Bioprospecting of Plant Biodiversity, 2021

Bioprospecting of Plant Biodiversity
for Industrial Molecules

Bioprospecting of Plant Biodiversity
for Industrial Molecules
Edited by
Santosh Kumar Upadhyay

Department of Botany, Panjab University, Chandigarh, India
Sudhir P. Singh
Center of Innovative and Applied Bioprocessing (CIAB), Mohali, India
This edition first published 2021
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Hardback ISBN: 9781119717218
Cover Design: Wiley
Cover Image: © Bernard Radvaner/Corbis/Getty Images
Set in 9.5/12.5pt STIXTwoText by Straive, Pondicherry, India
10 9 8 7 6 5 4 3 2 1
v
Contents
List of Contributors xv
Preface xxi
About the Editors xxiii
Acknowledgments xxv
1 An Introduction to Plant Biodiversity and Bioprospecting 1

Ramya Krishnan, Sudhir P. Singh, and Santosh Kumar Upadhyay
1.1 Introduction 1
1.2 What is Bioprospecting 1
1.2.1 Chemical Prospecting 3
1.2.2 Gene Prospecting 3
1.2.3 Bionic Prospecting 4
1.3 Significance of Plants in Bioprospecting 4
1.4 Pros and Cons of Bioprospecting 5
1.5 Recent Trends in Bioprospecting 6
1.6 Omics for Bioprospecting and in silico Bioprospecting 7
1.7 An Insight into the Book 8
References 10
2 Entomotoxic Proteins from Plant Biodiversity to Control the

Crop Insect Pests 15
Surjeet Kumar Arya, Shatrughan Shiva, and Santosh Kumar Upadhyay
2.1 Introduction 15
2.2 Lectins 16
2.3 Proteinase Inhibitors 21
2.4 α-Amylase Inhibitors 24
2.5 Ribosome-Inactivating Proteins (RIPs) 27
2.6 Arcelins 30
2.7 Defensins 32
2.8 Cyclotides 32
2.9 Canatoxin-Like Proteins 33
2.10 Ureases and Urease-Derived Encrypted Peptides 33
2.11 Chitinases 36
2.12 Proteases 36
vi Contents
2.13 Conclusions 37
References 37
3 Bioprospecting of Natural Compounds for Industrial and Medical Applications:

Current Scenario and Bottleneck 53
Sameer Dixit, Akanchha Shukla, Vinayak Singh, and Santosh Kumar Upadhyay
3.1 Introduction 53
3.2 Why Bioprospecting Is Important 54
3.3 Major Sites for Bioprospecting 54
3.4 Pipeline of Bioprospecting 55
3.5 Biopiracy: An Unethical Bioprospecting 55
3.6 Bioprospecting Derived Products in Agriculture Industry 56
3.7 Bioprospecting Derived Products for Bioremediation 57
3.8 Bioprospecting for Nanoparticles Development 59
3.9 Bioprospecting Derived Products in Pharmaceutical Industry 60
3.10 Conclusion and Future Prospects 63
Acknowledgments 64
References 64
4 Role of Plants in Phytoremediation of Industrial Waste 73

Pankaj Srivastava and Nishita Giri
4.1 Introduction 73
4.2 Different Toxic Materials from Industries 75
4.2.1 Fly Ash from Thermal Power Plants 75
4.2.2 Heavy Metals and Pesticides in Environment 75
4.2.2.1 Cadmium 75
4.2.2.2 Arsenic 76
4.2.2.3 Chromium 76
4.2.2.4 Pesticide in Environment 76
4.2.3 Phytoremediation Technology in Present Scenario 77
4.2.4 Conclusion 80
References 81
5 Ecological Restoration and Plant Biodiversity 91

Shalini Tiwari and Puneet Singh Chauhan
5.1 Introduction 91
5.2 Major Areas of Bioprospecting 92
5.2.1 Chemical/Biochemical Prospecting 92
5.2.2 Gene/Genetic Prospecting 92
5.2.3 Bionic Prospecting 93
5.3 Bioprospecting: Creating a Value for Biodiversity 93
5.4 Conservation and Ecological Restoration for Sustainable Utilization
of Resources 94
5.5 Biodiversity Development Agreements 95
5.6 Conclusions 96
References 96
Contents vii
6 Endophyte Enzymes and Their Applications in Industries 99

Rufin Marie Kouipou Toghueo and Fabrice Fekam Boyom
6.1 Introduction 99
6.2 The Rationale for Bioprospecting Endophytes for Novel Industrial Enzymes 100
6.3 Endophytes as a Source of Industrial Enzymes 101
6.3.1 Amylases 104
6.3.2 Asparaginase 105
6.3.3 Cellulases 107
6.3.4 Chitinases 109
6.3.5 Laccases 110
6.3.6 Lipases 111
6.3.7 Proteases 113
6.3.8 Xylanases 115
6.3.9 Other Enzymes Produced by Endophytes 116
6.3.9.1 AHL-Lactonase 116
6.3.9.2 Agarase 116
6.3.9.3 Chromate Reductase 116
6.3.9.4 β-Mannanase 117
6.4 Overview of the Methods Used to Investigate Endophytes as Sources
of Enzymes 117
6.5 Strategies Applied to Improve the Production of Enzymes by Endophytes 118
6.6 Conclusion 119
Acknowledgements 122
References 122
7 Resource Recovery from the Abundant Agri-biomass 131

Shilpi Bansal, Jyoti Singh Jadaun, and Sudhir P. Singh
7.1 Introduction 131
7.2 Potential of Agri-biomass to Produce Different Products 133
7.2.1 Conversion of Agri-biomass into Valuable Chemicals 133
7.2.2 Energy Production Using Agri-biomass 134
7.2.3 Role of Agri-biomass in Heavy Metal Decontamination 135
7.2.4 Manufacturing of Lightweight Materials 137
7.3 Case Studies 138
7.3.1 Utilization of Paddy Waste 138
7.3.2 Utilization of Mustard Waste 140
7.3.3 Utilization of Maize Waste 140
7.3.4 Utilization of Horticulture Waste 142
7.4 Conclusion and Future Perspectives 144
References 144
8 Antimicrobial Products from Plant Biodiversity 153

Pankaj Kumar Verma, Shikha Verma, Nalini Pandey, and Debasis Chakrabarty
8.2 Use of Plant Products as Antimicrobials: Historical Perspective 154
8.3 Major Groups of Plants-Derived Antimicrobial Compound 156
viii Contents
8.3.1 Simple Phenols and Phenolic Acids 156

8.3.1.1 Flavonoids 156
8.3.1.2 Quinones 160
8.3.1.3 Tannins 160
8.3.1.4 Coumarins 161
8.3.2 Terpenes and Essential Oils 162
8.3.3 Alkaloids 163
8.4 Mechanisms of Antimicrobial Activity 163
8.4.1 Plant Extracts with Efflux Pump Inhibitory Activity 164
8.4.2 Plant Extracts with Bacterial Quorum Sensing Inhibitory Activity 164
8.4.3 Plant Extracts with Biofilm Inhibitory Activity 165
8.5 Conclusions and Future Prospects 165
References 166
9 Functional Plants as Natural Sources of Dietary Antioxidants 175

Ao Shang, Jia-Hui Li, Xiao-Yu Xu, Ren-You Gan, Min Luo, and Hua-Bin Li
9.2 Evaluation of the Antioxidant Activity 176
9.3 Antioxidant Activity of Functional Plants 176
9.3.1 Vegetables 176
9.3.2 Fruits 177
9.3.3 Medicinal Plants 181
9.3.4 Cereal Grains 181
9.3.5 Flowers 181
9.3.6 Microalgae 181
9.3.7 Teas 182
9.4 Applications of Plant Antioxidants 182
9.4.1 Food Additives 182
9.4.2 Dietary Supplements 183
9.5 Conclusions 183
References 184
10 Biodiversity and Importance of Plant Bioprospecting in Cosmetics 189

K. Sri Manjari, Debarati Chakraborty, Aakanksha Kumar, and Sakshi Singh
10.1 Biodiversity, Bioprospecting, and Cosmetics – A Harmony of Triad 189
10.2 The Fury of Synthetic Chemicals in Cosmetics on Health 191
10.3 India’s Biodiversity and Its Traditional Knowledge/Medicine in Cosmetics 191
10.3.1 Herbal Cosmetics 194
10.4 Use of Plant-Based Products in the Cosmetic Industry 194
10.5 Green Cosmetics – Significance and Current Status of the Global Market 196
10.5.1 Sustainable Development Goals (Economic, Ecological Benefits) in Cosmetic
Industry – How Bioprospecting and Green Cosmetics Can Help? 199
10.6 Ethical and Legal Implications of Bioprospecting and Cosmetics 200
10.6.1 International Laws Regulating Bioprospecting 201
10.6.2 Indian Law Regulating Bioprospecting 202
10.6.3 Access and Benefit Sharing (ABS) 202
Contents ix
10.6.4 World Intellectual Property Organization (WIPO) 203

10.6.5 Intergovernmental Committee on Intellectual Property and
Genetic Resources, Traditional Knowledge, and Folklore (IGC) 203
10.7 Laws Regulating Cosmetics 203
10.8 Role of Biotechnology in Bioprospecting and Cosmetics 204
References 205
11 Therapeutic Lead Secondary Metabolites Production Using Plant

In Vitro Cultures 211
Vikas Srivastava, Aksar Ali Chowdhary, Skalzang Lhamo, Sonal Mishra,
and Shakti Mehrotra
11.2 Secondary Metabolites and Pharmaceutical Significance 212
11.3 Plant In Vitro Cultures and Strategies for Secondary Metabolite Production 214
11.3.1 Precursor Feeding 214
11.3.2 Metabolic Engineering 215
11.3.3 Elicitation 216
11.3.4 Bioreactor Up-scaling 216
11.4 Exemplification of the Utilization of Different Types of Plant In Vitro Cultures
for SMs Production 217
11.4.1 Shoot Culture 217
11.4.2 Adventitious Root Culture 220
11.4.3 Callus and Cell Suspension Culture 220
11.4.4 Hairy Root Cultures 221
11.5 Conclusion 221
References 222
12 Plant Diversity and Ethnobotanical Knowledge of Spices and Condiments 231

Thakku R. Ramkumar and Subbiah Karuppusamy
12.2 Habitat and Diversity of Major Spices and Condiments in India 232
12.3 Ethnobotanical Context of Spices and Condiments in India 241
12.4 Major Spices and Condiments in India 243
12.4.1 Black Pepper 243
12.4.2 Capsicums 243
12.4.3 Cinnamomum 244
12.4.4 Coriander 244
12.4.5 Cumin 244
12.4.6 Cardamom 245
12.4.7 Fennel 245
12.4.8 Ginger 245
12.4.9 Mustard Seed 246
12.4.10 Nutmeg 246
12.4.11 Saffron 246
12.4.12 Turmeric 246
12.4.13 Vanilla 247
x Contents
12.5 Importance of Indian Spices 247

12.6 Spice Plantation and Cultivation in India 249
12.7 Cultivation Technology of Caper Bud in India 250
12.8 Export of Indian Spices 251
12.9 Conservation Efforts Against Selected Uncultivated Wild Spices
and Condiments 254
12.10 Institutions and Organization Dedicated for Research and Development
in Spices and Condiments in India 254
12.11 Recent Researches on Spices and Condiments 255
12.12 Conclusion and Future Perspectives 256
Acknowledgments 256
Authors’ Contribution 256
References 257
13 Plants as Source of Essential Oils and Perfumery Applications 261

Monica Butnariu
13.1 Background 261
13.2 Biochemistry of Essential Oils 262
13.2.1 The Physiological Mechanism of Biosynthesis of Essential Oils 262
13.2.2 The Role of Terpenes in Plants 263
13.2.3 The Prevalence Essential Oils in Plants 264
13.2.4 Paths of Biosynthesis of Volatile Compounds in Plants 265
13.2.4.1 Metabolic Cycles Involved in the Biosynthesis of Different Groups of Secondary
Metabolites 265
13.2.4.2 Metabolic Cycles of Biosynthesis of Phenolic Compounds 266
13.3 The Metabolism Terpenes 269
13.3.1 Metabolic Cycle of Mevalonic Acid Biosynthesis 271
13.3.2 Metabolic Cycle of Methylerythritol Phosphate Biosynthesis 272
13.4 The Role of Essential Oils and the Specificity of Their
Accumulation in Plants 272
13.5 Essential Oils from Plants in Perfume 281
13.5.1 Linalool (3,7-dimethylocta-1,6-dien-3-ol), C10H18O 286
13.5.2 Camphor (1,7,7-trimethylbicyclo [2.2.1] heptan-2-one), C10H16O 286
13.5.3 Cedrol (1S, 2R, 5S, 7R, 8R)-(2,6,6,8-tetramethyltricyclo [5.3.1.01,5]
undecan-8-ol or cedran-8-ol), C15H26O 286
13.5.4 Eugenol (2-methoxy-4-allylphenol; 1-hydroxy-2-methoxy-
4-allylbenzene), C10H12O2 287
13.5.5 Citral (3,7-dimethyl-2,6-octadien-1-al), C10H16O 287
13.5.6 Vanillin (4-hydroxy-3-methoxybenzaldehyde) C8H8O3 287
13.5.7 Syringe Aldehyde (4-hydroxy-3,5-dimethoxybenzaldehyde) C9H10O4 288
13.6 Conclusions and Remarks 289
References 290
14 Bioprospection of Plants for Essential Mineral Micronutrients 293

Nikita Bisht and Puneet Singh Chauhan
14.2 Plants as a Source of Mineral Micronutrients 293
Contents xi
14.3 Bioavailability of Micronutrients from Plants 294

14.3.1 Bioavailability of Fe and Zn 294
14.3.2 Impact of Food Processing on Micronutrient Bioavailability
from Plant Foods 295
14.4 Manipulating Plant Micronutrients 296
14.4.1 Improving Bioavailability of Micronutrients from Plant Foods 296
14.4.2 Metabolic Engineering of Micronutrients in Crop Plants 297
14.5 Microbes in the Biofortification of Micronutrients in Crops 298
14.6 Conclusions 299
References 299
15 Algal Biomass: A Natural Resource of High-Value Biomolecules 303

Dinesh Kumar Yadav, Ananya Singh, Variyata Agrawal, and Neelam Yadav
15.2 Carbon Dioxide Capture and Sequestration 304
15.3 Algae in High-Value Biomolecules Production 306
15.3.1 Proteins, Peptides, and Amino Acids 310
15.3.2 Polyunsaturated Fatty Acids (PUFAs) 311
15.3.3 Polysaccharides 312
15.3.4 Pigments 313
15.3.4.1 Chlorophylls 313
15.3.4.2 Carotenoids 314
15.3.4.3 Phycobilliproteins (PBPs) 315
15.3.5 Vitamins 316
15.3.6 Polyphenols 316
15.3.7 Phytosterols 317
15.3.8 Phytohormones 318
15.3.9 Minerals 318
15.4 Algae in Biofuel Production/Generation 319
15.4.1 Thermochemical Conversion 319
15.4.2 Chemical Conversion by Transesterification 321
15.4.3 Biochemical Conversion 322
15.4.4 Photosynthetic Microbial Fuel Cell (MFC) 324
15.5 Algae in Additional Applications 325
15.5.1 Algae as Livestock Feed and Nutrition 325
15.5.2 Algae as Feed in Aquaculture 326
15.5.3 Algae as Bio-Fertilizer 326
15.6 Conclusion and Future Prospects 326
References 327
16 Plant Bioprospecting for Biopesticides and Bioinsecticides 335

Aradhana Lucky Hans and Sangeeta Saxena
16.2 Current Scenario in India 336
16.3 Plants-Based Active Compounds 337
16.3.1 Azadirachtin 337
16.3.2 Pyrethrins 338
xii Contents
16.3.3 Rotenone 338

16.3.4 Sabadilla 339
16.3.5 Ryania 339
16.3.6 Nicotine 339
16.3.7 Acetogenins 339
16.3.8 Capsaicinoids 339
16.3.9 Essential Oils 340
16.4 Advantages and Future Prospects of Bioinsecticides 340
Acknowledgment 343
References 343
17 Plant Biomass to Bioenergy 345

Mrinalini Srivastava and Debasis Chakrabarty
17.2 Plant Biomass 346
17.2.1 Types of Biomass (Source: [17]) 347
17.3 Bioenergy 347
17.4 Biomass Conversion into Bioenergy 348
17.4.1 Cogeneration 349
17.5 The Concept of Biomass Energy (Source: [27]) 349
17.5.1 Thermochemical Conversion 349
17.5.1.1 Direct Combustion 349
17.5.1.2 Pyrolysis 349
17.5.1.3 Gasification 349
17.5.2 Biochemical Conversion 350
17.5.2.1 Anaerobic Digestion 350
17.5.2.2 Alcohol Fermentation 350
17.5.2.3 Hydrogen Production from Biomass 350
17.6 Use of Biofuel in Transportation 350
17.7 Production of Biogas and Biomethane from Biomass 350
17.8 Generation of Biofuel 351
17.8.1 Bioethanol 351
17.8.2 Biodiesel 352
17.9 Advanced Technologies in the Area of Bioenergy 352
17.10 Conclusion 353
Acknowledgment 354
References 354
18 Bioenergy Crops as an Alternate Energy Resource 357

Garima Pathak and Shivanand Suresh Dudhagi
18.2 Classification of Bioenergy Crops 358
18.2.1 First-Generation Bioenergy Crops 358
18.2.1.1 Sugarcane 359
Contents xiii
18.2.1.2 Corn 359

18.2.1.3 Sweet Sorghum 359
18.2.1.4 Oil Crops 360
18.2.2 Second-Generation Bioenergy Crops 360
18.2.2.1 Switchgrass 360
18.2.2.2 Miscanthus 361
18.2.2.3 Alfalfa 361
18.2.2.4 Reed Canary Grass 361
18.2.2.5 Other Plants 361
18.2.3 Third-Generation Bioenergy Crops 362
18.2.3.1 Boreal Plants 362
18.2.3.2 Crassulacean Acid Metabolism (CAM) Plants 362
18.2.3.3 Eucalyptus 362
18.2.3.4 Agave 362
18.2.3.5 Microalgae 363
18.2.4 Dedicated Bioenergy Crops 363
18.2.5 Halophytes 363
18.3 Characteristics of Bioenergy Crops 364
18.3.1 Physiological and Ecological Traits 364
18.3.2 Agronomic and Metabolic Traits 364
18.3.3 Biochemical Composition and Caloric Content 365
18.4 Genetic Improvement of Bioenergy Crops 365
18.5 Environmental Impacts of Bioenergy Crops 366
18.5.1 Soil Quality 366
18.5.2 Water and Minerals 367
18.5.3 Carbon Sequestration 367
18.5.4 Phytoremediation 367
18.5.5 Biodiversity 368
18.6 Conclusion and Future Prospect 369
References 369
19 Marine Bioprospecting: Seaweeds for Industrial Molecules 377

Achintya Kumar Dolui
19.2 Seaweeds as Nutraceuticals and Functional Foods 378
19.3 Seaweeds in the Alleviation of Lifestyle Disorders 380
19.4 Anti-Inflammatory Activity of Seaweeds 381
19.5 Seaweed Is a Source of Anticoagulant Agent 381
19.6 Anticancer Property of Seaweed 382
19.7 Seaweeds as Antiviral Drugs and Mosquitocides 384
19.8 Use of Seaweeds in the Cosmeceutical Industry 385
19.9 Use of Seaweed as Contraceptive Agents 386
19.10 Extraction of Active Ingredients from Seaweed 388
19.10.1 Supercritical Fluid Extraction (SFE) 388
19.10.2 Ultrasound-Assisted Extraction (UAE) 389
xiv Contents
19.10.3 Microwave-Assisted Extraction (MAE) 389

19.10.4 Enzyme-Assisted Extraction (EAE) and EMEA 390
19.11 Market Potential of Seaweeds 390
19.12 Conclusion 391
References 391
20 Bioprospection of Orchids and Appraisal of Their Therapeutic Indications 401

Devina Ghai, Jagdeep Verma, Arshpreet Kaur, Kranti Thakur,
Sandip V. Pawar, and Jaspreet K. Sembi
20.2 Orchids as a Bioprospecting Resource 402
20.3 Orchids as Curatives in Traditional India 403
20.4 Therapeutics Indications of Orchids in Asian Region 403
20.5 Evidences of Medicinal Uses of Orchids in Ethnic African Groups 404
20.6 Orchids as a Source of Restoratives in Europe 405
20.7 Remedial Uses of Orchids in American and Australian Cultures 405
20.8 Scientific Appraisal of Therapeutic Indications of Orchids 406
20.8.1 Orchids as Potent Anticancer Agents 406
20.8.2 Immunomodulatory Activity in Orchids 412
20.8.3 Orchids and Their Antioxidant Potential 412
20.8.4 Antimicrobial Studies in Orchids 412
20.8.5 Orchids and Anti-inflammatory Activity 413
20.8.6 Antidiabetic Prospects in Orchids 413
20.8.7 Other Analeptic Properties in Orchids 414
Acknowledgments 415
References 415
Index 425
xv
L
ist of Contributors
Variyata Agrawal Fabrice Fekam Boyom

Molecular Biology and Genetic Antimicrobial and Biocontrol Agents Unit
Engineering Laboratory (AmBcAU)
Department of Botany Laboratory for Phytobiochemistry and
University of Allahabad Medicinal Plants Studies
Prayagraj Department of Biochemistry
India Faculty of Science
University of Yaoundé I
Surjeet Kumar Arya Yaoundé
Department of Entomology Cameroon
College of Agriculture, Food and
Environment Monica Butnariu
University of Kentucky Chemistry & Biochemistry Discipline
Lexington Banat’s University of Agricultural Sciences
KY and Veterinary Medicine “King Michael I
USA of Romania” from Timisoara
Timis
Romania
Shilpi Bansal
Vegetable Science Division
Debasis Chakrabarty
ICAR‐Indian Agricultural Research Institute
Molecular Biology and Biotechnology
Pusa
Division
New Delhi
Tissue Culture and Transformation Lab
India
CSIR‐National Botanical Research Institute
Lucknow
Nikita Bisht
India
(CSIR‐NBRI)
Debarati Chakraborty
Lucknow
Department of Molecular Biology and
India
Biotechnology
Academy of Scientific and Innovative University of Kalyani
Research (AcSIR) Kalyani
Ghaziabad West Bengal
India India
xvi List of Contributor
Puneet Singh Chauhan Institute of Urban Agriculture

CSIR‐National Botanical Research Institute Chinese Academy of Agricultural Sciences
(CSIR‐NBRI) Chengdu
Lucknow China
India
Devina Ghai
Academy of Scientific and Innovative
Department of Botany
Research (AcSIR)
Panjab University
Ghaziabad
Chandigarh
India
UT
India
Aksar Ali Chowdhary
Nishita Giri
Central University of Jammu
ICAR‐Indian Institute of Soil and Water
Samba
Conservation (ICAR-IISWC)
Jammu and Kashmir
Dehradun
India
Uttarakhand
India
Sameer Dixit
Department of Biology
Aradhana Lucky Hans
University of Western Ontario
Department of Biotechnology
London
Babasaheb Bhimrao Ambedkar University
Ontario
Lucknow
Canada
India
Achintya Kumar Dolui

Jyoti Singh Jadaun
Department of Lipid Science
CSIR‐Central Food Technological Research
Dayanand Girls Postgraduate College
Institute
Kanpur
Mysuru
Uttar Pradesh
Karnataka
India
India
Academy of Scientific and Innovative Subbiah Karuppusamy
Research Department of Botany
Ghaziabad Botanical Research Center
Uttar Pradesh The Madura College
India Madurai
Tamil Nadu
Shivanand Suresh Dudhagi India
Lucknow Arshpreet Kaur
India Department of Botany
Panjab University
Ren‐You Gan Chandigarh
Research Center for Plants and UT
Human Health India
List of Contributor xvii
Ramya Krishnan School of Public Health

CSIR‐National Institute of Interdisciplinary Sun Yat‐Sen University
Science and Technology Guangzhou
Thiruvananthapuram China
Kerala
India K. Sri Manjari
University College for Women
Current: Accubits Technologies
Osmania University
Thiruvananthapuram
Hyderabad
Kerala
Telangana
India
India
Aakanksha Kumar Shakti Mehrotra

Bioclues Department of Biotechnology
Hyderabad Institute of Engineering and Technology
Telangana Lucknow
India Uttar Pradesh
India
Skalzang Lhamo
Department of Botany Sonal Mishra
Central University of Jammu School of Biotechnology
Samba University of Jammu
Jammu and Kashmir Jammu
India Jammu and Kashmir
India
Hua‐Bin Li
Guangdong Provincial Key Nalini Pandey
Laboratory of Food Department of Botany
Nutrition and Health University of Lucknow
Department of Nutrition Lucknow
School of Public Health Uttar Pradesh
Sun Yat‐Sen University India
Guangzhou
China Garima Pathak
B.D. College – A Constituent Unit of
Jia‐Hui Li Patiliputra University
School of Science Patna
The Hong Kong University of Science and India
Technology
Hong Kong Sandip V. Pawar
China University Institute of Pharmaceutical
Sciences
Min Luo Panjab University
Guangdong Provincial Key Laboratory of Food Chandigarh
Nutrition and Health UT
Department of Nutrition India
xviii List of Contributor
Thakku R. Ramkumar Akanchha Shukla

Agronomy Department Department of Biology
IFAS, University of Florida University of Western Ontario
Gainesville London
FL Ontario
USA Canada
Sangeeta Saxena Ananya Singh

Department of Biotechnology Molecular Biology and Genetic
Babasaheb Bhimrao Ambedkar University Engineering Laboratory
Lucknow Department of Botany
India University of Allahabad
Prayagraj
Jaspreet K. Sembi India
Panjab University Sakshi Singh
Chandigarh Department of Molecular Biology and
UT Human Genetics
India Banaras Hindu University
Varanasi
Ao Shang Uttar Pradesh
Guangdong Provincial Key India
Laboratory of Food
Nutrition and Health Sudhir P. Singh
Department of Nutrition Center of Innovative and Applied
School of Public Health Bioprocessing (CIAB)
Sun Yat‐Sen University Mohali
Guangzhou India
China
Vinayak Singh
Shatrughan Shiva Department of Biology
Department of Plant Molecular Biology University of Western Ontario
and Genetic Engineering London
CSIR‐National Botanical Research Ontario
Institute Canada
Council of Scientific and Industrial
Research Rana Pratap Marg
Mrinalini Srivastava
Lucknow
India
Division
Academy of Scientific and Innovative Tissue Culture and Transformation Lab
Research (AcSIR) CSIR‐National Botanical Research Institute
Ghaziabad Lucknow
India India
List of Contributor xix
Pankaj Srivastava Jagdeep Verma

ICAR‐Indian Institute of Soil and Water Department of Botany
Conservation, (ICAR-IISWC) Government College
Dehradun Rajgarh
Uttarakhand Himachal Pradesh
India India
Pankaj Kumar Verma

Vikas Srivastava Department of Botany
Department of Botany University of Lucknow
Central University of Jammu Lucknow
Samba Uttar Pradesh
Jammu and Kashmir India
India
Shikha Verma
Kranti Thakur
Division
CSIR‐National Botanical Research
Shoolini Institute of Life Sciences and
Institute
Business Management (SILB)
Lucknow
Solan
Uttar Pradesh
Himachal Pradesh
India
India
Xiao‐Yu Xu
Guangdong Provincial Key Laboratory
Shalini Tiwari
of Food
Nutrition and Health
(CSIR-NBRI)
Department of Nutrition
Lucknow
School of Public Health
India
Sun Yat‐Sen University
Guangzhou
Rufin Marie Kouipou Toghueo
China
Antimicrobial and Biocontrol Agents Unit
(AmBcAU) Dinesh Kumar Yadav
Laboratory for Phytobiochemistry and Molecular Biology and Genetic
Medicinal Plants Studies Engineering Laboratory
Department of Biochemistry Department of Botany
Faculty of Science University of Allahabad
University of Yaoundé I Prayagraj
Yaoundé India
Cameroon
Neelam Yadav
Santosh Kumar Upadhyay Molecular Biology and Genetic
Department of Botany Engineering Laboratory
Panjab University Department of Botany
Chandigarh University of Allahabad
UT Prayagraj
India India
xxi
Preface
Nature has the reservoir for all the desired molecules in the form of biodiversity that
includes microbial, animal, and plants. Bioprospection is very well‐established method for
the identification and isolation of new active molecules of desired activity. Researches are
being conducted to exploit the biological resource for obtaining biomolecules of pharma-
ceutical, bioceutical, agricultural, bioremediation, etc. significance. The exploitation of
bioactive significance in the natural compounds in the biosphere is required to be intensi-
fied with systematic and sustainable approaches. The expedition and validation of the
scientific parameters in the ethnic knowledge, preservation of bioresource, and biotechno-
logical advancement in the generation of efficient biological systems, keeping in mind the
approach of societal development exploitation with nature’s protection, are the current
demand in scientific investigations.
Bioprospection is the exploration of economic potential in the biological resource mostly
in terms of nutraceutical value. In recent decades, substantial attention has been given on
a variety of bioresources for bioprospecting. For example, macro‐ and microalgae have
been demonstrated to be a biomass value of neutraceutical, pharmaceutical, food, biomedi-
cal, bioenergetic importance.
Plants are a crucial biological component of the biosphere in the earth. The plant resource
has served the humankind in several ways by providing food, feed, medicine, nutraceuti-
cals, shelter, etc. About 3.9 lakh known plant species make the animals and other organ-
isms’ life possible at the earth. Plant bioprospecting is being performed since the existence
of human life on the earth. Extensive investigations have been done to explore several phy-
tochemicals, pharmaceuticals, antioxidants, etc. There is a need to develop plant products
with prebiotic properties and with high bioavailable mineral micronutrients. A rich cul-
tural knowledge associated with multifarious health beneficial aspects of plants is available
in different parts of the world. Many plants of cosmetic and perfumery importance have
been shown to be of great economic value. The plants grown for production of spices and
condiments have significant societal and medicinal merits. Many lower plants have been
demonstrated to exhibit potential in biopesticide development. Furthermore, this is an era
of secondary agriculture by the valorization of the abundant residual plant biomass.
We firmly believe that this book will be an essential repository in obtaining the holistic
knowledge of plants bioprospecting. The compiled information will be useful to academi-
cians and researchers in augmenting their understandings on the aspects mentioned
earlier.
xxiii
About the Editors
Dr. Santosh Kumar Upadhyay is

currently working as an Assistant
Professor in the Department of Botany,
Panjab University, Chandigarh, India.
Prior to this, Dr. Upadhyay was DST‐
INSPIRE faculty at the National Agri‐
Food Biotechnology Institute, Mohali,
Punjab, India. He did his doctoral work
at the CSIR–National Botanical
Research Institute, Lucknow, and
received his Ph.D. in Biotechnology
from UP Technical University,
Source: Santosh Kumar Upadhyay Lucknow, India. He has been working
in the field of Plant Biotechnology for
more than 14 years. His present research focuses in the area of functional genomics. He is
involved in the bioprospecting and characterization of various insect toxic proteins from
plant biodiversity and defense and stress signaling genes in bread wheat. His research
group at PU has characterized numerous important gene families and long noncoding
RNAs related to the abiotic and biotic stress tolerance and signaling in bread wheat. He has
also established the method for genome editing in bread wheat using CRISPR–Cas system
and developed a tool SSinder for CRISPR target site prediction. His research contribution
led the publication of more than 58 research papers in leading journals of international
repute. Further, there are more than 5 national and international patents, 22 book chapters,
and 6 books in his credit. In recognition of his substantial research record, he has been
awarded NAAS Young scientist award (2017–2018) and NAAS‐Associate (2018) from the
National Academy of Agricultural Sciences, India, INSA Medal for Young Scientist (2013)
from the Indian National Science Academy, India, NASI–Young Scientist Platinum Jubilee
Award (2012) from the National Academy of Sciences, India, and Altech Young Scientist
Award (2011). He has also been the recipient of the prestigious DST‐INSPIRE Faculty
Fellowship (2012) and SERB‐Early Career Research Award (2016) from the Ministry of
Science and Technology, Government of India. Dr. Upadhyay also serves as a member of
the editorial board and reviewer of several peer‐reviewed international journals.
xxiv About the Editors
Dr. Sudhir P. Singh is currently Scientist at the Center of Innovative and Applied
Bioprocessing (CIAB), Mohali, India. He has been working in the area of molecular biology
and biotechnology for more than a decade. Currently, his primary focus of research is gene
mining and biocatalyst engineering for the development of approaches for transformation
of agro‐industrial residues and under or unutilized side‐stream biomass into value‐added
bio‐products. He has explored the metagenomic resource from diverse habitats and devel-
oped enzyme systems for catalytic biosynthesis of functional sugar molecules such as d‐
allulose, turanose, fructooligosaccharides, glucooligosaccharides, 4‐galactosyl‐Kojibiose,
xylooligosaccharides, levan, dextran biosynthesis, etc. Dr. Singh has published over 55
research articles, 4 review articles, and 4 books (edited). Further, he has five patents
(granted) to his credit as an inventor. He has been conferred International Bioprocessing
Association–Young Scientist Award (2017), School of Biosciences–Madurai Kamraj
University (SBS‐MKU) Genomics Award (2017), Professor Hira Lal Chakravarty Award,
ISCA, DST, (2018), and Gandhian Young Technological Innovation Award to team (2019).
0005092138.INDD 24 06-08-2021 18:41:21

xxv
Acknowledgments
We are thankful to the Panjab University, Chandigarh, and Centre of Applied and
Innovative Bioprocessing (CIAB) for providing facility to complete this book. We are grate-
ful to all the esteemed authors for their exceptional contributions and reviewers for their
critical evaluation and suggestions for the quality improvement of the book.
We would like to thank Miss Rebecca Ralf (Commissioning Editor), Miss Kerry Powell
(Managing Editor), and Shyamala Venkateswaran (Production Editor) from John Wiley &
Sons, Ltd for their excellent management of this project and anonymous reviewers for their
positive recommendations about the book.
We also appreciate the support of our friends and research students, whose discussion
and comments were beneficial to shape this book. We thank our numerous colleagues for
direct or indirect help in shaping this project.
SKU wishes to express gratitude to his parents, wife, and daughter for their endless sup-
port, patience, and inspiration. SPS is grateful to his parents and family for consistent moral
support. SPS acknowledges the support from CIAB and the Department of Biotechnology,
Government of India.
We would like to warmly thank faculties and staffs of the department and university for
providing a great working environment.
1
An Introduction to Plant Biodiversity and Bioprospecting

Ramya Krishnan1,*, Sudhir P. Singh2, and Santosh Kumar Upadhyay3*
1
CSIR-National Institute of Interdisciplinary Science and Technology, Thiruvananthapuram, Kerala, India
2
3
Department of Botany, Panjab University, Chandigarh, UT, India
1.1 Introduction
There is an extensive diversity in life that has been disentangled and organized into coherent
units called taxa. The five kingdom system of classification has simplified the life forms into
five groups. These groups orchestrate information concerning a wide variety of characteris-
tics such as morphological, genetic, metabolomic, ecological, etc. Kingdom Plantae is one of
these five kingdoms that consists of all the plant forms on earth and is rich in its metabolomic
characteristic. This kingdom is highly diverse and is composed of both seed bearing
(Phanerogams) and seedless (Cryptogams) plants forming five broadly classified groups, i.e.
algae, bryophytes, pteridophytes, gymnosperms, and angiosperms, which are evolutionarily
related. Each of these groups consists of hundreds of thousands of known species, which in
turn consist of a variety of chemicals called metabolites or more specifically secondary
metabolites. These secondary metabolites or natural products are believed to possess certain
biological activities that are used by the producer for their environmental and competitive
fitness. Progressively, it became a paradigm that all the plants possess some potent biologi-
cally active substance/s that could have great commercial/therapeutic value to humans. It
has been often argued that the currently available knowledge regarding the chemical diver-
sity of the plant biome represents only a fraction of that diversity, hence paving way toward
further explorations. Thus, their rich metabolomic diversity and its knowledge increase the
opportunity for humans to utilize plants as a key resource for bioprospecting.
1.2 What is Bioprospecting
Let us widen our imaginations and visualize an underdeveloped rural village in India,
where an old wise man is treating a sick man with his self‐made herbal concoction comprising
*Current: Accubits Technologies, Thiruvananthapuram, Kerala, India
Bioprospecting of Plant Biodiversity for Industrial Molecules, First Edition.

Edited by Santosh Kumar Upadhyay and Sudhir P. Singh.
© 2021 John Wiley & Sons Ltd. Published 2021 by John Wiley & Sons Ltd.
2 1 An Introduction to Plant Biodiversity and Bioprospecting
the roots of some wild plants, or, let us visualize him trying to squeeze a stream of juice
from a bunch of leaves upon a snake‐bitten area of a person’s leg. Just after a few days, the
concoction healed the patient of his fever and inflammations, and the juice rescued the
patient from snake venom. Therefore, it could be assumed that the concoction made from
the herbs might consist of metabolites having antimicrobial properties, and the juice of the
leaves might consist of chemical/metabolite that had antivenom properties. Now again, let
us visualize a group of scientists walking inside the same village, talking to this old wise
man, collecting these herbs, and returning to their respective laboratories, where they try
to screen these herbs for the presence of active compounds having antimicrobial or
antivenom properties using modern technologies. This whole procedure of exploring bio-
logically important/useful compounds from natural resources lays down foundations to
the science of “bioprospecting.” The former half of our visualization could be considered as
“traditional bioprospecting,” and the latter half of it could be considered as “modern bio-
prospecting.” Traditional bioprospecting can even be traced back to as old as the bronze
age. In 1991, a 5300‐year‐old corpse of an iceman “Otzi” was discovered in the Tyrolean
Alps and was found to have a whipworm (Trichuris trichiura) infection. Surprisingly he
was already equipped with the corresponding anthelmintic medicine, which is the fruiting
body of the fungus Piptoporus betulinus [1, 2]. Thus, the utilization of natural resources for
the interest of humans is as old as humankind itself, and what we follow today is just a
modern and sophisticated version of this science.
The term “bioprospecting” was initially described by Reid et al. [3] as the science, where
biological systems are screened for novel components that are of industrial, commercial, or
scientific value. It includes the hunt for biological products that possess characteristics inter-
esting to humankind. These characteristics could be considered to have great potentials in
the field of therapeutics, agriculture, cosmetics, etc. Although the utilization of the biologi-
cally active properties of plant/animal extracts for various purposes was seen even before
thousands of years, bioprospecting as a science for commercial and economic gains was
introduced and progressed in and around the twentieth century. In 1958, vinblastine and
vincristine, two therapeutic agents for cancer, were developed from the rosy periwinkle plant
in Madagascar. These therapeutic agents were researched and manufactured by the company
Eli Lilly with cues from the local shaman spiritual herbalists [4]. Further, prospecting in the
wild has warranted many therapeutic agents, such as antibiotics and several other anticancer
drugs. The modern biochemists and pharmacologists have been busy seeking ways to block
or enhance the function of a target protein molecule for a cure to a particular disease. The
classical combinatorial chemistry has its limits in the synthesis of new compounds when it
comes to the exceedingly large and diverse number of the target proteins that are being iden-
tified. The diverse and continuously evolving structures of the natural products of Mother
Nature may be a possible solution to these problems. Even as the rational drug designing with
the help of combinatorial chemistry is becoming more important, natural products have been
valuable for pharmaceutical companies owing to their wide structural diversity and their
excellent adaptation to biological active structures [4]. A fast exploration of any medicine
cabinet or a cosmetic shop directly indicates the share of bioprospecting natural products, by
astute businessmen in building the global economy.
Chemicals, genes, and designs are the three major sources of motivation that biodi-
versity extends to contemporary scientists. Thus, the science of bioprospecting finds its
1.2 What is Bioprospectin 3
applications with respect to these three domains and are called chemical prospecting, gene
prospecting, and bionic prospecting respectively.
1.2.1 Chemical Prospecting

Nature and natural resources are a combination of diverse and repeatedly evolving
systems that give rise to varying chemicals. The major defense mechanisms of the herbi-
vores rely mainly on the chemicals synthesized by the plant [5, 6]. Communication,
intraspecies and interspecies competition, attraction toward opposite sex, and pollina-
tion are also based to a great extent on chemistry and have accorded to the development
of diversity [7, 8]. The scan of nature by humans for such useful chemicals has been
termed as “Chemical prospecting” by Thomas Eisner [9], who in subsequent years had
been tirelessly busy promoting it [10]. Although humans have been busy creating novel
and diverse chemicals in the laboratory for different purposes, the contribution of the
chemical diversity present in nature toward these creations has been admirable. The
extent of chemical diversity found in nature has always found a role in our day‐to‐day
lives either as a lead molecule inspiring the chemists to create certain novel compounds
or the lead molecule used as common drugs. There have been many examples of natural
compounds used as therapeutic agents, which later have been synthesized commercially
and have led to economic gains. Most of these natural compounds were derived from
either wild plants, animals, or microorganisms. Snake venom, for example, has also been
a source of a number of neurological drugs. The peptides in the venom of snake Bothrops
jararaca were responsible for the antihypertensive medicines enalapril and lisinopril
[11]. This peptide in the snake venom was responsible for the inhibition of an enzyme in
the human blood, which converts the enzyme angiotensin I to a hypertensive form angio-
tensin II. The analysis of the bioactive compounds present in the snake venom finally led
to the formulation of antihypertensive drugs captopril and enalapril, etc. The nonsteroi-
dal anti‐inflammatory drug, diclofenac, was derived from the lead molecule salicin
obtained from the bark of willow tree Salix sp. [12]. The antiviral drug vidarabine and
anticancer drug cytarabine were obtained from the marine sponge [13]. Similarly, the
antiviral drug acyclovir was prepared using prior knowledge of cytosine arabinoside,
which was isolated from a Florida sponge [14].
The screening of the natural chemicals can be either random where materials are col-
lected from random plants and animals or is based on the traditional knowledge, where
materials are collected from plants and animals with a known function. These materials
are subjected to extract preparation and bioassays to discover the bioactivity. The bioactives
are further extracted and purified using automated systems. Many of the modern pharma-
ceutical industries have become huge economic giants, utilizing the ethnobotanical rich-
ness and diversity of nature for drug explorations.
1.2.2 Gene Prospecting

The advent of modern gene technology offers many opportunities for the selection and
propagation of efficient traits. There have been many products from nature that are in the
market or are close to entering the market. One of the examples of the gene prospecting is
the protein hirudin from the leeches, which has been used as an anticoagulant of blood,
based on the traditional knowledge of the use of leeches in thrombosis and hypertension.
Another example of such protein is from the saliva of a bat named Desmodus rotundus,
which uses this protein to inhibit the coagulation of blood in its preys, preventing throm-
bolytic blood clots and allowing clot‐free drinking. Enzymes have also been used for a
number of purposes by humans such as cheese making, meat tenderization, etc. In the
present world these enzymes have grown their importance in several industries such as
food, textiles, paper, etc. Gene prospecting searches for novel enzymes from natural sources,
and the modern gene technology helps in the production and propagation of these enzymes
at low cost and in abundance. Several important enzymes encoding protein families have
been identified by gene prospecting in recent years which have diverse catalytic potentials.
For instance, a number of studies reported the identification of antioxidant enzymes
encoding gene families from plants including crop plants [15, 16, 17, 18, 19]. The introduc-
tion of novel technologies such as metagenomics, metaproteomics, and metatranscriptom-
ics have allowed the isolation and production of important and useful enzymes even
without the conventional cultivation of the microorganisms.
1.2.3 Bionic Prospecting

