

Tian et al. experimentally proved the importance of magnesium oxide nanoparticle

1.4.1.18 Effect of Temperature on Secondary Metabolite

Ulaganathan et al. experimentally suggested the effect of temperature on secondary

1.4.1.19 Effect of Polyunsaturated Fatty Acids on Secondary

Wu et al. experimentally proved the effect of polyunsaturated fatty acids (linoleic

1.4.2 Production of Secondary Metabolites Using Biotic

1.4.2.1 Plant Growth Regulating Rhizobacteria Stimulated Secondary