Bionics can be described as biologically influenced engineering. It relates to the construc-
tion and building of materials or systems inspired by natural systems. Most of the bionic
prospecting was earlier based on biorational approaches. For example, the waxy coat of the
Lotus flower is presumed to have a role in its self‐cleaning mechanism, inspired by the
flower, similar mechanisms have been used in the buildings and cars to prevent dirt. Novel
architectures, bioengineering, sensor technologies, and bio‐modeling constitute impres-
sive areas of bionic prospecting.
1.3 Significance of Plants in Bioprospecting
Biodiversity, the heterogeneity of life, is multifariously distributed across the globe. Among
all the living beings, plants are the fundamental structural aspects of the terrestrial as well
as marine ecosystems and also are the basis of all food webs. With an estimated 300 000 spe-
cies spread across the world, plants provide key ecosystem organization and primary pro-
duction structure. High plant diversity is presumably linked to a high biotic heterogeneity,
which further leads to a higher probability of specialization potential in different groups [20].
Natural products have been found to be a continuous source of novel pharmaceutical or
other commercial products. Especially notable among these are the natural products from
plants, which remain an everlasting source of many drugs and cosmetics. It is worth men-
tioning that a quarter of all the drugs prescribed today by medical practitioners come from
plants [21]. This approximation indicates the significance of plant‐derived natural products
in the pharmaceutical industry. These plant‐derived natural products have been termed as
botanical therapeutics, which are compounds used to maintain good health and prevent/
treat diseases. Botanical therapeutics can be further classified into a range of plant‐derived
products for several purposes, such as commercial drugs, botanical drugs, dietary supple-
ments, food additives, medicinal and special dietary‐use foods, and cosmaceuticals.
1.4 Pros and Cons of Bioprospectin 5
Commercial drugs include plant‐based single compounds (such as aspirin, paclitaxel and
morphine, etc.), that are used for the diagnosis, cure, mitigation, or treatment of diseases.
Botanical drugs are plant‐based extracts (such as the topical drug polyphenon E, Senokot
etc.) that are used for the diagnosis, cure, and mitigation of diseases. Carrageenan and
garlic extract are the food additives and dietary supplements, respectively, with health
claims. Flavocoxid and Aloe cream are the special dietary‐use food and cosmaceutical,
respectively, which are also derived from plants.
Botanical therapeutic finds its roots in the indigenous knowledge of certain communities
or places. A shrub Tripterygium wilfordii, also known as Thundergod vine was initially used
in the Chinese medicine for providing relief to certain weather‐generated or activity‐
generated symptoms such as joint pains. Later it was found that these plants consisted of
certain inhibitors that prevented the production of certain inflammatory agents and
cytokines, thus preventing inflammation or pain [22]. This effect was later found to be aris-
ing because of the downregulation of certain transcription factors caused by the action of
two diterpenoids [23]. Another plant Artemisia dracunculus also known as Russian tarra-
gon, has been used in culinary and as a medicinal herb since a long time. The ethanolic
extract showed antidiabetic action in mice with type II diabetes. The extract was reported
to enhance insulin‐stimulated glucose uptake in case of insulin‐resistant mice. Further
researches revealed that the extract enhanced the building up of insulin receptor substrate‐2
and a protein kinase (specific for serine‐threonine) while decreased protein tyrosine phos-
phatase (PTP‐1B) levels, both activities are known to correlate with the increase in insulin
sensitivity [24]. Thus, plants have been considered as priceless sources of bioprospecting.
1.4 Pros and Cons of Bioprospecting
Bioprospecting has been a significant method of discovering new drugs and other commer-
cially important compounds since the beginning of the scientific world. A wide range of
drugs have been discovered from plants that are prevalent in the medical field [24]. With
the advent of novel scientific technologies employed in the screening and extraction of
commercially useful compounds from plants and other sources, bioprospecting has pro-
gressed a lot. This rapid progress has been responsible for the immense flow of certain
life‐saving drugs and other commercial or dietary products into the world market. Certain
drugs obtained through bioprospecting have proved to be a boon to the drug industry and
the medical field as well. Drugs such as vinblastine, vincristine, enalapril, etc. have proved
to be significant supports to the respective patients. The recent outbreak of the SARS‐CoV‐2
has also triggered a massive movement in the scientific community to search for efficient
and potential drugs through bioprospecting. Not only does bioprospecting help in obtain-
ing useful products and/or drugs but is also responsible for the economic gains to the host
country holding the indigenous knowledge on which the search was based upon. Apart
from the economic gains to the host country, it is also responsible for economic gains to the
business company and the country hosting the product. The market value of bioprospecting‐
derived herbal medicines alone has been found to have surpassed US$30 billion in the year
2000 [25]. Along with the dissemination of a specific indigenous knowledge to the entire
world, it also provides enormous employment opportunities in different avenues. Thus, on
the whole, bioprospecting contributes to the economic status of the world.
Although bioprospecting has a huge list of advantages, it is not immune to certain

l imitations. Biodiversity and the related indigenous information systems are the actual
foundations of highly biodiverse countries such as India. The embezzlement of the indig-
enous knowledge has increased by the introduction of certain international laws (the
Intellectual Property Rights [IPR] etc.). These traditional knowledge are sometimes stolen
from indigenous communities, countries, or individuals. The term Biopiracy refers to the
violation of the contractual agreement on the control and use of indigenous bioresources
and knowledge without the consent of the local community. Although these international
laws against stealing of traditional knowledge prevail, it often fails to offer sufficient pro-
tection to the traditional knowledge of the underdeveloped and biodiversity‐rich countries.
1.5 Recent Trends in Bioprospecting
Bioprospecting finds its foundations in the traditional knowledge about the uses of cer-
tain natural products. However, in due course of time, it has progressed a lot involving
the current sophisticated technologies in the identification of active compounds, its
extraction and purification, etc. The advent of molecular biology techniques and biotech-
nology has also paved way for the production of these active compounds on a large scale
in industries. Although numerous compounds have been identified from plants that have
bioprospecting values, the immense wealth of nature still remains unexplored, and with
each passing day researchers are busy proving their caliber in discovering these natural
compounds.
Plant itself is a vast kingdom, which includes different subkingdoms and families of
plants that are much more diverse and rich with respect to bioprospecting. Recent stud-
ies from the Angiosperms have revealed certain novel pharmacological aspects of the
compounds present in them. Gallic acid, quercetin and catechin from Psidium guajava
were reported to possess anti‐dengue properties in vitro [26]. A bioactive ingredient of
black pepper, piperine, was reported to have novel pharmacological activities [27]. The
antioxidant and anticancer properties of the aqueous extract of Stryphnodendron
adstringens were also evaluated and reported recently [28]. A genetic and metabolomic
approach has also been sought in recent bioprospecting trends. Recently, the potential
functions of a predicted biosynthetic gene cluster of plants were evaluated by superim-
posing their locations on metabolite quantitative trait loci [29]. Apart from medicinal
aspects, angiosperms were also bioprospected for their insecticidal activities. Several
studies suggested the potential applications of plant lectins as insecticides [30–35].
Plant latex was also reported for its insecticidal or antimicrobial potential [36] and
anthelmintic and antifungal actions [37].
Gymnosperms have also been a useful resource for bioprospecting. The genus Ephedra
from the family Ephedraceae has been reported by many researches to have potential
medicinal, economic, and ecological aspects [38]. Further, the antioxidant, antiprolifera-
tive [39], and antimicrobial effects [40] of Ephedra were also reported The male flowers of
Ginkgo biloba were reported to possess anticancer effects [41]. Inhibitory effects of oligos-
tilbenes in Gnetum latifolium upon neuroinflammation are considered to be a boon for
medicinal sciences [42].
1.6 Omics for Bioprospecting and in silico Bioprospectin 7
Even the liverworts have been found to be useful resources in bioprospecting [43]. Several
studies have also reported liverworts for their commercial importance. A cannabinoid‐like
compound in the genus Radula has been reported to have a similar structure to that of can-
nabis and has been termed as legal high [44]. A novel epi‐neoverrucosane‐type diterpenoid
was also reported in the liverwort Pleurozia subinflata [45].
1.6 Omics for Bioprospecting and in silico Bioprospecting

“Omics” represent a conjugative strategy to high‐throughput analysis of biological entities
for multifarious objectives. It mainly involves lipidomics, genomics, transcriptomics, prot-
eomics, and metabolomics. Plants or their endophytic microorganisms are prone to the
dynamicity of their immediate environment and thus manifest an immense genetic, meta-
bolic, and accompanying physiological variations. Traditional methods employ plant or
microbe collection/isolation, their extractions and performing numerous chemical analy-
ses making the bioprospecting process toilsome, exhaustive, and expensive. Progresses in
modern technologies such as next‐generation sequencing (NGS) have offered a platform
that allows the prediction of the important compounds with the help of the gene sequence.
Analysis of the proteome and metabolome could enhance our understanding of the molec-
ular and biochemical attributes of the plant/microbe under study.
It is of great interest in bioprospecting to describe the genes coding for enzymes having a
significant role in a biosynthetic pathway. Modern NGS has made gene ontology more compel-
ling, that the molecular interactions between the members of the microbiome and the host
can be easily predicted [46]. Elucidation of whole‐genome sequences has enabled us to deci-
pher the biosynthetic gene clusters (BCGs) and provide insights into the potentials of the
genome. It has also provided opportunities to explore various important gene families in
important plants including cereal crops [47, 48, 49]. The metagenome of a decaying wood
biomass was helpful in finding and isolating certain novel glycosyl hydrolases in a recent study
[50, 51]. Müller et al. [52] summarize the discovery of certain bioactive molecules from plant‐
associated microorganisms that have been deciphered with the help of plant metagenomics.
It is vital to gather knowledge on the expression patterns of the genes encoding a signifi-
cant protein involved in a biosynthetic pathway in bioprospecting. Transcriptomic analysis
of the biological entity renders information on the genes expressed under a particular con-
dition. The importance of the transcriptomic information was clearly depicted in the
closely related strains of a halophilic bacterium Salinibacter ruber, where the environmen-
tal sensing genes were downregulated in independent cultures and upregulated in cocul-
ture conditions [53]. Similarly the expression of an antitumor compound astin was found
to occur in a fungal endophyte Cyanodermella asteris residing in Aster tataricus only in
symbiotic conditions, where the plant signals were responsible for triggering the biosyn-
thesis [54]. Another study on the actinobacterium Streptomyces davawensis reported the
involvement of the cre gene homologs (creE and creD) in the production of desferrioxam-
ine only under coculture conditions [55].
Transcriptomics renders information on the expression of a set of genes under certain con-
ditions while whether it is translated to active protein products or not remains doubtful.
Further, the transcriptome data enabled the identification of numerous important long-non
coding RNAs in important plants, which could not be identified earlier [16, 56, 57, 58, 59].
Another omic approach, i.e. proteomics solves this dilemma and allows to know the active
protein products that are translated from the expressed genes. In addition, proteomics using
mass spectroscopy (MS) coupled with certain other techniques allows the researcher to know
the posttranslational modifications in the active protein products [60, 61].
A promising tool used in molecular biology today is the clustered regularly inter-
spaced short palindromic repeats (CRISPR). A strong tool for manipulation of gene
expression and genome editing, CRISPR are a group of bacterial or archaeal sequences.
The CRISPR‐Cas system consists of a CRISPR RNA (crRNA) that binds to a Cas protein
(Cas9 etc.), to form a CRISPR‐Cas complex, which directs cleavage of the DNA or RNA
target sequences. The number and type of CRISPR‐Cas systems are varying in different
organisms [62]. Hence an understanding and identification of such systems in microor-
ganisms can support in the prediction as well as modification of gene expressions for
secondary metabolite production.
Metabolomics is an omic approach where it can be used independently or as a conjuga-
tive technology along with proteomics, lipidomics, or glycomics, where it helps in the elu-
cidation of the respective results obtained. The small molecules known as metabolites
present within the cells, tissues, or organisms interact with one another in a biological
system and are known as the metabolome of the respective biological entity. Metabolomics
aims at deciphering the composition and interactions of the metabolome and its constitu-
ents. Unlike other omic approaches such as genomics, transcriptomics, and proteomics,
metabolomics faces an analytical challenge due to the varying physical properties of the
small molecules [63]. These challenges are overcome with the help of chemistry and other
integrated technologies (such as high‐resolution mass spectroscopy (HR‐MS) and nuclear
magnetic resonance (NMR)), which couple metabolomics [64]. A genetic metabolomic
approach method for bioprospecting plant biosynthetic gene cluster was evaluated recently
by superimposing the BCG location on metabolite quantitative trait loci [29].
On the whole, the bioprospecting territory has seen advances in the technologies involv-
ing single cell and single molecule, which has led to the progress and advancement of bio-
prospecting itself. Recent trends have a bias toward the computational sciences approach
as they are easy, reliable, and time‐saving. This has led to the revolutionization of the mul-
tidisciplinary approach involving all the areas of sciences to decipher the mechanisms of a
single biological entity. Due to the large databases of diverse metabolome and proteome
available today, there is a higher probability of discovering novel drugs or molecules of
commercial importance through the dissemination of this information.
1.7 An Insight into the Book

This book consists of chapters that deal with different aspects of bioprospecting in plants.
Our world is highly biodiverse, and this vast biodiversity unfurls an array of opportunities
for its exploitation commercially, medically, or scientifically. While on the one hand the
higher utilization and exploitation of natural resources for its bioprospecting aspects are
considered a progressive step toward the upliftment of both the scientific knowhow and
economy, on the other hand it can cause an adverse effect on the environment. Therefore,
it is necessary to conserve the resources that are utilized for bioprospecting. The chapter
1.7 An Insight into the Boo 9
titled “Ecological Restoration and plant biodiversity” deals with the need of conservation
and restoration of biodiversity for sustainable development through biochemical and bio-
technological approaches. It is very well known that plants are holobionts and not stand‐
alone entities, which consists of numerous microorganisms residing on or within them.
Among these, the endophytes that reside within the plants have tight associations to them
and also consist of several enzymes that may be useful in their maintenance within their
host plant. These enzymes have also been useful to the human kind in various aspects and
have been a subject of exploration since a long time. Thus, the chapter “Endophyte
enzymes and their application in industries” discusses the enzymes characterized from
diverse endophytes, their current industrial application, and the strategies applied to
increase their yield. Although plants are holobionts and consist of numerous microorgan-
isms residing along with them, not all microorganisms are beneficial to them. Some
microorganisms act as plant pathogens and cause harm to them. Plants have their own
immune system to recognize and fight against the entry of these pathogens. The fight
against the pathogen may trigger in the plants the release of certain secondary metabo-
lites, which have antimicrobial properties and kill the pathogens. The exploration of these
antimicrobial compounds may further be useful in combating several diseases in the
human population. The chapter “Anti‐microbial products from plant diversity” provides
recent insights into the possibilities of the important plant‐derived antimicrobial com-
pounds useful as an alternative to combat infections. Another chapter “Plant bioprospect-
ing for biopesticides and bioinsecticides” discusses the plants that consist of natural
mechanisms to evade pests and insects and further discusses its application in the produc-
tion of biopesticides and bioinsecticides. The primary immune system and the production
of secondary metabolites in plants protect them from biotic stresses such as microbial
pathogens or insects and pests. In addition to these primary defenses, plants have mecha-
nisms to overcome abiotic stresses as well. These include active compounds to fight
against the reactive oxygen species (ROS) compounds such as antioxidants, etc. These
active compounds are present in the plant products and, when included in human diet,
perform the same actions as in plants. The chapter titled “Functional plants as natural
sources of dietary antioxidants” describes the in vitro antioxidant activity of different
kinds of functional plants, including vegetables, fruits, medicinal plants, cereals, flowers,
and microalgae. Another interesting and most popular plant‐derived products that are
prevalent in the dietary intake all over the world are the spices and condiments. The chap-
ter “Plant diversity and ethnobiological knowledge of spices and condiments” discusses
the important chemical constituents that are responsible for the value of the spices and
the current research prevalent in India with respect to spices. In addition to these, plants
and plant‐derived compounds are utilized for various other purposes such as in the phar-
maceutical industry, food industry, and cosmetic industry. The chapter titled “Biodiversity
and plant bioprospecting in cosmetics” outlines various aspects of bioprospecting in the
cosmetic industry addressing the need to keep in line with various ethical guidelines
and enlists different natural products that are in use in the cosmetic industry. Similarly
the chapters “Plants as sources of essential oils and perfumery applications” and
“Bioprospection of plants for essential mineral micronutrients” discuss the utilization of
plant and plant products in obtaining the essential oils and essential mineral micronutri-
ents, respectively. Plants are a part of not only the terrestrial ecosystem but also the marine
ecosystems. In marine systems plants occur in the form of seaweeds and other micro and
macroalgae. For a long time marine sources have been considered as the richest in several
active compounds or commercially important compounds. The chapter “Marine bio-
prospecting: seaweeds for industrial molecules” provides a remarkable insight into the
high value and multimodal activity profile of seaweed extracts or seaweed‐derived mole-
cules, especially industrial molecules. Furthermore, plants are not only used as sources of
food, fodder, medicine, industrial molecules, etc. but are also used as an alternate source
of energy. The chapters “Bioenergy crops as an alternate energy source” and “Biomass to
bioenergy” discuss the utilization of energy crops as an alternate source of energy.
R
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15
Entomotoxic Proteins from Plant Biodiversity to Control

the Crop Insect Pests
Surjeet Kumar Arya1, Shatrughan Shiva2,3, and Santosh Kumar Upadhyay4
1
Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, USA
2
Department of Plant Molecular Biology and Genetic Engineering, CSIR-National Botanical Research Institute, Council of
Scientific and Industrial Research Rana Pratap Marg, Lucknow, India
3
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India
4
2.1 Introduction
Plant biodiversity is in danger due to many natural and man-made materials. We need to
protect its existence from all these culminating threats against the plant growth. These
materials could be predators, pathogens, along with man-made chemicals like pesticides.
New strategy should be designed to limit the use of pesticides and protect plant crops
against these predators and pathogens. In due course of this, the introduction of Bacillus
thuringiensis entomotoxic proteins expressing genes is one of those. This strategy found to
have positive impact in limiting the spread of lepidopteran pest could also have limited
usage due to its antinatural product label by the consumers and biosafety issues in mam-
mals [1]. There was a good review on agricultural land usage by Carlini and Grossi-de-Sá [1]
in which they explained continuous reduction in the average cultivated land per capita and
demands high protein’s production and supplements its need from other animal sources.
In this review, they have also explained the decrease in the agricultural exploitation but
presents a loss as high as 45%, before and after harvesting, due to the attack of a variety of
emerging pests, including nematodes, insects, and virus- and bacteria-induced diseases,
and estimated the loss to cost around 100 billion dollars [1, 2]. The major losses are reported
from arthropods that cause estimated losses around 17.7 billion dollars every year and
methods available to limit its spread are heavily dependent on chemical pesticides [3, 4].
These insect pests cause tremendous losses in agriculture worldwide, which are detrimen-
tal to several crops. In a broad way, the major insects that damage various economically
important crop plants are lepidoptera, coleoptera, and diptera and homoptera. Pesticide
application to control the spread of these pests is costly and has environmental hazardous
effects. So, there is a necessity to perform research and development work to identify the
alternate approach in combating the pests to reduce crop damages and demands for

16 2 Entomotoxic Proteins from Plant Biodiversity to Control the Crop Insect Pests
naturally occurring cheaper and more eco-friendly biopesticides. One of the approaches
that have gained good attention in recent years is the introduction of genes from one source
to the plant system through recombinant DNA techniques. This technique becomes techni-
cally feasible in producing the engineered crops having improved nutritional value and
production of vaccines against the variety of plant diseases. Introduction of efficient
biomolecules into crop plants through this genetic engineering technology made it easier
for providing protection against the insect damages.
This landmark was entrenched about 15 years ago where tobacco plant was engi-
neered to express entomotoxic protein from bacterium Bacillus thuringiensis [5]. There
are about over 180 Bt products registered in U.S. Environmental Protection Agency [6]
and about more than 276 Bt microbial formulations registered in China [7]. In spite of
the robust usage in some of the countries, it has also faced challenges in acceptance
due to biosafety issues in mammals as well as question raised on the ecological impacts
of this newly introduced biopesticide. The consumers are considering it as an antinatu-
ral products mostly due to the crossing of the species barrier [1]. An alternate planning
could be the manipulation of the plant own defense system and introduction of an
insect resistance gene from other plant source into desired damaged crop plant.
Inadequacy of the proper immune system in the plant system compared to animals
made them to produce several structural and chemical defense molecules to combat
the attack of the pest. In this chapter, we have discussed about some of the important
plant defense arsenals known to cause toxic effects against the pest when administered
orally or through transgenic approach. Some of these include, lectins, proteinase inhib-
itors, ribosome-inactivating proteins (RIPs), arcelins, α-amylase inhibitors, and plant
peptides that include defensins, cyclotides, canatoxin-like proteins, ureases and
urease-derived encrypted peptides, chitinases, and proteases.
2.2 Lectins
Plants have evolved with different defense strategies to cope up with the threat from phy-
tophagous insects. The morphological and structural features are the part of defense arse-
nals against the pest. Among these known barriers, the release of chemical compounds
also plays major role as protecting mechanism. These chemical products range from low
molecular weight compounds called secondary metabolites to peptides and proteins [1, 8].
Such defense proteins include plant lectins. They are considered as nonimmune origin
heterogeneous group of proteins with noncatalytic domain that can bind specifically to
carbohydrate moiety and RIPs. It is also considered as multivalent proteins that can agglu-
tinate cells. Distribution of these lectins is from various groups ranging from plants, viruses,
bacteria, invertebrate, and vertebrates, including mammals. These lectins are highly con-
served in the plant kingdom and purified from various plant sources. Seeds are considered
as an importance reservoir of lectins. There are about more than 200 three-dimensional
structures deposited in the 3D Lectin databank, mostly from legume seeds [8]. Different
plant sources lectins exhibited structural similarity in the amino acid homologies that exist
from Canavalia ensiformis (concanavalin A) and from other leguminous seeds, despite dif-
ferences in their carbohydrate-binding specificities.
2.2 Lectin 17
The research on plant lectins was dated 130 years ago. Many biochemist and molecular
biologist started taking interest on carbohydrate-binding ability of this molecule. The first
discovery of a plant lectin was reported by Stillmark from the seeds of castor bean (Ricinus
communis L.) and named as “ricin.” This ricin is shown to agglutinate red blood cells, and
the term hemagglutinin was introduced. This finding gave birth to the word “lectin” which
is derived from “legere,” the larin verb for “to select” [8].
Previously, efforts were made to organize this very heterogenous group of plant lectins
and classified lectins into several specificity group called “natural groups.” This classifica-
tion was based on their ability to recognize and bind-specific sugars [8]. Later this classifi-
cation was considered irrelevant and artificial with respect to evolutionary relationship
between plant lectins. New classification of the lectins was taken under consideration
related to sequence information that have become available in the last decades. Based on
available genome/transcriptome information, lectins are now classified into twelve distinct
families of evolutionary and structurally related lectin domains. These binding domains
were arranged in an alphabetical order staring from Agaricus bisporus agglutinin homologs,
amaranthins, V chitinase homologs, cyanovirin family, Euonymus europaeus agglutinin
family, Galanthus nivalis agglutinin (GNA) family, proteins with hevein domains, jacalins,
proteins with a legume lectin domain, LysM domains, agglutinin family, and andricin-B
family. Each individual lectin domain bears its own characteristics with one or more bind-
ing sites. Moreover, most of these domains are spread all over the plant kingdom [8].
There are many lectins that are present abundantly in seeds or numerous storage tissues
which include bulbs, tubers, bark, or rhizomes [8]. Common examples are different iso-
forms of the Phaseolus vulgaris agglutinin (PHA) produced during seed development
which can accommodate 10% of total seed protein [8]. Other examples of storage tissue
lectins are GNA present in the bulbs of the snowdrop or the Urtica dioicaagglutinin (UDA).
These known lectins accumulate in a certain plant tissues or organs and their synthesis is
independent of external stimulus. One more lectin named NICTABA is shown to be
expressed in tobacco (N. tabacum) leaves when treated with plant hormone methyl jas-
monate and insect herbivory [9–11].
In the last few decades, many nonstorage tissue lectins have been described whose
expression does not involved developmental regulation instead controlled by environmen-
tal stimulus like certain biotic and abiotic stress conditions such as insect herbivory, cold or
high salt concentration, wounding, and drought. This group of lectins is now referred as
“inducible plant lectins,” and their non-storage tissues include leaves, roots, or flowers.
Mannose-specific jacalin-related lectin is the first example of inducible lectin which accu-
mulates from salt-induced rice seedlings (Oryza sativa) called ORYSATA [12, 13].
Basically, plant lectins are classified into four major groups due to the presence of domain
architecture and differentiated as merolectins, hololectins, chimerolectins, and superlec-
tins [8]. Hololectins are majorly isolated, and well-characterized lectins that are composed
of two or more than two identical carbohydrate-binding domains which agglutinate cells.
In chimerolectins, carbohydrate domain fused with each other and shows biological activi-
ties irrespective of domain organizations. Sequences analysis of plant genomes reveals
abundant presence of chimerolectinsOpposed to other lectins, superlectins recognize
structurally nonrelated structures with their more than two carbohydrate domains.
Many lectins reported to be toxic to numerous insect orders belongings to economically

important pest groups such as Lepidoptera, Coleoptera, Diptera, or Hemiptera. Since from
last two decades, insecticidal properties of the lectins have been exploited to render crop
plant resistance to insect attack. One example of these include extraction and expression of
the lectin in crop plants in the development of transgenic tobacco expressing pea lectin that
showed reduced growth of the Heliothis virescens (Fabricius) [1, 14]. Other examples
include the mannose specific lectin isolated from pea expressed in nine food crops found to
be toxic against C. maculatus [15]. Previously, experiment was conducted to test the insec-
ticidal properties of the lectins on artificial diet delivering the purified lectins. Examples of
these include screening of the 25 lectins extracted from 15 different plant families and
tested against the legume pod borer [16].
The lectins purified from bulbs of Amaryllidaceae species such as snowdrop (Galanthus)
or daffodils (Narcissus) showed protection of bulbs against the pest. The best studied
plant lectin, GNA which was purified from snowdrop bulbs shown to have toxicity
against wide range of insect orders especially, Hemiptera. This GNA has been engineered
successfully in many varieties of crops like as rice, wheat, potatoes, or tobacco to provide
resistance against the agricultural important pest. Pea lectin purified from PSA was eval-
uated on the growth and survival of the pollen beetle (Meligethes aeneus) [17]. This pea
lectin reported to have caused reduced in mass gain for larvae but had no effect on the
adult beetles as was reported by Lehrman et al. [18]. To further confirm the toxicity of
several legume lectins for Coleoptera, legume lectin purified from jackbean concanavalin
A (ConA) has shown toxicity against Hemipteran pea aphid (Acyrthosiphon pisum) [19,
20]. This result clearly indicates that selectivity of a plant lectin against the pest irrespec-
tive of binding specificity.
In the past decades, there are many other lectins that have showed entmotoxic
properties apart from lectins discussed above. Examples of these plant lectins include
leaf lectin, NICTABA expressed after folivory have toxic effect against insect’s pest.
Ectopical expression studies of NICTABA using transgenic tobacco based on feeding
trials clearly explained that NICTABA is toxic against the larvae of two Lepidopteran
insect, the tobacco hornworm (Manduca sexta) and cotton leafworm (Spodoptera litto-
ralis) [11]. Another protein belonging to the NICTABA family named as protein 2 (PP2)
reported to show entomotoxic effect [21, 22]. Group of lectins with a ricin domain
called cinnamomin expressed in seeds of the camphor tree (Cinnamomum camphora)
shown to have toxic effect on mosquito (Culex pipienspallens) and bollworm (Helicoverpa
armigera) [23]. There are other lectins that shown to have toxic effect as insect control
agents against sap-sucking pest insects are amaranthins and the jacalin-related lectins.
There was report of ectopic expression of Amaranthus caudatus agglutinin (ACA)
expressed under phloem-specific promoter showed enhanced resistance against the
nymphs of cotton aphid (Aphis gossypii) [24].
Another promising jacalin-related lectin was used in development of transgenic crop
plants, called HFR1 shown to produce entomotoxic effect against Hessian fly during
infestation on wheat (Triticum aestivum) [13, 25]. Various Allium species have also been
screened for the isolation of mannose-binding lectins having insect toxic activities
[26–29, 208]. There are several other important lectins having entomotoxic potential
against various important insect pests of agriculture which are listed in Table 2.1.
Table 2.1 List of plant lectins having insecticidal properties.
Nature Plant Plant Common

Lectins of protein biological name common name Tissue Plant family Target insect name-insect Insect order Application Reference
Wheat germ GlcNac Triticum Bread wheat Seeds Poaceae Callosobruchus Cowpea seed Coleoptera Effects on both [30]
agglutmm aestivum maculatus beetle developmental
(WGA) time and causes
larval mortality
Recinous lectin GalNac/Gal Ricinus Castor bean Seeds Euphorbiaceae Diabrotica Spotted Coleoptera Inhibit larval [31]
communis undecimpunctata cucumber growth and highly
beetle causes mortality
Eranthis lectin Eranthis Winter Bulbs Ranunculaceae Diabrotica Spotted Coleoptera Inhibits insect [32]
(EHL) hyemdis aconite undecimpunctata cucumber larval growth
beetle
Griffonia Glc/Nac Griffonia Griffonia Seeds Fabaceae Callosobruchus Cowpea seed Coleoptera Proteolytic [33]
simplicifolia leaf simplicifolia maculatus beetle degradation by
lectin II (GSII) Cowpea bruchid
midgut extracts
Galanthus nivalis Galanthus Snowdrop Seeds Amaryllidaceae Sitobion avenae English Homoptera Decrease insect [34]
agglutinin nivalis grain aphid fecundity but not
(GNA) affect survival
Artocarpus Artocarpus Wild jack Seeds Moraceae Tribolium Red flour Coleoptera Grorth inhibition [35]
lectin hirsutus castaneum beetle of insect larvae
ASA (Allium Mannose- Allium sativum Garlic Bulbs Amaryllidaceae Dysdercus Red cotton Hemiptera Highest mortality [36]
sativum) binding lectin cingulatus bug in insect larvae
(WsMBP1)
CEA (Colocasia Colocasia Taro Leaves Araceae Dysdercus Red cotton Hemiptera Affect insect [36]
esculenta) esculenta cingulatus stainer larval growth and
development
DEA Differenbachia Dumbcane Leaves Araceae Dysdercus Red cotton Hemiptera Affect insect [36]
(Differenbachia sequina koenigii stainer larval growth and
sequina) development
(Continued)
0005092140.INDD 19 06-03-2021 18:49:59

Table 2.1 (Continued)
Nature Plant Plant Common

Lectins of protein biological name common name Tissue Plant family Target insect name-insect Insect order Application Reference
Parigidin-br1 Bracelet Palicourea Bate-caixa Leaves, Rubiaceae Diatraea Sugarcane Lepidoptera Disrupts insect [37]
subfamily of rigida inflorescences, saccharalis borer cell membranes
cyclotides and peduncles
Wheat germ Triticum Bread wheat Seeds Poaceae Helicoverpa zea Corn Lepidoptera WGA causes [38]
agglutinin aestivum earworm growth inhibition
(WGA)
PF2 Lectin Olneya tesota Desert Seeds Fabaceae Zabrotes Mexican Coleoptera PF2 interferes with [39]
ironwood subfasciatus bean weevil starch digestion in
insect gut
Tma12 Chitin- Tectaria Fronds and Tectariaceae Bemisia tabaci Whitefly Hemiptera Number of eggs, [40]
binding and macrodonta rhizomes egg-laying pattern
chitinase and nymphal
activity development were
severely affected
Withania Mannose- Withania Ashwagandha Leaves Solanaceae Hyblaea puera Teak Lepidoptera Delay in growth [41]
somnifera lectins binding lectin somnifera defoliator and
(WsMBP1) metamorphosis,
decreased larval
body mass and
increased mortality
Withania Withania Ashwagandha Leaves Solanaceae Probergrothius Indian red Hemiptera Delay in growth [41]
somnifera lectins somnifera sanguinolens bug and
metamorphosis,
decreased larval
body mass and
increased mortality
Soursop lectin Carbohydrate- Annona Soursop Seed kernel Annonaceae Chironomus Lake flies Diptera Mosquito [42]
binding muricat costatus larvicidal
proteins
0005092140.INDD 20 06-03-2021 18:49:59

2.3 Proteinase Inhibitor 21
2.3 Proteinase Inhibitors
Plant proteinases belongs to family Leguminosae (legumes), Solanaceae (nightshades), and

Poaceae (Grasses) and considered as an important components of plants secondary metabo-
lites. These proteinase inhibitors (PIs) target insect gut proteases upon insect herbivory. Plants
engineered with PI defensive gene under inducible or constitutive promoter could be a valuable
methods in comparison to genes identified from complex pathways which enhances resistance
to insect pests [43, 44]. It has been reported that plant PIs target almost all the insect digestive
proteases of different classes. During insect herbivory, the expression of plant PIs found to be
upregulated even in the presence of endogenous plant proteases [45–47].
The classification of PIs based on reactive amino acid residue in the active site that
include (i) serine protease inhibitors or Serpin present in the Bowman-Birk inhibitors
(BBIs) family, squash inhibitors, Kunitz family, cereal trypsin/amylase inhibitors, (MSI),
potato type I (PI 1), and potato type II protease inhibitors (PI 2), mustard (Sinapis) trypsin
inhibitors; (ii) cysteine protease inhibitors (Cystatins); and (iii) aspartyl and metallocar-
boxypeptidase inhibitors [44, 48].
These PIs played a crucial role of plant defense against insect herbivory [44, 49, 209].
Green and Ryan proposed around four decades ago that during attack of potato beetles, a
rapid accumulation of PIs in potato or tomato was observed locally as well as systemi-
cally [45]. Few years later, [50] reported transgenic tobacco expressing PIs by using plant
genetic transformation methods. In this transgenic crop, plant was transformed with con-
struct having PI-encoding genes from cowpea (Cowpea trypsin inhibitor, CpTI) linked to
CaMV 35S promoter. After that, there was lot of research papers coming out that estab-
lished the concept of overexpression of both native and foreign PI in plants that propelled
such studies of transgenic plant development upon insect feeding [51–53].
There is a diverse group of plant PIs that differ mechanistically in their structure against
the proteases [54]. Previously, the method of PIs classification was based on their protease
specificity. Now a days, it has been added more with sequence information and their
3D-structures [55] In plants, numerous roles of PIs have been described that include their
action as storage proteins, endogenous proteolytic activity regulators [43], part of develop-
ment processes including programmed cell death [56], and as resistance moiety against
insect and pathogens to protect plants [1, 57, 58].
Setting up insecticidal bioassay comprising of dietary supplementation of selected PIs
which involved either uptake by feeding of purified PIs to insect in combination with arti-
ficial diet or by overexpression of the construct in the development of transgenic plants.
Increased mortality was observed while delivering the PIs in an artificial diet or transform-
ing plants with overexpressing construct [59, 60] that resulted in retarded growth and
development of larvae belonging to various insect orders [44, 54, 61–64].
These plant PIs are known to inhibit the proteolytic enzymes of insect guts resulting in
reduced fecundity, extended developmental period, and increased mortality due to the
absence of desired amino acid residues. Upon dietary PIs feeding, it is known to start feed-
back mechanism that leads to compensatory enzymatic hyperproduction of digestive pro-
teases that have caused reduced amount of essential amino acid intake by the insects [43,
44, 65]. Biochemical examination of protease activity reveals substantial molecular adjust-
ments in the midgut cells, to counter dietary stress caused on PI feeding [44, 66, 67]. Other
protease inhibitors have also played a critical role against the insect to cause significant
mortality and growth hindrance (Table 2.2).
Table 2.2 List of plant protease inhibitors with insect toxic activity.
Plant protease Nature Plants Plant Action Insect

inhibitor of protein Plant species common name part used Plant family against insect common name Order Application Reference
Potato type I Serine protease Solanum tuberosum Potato Leaves Solanaceae Manduca sexta Carolina Lepidoptera Inhibit insect laeval [52]
inhibitor inhibitor sphinx moth growth and
(StPin1A) + potato development
type II inhibitor
(NaPI)
Potato proteinase Serine protease Solanum tuberosum Potato Leaves Solanaceae Sesamia Pink stem Lepidoptera Inhibit insect laeval [51]
inhibitor II (PINII) inhibitor inferens borer growth
gene (pin2)
Cowpea protease Serine protease Fragaria × ananassa Strawberry Stem Rosaceae Otiorhynchus Vine weevi Coleoptera Weevils had not [68]
trypsin inhibitor inhibitor tissue sulcatus developed to the adult
gene (CpTi) stage
Mustard trypsin Serine protease Brassica napus Oilseed rape Seeds Brassicaceae Spodoptera Cotton Lepidoptera Growth inhibition of [69]
inhibitor (MTI-2) inhibitor littoralis leafworm insect larvae
Maize proteinase Serine protease Glycine max Soybean Seeds Fabaceae Chilo Striped rice Lepidoptera Affect larval growth and [70]
inhibitor MPI inhibitor suppressalis stemborer insect gut proteinases
Arabidopsis Serine protease Nicotiana alata Jasmine Leaves Solanaceae Epiphyas Light brown Lepidoptera Affects the growth and [71]
thaliana Serpin1 inhibitor tobacco postvittana apple moth development of larvae
(atserpin1)
Soybean PI (Kunitz Serine protease Variety conquista Soybean Seed Fabaceae Scheloribates Soil mite Oribatida Reduce insect survival [72]
and BBPI) inhibitor praeincisus rate
N. attenuata trypsin Trypsin Nicotiana attenuata Coyote tobacco Seeds Solanaceae Manduca sexta Tobacco Lepidoptera Inhibit insect gut [73]
proteinase proteinase hornworm proteinase which
inhibitors (NaTPIs) inhibitors significantly reduce
larval performance
SaPIN2a Serine protease Solanum American Leaves Solanaceae Helicoverpa Cotton Lepidoptera Reduction in the larval [74]
inhibitor americanum black and stems armigera bollworm weight of H. armigera
nightshade
BWI-1a (ISP) Serine protease Fagopyrum Buckwheat Seeds Polygonaceae Phalaena Tortrix moth Lepidoptera Inhibit growth of larvae [75]
inhibitor esculentum viridana
0005092140.INDD 22 06-03-2021 18:50:00

AtSerpin1 Serine protease Arabidopsis Arabidopsis Leaves Brassicaceae Spodoptera Cotton Lepidoptera Inhibit insect digestive [76]
inhibitor thaliana littoralis leafworm proteases
PA1b (Pea Albumin Inhibitory Pisum sativum Pea Seeds Fabaceae Sitophilus Grain weevil Coleoptera Ability to kill cereal [77]
1, subunit b) cysteine-knot granarius weevils
(ICK)
Barley cystatins Cysteine protease Hordeum vulgare Barley Leaves Poaceae Myzus persicae Green peach Hemiptera Causes proteolytic [78]
(HvCPI-6) inhibitor aphid digestion in aphids
L. bogotensis Cysteine protease Lupinus bogotensis Lupinus Seeds Fabaceae Meloidogyne Southern Tylenchida Inhibit growth and [79]
aspartic protease inhibitor incognita root-nematode development
inhibitor (LbAPI)
oryzacystatin II Serine protease Oryza sativa Rice Leaves Poaceae Leptinotarsa Colorado Coleoptera Reduce larval weights [80]
proteinase inhibitor inhibitor decemlineata potato beetle but mortality
significantly not affected
Capsicum annuum Serine protease Capsicum annuum Bell peppers Leaves Solanaceae Helicoverpa Cotton Lepidoptera Growth retardation in [81]
proteinase inhibitor inhibitor armigera bollworm insect larvae
(CanPI7)
Kunitz trypsin Serine protease Arabidopsis Arabidopsis Leaves Brassicaceae Tetranychus Red spider mite Trombidiformes Increase mite mortality [82]
inhibitor (AtKTI4 inhibitor thaliana urticae and reduce mite
and AtKTI5) reproduction
Protease inhibitor Trypsin Allium sativum Garlic Peeled Amaryllidaceae Aedes aegypti Yellow fever Diptera Inhibition of gut [83]
from Allium sativum proteinase garlic mosquito proteases
“garlic” (ASPI) inhibitors bulbs
Barley protease Serine protease Hordeum vulgare Barley Leaves Poaceae Myzus persicae Green peach Hemiptera Reduce nymph [84]
inhibitor CI2c inhibitor aphid production per aphid
during the lifespan
Cystatin TaMDC1 Cysteine protease Triticum aestivum Wheat Seedling Poaceae Leptinotarsa Colorado Coleoptera Inhibit insect larval [85]
inhibitor decemlineata potato beetle growth
Cowpea trypsin Serine protease Vigna unguiculata Cowpea Seeds Fabaceae Callosobruchus Cowpea weevil Coleoptera Act on the digestive [59]
inhibitor (CpTI) inhibitor maculatus enzymes of insects
0005092140.INDD 23 06-03-2021 18:50:00

α-Amylase Inhibitors
2.4
Being expressed in seeds as storage protein plant proteinases inhibitors considered to be

part of constitutive and inducible defense mechanism against the attack of various biotic
factors like pests and pathogens [86, 87]. These inhibitors work by regulating its action
on insect gut proteinases and α-amylases by disrupting the proper digestion of plant pro-
teins and starch. The role of α-amylases from insect and mammals has been well described
in the point of biochemical, molecular, and structural view in many reported litera-
tures [1, 88–90]. These enzymes present in the gut of insect when they feed on seed prod-
ucts. It is found to be highly conserved as an important molecule. When the action of
these molecules is impeded by any inhibitors could cause decrease in the energy of the
insect. These inhibitors are particularly abundant in various plant sources like [91–94]
legumes [88, 95, 96]) as a part of plant defense mechanism. In addition to that, plant
inhibitors are gaining interest now a days as tool to engineered resistance against pests in
transgenic plants [1, 97, 98].
In the last decades focused has been given to lectin-like inhibitors identified in the common
bean P. vulgaris seeds, which was found to have toxic effect against the insects [1, 99, 100]. aAIs
identified in Phaseoulus seeds belongs to protein family which already have two other defensive
proteins called phytohemagglutinin (PHA) and arcelins (Arc) [1, 101].
Diversity of a-AIs is very vast with different sources and reported to block the diges-
tive enzyme action on mammals and insects [102]. These molecules known to inhibit
the action of alpha-amylases are identified from various sources like wheat, Indian fin-
ger millets, barley, and Amaranthus paniculatus against the insect of particular order
Coleoptera [103, 104]. Many transgenic crops expressing a-AIs have been developed that
known to inhibit digestion of alpha amylases against three burchids, Bruchus pisorum
(pea weevils), Callosobruchus maculatus (cowpea weevils), and C. chinensis (adzuki
weevils) [105, 106].
There are many types of α-AIs that have been reported are known to classify as proteina-
ceous and non-proteinaceous. In the case of proteinase inhibitors, six classes were coming
from higher plants and two were reported as Tendamistat from Streptomyces tendae and
Helianthamide from Stichodactyla helianthus. Six different α-Ais classes include lectin-
like, knottin-like, cereal-type, Kunitz-like, γ-purothionin-like, and thaumatin-like [93,
107]. These reported classes of inhibitors have diversified action on alpha amylase based on
their different structure and mode of action. In the case of non-proteinases inhibitors, they
are mostly organic compounds that can act as substrate analogs and inhibit α-amylases,
e.g., acarbose.
Based on biochemical studies, these inhibitors reported to possess diverse specificities
with various amylases. There are several factors involved to cause differential action and
interactions. These differences could be assigned to variations in the sequences and struc-
tural modification in α-amylases and α-AIs.
The plant α-AI diversity was also found across different species, most of them are par-
ticularly specific to one family. Examples of this is Ragi bifunctional inhibitor (RBI), which
is mostly expressed in cereals and could have the ability to inhibit mammalian as well as
insect α-amylases. Examples of many more α-AI are present in Table 2.3.
Table 2.3 Amylase inhibitors from various plants having insect toxic potential.
Nature Plants Plant Action Insect

Protein name of protein Plant species common name part used Plant family against insect common name Order Application Reference
AmI1 and AmI2 α-Amylase Triticum Wheat Grains Poaceae Tenebrio molitor Mealworm Coleoptera Affect larval midgut [108]
inhibitor aestivum
Wheat 0.28 Monomeric Triticum Wheat Seeds Poaceae Tenebrio molitor Mealworm Coleoptera Affect growth of insect [109]
proteins aestivum beetle
inhibitor
WDAI-3 Dimeric Triticum Wheat Grains Poaceae Tenebrio molitor Mealworm Coleoptera WDAI-3 more active [109]
inhibitors turgidum beetle against Tenebrio
molitor than other
insect
SIα1, SIα2 and SIα3 Sorghum bicolor Sorgham Seed Poaceae Melanoplus Locust Orthoptera Inhibit insect gut [110]
differentialis α-amylases
Wheat WRP24 Dimer Triticum Bread wheat Wheat Poaceae Tribolium Red flour beetle Coleoptera WRP24 suppressed [92]
protein aestivum kernels castaneum larval growth by more
inhibitor than fourfold
Wheat WRP25 Triticum Bread wheat Wheat Poaceae Sitophilus Rice weevil Coleoptera Substantial weight [92]
aestivum kernels oryzae loss
Wheat WRP26 Triticum Bread wheat Wheat Poaceae Tenebrio molitor Mealworm Coleoptera Inhibite α-amylases of [92]
aestivum kernels Mealworm beetle
Wheat WRP27 Monomeric Triticum Bread wheat Wheat Poaceae Sitophilus Rice weevil Coleoptera Strongly inhibiteonly [92]
proteins aestivum kernels oryzae the rice weevil
inhibitor a-amylases
PAI Cajanus cajan Pigeonpea Seeds Fabaceae Helicoverpa Cotton bollworm Lepidoptera Increased mortality [111]
armigera and adverse effects on
larval growth and
development
AMY2 Hordeum vulgare Barley Malt Poaceae [112]
Wheat 0.19 Triticum Wheat Seeds Poaceae Acanthoscelides Bean weevil Coleoptera Inhibit insect [93]
aestivum obtectus α-amylase activity
(Continued)
0005092140.INDD 25 06-03-2021 18:50:00


Protein name of protein Plant species common name part used Plant family against insect common name Order Application Reference
Wheat 0.53 Triticum Wheat Seeds Poaceae Tenebrio molitor Mealworm Coleoptera Inhibit Tenebrio [93]
aestivum beetle molitor α-amylase
activity
BIII (rye) Bifunctional Secale cereale Rye Endosperm Poaceae Zabrotes Mexican bean Coleoptera More effective against Iulek
α-amylase/ subfasciatus weevil insect α-amylases than et al. [113]
trypsin against mammalian
inhibitors enzymes
Zeamatin α-amylase Zea mays Corn Poaceae Seeds Tribolium Red flour beetle Coleoptera Inhibit α-amylase [114]
and trypsin castaneum activity
inhibitor
α-AI1 Endo- Phaseolus Common bean Seeds Fabaceae Callosobruchus Cowpea seed Coleoptera Enzyme inhibitors [115]
amylases vulgaris maculates beetle impede insect
digestion through
action on insect gut
digestive α-amylases
α-AI2 Endo- Phaseolus Common bean Seeds Fabaceae Zabrotes Mexican bean Coleoptera Enzyme inhibitors [115]
amylases vulgaris subfasciatus weevil impede insect
digestion through
action on insect gut
digestive α-amylases
RBI Inhibits both Eleusine Finger millet Seeds Poaceae [116]
α-amylase coracana
and trypsin
A. hypochondriacus α-Amylase Amaranthus Prince-of-Wales Leaves, Amaranthaceae Callosobruchus Adzuki bean Coleoptera Affect the growth of [103]
α-amylase inhibitor inhibitor hypochondriacus feather inflorescence chinensis weevil C. chinensis
(AhAI)
0005092140.INDD 26 06-03-2021 18:50:00

2.5 Ribosome-Inactivating Proteins (RIPs 27
2.5 Ribosome-Inactivating Proteins (RIPs)
RIPs found widely in plant species and within different tissues, and it is considered to have
toxic effect due to the presence of N-gulcosidases [117]. This N-glycosidase depurinate the
eukaryotic and prokaryotic rRNAs, thereby arresting the synthesis of protein during trans-
lation. RIPs are demonstrated to have antifungal, antibacterial, antiviral, and insecticidal
activities in various in vivo; and transgenic plant experiments [117–121]. These studies
have provided a vast knowledge to comprehend the biochemical and medicinal properties
of RIPs. There were many research articles, and reviews were published that explained its
importance [117, 122–124].
RIPs have been divided into three main types on the basis of physical properties such as
type I, type II, and type III [117, 125, 126]. There are about various kinds of RIPs that have
been reported that cover around 17 plant families and showed its presence in bacteria,
fungi, and algae as well [117, 122]. Many identified RIPs belong to numerous small group
of plant species, such as Rosaceae, Caryophyllaceae, Euphorbiaceae, Cucurbitaceae,
Sambucaceae, Poaceae, Phytolaccaceae, and Rosaceae [117, 122, 127, 128].
Trichosanthrip, was designated as novel RIP, was initially purified from mature seeds
Trichosanthes kirilowii. This RIP effectively inhibits cell-protein synthesis [129]. RIPs
have been identified to be located in various plant tissues like leaves, seeds, roots, and
tubers [129]. Certain bacteria produces RIPs have shown to have enzymatic activity
similar to plant analogs like type II RIPs Shiga toxin type 1 (Stx1) and Shiga toxin type 2
(Stx2) [117, 130, 131]. Many studies have proven that RIPs possess insecticidal activities
upon insect attack that includes Lepidoptera [117, 132–135] and Coleoptera [117, 136].
This RIPs reported to enhance plant resistance against the insects [117, 137]. Artificial
diet experiment was conducted to confirm the insecticidal activity of RIPs against the
pest, by supplementing the diet with variable concentration of RIPs, such as type II RIP
from Sambucus nigra, which have caused decreased in the fecundity and survival of
Acyrthosiphon pisum [138]. There was another experiment in which transgenic plant
overexpressing the RIPs SNA-I had caused the retarded development and decreased sur-
vival upon insect feeding by Myzus nicotianae [138]. In addition to this, when diet was
supplemented with different type-1 RIPs had caused decreased survival and fecundity in
Anticarsia gemmatalis Hübner and Spodoptera furgiperda [117, 139]. In 2016, there was
a study that showed type-I and type-II RIPs insecticidal properties from the Apple
(Malus domestica Borkh) against the pea aphids which have caused reduction in nymph
survival [140]. RIPs overexpression have also detected to cause resistance against
Helicoverpa zea [134]. More to this, report from maize resistance to feeding by Spodoptera
frugiperda and corn earworms (Helicoverpa zea) was dedicated to maize ribosome-inac-
tivating protein (MRIP) and wheat germ agglutinin (WGA) [38]. Overexpression of type
I and type II RIPs has also produce resistance in tobacco plants against the insect pest,
Spodoptera exigua [141].
The mechanism of RIPs action is still now clear, and several studies explained that RIPs
can also take part in the apoptosis process 117, 142, 143]. Examples of this were reported
from feeding experiment on A. pisum supplemented with SNA-I-induced apoptosis in the
midgut through caspase-3 activation [117, 144]. Other important RIPs are shown in
Table 2.4.
Table 2.4 List of ribosome inactivating and other related proteins with insect toxic activity.
Plants Plant Action Insect

Proteins Nature of protein Plant species common name part used Plant family against insect common name Order Application Reference
Ricin Carbohydrate- Ricinus Ricinus Seeds Euphorbiaceae Callosobruchus Cowpea seed Coleoptera Highly potent toxin to [136]
binding protein communis maculatus beetle cowpea seed beatle
Cinnamomin rRNA N-glycosidase Cinnamomum Camphorwood Seeds Lauraceae Helicoverpa Cotton Lepidoptera Inhibition of protein [23]
activity camphora armigera bollworm synthesis in bollworm
larvae
Culex Mosquito Diptera Inhibit protein [23]
pipinespallens synthesis in insects
Maize RIP rRNA N-glycosidase Zea mays Maize Seeds Poaceae Helicoverpa zea Corn earworm Lepidoptera Increase mortality and [134]
activity reduced weights
Type II RIP Ribosome- Cinnamomum Camphorwood Seeds Lauraceae Bombyx mori Silkworm Lepidoptera Inhibit protein [135]
inactivating protein camphora synthesis in insects
Culex Northern house Diptera Inhibit protein [135]
pipienspallens mosquito synthesis in insects
Maize RIP rRNA N-glycosidase Zea mays Maize Seeds Poaceae Lasioderma Cigarette beetle Coleoptera Increase mortality and [132]
activity serricorne reduction in feeding
SNA-I Ribosome- Sambucus Black elder Bark Adoxaceae Acyrthosiphon Pea aphid Homoptera Reduced survival and [138]
inactivating protein nigra L pisum fecundity
Myzus nicotianae Tobacco aphid Hemiptera Reduced survival and [138]
fecundity
Saporin Ribosome Saponaria Bouncing Betty Seeds Caryophyllaceae Anticarsia Lelvetbean Lepidoptera Highly toxic and [139]
inactivating protein officinalis gemmatalis caterpillar induced mortality
Spodoptera Fall armyworm Lepidoptera Highly weight loss [139]
frugiperda
PAP-S rRNA N-glycosidase Phytolacca American Leaves Phytolaccaceae Anticarsia Lelvetbean Lepidoptera Highly toxic and [139]
activity americana pokeweed gemmatalis caterpillar induced mortality
frugiperda
0005092140.INDD 28 06-03-2021 18:50:00

Lychnin Ribosome- Lychnis Flower of Seeds Caryophyllaceae Anticarsia Lelvetbean Lepidoptera Highly toxic and [139]
inactivating protein chalcedonica Bristol gemmatalis caterpillar induced mortality
frugiperda
Gelonin rRNA N-glycosidase Gelonium False lime tree Seeds Euphorbiaceae Anticarsia Lelvetbean Lepidoptera Highly toxic and [139]
activity multiflorum gemmatalis caterpillar induced mortality
frugiperda
Momordin Ribosome Momordica Bitter melon Seeds Cucurbitaceae Anticarsia Lelvetbean Lepidoptera Highly toxic and [139]
inactivating protein charantia gemmatalis caterpillar induced mortality
frugiperda
Maize Ribosome- Zea mays Maize Seeds Poaceae Spodoptera Fall armyworm Lepidoptera High mortality to [38]
ribosome- inactivating protein frugiperda spodoptera
inactivating
protein
(MRIP)
Type-1 RIP rRNA N-glycosidase Malus Apple Leaves Rosaceae Acyrthosiphon Pea aphid Homoptera Reduction in [140]
activity domestica pisum fecundity, intrinsic
rate of increase, net
reproductive rate and
doubling time of the
insect population
Type-2 RIP Catalytic activity Malus Apple Leaves Rosaceae Myzus persicae Green peach Hemiptera Reduction in [140]
and lectin-binding domestica aphid fecundity, intrinsic
properties rate of increase, net
reproductive rate and
doubling time of the
insect population
Type-1 RIP Malus Apple Leaves Rosaceae Spodoptera Beet armyworm Lepidoptera Highly entomotoxic [140]
and Type-2 domestica exigua activity causes 78%
RIP mortality during the
larval stage
0005092140.INDD 29 06-03-2021 18:50:00

2.6 Arcelins
Arcelin (Arc) is a carbohydrate-binding insecticidal proteins mostly found in noncultivated

wild type accessions of the common beans. These insecticidal proteins shown to confer
resistance against burchid beetles. In some of the tropical and subtropical countries, the
common beans suffer post-harvest losses which are primarily caused by the bruchids pests,
Acanthoscelides obtectus and Zabrotes subfasciatus. It is considered as the novel storage
protein in the common bean seeds in addition to phaseolin and phytohemagglutinin. Till
now, seven arcelin variants have been identified that are designated as arcelin 1 to arcelin
7, among these 1 and 5 shown to provide resistance in leguminous crops [145–148]. Highest
resistance to the Mexican bean weevil in wild type, Phaseolus vulgaris accessions were
observed due to the presence of these two variants [1, 149]. These types of high resistance
accession were only maintained in lines generated from crossing between arcelin-1 or arce-
lin-5 parents.
Arcelin 1 shown to have insecticidal activity observed during feeding experiments with
artificial seeds [150]. It was realized that the composition of artificial diets differs to several
parameters from that of arcelin-containing beans, the amount of arcelin as a percentage of
total protein, and the arcelins by phaseolin ratio. The effectiveness of arcelin-5 was tested
through artificial diet against Z. subfasciatus larval development or through transgenic
seeds in which high expression of arcelin-5 was observed.
In comparison to other arcelin, Arcelin-1 and 5 variant was discovered later and not
considered for first series of the breeding experiments [1] and artificial seed assays [150].
Arcelin-5 was to shown be present in the wild-type G02771 accession and consists of three
polypeptides (Arc5a, Arc5b, and Arc5c), which are organized as monomers and dimers in
their native states [151, 152]. Arcelin-5 is released due to the expression of two genes,
namely, arc5-1, that codes for Arc5a and arc5-II which is further encoded by the two genes,
namely Arc5b and Arc5c [151, 153]. The insecticidal activity of arcelin-5 was checked
in vivo; and feeding assay indicated the presence of arcelin-5. Arcelin considered to be
weak lectins despite having sequence homology. The sugar-binding specificity of the arce-
lin-1 was significantly differs from those of PHA-L and PHA-E. These differences between
arcelin-1 and lectin were reported to be come from substitution or deletions of essential
amino acid residues during metal and sugar recognition [154].
Arcelin usage as insecticidal has gain attention toward bruchids pests as well as its inhib-
itory role on larval development of Z. subfasciatus was highly appreciated [1, 150, 155].
There are other wild accessions that have showed little resistance against some of impor-
tant bean burchid, like Acanthoscelides obtectus [155, 156]. The mechanism of action of
arcelin still contentious, despite having good research on it, was considered toxic by
Osborni et al. [150] and could be referred indigestible that caused larval starvation [1, 149].
It was also postulated that Asn-linked glycans of arcelin-1 could be the reason of toxicity
through its binding affinity toward lectins [154]. Few research were conducted to under-
stand why arcelin is an insecticidal moiety against Z. subfasciatus but not for A. obtectus.
The experiment explained that arcelin-1 disrupted the midgut epithelial structures in
Z. subfasciatus which was not possible in midgut of A. obtectus [1, 156]. There are many
other reported arcelins that are present in Table 2.5.
Table 2.5 Various plants arcelins with insect inhibitory functions.

Arcelins of protein Plant species common name part used Plant family against insect common name Order Application Reference
Bean arcelin Phaseolus Common bean Seeds Fabaceae [147]

vulgaris
Arcelin-1 One of the four Phaseolus Common bean Seeds Fabaceae Callosobruchus Cowpea weevil Coleoptera Inhibited the [101]
electrophoretic vulgaris maculatus development of larvae
variants of of Callosobruchus
arcelin maculatus
Arcelin-4 Phaseolus Common bean Seeds Fabaceae Acanthoscelides Bean weevil Coleoptera Causes larval growth [157]
vulgaris obtectus inhibition and
mortality
Arcelin-5 Glycoproteins Phaseolus Common bean Seeds Fabaceae Zabrotes Mexican bean Coleoptera Inhibit insect growth [151]
vulgaris subfasciatus weevil and development
Native arcelin-1 Dimeric Phaseolus Common bean Seeds Fabaceae Zabrotes Mexican bean Coleoptera Inhibit insect growth [158]
glycoprotein vulgaris subfasciatus weevil and development
Arc5-III Phaseolus Common bean Seeds Fabaceae Zabrotes Mexican bean Coleoptera Highest inhibitory effect [159]
vulgaris subfasciatus weevil on the development of
Zabrotes subfasciatus
larvae
L. purpureus Arcelin Lablab Hyacinth bean Seeds Fabaceae Spodoptera Asian Lepidoptera Inhibitory effect on the Malaikozhundan
purpureus litura armyworm larval development et al. [160]
Tepary bean Arcelin Phaseolus Tepary bean Seeds Fabaceae Zabrotes Mexican bean Coleoptera Provides resistance [161]
acutifolius subfasciatus weevil towards the Mexican
bean weevil
L. purpureus Arcelin Lablab Hyacinth bean Seeds Fabaceae Rhyzopertha Lesser grain Coleoptera 5% Dose of the L. [162]
purpureus dominica borer purpureus fraction
resulted in complete
mortality of all larvae
Arcelin, Phaseolus Common bean Seeds Fabaceae Zabrotes Mexican bean Coleoptera Inhibit insect growth [163]
phytohemagglutinin vulgaris subfasciatus weevil and development
and α-amylase
inhibitor (ABA)
L. purpureus Arcelin Lablab Hyacinth bean Seeds Fabaceae Callosobruchus Cowpea weevil Coleoptera Inhibit larval growth [164]
purpureus maculatus
P. lunatus Arcelin Phaseolus Lima bean Seeds Fabaceae Callosobruchus Cowpea weevil Coleoptera Inhibit larval growth [165]
lunatus maculatus
0005092140.INDD 31 06-03-2021 18:50:00

2.7 Defensins
These are considered as a small cationic peptide that have 44–54 amino acid residues which
are stabilized by three to four disulfide bridges having molecular mass of approximately
5 kDa [166, 167]. Till date, there are several defensins molecules that have been extracted from
various plant tissues like leaf, stem, root, and endosperm. These isolated defensins possess
numerous antibacterial, antifungal, and insecticidal properties [167]. The role of defensins is
primarily reported to inhibit insect enzymes, importantly α-amylases and proteases. The first
reported defensin molecules were extracted from plant sorghum (Sorghum bicolor) [110, 166].
It showed toxic effects against insect, Periplaneta americana alpha amylases but does not cause
any effect on the mammalian digestive enzymes [110].
The mung bean isolated defensins have been thoroughly studied and well characterized
in terms of its structure and function and referred as VrD1. Many studied reported the
expression of VrD1 in the yeast was found to have inhibitory effect against the Callosobruchus
chinensis in the bioassay [168]. The defensins, VuD1, isolated from cowpea have showed
inhibitory effect against A. obtectus and Z. subfasciatus, insect α-amylases but had not pro-
duce effect against weevil Callosobruchus maculatus [169]. Later studies have shown inhib-
itory action of VuD1 against C. maculatus, α-amylases at micromolar concentration,
without causing any effect on the mammalian enzymes [166, 170]. Many studies in the
identification of defensins have been carried out which elaborated the usage of these pep-
tides in developing transgenic plants resistance to insect pests. Examples of some of these
include defensins, BrD1, which were isolated from turnip (Brassica rapa) expressed in GM
rice cultivars. These developed transgenic crops exhibited increased resistance against the
insect, N. lugens compared to the nontransformed plants. Other defensins molecules found
toxic against insect is presented in Table 2.6.
2.8 Cyclotides
These are the peptide molecules that are like as defensins and have low molecular mass
cationic peptides that is approximately 30 amino acid residues in length. Unlike defensins,
they do not have N- and C-termi [166, 185]. Presently, most of cyclotides have been extracted
and well characterized, these peptides possess many antibacterial, antiviral, insecticidal,
and hemolytic properties [185]. First reported cyclotides extracted from African plant,
Oldenlandia affinis, was kalata B1 and was found to have insecticidal action against lepi-
dopteran Helicoverpa punctigera [186]. Kalata B1 was recently expressed in the transgenic
crop plant, Nicotiana benthamiana, to study the cyclization of this peptide. Three mostly
conserved regions that were considered for posttranslational modifications identified to be
at C-terminal region of kalata B1 [166, 187]. Apart from kalata B1, the insecticidal activity
of kalata B2 was identified that have inhibitory affect against H. armigera larvae which was
isolated from O. affinis [175]. These inhibitory effects were due to less consumption of food
by the H. armigera larvae rather than due to toxicity [188]. There was another cyclotide
identified from blue pea (Clitoria ternatea) that has shown inhibitory effect against
Z. subfasciatus and A. obtectus when fed on an artificial diet [189]. After that there was
2.10 Ureases and Urease-Derived Encrypted Peptide 33
another study that has extracted cyclotides from the Brazilian Savannah Rubiaceae flower
plant, Palicourea rigida referred as paragidin-BR1, that have caused mortality more than
60% against Diatraea saccharalis larvae. In in vitro; assay, the efficacy of paragidin-BR1 was
also checked at micromolar concentration against the Sf-9 cell line of S. furgiperda [37, 166].
There is tremendous potential in the use of cyclotides in the future application in devel-
oping transgenic plants against the insect. However, till date, no transgenic plants express-
ing cyclotides genes against the insect pests have been reported.
2.9 Canatoxin-Like Proteins
The first protein ever crystallized from the jackbean, C. ensiformis, is lectin concanavalin A [1].
Jacbeans also contain a potent neurotoxic protein named canatoxin [1, 171]. This potent toxic
protein is a noncovalently linked dimer of a 95 kDa polypeptide chain, which accounts for
0.5% of the dry seed weight. When this toxin ingested by insect is cleaved by cathepsin-like
enzymes from gut releasing entomotoxic peptide named pepcanatox. Other types of digestive
enzymes like trypsin are not susceptible from the attack of this toxin [190]. Diversified pres-
ence of this canatoxin-like proteins in other plant sources was also reported which includes
ureases and urease-derived encrypted peptides. The role of this toxin on different insects was
tested to predict its role as a plant defense molecule [190]. The insects, C. maculatus and R. pro-
lixus, that relied on cathepsins B and D as main digestive enzymes were found to be have lethal
effects due to canatoxin. The two major pests of agricultural importance, Dysdercus peruvianus
(cottonsucker bug) and Nezara viridula (Southern greensoybean stinkbug) are also susceptible
to the toxic effect of canatoxin. There was a report that confirmed the toxicity effect of entomo-
toxic protein, canatoxin, in the presence of insect cathepsin-like enzymes to produce entomo-
toxic peptide(s) [191]. Canatoxin is found to be less toxic in comparison to α-amylase inhibitors,
proteinase inhibitors, and some lectins but was found to be 40-fold more toxic than arcelins
against insect, Z. subfasciatus [1]. Other canatoxins-like proteins have also played a critical role
against the insect, which is represented in Table 2.6.
2.10 Ureases and Urease-Derived Encrypted Peptides
The ureases are the metalloenzymes that has role to hydrolyze urea into ammonia and
carbon dioxide. Their presence was reported in plants, fungi, and bacteria [1, 178]. This is
a first enzyme extracted from jack beans to be crystallized that consists of a 90.7 kDa
homohexamer chains [1].
The important role of plant ureases is to utilize urea as a nitrogen source internally or exter-
nally. These are found to be highly expressed within the seeds. The stored ureases in seeds help
in early germination by utilizing nitrogen sources [178]. Apart from this, it also exerts insecti-
cidal and antifungal properties. Importantly, insects that produce cathepsin-like enzymes dur-
ing digestion are found to be very susceptible to ureases, whereas insect having trypsin-like
digestive enzymes does not found to susceptible to ureases [178]. The insects that are suscep-
tible to ureases are C. maculatus and Rhodnius prolixus (kissing bug) and unsusceptible insects
include, such as Schistocerca americana (locust), Manduca sexta, and Drosophila melanogaster
Table 2.6 List of other proteins with insect toxic activities.
Plants Plant Action Insect

Proteins Nature of protein Plant species common name part used Plant family against insect common name Order Application References
Canatoxin-CNTX Canavalia Jack bean Seeds Fabaceae [171]

ensiformis
Soybean urease Urea Glycine max Soybean Seeds Fabaceae Dysdercus Peruvian larder Coleoptera Ubiquitous [172]
(SBU) amidohydrolase peruvianus beetle enzyme
Potato urease Ubiquitous enzyme Solanum Potato Leaves Solanaceae [173]
tuberosum
JBURE-II Canavalia Jack bean Seeds Fabaceae Nezara viridula Green stink bug Hemiptera 100% lethality after [174]
ensiformis 72 h of second
instar larvae
Kalata B2 Subfamily of Oldenlandia Hedyotis affinis Leaves Rubiaceae Helicoverpa Cotton Lepidoptera Inhibits the growth [175]
cyclotides affinis Roem armigera bollworm and development
of Helicoverpa
armigera larvae
G. hirsutum seed Gossypium Cotton Seeds Malvaceae Dysdercus Cotton stainer Hemiptera Degradation of [176]
urease (GHU) hirsutum peruvianus bug digestive enzymes
present in the
target insects
Momordica Momordica Bitter melon Seeds Cucurbitaceae [177]
urease charantia
Jack bean Nickel-dependent Canavalia Jack bean Seeds Fabaceae Rhodnius Triatomid bug Hemiptera 96% mortality after [178]
urease – JBURE-I enzymes ensiformis prolixus 24 h and reduced
body weight gain
Jaburetox-2Ec Canavalia Jack bean Seeds Fabaceae Dermestes Peruvian larder Coleoptera 100% mortality [178]
ensiformis peruvianus beetle after 11 days
Pigeon pea Cajanus cajan Pigeonpea Seeds Fabaceae Callosobruchus Kdzuki bean Coleoptera Affect insect [179]
urease (PPU) chinensis weevil digestive system
0005092140.INDD 34 06-03-2021 18:50:01

Cyclotides Binds to Clitoria ternatea Butterfly pea Leaves Fabaceae Spodoptera Fall armyworm Lepidoptera Cytotoxicity Oguis
phospholipid frugiperda against Sf9 cell et al. [180]
membranes
Concanavalin A Carbohydrate- Canavalia Jack bean Seeds Fabaceae Bactericera Potato psyllid Hemiptera Apoptotic response [181]
(ConA) binding protein ensiformis cockerelli was induced by
ConA in psyllid
midgut cells
Jaburetox (JBTX) Ureases activity Canavalia Jack bean Seeds Fabaceae Oncopeltus Large milkweed Hemiptera Ureases activity [182]
ensiformis fasciatus bug
ϒ1-hordothionin Hordeum vulgare Barley Seeds Poaceae [183]
ϒ1-purothioni Triticum aestivum Wheat Seeds Poaceae [183]
VrCRP A cysteine-rich Vigna radiata Mung bean Seeds Fabaceae Callosobruchus Chinese Coleoptera Lethal to larvae of [9]
protein chinensis bruchid the bruchid
AtPDF1.1 Arabidopsis Arabidopsis Leaves Brassicaceae [184]
thaliana
AtPDF1.2a Arabidopsis Arabidopsis Leaves Brassicaceae [184]
thaliana
0005092140.INDD 35 06-03-2021 18:50:01

(fruit fly) [1, 178]. The differential action of plant ureases by insect digestive enzymes in differ-
ent developmental stages affects susceptibility status of adult and nymph pest [1, 178]. The
toxic effect of JBURE-1 isoform isolated from major jack bean depends on the release of ent-
motoxic-derived encrypted peptide called pepcanatox and cathepsin-like enzymes [191].
Based on sequence information of pepcanatox, another recombinant peptide was also pro-
duced named, Jaburetox [192]. This recombinant peptide has a molecular weight of 11 kDa,
which is toxic to various insect pests that are not susceptible to native urease, JBURE-I [178].
The insects Dysdercus peruvianus (cotton stainer bug), Oncopeltus fasciatus (large milk-
weedbug), and R. prolixusare are susceptible to the toxic effect of JBURE-I [178, 193, 194].
JUBRE-II isoform exerts toxic effect against the insect R. prolixus [195]. S. furgiperda was
susceptible to Jaburetox [178, 192, 196].
2.11 Chitinases
As we know, chitin is the extracellular layer of insect exoskeleton and could be considered
as important target for the pesticidal action [166, 197]. Apart from lectins, plant also pro-
duces chitinases, a hydrolytic enzyme that can interact with chitin monomers to disrupt
the action of insect chitin synthase [197].
These chitinases help in the hydrolysis of chitin that present β-1,4-linked N-acetylglucosamine
residues [198, 199]. Chitin is considered as monomeric protein having a molecular mass of
25–35 kDa [166, 198]. Plant chitinases were classified as endo- and exo-chitinases based on
the specific cleavage site in the target chitin moiety. According to primary structure, plant
chitinases are divided into four different groups that include, class I, class II, class III, and
class IV. In class I, N-terminal cysteine-rich domain was present with having near about 40
amino acid residues in length that is found to be highly conserved in the main structure [166].
In class II, these chitinases do not have cysteine-rich domain at the N-terminus. The Class III
include enzymes with no sequence similarity with class I or II, but in class IV, sequence simi-
larity was observed with class I that contain cysteine-rich domain [166, 198].
There was report that described the toxic effect of chitinase, when engineered into
tomato plants against the pest, Coloradopotato beetle larvae (Leptinotarsa decemlineata),
named WIN6 was isolated from popular plants (Populus trichocarpa). Another report came
from D. melanogaster where they have shown toxic effect of two chitinases, namely, LA-a
and LA-b when administered in an artificial diet [200].
2.12 Proteases
These are classified as peptidases or proteinases that are present in animals, plants, bacte-
ria, archaea, and viruses that hydrolyze the covalent bonds present in the polypeptide
chains. Many of them have emerged as a protective moiety against herbivorous pest.
However, those proteases that do not evolve as enterotoxins could also have insecticidal
effect when administered ectopically [166, 201].
There is a very limited study that described behavior of proteases as toxic against insect pests.
One example of these types include Mir1-CP, which is also known as papain-like cysteine
isolated from maize lines and have shown resistance against S. furgiperda [202–204].
Reference 37
Engineered GM plant calluses expressing Mir1-CP exerts growth inhibition when insect
fed on it [205]. In addition to this, purified Mir1-CP recombinant is known to enhance the
toxic effect of Bt Cry toxin on peritrophic matrix of S. furgiperda and many other
insects [206]. Papain, which is another protease, found to be present in the latex papaya
(Carica papaya) and one more cysteine protease called ficin identified in the wild fig (Ficus
virgata) caused to have detrimental effect against three important lepidopteran species,
namely, Samia ricini (Indian erisilkmoth), Mamestra brassicae (cabbage moth), and
Spodoptera litura (tobacco cutworm) [166, 207]. Therefore, these plant proteases could be
one of the unexplored promising agents in the development of transgenic plants against
insect pests [201].
2.13 Conclusions
Here, we have discussed about the plant defense systems that cause detrimental action
against insect growth and phenotype. Different plant systems have variable effect on insect
growth established through feeding bioassay and transgenic approach. We could manipu-
late the selected defense proteins against the insect either by overexpressing the gene of
interest or introducing the plant genes into different plant species. These plant proteins
have tremendous potential in controlling the insect spread and saving the loss happened in
the agricultural field to food productions.
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53
Bioprospecting of Natural Compounds for Industrial

and Medical Applications
Current Scenario and Bottleneck
Sameer Dixit1, Akanchha Shukla1, Vinayak Singh1, and Santosh Kumar Upadhyay2
1
Department of Biology, University of Western Ontario, London, Ontario, Canada
2
3.1 Introduction
All substances produced by biosynthetic pathways inside living organisms that are not
essential for energy metabolism but required for their ecological fitness are termed as natu-
ral products, for example, secondary metabolites. Evolutionary pressure on biosynthetic
pathways resulted in the development of such chemically diverse and biologically potent
molecules [1]. These natural products can be attained from enormous biodiversity that is
natural sources or organisms from diverse ecosystems and ecological complexes. Thus, the
process of exploration of biodiversity for bioactive compounds with agricultural, indus-
trial, and medical applications is known as bioprospecting [2, 3]. Since natural resources
are limited, protecting genes, species, and habitats in ecosystems became necessary to pre-
vent long-term depletion of biodiversity. A Convention on Biological Diversity agreement
was made obligatory on 29 December 1993 for preservation and sustainable use of biodi-
versity, as well as ethical sharing of benefits with independent states and local communi-
ties [4]. Thus, a series of ethical and legal issues like patents, intellectual property rights,
cultural and individual rights to privacy, cross-cultural understanding, and other aspects
must be dealt before starting bioprospecting ventures [5, 6]. A successful bioprospecting
program involves scientific and economic activities enabling sustainable development and
economic growth by the revenue generated from royalties contributing to biodiversity con-
servation and safeguarding traditional medicine knowledge [2].
Due to changes in globalization and evolving environmental problems, there is increased
economic relevance of biological diversity. It is difficult to estimate the actual overall biodi-
versity value in financial terms for all ecosystems [7]. However, it was recently estimated to
be about US$2.9 trillion for the whole world [8]. Bioprospecting has contributed
#
Authors have contributed equally.

54 3 Bioprospecting of Natural Compounds for Industrial and Medical Applications
enormously in drug discovery to treat variety of infectious diseases, cancer, autoimmune

diseases, etc. [9]. In agricultural field, bioprospecting leads to the development of various
pesticidal (Bacillus thuringiensis toxin and annonins from Annona squamosal plant) and
herbicidal compounds and bio-fertilizers (rhizobium) enhancing the plant growth, produc-
tivity, and ability to withstand adverse environmental conditions [10, 11]. Apart from phar-
maceutical and agricultural applications, bioprospecting products are also used in
bioremediation (laccase enzyme from Phanerochaete chrysosporium), cosmetics (zeaxan-
thin from Xanthobacter autotrophicus, collagenases from Clostridium histolyticum, and
keratinases from Microsporum), and biosensors (laccase containing electrodes) [12–14].
In this chapter, we will discuss about bioprospecting, rational bioprospecting processes,
their drawbacks, and new approaches to advance utility of natural products in agricultural,
medical, and industrial fields.
3.2 Why Bioprospecting Is Important
The sole purpose of bioprospecting is to explore bio-diversity to find natural product/

organisms that can benefit humans. After the Convention on Biological Diversity 1992–1993,
the useful natural product/organisms not only benefited industries involved in identifica-
tion or production but also the indigenous community or host country. Thus, local agency
has the control and sovereignty on identified biological resources and also expect adequate
economic compensation for resource. This will enhance the cooperation between indus-
tries and local agencies and also increases the employment opportunity resulting in the
improvement of socioeconomic status. It is typically considered that the impact of bio-
prospecting benefits will be much better if it is based on the knowledge and information
from local people [15].
3.3 Major Sites for Bioprospecting
Bioprospecting can be performed anywhere with rich biological diversity such as forest,
conservation areas, hotspots, ocean, etc. [16]. Extreme environments such as polar regions
and hot water springs can also serve as potential site for bioprospecting [16]. Statistically, it
is considered that 1 in 30,000 to 40,000 is the chance to find useful natural product [17].
Terrestrial region is most commonly used for bioprospecting programs. A classic exam-
ple is the discovery of Artemisinin by TuYouyou, a nobel laureate [18]. Globally, scientists
test ~240,000 compounds without any breakthrough success. TuYouyou inspired by “The
handbook of prescriptions for emergency treatments,” a Chinese medical handbook writ-
ten by Ge Hong around 340 ce during Jindynasty. She screened ~2000 biological samples
and identified that ethyl ether extract of Qinghao (Chinese common name Artemisia plant)
significantly inhibited malaria parasites; this led to the successful discovery of
Artemisinin [18]. Many national and international terrestrial bioprospecting programs
have started in recent years, such as INBio-Merck (between INBio-National Biodiversity
Institute of Costa Rica and Merck & Co. Ltd), Peruvian (between ICBG, USA; Bristol-Myers
Squibb, Monsanto and Glaxo-Wellcome), and The TBGRI-Kani contract (between TBGRI
3.5 Biopiracy: An Unethical Bioprospectin 55
and Arya Vaidya Pharmacy along with the help of Kani community) are some of
them [19–21].
After land, oceans are the second most explored site for bioprospecting programs. About
70% of the earth is covered from ocean with one of the richest biological diversity on planet.
In last four decades, ~15,000 marine bioactive natural products are identified [22]. Omega-3
fatty acid from fish oil, Cytarabine and Vidarabine from Sponge Tethyacrypta, Cephalosporin
from Marine fungi are some of the few examples of bioactive compounds derived from
marine biodiversity [23].
Polar region and Hot Spring Lake are the other important sites that are exploited for bio-
prospecting programs [16]. These regions have harsh climate during most of the year, tem-
peratures reach minimum of −50 °C (Artic) to maximum 100 °C (Hot Spring). These
extreme environments lead to the local and unique adaptations in biological organisms
which are living in these regions [24]. Sometimes these adaptations are due to the accumu-
lation of new compounds that can have bioactive capabilities. Due to the global warming
and climate change, the ice in polar region is melting, thus proving more area to explore or
may exploit. The recent example is MabCent project, a team of researchers spent a year in
extreme environment and collect ~3000 pounds of biological sample (consisting microal-
gae, invertebrates, and many others) from the 1000 different sites around Norway’s Svalbard
archipelago that are being screened for their bioactive properties [24]. Identification of
New Biofuel-producing bacteria from Iceland’s famed hot springs, New lipase for the pro-
duction of biodiesel from Taptapani Hot Spring (Odisha), and phytase-producing thermo-
philic fungi from West Anatolia are some of the recent examples of hot springs
bioprospecting expedition [25–27].
3.4 Pipeline of Bioprospecting
Bioprospecting process is generally divided in to four major phases which starts from the
collection of sample to commercialization of the product [28, 29]. Phase I consists of the
identification of Bioprospecting site followed by onsite collection and preservation of bio-
logical sample. Initial processing such as washing/cleaning/freezing of biological sample
also lies in this phase. Phase II starts with the chemical processing of sample followed by
isolation and identification of bioactive compound/organism. If required, this phase also
consists of the characterization and mass production of specific bioactive material. Phase III
begins with the detailed characterization of specific compound/organism and in-depth
screening or validation of biological property. Phase IV is the last stage in which bioactive
compound/organism goes for product development and ready for commercialization [28, 29].
3.5 Biopiracy: An Unethical Bioprospecting
Biopiracy is considered as the exploitation or monopolization of biological material or

indigenous knowledge without informing/compensating the local community or country
from where the biological material or knowledge is procured [30]. Biopiracy imposes many
negative effects on local biodiversity like overexploitation of endemic biological material,
reduction in biodiversity or local niche, and illegal privatization of biological material,

which results in adversely affecting the cultural, regional, and traditional identity or knowl-
edge of indigenous people [31]. Biopiracy is illegal by all means but due to its commercial
and financial profit, many private companies prefer it [31]. There are many international,
national, and provisional law or rights are available to counter biopiracy such as Intellectual
property rights, Plant breeders’ rights, Plant Variety Protection Act, Convention on biologi-
cal diversity act, and many more [21, 32, 33]. The well-known case of biopiracy in the
context of India is Neem tree (India) against W. R. Grace and Company. Neem (Azadirachta
indica) tree is found throughout in India and its neighboring country and traditionally
known for its antifungal properties [34]. W. R. Grace and Company and USDA obtained a
European patent (EP0436257B1) on Method for controlling fungi on plants by the aid of a
hydrophobic extracted neem oil in 1994 without compensating to the local community or
country [35]. Many groups from India and Europe mainly Vandana Shiva, EU Green Party,
and the International Federation of Organic Agriculture Movements opposed this patent
which is finally revoked in 2000 [35]. The similar case is happened in India–US basmati
rice dispute. USA-based corporation RiceTec acquired a patent (US5663484C1) of Basmati
Rice hybrid lines bas 867 Rt1117 and Rt112 in 1997 [36]. In 2002, Government of India
challenged this patent and found that 15 of the 20 claims are not valid [37]. Under-
developed countries like African countries are the most adversely affected countries. There
is a proper need to strengthen the policy, law, and institutional framework in these
countries.
3.6 Bioprospecting Derived Products in Agriculture Industry
To provide a balanced diet and nutrition-rich food products for global population is a major
challenge in this era. Unfortunately, nutrition in human diet is not rich because of non-
plant-based food product. So, deficiency causes malnutrition in human beings. There is a
strong need of protein-rich food for human beings to combat through malnutrition. So, in
this way bioprospecting is an important field for enhancing the agricultural products.
Bioprospecting in agriculture industry is utilized in different ways. Some of these are
neglected and underutilized wild plants for nutritional enhancement, endophytes to
enhance the production of crops, and nutrient enrichment through transgenic approach.
There are many crops having nutritional-rich value, but they are treated as wild,
neglected, and underutilized, although there are thousands of wild and neglected plant
species that are reported to have nutritional value. Therefore, presently, food production
depends only on limited plant species. Different underutilized crops have been studied.
There are many genera of legumes species for important grains and agroforestry species [38].
In these, genera of legumes, chick pea (Cicer arietinum), cowpea (Vigna unguiculata), and
pea (Pisum sativum) are utilized as major food source in case in human beings. Apart from
these legumes, some are used as orphan crop like Psophocarpus tetragonolobus (L.) Parkia
roxburghii, and Canavalia sp. Psophocarpus tetragonolobus is also called a wonder legume
as it has high protein content in the seeds and therefore considered as a versatile legume. It
is also known as “Soybean of tropics” due to its high protein content in each part of this
legume. But there is also the presence of anti-nutrient as condensed tannin, which binds
with protein of seed and edible part of plant. That creates absorption problem at the time
3.7 Bioprospecting Derived Products for Bioremediatio 57
of digestion. So to identify the best germplasm, Singh and others identified less condensed
tannin (0.265 mg/g of dry weight) winged bean from 100 different global germplasm line of
Psophocarpus tetragonolobus on the basis of physiochemical, biochemical, and genetic
basis [39]. Along with this legume Parkia sp. and Canavalia sp. bioprospection is under
process to develop the best variety for sustainable development [40, 41].
Endophytes secrete bioactive compound in host plant in which they survive. Those com-
pounds are necessary for plant growth and protection. They have numerous functions to
increase agricultural productivity. They sustain the agriculture industry in a proper way in
terms of nutrition richness. There are many bacteria, fungi, and actinomycetes involved in
the synthesis of bioactive compounds. You and others identified the Penicillium sp. in
Suaeda japonica plant, which synthesize Giberellic acid and help in seed germination [42].
Aspergillus sp., Cladosporium sp., Penicillium sp., and many more fungi live with panax
ginseng plant and synthesize triterpenoid- and saponin-type secondary metabolites, which
provide protection and root growth to plant [43]. Seed germination in Phaseolus vulgaris is
enhanced by bioactive compounds (proteolytic enzymes, phosphate solubilization factors,
active volatile and non-volatile metabolites) synthesized by endophytes Trichoderma atro-
viridae, T. polysporum, and T. harzianum [44]. Along with these there is a long list of bac-
teria that are involved in plant growth and nutrition uptake. Plant bioprospecting has also
been performed for the isolation of numerous insect toxic proteins for their use in agricul-
tural industries [129] [128] [127] [126] [130].
Transgenic approach is also applied for bioprospection of value-added products. In this
approach, first one is insect pest management in crop plant to avoid loss of crop production
by pest. Second, to increase the level of particular micronutrients in particular plant part
that is edible. For instance, protein disorder micronutrients deficiency is another major
nutritional disorder in women and young children [45]. Iron is a type of micronutrient
whose deficiency causes anemia in a large population in the world. To eliminate iron defi-
ciency, there is a need of iron-rich food. Genetic engineering approaches have been taken
care to Fe-fortified plants to increase iron content in rice and maize [46].
ZmZIP5 and OsIRT1 genes are involved in iron uptake and translocation in maize and
rice, respectively [47, 48]. There are many other genes, which are overexpressed/ knock-
down for bio-fortified minerals in plant.
3.7 Bioprospecting Derived Products for Bioremediation
In the twenty-first century, the pollution of water and soil with toxic elements due to indus-
try revolution is one of the major concerns. So, the removal of these pollutants from the
environment is a major hurdle for sustainable development. Apart from conventional
method to remove the pollutants, bioremediation has evolved as eco-friendly, cost-effective,
and sustainable approach. It is processed through microorganism and plants. Bioprospection
of these microorganisms and plants with better activity have been performed by several
researchers. It means use of new beneficial functions from microorganisms, such as new
enzymes for chemical and biochemical reactions of interest, processes to increase
bioremediation.
Several microorganisms are well known for bioremediation of heavy metal pollutants
such as Cd, Hg, Pb, Zn, and U. But genetically engineered bacteria draw more attention
these days. Genetically modified bacteria have performed more efficient and target-based
bioremediation. Lee et al reported that, Pseudomonas putida 06909, an antifungal rhizos-
phere bacterium was engineered for the purpose of producing metal binding peptides. This
peptide has very high affinity with Cd [49]. An Hg (II) resistance gene (merA) has been
expressed in Deinococcus radiodurans strains to remove radioactive pollutant [50].
Mesorhizobium huakuii B3 bacteria was genetically modified with Phytochelatin synthase
(PCS) gene expression, which eradicate Cd2+ from rice field [51]. Similarly the B. subtilis
BR151 (pTOO24) has been modified with the expression of luminescent Cd sensor gene
which remove Cd from polluted soils [52].
These results imply that a modified microbial load in soil can be a promising strategy for
heavy metal cleanup. This could be a crucial factor for sustaining the growth of the engi-
neered strain in the presence of the native bacterial population.
Endophytes are the most diverse group of microorganisms found in every plant on earth.
They may be mainly fungi or bacteria. Entophytes were reported to be mainly isolated from
plants ranging from herbs to large trees, marine grasses, and lichens. Pseudomonas bacteria
live in Lolium multiflorum, a type of Italian ryegrass which releases bioactive compound
alkanes [53]. Alkanes are used for diesel degradation at the site of pollution. Burkholderia
cepacia is present in Zea maize plant. They release phenol and toluene, which is used in
Petroleum tolerance and degradation [54]. The Co-contamination of nickel and trichloro-
ethylene tolerance and degradation were performed by bioactive compound
(Trichloroethylene) released from Pseudomonas putida in poplar plant [55]. Similarly, the
pyrenes bioactive compounds released by Enterobacter, which lives within Alium mac-
rostemon and function as Pyrenes tolerance and degradation [56].
Apart from microorganisms and bioactive molecules, some plants also perform bioreme-
diation against toxic elements. Along with these, the transgenic plants are also designed to
degrade the particular pollutant from environment.
Sarah Jamil and others revealed that Jatropha curcas plant helps in remediation of fly ash
(FA), which is produced in thermal power plants [57]. They showed that the Fe and Mn
accumulate in roots and Cu, Al, and Cr were translocated more to the shoot, when the
plants were treated with fly ash of thermal decomposition.
Environmental pollution with pesticides, pharmaceuticals, and petroleum compounds is
not decontaminated through conventional methods. So, transgenic strategy has been
enlightened to develop the transgenic plant with Insertion of CYP450 in plants to increase
the xenobiotics metabolism [58]. Over expression of CYP450 isoenzymes (CYP1, CYP1,
CYP3 isolated from human and mammals) in plants has been done mainly in Solanum
tuberosum, Oryza sativa and Nicotiana tabaccum. The main objective of these transgenic
developments is to produce herbicide resistant plant and plants capable of enhanced
metabolization of foreign metabolites. The CYP1A1 gene from human expressed in Oryza
sativa, the transgenic plant enhanced the metabolism of chlorotoluron, norflurazon in
soil [59, 60]. Similarly onr gene isolated from Enterobacter cloaceae and expressed in
Nicotiana tabaccum. The result showed enhancement of denitration of glycerol trinitrate
(GTN) and TNT [61]. But when CYP1A1 gene with CYP2B6 gene from human was
expressed in Solanum tuberosum, it showed different result than Oryza sativa transgenic
with CYP1A1 gene. Transgenic Solanum tuberosum has performed resistance to sulfonylu-
rea and other herbicides [62].
3.8 Bioprospecting for Nanoparticles Developmen 59
3.8 Bioprospecting for Nanoparticles Development
Nanoparticles revolutionized science and technology in recent years. They have already
proven their vast application in medical, electrical, wastewater processing, construction,
military and many more industries [63, 64]. Many plants and micro-organism have poten-
tial to synthesis metallic nanoparticles (Table 3.1), [64, 89, 90]. Biological organism mainly
utilized two mechanisms for the synthesis of nanoparticles: (i) bioreduction and (ii)
biosorption [89, 90]. In bioreduction, metal ions are biochemically reduced into inert and
stable nano form. This bioreduction is mostly couple with the oxidation of specific enzymes
Table 3.1 List of nanoparticles producing bio-organism.
Nanoparticle Bio-organism Name Method Localization References
Au Bacteria Thermomonospora sp. Reduction Extracellular [65]

Bacteria Rhodococcus sp. Reduction Intracellular [66]
Bacteria Rhodopseudomonas Reduction Extracellular [67]
capsulata
Bacteria Pseudomonas aeruginosa Reduction Extracellular [68]
Bacteria Delftia acidovorans Reduction Extracellular [69]
Fungi Fusarium oxysporum Reduction Intracellular [70]
Fungi Verticillium sp. Reduction Intracellular [71]
Plant extract Cymbopogon flexuosus Reduction Extracellular [72]
Live plant Medicago sativa — Intracellular [73]
Ag Bacteria Bacillus licheniformis Reduction Intracellular [74]
Bacteria Bacillus sp. Reduction Extracellular [75]
Bacteria Klebsiella pneumonia Reduction Extracellular [76]
Bacteria Escherichia coli Reduction Extracellular [76]
Bacteria Enterobacter cloacae Reduction Extracellular [76]
Bacteria Lactobacillus sp. Both Extracellular [77]
Bacteria Enterococcus faecium Both Extracellular [77]
Bacteria Lactococcus garvieae Both Extracellular [77]
Fungi Pediococcus pentosaceus Both Extracellular [77]
Fungi Fusarium oxysporum Reduction Extracellular [70]
Fungi Aspergillus fumigatus Reduction Extracellular [78]
Fungi Aspergillus flavus Reduction Extracellular [79]
Fungi Coriolus versicolor Reduction Both [80]
Plant latex Jatropha curcas Reduction Extracellular [81]
Leaf extract Acalyphaindica Reduction Extracellular [82]
Seed exudate Medicago sativa Reduction Extracellular [83]
Leaf extract Magnolia kobus Reduction Extracellular [84]
(Continued)
Nanoparticle Bio-organism Name Method Localization References
Pt Bacteria Escherichia coli Reduction Extracellular [85]

Fungi Fusarium oxysporum Reduction Extracellular [70]
Fungi Neurospora crassa Reduction Both [86]
Pd Bacteria Escherichia coli Reduction Extracellular [85]
Bacteria Desulfovibrio Reduction Extracellular [87]
desulfuricans
U, Cu, Pb, Bacteria Bacillus sphaericus Both Extracellular [88]
Al, Cd JG-A12
Au, gold; Ag, silver; Pt, platinum; Pd, palladium; U, uranium; Cu, copper; Pb, lead; Al, aluminium; Cd,
cadmium; both (method), reduction and absorption; both (localization), intra and extracellular.
and may also require biological energy. During biosorption, metal ions are absorbed into
the bioorganism itself, such as bacterial, fungal, or plant cell. These ion–cell complexes are
stable complexes and able to perform biological reaction. These bio-nanoparticles can be
used in biosensors for the detection of heavy metal in soil, polyphenolic in wine, phenols
and lignins in wastewater, and many more [89, 90].
3.9 Bioprospecting Derived Products in Pharmaceutical Industry
Current environmental and global hazards and the emergence of infectious diseases
demand new initiatives in the development of therapeutics and their commercializa-
tion [91]. Bioprospecting can be a promising approach for drug discovery that utilizes the
biodiversity. This multidisciplinary approach exploits ecology, pharmacology, and thera-
peutics to explore new chemical compounds from terrestrial, marine, and other ecosys-
tems. The pharmaceutical industry depends the most on bioprospecting. Food and Drug
Administration (FDA) approved approximately 30% of the new drugs that originated from
natural sources from 2008 to 2012 [92]. Bioprospecting for pharmaceuticals involves iden-
tification of a disease state, development of an extrapolative biological assay that can either
generally or specifically test the efficacy of compounds followed by identification, collec-
tion, and testing of biodiversity samples. New compounds can be identified through two
major strategies: first, the random collections that are relatively large in number, and sec-
ond, focused collections that follow phylogenetic or cultural clues, where the likelihood of
success is maximum with a minimum number of samples [93].
Bioprospecting has given numerous drugs that are used in the treatment of variety of
diseases and ailments (Table 3.2). Traditionally, compounds isolated from terrestrial
sources, mainly plants, have contributed to the pharmaceutical industry [94]. The bioactive
entities obtained from plants may be purified natural products or semi-synthetic/synthetic
derivatives that show high efficacy and decreased side-effects [93]. Bioprospecting from
terrestrial sources have generated multiple drugs approved by the FDA to treat various
forms of cancer [95–97]. For example, taxol (isolated from the Pacific yew tree
3.9 Bioprospecting Derived Products in Pharmaceutical Industr 61
Table 3.2 List of drugs obtained from bio-organism.
Name Source Function
Drugs derived from plants

Taxol Taxus brevifolia Anticancer
Vincristine Catharanthus roseus Anticancer
Vinblastine
Harringtonine Cephalotaxus harringtonia Anticancer
Homoharringtonine Cephalotaxus harringtonia Anticancer
Topotecan Camptotheca acuminate Anticancer
Irinotecan Camptotheca acuminate Anticancer
Artemether Artemisia annua L Antimalarial
Quinine Cinchona ledgeriana Antimalarial, antipyretic
Crofelemer Croton lechleri Treatment of diarrhea associated with
antiretroviral HIV/AIDS therapy
Prostratin Homalanthus nutans Antiviral
Morphine Papaver somniferum Analgesic, antitussive
Codeine
Atropine Atropa belladonna Anticholinergic
Hyoscyamine
Digoxin Digitalis purpurea Cardiotonic
Digitalin
Digitoxin
Convallatoxin Convallaria majalis Cardiotonic
Acetylsalicylic acid Salix alba Analgesic
Sinecatechins Camellia sinensis Treatment of genital warts caused by
human papillomavirus (HPV)
Colchicine Colchicum autumnale Anti-inflammatory
Rivastigmine Physostigma venenosum Cholinesterase inhibitors to treat
Alzheimer’s disease
Galantamine Lycoris radiata Cholinesterase inhibitors to treat
Alzheimer’s disease
Agrimophol Agrimonia supatoria Anthelmintic
Danthron Cassia species Laxative
Scopolamine Datura species Sedative
Tetrahydrocannabinol Cannabis sativa Antiemetic, decreases occular tension
Thymol Thymus vulgaris Topical antifungal
Theophylline Theobroma cacao Diuretic, bronchodilator
Drugs derived from microbes
Bleomycin Streptomyces verticillus Anticancer
Daunorubicin Streptomyces species Anticancer
Doxorubicin
(Continued)
Name Source Function
Griseofulvin Penicillium griseofulvum Antifungal

Amphotericin B Streptomyces nodosus Antifungal and antileishmanial
Ivermectin Streptomyces avermitilis Antihelminthic
Ciclosporin Tolypocladium inflatum Immunosuppressant
Compactin Penicillium compactum Cholesterol-lowering agent
Penicillin Penicillium notatum Antibacterial
Fusidic Acid Fusidium coccineum Antibacterial
Cyclosporin A Tolypocladium inflatum Immunosuppressant
Lentinan Letinula edodes Anticancer, cholesterol-lowering
agent, anti-infectious agent
Ergotamine Claviceps purpurea Migraine headaches
Drugs derived from marine sources
Cytarabine Tethya crypta Anticancer
Trabectedin Ecteinascidia turbinate Anticancer
Eribulin mesylate Halichondria okadai Anticancer
Vidarabine Tethya crypta Antiviral
Ziconotide Conus magus Analgesic
Taxusbrevifolia), vincristine, and vinblastine (from Madagascar periwinkle

Catharanthusroseus) as well as their semi-synthetic and synthetic derivatives. A variety of
gymnosperm species have been studied for taxol-like activity. Bioactive compounds
harringtonine and homoharringtonine were from the gymnosperm, Cephalotaxus har-
ringtonia [98]. Smith-Kline Beecham and Pharmacia & Upjohn developed topotecan and
irinotecan, respectively, that are semi-synthetic derivatives of the natural compound
Camptothecin isolated from Camptotheca acuminate for the treatment of ovarian cancers
and rectal cancer [93]. New Earth Biomed (NEBM), a not-for-profit cancer research organi-
zation, is currently evaluating alternative research programs to analyze complex mixtures
of plant-derived entities and other natural substances used in Indian Ayurveda and tradi-
tional Chinese medicine for new anticancer treatments [99].
Besides anticancer drugs, terrestrial bioprospecting has yielded antimalarial drug
Artemether, a semi-synthetic derivative of artemisinin [100, 101] isolated from the Chinese
medicinal plant Artemisia annua L. Crofelemer (Fulyzaq™) developed and approved by
FDA for the treatment of diarrhea associated with antiretroviral HIV/AIDs therapy.
Crofelemer is a purified compound from the latex of Croton lechleri (Euphorbiaceae) [99].
Another promising drug Prostratin is isolated from a Homalanthus nutans tree. Prostratin
is able to clear laboratory animals of virus [99]. Other examples of drugs from plants
include morphine and codeine from Papaver somniferum, atropine and hyoscyamine from
Atropa belladonna, digoxin from Digitalis spp., acetylsalicylic acid (ASA) (painkiller
derived from willow bark, Salix alba), Sinecatechins obtained green tea leaves from
3.10 Conclusion and Future Prospect 63
Camellia sinensis (active ingredient in an ointment to treat genital wart caused by human
papilloma virus), colchicine (anti-inflammatory drug) to treat gout flares (obtained from
Colchicum autumnale), the cholinesterase inhibitors rivastigmine (from Physostigma
venenosum) and galantamine (from Lycoris radiata) used to treat Alzheimer’s disease [93,
102–104].
Besides plants, drugs obtained from terrestrial microbial sources include bleomycin
(obtained from the soil bacterium Streptomyces verticillus), Daunorubicin, and its
14-hydroxylated form doxorubicin (from Streptomyces spp.) that are used as anticancer
drugs [98]. Numerous antibacterial drugs were also discovered by bioprospecting like
β-lactam antibiotics, rifamycins, tetracyclines, polymyxins, aminoglycosides, and phospho-
nic acid antibiotics [105]. In addition to antifungal drug griseofulvin (from the soil fungus
Penicillium griseofulvum) [106], amphotericin B (from the soil bacterium Streptomyces
nodosus) is the antifungal and antileishmanial drug [107], ivermectin (from Streptomyces
avermitilis soil bacterium) used as the antihelminthic drug [108], and ciclosporin (from the
Tolypocladium inflatum soil fungus) immunosuppressant drugs used to treat rheumatoid
arthritis and psoriasis [109] were also obtained from microbial sources from terrestrial
microbial bioprospecting.
Marine organisms usually produce more varieties and amounts of metabolites than
are presently identified in other sources. Seaweeds, salt marsh plants, and marine worms
survive in the extreme temperatures, wide-ranging pressures, low energy, and absence
of sunlight favoring evolution of highly developed defense system [28]. Thus, these
organisms possess unique genetic pools that may have the potential of treating several
diseases or ailments [110, 111]. Several FDA-approved drugs have been discovered from
marine bioprospecting like anticancer drugs Cytarabine (Ara-C) trabectedin and eribu-
lin mesylate from Sponge Tethya crypta, Marine tunicate Ecteinascidia turbinate, and
Sponge Halichondria okadai, respectively, antiviral drug vidarabine (Ara-A) from
Sponge Tethyacrypta, analgesic ziconotide from Cone snail Conus magus, etc. [112, 113].
Omega-3 fatty acids play an important role in the human diet and in human physiology
preventing cardiovascular, atopic, and inflammatory diseases [114–117].
Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) omega-3 fatty acids are
commonly found in marine fish oils [28]. Recently, the arctic having extreme environ-
mental conditions favoring significant and exceptional adaptations in organisms that
inhabit the region have emerged as promising bioprospecting site that may hold future
new medicines.
3.10 Conclusion and Future Prospects
Bioprospecting is exploration of biodiversity and ethnic knowledge for the production of

commercially valuable products for medicine, agriculture, and other industries [118].
Initially bioprospecting focused on plant-based entities, but now other forms of biodiver-
sity like insects, algae, micro-organisms have been explored with significant suc-
cess [119]. Experimentation of new approaches like metabolomics or genetic manipulation
for the quantification of metabolites, understating of metabolic profiles of compounds
associated with diverse metabolic pathways, metabolomic fingerprinting to categorize
the biological sample, and relationship of metabolite biosynthesis with a particular

genomic sequence have made significant developments in this field [120, 121]. Moreover,
bioprospecting programs are also including nanotechnology for natural products drug
delivery systems to augment their bioavailability, therapeutic effects, and reducing the
multiple administration [122, 123]. Bioprospecting has benefitted the society by provid-
ing various lifesaving drugs and agricultural products; there is growing concerns of
biodiversity overexploitation and biopiracy [124]. Despite various sanctioned agreements
by biodiversity convention industries resort to malpractice and unethical means [124].
This is due to lack of effective monitoring and enforcement of such guidelines [91]. It is
worth noting that the proper terms and conditions of bioprospecting agreements must
follow transparency and clarity without ambiguity [124]. Sharing of information and
accessibility to natural resources between companies/scientific organizations and bio-
prospecting site nations can be helpful to reduce biopiracy [91]. Besides equal profit
sharing with indigenous communities, multi-national companies can also benefit local
people at bioprospecting sites by providing jobs, trainings, and expertise [124, 125]. Thus,
the principal aim of bioprospecting programs should be sustainable growth of the com-
munities, balanced ecosystems, biodiversity conservation, and social benefits of the
developed products [16].
Acknowledgments
We wish to apologize to all colleagues whose work, because of lack of space, could not be
cited. SD is also thankful to MITACS for postdoc funding. We thank all the lab members of
the Grbic laboratory situated in western university, Canada, for helpful discussions.
R
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Role of Plants in Phytoremediation of Industrial Waste

Pankaj Srivastava and Nishita Giri
ICAR-Indian Institute of Soil and Water Conservation (ICAR-IISWC), Dehradun, Uttarakhand, India
4.1 Introduction
In order to increase our agriculture production, farmers, industries and other communities
are using huge amount of environmental hazardous materials that are discharged into the
soil, air and water. Moreover, the emergence of new industries such as electrical, pharma-
ceuticals and materials science industry leads to pollute our biosphere with new emerging
pollutants such as cosmetics, disinfectant, plasticisers and phthalates, wood preservatives,
paint additives, analgesics, nanoparticles, etc. [1–11]. The trace metals are important part
of our ecosystem, but the accumulation of harmful metals in plants and human beings may
be dangerous and have serious health problems. The toxic metals are difficult to remove
from the biosphere as they cannot be biologically or chemically degraded in the environment.
In phytoremediation system, there is a close relation between plants, microbes and soil
system for a long duration. This concept makes use of plants’ ability to remove, activate
and collect the materials from the atmosphere in different parts of the plants [12].
Phytoremediation is an integrated term that covers plant-related strategies and
approaches for remediation of contaminated biosphere [13]. Phytoremediation includes
(i) phytoextraction – the accumulation of heavy metals in plant biomass with high concen-
tration, (ii) rhizofilteration – the removal of metals from aqueous waste streams through
adsorption in plant parts, (iii) phytovolatalization – the process of volatilization includes
through air and plants, (iv) phytodetoxification – where plants change the chemical
species into less toxic form, (v) phytostabilization – in this process plants immobilize the
contaminants physically and chemically at the site and reduce the chances of movement
in surrounding areas [14]. The phytoremediation technology is still in research and
advancement phase and there are some methodological barriers, which need to be still
addressed. Most heavy metal accumulating plants have a small biomass and are slow
growing. To make phytoremediation a feasible technique, there is an urgent need to

74 4 Role of Plants in Phytoremediation of Industrial Waste
Table 4.1 Naturally growing plant species capable of capturing fly ash dust [17, 18].
English name Botanical name Family Habit
Opposite leaved fig Ficus hispida Moraceae Tree

Gigantic swallow wort Calotropis gigantean Asclepediaceae Shrub
Sebastian plum Cordia dichotoma Boraginaceae Tree
Banyan tree Ficus benghalensis Moraceae Tree
Jackfruit Artocarpus heterophyllus Moraceae Tree
Wild sage Lantana camara Verbenaceae Shrub
Country Mallow Abutilon indicum Malvaceae Herb
Sweet basil Ocimum basilicum Labiatae Herb
Jujube tree Ziziphus mauritiana Rhamnaceae Tree
African Tulip Spathodea campanulata Bignoneaceae Tree
Coral Jasmine Nyctanthus arbor-tristis Oleaceae Shrub
Fig Ficus glomerata Moraceae Tree
Vasan vel Cocculus hirsutus Manispermaceae Shrub
French mulberry of ghats Calicarpa tomentosa Verbenaceae Shrub
Indian Tulip tree Thepesia populnea Malvaceae Tree
Bastard cedar Guazuma ulmifolia Sterculiaceae Tree
Monkey face tree Mallotus philippinensis Euphorbiaceae Tree
Teak Tectona grandis Verbenaceae Tree
Gunpowder tree Trema orientalis Ulmaceae Tree
either find fast growing hyperaccumulators or transgenic plants with better genes in
hyperaccumulators for high metal accumulation [15].
Several plant genes encoding metal transporters have been already identified and charac-
terized [16]. The selection of plant species is a vital task in the success of phytoremediation
technology (Table 4.1). The species selected should be able to grow in high concentration
of trace metals. Some of the plants belonging to Brassicaceae like Alyssum species, Thlaspi
species and Brassica juncea, Violaceae such as Viola calaminaria, Leguminosae such as
Astragalus racemosus are known to take up high concentrations of heavy metals and radio-
nuclides. To date, there are approximately 400 known metal hyperaccumulators in the
world that can be used for phytoremediation [19].
Some new concept of symbiosis between nitrogen fixing plant species and rhizobia was
also developed. This system uses both advantages of plants and microbes, particularly
engineered genes can be transformed to plants through infection with recombinant
microbes [13]. Our atmosphere recently polluted with various pesticides, pharmaceuti-
cals, petroleum compounds, PAHs, PCBs, etc. is a worldwide issue, and the progress of
inventive phytoremediation for the decontamination of sites is therefore of paramount
importance. The different mechanisms can be used for this purpose [20–28]; however, the
technology has effective mechanism in decontamination of the affected areas [29–31].
4.2 Different Toxic Materials from Industrie 75
Several authors demonstrated that phytoremediation potential in their studies and

proved that plants have excellent mechanism in phytoremediation process [32–55], and the
technique requires more wide application.
However, the process of detoxification in plants is somewhat slow, but the metals or
pesticides could be released into the atmosphere [56]. Some biofuel plants like Ricinus
communis [57], Jatropha curcas [58], Miscanthus giganteus [59], etc. have great potential to
reduce the contamination and sustainable ecosystem services.
4.2 Different Toxic Materials from Industries
4.2.1 Fly Ash from Thermal Power Plants

The thermal power plants are the major source of fly ash that creates environmental
problem in nature. The coal used in Indian power plants has more ash (34–35%) [60]. Fly
ash particles contain the small sized particles ranging from 0.01 to 100 μm [61, 62]. The
thermal power plants generate more than 100 million tons of fly ash each year, and close to
90% of this is dumped in landfills or settling ponds. Distribution of fly ash in the surrounding
areas may occur either during crude transportation or from the disposal site like ash ponds
or landfills where the dry fly ash become airborne along the direction of the wind [10]. The
distribution of particulate matter (PM) in the surrounding area of thermal power plants is
enormous when the disposal of fly ash is going near dumping sites. The finer particles
deposits on the surface of the materials and plants. A number of health studies have proven
adverse effect on humans due to the presence of particulate matter (PM < 10 μm). Some
smaller sized particles (<2.5 μm) have serious effects on the respiratory system [63–67].
Fly ash has been recognized as a great environmental issue due to large consumption of
coal in thermal power plants which need more than 1000 km2 area in India. Globally, it
would require more than 3235 km2 of area for fly ash disposal and remediation [68].
Therefore, phytoremediation of fly ash disposal is the need of the hour to reduce environ-
mental contamination and sustainable development of the community and to reduce
health hazards [18, 58, 69–71]. Several plants from the families Brassicaceae, Chenopodiaceae,
Fabiaceae, Leguminoceae and Poaceae are growing in fly ash with reduction in contamina-
tion. Acacia sp. and Leucaena leucocephala were established to have a high tolerance and
survival in waterless, barren and metal-contaminated areas [72].
4.2.2 Heavy Metals and Pesticides in Environment

4.2.2.1 Cadmium
Cadmium (Cd) is dangerous to our environment and released into the environment though
different industries waste like electroplating, plastic manufacturers, paint industry, alloy
and battery preparations. One of the important toxic metals in storm water is cadmium
(Cd), which may be unsafe even in very low concentration and harm human kidney and
have severe health effects [73]. The appliances used in houses, automobiles and heavy
trucks, agricultural, industrial and aeroplane tools and fasteners of all kinds (like nuts,
bolts, screws and nails) are mostly found in Cd coating, which is available for photography,
rubber curing, fungicides and for luminescent dials [74].
4.2.2.2 Arsenic
The problems of toxicity and occurrence of Arsenic (As) have drawn much attention in
Indian scenario. The issue of release of As from fly ash has increased from several thermal
power plants and produces environmental pollution. The heavy metal As exists in the −3,
0, +3 and +5 oxidation states [75]. Due to the existence of different oxidation speciations,
the arsenic may be found in many different chemical states and forms. Therefore, the
health effects of As may vary widely depending on the chemical form of the As. The burn-
ing of coal and smelting of metals are major sources of As in the air. The most probable
As species are elemental (As) and oxide forms (As2O3) in the oxidizing flue gas in coal
combustion process. However, As2O3is much more volatile than the elemental As, and
researchers have concluded that As could only be present in an oxide form in the flue
gas [76, 77]. In coal gasification, As4 with traces of arsine (AsH3) is the most probable spe-
cies [78, 79]. As concentrations in the combustion residues vary widely depending on the
coal quality, pH of ashes and the ignition conditions, ranging from 2 to 240 ppm in the fly
ash and from 0.02 to 168 ppm in bottom ash [80]. Arsenic (As) represents an important
environmental problem due to the toxic effects of this metalloid, and its accumulation
through the food chain that poses long-term risks to human being [81].
4.2.2.3 Chromium
Chromium is present in different states, ranging from Cr+2 to Cr+6, but in soils the most stable
and popular are trivalent Cr (III) and hexavalent Cr (VI) forms [82], which display quite
different chemical properties and affect organisms in different ways. In fact, in contrast to other
metals, the hazard of chromium is dependent on its oxidation state. Hexavalent chromium
is most dangerous to human health and carcinogenic in nature, while trivalent chromium is
essential (in low concentrations) for human and animal nutrition, relatively water insoluble
and less toxic than Cr (VI) [83]. In many countries, chromate, which is the most prevalent form
of Cr (VI), is present in solid/liquid waste due to anthropogenic activities [84].
4.2.2.4 Pesticide in Environment

Chemical pesticides have contributed greatly to the increase of yields in agriculture by
controlling pests and diseases and also towards checking the insect borne diseases (malaria,
dengue, encephalitis, filariasis, etc.) in the human health sector [85, 86]. The world’s
increasing population has facing the risk of food security [87, 88]. One of the best options
to increase the food production is the use of pest management practices because most of
the food degraded due to pests. Due to variations in climate conditions, the tropical coun-
tries are facing more reduction in food grain production [89, 90].
However, the sporadic use has been leading to significant consequences not only to pub-
lic health but also to food quality resulting in an impact load on the environment and hence
the development of pest resistance [91]. Through over use and misuse there is considerable
waste, adding to the cost and contributing to the adverse environmental and health conse-
quences. Inappropriate application of pesticides affects the whole ecosystem by entering
the residues in food chain and polluting the soil, air, ground and surface water [91–93].
Some important insecticides like aldrin, Orgnochlorine, DDT hexachlorocyclohexane
(HCH) and dieldrin, are among the frequently used pesticides in the developing countries
of Asia because of their low cost and versatility against various pests ([88, 94, 95]).
Nevertheless, because of their potential for bioaccumulation and biological effects,

these compounds were banned in developed nations two and half decades ago [96–98].
Their resistance to degradation has resulted in contamination universally and found in
many environmental compartments. Such residues may be composed of many substances,
which include any specified derivatives such as degradation products, metabolites and
congeners that are considered to be of toxicological significance. According to the Food
and Agriculture Organization (FAO) inventory [99], more than 500,000 tons of unused
and obsolete pesticides are threatening the environment and public health in many coun-
tries. Public concern over pesticide residue has been increasing during the last decade.
Recovering from the euphoria of green revolution, India is also now battling from
residual effects of extensively used chemical fertilizers and pesticides such as HCH, DDT,
endosulfan, phorate, etc. [86, 87, 100].
4.2.3 Phytoremediation Technology in Present Scenario

Several phytoremediation strategies have been initiated by the different industries and
organizations working in the field of phytoremediation. The concept of using green plants
for phytoremediation like green belts came initially from three nations such as Kenya,
Britain and USA [101–103].
The green belt has been defined as “a strip of trees of such species and such geometry
that when planted around a source would considerably attenuate the pollution in air by
intercepting and accumulating the toxic elements in sustainable manner” [103]. Sometimes
green belts are simply called as trees; in mainstays of green belts are trees with some shrubs
and ground vegetation. The purpose of green belt may vary for from industry to industry. It
is important to capture several factors, viz. selection of suitable plant species depending on
the agro-climatic environment of the province; nature of the pollutant to be ameliorated;
tolerance of plant species against the pollutant and general landscape and topography of
the area [103, 104]. Plants have taken the toxic elements form atmosphere by three meth-
ods such as through leaves where absorption and deposition of elements takes place and
particulate matter on downwind side due to slow movement of air [105–110]. The green
belt plantation can help in removing the air pollutants and alter the urban environment by
lowering the temperature, and evapotranspiration can help in the improvement of healthy
life for humans [111–113]. Vegetation in the landfills seems to be the most viable in in situ
remediation technique that will lead to stabilization against wind and water erosion;
decontamination of toxic heavy metals by plants and development of aesthetically pleasing
landscape that will provide shelter and habitat for wildlife [72]. Phytoremediation of heavy
metal-contaminated soil is a developing technology and has attracted much attention
because it is an environment friendly and relatively cheap technique. One of the process of
phytoremediation the phytoextraction in which green plants uptake the metals and pesti-
cides from the soil and accumulate in their parts.
The fly ash grown species are recommended by various researchers are like Calotropis
procera, Cassia tora, Chenopodium album, Sida cardifolia, Blumea lacera [114]; Prosopis
juliflora [115]; Acacia auriculiformis, Leucaena leucocephala [72]; Cicer arietinum [116];
Sesbania cannabina [117]; Cassia siamea [118]; Agropyron elongatum, Festuca arundina-
cea, Melilotus officinalis [119]; Liquidambar styraciflua, Platanus occidentalis [120];
Mesembryanthemum nodiflorum, Enchylaena tomentosa, Halosarcia halocnemoides,

Halosarcia pergranulata [121]; Melilotus alba [122]. Phytoremediation technique offers a
useful way to check leaching of metals from fly ash dykes and growing of multipurpose tree
species (Table 4.1) on these soils.
Another phenomenon, the “phytostabilization,” involves the use of plants to arrest the
contaminant in the rhizosphere and thus reduce its bioavailability in the environment [123,
124]. In several cases, it has been observed that despite the presence of high concentration
of metals in soil, they have low mobility into the plant system [125]. For such cases, che-
late-induced phytoextraction was developed, with the objective of desorbing heavy metals
from soil matrix into soil solution to facilitate the transport of metals into xylem and
increase the translocation of metals from the roots to shoots of some fast growing, high
biomass producing plants [126, 127].
Although the latest advent of phytoremediation has provided state-of-art molecular biol-
ogy to genetically modify suitable plants for enhanced tolerance, uptake and remediation
of pesticides, lot of innovations and additional researches are required to establish real-
time onsite phytoremediation technologies (Figure 4.1). Taking all these factors into con-
sideration, Jatropha curcas tested its suitability for phytoremediation of fly ash. J. curcas is
a large shrub or small tree belonging to the family Euphorbiaceae. It is regarded as a poten-
tial biofuel crop for future due to its low moisture demands, pure hardiness and stress
Agro biotechnological practices

Other benefits
CO2
Genetic manipulations fixation
Biomass for bioenergy

production, paper and
pulp, plywood making,
charcoal, etc.
Rhizospheric modifications Lindane
Dioxin
Microbial interventions
Exploiting catabolic and plant growth
promoting rhizospheric microbial diversity
Chemical interventions
(modifying rhizosphere by
the addition
of nanoparticles which
enhances the
plant microbe signalling)
Figure 4.1 Strategies for enhancing the onsite remediation potential and economic benefits of
phytoremediation [128].
handling ability [129, 130]. It grows fast with little maintenance and can reach a height of
3–8 m [129–131].
It has been identified in India as the most suitable oil bearing plant and has been recom-
mended for plantation on wasteland as it requires minimal inputs for its establishment
[130, 131]. Fly ash landfills are vast wastelands in India. Our aim is to exploit this plant for
phytoremediation. First, for phytostabilization of fly ash, to reduce its blowing in nearby
crop fields; and second, for phytoextraction of heavy metals through the use of chelator.
This would restrict the leaching in the water bodies.
Sinha et al. [131] suggested some species for phytoremediation like Elodea canadensis,
Ceratophyllum demersum, Potamogeton spp., Myriophyllum spp., Spartina alterniflora,
Pinus sylvestris, Poa alpine, Bouteloua gracilis [133, 134].
Some best suited plants for phytoremediation are from the family related to Gramineae
and Cyperaceae and some members such as genera Brassica, Alyssum and Thalapsi of
families Brassicaceae, and Salicaceae (willow and poplar trees). Several grass species like
vetiver, rye, Bermuda and tall fescues have important role in phytoremediation of metal
and oil pollution [135].
Phytoremediation by vetiver (grass) is a low-cost technology as compared with
conventional (engineering) methods for site remediation. It is also virtually maintenance
free, the grasses regrow very quickly and its efficiency improves with age [136]. So many
green plants are used in phytoremediation task. Important among them are other grasses
like the Bermuda grass (Cynodon dactylon), Bahia grass (Paspalum notatum), Rhodes
grass (Chloris guyana), the tall wheat grass (Thynopyron elongatum), common reed
grass (Phragmites australis), the munj grass (Sachharum munja) and Imperata cylindrica.
Other plants are the marine couch (Sporobolus virginicus), cumbungi (Typha domingensis)
and Sarcocrina spp.
Although various remediation methods such as containment, solidification and stabili-
zation etc. have been proposed for the decontamination of onsite As-contaminated soils, all
of these methods require appropriate controls and long-term monitoring to ensure the
behaviour of As through soil column [137]. In some studies, it has been reported that
hyperaccumulators growing in As-contaminated soils have the ability of accumulating
high concentration of arsenic [138–142]. However, the field utilization potential of most
of these plants are very less due to their small biomass and slow growth rate ([143]).
Rhizoremediation, involving both plants and the rhizospheric microbes, is an efficient
bioremediation process for contaminant degradation, and different metal tolerance mecha-
nisms have also been discovered in various microbes [144, 145]: exclusion, active removal,
biosorption, precipitation or bioaccumulation, both in external and intracellular spaces.
Some bacterial strains are also known to play an important role in the biochemical cycle of
As, through its conversion to species with different solubility, mobility, bioavailability and
toxicity [146, 147].
Variety of technologies (including chemical physical and biological treatment methods)
are available for storm water treatment. However, these methods present different
efficiencies for different metals, and they can be very expensive for the treatment of low
level metal-contaminated water. In contrast, the phytofiltration has been proposed as a
promising, environmentally friendly technology for removing the heavy metal concentration
of the contaminated water. In phytofiltration, high metal-accumulating plants function as
biofilters, which can be remarkably effective in sequestering metals from polluted

waters [9]. Many aquatic plants, usually those found in polluted water bodies, have been
suggested for waste water treatment, i.e. they have the ability to accumulate unusually high
concentration of heavy metals, without impact on their growth and development. However,
most species identified so far are not suitable for onsite phytofiltration due to their small
root and shoot biomass and slow growth rate. In contrast, plants with good growth usually
show low metal accumulation as well as low tolerance to heavy metals [74].
The advantage of this free-floating culture system is its easy harvesting. Further, more
studies are needed to evaluate the onsite application of this free-floating phytofiltration
technique [1]. There is a report that Brassica (mustard) species or varieties of Brassica jun-
cea (Indian mustard) have an enhanced ability to accumulate metals from hydroponics
solution into their above ground (harvestable) parts. These plants reduce the toxicity of the
metals through accumulation in shoot biomass [148].
The remediation of Cr(VI)-contaminated soils, today, is essentially based on physical and
other approaches, which consist of mine or pumping of infected material and added reduc-
ing agents that lead to the precipitation and sedimentation of reduced chromium [Cr(III)],
less toxic than Cr(VI) and greatly insoluble. Cr(VI) remediation strategies using traditional
technologies have been dealt in depth by Higgins et al. [149].
The ability of several microbial groups (bacteria, fungi, microalgae) to reduce Cr(VI) to
Cr(III) has been considered of much interest in order to clean up soil/water polluted with
chromate. In fact, there is no doubt that the development of an effective biological system
to alleviate the environmental problems associated with hexavalent chromium is highly
desirable. The ability of a bacterial strain to reduce hexavalent chromium, although the
mechanism of Cr(VI)-reduction may differ from strain to strain, is an attractive feature in
order to plan a biological strategy for effective chromate detoxification, but high concentra-
tions of chromate in the environment often can repress the microbial activity and
growth [150, 151]. The ability of direct Cr(VI) reduction has been found in many bacterial
genera including Pseudomonas, Micrococcus, Bacillus, Achromobacter, Microbacterium
Arthrobacter, Corynebacterium [151–154].
4.2.4 Conclusion
Phytoremediation is an emerging technology because conventional methods to clean up
the environment are cost intensive and eco-unfriendly. In addition, microbes can modify
chemical reactions in toxic metals and enhance their uptake in root zone areas to be taken
up by metal hyperaccumulating plants. This technology is termed as phytoremediation and
has received a lot of attention in recent years due to its cost-effectiveness, solar driven and
high efficiency.
Some molecular techniques have the potential to increase the phytoremediation process
with the genetic manipulations from low biomass accumulating metal hyperaccumulator
plants to high biomass yielding non-accumulating plants. Phytoremediation by vetiver is a
low cost technology as compared with conventional methods for phytoremediation.
However, phytoremediation, especially phytoextraction, is getting popularity as a low cost
and inventive method of remediation. Phytoextraction is a solar-driven technology so that
it can be successfully deployed for the cleaning up of the As-contaminated soils.
Reference 81
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91
Ecological Restoration and Plant Biodiversity

Shalini Tiwari1 and Puneet Singh Chauhan1,2
1
CSIR-National Botanical Research Institute (CSIR-NBRI), Lucknow, India
2
5.1 Introduction
An organized and systematized exploration of socially and economically valuable products

obtained from biological resources, viz. microorganisms, plants, animals, etc., is called
bioprospecting. It involves the search, collection and derivation of genetic material from biodi-
versity samples that can be further developed for commercialization of pharmaceutical, agri-
cultural, industrial products and hence, overall for societal benefits [1, 2]. It is carried out by a
diverse range of industries including botanical medicines, pharmaceuticals, crop protection,
agricultural seeds, horticulture, cosmetics, environmental monitoring and manufacturing [3].
Biodiversity is variable between living organisms from all sources including terrestrial and
aquatic ecosystems. This includes intraspecific and interspecific diversity and thus can be
divided into three parts: (i) Genetic biodiversity: difference in the types and number of genes
and chromosomes present in different species; (ii) Species biodiversity: variation in the num-
ber as well as species richness within a region and (iii) Ecosystem biodiversity: interaction
among species that live together in a given area [4]. Considering terrestrial habitat, 5–30 mil-
lion species contains thousands of genes. Still, approximately known two million species have
limited knowledge of the global distribution [2]. Therefore, it is rational to use the application
of new technologies to explore the largely unidentified and unexplained species that will pro-
vide even more benefits to mankind. Hence, it can be assumed that for bioprospecting, biodi-
versity is the essential resource, but it is hardly ever possible to predict the specific genes,
species as well as ecosystems will turn out to be valuable in the future for bioprospecting.
Bioprospecting thus has generated numerous valuable products from different various
ecosystems, viz. tropical forests, temperate forests, temperate grasslands, arid and semiarid
regions, freshwater ecosystems, along with cold and warm oceans. Among these ecosys-
tems, tropical forests are a major source for countless products due to the species richness
in its environment. Unfortunately, for many diverse purposes, unregulated and rigorous
collection of samples by humans for hundreds of years, the United Nations has established

92 5 Ecological Restoration and Plant Biodiversity
a framework to preserve the world’s biodiversity while promoting the sustainable use of
biodiversity. Consequently, the conservation and restoration of biodiversity present in all eco-
systems would provide various prospects for future bioprospecting. Therefore, this chapter
includes the area of bioprospecting, followed by its conservation and ecological restoration.
5.2 Major Areas of Bioprospecting
Bioprospecting comprises systematic search for genes, natural compounds, as well as

organisms of wild resources to develop product by bio-physicochemical and genetic meth-
ods without disrupting nature and natural resources. Thus, bioprospecting includes three
major areas [3]: (i) Chemical prospecting, (ii) Gene prospecting, and (iii) Bionic prospect-
ing (Figure 5.1).
5.2.1 Chemical/Biochemical Prospecting

Biochemical prospecting includes investigation of novel and valuable chemicals from
organisms [5]. Recently, high-performance chemical screening and automated bioassay
platforms for identification, isolation and characterization of novel bioactive compounds
from wild bio-resources (members from lower to higher plant kingdom, insects and inver-
tebrates from animal kingdom) have shed the light in the area of natural product, drug and
pharmaceuticals research [6]. Chemical prospecting from wild-type plant species is widely
applicable in agro-chemistry (bio-pesticides), pharmaceuticals, enzymes, proteins, food
additives, cosmetics and other industrially useful chemical products.
5.2.2 Gene/Genetic Prospecting

Molecular systematics aims to identify and characterize genetic variants within a species
or a population at the DNA level by widely using DNA fingerprinting techniques [3].
Such types of approaches are gaining momentum nowadays and are useful for gene- and
Bioprospecting
Chemical Drug and pharmaceuticals, pesticides,

prospecting cosmetics/cosmaceuticals, food additive/
nutraceuticals, other industrial valuable products
Gene Genetic engineering, crop development,

prospecting fermentation, cell culture
Bionic Designs, sensors technologies, architecture,

prospecting bioengineering, bio-modeling
Figure 5.1 Major areas of bioprospecting.

5.3 Bioprospecting: Creating a Value for Biodiversit 93
biodiversity-rich poor countries. But due to the new patent laws, development of geneti-
cally modified organisms, hybrids and transgenic plants may rise serious implications on
the jurisdiction of biodiversity-rich poor nations. Therefore, it is essential for such coun-
tries to develop appropriate research programmes to identify, characterize and evaluate
their own genetic bio-resources, specifically of endemic species and perform molecular
characterization to prevent biopiracy or genepiracy issues [7]. Modern molecular
approaches such as DNA recombinant techniques and transgenic technologies facilitate
transfer of the desirable agronomic traits or chemicals from plant to bacteria. The resulting
transgenic bacteria are potential candidate for chemical factories by synthesizing specific
products such as proteins, enzymes and other related biomolecules at large scale.
5.2.3 Bionic Prospecting

Bionics also known as biologically inspired engineering is a new area of bioprospecting in
which new patterns, models, designs and techniques are developed and built on natural
biodiversity. Bionic prospecting also includes sensor technologies, bio-engineering, bio-
modelling and architecture.
5.3 Bioprospecting: Creating a Value for Biodiversity
The nature’s library has potential to abundantly provide food, nutrients, medicines and
various valuable industrial products. Apart from this information there are still various
unknown potential in genes, species, population as well as ecosystems that embodies
incredibly never-ending biological horizons. Years ago researchers focused only on the
domesticated plant species of biodiversity, and the information about most of the worthy
bioresources was challenging and unavailable. But, with the help of bioprospecting, the
majority of the bioresources have been explored and their genetic, chemical as well as eco-
nomic potential has been evaluated using modern technologies. These recent approaches
thus have capability to identify and isolate potent and essential bioactive compounds at
massive scale.
Biochemical techniques are one of the approaches that help to isolate and identify
bioactive chemicals from plant extracts. It includes initial screening on the basis of bio-
logical activity of compounds using in vitro enzyme methodology and further isolation
and characterization of chemicals via modern chromatographic techniques such as high-
performance liquid chromatography (HPLC), high-performance thin-layer chromatogra-
phy (HPTLC), gas liquid chromatography (GLC), medium pressure liquid chromatography
(MPLC) and liquid chromatography mass spectrometry (LCMS).
Apart from biochemical techniques, biotechnological approaches are also powerful tools
that not only help to comprehend the structure and function of living cells, tissues or
organisms but also help in manipulating or editing the genome of the organism to produce
modified or novel products. Hence, it called the most promising area of bioprospecting
named gene prospecting. It provides facts about the retaining and transmitting of genetic
information of living beings, their response against environmental, physical and chemical
changes, etc. The improvements made in older biotechnology tools include DNA
fingerprinting; it has now become possible to study the species genetic diversity in an easy
manner. Hence, biotechnology applications provide various benefits to humankinds in the
form of foods, medicines, drugs and other healthcare products as well as economic goods.
5.4 Conservation and Ecological Restoration

for Sustainable Utilization of Resources
Uncontrolled exploitation of biodiversity for food, fodder, fibre, fuels, medicines, oils, per-
fumes, dyes, gums, resins, pesticides, insecticides, phytochemicals, proteins and genes
causes biodiversity loss. Hence, special attention is required to protect economic, ecological
and existence value of biodiversity. Conservation of biodiversity needs greater importance
as humans will confront serious threats in near future due to the problems that arise via
environmental degradation, including loss of biological diversity. Approximately 60,000
plant species out of the total 2,87,655 known species are on the verge of extinction due to
countless reasons, in which overexploitation is one of the major issues [8]. Being a priceless
and renewable resource, biodiversity exhibit potential values to humankind and interest-
ingly have self-sustaining ecological system for maintaining the integrity and potency of
biosphere. Thus, this devastating condition of biodiversity contrives numerous national
and international initiatives, strategies, action plans, policies and legal frameworks aiming
to prevent the continuing damage and loss of plant genetic resources [7]. The Global
Strategy for Plant Conservation (GSPC) is among those strategies introduced by conference
of parties (COP) of CBD at its sixth meeting held in Hague, 2002. Its aim is to conserve
plant diversity by in situ and ex situ or by combination of both methods. In recent years, the
GSPC committee members updated their strategies of plant conservation for the year
2011–2020 (https://www.cbd.int/gspc/). There are several other plant conservation pro-
grammes organized by government officials at the national level to halt the loss of plant
resources. In India, the National Biodiversity Strategy and Action Plan identifies ex situ
plant diversity conservation as a priority area of action in India and suggests strengthening
and enhancing the role of botanical gardens, home gardens and other ex situ conservation
networks.
The National Biodiversity Strategy and Action Plan (NBSAP) defines ‘ex situ plant diver-
sity conservation’ as a priority action to ensure the social, environmental and economic
development of the country (https://sustainabledevelopment.un.org/). It emphasizes and
enhances the role of botanical gardens, home gardens and other ex situ conservation setups
within individual countries as effective ways for biodiversity conservation. Recently in
2019, NBSAP prepared the first Global Sustainable Development Report (GSDR) by the
independent group of researchers and scientists appointed by the United Nations Secretary-
General and laid the foundation for 2030 Agenda and the Sustainable Development Goals
(SDGs). In India, a special assistance program is being implemented at huge level by the
Ministry of Environment & Forests (MOEF) to provide fund to botanical gardens to develop
and maintain their infrastructure and other resources to support ex situ plant diversity
conservation programmes as India supports 8.1% of the world’s biodiversity with its 2.4% of
world landmass (http://moef.gov.in/wp-content/uploads/2019/04/EFC.pdf). India has a
strong network of research and development institutions funded by the ministries and
5.5 Biodiversity Development Agreement 95
departments of the central as well as the state government. Research programmes carried
out by both national and international institutes to conserve plant resources include main-
tenance of catalogue of rare endangered threatened (RET) species, their assessment and
distribution, in situ and ex situ conservation of RET species via micro-propagation, genetic
diversity assessment, gene bank, bioprospecting and sustainable utilization [6].
Ecological restoration varies from conservation as its effort is to restore the ecosystem in
its original appearance. The conservation focuses on sustainable use and preservation of
the original form, while the restoration, on the other hand, involves restoring the ecosys-
tem to its original form that once existed. Although, the diversity of the restored species is
usually reduced in comparison to the former habitat [3]. Hence, restoration is strictly nec-
essary across the globe, as a result of national and local government policies demanding
the repair and maintenance of destroyed habitats such as abandoned industrial and mining
sites, polluted and contaminated farm land as well as surface water by rigorous human
activities. Ecological restoration is possible by proper harvesting or offsite plantation and
further transfers to the restored site of plants (species) of neighbouring or comparable eco-
systems [6, 9, 10]. It requires an in-depth knowledge of species, their ecological behaviour
and their interaction with each other and within the ecosystem. Sometime, species seems
extinct at a particular location, but in actual they survive in dormant or hidden forms.
These species could be easily restored via removing the inhibiting factors [11]. For exam-
ple, via preventing excessive burning or grazing, the woody plants of degraded land of
tropical savanna restored by their own due to the presence of hidden rootstocks in the soil.
Another species that reappeared after an absence of almost 70 years at wetland site of
Britain is fen violet Viola persicifolia [12]. It became possible due to long dormancy period
of its seeds. Alternatively, agroforestry is also an example of ecological restoration and
nowadays it became an important practice in this field as the presence of trees in land con-
fers several advantages. Numerous agroforestry approaches have been performed till date
to restore and increase the productivity of farm land. For example, trees stabilize the soil,
and used in terracing, strip-cropping and contour cultivation to prevent soil erosion and
increase water availability. Trees are also able to fix nitrogen and thus increase soil fertil-
ity [13]. These suitable soil conditions help species in their restoration and conservation of
biodiversity. Food and non-food products obtained from trees also helps farmers by provid-
ing nutritional security, enhancement in income, better livelihoods and hence combating
poverty [14]. About concerning the land degradation and biodiversity loss, an international
committee takes a global initiative in 2011 to restore 150 mha area of deforested land by
2020. The New York Declaration on Forests in 2014 promises and raised up the restoration
target up to 350 mha area by 2030 [15].
5.5 Biodiversity Development Agreements

Besides several advantages of bioprospecting, there are some demerits of it. Bioprospecting
needs to evaluate the frequent occurrence of biopiracy and most importantly the enhance-
ment in biodepletion that might led to mass extinction [16]. Biodiversity development
agreements (BDA) intend to distribute the benefits of biodiversity to those who bear the
conservation costs through bioprospecting efforts. BDAs are agreements between
biodiversity holders (usually of developing country) and biodiversity users (usually of pri-
vate firm) to share bioresources and the benefits from the development of new products [7].
In general, the prospecting efforts for biodiversity have two objectives: (i) to discover and
use certain genetic resources of unknown species that could have potential benefit for agri-
culture and industry and (ii) to offer local residents a return on biodiversity conservation.
These agreements are proposed for the help of countries as well as local people that have
right to share greater benefits of resource conservation. Unfortunately, compared with the
different opportunity aspects, necessary attention is not paid to the potential of BDAs. For
example, the consequences of food prices, demographic demand, hunger and land-use
policies by the government have a significant impact on the returns to competing forest
land use. From this perspective, excessive demand and exploitation for agricultural land
leads to loss of species richness and biodiversity.
5.6 Conclusions
Bioprospecting will accomplish several goals such as (i) generate revenues for local com-
munities, lands and environmental projects; (ii) develop scientific and technological
approaches to maintain biodiversity; (iii) raise awareness for the commercial and non-
commercial value of biodiversity and (iv) sustainable resource management. Overall, the
biodiversity is a valuable natural resource that must be protected and used sustainably
for the good of both present and future human generations. But excessive exploitation of
biodiversity for benefits of humankind causes biodiversity loss at mass level. At present,
a pandemic disease COVID-19 is negatively impacting human societies of most of
the countries in the world, however reduced human activities due to lockdown in most
of the countries is benefiting biodiversity [17]. This situation also points towards the
need of conservation and restoration of biodiversity in present. Hence, the conservation
and ecological restoration is an optimistic, prosperous vision in which human activities
promote the sustainability of plant life such as maintenance of plant genetic diversity,
endurance of plant species and their habitat as well as related ecosystems which in turn
strengthen our livelihoods and well-being. Thus, the remarkable capability of humans
will definitely help in the field of biodiversity by deciphering the complexities of nature,
developing novel technologies and controlled utilization for the sustainable use of the
limited bioresources.
R
eferences
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BioScience 56 (7): 560–563.
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biodiversity. The Pharma Innovation Journal 8 (4): 256–265.
bioprospecting, traditional knowledge. sustainable development and value added products:
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what happens when discoveries are made. Arizona Law Review 50: 545.
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restoration: exploring the potential of agroforestry to enhance the sustainability and
resilience of degraded landscapes. Food and Agriculture Organization of the United
Nations (FAO), www.fao.org/publications.
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through agroforestry. Journal of Agriculture and Environment 10: 133–143.
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17 Corlett, R.T., Primack, R.B., Devictor, V. et al. (2020). Impacts of the coronavirus pandemic
on biodiversity conservation. Biological Conservation 246: 108571.
99
Endophyte Enzymes and Their Applications in Industries

Rufin Marie Kouipou Toghueo and Fabrice Fekam Boyom
Antimicrobial and Biocontrol Agents Unit (AmBcAU), Laboratory for Phytobiochemistry and Medicinal Plants Studies,
Department of Biochemistry, Faculty of Science, University of Yaoundé I, Yaoundé, Cameroon
6.1 Introduction
Produced by living cells to bring about specific biochemical reactions, enzymes are biocata-
lysts, providing the lower-energy pathway between reactants and products. These biomol-
ecules are the primary instruments for the expression of gene action, required for both
syntheses and breakdown reactions. Enzymes are highly specific in their action on sub-
strates and often act as a consortium to catalyse, by concerted action, the sequence of meta-
bolic reactions performed by the living cells [1–3].
The use of enzymes by humans is thousands of years old. The earliest known record of
enzymes is found in Homer’s Greek epic poems dating from about 800 BCE, mentioning
their use in the production of cheese. In Japan, documents from more than a 1000 years
ago denote the use of naturally occurring enzymes in the production of fermented products
such as sake (Japanese rice wine). Other ancient documents mention the use of enzymes
for brewing, baking and alcohol production [3, 4]. Nowadays, the enzyme industry has a
great market because of their increasing demand in multiple industries, including food
manufacturing, animal nutrition, cosmetics, as well as for research and development and
mostly as therapeutic agents against many diseases, to name but a few [5, 6]. These biomol-
ecules are found in animals, plants and particularly microbes which are the source of most
commercial enzymes [7].
Indeed, microorganisms are the greatest source of enzymes, bacteria and fungi being
the best suited for industrial-scale production of most of the enzymes used in various
industries [3, 5]. Their favourable features include the ability to be cultured in large quan-
tities in simple and inexpensive substrates, to be easily handled and grown in huge tanks
without light, to have a very high growth rate and shorter generation times, and to be
genetically modified more easily to increase enzyme production yield [8, 9]. Moreover,
microbial enzymes are preferred in multiple industries because they are biochemically
diverse and have a broad range of activities inherent to their resilience towards variation

100 6 Endophyte Enzymes and Their Applications in Industries
of environmental parameters such as pH, temperature and salinity. In addition, compared

to some plant and animal enzymes, they are more active and stable and remain active
under a wide range of varied physicochemical conditions [9–11]. However, the absolute
and relative amounts of the various individual enzymes produced vary markedly between
genus and even between strains of the same species. Therefore, selecting the appropriate
microbial strains with the capacity for producing the highest amounts of particular or
desired enzymes is of great importance. Of note, industries are nowadays looking for new
microbial strains with abilities to produce specific enzymes to fulfil the current enzyme
requirements. The search for novel active enzymes is currently concentrated on microor-
ganisms living in diverse environmental conditions, or inhabiting unique and exceptional
biotopes. Endophytic microorganisms able to survive under quite extreme and inhospita-
ble conditions have recently seen a huge jump in scientific attention and interest [12].
Living asymptomatically inside plants for at least part of their life cycle, endophytes have
emerged as a valuable source of novel metabolites and industrially important enzymes [13].
This group of microorganism has been meticulously investigated and used as a base for
sustainable research and development of new enzymes [14]. Within this scope, multiple
enzymes, including amylase, asparaginase, cellulase, lipase, laccase, protease, xylanase,
etc., have already been reported from endophytes [15, 16]. This chapter presents recent
updates on the potential of endophytes as enzyme sources, with particular focus on inves-
tigations reporting the purification and characterisation of enzymes produced by endo-
phytic microbes.
6.2 The Rationale for Bioprospecting Endophytes for Novel

Industrial Enzymes
The rationale behind investigating endophytes for novel industrial enzymes is guided by
the recognition that endophytic microorganisms are capable of producing enzymes for
their inherent natural needs. Of note, endophytes produce enzymes to (i) facilitate their
penetration in their host plant tissues, (ii) evade the host defence mechanism and (iii) pro-
tect and promote the growth of the host plant (Figure 6.1).
Indeed, for the successful infection of their host plant, endophytes produce a series of
hydrolytic and oxidative enzymes that are necessary for the degradation of lignocellu-
losic components from the cells of the host plant to facilitate the penetration and
Endophytes
Out of the host plant Inside the host plant

Facilitate their Protect and
Enzymes
Enzymes
Enzymes
Escape the
penetration promote the
host defence
into their host growth of the
mechanism
plant host plant
Figure 6.1 Attributes of endophytes making them suitable for enzymes bioprospecting.
6.3 Endophytes as a Source of Industrial Enzyme 101
colonization. Endophytes have been reported to possess a hydrolytic system responsible

for polysaccharides degradation consisting mainly of xylanases and cellulases and a
unique oxidative ligninolytic system comprising mainly laccases, ligninases and peroxi-
dases which degrade lignin and open the phenyl rings [14]. On the other hand, it is
widely recognised that to enter the host plant, endophytes have to pass through the first
line of defence of the plant’s immune system. To this end, endophytes produce enzymes
such as amylases, lipases and proteases, as part of their mechanism to overcome the
defence set in place by the host against any microbial invasion [17]. Fungal endophytes
for instance have been reported to modulate the plant’s immune system by secreting
chitin deacetylases, which deacetylate chitosan oligomers and, hence, prevent from
being recognised by chitin-specific receptors [18, 19]. Moreover, to counteract the oxida-
tive burst of reactive oxygen species (ROS) generated by the plant as a defence mecha-
nism, endophytes also produce multiple enzymes such as superoxide dismutases,
catalases, peroxidases, alkyl hydroperoxide reductases and glutathione S-transferases [20].
In case the host plant produces antimicrobial secondary metabolites as a defence mecha-
nism against microbial colonisation, endophytes might secrete the matching detoxifica-
tion enzymes, such as cellulases, lactases, xylanases and proteases, to decompose these
secondary metabolites before penetrating through the defence systems of the resided
host plants [21].
Besides helping endophytes to escape the plant defence system, the production of enzymes
is also needed to help initiate the host symbiosis process [22].Once inside the host plant, the
symbiosis very well established between endophytes and their host might condition the sur-
vival and health of plants [23, 24]. Some endophytes play an imperative role in protecting or
preparing the plant against abiotic and biotic stresses and also help enhance the growth and
yields [25, 26]. Chen et al. [27] reported that some endophytic fungi could promote the
growth and fitness of the host plants by activating the expression of certain enzymes and
genes. Corroborating this statement, Sherameti et al. [28] showed that the endophyte
Piriformospora indica increased the growth of tobacco roots by stimulating the expression of
nitrate reductase and the starch-degrading enzyme (glucan-water dikinase). These extracel-
lular enzymes produced by endophytes target various macromolecules such as carbohy-
drates, lignin, organic phosphate, proteins and sugar-based polymers to breakdown into
transportable products throughout the cells and to continue heterotopic metabolism [29].
Overall, the obvious ability of endophytes to produce a wide variety of enzymes, including
lipases, phytases, amylases, cellulases, proteases, xylanases, etc., that are capable of degrad-
ing complex macromolecules make them an attractive source for bioprospecting for industri-
ally important enzymes.
6.3 Endophytes as a Source of Industrial Enzymes

Endophytic microbes inhabiting plants from various environmental settings have been
reported to produce various kinds of extracellular enzymes (Table 6.1). In the present sec-
tion, we are discussing enzymes characterised from diverse endophytes and their current
industrial application. This is an attempt to catalogue endophytic enzyme producers that
have the potential to be further investigated for practical application.
Table 6.1 Selected enzymes, endophyte-producing strains and the optimal conditions
for production of enzymes.
Enzymes Endophytes Host plant Fermentations condition Yields References
Amylases G. pulicaris NM Medium composition: 2 g 260 U/mg [30]

NaNO3, 0.4 g yeast extract, 0.5 g
KH2PO4, 0.5 g KCl, 0.5 g
MgSO4 · 7H2O, 0.01 g
FeSO4 · 7H2O and 60 g raw
potato; T: 30 °C; Ip: 5 days;
shake: 120 rpm
Cylindroce A. calcarata Medium composition: g/L (3 g 0.035– [31]
phalum sp. NaNO3; 0.5 g MgSO4 ∙ 7H2O; 5 g 0.25 U/mL
KCl; 1 g KH2PO4; 0.01 g
FeSO4 ∙ 7H2O; 0.1 g CaCl2) with
1.5% maltose and 0.3% sodium
nitrate; pH: 7.0; T: 30 °C; Ip:
7 days
P. minima E. longifolia Medium composition: 4% 212 U/mL [32]
soluble starch-asparagine; pH:
5.0; T: 25 °C; Ip:5 days; shake:
180 rpm
T. cecidicola T. catimb Medium composition: Czapex 2.30 U/g [33]
URM7826 auensis Dox’s brothsupplemented with
Penicillium l-asparagine; T: 30 °C; IS: 108 1.28 U/g
sp. 4URM7827 spores/mL; pH: 6; Ip: 4 days;
shake 120 rpm
F. proliferatum A. altissima Medium composition: modified 0.492 U/ [34]
Czapex dox’s broth (glucose mL
(2.0 g/L), l-asparagine
(10.0 g/L), KH2PO4 (1.52 g/L),
KCl (0.52 g/L), MgSO4 · 7H2O
(0.52 g/L), CuNO3 · 3H2O
(0.001 g/L), ZnSO4 · 7H2O
(0.001 g/L), FeSO4 · 7H2O
(0.001 g/L)); T: 36 °C; Ip: 5 days;
shake 120 rpm
Cellulases P. glabrum Espeletia Medium composition: w/v (1% 0.04– [35]
spp. NH4NO2, 1% K2HPO4, 0.5% 1.13 U/mL
MgSO4, 0.1% NaCl, 0.13% CaCl2,
0.01% MnSO4, 0.01% boric acid,
0.001% CuSO4, 0.2% FeSO4,
0.001% ZnSO4, 10 g/L palm oil
empty fruit bunch, 5 g/L glucose
and 6 g/L peptone); T: 25 °C;
shake: 150 rpm and Ip: 20 days
Strain L. corticata Medium composition: 450 U/gds [36]
Tahrir-25 sugarcane bagasse-corncob
(2 : 1); pH: 5.5; T: 30 °C; Mc:
60%; and IS:106
Laccases M. verrucaria Cajanus Medium composition: g/L (200 16.52 U/ [37]
cajan L. potato; 3 KH2PO4; mL
1.5 MgSO4 · 7H2O; 0.01 VB1; and
0.25 CuSO4 · 5H2O) with 10 g/L
peptone and 2% glucose; Ip:
5 days, T: 30 °C; and pH: 6.22
Enzymes Endophytes Host plant Fermentations condition Yields References
N. luteum Eucalyptus Medium composition: modified 0.28 U/mL [38]

trees Czapek Dox broth (KH2PO4,
1 g/L; C4H12N2O6, 2 g/L; yeast
extract, 1 g/L; MgSO4 · 7H2O,
0.5 g/L; KCl, 0.5 g/L; and 1 mL of
a mineral solution with trace
elements: Na2B4O7, 0.092 mg/L;
MnSO4 · H2O, 0.1 mg/L;
(NH4)6Mo7O2 · 4H2O, 0.01 mg/L;
FeSO4 · 7H2O, 0.5 mg/L;
ZnSO4 · 7H2O; 0.1 mg/L; and
CuSO4 · 5H2O, 0.01 mg/L) with
1% (v/v) ethanol and 0.30 mM
CuSO4; T: 22 °C; shake: 120 rpm;
Ip: 20 days
Lipases C. kikuchii Tithonia Medium composition: Vogel’s 14.87 U/ [39]
diversifolia minimum medium with 2% mL
soybean oil; T: 30 °C; shake:
120 rpm; Ip: 6 days
F. oxysporum C. oblongi Medium composition: (w/v) (1% 4.3 U/mL [40]
PTM7 folius peptone, 0.15% KH2PO4, 0.5%
NaNO3, 0.05% NaCl, 0.05%
MgSO4, 0.025% CaCl2, 0.0001%
FeSO4, 0.0001% ZnSO4, 0.0001%
CuSO4 and 1% (v/v) olive oil); T:
30 °C; pH:7; Ip: 6 days
C. guillermondi R. communis Medium composition: 15 g/L 18 U/mL [41, 42]
(NH4)2SO4, 0.15 g/L manganese
sulfate, 10 g/L brewer’s yeast,
80 g/L Tween, 15 g/L glycerol
and 30 g/L soybean oil, dissolved
in 25 mmol/L phosphate buffer;
pH: 6.5; T: 30 °C; Ip: 3 days and
shake: 180 rpm
Proteases Penicillium sp. D. hemprichi Medium composition : rice 3560 U/g [43]
Morsy1 straw supplemented with g/L
(0.3 (NH4)2SO4; 0.5 NaCl;
0.5 KH2PO4; 0.4 CaCl2); T: 26 °C,
IS: 105 spores/mL; pH: 6; Ip:
5 days and Mc: 80%
Acremonium Saraca asoca Medium composition: (g/L) 3.34 U/mL [44]
sp. (skimmed milk 20, yeast extract
0.7, 1.9 KH2PO4, KH2PO4 0.85,
0.0015 MgSO4 ∙ 7H2O) with
fructose and (NH4)2SO4; T:
37 °C; pH: 7; Ip: 3 days
Xylanases A. terreus M. peregrina Medium composition: 1% wheat 54 U/mg [45]
bran as a carbon source; T:
30 °C; pH: 6; shake: 100 rpm and
Ip: 48 h
IS, inoculum size (spores/mL); Mc, moisture content (v/w); Ip, incubation time; T, temperature; rpm,
rotation per minute; NM, not mentioned.
6.3.1 Amylases
Amylases including α-amylase (1,4 α-glucan glucanohydrolase, EC 3.2.1.1), β-amylase (1,4
α-glucan maltohydrolase, EC 3.2.1.2), and glucoamylase (1,4 α-glucan glucohydrolase, EC
3.2.1.3) represent one of the most important groups of enzymes for biotechnological appli-
cation [46, 47]. In a variety of industries (e.g. food, chemical, detergent and textile), micro-
bial amylolytic enzymes are used to convert starch into different sugar solutions through
the hydrolysis of α-1,4-glycosidic linkages. Indeed, in today’s processing industries, micro-
bial amylases have completely replaced the chemical hydrolysis of starch, because of their
multiple advantages, such as higher yield and specificity, the better control over amylolysis
with the complete elimination of the neutralisation steps and undesirable reactions, as well
as the reduction of the probabilities of contamination because of the thermostability of
these enzymes. In addition, the products generated are also very stable and the overall pro-
cess is cost-effective [7]. Among the amylases produced by diverse microorganisms, the
α-amylases from Bacillus sp. have been the most extensively studied given especially their
extreme thermostability [48], while Aspergillus sp. and Rhizopus sp. are often used as
sources of industrial production of glucoamylases [46].
There is a growing interest in exploring endophytes for the production of amylolytic
enzymes that are active at low temperatures and alkaline conditions. In this respect, Marlida
et al. [49] screened endophytic isolates of Gibberella pulicaris, Acremonium sp., Synnematous
sp. and Nodilusporium sp., for their ability to produce raw starch degrading enzymes in sub-
merged fermentation. Their results showed that enzymes produced by Acremonium sp. had
a broad activity towards both small and large granule sized raw starches, whereas the
enzyme from the other isolates showed high activity only toward starches of smaller granule
size. Moreover, the thin layer chromatography analysis of the end products revealed that
enzymes from G. pulicaris, Acremonium sp. and Nodilusporium sp. hydrolysed raw sago
starch to produce solely glucose, while the amylase from Synnematous sp. produced glucose
and maltose. In subsequent investigations, they studied the effect of different nitrogen and
carbon sources on the growth of Acremonium sp. and the yield of amylase enzyme. Their
data showed that using peptone and sodium nitrate as nitrogen sources and raw sago starch
as carbon source each at the optimum concentration of 0.5, 3 and 20 g/L, respectively, the
fungal growth and enzyme activity levels significantly improved. Moreover, at the optimum
pH of 5.0 and incubation temperature of 30 °C, the enzyme production significantly
increased by 19- to 22-fold compared to the activity obtained in the original basal
medium [50]. Further investigation of G. pulicaris led Marlida et al. [30] to show the suita-
bility of raw potato for amylase production and the highest amylase production (260 U/mg
protein) to be achieved when the concentration of raw potato starch was increased to
60 g/L. After several endophytic fungi were screened for amylolytic activity, Fusicoccum sp.
BCC4124 exhibited great potential when grown on multi-enzyme induction-enriched media
and agro-industrial substrates. It was also found that both α-amylase and α-glucosidase
activities were highly induced in the presence of maltose and starch as carbon sources. An
α-amylase with strong hydrolytic activity (kcat/Km:6.47 × 103 min/(mL/mg)) on soluble
starch was purified and exhibited characteristics such as high activity at 70 °C and resistance
to inhibition by glucose of up to 1 mol/L, making it a very good candidate for potential bio-
technological applications [51]. An endophytic Cylindrocephalum sp. strain isolated from
the medicinal plant Alpinia calcarata (Haw.) Roscoe was also reported to exhibit maximal
amylase production when cultured in submerged fermentation at 30 °C and pH 7.0. Various
carbon and nitrogen sources were tested to identify maltose (1.5%) and sodium nitrate (0.3%)
as the best combination of carbon and nitrogen sources for the production of enzymes [31].
An α-amylase produced by the endophytic Preussia minima from the Australian native
plant, Eremophilia longifolia, was purified to homogeneity through a fractional acetone pre-
cipitation and Sephadex G-200 gel filtration, followed by DEAE-Sepharose ion-exchange
chromatography. This enzyme with a molecular weight of 70 kDa exhibited maximum activ-
ity at optimum temperature and pH of 25 °C and 9, respectively, and was activated and sta-
bilised by Mn2+ and Ca2+. The investigation of the effect of different carbon and nitrogen
sources on the enzyme production revealed that with starch as the carbon source and
l-asparagine as the nitrogen source the highest production of 138 U/mg was achieved.
Moreover, bioreactor studies showed that the enzymatic activity was comparable to that
obtained in shaker cultures, which indicates likelihood of scale-up fermentation for the
mass production of this enzyme [32].
6.3.2 Asparaginase
l-Asparaginase also known as l-asparagine amidohydrolase (EC 3.5.1.1) is an enzyme cat-
alysing the deamidation of l-asparagine to l-aspartic acid and ammonia. Both the sub-
strate and product of this enzyme are necessary to living organisms because of their
important role in various metabolic processes. l-Asparagine is essential for a cell’s exist-
ence. For instance, l-asparagine is the most abundant metabolite for the storage and trans-
port of nitrogen in plants [52]. This amino acid is required for the fast growth of cells and
can be synthesised by the human body when needed without any external supplementa-
tion required [7]. Some cancer cells, especially lymphocytes, do not have the ability to syn-
thesise l-asparagine because their lack of the enzyme aspartate ligase. Therefore, their
survival entirely depends on the external uptake of asparagine. The depletion of l-aspara-
gine from the surrounding environments of cancer cells by the l-asparaginase supplied
exogenously has proven its efficacy as a therapeutic strategy for cancer treatment [7, 52].
Over the past decades, l-asparaginase became a powerful anti-tumour medicine and
researchers are looking for microbial strains able to produce more potent and safer
l-asparaginase at the industrial level [53]. Therefore, endophytic microbes have been
investigated as a potential source of this important therapeutic enzyme.
Fifty endophytic microbes (14 bacteria, 22 actinomycetes and 14 fungi) isolated from
rhizomes of five medicinal plants of the Zingiberaceae family, including Alpinia galanga,
Curcuma amada, Curcuma longa, Hedychium coronarium, and Zingiber officinale, were
screened for l-asparaginase activity. Of these microorganisms, 31 (62%) exhibited l-aspar-
aginase activity in the range of 54.17–155.93 U/mL in an unoptimised medium.
Unfortunately, further experiments with an isolate Talaromyces pinophilus from Curcuma
amada showed a decrease in enzymatic activity from 108.95 to 80 U/mL due to strain
degeneration [54] possibly caused by the inadaptation of the endophyte isolate to the labo-
ratory medium. Twenty endophytic isolates from Tillandsia catimbauensis were screened
for l-asparaginase activity to identify 10 (50%) potential enzyme (0.50–2.30 U/g) producers.
Two isolates of Talaromyces cecidicola URM7826 (2.30 U/g) and Penicillium sp. 4URM7827
(1.28 U/g) were the most potent [33]. Similarly, 20 endophytic fungi isolated from leaves of
the Brazilian medicinal plant Myracrodruon urundeuva were screened for l-asparaginase
production out of which 17 exhibited l-asparaginase activity ranging from 0.57 to 2.41 U/g.
The fungus Diaporthe sp. URM 7793 (2.41 U/g) was the best l-asparaginase producer, fol-
lowed by Diaporthe sp. URM 7779 (2.0 U/g), Talaromyces sp. URM 7785 (1.91 U/g) and
Diaporthe sp. URM 7792 (1.47 U/g) [55].
In another screening of bacterial and fungal endophytes isolated from Ocimum tenuiflo
rum (Tulasi) from NIT Warangal, Telangana, India, four strains produced a high amount of
l-asparaginase. The enzyme produced by the most potent strain, Bacillus stratosphericus,
was further purified. The docking studies showed that in comparison to l-Glutamine hav-
ing 67.7 kcal/mol, the free energy binding efficiency of receptor towards l-asparagine was
good with lesser energy −71.6 kcal/mol, revealing the high affinity of this enzyme to
l-asparagine [56]. In another in silico study to investigate l-asparaginase-producing endo-
phytic bacteria, an approach consisting of protein-protein basic local alignment search tool
with Escherichia coli and Erwinia chrysanthemi asparaginase enzyme sequences against
262 endophytic bacteria was adopted. The results showed that some of the asparaginase
enzymes produced by endophytic bacteria possess more suitable immunological indices
when compared with asparaginase enzymes of E. coli and E. chrysanthemi. For instance,
Herbaspirillum rubrisubalbicans were predicted to produce a non-allergen and non-antigen
asparaginase enzyme, and the number of antigenic determinants was also predicted to be
lower in asparaginase enzymes produced by Bacillus amyloliquefaciens, H. rubrisubalbi
cans and Herbaspirillum seropedicae. Moreover, the number of high-scored B-cell epitopes
was lower in enzyme sequences related to the aforementioned bacteria and Paenibacillus
polymyxa. The number of discontinuous epitopes and the number of T-cell epitopes were
lower in B. amyloliquefaciens-produced enzymes. Therefore, the therapeutic use of these
enzymes is possible because they are predicted to be very potent with lesser or no side
effect [57].
Endophytic fungi isolated from four anticancer medicinal plants, including Murraya
koenigii, Oldenlandia diffusa, Pereskia bleo and Cymbopogon citratus, were investigated for
their ability to produce l-asparaginase. Four isolates showed potency among which
Fusarium oxysporum and Penicillium simplicissimum were the most potent with mean
l-asparaginase activity of 0.013 and 0.019 μM/mL/min, respectively [58]. In another
screening of 33 endophytic fungi from plants collected in moist and evergreen forests of
Western Ghats, India, 31 isolates were l-asparaginase positive, with 19 isolates able to pro-
duce glutaminase-free l-asparaginase. Further investigation led to the identification of 11
promising fungi belonging to Acremonium, Alternaria, Aspergillus, Botrytis, Cylindrocladium,
Cladosporium, Corynespora, Fusarium, Sordaria, Lasiodiplodia and Pestalotiopsis genera.
The most potent isolate was Alternaria sp., exhibiting a glutaminase-free l-asparaginase
specific activity of 1.65 U/mg [59]. Similarly, the l-asparaginase screening of endophytic
fungi from the cactus plant Cereus jamacaru showed that species belonging to Aspergillus,
Fusarium and Penicillium exhibited l-asparaginase activity with Aspergillussydowii
(29.02 U/mL) as the most potent [60]. In a recent study conducted to isolate l-asparagi-
nase-producing endophytic fungi from medicinal plants of the Asteraceae family, a total of
84 isolates were screened using a qualitative plate assay on modified Czapex dox’s agar
medium and l-asparaginase further quantified by the nesslerisation method. Of the 84

isolates screened, 38 were able to produce l-asparaginase and their activities ranged
between 0.019 and 0.492 U/mL with Fusarium proliferatum being the most potent followed
by Plenodomus tracheiphilus isolated from Anthemis altissima (0.481 U/mL). Overall, this
study indicated that l-asparaginase-producing endophytes belong to Plectosphaerella,
Fusarium, Stemphylium, Septoria, Alternaria, Didymella, Phoma, Chaetosphaeronema,
Sarocladium, Nemania, Epicoccum, Ulocladium and Cladosporium genera. This study fur-
ther demonstrates the diversity of fungi that can be investigated as alternative source for
the production of this anticancer enzyme (l-asparaginase) [34].
6.3.3 Cellulases
In nature, cellulose is found in combination with lignin and hemicellulose resulting in a
complex and recalcitrant solid called lignocellulose. Cellulases namely endoglucanase
(1,4-d-glucan-4-glucanohydrolase, EC 3.2.1.4), exo-cellobiohydrolase (1,4-d-glucan gluco-
hydrolase, EC 3.2.1.74) and β-glucosidase (d-glucoside glucohydrolase, EC 3.2.1.21) play a
predominant role in the hydrolysis and conversion of cellulosic substrates into monomeric
products. In general, while the endoglucanase hydrolases the glycosidic bonds of cellulosic
substrates, exo-cellobiohydrolase is attacking the crystalline ends to yield cellobiose [61]
which under the action of β-glucosidase is converted into glucose molecules [62]. These
enzyme-based conversions have the advantage of using low-cost substrates mainly consist-
ing of cellulose and hemicelluloses (75–80%).In fact, cellulosic biomass, deriving from non-
food sources, such as trees and grasses, is being explored as raw material for the production
of biofuels, especially bioethanol for the transportation sector, with cellulases being the
main group of enzymes used [63]. In addition, cellulases can also be used for the extraction
of olive oil, treatment of wines and the clarification of fruit juices and to improve the qual-
ity of bakery products in food industries. In the textile industry, they are used for the ston-
ing of denim and the polishing of cellulosic fibre. Cellulases are also used for the recycling
of waste paper in the paper and pulp industry and to improve the digestibility of animal
feed [64]. Therefore, investigations have been carried out to identify new microbial species
capable of producing cellulases with high yield and specificity.
Two endophytic fungi isolates, Colletotrichum sp. and Alternaria sp., were evaluated for
their potential as biodiesel feedstock. The results showed that in solid-state fermentation,
the two endophytes grew successfully on a combined rice straw and wheat bran as substrate
leading to the production of cellulase (1.21–2.51 U/g dry substrate (gds)) and a substantial
amount of lipid (60.32–84.30 mg/gds) [65]. Earlier, Peng and Chen [66] screened 149
endophytic fungi isolated from 7 plants for their ability to contain intercellular lipid bodies
and 26 showed great potency. In solid-state fermentation, these isolates produce cellulase
enzymes and microbial oil with the yields of 0.31–0.69 filter paper unit and 19–42 mg/g
initial dry substrate, respectively. This study also demonstrated the ability of endophytes to
produce simultaneously a substantial amount of lipid and cellulase enzymes. In another
study, 100 endophytes isolated from Espeletia spp. were evaluated for their cellulolytic
potential on the saccharification of the palm oil empty fruit bunch using filter paper (FPA)
followed by the assessment of carboxymethyl cellulase (CMCase), exoglucanase and
β-glucosidase activities. The results showed that four isolates were potently active among
which Penicillium glabrum had the highest cellulolytic activity (CMCase (1.87 U/mL), FPA
(0.035 U/mL), β-glucosidase (0.06 U/mL) and exoglucanases (0.87 U/mL)). The partially
purified enzymes exhibited maximal CMCase, exoglucanase and β-glucosidase activities of
44.52, 48.36 and 0.45 U/mL, respectively [35]. Another study reported the ability of β-1,4-
endoglucanase from a citrus endophytic Bacillus pimulus to hydrolyse cellulose under
in vitro conditions. This enzyme was reported to have a specific activity of 8.25 U/mg pro-
tein, an optimum pH range of 5–8 and remarkable heat stability (50–60 °C), retaining more
than 85% activity even after a 24-hour incubation at pH 6–8.6. Ions Ca2+, Na+, Mn2+ and
Mg2+ slightly stimulated, while Zn2+, Cu2+, K+, Li+ and Co2+ inhibited its activity [67].
Another endophytic Bacillus isolate, Bacillus amyloliquefaciens, was also reported to pro-
duce β-1,3-glucanase[68].
The cellobiohydrolase encoding gene from a fungal endophyte Fusiccoccum sp.
BCC4124 identified as a potent cellulase producer was expressed in Pichia pastoris KM71.
Further investigations showed that the recombinant enzyme obtained could exhibit the
ability to degrade Avicel, filter paper and 4-methyl umbelliferyl β-d-cellobioside but was
unable to hydrolyse carboxymethylcellulose. This enzyme exhibited optimal activity at
temperature and pH of 40 °C and 5.0, respectively, and had activity parameters (Km and
Vmax) values of 0.57 mM and 3.086 nmoL/min/mg protein, respectively. The enzyme was
stable at pH ranging from 3 to 11 and retained 50% of its activity at 70–90 °C for 30 min-
utes [69]. In a similar investigation by Harnpicharnchai et al. [70], a thermotolerant
β-glucosidase enzyme was reported from an endophytic fungus, Periconia sp. BCC2871,
and its full-length gene was cloned in Pichia pastoris KM71 strain. The recombinant
enzyme obtained showed optimal activity at pH 5 and 6 and a temperature of 70 °C. More
interestingly, the enzyme maintained 60% activity after a 90-minute incubation at 70 °C,
remaining 100% active after 120 minutes incubation at pH 8. Both investigations [69, 70]
showcase the enzymatic ability of recombinant enzymes and their stability in a wide range
of pH and temperatures and encourage further investigations for various biotechnological
applications. In another study, several endophytes from Egyptian marine sponge
Latrunculia corticata were screened for cellulase activity, and Trichoderma sp. Merv6,
Penicillium sp. Merv2 and Aspergillus sp. Merv70 exhibited high cellulase activity while
using different lignocellulosic substrates in solid-state fermentation. By applying mutagen-
esis and intergeneric protoplast fusion, the authors obtained a recombinant strain named
Tahrir-25 that exhibited higher cellulases (Exo-β-1,4-glucanase, endo-β-1,4-glucanase and
β-1,4-glucosidase) activity. Moreover, by optimising parameters such as initial moisture
content at 60% (v/w), inoculum size at 106 spores/mL, the average substrate particle size at
1.0 mm, carbon source (a mixture of sugarcane bagasse and corncob (2 : 1) supplemented
with carboxymethyl cellulose and corn steep solids), fermentation time (7 days), medium
pH (5.5) and temperature (30 °C), El-Bondkly and El-Gendy [36] afforded an activity of
450, 191 and 225 U/g dry substrate (gds) of CMCase, filter-paperase (FPase) and
β-glucosidase, respectively. The authors suggested that these marine endophytes could be
exploited for the cost-effective production of cellulases for second-generation bioethanol
processes.
Fifty endophytic fungi from Cameroonian medicinal plants were also screened for their
cellulase activity using a qualitative assay. A promising fungus named Penicillium sp. 51
exhibited FPase and CMCase activities of 0.36 and 0.44 U/mg. respectively, with the
optimal pH and temperature for the activity being, respectively, 5–6 and 40–50 °C [71].
From the screening of 20 endophytic fungi isolated from leaves of the ornamental plant
Pachystachys lutea for cellulase activity, five endophytes including Diaporthe anacardii
PL01, Diaporthe sp. PL03, fungal endophyte PL35, Xylaria berteroi PL36 and Diaporthe sp.
PL67 were active with cellulase production ranging between 0.87 and 1.60 μmoL/min [72].
In another investigation, four endophytes Hypoxylon sp. CI4A, Hypoxylon sp. EC38,
Hypoxylon sp. CO27 and Daldinia eschscholzii EC12 were evaluated for their consolidated
bioprocessing (CBP) potential. The analysis of their genomes indicated that these endo-
phytes have a rich reservoir of biomass-deconstructing carbohydrate-active enzymes
(CAZys), which includes enzymes active on both polysaccharides and lignin, as well as
terpene synthases (TPSs), enzymes that may produce fuel-like molecules. In addition, the
analysis of their cellulase activity revealed that these endophytes actively produce endoglu-
canases, exoglucanases and β-glucosidases [73]. The authors concluded that looking at the
richness of CAZymes as well as TPSs identified in these four endophytic fungi, they are
great candidates to pursue development into platform CBP organisms. More recently,
Lysinibacillus xylanilyticus, a Gram-positive cellulose-decomposing endophytic bacterium
was isolated from medicinal plant Chiliadenus montanus. Further analysis showed that
this isolate was capable of degrading 58% of cellulosic filter paper (100 g/L) after 15 days of
incubation. In another experiment, the authors showed that maximum cellulase activity
(0.18 U/min) was detected after 12 days of incubation, while the maximum release of solu-
ble sugars (11.85 mg/mL) was detected after 15 days of incubation [74].
6.3.4 Chitinases
Chitinases are enzymes belonging to the glycosyl hydrolase family. They are used to reduce
chitin into monomer and oligomer units via the hydrolysis of the 1 → 4 β-glycoside bonds
of N-acetyl-d-glucosamine. They are classified into two categories, endochitinases and
exochitinases. Endochitinases (EC 3.2.1.14) are known to randomly cleave chitin at inter-
nal sites to produce chitotriose, chitotetraose and diacetylchitobiose. Exochitinases on the
other hand are further classified into two subcategories including (i) chitobiosidases (EC
3.2.1.29) and (ii) β-1,4-N-acetyl glucosaminidases (EC 3.2.1.30). The former release the dia-
cetylchitobiose from the non-reducing end of chitin, while the latter hydrolases the prod-
ucts of endochitinases and chitobiosidases, to generate units of N-acetyl-d-glucosamine [7].
This group of enzymes has a huge pharmaceutical and agricultural potential since they are
used in morphogenesis of insects and yeasts, to potentiate the activity of antifungal drugs,
and as fungicide and insecticide agents [75]. They are also used for the production of
proteins from shellfish waste, chitooligosaccharides and glucosamines, which have wide
applications in the pharmaceutical industry [76].
In a search for chitinases, Purushotham et al. [77] reported the presence of four chitinases
named SpChiA, SpChiB, SpChiC and SpChiD from a genome analysis of the endophyte
Serratia proteamaculans 568. The heterologous expression and characterisation of SpChiA,
SpChiB and SpChiC revealed their optimal activity at pH 6.0–7.0 and 40 °C. Additional
investigation showed that the three S. proteamaculans chitinases displayed the highest
activity binding to β-chitin and showed a broad range of substrate specificities. In fact,
these enzymes released dimers as major end products from oligomeric and polymeric
substrates, and tetramers from colloidal chitin substrates. However, the longest incubation
time was required for the hydrolysis of the trimer. Overall, enzymes SpChiA and SpChiB
were processive chitinases, while SpChiC was a non-processive chitinase. This study is
encouraging and suggests that further investigation of endophytes could lead to the identi-
fication of several and more potent chitinase enzymes.
6.3.5 Laccases
Laccases (EC 1.10.3.2) are phenol oxidases which oxidise phenolic and aromatic com-
pounds, including some amines, ethers and esters. This multicopper blue oxidase couples
the electron transfer, causing the oxidation of water molecules. Laccases have been investi-
gated for use in various biotechnological processes because of characteristics such as the
wide range of substrate specificity, the use of oxygen as a final electron acceptor, and no
requirement for a cofactor or peroxide for their catalytic activity [6, 7]. In fact, laccases are
currently used in various industries such as food and beverage stabilisation, cosmetics,
pulp and paper, textile for polymer synthesis, petrochemical, pharmaceutical as catalysts
for the manufacture of anticancer drugs, and so forth [6]. They are also used for the degra-
dation of pesticides and herbicides from agricultural fields [78], the treatment of wastewa-
ter that contains dyes from textile industries [78], the bioremediation of soil contaminated
with oil hydrocarbons [79] in order to protect the environment from hazardous compounds,
and for bio pulping and biobleaching processes [80, 81]. This wide spectrum of applications
has raised the interest of researchers to investigate new sources of laccase enzymes.
In this scope, the endophytic fungus Myrothecium verrucaria was isolated from pigeon
pea. Using response surface methodology to optimise several fermentation parameters, lac-
case activity reached 16.52 U/mL after five days, at 30 °C and pH 6.22. This enzymatic activ-
ity was relatively stable within pH (4.5–6.5) and in the temperature range of
35–55 °C. Additionally, the laccase showed effective decolourisation capability towards sev-
eral synthetic dyes such as Congo red, Methyl orange, Methyl red and Crystal violet in the
presence of the redox mediator ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid)), with more than 70% of dyes decolorising after 24 hours of incubation [37]. In
another investigation, the in vitro culture of adventitious roots of Ipomoea hederifolia and
its endophytic fungus Cladosporium cladosporioides successfully decolourised Navy Blue
HE2R at a concentration of 20 ppm up to 83.3 and 65%, respectively, within 96 hours.
Whereas the combination of C. cladosporioides and adventitious roots decolourised the dye
more efficiently and gave 97% removal within 36 hours. Further analysis showed the sig-
nificant induction of enzymes tyrosinase, laccase and riboflavin reductase in C. cladospori
oides. The combination was tested in a rhizoreactor leading to 97% decolourisation after 36
hours [82]. These findings suggest that laccase-producing endophytes could provide a
promising alternative for future industrial and environmental applications, including eco-
friendly remediation purposes.
New laccase-producing endophytic fungi isolated from eucalyptus trees in Spain were
assessed for their ability to enhance the saccharification of Eucalyptus globulus wood.
Overall, endophytic Ulocladium sp. and Hormonema sp. produced greater enhancements
in saccharification of E. globulus than Trametes sp. I-62 with an increase in sugar yields of
8.5, 8.0 and 6.0 fold, respectively. This study demonstrated the potential of these new
laccase-producing fungi to enhance saccharification [83]. In another investigation, 127

endophytic fungi isolated from Eucalyptus trees were screened for laccases activity. From
all the studied fungal strains, only five, namely Neofusicoccum luteum, Neofusicoccum aus
trale, Pringsheimiasmilacis, Hormonema sp. and Ulocladium sp., were able to exhibit lac-
case activity, reaching maximum of 0.01–0.04 U/mL by the fifth day in all cases. At room
temperature and pH 4, the laccase activity was 0.25, 0.15, 0.15 and 0.05 U/mL for N. luteum,
N. australe, Hormonema sp. and P. smilacis, respectively. The maximum laccase activity
was obtained at pH 2 for Hormonema sp., N. australe and N. luteum, while the enzymatic
activity decreased significantly at pH values higher than 4 for N. australe and Hormonema
sp. The investigation of the effect of temperature showed that the higher the temperature,
the greater laccase activity was for the four strains. At 80 °C, Hormonema sp. reached maxi-
mum activity, while P. smilacis showed an abrupt decrease in laccase activity [38].
6.3.6 Lipases
Lipases (EC 3.1.1.3) are hydrolytic enzymes also called triacylglycerol ester hydrolases,
catalysing the hydrolysis of triglycerides to glycerol and free fatty acids [84]. Besides the
hydrolysis reaction, these enzymes are also able to catalyse esterification, interesterifica-
tion, transesterification, acidolysis, alcoholysis and aminolysis reactions under proper con-
ditions [85, 86]. They possess broad substrate specificity with optimal activity over a wide
range of temperatures. This makes lipases a most versatile biocatalysts. In fact, interests for
lipases are predicted to be the fastest-growing given their wide range of applications in vari-
ous industrial sectors [7]. Microbial lipases that are mainly obtained from certain species of
bacteria and filamentous fungi are the most sought after because of their outstanding char-
acteristics including high stability in organic solvents, non-requirement of cofactors and
their ability to act over an ample variety of substrates [84, 87, 88]. Their Industrial applica-
tions are various including detergent processing, flavour improvement by the synthesis of
esters of short-chain fatty acids and alcohols, resolution of racemic mixtures and amino
acid derivatives, and also the construction of biosensors that are used as diagnostic tools for
the detection of various diseases [87]. Therefore, the identification of novel microorgan-
isms capable of producing highly potent lipases with desirable physicochemical properties
suitable for industrial applications is of interest. Endophytes have been investigated for
such purpose and interesting findings were reported.
Within this framework, Torres et al. [89] screened endophytic fungi isolated from several
Mediterranean plants and identified lipase from Rhizopus oryzae fungus with the ability to
catalyse the esterification of fatty acids in isooctane. Further investigation of various fac-
tors such as water content, temperature and pH on the enzyme activity reveals that the
catalytic activity inversely correlates with the water content. Moreover, the enzyme was
active over a wide pH range (3–8) with maximal activity at pH 4 and 7. The enzyme was
also thermostable, with maximal activity at 60 °C. In another investigation performed by
Costa-Silva et al. [39] to evaluate the effect of various conditions on the production and
stabilisation of a lipase enzyme produced by the endophytic fungus Cercospora kikuchii, it
was noted that the maximum enzyme production (14.87 U/mL) was obtained after six days
in a medium supplemented with 2% soybean oil. As well, after drying lipases with 10%
(w/v) of each different adjuvants, including lactose, β-cyclodextrin, maltodextrin,
mannitol, gum arabic and trehalose, the residual enzyme activity ranged from 63 to 100%.
However, the absence of adjuvant led to the loss of the enzyme activity. After eight months
of storage, the enzymatic activity was maintained to at least 50% at 5 °C and 40% at 25 °C
with the lipase dried with 10% of β-cyclodextrin retaining 72% of its activity at 5 °C. Finally,
the lipases were separated in a butyl-Sepharose column into four pools, among which one
pool was partially purified (33.1%; 269.5 U/mg protein). This fraction was also spray-dried
in 12% maltodextrin De10 that maintained its activity (27 U/mL) at 100%.
Sixty-five endophytic fungi were isolated from Croton oblongifolius Roxb. (Plao yai) and
also screened for extracellular lipase activity. Ten out of the investigated fungi exhibited
enzyme activity, with Fusarium oxysporum PTM7 isolated from the plant leaf being the
most potent. Further investigation indicated that carbon, organic and inorganic nitrogen
sources such as 1% (v/v) olive oil, 1% (w/v) peptone and 0.5% (w/v) sodium nitrate, respec-
tively, could elicit increased lipase activity of F. oxysporum. The purified lipase had a
molecular mass of 37.4 kDa and showed optimal activity at pH 8 and 30 °C, with reasonable
stability at 40 °C over a wide range of pH (8.0–12). Further analysis showed that divalent
cations such as Ca2+, Mg2+ and Mn2+ could stimulate the enzyme, while Cu2+, Fe2+, Hg2+
and Zn2+ inhibited its activity. Further determination of activity parameters showed that
the enzyme has a Km and Vmax values of 2.78 mM and 9.09 μmoL/min/mg protein, respec-
tively, for the substrate p-nitrophenyl palmitate. However, the enzyme presented low trans-
esterification activity [40].
The endophytic yeast Candida guillermondi was isolated from castor leaves (Ricinus com
munis L.) and used for the production of lipase under submerged fermentation in a medium
containing soybean oil as the main nutrient. The obtained enzyme was partially purified
and freeze-dried before immobilisation on agarose and silica gel supports [41]. In further
investigations, the partially purified enzyme was characterised and showed to present high
activity (26.8 U/mL) in the presence of 5 mmol/L NaCl at 30 °C and pH 6.5. The scale-up
production of this enzyme in submerged fermentation in a 14-L bioreactor led to an activity
of 18 U/mL. The enzyme was applied in the oleic acid esterification in different solvents
(hexane, cyclohexane and cyclohexanone) and different acid: alcohol molar ratios. As
results, higher ester conversion rate (81%) was achieved using hexane and methanol with a
molar ratio of 1 : 9 [42]. These studies suggest the potential for the development of endo-
phytic yeast to produce biocatalysts via the submerged fermentation using agro-industrial
residues as culture media.
In a recent study, Lasiodiplodia theobromae VBE1, the coconut kernel-associated fungus,
was grown on coconut cake with added coconut oil as lipase inducer under solid-state fer-
mentation conditions. Two extracellular lipases were purified including lipase A (68 kDa)
and lipase B (32 kDa). Both lipases were shown to exhibit optimal activity at pH 8.0 and
35 °C, and when activated by Ca2+, they exhibited the highest specificity towards coconut
oil and p-nitrophenyl palmitate, and were stable in isooctane and hexane. Ethanol sup-
ported higher lipase activity, while n-butanol inactivated both lipases. Additional studies
showed that crude lipase immobilised by entrapment within 4% (w/v) calcium alginate
beads was more stable than the crude-free lipase preparation within the pH and tempera-
ture range of 2.5–10.0 and 20–80 °C, respectively. The immobilised lipase preparation was
used to catalyse the transesterification/methanolysis of coconut oil to biodiesel (fatty acyl
methyl esters (FAMEs)). The principal FAMEs were laurate (46.1%), myristate (22.3%),
palmitate (9.9%) and oleate (7.2%), with trace amounts of caprylate, caprate and stearate.
The FAME profile was comparatively similar to NaOH-mediated transesterified biodiesel
from coconut oil but distinctly different from petroleum-derived diesel [90]. The authors
concluded that Lasiodiplodia theobromae VBE1-lipases have the potential for biodiesel pro-
duction using coconut oil as a substrate. Previously, 212 endophytic fungi isolated from
leaves and fruits of Amazonian plants were screened for lipase activity. The results showed
that about 87% of the isolates could hydrolyse a tributyrin substrate, among which 30%
were able to growth in lipase amended media. The evaluation of the esterification and
transesterification ability of these isolates in organic solvents led to the selection of nine
isolates that were further submitted to lipase activity in (R, S)-2-octanol resolution reac-
tion. One isolate named endophyte UEA_115 was the most ingenuous biocatalyst, demon-
strating a conversion ability of more than 90% in esterification reactions and enhanced
aptitude for the resolution of (R, S)-2-octanol (ees 29%; eep 99%; c 22%; E > 200). This
investigation revealed the potent nature of endophytic fungi as lipase suppliers in lipid
biocatalysis [91].
6.3.7 Proteases
Proteases also called proteinases, or proteolytic enzymes, are a group of enzymes belonging
to the hydrolases family, hydrolysing peptide bonds of proteins. They are subdivided into
two major groups thatare, exo- and endopeptidases. Proteases are ubiquitous enzymes
present in all living organisms and generate small peptides and amino acids that are essen-
tial for cell signalling, differentiation, cell growth and metabolism [7]. In addition to their
physiological importance, proteases are also widely employed for commercial, industrial
and health purposes [92, 93]. In fact, proteases are widely used in bioremediation processes
and recycling of waste [94, 95], wastewater treatment [96], catalysis of peptide synthesis in
organic solvents [97, 98], fortification of fruit juices and preparation of protein-enriched
diets [99], leather processing [100], manufacturing of laundry detergents [101], meat ten-
derisation and cheese preparation [10], production of methionine-enriched protein [102],
and treatment of diseases such as inflammation and cancer [103]. Industrially, microbial
proteases have very high commercial value compared to animal and plant proteases.
Therefore, there is a growing interest in investigating microorganisms as a source for novel
proteases for industrial needs. Few studies have reported the ability of endophytes to
produce proteolytic enzymes.
Among those, the screening of endophytic fungi from Dendronephthya hemprichi plant
led to the identification of fungal strain Penicillium sp. Morsy1, exhibiting the highest
keratinase activity (1600 U/g) when grown on different agricultural and poultry wastes
using solid-state fermentation technology. The maximum production of keratinase was
observed when the fungus was grown for five days on rice straw as the carbon source, at
26 °C, pH 6 and moisture content of 80%. Two types of keratinases named Ahm1 and
Ahm2 with a respective molecular weight of 19 and 40 kDa were purified and characterised
by precipitation with ammonium sulphate, DEAE-Sepharose and gel exclusion chromatog-
raphy in a sequential manner. The optimal condition for the hydrolysis of azokeratin was
pH range 7.0–8.0, at 50 °C, stable in the pH range of 6.0–8.0 for Ahm1 and pH range
10.0–11.0, at 60–65 °C, stable in the pH range of 6.0–11.0 for Ahm2. Both enzymes were
inhibited by chelating agents such as EGTA and ethylenediaminetetraacetic acid (EDTA)

and activated by Ca2+, Mg2+ and Mn2+ [43]. The endophytic fungus Fusarium sp. CPCC
480097 isolated from chrysanthemum stems produces a very potent fibrinolytic enzyme,
characterised by a molecular weight of 28 kDa, an isoelectric point of 8.1 and maximal
activity at 45 °C and pH 8.5 [104]. In another investigation, a novel fibrinolytic enzyme
from the endophytic bacterium Pa. polymyxa EJS-3 was purified with ammonium sulphate
precipitation, hydrophobic chromatography, ion-exchange and gel filtration chromatogra-
phy. This new enzyme has a molecular weight of 63.3 kDa with the optimum temperature
and pH value of 37 °C and 7.5, respectively. Additional investigations revealed that the
enzyme rapidly hydrolysed the α-chain of fibrinogen, followed by the β-chains, and also
hydrolysed the γ-chains, but more slowly. The enzyme was also activated by metal ions
such as Zn2+, Mg2+ and Fe2+ and was inhibited by Ca2+ and Cu2+. Further studies showed
that the enzyme exhibited a higher specificity for the synthetic substrate N-succinyl-Ala-
Ala-Pro-Phe-pNA for chymotrypsin, indicating that the enzyme is a chymotrypsin-like ser-
ine protease and displayed significant anticoagulant activity in vitro [105]. These proteases
may have potential applications in thrombolytic therapy and in thrombosis prevention. In
fact, although Bacillus from traditional fermented foods has been so far the most important
source of fibrinolytic enzymes discovered from microorganisms [106], outcomes from the
above two investigations demonstrate the potential of endophytic fungi and bacteria as
fibrinolytic enzymes-producing organisms.
An endophytic Acremonium sp. isolated from leaves of Saraca asoca exhibited protease
activity when grown on skim milk medium. For the highest protease production, fructose
and ammonium sulphate were the preferred carbon and nitrogen sources. The evaluation
of the enzymatic activity of the partially purified enzyme showed that the optimum enzyme
activity of 3.34 U/mL was recorded at pH 7.0 with the protein content of 20 μg/mL, and the
specific activity being 0.167 U/μg [44]. Zaferanloo et al. [107] investigated the protease-
producing potential of three fungal endophytes Alternaria alternata, Phoma herbarum and
an unidentified fungus, isolated from the Australian native plant, E. longifolia. From the
results, A. alternata showed the greatest protease activity in a wide range of pH (3–9), while
the broadest activity between 9 and 50 °C was observed at pH 7. Overall, the optimum con-
ditions were 37 °C and pH 7 with a maximum specific activity value of 69.86 U/mg.
Mayerhofer et al. [108] also reported the enzymatic ability of roots endophytes Umbelopsis
isabellina and Meliniomyces variabilis from Birch tree (Betula spp.) and Phialocephala for
tinii from Pseudotsuga menziesii plant. U. isabellina was shown to efficiently utilised pro-
tein, while M. variabilis and Ph. fortinii exhibited intermediate levels of protein utilisation,
demonstrating their ability to produce proteolytic enzymes. In a more recent investigation,
Bacillus halotolerans isolated from Tunisian potatoes was identified as a protease-produc-
ing strain. The proteolytic enzyme was purified by ammonium sulphate precipitation,
sephacryl S-200 gel filtration and SP-Sepharose cation-exchange chromatography and
demonstrated an optimal activity at pH 9 and temperature of 50 °C. The proteolytic activity
was enhanced by Ca2+ and Mn2+ ions and exhibited high stability in the presence of com-
mercial detergents including liquid (Auchan, Ariel, Nadhir and Det) and powder (Ariel,
Dixan and Omo) formulations. This enzyme showed broad substrate specificity towards
both synthetic and natural substrates, with a high capacity to hydrolyse legume seed
proteins. The electrophoretic analysis reveals a molecular weight of around 250 kDa with
two major subunits, showing important homologies with serine proteases belonging to the
subtilisin-like serine proteases. The Km and Vmax values were 10 mg/mL and 50,000 U/
mg, respectively. The Kcat and Kcat/Km were also 5263.15 × 103/min and 526.31×103/min/
(mg/mL), respectively [109]. The authors concluded that this new enzyme displayed rele-
vant properties suitable for various industrial and biotechnological applications.
In an investigation designed to identify microorganisms with ability to efficiently degrade
polyester polyurethane in both solid and liquid suspensions, Russell et al. [110] screened
several endophytic fungi and identified two Pestalotiopsis microspora isolates capable of
growing on polyester polyurethane as the sole carbon source under both aerobic and anaer-
obic conditions. From their investigation, the authors concluded that since the main
enzyme responsible for the degradation of polyester polyurethane is a serine protease,
these endophytes can be used to produce these enzymes at the industrial level for bioreme-
diation purposes.
6.3.8 Xylanases
Xylanases (EC 3.2.1.8), also named as endo-β-1,4-xylan-xylanohydrolase, belong to the gly-
coside hydrolases family, catalysing the hydrolysis of xylan into xylooligosaccharides, xylo-
biose and D-xylose. In general, three major enzymes, including endoxylanases, exoxylanases
and β-xylosidases, act synergistically to breakdown the xylan backbone into hemicellulose.
In fact, endoxylanases (EC 3.2.1.8) cleaves the β-1,4 bonds of xylan backbone, while exoxy-
lanases (EC 3.2.1.37) hydrolyse the β-1,4 bonds of xylan from the non-reducing ends to
release xylooligosaccharides and β-xylosidases release xylose from the cleavage of xylobi-
ose and xylooligosaccharides [111]. In nature, xylanases are produced by microorganisms
to cleave xylans, a major constituent of hemicellulose. Xylanases have received a great deal
of attention because of their biotechnological potential in various industrial sectors.
Xylanases are used to improve the quality of bread. In fact, some evidence showed that the
use of xylanases decreases the water absorption and thus reduces the amount of added
water needed in baking leading to a more stable dough [3]. Xylanases are applied in bleach-
ing of kraft pulps, which liberate lignin fragments by hydrolysing residual xylan and
improve the pulp fibrillation, and restoration of bonding, which increases the freeness in
recycled fibres and improves the bleaching of wood pulp. Overall, this considerably reduces
the need for chlorine-based bleaching chemicals [112]. Xylanases are also used to treat
hemicelluloses waste and clarify fruit and vegetable juices, for the pre-treatment of forage
crops to improve the digestibility of ruminant feed, for the production of ethanol and
xylitol, and for the degumming of bast fibres such as flax, hemp, jute and ramie [113]. One
of the industrial producing organisms is Trichoderma sp. from which xylanase has already
been purified, crystallised and improved by design-oriented mutagenesis [114]. To supply
the industry with novel and more potent xylanase-producing strains, microorganisms from
diverse ecological niches including endophytes have been investigated.
In this respect, 54 endophytic fungi isolated from leaves of C. oblongifolius were screened
for xylanase activity. Thirty isolates were positive at the primary and secondary screenings.
The most potent isolate, named PTRa9, was further investigated for purification and char-
acterisation of the enzyme produced. The optimal conditions for xylanase production were
96 hours of incubation in a medium composed of 2% (w/v) rice bran and 0.1% (w/v)
ammonium sulfate ((NH4)2SO4), as sources of carbon and nitrogen, respectively. The

enzyme had a molecular weight of 54.8 kDa and specific activity of 161.1 U/mg protein. It
was active at a broad range of temperature (−20 to 45 °C) and pH (3–11) with the optimum
being 45 °C and 5.0, respectively. The enzyme exhibited a Km of 0.421 μg/mL and Vmax of
0.826 U/mg protein and was sensitive to Cu2+, Hg2+ and EDTA [115]. In another investiga-
tion, a thermotolerant xylanase produced by Aspergillus terreus isolated from Memora per
egrina was purified and characterised. When grown on wheat bran as a carbon source at
30 °C for 48 hours, the production yield was higher. The xylanase with a molecular weight
of 23 kDa exhibited optimal enzyme activity at 55 C and pH 4.5. The enzyme was tolerant
to a temperature of 45 and 50 °C, with a half-life of 55 and 36 minutes, respectively, and was
activated by ions K+ and Mn2+. Enzyme kinetic studies revealed the Km and Vmax values
of 22 and 625 μg/mL, respectively [45].
6.3.9 Other Enzymes Produced by Endophytes

6.3.9.1 AHL-Lactonase
Biofilm formation is one of the strategies used by pathogenic bacteria and fungi to resist
antibiotic treatment. Several approaches have been investigated to fight against pathogens
biofilms, including the use of enzymes. In their investigation of the antibiofilm potential of
endophytes, Rajesh and Rai [116] reported that the ability of the endophytic bacterium
Enterobacter aerogenes VT66 to quench the N-acyl homoserine lactone (AHL) molecules
produced by Pseudomonas aeruginosa PAO1 was due to the production of an AHL-lactonase
enzyme. The purified AHL-lactonase with a molecular weight of about 30 kDa was able to
inhibit in vitro more than 70% of the biofilm formed by P. aeruginosa PAO1. These results
are encouraging and suggest that the AHL-lactonase enzyme produced by E. aerogenes
VT66 could be further developed as an antibiofilm agent for biomedical applications.
6.3.9.2 Agarase
Agar is a polysaccharide extracted from the cell walls of some macro-algae. Most of the
reported agarases come from the marine environment. There is a need to investigate new
sources of this enzyme. In this line, Song et al. [117] reported the identification of seven
agar-degrading endophytic bacteria belonging to three genera, viz. Paenibacillus,
Pseudomonas and Klebsiella. Further studies showed that the seven endophytes could grow
on several polysaccharides such as araban, carrageenan, chitin, starch and xylan and could
also produce agarase in the presence of different polysaccharides other than agar. Further
analysis of the extracellular agarase produced revealed a molecular weight of 75 kDa,
which greatly differentiated from previously reported agarases.
6.3.9.3 Chromate Reductase

The phytoremediation industry is in continuous expansion. Endophytic bacteria from
Albizzia lebbeck, a chromium hyperaccumulator plant, were investigated for their chro-
mium detoxification ability. Out of four different groups of endophytic bacteria investi-
gated, comprising Pseudomonas, Rhizobium, Bacillus and Salinicoccus, three Bacillus spp.
exhibited not only remarkable chromium accumulation ability but also high chromium
reductase activity. The most potent isolate Bacillus sp. DGV 019 was selected to purify the
6.4 Overview of the Methods Used to Investigate Endophytes as Sources of Enzyme 117
enzyme chromate reductase to homogeneity. The enzyme had a molecular weight of

34.2 kDa and was stable at temperatures and pH ranging from 20 to 60 °C and 4.0 to 9.0,
respectively. Further analysis showed that the enzyme activity was enhanced with the elec-
tron donors NADH, followed by NADPH, and not affected by glutathione and ascorbic
acid. In addition, ion Cu2+ enhanced its activity, while Zn2+ and EDTA inhibited it. This
chromate reductase enzyme showed versatile adaptability that can be used for chromium
remediation[118].
6.3.9.4 β-Mannanase
The bacterial endophyte Paenibacillus sp. CH-3 isolated from Robinia pseudoacacia was
identified as a highly potent β-mannanase producer. The β-mannanase gene (manB) from
this bacterium was cloned, expressed, purified and characterised to reveal that the gene
encoding 327 amino acid residues, which belongs to family 5 of glycosyl hydrolase. The
enzyme with a molecular weight of 50.4 kDa had an optimal activity level at pH 7.0 and
45 °C and was stable in the pH range of 4–9. Further study revealed that the optimal tem-
perature, isopropyl β-d-thiogalactoside concentration and induction time were 28 °C,
0.05 mM and 12 hours, respectively, with the highest activity reaching 1054.17 U/mL. In
addition, the enzyme has Km and catalytic efficiency (kcat/Km) values, respectively, of 7.30
and 176.31 mL/(mg/s) for locust bean gum, 8.69 and 116.69 mL/(mg/s) for guar gum, and
27.17 mg/mL and 115.49 mL/(mg/s) for konjac glucomannan. The hydrolysis of glucoman-
nans by this enzyme produced mannobiose, mannotetrose and a higher manno-oligosac-
charide [119]. The authors suggested that this enzyme could be useful as an additive in the
animal feed industry.
6.4 Overview of the Methods Used to Investigate Endophytes

as Sources of Enzymes
The investigation of endophytes for the production of novel products including but not
limited to bioactive secondary metabolites and enzymes starts with the collection of plant
material based on relevant criteria, followed by the isolation and purification of endophytic
fungi or bacteria on agar plates. To achieve microbial enzyme isolation, fungal or bacterial
isolates are subsequently cultured on agar medium supplemented with a specific discrimi-
natory substrate as a function of the enzyme of interest. For instance, starch is used as
substrate for amylases, carboxymethylcellulose is used for cellulases, casein or gelatin for
proteases, etc. Potent microbial isolates identified based on their ability to hydrolyse the
specific substrate and form a measurable inhibition halo are thereafter selected for sub-
merged or solid-state fermentation[15]. At this stage, several agro-industrial residues, such
as raw potato, cassava bagasse, sugarcane bagasse, sugar beet pulp/husk, orange bagasse,
oil cakes, apple pomace, grape juice, grape seed, coffee husk, wheat bran, coir pith, etc., are
often used as adequate substrates for fermentation. The successful fermentation is directly
followed by extraction, concentration and purification of enzyme. Methods reported to be
adequately used for enzyme purification in several investigations include precipitation,
centrifugation, ion-exchange chromatography, affinity chromatography, hydrophobic
chromatography, electrophoresis, and ultrafiltration and gel filtration [3]. The proof of
concept of the purified enzymes activity is subsequently achieved using a broad range of
substrates and diverse experimental conditions, including varied pH, temperature, divalent
cations, different adjuvants (e.g. lactose, β-cyclodextrin, maltodextrin, mannitol, gum ara-
bic and trehalose), etc., to identify the optimal conditions for enzyme activity. Parameters
for enzymatic activity such as Km, Vmax and catalytic efficiency (kcat/Km) constant are
thereafter determined.
6.5 Strategies Applied to Improve the Production of Enzymes

by Endophytes
It is obvious from the data presented in this chapter that endophytic microorganisms have
the ability to produce a wide range of industrially relevant enzymes. Despite their out-
standing potential as candidates for enzyme production, it is noted that the amount of
enzyme produced is generally low, not allowing comprehensive enzyme profiling.
Moreover, some endophytes often lose the vigour of enzyme production in axenic cultures
under laboratory conditions as exemplified by the investigation of Krishnapura and
Belur [54]. This could be due to the absence of native environmental conditions found in
the host plants where the production of metabolites is mediated by multiple signals from
the surrounding environment [120]. To overcome these limitations, several methodologies
have been developed to improve the production of enzymes by endophytes.
One of such approaches that are currently used with some extent of success is the varia-
tion of culture conditions, including but not limited to composition of culture media (car-
bon and nitrogen sources), pH and temperature of incubation, aeration rate, agitation or
not [121]. For instance, Marlida et al. [30, 50] used this strategy to improve the production
yield of amylase by Acremonium sp. by 19- to 22-fold and also reported an optimal amylase
production with raw potato starch as carbon source. Similarly, Sunitha et al. [31] optimised
the production of amylase by Cylindrocephalum sp. by varying temperature of incubation,
pH of growth medium and carbon and nitrogen sources. Although time-consuming, this
approach is easily applicable and has the potential to improve the yield of enzymes produc-
tion by endophytes when the growth conditions are successfully identified.
In addition to varying growth conditions, the supplementation of the culture medium
with inducers such as metals ions, small organic chemicals or organic solvents [122] has
also been proposed as a vital strategy for improving the yield of endophytic enzyme produc-
tion. In fact, Fillat et al. [38] have demonstrated in their study that the production yield of
laccases by endophytes N. luteum, N. australe and Hormonema sp. significantly improved
with the addition of 1% ethanol (v/v) and 0.30 mM CuSO4 in the growth medium. Several
other authors have reported similar trends with microorganism growth media supplemen-
tation ethanol [123–125]. However, the addition of inducers has not shown to elicit the
same yield trend in every endophytic strain tested. In fact, the addition of ethanol in the
growth medium of P. smilacis did not improve the production of laccase but rather reduced
the yield when the incubation time was prolonged [38]. This observation reveals that the
influence of inducers on enzyme production is highly dependent on the microorganism
under study. This further strongly denotes the absolute necessity to carefully plan screen-
ing to identify a proper inducer for each microorganism that is being investigated [122].
6.6 Conclusio 119
Finally, besides culture-dependent approaches used to improve the production of

enzymes by endophytes, genetic manipulation of producing strains has also been applied
with great success. This approach has been used to improve the production of cellulases by
the endophytic fungi Fusiccoccum sp. [69] and Periconia sp. BCC2871 [70]. Moreover,
El-Bondkly [126] used a shuffling of the genome to improve xylanase production by endo-
phyte Aspergillus sp. NRCF5. Genetic variability was induced on this isolate using different
combinations and doses of mutagens. Ultraviolet irradiation (five minutes) and N-methyl
N-nitro-N-nitrosoguanidine (NTG, 100 mg/mL) for 30 (UNA) and 60 (UNB) minutes along
with NTG (100 mg/mL) and ethidium bromide (250 mg/mL) for 30 (NEA) and 60 (NEB)
minutes were used in combination as mutagens which led to the increase in xylanase activ-
ity of mutants strains. Four rounds of genome shuffling led to the rise of seven high xyla-
nase producing fungal strains. The recombinant xylanase obtained from the most potent
strain showed 6.13 higher folds of xylanase activity when compared with starting strain
NCRF5 with 427.5 U/mL xylanase [126]. Mutation technology has been used in industry to
generate hyper-producer strains including Trichoderma reesei RUT C-30, one of the best
cellulase producers for decades [3]. Moreover, recombinant enzymes from various micro-
organisms are used today for the production of a wide range of products at various indus-
trial scale [7]. Overall, different actions could be performed depending on available facilities
to efficiently improve the production of enzymes by endophytic microbes at the laboratory
or industrial levels. These methods can be fermentation media optimisation using multiple
micronutrients or genetic modification of strains or the combination of both methods.
6.6 Conclusion
Microorganisms represent a reservoir of important enzymes with multiple applications in

various industries. Their multiple characteristics including the easy multiplication under
controlled conditions, short generation times, easy genetic manipulation and high produc-
tion yield make microorganisms the first choice for industrial production of enzymes.
Discovering novel of such sources of enzymes is of high interest for various industrial sec-
tors. In this chapter, an attempt was made to share an update regarding the investigation of
endophytic microbes as a potent source of industrial enzymes. An emphasis was made on
the producing strains, the characteristics of enzymes reported so far (Table 6.2) and the
possible industrial application. This insightful look into literature revealed that a huge
number of endophytic microbes are still to be examined for their enzymatic ability. In fact,
only limited number of bacteria and actinomycetes endophytes have already been reported
as enzymes producers. This leaves the room for a wide area of investigation and highlight-
ing the need to expand such investigations to increase the possibility for new discoveries. It
also came out from this survey that the strategies such as variation of growth conditions,
supplementation of growth media with inducers and genetic manipulation have been suc-
cessfully applied to improve the production of enzymes by endophytes. Indeed, these strat-
egies are already very well established as necessary tools to activate silent genes clusters in
endophytic microbes and induce the production of structurally diverse secondary metabo-
lites exhibiting various biological activity. Therefore, investigations in this direction are
urgently needed to fully harness the potential of endophytic microbes to produce useful
Table 6.2 Selected endophyte enzymes and their characteristics.
Enzyme
Enzymes Endophytes MW (kDa) activity Optimal pH Optimal T (°C) Km Vmax Other characteristics References
Amylases Fusicoccum sp. 52.58 838.9 U/mg 7 70 5.2 mg/mL 640.8 U/mg Resistance to inhibition by [51]
BCC4124 protein glucose of up to 1 mol/L and
activated by Ca2+
P. minima 70 138 U/mg 9 25 ND ND Activated by Mn2+ and Ca2+ [32]
Cellulases Bacillus pumilus 71.3 8.25 U/mg 5–8 60 ND ND Retain 85% of activity after a [67]
protein 24 h at pH range of 6–8.6
Fusiccoccum sp. 50 4.37 U/mg 5.0 40 0.57 mM 3.086 nmol/ Stable at pH (3–11) and [69]
BCC4124 protein min/mg retained 50% of its activity at
70–90 °C for 30 min
Penicillium sp. 51 55 0.44 U/mg 5–6 40–50 ND ND None [71]
Laccases M. verrucaria ND 16.52 U/mL 6.22 30 ND ND Stable at pH (4.5–6.5) and [37]
35–55 °C
Lipases F. oxysporum 37.4 156.3 U/mg 8 30 2.78 mM 9.09 μmoL/ Activated by Ca2+, Mg2+, and [40]
PTM7 protein min/mg Mn2+
L. theobromae 68 1981 U/mg 8.0 35 ND ND Activated by Ca2+, Mg2+ and [90]
VBE1 protein Mn2+
32 1440 U/mg 8.0 35 ND ND
protein
Proteases Penicillium sp. 19 8.06 U/mg 7–8 50 ND ND Activated by Mn2+, Ca2+ and [43]
Morsy1 40 39.593 U/ 10–11 60–65 ND ND Mg2+ and inhibited by EGTA
mg and EDTA
Fusarium sp. 28 76,111 U/ 8.5 45 ND ND Isoelectric point of 8.1; [104]

CPCC 480097 mg activated by Ca2+ and inhibited
by Co2+, Cu2+, and Zn2+
Acremonium sp. ND 3.4 U/mL 7.0 37 ND ND Specific activity: 0.167 U/μg [44]
0005092144.INDD 120 6/2/2021 9:39:41 AM

Enzyme
Enzymes Endophytes MW (kDa) activity Optimal pH Optimal T (°C) Km Vmax Other characteristics References
B. halotolerans 250 773.4 U/mg 9 50 10 mg/mL 50,000 U/mg Enhanced by Ca2+, Mn2+; 10% [109]
ethanol and hexane
Xylanases Strain PTRa9 54.8 161.1 U/mg 5.0 45 0.421 μg/ 0.826 U/mg Sensitive to Cu2+, Hg2+, and [115]
mL EDTA
A. terreus 23 6.92 U/mg 4.5 55 22 mg/mL 625 U/mg/ Activated by K+ and Mn2+ [45]
min
Chromate Bacillus sp. 34.2 ND 4–9 20–60 ND ND Enhanced by NADH, NADPH [118]
reductase and Cu2+
2+ 2+ 2+
β-Mannanase Paenibacillus sp. 50.4 1054.17 U/ 7.0 45 7.30– 6.09– Activated by Ca , Ba , Mg , [119]
CH-3 mL 27.17 mg/ 18.83 μmol/ Mn2+ and Co2+ and inhibited
mL min by Cu2+, Zn2+ and EDTA
ND, not determined; T, temperature; MW, molecular weight.
0005092144.INDD 121 6/2/2021 9:39:41 AM

enzymes. Therefore, available technologies such as metagenomics, genomics, proteomics,

efficient expression systems and recombinant DNA technology should be used as tools to
improve endophyte enzymes already identified and find new enzyme-producing strains.
Moreover, attempts should be made to further the investigation on potent endophyte strains
identified so far to make them suitable for the industrial production of enzymes. This can
only be achieved by the combination of research efforts in a strong and open partnership
between academics and industries.
Acknowledgements
The authors are grateful to Íñigo Zabalgogeazcoa, Senior scientist at the Instituto de Recursos
Naturales y Agrobiología de Salamanca (IRNASA), Consejo Superior de Investigaciones
Científicas (CSIC), Salamanca, Spain, for his critical review of the manuscript.
References
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131
Resource Recovery from the Abundant Agri-biomass

Shilpi Bansal1, Jyoti Singh Jadaun2, and Sudhir P. Singh3
1
Vegetable Science Division, ICAR-Indian Agricultural Research Institute, Pusa, New Delhi, India
2
Department of Botany, Dayanand Girls Postgraduate College, Kanpur, Uttar Pradesh, India
3
7.1 Introduction
Unpredictable increase in human population leads to a rapid depletion of natural resources

and consequently load of waste products is also expanded on the earth. Globally, a massive
increase in waste quantity is becoming a threat to maintaining a worthy living environment
for the future generation. Before the onset of the difficult adverse situation, we have to
think over the management of different waste sources by using a sustainable development
approach for maintaining the balance between economy, society and environment.
Bio-economy may be a suitable alternative for waste or biomass valorization, which will
support in securing the need of the growing population. Bio-economy is defined as the
process in which renewable biological resources are utilized for generation of food, feed,
bioenergy and other value-added products [1]. Renewable biological resources include
crops, forestry, algae, microorganism and also waste products generated by them [2]. Use
of biomass for production of bioenergy and other valuable products will reduce the nega-
tive impact on the environment as it will help to minimize the problem of landfill disposal
and emission of greenhouse gases due to combustion of crop residues [3]. Simultaneously,
it will boost up the economy, and it will be a fundamental step towards achieving sustain-
able development goals. Agri-biomass includes all the waste materials produced through
agricultural practices, for example, cereal straw, rice husk, corn stover, sugarcane bagasse,
crop residues and animal manure, etc. Each year approximately 140 Gt of agricultural resi-
dues are produced [4]. Crop straw constituted more than half of total crop residues, and
burning of crop straw is becoming a significant challenge for environmentalist [5, 6].
Different technologies have been applied to convert these renewable sources into profitable
products for society, but the advent of synthetic biology built a more suitable platform for
#
Equal authorship

132 7 Resource Recovery from the Abundant Agri-biomass
this purpose [7, 8]. There are several successful case studies showing the synthesis of valu-
able products from low-cost substrates [9–11]. The expanding market of bio-based products
is now focusing on End-of-Use (EoU)/End-of-Life (EoL) alternatives which will support
resources for establishing the circular economy [12]. Agri-biomass is mainly composed of
cellulose, hemicellulose and lignin, and in the near future, it will be an appropriate alterna-
tive for the production of bio-energy and industrially valuable chemicals [13]. Lactic acid,
a valuable chemical, is produced through saccharification and fermentation process of
various agriculture feedstocks such as alfalfa fibre and soya fibre corncob (soft) and wheat
straw by applying Lactobacillus delbrueckii and Lactobacillus plantarum [14]. Lignocellulosic
part of different agri-residues such as corn cob, rice straw, rice husk, wheat straw, bagasse,
sorghum stalk, jatropha pruning cotton stalk, mustard stalk, corn stover have good poten-
tial for generation of bioenergy [15, 16]. Use of biomass for bioenergy production serves a
dual purpose as it can combat energy crises in a sustainable manner with environment
protection [17]. Biofuels have the capability to displace fossil fuels like gasoline, coal and
natural gas, etc. [18]. Lignin shows resistance to chemical and biochemical degradation;
therefore, pre-treatment either through physical, chemical or biological methods is required
(Figure 7.1). Thereafter, cellulose and hemicellulose are hydrolyzed into constituent sugars
which undergo fermentation reaction to produce biofuel [19]. This is the peak time to think
over the problem of agri-biomass utilization in an advantageous manner; globally research-
ers are trying to develop sustainable technologies for consuming the agri-biomass in a crea-
tive manner with minimization of its negative impact on environment and society.
Although technologies are progressing vigorously in this direction, but still we have to walk
a long distance for implementation of these technologies globally.
In this chapter, we have described different ways to utilize the agricultural residues for
the recovery of valuable resources. Globally, utilization of agri-biomass is performed to
Crop harvesting
Crop cultivation
Crop residues generated

Field leftover residues during harvesting process
Forest residues
Utilization as bio- Utilization in

sorbents in extraction Ligno-cellulosic biomass preparation of light
of heavy metals weight materials
Pre-treament
Physical Chemical
Microbial Enzymatic
treatment treatment • Biochemical conversion
Fermentable sugars and Biofuels
• Thermochemical conversion chemicals
other
intermediate chemicals bioenergy
Figure 7.1 Schematic illustration of different routes for utilization of agri-biomass to produce
valuable products.
7.2 Potential of Agri-biomass to Produce Different Product 133
generate energy. Recent technologies are gaining attention to develop methodologies for
the extraction of valuable chemicals from crop residues. We have also discussed the novel
utility of agri-biomass in manufacturing of light-weighted materials that are useful in
construction work and other industrial products.
7.2 Potential of Agri-biomass to Produce Different Products

7.2.1 Conversion of Agri-biomass into Valuable Chemicals
Agri-biomass serves as a reservoir of chemicals/metabolites that possess large scale
applicability. The so-called waste has immense potential to be used as a source of sugars,
fibres, vitamins, minerals, pharmaceuticals, nutraceuticals, etc. [20]. Preference of people
towards natural products over synthetic products triggered the development of new
techniques and new resources (wastes) for the production of bioactive compounds [21].
One such compound includes dietary fibres that are complex and heterogeneous polysac-
charides which impart various health benefits. Discarded peel, pulp, pomace of fruits
such as apple, banana, guava, etc. are known to be a rich source of dietary fibres [22]. By
utilization of a novel technique, dietary fibres are extracted from olive mill wastewater, a
by-product from olive industry [23]. Bioactive molecules are another important class of
chemicals that hold importance as therapeutics and are produced as secondary metabo-
lites via different pathways. Regardless of the mode of extraction (chemical, physical,
biological), agri-biomass provides a wide range of bioactive compounds. Several reports
ensure the presence of various carotenoids, flavonoids, polyphenols, solanesol, saponarin,
glucosinolates, capsaicin, isovitexin, etc. from the horticulture crop-based residues
(broccoli, pepper, cucumber, tomato) which possess anti-oxidant and antimicrobial
properties [24–26]. Thousand tons of starch waste is produced in the form potato peel, rice
bran and husk, wheat bran, maize bran and barley bran. Utilizing different extraction
protocols and solvents, research community successfully isolated a pool of compounds
which included vitamin E, tocopherols, β-glucan, carotenoids (lutein, zeaxanthin,
β-carotene), phenolic acids (p-couramic acid, ferulic acid, protocatechuic acid, vanillic
acid, gallic acid), steroidal alkaloids (solanidine, demissidine) from these starch waste [27,
28]. Phenolics and flavonoids isolated from waste have been known to increase the shelf
life of foods. It prevents them from browning and getting spoiled by the action of microbes.
As a natural additive, these molecules increase the antioxidant capacity and prevent
freshly cut fruits and vegetables against microbial attack [29]. Conte et al. showed that the
application of lemon extracts on mozzarella cheeses increased its longevity without affect-
ing its natural properties [30].
These by-products/waste have the potential to replace synthetic additives used in food
items. They can be used as colouring, flavouring, anti-browning agents. Additives
produced from the by-products offer the advantage of being non-toxic, easily available and
cost effective. Anthocyanin belongs to an important class of food colourants that can be
extracted from the wastes of red cabbage, red reddish, purple sweet potato, aronia, cherry,
blackberry and red-fleshed potato [31]. Another potential candidate for food colour is
lycopene. It has been established that pomace and lyco-oleoresin extract from tomato is
rich in lycopene that may impart yellow to red colour to food items including baked goods,
spreads, dairy products, beverages, etc. and is approved by European food safety
authority [32].
Enzymes are the proteins which act upon the substrate to catalyse the biological reaction
to yield products. Enzyme accounts for widespread application at home and industrial
level, which involves brewing, baking, fruit juice and beverage processing, meat industry,
in oil and fats and dairy industry. Despite its excessive utility, enzymes are highly expen-
sive, and raw materials required for its production is one of the critical factor determining
the overall production cost. Hence, agricultural waste from rice, wheat, barley, potato, etc.
which are rich sources of carbon is being immensely used for enzyme production in order
to lower the production cost. Alpha amylases are continuously being used in paper, pulp,
pharmaceutical, bakery industry and has been successfully produced from Bacillus
amyloliquefaciens using wheat bran, rice bran and potato peel as substrates with maximum
enzyme production from wheat bran with specific activity 1.2 U/μg [33]. Similarly, experi-
ments conducted to optimize alpha-amylase production by Bacillus subtilis using banana
peel yielded maximum activity (9.06 IU/mL/min) at 35 °C, pH 7 [34]. Molasses and sugar
beet pulp were employed by a scientist for beta-amylase production by a novel strain
Paenibacillus chitinolyticus CKS1 [35]. Increase in dependency on bioethanol demands
cheaper methods for its production, which implies in the identification of novel methods
for cellulase production, an enzyme involved in bioethanol synthesis. Different waste prod-
ucts like wheat straw, paddy straw, corn straw, sorghum husk, water hyacinth and sugar-
cane bagasse were evaluated for their ability to produce cellulases from a bacteria
Amycolatopsis sp. GDS, among which maximum yield was obtained with wheat straw.
Further analysis showed that the enzyme is thermostable up to 70 °C and expression
increases on the addition of NaCl and ionic liquid (10%) [36]. Trichoderma reesei produced
significant levels of cellulase when the pH of the medium was adjusted in the range of 5–6
by supplying the furfural residues as the carbon source. Treatment of furfural residues with
H2O2 to remove lignin further improved the yield of the enzyme [37]. Substantial amount
of xylanase was procured from different bacterial and fungal strains by utilizing different
agri-waste as carbon source such as Ncosartorya spinosa D19, Fomes selerodemeus (wheat
bran), Aspergillus terrcus (palm waste), Coprinellus disseminates (wheat bran), A. carneus
M34 (soyabean shell, rice bran), A. awamori (grape pomace), T. harzianum 1073 D3 (apple
pomace, melon peel and hazelnut shell), etc. [38, 39].
7.2.2 Energy Production Using Agri-biomass

Since ancient times energy is the fundamental requirement of humans to sustain. Energy
sources can be classified as renewable and non-renewable. Fast rate of depletion of non-
renewable sources and lack of acceptance and economic factors associated with renewable
sources have led to a surge for alternate sources of energy. The alternate sources should
generate energy that is low at cost, high at productivity and can be managed within the
limited land area available to meet the demands of the vast population. This marks the
concept of waste reuse. Agro waste has enormous potential to be exploited as a source of
energy when linked with proper technological support which otherwise would be discarded
as waste only causing severe environmental issues.
Both the field-based residues (corn corb, stalks, cotton sticks, mustard stalks, wheat
straw and husks) and process-based residues (bagasse, rice husks and hulls, coconut shells,
fibre, by-products from olive oil industry, potato peel) are extensively used for energy. Fuels
generated by these sources are categorized as first- and second-generation fuels. The carbo-
hydrate present in the by-product is of three types: lignocellulosic, starch and sugar.
Different processing techniques are applied for energy generation (i) physical, (ii) thermo-
chemical (incineration, gasification, pyrolysis) and (iii) biological (fermentation, anaerobic
digestion) (Figure 7.1).
Fermentation of sugar and starch is easier; however, fermentation of lignocellulosic
biomass requires an additional step of pre-treatment prior to fermentation as lignocellu-
losic waste is more stable. This is mediated by the process of hydrolysis either acid or enzy-
matic, which converts cellulose to glucose and hemicellulose to pentose and hexoses. These
simple sugars are acted upon by microbes to produce biofuels [40].
It was observed that when banana pseudostem underwent alkali and microbial treatment
by A. ellipticus and A. fumigatus under co-culture fermentation to release simple sugar
molecules the hydrolysate (4.1 g %) so obtained produced ethanol (17.1 g/L) on fermentation
by Saccharomyces cerevisiae NCIM 3570 after 72 hours [40]. Clostridium beijerinckii P260-
mediated fermentation of corn stover hydrolysate produced 16.00 and 18.04 g/L of acetone–
butanol–ethanol (ABE) on dilution with water (2 : 1) and wheat straw hydrolysate (1 : 1),
respectively. Further treatment of hydrolysate with calcium hydroxide to remove inhibitors
elevated the production level up to 26.27 g/L which was much higher than the control reac-
tion with glucose as carbon source that yielded 21.06 g/L ABE [41]. An ample amount of
study has been conducted on sugarcane bagasse that reveals that the by-product has huge
potential to produce ethanol using different micro-organisms such as E. coli, S. cerevisiae
D5A, Candida tropicalis JH030, Pachysolen tannophilus DW06, Zymomonas mobilis under
varying hydrolysis and fermentation conditions [42]. Cai et al. demonstrated the impor-
tance of every part of corn stalk in ABE production [43]. NaOH pretreated individual parts
were subjected to enzymatic hydrolysis followed by fermentation with C. acetobutylicum
ABE 1301. It was shown that 1 kg of stem, leaf, cob, flower and husk yields 116.4, 126.3,
169.1, 143.7, and 107.7 g butanol, respectively.
7.2.3 Role of Agri-biomass in Heavy Metal Decontamination

Need not to mention that water is indispensable for humans. However, the significant
population of the country is still fighting for it due to the fast depletion of water resources.
The condition is further worsened due to the pollution of water. The major factors responsi-
ble for water pollution are heavy metals released by various industries. The industries releas-
ing these contaminants directly or indirectly to water bodies include textile, fertilizers,
pesticides, metallurgy, tanneries, aerospace, photography, mining, electroplating, batteries,
finishing, energy and fuel, refining ores, etc. The most common metals that are responsible
for water toxicity include arsenic, mercury, cadmium, lead, chromium, nickel, zinc, cobalt
and copper. The heavy metals accumulate in an individual’s body system and impart various
detrimental effects. These are non-biodegradable, have the ability of bio-magnification,
interferes with various regulatory systems of the body such as the cell cycle, circulatory
system, nervous system, etc. Thus poses critical health issues that many times prove to be
fatal. Chemical precipitation, ion-exchange, reverse osmosis, ultra-filtration, reverse dialysis

are the conventional techniques opted for heavy metal removal. However, they have their
own constraints linked with them that involve high cost, lower efficiency, large scale pro-
duction of sludge and its disposal, etc. To overcome these problems, a novel concept of
biosorption is rapidly emerging, which involves the binding of metals to biological adsor-
bents. The literature cites numerous examples of agricultural waste as a biosorbant compris-
ing of rice husk and bran, wheat husk and bran, fruits peel, sugar beet pulp, cottonseed
hulls, corn cob, sugarcane bagasse and many more. Chromium (IV) adsorption efficiency of
naturally occurring rice husk and its activated form showed promising results which were
comparable to commercially available adsorbents [44]. These biosorbants are primarily
composed of cellulose, hemicellulose, lignin along with lipids, starch, sugars, ash and many
more [45]. These sequester metals with the aid of functional groups (hydroxyl, carbonyl,
carboxyl, amino, amido, acetamido, sulphydryl, phenolics, esters) attached to it which either
replaces the hydrogen ion with metal ions or donate lone electron pair to form complexes
with metal ions [45, 46]. Copper is yet another toxic metal for which various agricultural
waste product has been investigated for their adsorption potential under optimized condi-
tions; wheat shell, peanut hulls, barley straw, rice husk, fly ash are among the few candi-
dates [47–49]. Experiments have been conducted to validate the finding, and in one such
study, it was observed that the functional groups influenced the copper-binding ability of
activated carbon obtained from walnut shell. It was observed that the presence of oxygen-
containing groups facilitated the copper uptake (77.68% increase), which was further
enhanced by oxidation reaction, whereas amino groups have a lesser role to play [50].
Similarly, nitric acid-mediated oxidation of carbon in wheat and barley straw led to increas-
ing in carboxylic groups by 11-folds as seen by FITR spectra and Boehm titration. Comparison
of oxidized and unoxidized carbon for their Cu binding capacity showed better performance
than oxidized carbon [51]. Blockage of carboxyl group led to 32.8, 58.5, 65.3% decrease in
Cd(II), Cu(II) and Zn(II) adsorption capacity while blockage of hydroxyl group caused 30.9,
27.5, 46.1% decline in adsorption for the three metals thereby confirming the importance of
functional groups [52]. However, these functional groups do not hold the sole responsibility
for the efficacy of metal sequestration. They function well in association with various other
factors including physical and chemical properties of biosorbant and metal ions, availability
of binding sites, ease of access to these sites, the affinity between the binding site and metal
ion, pH, temperature, initial concentration of both and contact time, etc. [53].
The rate of biosorption is higher during the initial period of contact that could be attrib-
uted to the presence of a large number of vacant active sites initially and, thereafter, a satu-
ration stage reaches where the metal needs to be diffused into the inner sections of the
biomass [54, 55]. Another determining factor is the pH that alters the surface charge of
biosorbant and regulates the competition between the hydrogen ion and metal ion. At
lower pH due to higher concentration of hydrogen ion, most of the active sites are occu-
pied. Increase in pH shifts the sites towards negative charge, making functional groups
available for binding of metal ions. Further higher pH leads to metal precipitation, thereby
restricting the biosorption [52–56]. In the case of maize stalk sponge, the adsorption of Pb
increased with increasing pH with maxima at 6.0 ± 0.2 becomes stable up to pH 8.0 and
then Pb precipitation started [54]. The effect of temperature is controversial as certain
reports favour an increase in temperature and relate it to improve in its surface activity
while other reports are reverse to previous studies and link it to structural damage of active
sites. Thus, the selection of optimum temperature is preferential that is dependent on the
type of functional group, metal ion and other factors [53]. Increase in initial metal ion con-
centration above a threshold level is inversely proportional to the removal efficiency of
metal ions, and this may be attributed to the non-availability of active binding sites as it has
been already occupied by metal ions and a state of saturation is achieved. Metal ion removal
by the banana peel is a good example of it where metals at a concentration of 150 mg/L, the
removal efficiencies of Cu(II), Pb(II), Zn(II) and Ni(II) were noted as 92.52, 79.55, 63.23
and 68.10% while at a lower concentration of 25 mg/L the corresponding values were 94.80,
86.81, 84.63 and 82.36%, respectively [57].
This issue could be resolved by the addition of more of the biosorbants. However, the
increase in biosorbants elevates the removal percentage of metal ions but decrease the max-
imum adsorption capacities due to a decrease in the total surface area caused by the overlap
of adsorption sites.
Agricultural waste biomass and metal ions each have their individual properties. It is
their amalgamation under optimized conditions that leads to successful metal adsorption.
7.2.4 Manufacturing of Lightweight Materials

The construction and building materials sector is the largest user of natural resources and
concrete forms the backbone of the sector. Generally, concrete consists of 12% cement, 80%
aggregates and 8% water by mass. Both water and aggregates are natural resources, whereas
cement is produced by a cement manufacturing plant. The cement industry is one of the
most polluting industries of the world, wherein 480 kg of CO2 is released in producing 1 m3
of cement. One strategy is to reduce the cement dosage in concrete formation by using fly
ash which is a waste generated by the burning of coal used in the power generation sector
or any other industry that uses boilers for steam formation [58]. To reduce the impact of
construction on natural resources, the best option is to use materials that are produced
from agricultural waste. Some of the agricultural waste that can be used as an aggregate in
the concrete are groundnut shell, sugarcane bagasse ash, sawdust, rice husk ash, giant reed
ash, tobacco waste and cork. The major difference among these wastes is the place from
which they are collected and the process by which they are converted into aggregates.
Many studies have been conducted by using these wastes in concrete. By adding 10% of
sugarcane bagasse ash in concrete, its compressive strength increases, but further addition
of the ash resulted in a decrease in strength [59]. Almeida et al. found that the strength of
mortar was not affected by adding sugarcane bagasse ash [60]. When groundnut shell was
added to the control concrete, the maximum compressive strength could be achieved with
5% mix. The authors also found that the concrete mixed with groundnut shell was not suit-
able for structural members exposed to water as the moisture affects the strength and
weight [61]. With giant reed ash, the compressive strength of the concrete increased till
7.5% replacement of further aggregate increase in percentage led to decrease in strength,
but it was still higher than the minimum requirement. The authors argue that silica present
in the ash is the reason behind the increase in strength [62]. Rice husk ash and limestone
were used to form cylindrical concrete test samples [63]. The authors found very low
compressive strength for 100% replacement of rice husk ash, whereas the strength was
maximum for limestone at 10% replacement. The decrease in compressive strength was
attributed to greater porosity of rice husk ash and limestone, which was indicated by higher
water requirement. Moreover, the authors found that rice husk ash was able to fill the
microvoids in the cement particle in an improved manner. Nóvoa et al. found that the com-
pressive strength of cork containing polymer mortar was higher than normal mortar [64].
Moreira et al. found the compressive strength of screed containing expanded cork. The
authors observed that the strength increases as the cement content increases, and as the
cork granules percentage increases the strength decreases [65]. Öztürk and Bayrakl found
that using a small amount of tobacco waste increased in compressive strength of con-
crete [66]. The authors suggested that the lightweight concrete can be used as an insulating
material for coating and separating materials in construction.
Shafana and Venkatasubramani studied the tensile strength of concrete with the addition of
sugarcane bagasse ash and found optimal strength at 10% replacement of aggregate with
bagasse ash [67]. Similarly, Mageswari and Vidivelli found improvement in tensile strength up
to 20% replacement of aggregate with sawdust ash further addition resulted in a reduction in
tensile strength [68]. The flexural strength of reed-based concrete was carried out by Ismail and
Jaeel [62]. The authors found improvement in strength till 7.5% replacement of aggregate with
reed further replacement resulted in a decrease in strength even below the concrete strength
levels. The authors attributed the increase in strength to the presence of organic bindings in the
reed in acidic or alkali environment, which decomposes to pentose and hexose. These mono-
saccharides are easily dissolved in water resulting in the formation of hydrophilic adsorption
layers on cement grains. Similarly, the addition of cork to mortar resulted in an increase in
flexural strength and higher percentage resulted in a decrease in the flexural strength [64].
Ismail and Jaeel [62] also studied the effect of alkali on concrete containing reed ash and
reed fibre. The authors found that the concrete containing giant reed fibre was affected
faster than the control concrete. The authors argued that the complex lignin molecules in
the fibre were affected, causing it to fracture. Almeida et al. used colorimetric treatment for
studying the effect of chloride penetration in bagasse ash containing mortar [60]. The
authors observed higher resistance to chloride penetration with bagasse containing mortar
in comparison to control concrete. The water absorption and porosity of hardened concrete
is an important indicator of its durability. If these values are low, then the life and service-
ability of concrete will increase many folds. Modani and Vyawahare found that the sorptiv-
ity coefficient increases with an increase in bagasse ash content in the concrete [59]. Ismail
and Jaeel found a higher percentage of water absorption with reed fibres containing con-
crete as compared to reed ash and control concrete [62]. Moreover, the increase in the reed
ash percentage in concrete the water absorption percentage also increases.
7.3 Case Studies
7.3.1 Utilization of Paddy Waste
Rice is the second most eaten staple food after wheat with annual growth around
680 million tons. It is rich in vitamins, minerals and many other nutrients. Viable rice
growth is dependent on the continuous availability of nitrogen, silicon and water. It is
cultivated all over the world with distinct genetics, morphology and nutrient
7.3 Case Studie 139
composition. Not all the rice grown in the field is as such ready for consumption, but
approximately 40% of it is total rice produced is lost in the form of by-products. During
its journey from field to house, the rice undergoes a series of processes which includes
harvesting, transport, reception and pre-cleaning, drying, storage, shelling, milling/pol-
ishing, selection and classification. The amount and type of by-product produced is a
function of various factors such as type of rice, milling techniques, processing techniques
used which results in the production of husk, straw, bran, broken rice and ash. These
products previously treated as waste has now high economic and nutritional value as it
embraces a plethora of useful compounds in itself. Rice straw is produced during harvest-
ing and accounts for approximately 50% of the dry weight of rice. It is composed of low
levels of lignin but a high concentration of silica oxide [69]. It has been established that
rice straw is a good source of nitrogen yielding about 2.5 kg nitrogen per ton straw and
can serve as a matrix for the cultivation of numerous vegetable crops like tomato, cucum-
ber and okra [70]. Addition of paddy straw in soil positively affects the physicochemical
properties of soil. It helps in retaining Fe, Cu, Mn, Zn content and prevents nitrate leach-
ing. It promotes the growth of microbial fauna, thereby activating enzymatic machinery
in soil and alleviating the levels of macro and micronutrients [71]. Rice husk or rice hull
is a protective coating developed during the course of seed formation [72]. Primarily it
constitutes of cellulose (40–50%), hemicellulose (25%), lignin (20–30%) and ash (17–18%).
It is rich in organic compounds (94.99% by weight) than inorganic compounds with ash
contributing around 94% of total silica. High silica content lowers its decomposition rate
and allows recovery of rice husk ash [73, 74]. In today’s era of technology, the role of
silicon oxide cannot be negated as it is the basic component of electronic circuits, diodes,
insulators, solar panels, semi-conductors and also has broadened its area of application
to polymers and ceramics. Synthetic production of silica is expensive, thereby generating
a need for alternate sources of silica production. Methods have been developed to extract
silica from rice husk ash as it is rich in silica [75]. This extracted silica has been used as a
support for the synthesis of a number of catalysts where metals usually transition metals
are impregnated onto the silica surface via different techniques (sol–gel, ion exchange,
deposition–precipitation) [76]. In a developing country like India, energy generation is
always an issue leading to exhaustion of natural non-renewable energy sources. Keeping
this in mind, rice husk has been used by researches to identify alternate sources of energy
production which includes power, fuels, heat, syngas, etc. In order to utilize husk for
power generation, the rice husk was heated at high temperature causing decomposition
of material and production of combustible gases which was then burnt to produce steam/
heat which leads to activation of turbines producing electricity. In many countries, most
of the rice husk is consumed by rice processing companies for electricity, making it eco-
nomical and environment friendly [77, 78]. Roy et al. determined the use of rice husk
fuel in the process of parboiling of rice. In a study, for the production of bioethanol, rice
husk in combination with orange peel wastes were used [79]. To it, water was added, and
substrate (11 kg) was prepared for hydrolysis by sulfuric acid to produce sugar at
121 °C. Further Saccharomyces cerevisiae-mediated fermentation led to the production of
22.77 g of ethanol per liter of the substrate [80].
Rice bran constitutes an important class of by-product from rice due to its richness in
carbohydrates, lipids, proteins, fibres, minerals and vitamins. About 10% of rice grain mass
comes from bran which contains 15–20% of bran oil. Similar to any medicinal plant rice
bran exhibits a number of therapeutic properties. Compounds like ferulic acid and tricin
induce apoptosis and inhibit cancer cell proliferation [81].
7.3.2 Utilization of Mustard Waste

Mustard commonly called as rai or kali sarso belongs to the member of Brassicaceae family.
It is mainly cultivated in northern plains of India including Rajasthan, Madhya Pradesh,
Gujarat, Haryana, Uttar Pradesh, etc. India is the third largest producer of mustard in the
world. The seeds of mustard are used for oil extraction and seasoning in food while the
leaves are used as a vegetable. The large scale cultivation of mustard and its utilization lead
to large scale production of waste from it. However, in the present time, these waste prod-
ucts are no more a waste product but are efficiently utilized for different purposes. Higher
levels of astaxanthin, a keto-carotenoid with a beneficial role in food, cosmetics and medi-
cal industry, were produced by using mustard waste media in comparison with standard
media in yeast strain Xanthophyllomyces dendrorhous [82]. Further mustard stalk and
straw was used as a substrate by fungus Termitomyces clypeatus to produce various lignocel-
lulolytic enzymes like beta –glucosidase, beta xylanase, beta xylosidase. Also, this waste,
after saccharification with the enzymes of T. clypeatus, was used for the production of
bioethanol [83]. Sahoo et al. showed the isolation of lipase, which was thermostable in dif-
ferent conditions from Anoxybacillus sp. ARS-1 using mustard cake by solid substrate fer-
mentation [84]. Similarly, protease, an important enzyme used widely in industries, was
produced from the mustard cake with a concentration of 4% under optimized conditions of
pH-11, 40 °C and submerged fermentation for 70 hours [85]. Pollution caused by heavy
metals is a major concern nowadays, which poses a threat to all forms of living beings.
Waste biomass obtained after extraction of oil from seeds of white mustard have shown to
possess biosorption property for heavy metals. It was seen that biosorbant at a concentra-
tion of 5 g/L at 5.5 pH and 55 °C was very effective in adsorption of zinc, lead and cad-
mium [86]. Husk and sticks from mustard are continuously supplied to the bricks industry
by farmers. Also, a combination of mustard waste along with leaves and grasses and wood
waste leads to the formation of briquettes which is a potential source of energy [87].
Deforestation has led to a shortage of wood supply which in turn is affecting the furniture
industry. Substitution of wood chips with white mustard straw wholly or partially has pro-
vided a solution to this problem. General boards and interior boards which are used under
dry conditions can be prepared using mustard waste [88].
7.3.3 Utilization of Maize Waste

Maize, also known as corn, belongs to the family of Gramineae. It is believed that maize
first originated in central Mexico 7000 years ago in the form of wild grass. The natives
transformed maize into a better source of food. It contains nearly 72% starch, 10% protein
and 4% fat, supplying an energy density of 365 kCal/100 g [89]. Maize is grown throughout
the world, according to the Food and Agricultural Organization of the United Nations in
the year 2018, nearly 193 million hectares were harvested by the world and produced nearly
1147 million tons of maize. United States of America, China and Brazil were the top
7.3 Case Studie 141
producers of maize. United States produced nearly 392 million tons, China produced
257 million tons and Brazil produced 82 million tons of maize in the year 2018 [90]. Apart
from food and fodder, it is possible to process maize into a variety of food and industrial
products such as sweeteners, starch, corn oil, glue, beverages and ethanol. In the last ten
years, nearly 40% of the maize produced in the United States is utilized in producing fuel-
grade ethanol [89].
Apart from the grain, the maize stover consisting of leaves, stalks, cobs and husk is left in
the field after harvesting [91]. After harvesting of the grain, the stover contains nearly 15%
cob, 8% husk, 21% leaf and 56% stalk. The stover can be used as a litter for animals, fuel, soil
conditioner and fodder for ruminants. In the United States, the maize stover is also used for
producing ethanol due to its higher lignocellulose content [92]. Maize stalks can be used as
a fuel; it has a higher heating value of 17.2–18.5 MJ/kg. It has moderate ash content and
low moisture content. As compared with other solid fuels, its sulphur content is low, and
oxygen content is high [93]. Maize stalks can be used as fodder for ruminants, but its qual-
ity is low due to insufficient protein content. To increase the protein content, various meth-
ods are available such as fermentation with yeasts and fungi, alkali treatment and
ensilage [94]. The stalks can also be used as soil conditioners for replenishing the organic
matter in the field for the next crop. The maize stalks contain hydroxycinnamic acids,
namely ferulic and p-coumaric acids, as they are rich in cell wall materials. These acids are
the main components for cell wall shape, integrity and defence against pathogenic ingress.
As bi-functional molecules with phenolic and carboxylic bonding sites, the ferulic acid
provides the linkage between esters in the primary cell wall polysaccharides and the ethers
linking the lignin content [95]. With alkaline treatment, the lignin is dissolved by the cleav-
age of ester linkages in the lignin-polysaccharides complex, thereby releasing the phenolic
acids. Both the hydroxycinnamic acids have great importance in the form of antioxidants
as they can preserve food due to their ability to inhibit fatty acid peroxidation, as flavoring
and aroma compounds, as feedstock for high-value vanillin bio-production and as anti-
cancer or a potent anti-inflammatory substance [96, 97].
Maize cob left after extracting the grain can also be used as a fuel, as its higher heating
value is close to the maize stalk. It also has low protein content, but it has high fibre con-
tent, due to their wide availability they are also used as fodder for ruminants. Compared
with maize stalks they have rather higher hydroxycinnamic acid content. Therefore,
maize cob is considered as an important source of ferulic and p-coumaric acids along
with wheat bran and rice husk [98]. Due to their higher abrasiveness and absorbency,
they are an ideal biomass-based material for industrial applications such as absorbing
oils and chemicals, cleaning industrial and environmental spills and also for polishing
and blasting of materials. Foo attempted to utilize corn cobs as an absorbent of carbo-
furan [99]. Carbofuran is a pesticide used against a wide range of insects. However, its
incessant use has resulted in its reaching the potable drinking water causing a wide vari-
ety of human health issues, including reproductive failure, teratogenicity, neurotoxicity,
carcinogenicity and mutagenicity. Tsai et al. converted corn cobs into activated carbon
using potassium hydroxide/potassium carbonate and carbon dioxide [100]. The authors
studied the efficacy of using the activated carbon as an absorbent of organic compounds
and found the adsorbent had large surface area and higher pore volume. Synthetic
organic dyes used in textiles, printing, leather tanning, food processing, rubber,
cosmetics, photography, etc. are a big source of water pollution. Berber-Villamar et al.
assessed the potential of removing Direct Yellow 27 dye from the wastewater using corn
cobs [101]. The authors found that the adsorption capability of the cob was dependent
upon the particle size of corn cob, pH of the solution, contact time and initial dye con-
centration, and most of the dye could be easily removed from the solution. Patil et al.
used corn cob for treating sewage water and found that the biochemical oxygen demand
reduced from 460 to 187 mg/L, chemical oxygen demand reduced from 466 to 194 mg/L,
total suspended solids reduced from 514 to 220 mg/L. Silica extracted from corn cob can
also be used for wastewater treatment [102]. Shim et al. found that 84–88% copper ion
and 83–87% cadmium ion can be removed within 24 hours from the contaminated
water [103]. Corn cob can be used for making particle boards, which can provide acoustic
insulation [104] and thermal insulation [105]. It can also be used as an agglomerate for
concrete production [106].
7.3.4 Utilization of Horticulture Waste

Fruits and vegetables as compared with edible content have a higher proportion of residues
and wastes. Table 7.1 shows the type of waste and the percentage of waste in various fruits
and vegetables after processing. It is seen that the citrus fruits contain the maximum waste
of 50%, followed by mango, which has 45% waste and onion and guava processing gives the
lowest waste of 10%. Banana peels account for 30% of the fruit, and it contains high levels
of flavonoids, phenolics, proanthocyanidins and other antioxidants [108, 109]. It was found
that the seed fractions and peel of some fruits such as grape seed, hawthorn peel, pome-
granate peel, lychee seeds are a rich source of natural antioxidants and have high antioxi-
dant activity [110]. In some materials, it is seen that the by-products contain a major
amount of bioactive compounds in comparison to the major counterparts. For example,
cashew apple and Surinam cherry by-products contain a high amount of total phenolic
Table 7.1 Fruits and vegetable processing wastes [107].
Fruit/vegetable Nature of waste Approximate waste percentage
Citrus Peel, rag, seed 50

Banana Peel 35
Pineapple Skin, core 33
Mango Peel, stones 45
Apple Peel, pomace, seed 25
Grape Stem, seed, skin 20
Tomato Skin, core, seed 20
Guava Peel, core, seed 10
Pea Shell 40
Potato Peel 15
Onion Outer leaves 10
7.3 Case Studie 143
compounds and anthocyanins as compared to their pulps [111]. Therefore, processing

waste vegetables and fruits can help recover natural antioxidants that can replace synthetic
antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT)
as sources of health benefits [112, 113]. Some of the bioactive compound resources are
shown in Table 7.2.
In the laboratory, microorganisms are grown for microbial studies with the help of a
suitable culture media. Commercially available media such as MacConkey agar, Nutrient
agar and Cetrimide agar are used for the growth of microorganism; however, these
media are highly costly [121]. For the culture of both fungi and bacteria, nutrient agar
has been formed from carrot, cabbage, tomato, gooseberry, pumpkin, etc. [122]. To
reduce the cost of microbial media, cowpea, black gram and green gram have also been
used as starch and protein substitutes. Agar made from the peel of Dragon fruit has also
been used as a media for growing microbes [123]. Banana, grapefruit and melon peels
contain carbohydrates that can be used as a substrate for growing amylase [124]. Fungi
growth has also been carried out using banana peel which is an economical and effec-
tive media [125].
Fruits and vegetable peels can also be used to make biosorbents that can remove heavy
metals present in the water [126]. Malachite green was removed in batch mode using
sponge gourd peel which is an inexpensive natural biosorbent [127]. Rhodamine-B is a
cationic water-soluble dye which was removed using banana peels [128]. A study was
also carried out in which a biosorbent was developed using banana peel-activated carbon,
methylated banana peel and natural banana peel for treating palm oil mill
effluent [129].
Table 7.2 Vegetables and fruit wastes containing bioactive compounds.
Source Residue Bioactive compounds References
Papaya, olive Leaves Flavonoids, phenolic compounds, saponins, [114, 115]

proanthocyanidins, tyrosol, gallic acid,
oleuropein
Grape Stalks Flavonol glycosides, anthocyanins, catechins, [116]
procyanidins, phenolic acids
Guava, mango, Peels or Syringic acid, Gallic acid, mangiferin, gentisyl- [108, 117, 109]
apple, cashew, skins protocatechuic, anthocyanins, quercetin, ellagic
sapodilla, papaya, acid, caffeic acids, carotenoids, chlorogenic,
macadamia, potato flavonoids, proto-catechuic, phenolics,
proanthocyanidins
Acerola, olive, grape, Seeds Hydroxybenzoic acid, protocatechuic acid, [111, 109]
soursop, guava, caffeic acid, ferulic acid, syringic acid, sinapic
papaya, apricot, acid, quercetin, rutin, cinnamic acid,
passion fruit hesperidin, persipan
Pistachio Hulls Phenolic compounds [118]
Apple, grape, olive, Pomace Flavonol glycosides, bioactive polar lipids, [109, 119, 120]
apple, tomato anthocyanins, catechins, phenolic acids,
stilbenes and lycopene, syrigin and quercetin
7.4 Conclusion and Future Perspectives
Recent studies revealed that agriculture residues could be a source of valuable chemicals
that can be used pharmaceuticals, nutraceuticals industry. Organic matter of biomass can
be diverted into various ways to produce desired compounds as after hydrolysis of cellu-
losic biomass, glucose is produced, which is a platform chemical for generation diverse
sugars and alcohols, etc. Novel technology shows the use of agri-residues in the prepara-
tion of construction materials also. Paddy straw and corn cobs are actively utilized in the
production of bioenergy in many countries, and this process significantly supports the
energy supplies to society. Many agri-residues have adequate capability to extract heavy
metals from industrial effluents and contaminated water. Sustainable use of agricultural
residues will support a circular economy as well as it can avoid several issues related to the
environment, so it will provide dual benefit to society.
As rapid depletion of non-renewable resources is a major challenge for any country so
globally, scientists are looking for the development of renewable resources for fulfilling the
need of the growing population. In future, we will see more investment in this sector as
bio-economy is becoming a trend in the global market. To maintain the criteria of sustain-
able development, constructive use of biomass is the urgent requirement to support the
economy of a country.
R
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153
Antimicrobial Products from Plant Biodiversity

Pankaj Kumar Verma1, Shikha Verma2, Nalini Pandey1, and Debasis Chakrabarty2
1
Department of Botany, University of Lucknow, Lucknow, Uttar Pradesh, India
2
Molecular Biology and Biotechnology Division, Tissue Culture and Transformation Lab, CSIR- National Botanical Research
Institute, Lucknow, India
8.1 Introduction
Medicinal plants are natural resources and contain a number of products that perform
significant roles in human health benefits [1]. In the current scenario, only a few plants (about
1–10% of the total estimated plants on earth 2.5 × 104 to 5.0 × 104) were identified for their
medicinal properties and used to treat human diseases. However, the demand for medicinal
plants to isolate the bioactive compounds has been increased rapidly. In past few years,
researcher shifted their attention toward medicinal plants to demonstrate the promising role of
plant-derived antimicrobial substances to treat many diseases caused by microbes. These plant
extracts may have antimicrobial properties and may be used for various applications, including
natural therapeutics, processed food preservation, alternative medication, and pharmaceuti-
cals. Several communities of Asian countries widely used plant products to treat microbial
infection as traditional medicine. For the preparation of drugs from phytochemical compounds
extracted from plants, it is important to understand the structural composition and its pharma-
cological properties. Hence, the development of novel scientific approaches for the identifica-
tion, characterization, and validation of different plant-derived antimicrobial components,
extracts, and chemical or biological compounds is important in order to develop efficient and
cost-effective herbal medicines. Therefore, attention must be focused on the propagation and
preservation of medicinally important plant populations, which may be active sources for more
effective herbal medicine. The medicinal use of plants is studied under ethnopharmacology for
a long time. It involves the identification of raw materials for the preparation of advanced medi-
cines and the examination of bioactive isolates from natural resources. Natural compounds
such as secondary metabolites may constitute a vital source of chemical diversity. Various stud-
ies described the significance of natural plant isolates to cure human diseases. Due to the effec-
tiveness of these natural compounds, around 69% new antibacterial and 21% antifungal drugs
were approved between 1981 and 2006 to treat various diseases caused by microbes.

154 8 Antimicrobial Products from Plant Biodiversity
According to a recent study, the utilization of plant-derived medicines may be classified as a

three-generation of the progressive development of plant-derived medicines. Since the ancient
age, the crude extract of medicinal plants was used by traditional societies to manage infectious
disease grouped in the first generation; progressively the active constituents were extracted from
plants through scientific processing grouped as the second generation, and the third generation
of phytotherapeutic agents was examined for comprehensive pharmacological/biochemical
studies. Different plants of Dracenaceae, Anacardiaceae, Pinaceae, Apiaceae, Burseraceae,
Palmaceae, Cupressaceae, Euphorbiaceae, and Fabaceae families are considered a rich source of
secondary metabolites. These metabolites may have antimicrobial activity, thus may be used as
potential antimicrobial agents to treat a number of virulent including drug-resistant microbes,
therefore used as a superior and cost-effective substitute for antibiotics. For example, various
species of Hypericum spp. (Common: St John’s Wort, Hindi: Choli phulya) known as the world’s
leading herbal plant, which contained several medicinal compounds including hyperenone A,
hypercalin B, acylphloroglucinol hyperforin, xanthones, and naphthodianthrone, etc., having an
antibacterial, antiviral, antifungal, anti-inflammatory, anticancer, and antioxidant activity [2].
Similarly, Moringa oleifera contains flavonoids, alkaloids, gallic acid, catechol-type saponins, and
tannins, which have antimicrobial and anti-inflammatory properties and thus helps in main-
taining cholesterol level and blood pressure [3]. Additionally, extracts derived from medicinal
plants such as Kaempferia pandurata and Senna alata possess antimicrobial properties for
Staphylococcus aureus [4]. Similarly, Anogeissus schimperi and Anacardium occidentale against
Pseudomonas aeruginosa and Camellia sinensis against Listeria monocytogenes [5]. Further, phe-
nolics and alkaloids derived from Cameroonian plants own antimicrobial activity (MIC < 10 μg/
mL) [6]. Wangchuk and coworkers identified various classes of phytochemicals from a number
of Bhutanese medicinal plants having a broad range of biological activities [7]. They suggested
that these plants having flavonoids, alkaloids, and tannins that may be used to treat diseases
caused by bacterial, fungal, malarial parasites, and also used to treat African sleeping sickness
caused by Trypanosoma brucei rhodesiense. Additionally, an aromatic plant caper (Capparis sp.)
found in Mediterranean Sea Basin has been used as a traditional medicine to eliminate many
diseases, such as seeds extract of Capparis decidua possesses antileishmanial, antibacterial, and
antifungal activity due to its quaternary ammonium and glucosinolate [8]. Phytochemical stud-
ies indicated the presence of different compounds, such as carotenoids, spermidine, tocopherol,
rutin, campesterol, stigmasterol, quercetin, and kaempferol, which are responsible for antimi-
crobial, antifungal, antiviral, antioxidative, and anti-inflammatory properties.
Plants-derived antimicrobial compounds possess enormous therapeutic potential with
lesser side effects and very less chance to develop resistance for medicine. Therefore, there
is a need to investigate essential antimicrobial substances from plants and the development
of novel drugs to combat microbial infections. In this chapter, an attempt has been done to
summarize the current state of knowledge regarding the antimicrobial properties of essen-
tial substances derived from plants.
8.2 Use of Plant Products as Antimicrobials: Historical Perspective
Earth has a rich diversity of flora and fauna, around 350,000–500,000 plant species are recorded
on Earth. From ancient times, humans learn the use of plant products to cure a disease by
their experience. The oldest record for using medicinal plants was found in Indian holy books
8.2 Use of Plant Products as Antimicrobials: Historical Perspectiv 155
Vedas, which stated about the use of different plant species to cure various diseases. Similar
evidence also found from different countries, such as in China there was evidence of use of
dried plant parts to treat diseases; some are still in use such as Podophyllum, Rhei rhizoma,
Theae folium, Ephedra, Cinnamon bark, etc. The Ebers Papyrus written in 1500 BCE was con-
sidered as the most important medicinal book of ancient Egypt. It has a collection of 700 for-
mulas and remedies from plants to treat various diseases. Additionally, two Homeric epics,
the Iliad and the Odyssey (800 BCE) reported 63 plant species that were used for pharmaco-
therapy. In 459–370 BCE, Greek physician Hippocrates used 300 plants according to its phar-
macological behavior, such as Centaurium umbellatum Gilib was used to treat fever, garlic was
used for intestinal parasites and as a diuretic, also the deadly nightshade, henbane, opium,
and mandrake as narcotics. Theophrastus (371–287 BCE), “the father of Botany,” wrote two
books “De Causis Plantarium” and “De Historia Plantarium,” in which he classified and
described more than 500 medicinal important plants. Pedanius Dioscorides “The father of
pharmacognosy,” wrote his work “De Materia Medica” in 77 CE, in which he described
various plant-based drugs. He stated that, domestic plants such as garlic, chamomile, onion,
coriander, sea onion, ivy, parsley, and marshmallow could be used as drugs, for example,
chamomile used to treat wounds, burns, stings, and ulcers. Pliny the Elder described the use
of about 1000 medicinal plants in his book “Historia Naturalis” in 23–79 CE and covered all
the medicinally important plants from Germany and Spain. In 131–200 CE, Roman physi-
cian, Glen explained drugs identical to dioscorides, while introducing several new plants-
based drugs, such as Uvae ursi folium used as uro-antiseptic and diuretic even also using
currently. Slavic natives (seventh century CE) used several plants including Veratrum album,
Cucumis sativus, Rosmarinus officinalis, Allium sativum. Ocimum basilicum, Urtica dioica, and
Mentha viridis to treat several diseases. Charles the Great (742 CE–814) quoted 100 different
plants in his work “Capitularies,” which are also using in the modern world such as mint,
poppy, sage, iris, marshmallow, onion, etc. The Arabs added various new plants such as cof-
fee, aloe, ginger, saffron, cinnamon, pepper, etc. from India in pharmacotherapies, which con-
tinue to be used in the present world also. In the seventeenth century, many medicinal plants
were introduced in European countries, such as Cortex Chinae from the bark of Cinchona
succirubra Pavon. In the eighteenth century, Linnaeus described and classified the species in
his work “Species Plantarium,” a binominal system where each species consists of a genus
name written with an initial capital letter and a species name with an initial small letter [9].
In the early nineteenth century, use and understanding of medicinal plants were more
explored with the discovery, isolation, and validations of drugs. Alkaloids, glycosides, and qui-
nine isolation from poppy and other plants initiated the start of scientific pharmacy. With
improving a chemical process, other compounds were also discovered from medicinal plants
such as tannins, vitamins, hormones, saponosides, etc. In the early twentieth century, a study
was conducted for stabilization and cultivation of plants with medicinal properties. For the
physiological, chemical, and clinical studies, various drugs producing extinct plants were
restored for pharmacies such as Opium, Punica granatum, Filix mas, Colchicum, Stramonium,
Styrax, Ricinus, and many more. Some plant drugs have a long history such as quinine used in
the cure of malaria; currently, quinine analog chloroquine is a widely prescribed drugs for
malaria. Similarly, higher plants also used in cancer therapies, for example, antileukemic alka-
loids were isolated from Catharanthus roseus syn. Vinca roseus. There are a lot of examples of
this kind; therefore, to explore the utility of many more ancient plants-based drugs, there is a
need to study the old manuscripts and books to use as a potential source in pharmacotherapy.
8.3 Major Groups of Plants-Derived

Antimicrobial Compound
Plant-derived substances that are used as medicines are principally secondary metabolites
produced by the plants and the basic roles of these compounds are in defense mechanisms.
These compounds impart flavor, pigmentation, and medicinal properties as well as possess
a broad range of activities according to plant species, country origin climate, and topogra-
phy. Their antimicrobial action was modified by variations in their chemical composition.
Therefore, random screening of medicinal plants for active compounds is needed to evalu-
ate their pharmacological activity. Phytochemical extracted from medicinal plants can be
typically divided into several distinct categories, as described in the next sections (Table 8.1).
8.3.1 Simple Phenols and Phenolic Acids

Phenolic compounds possess a broad range of bioactive natural substances and are utilized
for various medicinal purposes. These substances perform a significant role in protecting
plants from diverse microbes and pathogens through several mechanisms [59]. They have
potential antimicrobial and antioxidative properties [60]. Phenols have a vast group of aro-
matic compounds including flavonoids, flavones, and flavonols typically consisting of one
carbonyl group, whereas quinines contain two carbonyl groups, tannins with polymeric
phenolic substances, and coumarins contain fused benzene and pyrone groups [61]. All
groups of phenolic compounds acquire antimicrobial properties [62]. Wide ranges of phe-
nylpropane-derived compounds have higher oxidation states such as cinnamic and caffeic
acids. Traditional herbs, thyme, and tarragon contain caffeic acid, which possesses antivi-
ral, antibacterial, and antifungal properties. Catechol and pyrogallol are hydroxylated phe-
nols, catechol has two hydroxyl groups, and pyrogallol contains three hydroxyl groups.
These hydroxylated phenols have antimicrobial properties based on-site and the number of
hydroxyl groups on the phenolic ring, with toxicity against microbes, increases with
increased hydroxylation. The possible mechanisms of action of these oxidized compounds
usually include microbial enzyme inhibition, mainly by reaction with sulfhydryl groups or
via nonspecific interaction with specific proteins. While low-molecular-weight phenolics
possess antimicrobial activity by diffusing across the microbial membrane, which leads to
cytoplasmic acidification, causing cell death.
8.3.1.1 Flavonoids
Flavonoids are hydroxylated phenolic structures found in photosynthetic plants such as veg-
etables, fruits, stem, flowers, seeds, etc. The structural composition of flavonoid is 2-phenyl-
benzopyrane or flavone nucleus, comprising two benzene rings connected through a
heterocyclic pyrene ring. On the basis of chemical composition and substituent position on
rings, flavonoids are divided into 14 classes. The flavonoid compounds can form a complex
with bacterial membranes as well as with both soluble and extracellular proteins result in
antibacterial properties [63]. They additionally maintain inhibitory action against various
viruses [64]. Several investigations demonstrated the efficiency of flavonoids such as glycyr-
rhizin, chrysin, and swertifrancheside against HIV. The studies found that flavonoid com-
pounds have inhibitory action toward respiratory syncytial viruses (RSV). Another study
Table 8.1 Major classes of antimicrobial compounds from plants.
Class Compounds Plant species Mechanisms of action Active against References
Phenolics Resveratrol Vitis vinifera, Vaccinium Efflux pump inhibitor, B. cereus, E. faecalis, P. aeruginosa, [10]
corymbosum anti-biofilm M. smegmatis, C. jejuni, MRSA
Tannin/ Sorghum spp. S. aureus, S. typhimurium, A. niger, A. flavus, [11]
tannic acid S. cerevisae
Baicalein Scutellaria baicalensis Georgi Efflux pump inhibitor anti M. smegmatis, C. albicans, S. epidermidis, [12]
biofilm P. aeruginosa, E. coli, MRSA
Biochanin A Trifolium pratense L. Efflux pump inhibitor Bifidobacterium spp., Clostridium spp. [13]
Luteolin Cuminum cyminum, Bacopa Biofilm inhibitor S. aureus, L. monocytogenes [14]
monnieri, Achillea millefolium
Kaempferol Polygonum tinctorium Lour Efflux pump inhibitor, S. aureus, H. pylori [15, 16]
biofilm inhibitor
Quercetin A. japonica Efflux pump inhibitor S. aureus [17]
Catechol Acacia catechu Antibiofilm E. coli, P. mirabilis, P. aeruginosa [18]
Coumarin Dipteryx odorata, Galium Quorum sensing and biofilm E. aerogenes, E. coli, S. aureus, [19, 20]
odoratum formation inhibition S. typhimurium
Gallic acid Hamamelis virginiana, Cell membrane disruption, E. coli, L. monocytogenes, P. mirabilis, [21]
Camellia sinensis biofilm inhibition P. aeruginosa
Caffeic acid Eucalyptus globulus Destruction of cellular B. cereus, E. coli, P. aeruginosa, S. aureus [22, 23]
membrane
Catechin Acacia catechu, Camellia Reducing antioxidant B. cereus, B. subtilis, S. aureus, E. faecalis, [24]
sinensis capacity E. coli, Salmonella
Apigenin P. oleracea L. P. aeruginosa, S. typhimurium, P. mirabilis, [25]
K. pneumoniae, E. aerogenes
Naringenin Citrus Genomic DNA-binding S. aureus [26]
Curcumin Curcuma longa Efflux pump inhibitor P. aeruginosa [27]
(Continued)
0005092146.INDD 157 06-03-2021 21:18:08

Alkaloids Reserpine Rauvolfia serpentina Biofilm inhibitor P. aeruginosa, S. aureus [28, 29]
Piperine Piper nigrum Efflux pump inhibitor B. subtilis, E. coli, K. pneumoniae, MRSA [30, 31]
Berberine Berberis vulgaris Cell division inhibitor, E. coli, M. luteus, P. aeruginosa, MRSA [32, 33]
protein and DNA synthesis
inhibitor
Conessine Holarrhena floribunda Efflux pump inhibitor P. aeruginosa [34]
Tomatidine Solanaceae Inhibitor of macromolecule S. aureus, B. cereus, B. subtilis, [35]
biosyntheses L. monocytogenes
Chelerythrine Chelidonium majus, Toddalia Destruction of bacterial cell B. subtilis, E. coli, K. pneumonia, S. pyogene [36, 37]
asiatica wall and cell membrane,
inhibition of protein
biosynthesis
Sanguinarine Sanguinaria canadensis Antibiofilm K. pneumonia, P. aeruginosa, S. pyogenes, [38–40]
C. albicans, MRSA
Coumarin Aegelinol Ferulago campestris DNA gyrase inhibitor S. enterica serovar Typhi, E. aerogenes, [41]
E. cloacae, S. aureus
Agasyllin Ferulago campestris DNA gyrase inhibitor S. enterica serovar Typhi, E. aerogenes, [41]
E. cloacae, S. aureus
4′-Senecioil- Prangos hulusii DNA gyrase inhibitor B. subtilis [42]
oxyosthol
Osthole Cnidium monnieri, Angelica DNA gyrase inhibitor B. subtilis, S. aureus, K. pneumoniae, MSSA [43]
pubescens
Novobiocin Citrullus colocynthis DNA gyrase inhibitor A. baumannii [44]
Bergamottin Grapefruit Efflux pump inhibitor MSRA [45]
epoxide
Galbanic acid Ferula species Efflux pump inhibitor S. aureus, E. coli [46]
0005092146.INDD 158 06-03-2021 21:18:08

Terpene Farnesol Usnea longissima Antibiofilm S. aureus [47]

Nerolidol Momordica charantia Antibiofilm S. aureus [48]
Dehydro- Picea abies, Larix decidua Antibiofilm S. aureus, E. coli [49]
abietic acid
Carvone Lippia alba, Mentha spicata L. Antibiofilm S. aureus [50]
Thymol Thymus vulgaris Efflux pump inhibition, cell C. albicans, C. glabrata, C. krusei, A. niger, [51, 52]
membrane disturbance, A. fumigatus, A. flavus, A. ochraceus,
inhibits H(+)-ATPase in the A. alternata, B. cinerea, Cladosporium spp.,
cytoplasmic membrane, P. citrinum, P. chrysogenum, F. oxysporum,
antibiofilm Rhizopus oryzae, E. coli, E. aerogenes,
S. aureus, P. aeruginosa, S. typhimurium,
S. enteritidis, S. saintpaul
Carvacrol Anabasis setifera Cell membrane disturbance, A. niger, A. fumigatus, A. flavus, [53]
efflux pump inhibition, A. ochraceus, A. alternata, B. cinerea,
antibiofilm Cladosporium spp., P. citrinum,
P. chrysogenum, F. oxysporum, Rhizopus
oryzae, E. coli, E. aerogenes, S. aureus,
P. aeruginosa
Eugenol Eugenia aromaticum, Ocimum Cell membrane disturbance, A. niger, A. fumigatus, A. flavus, [54–56]
sanctum L. binding to quorum sensing A. ochraceus, A. alternata, B. cinerea,
receptors Cladosporium spp., P. citrinum,
oryzae, P. aeruginosa, MRSA, MSSA
Menthol Mentha arvensis L. Cell membrane disturbance A. niger, A. fumigatus, A. flavus, [54]
A. ochraceus, A. alternata, B. cinerea,
Cladosporium spp., P. citrinum,
oryzae
Cinnam- Cinnamo-mum zeylanicum Cell membrane disturbance, Streptococcus spp., S. aureus, S. epidermidis, [57, 58]
aldehyde antibiofilm Campylobacter spp.
0005092146.INDD 159 06-03-2021 21:18:08

demonstrated the flavonoids’ inhibitory activity toward different viruses such as poliovirus
type1, RSV, simplex virus type 1(HSV-1), and parainfluenza virus type 3 and reported that
hesperetin, quercetin, and catechin hinder the viral activity in diverse ways. Quercetin and
catechin reduce the infectivity of the virus while hesperetin reduces the intracellular repli-
cation of viruses [65]. The study proposed that small structural differences played a critical
role in their activity. Some flavonoids such as phloretin possess activity to counter a massive
variety of microorganisms, Galangin from Helichrysum aureonitens showed activity against
a broad range of gram-positive bacteria, fungi, and viruses. Catechins comprise a reduced
form of C3 unit in flavonoid compounds and have been widely explored because of its
presence in green tea [66]. It was observed that tea consists of a mixture of catechin and
epicatechin, possess antimicrobial activity. These compounds suppressed Streptococcus
mutants, Vibrio cholera, Shigella, and other microbial activity. In vitro study demonstrated
that catechins inhibited glucosyltransferases in S. mutans and inactivated cholera toxin in
V. cholera, in vivo; activity also confirmed the same [67]. Flavonoids, chalcones displayed
diverse biological activities due to multiple substitutions in its structure. It is investigated
against E. coli and S. aureus and found that their antimicrobial activity is due to the energy
variation between two molecular orbitals. There is a conflict for the possible mechanism of
action of flavonoids. Several reports found that flavonoids lack –OH groups on their B rings
and have more antimicrobial activity than those that occupy two –OH groups; this supports
the findings that mechanism of action of these flavonoids is a microbial membrane. Whereas
various authors found contradict results, mean more hydroxylation, higher the antimicro-
bial activity. Therefore, it requires further study to correlate between the degree of hydroxy-
lation and the effectiveness of the antimicrobial activity of flavonoid compounds.
8.3.1.2 Quinones
Quinones are a group of secondary metabolites, derived from aromatic rings having two
ketone substitutions and contain important antimicrobial properties. These compounds
cause brown color reaction in cut or injured vegetables and fruits and also an intermediate
between melanin synthesis pathways in humans. The exchange between hydroquinone and
quinine takes place by oxidation and reduction reactions. In many biological systems, the
significant role of quinone–hydroquinone pair is very crucial, like the role of coenzyme Q
(ubiquinone) in mammalian electron transport system. Additionally, they form stable free
radicals that complex irreversibly with nucleophilic amino acids of microbial proteins, which
leads to inactivation and loss of protein function. This is the reason for a broad spectrum of
the antimicrobial effect of quinone. It targets the microbial cell’s surface-exposed adhesions,
membrane-bound proteins, and cell wall peptides, causing the death of microorganisms. A
report illustrated anthraquinone from Cassia italic, having bacteriostatic property on Bacillus
anthracis, P. aeruginosa, and Corynebacterium pseudodiphthericum, and bactericidal prop-
erty against Pseudomonas pseudomalliei [68]. Another anthraquinone, hypericin from
Hypericum perforatum, has antidepressant and antimicrobial properties [69].
8.3.1.3 Tannins
Tannins are a group of polymeric phenolic compounds found in all parts of plants such as
wood, bark, roots, leaves, and fruits [70]. The molecular weight of tannin ranges from 500
to 3000. Tannins are classified into two groups: hydrolyzable tannin and condensed tannin.
8.3 Major Groups of Plants-Derived Antimicrobial Compoun 161
Hydrolyzable tannins are multiple esters with D-glucose based on gallic acid while the
condensed tannins are called proanthocyanidins, derivative of flavonols. Tannins possess
antimicrobial activity by inactivating microbial adhesions proteins, transporter proteins,
and enzymes. A group of Gallotannin-rich plant extracts inhibited the diverse microbial
activity through the inactivation of membrane-bound proteins. In plants, the cytoplasmic
leaflet of endoplasmic reticulum synthesizes anthocyanidin and accumulated in vacuoles.
Typically two groups of proanthocyanidins (A and B) are discovered on the basis of the type
of bonds formed between the oligomer forming anthocyanidin molecules. In A-type proan-
thocyanidins, two bonds are present between carbons of 2b-7 and 4b-8, while in B type only
one 4b-8 bond is present between oligomer-forming molecules.
The positive results of anthocyanin have been very well-known from sixteenth century,
where blackberry juice was applied to cure eye and mouth infections in humans. Though,
relatively few studies have demonstrated the antimicrobial action of these substances. The
study described the anthocyanin profile of different berries, containing stilbenoid resvera-
trol, possessing activity on gram-positive bacteria. Corilagin extracted from Terminalia che-
bula showed antimicrobial activity for Acinetobacter baumannii, S. aureus, E. coli, and
Candida albicans [71]. Similarly, tannic acid, punicalagin, geraniin, and castalagin demon-
strated inhibitory action against E. coli, S. aureus, and Vibrio [72]. In addition, punicalin,
gallic acid, ellagic acid, and punicalagins isolated from P. granatum showed antimicrobial
activity against methicillin-resistant S. aureus (MRSA), Mycobacterium intracellulare,
E. coli, Aspergillus fumigatus, P. aeruginosa, Cryptococcus neoformans, and C. albicans [73].
Tannic acid contains significant antimicrobial action and is additionally used as a safe food
additive in cocoa beans and tea. It indicated inhibitory action by binding to iron to chelate
and make it unavailable for several microorganisms such as Enterobacter cloacae,
Bacteroides fragilis, E. coli, and Clostridium perfringens [74]. Hydrolyzable tannins also
showed antiviral activity such as casuarinin extracted from Terminalia arjuna showed anti-
HSV-2 activity through the prevention of attachment and penetration [75]. Chebulagic acid
and punicalagin isolated from T. chebula showed action against anti-HSV-1, measles virus,
cytomegalovirus, dengue virus, hepatitis C virus, and respiratory syncytial virus [76]. Silver
nanoparticles intrinsically possess superior anti-microbial property, in this way; tannic acid
modified with silver nanoparticles had higher anti-HSV-2 activity [77].
8.3.1.4 Coumarins
Coumarins are heterocyclic phenolic compounds naturally found in many plants and
microorganisms [78]. Coumarins are found free or as heterosides in various plant families
such as Fabaceae, Apiaceae, Rosaceae, Asteraceae, Moraceae, Solanaceae, Rubiaceae, and
Rutaceae, as well as Gramineae and orchids, which contain enormous amounts of cou-
marins. It is mostly synthesized in leaves, but its concentration is higher in fruits followed
by roots and stems. These compounds exhibit a broad range of biological activities such as
anticancer, antimicrobial, antiviral, antioxidant, antidiabetic, antihypertensive, antipara-
sitic, and anti-inflammatory activities. All compounds exhibited activity against E. coli,
S. aureus, and Bacillus subtilis. There are several studies that demonstrated the antimicro-
bial activity of both natural coumarins and synthetic derivatives of coumarins. Coumarins
and pyranocoumarins isolated from Ferulago campestris roots naturally have antimicrobial
and antioxidant activities against both Gram-negative and positive bacteria [19]. Warfarin,
a well-known coumarin, was the first used compound as oral anticoagulant and rodenti-
cide equally retaining antiviral activity [79]. Scopoletin, a well-identified coumarin and
two chalcones were extracted from Fatoua pilosa having antitubercular activity [80].
El-Seedi discovered new aryl coumarin glucoside, asphodelin A 4′-O-β-d-glucoside as well
as asphodelin A from Asphodelus microcarpus [81]. A study investigated clorobiocin, novo-
biocin, and coumermycin A1 derived from distinct species of Streptomyces, containing
antibiotic activity [82]. This study displayed that noviosyl sugar moiety in the compound is
essential for its biological activity, in addition to the coumarin portion [83]. They also dem-
onstrated that coumarins act as strong inhibitors of DNA topoisomerase type II, called
DNA gyrase. Coumarins’ antimicrobial activity is due to a passive diffusion mechanism to
facilitate cellular penetration, particularly for Gram-positive bacteria. Additionally, Sardari
and coworkers [84] demonstrated that in coumarin nucleus, free 6-hydroxyl and 7-hydroxyl
have significant role in antifungal and antibacterial activity, respectively. The study sug-
gested that coumarins having methoxy group at C-7, in addition to –OH moiety at either
C-6 or C-8 invariably possessed antibacterial activity against a broad spectrum of bacteria.
While the presence of aromatic dimethoxy array generated compounds against microor-
ganisms that involve special growth factors including Streptococcus pneumonia, beta-
hemolytic Streptococcus, and Haemophilus influenza. Collinin, which was extracted from
Zanthoxylum schinifolium exhibited anti-HBV (hepatitis B virus) activity [85]. The antima-
larial activity was observed in daphnetin, isolated from the plants of the genus Daphne,
and also found in dentatin and clausarin, extracted from Clausena harmandiana [86].
8.3.2 Terpenes and Essential Oils

Terpenes are compounds called isoprenoids and their chemical structure is C10H16.
Terpenes are also present as diterpenes, triterpenes, tetraterpenes (C20, C30, and C40), as
well as hemiterpenes (C5) and sesquiterpenes (C15). Terpenes biosynthesis occurs mainly
via two pathways, namely the Mevalonic acid pathway, which synthesized sesquiterpenes,
sterols, and ubiquinones; and Methyl erythritol phosphate pathway, which primarily pro-
duces diterpenes, triterpenes, and hemiterpenes. Typical examples of terpenoids are cam-
phor (monoterpenes), artemisinin (sesquiterpenoids) methanol, and farnesol. Artemisinin
and its derivatives are equally known as qinghaosu, currently used as an antimalarial drug.
Nerolidol, sesquiterpene found in several plants and used as scent products and food fla-
voring agent, exhibited, antimicrobial, anthelmintic, and antimalarial activity [87–90]. It
was found that approximately 60% of essential oil derivatives observed to date are able to
inhibit fungal growth, while 30% essential oils are able to inhibit bacteria growth. The
mechanism of action of terpenes involves membrane disruption by the lipophilic com-
pounds [90]. Terpenes showed activity against bacteria, fungi, viruses, and protozoa as well
as a triterpenoid, betulinic acid able to prevent HIV infection [91]. Cichewicz and Thorpe
showed that capsaicin improved the growth of Candida albicans but also acts as bacteri-
cidal to Helicobacter pylori [92]. Diterpene, a framodial from Cameroonian spice, is broad-
spectrum antifungal. Guimarães and coworkers screened 33 terpenes and found that only
16 had antimicrobial activity [93]. Eugenol displayed antimicrobial activity against
Salmonella enteric, and terpineol exhibited significant bactericidal action against S. aureus
strains. Furthermore, carveol, geraniol, and citronellol showed antimicrobial activity
8.4 Mechanisms of Antimicrobial Activit 163
against E. coli [94]. This report also demonstrated that terpenes containing hydroxyl groups
increase the antimicrobial activity, whereas hydrocarbons decrease its activity. Citrus, ber-
gamot oil, and terpenes also showed bactericidal action against various bacterial strains [95].
8.3.3 Alkaloids
Alkaloids are naturally occurring organic compounds that predominantly contain nitrogen
atoms, possessing antimicrobial activities. Morphine, isolated and identified in 1805, was
the first medically used alkaloid from Papaver somniferum [96]. Diterpenoid alkaloids iso-
lated from Ranunculaceae plants consist of antimicrobial properties [97]. Berberine, an
important alkaloid, extracted from the root and stem of Berberis species widely used as
traditional medicine, has antibacterial, antiviral, antifungal, and antiprotozoal activity [98].
It is an excellent DNA intercalator and target on nucleic acid, RNA polymerase, topoi-
somerase IV, and gyrase actively, thus showing activity against several microorganisms.
Ungeremine is methanol extract isolated from Pancratium Illyricum L. bulbs, possesses
antimicrobial activities [99]. Solamargine, a glycoalkaloid, and other alkaloids isolated
from Solanum aculeastrum berries might be useful against cancer-related to AIDS [100].
Lupanine, 13α-hydroxylupanine, albine, angustifoline, and 13α-tigloyloxylupanine iso-
lated from the Lupinus genus displayed excellent activity towards P. aeruginosa and
Klebsiella pneumonia [101]. Many alkaloids were extracted from Chelidonium majus
(Papaveraceae) exhibit antimicrobial activity, such as chelerythrine, which was mainly
effective toward P. aeruginosa and C. albicans, while sanguinarine showed activity against
S. aureus [102]. Quinoline alkaloids, such as kokusagine, dictamnine, and maculine,
extracted from bark of Teclea afzelii displayed effective antimicrobial activity [103].
Reserpine, an indole alkaloid isolated from Rauwolfia serpentina, possesses potent
antibacterial activity [104]. Tomatidine, a steroidal alkaloid extracted from plants of
Solanaceae displayed efficient antibacterial activity against S. aureus [35]. Apart from this,
chanoclavine is classified as tricyclic ergot alkaloid, identified from Ipomoea muricata,
exerts synergistic effects upon coadministered with tetracycline against E. coli [105]. H. anti-
dysenterica bark contains steroidal alkaloid conessine displayed possible antimicrobial
activity [106].
8.4 Mechanisms of Antimicrobial Activity
The antibacterial property of a substance is primarily based on two mechanisms; first,

including chemical interference in the production or function of fundamental components
of bacteria; and second, preventing traditional antimicrobial resistance mechanisms. These
compounds provide multiple targets on bacterial cells that include (i) cell wall biosynthe-
sis, (ii) cell membrane destruction, (iii) protein biosynthesis, (iv) metabolic-pathway inhi-
bition, and (v) DNA replication and repair mechanism. In response to this, bacterial cells
also show various resistance mechanisms against antimicrobial agents. Therefore, it is
critical to find out the mechanism responsible for evolution of antibiotic resistance, pri-
marily involving the activation of efflux pump, modifying, and destruction of antibacterial
reagents with the help of enzymes and alteration of the target structures. It is equally
significant that one sort of mechanism behind the resistance to antibacterial agents may
function alone or together with other mechanisms. So, in the next sections, different mode
of action of plants-derived products will be discussed.
8.4.1 Plant Extracts with Efflux Pump Inhibitory Activity

Bacterial multidrug resistance by the expression of the efflux pump represents a crucial
problem in clinical trials. Therefore, an effective approach should be required to identify the
molecules or compounds interfering with the bacterial efflux process. It is sufficiently
known that plant-derived compounds have antimicrobial activity against Gram-positive
bacteria by inhibiting the efflux pump while Gram-negative bacteria have innate multidrug
resistance to many antimicrobial compounds due to efflux pumps. Several studies described
a number of bacterial efflux pump inhibitors from different plants. Plant alkaloids such as
berberine, reserpine, isoflavones, and methoxylated flavones have putative efflux inhibitory
activity. Similarly, gallotannin extracted from Terminalia chebula demonstrated antimicro-
bial activity for E. coli and MDR. Additionally, an essential oil extract from the Corsican
plant, Helichrysum italicum, consists of geraniol, demonstrated activity against different
Gram-negative bacteria with chloramphenicol. Also, falcarindiol, isolated from Levisticum
officinale, displayed synergistic activity with ciprofloxacin against Gram-negative bacteria.
An efflux pump inhibitor, phenylalanine arginine b naphthylamide (PAbN) enhances
Dichrostachys glomerata extract activity against Klebsiella pneumonia, E. coli, and Providencia
stuartii. Acer saccharum extract showed efflux pump inhibitory potentials against P. aerugi-
nosa, E. coli, and P. mirabilis [107]. Ursolic acid isolated from Eucalyptus tereticornis acts as
a putative efflux pump inhibitor against MDR E. coli (KG4) [108]. Similarly, berberine and
palmatine extracted from roots and rhizomes of Berberis vulgaris showed efficient efflux
pump inhibitory efficacies against P. aeruginosa [109]. Most of the studies focused on iden-
tifying the efflux pump inhibitors against Gram-positive bacteria with very few reports on
Gram-negative strains because Gram-negative bacteria are difficult to target due to the pres-
ence of more powerful efflux pumps and other efficient membrane barriers [110]. A study
displayed the inhibition of EmrD3 pump-mediated drug efflux of Vibrio cholera by allyl
sulfide, a bioactive compound present in garlic extract [111]. Considerably, more investiga-
tion is required to target Gram-negative bacteria by exploring different plant-derived
compounds or molecules to act as potent efflux pump inhibitors.
8.4.2 Plant Extracts with Bacterial Quorum Sensing Inhibitory Activity

Bacterial populations collaborate and communicate through diffusible signal molecules
and detection of these signal molecules known as quorum sensing (QS). The quorum sens-
ing regulates the expression of specific bacterial genes, which modify the local host envi-
ronment favoring the invasion of a pathogen. Therefore, recognizing the fact that numerous
bacteria utilize QS to regulate their virulence, the QS system becomes a target for antimi-
crobial activity. The perfect QS inhibitor (QSI) could have low molecular weight, able to
decrease the expression of QS-controlled genes, and have high specificity toward the target
QS molecule. Several studies described the effect of plant-derived compounds on QS and
used in clinical approaches. For example, products extracted from marine red algae Delisea
pulchra, halogenated furanones were the first identified anti-QS compounds. In addition,
8.5 Conclusions and Future Prospect 165
plant extracts from garlic, Capsicum chinensis, carrot, yellow pepper, propolis inhibit P. aer-
uginosa QS [112]. This study also discussed that garlic extract contains three separate inhib-
itors of QS and reduced pathogenicity of Caenorhabditis elegans and P. aeruginosa.
Phytoalexin resveratrol (3,5,4′-trihydroxystilbene) has direct antimicrobial activity against
Neisseria meningitides and Neisseria gonorrhoeae [113]. The extracts isolated from Citrus
sinensis, Elettaria cardamomum, Laurus nobilis, Coriandrum sativum, and Allium cepa
exhibited inhibition of QS, so suppressing the virulence of P. aeruginosa [114]. Further,
polyphenol of Rosa rugosa tea extract inhibited the QS regulated violacein production in
Chromobacterium violaceum [114]. Currently, it was also observed that Apium graveolens
oleoresin has an anti-QS activity against Chromobacterium violaceum CV12472 and
Pseudomonas aeruginosa PAO1 [115]. Tannins isolated from Terminalia bellirica,
Phyllanthus emblica, S. cumini, Mangifera indica, Punica granatum, and Terminalia cheb-
ula exhibited broad-spectrum anti-QS activity [116].
8.4.3 Plant Extracts with Biofilm Inhibitory Activity

Microbial biofilms are cell arrangement, attached to the substrate and enclosed by self-
produced extra polymeric substance (EPS) matrix. Biofilm base infection causes various
diseases in human being and its eradication with typical antibiotics is extremely difficult.
For instance, Staphylococci can adhere and form biofilm on both abiotic surfaces and
eukaryotic cells and perform a significant role in virulence hence difficult to treat.
Compounds isolated from Macleaya cordata, Aesculus hippocastanum, Krameria lappacea,
and Chelidonium majus show clinically significant inhibitory action on Staphylococci.
Proanthocyanidin isolated from A. hippocastanum and chelerythrine extracted from
Macleaya cordata downregulate the different proteins involved in various pathways and
inhibit biofilm formation. The sanguinarine and chelerythrine act on bacterial proteins
involved in synthesis of heat shock response including surface-exposed lipids and meth-
oxymycolic acid. They also act as bacterial cytoskeletal protein inhibitor; therefore, they
function as a beneficial compound for the development of novel drugs [117]. Tannin,
hamamelitannin, isolated from Hamamelis virginiana, appreciably reduce biofilm meta-
bolic activity of diverse microbes. Carvacrol, a natural biocide, inhibits the biofilm forma-
tion of S. aureus and Salmonella enteric. Collectively with thymol, interaction with lipid
bilayer membranes causes loss of integrity of bacterial cells by affecting its structural and
functional properties. Similarly, A 1-deoxynoijirimycin isolated from Morus alba inhibited
biofilm formation activity of Streptococcus mutans, which lowers bacterial extracellular
polysaccharide secretion. Propionibacterium acnes, with the ability to form biofilm and
cause acne vulgaris, is susceptible to compounds such as salidroside, icariin, and resvera-
trol extracted from plants. Extract isolated from Piper betle, 4-chromanol acts as an effective
antibacterial and antibiofilm agent against oral pathogens [118].
8.5 Conclusions and Future Prospects
In the last few years, researchers explored the importance and effectiveness of plant-derived
compounds as antimicrobial agents. Plants are a rich source of a broad range of secondary
metabolites, such as alkaloids, flavonoids, phenols; tannins, etc., thus have great potential to
be a source of novel antimicrobial agents. To date, thousands of phytochemicals have shown

inhibitory activity against different types of microorganisms. Various compounds showed
both antibacterial and antibiotic modifying resistance activity. While some are not effective
as antibiotics alone, however, when co-administrated with antibiotics, they significantly
inhibited the antibiotic resistance mechanism in bacteria. Hence, it is essential to investigate
the exact molecular mechanism and synergetic effects of these compounds with antibiotics.
Furthermore, it is also important to know antimicrobial plant mechanisms for developing a
novel clinical approach and also to understand the safety of antimicrobial phytochemicals.
The spread of drug-resistant microbes withstands a massive challenge to the successful
treatment of microbial diseases. Therefore, there is an urgent need to discover novel com-
pounds characterized by varied chemical structures and mechanisms of action. The uses of
several plant-derived compounds as antimicrobial and antifungal agents represent an unu-
sual approach for discovering bioactive products that could become helpful as therapeutic
tools. Therefore, continued researches should be carried out to improve the understanding
of exact mechanisms and also explore the pharmacodynamic and pharmacokinetic proper-
ties of the molecules.
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175
Functional Plants as Natural Sources of Dietary

Antioxidants
Ao Shang1, Jia-Hui Li2, Xiao-Yu Xu1, Ren-You Gan3, Min Luo1,
and Hua-Bin Li1
1
Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Nutrition, School of Public Health,
Sun Yat-Sen University, Guangzhou, China
2
School of Science, The Hong Kong University of Science and Technology, Hong Kong, China
3
Research Center for Plants and Human Health, Institute of Urban Agriculture, Chinese Academy of Agricultural Sciences,
Chengdu, China
9.1 Introduction
Reactive oxygen species (ROS), or oxygen-free radicals, are produced in cell metabolism.
However, excessive production of ROS can lead to oxidative stress, which can cause the
destruction of normal functions of cellular lipids, proteins, and DNA [1]. Numerous stud-
ies have demonstrated that oxidative stress is associated with aging and the development of
a variety of diseases in human, such as neurodegenerative diseases, cardiovascular dis-
eases, cancers, liver diseases [2–6]. Antioxidants are substances that can prevent the gen-
eration of free radicals or capture and neutralize free radicals. Hence, using antioxidants is
considered as a strategy of eliminating harmful effects of oxidative stress on the body [7].
On the other hand, antioxidants contribute to the food preservation and shelf life extension
as the food additive. For instance, antioxidants can delay or reduce the oil oxidation during
food processing and preservation, which can prevent food from browning or loss of nutri-
ents due to oxidation [8, 9].
Plants are rich in polyphenols, polysaccharides, saponins, lycopene, carotenoids and
glucosinolates, and other bioactive compounds, and plants are also regarded as an impor-
tant source of dietary antioxidants [10–13]. According to the researches, many kinds of
vegetables, fruits, medicinal plants, cereals, flowers, microalgae, and teas have antioxidant
activity, which can be excellent participants of natural antioxidants [14–16].

176 9 Functional Plants as Natural Sources of Dietary Antioxidants
9.2 Evaluation of the Antioxidant Activity
The antioxidant activity needs to be evaluated by accurate scientific methods. Currently,

according to the mechanisms, the evaluation methods of in vitro antioxidant activity can
be divided into the free radicals scavenging and inhibiting capacity, reducing capacity,
metal ion-chelating capacity, and inhibiting oxidase capacity. The common methods of
detecting the antioxidant activity are 2,2′-azinobis-3-ethylbenzotiazoline-6-sulfonic acid
(ABTS▪+) free radical scavenging/Trolox equivalent antioxidant capacity (TEAC) assay and
ferric ion reducing antioxidant power (FRAP) assay.
In ABTS free radical scavenging method, ABTS▪+ free radicals are oxidized to blue-green
ABTS▪+ under the action of oxidants, and the presence of antioxidants can inhibit the gen-
eration of ABTS▪+, resulting in a decolorization. This reaction can be observed as the
change of absorbance at 743 nm, because 734 nm is the wavelength having the maximum
absorbance of the mixture [17].
FRAP assay is often used to measure the total antioxidant capacity, and it evaluates the
ferric ion reducing capacity of the antioxidants. At a low pH, the ferric iron (Fe(III)) and
tripyridyltriazine (TPTZ) form a complex, and it appears blue when it is reduced to a com-
plex of ferrous iron (Fe(II))-TPTZ, which has a maximum absorption at 593 nm [18].
In addition, phenols are compounds widely found in plants with potential health-
promoting effects, which are often found in the peels, roots, leaves, and fruits of plants.
The phenolic compounds in plants are known as natural antioxidants with free radical
scavengers’ capacity [19]. Therefore, the total phenolic content (TPC) can reflect the anti-
oxidant activity of plants to some extent, which is usually detected by Folin–Ciocalteu
method. The principle of this method is that the reaction of Folin–Ciocalteu reagent with
phenolic compounds exhibits blue color, and the depth of blue color is proportional to the
number of phenolic groups. The mixture has the maximum absorption at 760 nm [20, 21].
9.3 Antioxidant Activity of Functional Plants
9.3.1 Vegetables
Vegetables are one of the essential foods in the daily diet, which can provide a variety of
nutrients necessary for the human body, and are also rich in antioxidants. According to the
report of Deng et al., the antioxidant activity of 56 commonly consumed vegetables is
assessed by FRAP and TEAC assays, and the TPC of these vegetables is also measured [22].
The FRAP values of hydrophilic fraction, lipophilic fraction, and whole of these vegetables
respectively are in the range of 0.08–21.6, 2.44–39.30, 2.69–60.9 μmol Fe(II)/g, while the
total TEAC values of hydrophilic fraction, lipophilic fraction, and total are in the range of
0.05–19.01, 5.57–18.14, 6.93–33.63 μmol Trolox/g, respectively. The TPC of hydrophilic
fraction, lipophilic fraction, and whole of these vegetables are measured by Folin–Ciocalteu
method, and the values are in the range of 0.02–8.16, 4.83–15.11, and 4.99–23.27 mg gallic
acid equivalents (GAE)/g, respectively. The study demonstrates that the antioxidant capac-
ity of different vegetables varied greatly, among which the antioxidant effects of Chinese
toon bud, loosestrife, perilla leaf, cowpea, caraway, lotus root, sweet potato leaf, soy bean
9.3 Antioxidant Activity of Functional Plant 177
(green), pepper leaf, ginseng leaf, chives, and broccoli are the highest, while marrow squash
and eggplant (purple) have quite low values (Table 9.1). At the same time, there is a positive
correlation between antioxidant activity and TPC, suggesting that phenolic compounds
may be the main contributor to the antioxidant activity. The compounds such as chloro-
genic acid, gallic acid, and galangin are identified as prevalent in these vegetables.
In addition, the contents of antioxidants in vegetables are influenced by storage and pro-
cessing. For example, the phenolic compounds are greatly affected by the environmental
conditions around the harvest. The proper storage conditions are helpful to increase the
phenolic content, while vitamin C and phenols are more likely to lose during
processing [23].
9.3.2 Fruits
Fruits contain abundant nutrients that are beneficial for human health. The antioxidant
activity of 62 fruits is evaluated by FRAP and TEAC assays, and there are obvious differ-
ences in the antioxidant capacity of different fruits [24]. The FRAP values of all 62 fruits
range from 0.11 to 72.11 μmol Fe(II)/g, with an average of 8.76 μmol Fe(II)/g. The TEAC
values range from 0.84 to 80.68 μmol Trolox/g, with an average of 6.01 μmol Trolox/g. The
TPC values range from 0.12 to 5.85 mg GAE/g. The fruits with the highest antioxidant activ-
ity and TPC are Chinese date, pomegranate, guava, sweetsop, persimmon, Chinese wampee,
and plum. On the other hand, grape is also a commonly consumed fruit, and the antioxi-
dant property of 30 different grape varieties is measured by FRAP, TEAC assays, and their
TPC and total flavonoid contents (TFC) are also detected [25]. Some grape varieties includ-
ing Pearl Black Grape, Summer Black Grape, Pearl Green Grape, Seedless Green Grape,
and Seedless Red Grape possess the most significant antioxidant activity and highest
TPC. The antioxidants in those grapes are identified as caffeic acid, catechin gallate, epicat-
echin, gallic acid, protocatechuic acid, and rutin.
Another study paid attention to the antioxidant activity of 56 wild fruits from South
China [26]. Wild fruits have strong capacities of scavenging free radicals and reducing oxi-
dants, and there is a certain correlation between the FRAP value and TEAC values. The
FRAP, TEAC, and TPC values vary in the range of 1.28–502 μmol Fe(II)/g, 3.38–1140 μmol
Trolox/g, and 0.49–54.8 mg GAE/g, respectively. The wild fruits with the strongest antioxi-
dant activity and the highest TPC rank as Eucalyptus Robusta, Euryanitida, Melastoma san-
guineum, Melaleuca leucadendron, Lagerstroemia indica, Caryota mitis, Lagerstroemia
speciosa, and Gordonia axillaris. As a result, wild fruits appear to have higher antioxidant
capacity than common fruits, suggesting that they have the potential to be a source of natu-
ral antioxidants.
Moreover, some fruits wastes such as peels and seeds also show strong antioxidant activ-
ity, suggesting that the fruits without edible parts are also valuable [27]. The range of FRAP,
TEAC, and TPC values of 50 fruit peel extracts are 0.74–155.73 μmol Fe (II)/g, 1.72–93.10 μmol
Trolox/g, and 0.38–22.95 mg GAE/g, respectively. The antioxidant capacities and TPC of
the fruit seeds are approximate to those of peels, and the ranges are 0.34–181.39 μmol
Fe(II)/g, 2.45–92.62 μmol Trolox/g, and 0.30–22.95 mg GAE/g, respectively. Among them,
the antioxidant property of grape residues is higher, and the antioxidant activity and TPC
of 30 grape peels and seeds are also evaluated in another study [28].
Table 9.1 The FRAP, TEAC, and TPC values of several plants.
Antioxidant activity
Total phenolic
FRAP value TEAC value (μmol content
Plant Scientific name (μmol Fe (II)/g) Trolox/g) (mg GAE/g) References
Vegetable
Chinese toon bud Toona sinensis 60.90 ± 4.54 33.63 ± 1.59 23.27 ± 1.59 [22]
Cowpea Vigna unguiculata (Linn.) Walp 17.01 ± 0.74 21.01 ± 1.11 8.28 ± 0.14
Ginseng leaf Panax ginseng C. A. Mey. 16.80 ± 1.41 15.35 ± 0.44 11.78 ± 0.44
Loosestrife Lysimachia mauritiana Lam. 25.22 ± 0.70 25.44 ± 1.30 13.13 ± 0.70
Perilla leaf Perilla frutescens 44.50 ± 3.15 23.94 ± 0.48 14.37 ± 0.48
Fruit
Chinese date Ziziphus jujuba Mill. 72.11 ± 2.19 8.33 ± 0.12 5.85 ± 0.19 [24]
Guava Psidium guava 23.80 ± 1.44 15.18 ± 0.81 1.94.11 ± 0.07
Pearl Black Grape Vitis vinifera L. 11.77 4.84 1.40
Persimmon Diospyros kaki L. 16.97 ± 0.26 9.38 ± 0.25 1.12 ± 0.05
Pomegranate Punica granatum L. 25.57 ± 0.53 40.61 ± 0.11 1.47
Wild fruit [26]
Baiqianceng Melaleuca leucadendron 214 ± 8.86 461 ± 6.88 25.6 ± 1.59
Maoren Melastoma sanguineum 288 ± 10.4 404 ± 21.5 23.3 ± 0.49
Swamp mahogany Eucalyptus robusta 502 ± 6.41 1140 ± 7.93 54.8 ± 3.05
Xichiyeling Eurya nitida 428 ± 12.0 478 ± 14.1 35.0 ± 0.96
Ziwei Lagerstroemia indica 254 ± 5.75 109 ± 5.13 12.1 ± 0.30
Fruit peel [25]
Blueberry Vaccinium corymbosum L. 104.55 ± 0.77 59.10 ± 3.85 8.92 ± 0.24
0005092147.INDD 178 6/2/2021 10:21:58 AM

Chinese olive Canarium album L. 96.17 ± 6.69 77.12 ± 2.43 13.16 ± 0.32
Hawthorn Crataegus pinnatifida 89.57 ± 6.54 66.76 ± 2.77 12.66 ± 1.09
Starfruit Averrhoa carambola 84.52 ± 3.19 70.15 ± 4.91 10.45 ± 0.97
Sweetsop Annona squamosa L. 155.73 ± 8.46 84.14 ± 2.91 17.77 ± 0.16
Fruit seed [25]
Avocado Persea americana Mill. 45.79 ± 3.01 42.63 ± 1.94 8.39 ± 0.27
Grape (USA) Vitis vinifera L. 181.39 ± 6.93 92.62 ± 0.37 22.95 ± 1.01
Longan Dimocarpus longan Lour. 86.39 ± 2.03 75.33 ± 2.76 2.37 ± 0.32
Mango Mangifera indica L. 31.17 ± 3.62 50.55 ± 2.52 7.54 ± 0.24
Jujube Ziziphus jujube 45.53 ± 1.21 56.03 ± 2.04 9.00 ± 0.94
Medicinal plants [29]
Chinese peony Paeonia lactiflora Pall. (red) 481.36 ± 7.42 365.72 ± 5.08 31.48 ± 1.52
Coltsfoot Tussilago farfara L. 529.48 ± 10.40 225.61 ± 1.25 34.50 ± 0.52
Danshen Salvia miltiorrhiza Bge. 601.37 ± 27.50 1544.38 98.88
Diyu Sanguisorba officinalis L. 1844.85 20.84 9.37 ± 0.22
Hongjingtian Rhodiola sacra Fu 541.22 ± 3.61 471.53 ± 4.63 51.06 ± 1.75
Peach Prunus persica (Linn) Batsch. 5.07 ± 0.68 793.13 ± 35.89 55.23 ± 0.61
Qinpi Fraxinus rhynchophylla Hance. 548.25 ± 3.68 401.35 ± 9.28 52.31 ± 3.26
Rhizoma cimicifugae Cimicifuga foetida L. 707.02 ± 23.19 349.48 ± 3.12 24.97 ± 0.09
Tuber fleeceflower Polygonum multiflorum Thunb. 506.83 ± 2.35 538.75 ± 5.39 45.24 ± 1.32
root (Stem)
Xueteng Sargentodoxa cuneata Rehd. et 521.62 ± 7.53 458.91 ± 8.74 65.28 ± 2.93
Wils.
Cereal grains
Black rice Oryza sativa L. indica 126.19 ± 2.91 30.03 ± 1.10 9.47 ± 0.48 [30]
Buckwheat Fagopyrum esculentum Moench 31.20 ± 0.93 9.43 ± 0.35 4.48 ± 0.46
(Continued )
0005092147.INDD 179 6/2/2021 10:21:58 AM

Total phenolic
FRAP value TEAC value (μmol content
Plant Scientific name (μmol Fe (II)/g) Trolox/g) (mg GAE/g) References
Corn Zea mays L. 11.66 ± 0.40 4.52 ± 0.10 1.97 ± 0.06

Millet Setaria italica (L.) Beauv. var. 11.29 ± 1.19 1.88 ± 0.11 2.05 ± 0.13
germanica (Mill.) Schred.
Oat Avena sativa Linn. 16.15 ± 1.06 1.57 ± 0.20 2.83 ± 0.16
Flower
Chinese rose Rosa hybrida 629.64 ± 24.61 175.39 ± 1.74 35.84 ± 1.67 [31]
Geranium Pelargonium hortorum 212.84 ± 3.94 132.18 ± 5.46 25.68 ± 1.02
Qinyeshanhu Jatropha integerrima 219.95 ± 10.08 115.01 ± 4.20 17.22 ± 0.77
Statice Limonium sinuatum 500.04 ± 70.78 157.42 ± 2.68 34.17 ± 1.17
Sweet osmanthus Osmanthus fragrans 163.57 ± 15.77 71.98 ± 3.13 16.00 ± 0.57
Microalgae
Synechococcus sp. FACHB 283 29.56 ± 1.24 10.56 ± 0.11 [32]
Nostoc ellipsosporum 21.09 ± 1.83 60.35 ± 2.27
CCAP 1453/17
Chlamydomonas nivalis 24.13 ± 0.47 15.07 ± 0.26
Chlorella pyrenoidosa #1 17.32 ± 0.66 10.46 ± 0.20
Chlorella protothecoides #7 9.22 ± 0.35 19.03 ± 0.14
Tea
Black teas Camellia sinensis (L.) O. Ktze. 1141.58 ± 13.92 724.28 ± 12.63 107.54 ± 1.80 [33]
Dark teas 1124.96 ± 23.87 589.43 ± 8.35 78.16 ± 1.33
Green teas 3621.75 ± 81.44 1964.50 ± 38.29 195.79 ± 5.45
Oolong teas 2013.37 ± 26.17 1460.46 ± 22.23 172.11 ± 2.09
Yellow teas 3182.34 ± 31.31 2087.81 ± 42.89 198.44 ± 5.39
0005092147.INDD 180 6/2/2021 10:21:59 AM

9.3 Antioxidant Activity of Functional Plant 181
9.3.3 Medicinal Plants

Antioxidants in food not only exist in fruits and vegetables, but also in medicinal plants.
The antioxidant capacity of infusions from 223 medicinal plants in China are demonstrated
by TEAC and FRAP assays, and the TPC values are tested by Folin–Ciocalteu method [29].
The range of FRAP values is 0.14–1844.85 μmol Fe(II)/g DW, the range of TEAC values is
0.99–1544.38 μmol Trolox/g DW, while the range of TPC values is 0.19–101.33 mg GAE/g
DW. Ten medicinal plants infusions with high antioxidant activity and TPC are Salvia milti-
orrhiza Bge., Polygonum multiflorum Thunb. (Stem), Rhodiola sacra Fu, Sargentodoxa
cuneata Rehd. et Wils., Fraxinus rhynchophylla Hance, Prunus persica (Linn) Batsch.,
Cimicifuga foetida L., Paeonia lactiflora Pall. (red), Tussilago farfara L., and Sanguisorba
officinalis L. Considering their high antioxidant activity and low toxicity, some of medici-
nal plants are expected to be developed as natural antioxidant products.
9.3.4 Cereal Grains

Cereal grains are an essential component of daily diet, and their antioxidant activities is
also a concern. A finding shows that the antioxidant activity and the TPC of 24 hydrophilic
and lipophilic fractions of cereal grains are determined [30]. The total FRAP values vary
from 5.23 to 126.19 μmol Fe(II)/g, the total TEAC values vary from 0.62 to 30.03 μmol
Trolox/g, and the TPC values vary from 1.35 to 9.47 mg GAE/g. The first five cereals with
the highest antioxidant activity and TPC value are black rice, organic black rice, red rice,
purple rice, and buckwheat. The results show that the antioxidant activity of pigmented
rice is stronger than that of normal white rice, indicating that the pigmented rice may have
better health benefits. Besides, gallic acid, kaempferol, quercetin, galangin, and cyanidin-
3-glucoside are the main phenolic compounds in those cereal grains.
9.3.5 Flowers
Flowers are not only ornamental plants, but also a special kind of food. The history of adding
flowers to the diet in Europe can be traced back to the sixteenth century, while some flowers
can also be used as traditional Chinese medicine in China. The antioxidant capacities of 51
edible and wild flowers are evaluated, and the results show that the FRAP values ranged from
0.17 to 629.64 μmol Fe(II)/g, the TEAC values ranged from 0.23 to 175.39 μmol Trolox/g [31].
The TPC values of water-soluble and fat-soluble fractions range from 0.63 to 35.84 mg GAE/g.
Combining the free radical scavenging capacity and ferric ion reducing capacity, it is con-
cluded that the edible and wild flowers with strongest antioxidant capacity and highest TPC
values are Rosa hybrid, Limonium sinuatum, Pelargonium hortorum, Jatropha integerrima,
and Osmanthus fragrans. In addition, the antioxidant capacity is highly correlated with TPC,
suggesting that phenols such as homogentisic acid, cyanidin-3-glucoside, protocatechuic
acid, catechin, gallic acid, and epicatechin may be the main antioxidants in these flowers.
9.3.6 Microalgae
Microalgae are a kind of autotrophic plants with abundant nutrition and high photosyn-
thetic utilization, which are widely distributed in river, lake, and sea. Li et al. measure the
antioxidant activity of 23 kinds of microalgae by TEAC essay and determine the TPC values
by Folin–Ciocalteu method [32]. The different species of microalgae have quite different
radical scavenging capacity, and the TEAC values vary from 1.33 to 29.56 μmol Trolox/g.
Several microalgae such as Synechococcus sp. FACHB 283, Chlamydomonas nivalis, and
Nostoc ellipsosporum CCAP 1453/17 are found to have the strongest antioxidant capacity.
The TPC values of these 23 microalgae range from 3.59 to 60.35 mg GAE/g. However, the
correlation between the antioxidant capacity and the TPC of microalgae is very low, so it
can be inferred that phenolic compounds are not the main antioxidants in microalgae,
while carotenoids, polyunsaturated fatty acids, and polysaccharides might be the main
antioxidants in microalgae.
9.3.7 Teas
Tea is a popular natural beverage with pleasant taste and health benefits. In a study, the
antioxidant activity and TPC values of the fat-soluble, water-soluble, and bound-insoluble
fractions of 30 teas from six categories (green, black, oolong, white, yellow, and dark teas)
were examined [33]. The range of total FRAP values is 611.18–5375.18 μmol Fe (II)/g DW,
the range of total TEAC values is 326.32 ± 0.48 to 3004.40 ± 112.89 μmol Trolox/g DW, and
the range of TPC values is 37.25 ± 0.16 to 254.29 ± 15.51 mg GAE/g DW. Among them, sev-
eral kinds of teas exhibit the highest antioxidant capacity, including Dianqing Tea, Lushan
Yunwu Tea, and Xihu Longjing Tea. In addition, the antioxidant activity of green tea, yellow
tea, oolong tea is higher than those of black tea, dark tea, and white tea. Green tea has a
stronger antioxidant activity than other types of teas, and it also contains more phenolic
compounds. Moreover, several phenolic compounds are isolated and identified from 30 teas,
including catechins (epicatechin, epigallocatechin, epicatechingallate, and epigallocatech-
ingallate), gallic acid, chlorogenic acid, ellagic acid, and kaempferol-3-O-glucoside.
Furthermore, the antioxidant activity and TPC values of the infusions of these 30 teas are
measured as well [34]. The antioxidant activities of green tea, yellow tea, oolong tea, black
tea, dark tea, and white tea decreased successively, and several phenolic compounds are
found in the infusions, including eight catechins, three phenolic acids, two flavonols, two
flavonol glycosides, and theaflavin. Overall, teas may be a promising source of natural anti-
oxidants, with phenolic compounds as major contributors.
9.4 Applications of Plant Antioxidants
9.4.1 Food Additives

Most natural foods and some processed foods tend to become unpalatable, browning, and/
or rotten during preservation, especially the oxidation of oils and fats. Oxidative rancidity
is an important factor in food deterioration, mainly due to the formation of potentially
toxic secondary compounds [35, 36]. Some plant antioxidants, such as phenolic com-
pounds, are excellent participants in food antioxidant additives because they are relatively
safe and efficient. For example, betanin is approved for use as a natural red colorant in food
and pharmaceutical production. It also has the capacity of scavenging peroxy-radicals in
pork, which can be used to maintain food quality [37]. The extracts of grape and olive pom-
aces can effectively replace the synthetic sodium ascorbate in raw lamb products [38].
9.5 Conclusion 183
Antioxidant dietary fiber from red grape pomace is rich in polyphenols, which can be
added to cooked chicken hamburgers to markedly improve its oxidative stability. Also, the
antioxidants in white grape pomace retard lipid oxidation in minced fish muscle [39].
Moreover, maclurin, a phenolic compound existing in white mulberry, is able to inactivate
anti-polyphenol oxidase, inhibit the enzymatic browning of in potato supernatant, and
improve its antioxidant capacity [40]. In addition, many other plants and their active ingre-
dients have the potential to be applied as antioxidant additives in foods, such as Carex dis-
tachya roots, eucalyptus leaf, pomelo peels, oregano essential oil, dihydromyricetin,
Andean berrypolyphenols, and olive biophenols [41–46]. Natural plant antioxidants are
effective in preventing food from rapid deterioration, while being relatively safe for humans
and environmentally friendly. Hence, the substitution of synthetic antioxidants with natu-
ral plant antioxidants is a promising direction for the research and development of food
additives.
9.4.2 Dietary Supplements

Oxidative stress is associated with the pathogenesis of many diseases, such as neurodegen-
erative diseases, cardiovascular diseases, cancers, liver diseases, and several metabolic dis-
eases [47]. Some plants and active compounds inhibit the progression of these diseases by
alleviating oxidative stress. Several antioxidants found in fruits, vegetables, legumes, and
medicinal plants, including flavone, apigenin, luteolin, kaempferol, piceatannol, esculetin,
vanillin, and ellagic acid, may contribute to the prevention and treatment of neurodegen-
erative diseases such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s dis-
ease [48]. According to studies, natural plant antioxidant polyphenols have a protective
effect on cardiovascular diseases, including quercetin, stilbenoids, blueberry phenolic acid,
phlorizin, anthocyanins, etc. [49–53]. In addition, some plant-derived dietary antioxidants,
including tea polyphenols, curcumin, genistein, resveratrol, lycopene, pomegranate, and
lupeol, can help prevent and manage several cancers without significant side effects [54].
Some antioxidant flavonoids, alkaloids and terpenoids in plants have been shown to pre-
vent alcoholic fatty liver disease, including anthocyanins, catechins, isorhamnetin, nariru-
tin, hesperidin, chlorogenic acid, berberine, curcumin, and lignans [55]. Moreover, plant
antioxidants are able to prevent and improve diabetes, obesity, and gout as well [56‑58].
The superior preventive and therapeutic effects of natural plant antioxidants on diseases
are worthy of further study. It will also be the development trend of modern medicine and
health care industry to search for antioxidant substances from plants.
9.5 Conclusions
The moderate amount of antioxidants is beneficial for human health, and it could be used
in the prevention and treatment of some diseases. The antioxidants are also important food
additives, which can prevent or delay the oxidation and spoilage of food ingredients, and
guarantee the quality and flavor of food. Plants are the treasure trove of natural antioxi-
dants, including vegetables, fruits, medicinal plants, cereals, flowers, microalgae, and teas.
According to the studies, the antioxidant capacity of teas is relatively high. However,
considering the limited consumption of tea, it is recommended to have a balanced diet to

ensure a reasonable intake of antioxidants. In addition to daily consumption, antioxidants
from plants can also be developed into the dietary supplements and food additives. The
antioxidants in the nonedible parts of plants, such as peels and seeds, also have potential to
be exploited. Furthermore, given the high antioxidant activity of wild fruits and wild flow-
ers, some unusual wild plants are also considered as an important source of natural anti-
oxidants worth developing.
It is reported that phenolic compounds are the main contributors to the antioxidant
activity of most plants. At present, several common phenolic compounds are isolated and
identified, such as gallic acid, kaempferol, and quercetin, etc. Meanwhile, more other anti-
oxidant compounds such as polysaccharides and carotenoids need further investigation. It
should be noted that the contents of antioxidants in plants can be changed due to the envi-
ronmental factors such as harvest, storage, and transportation conditions. With these issues
solved, the application of plant-derived antioxidants has a promising prospect on the devel-
opment of food additives to delay food oxidation and promote the production of functional
food, which can prevent oxidative-stress-related diseases.
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189
10
Biodiversity and Importance of Plant

Bioprospecting in Cosmetics
K. Sri Manjari1, Debarati Chakraborty2, Aakanksha Kumar3, and
Sakshi Singh4
1
University College for Women, Osmania University, Hyderabad, Telangana, India
2
Department of Molecular Biology and Biotechnology, University of Kalyani, Kalyani, West Bengal, India
3
Bioclues, Hyderabad, Telangana, India
4
Department of Molecular Biology and Human Genetics, Banaras Hindu University, Varanasi, Uttar Pradesh, India
10.1 Biodiversity, Bioprospecting,
and Cosmetics – A Harmony of Triad
Biodiversity, a natural repository of all living things on earth encompassing microbes to

humans has three levels – species, habitat, and gene. Biodiversity serves as the natural cur-
rency that contribute immensely toward the development of human civilization. Therefore,
it is extremely important that biodiversity be used in a sustainable manner [1]. India is a
land with diverse ethnic, cultural, and climate zones along with four biodiversity hot spots
making it for a mega biodiverse country. Traditionally, Indians are aware of this distin-
guished biodiversity and have inculcated several practices to conserve and economically
utilized these resources for medicine, food, agriculture, and other commercial products,
growing its market both nationally and internationally [2].
Bioprospecting, also known as biodiversity prospecting, is a dynamic and methodical
search for natural ingredients and genes in wildlife that have the potential to be turned
into industrial products by biological, genetic, and chemical manipulation and without
harming nature. Bioprospecting is applied in fields of cosmetics, pharmaceutical, agri-
culture biotechnology, enzymes, and proteins and sensor technology [3]. New methods
involve high-throughput screening and other automated programs to identify, screen,
and isolate novel wild bioactive compounds (plants, microbes, polyphenols, fungi, and
animals, etc.) [1]. There is an ocean of untapped potential in unknown bioresources
that can provide industries with novel developments. While bioprospecting leads to
discovery and utilization of natural products, biotechnology is needed to turn the natural
compound into a more accessible and potent product with higher efficacy. Biotechnology

190 10 Biodiversity and Importance of Plant Bioprospecting in Cosmetics
can also contribute toward conserving and sustaining the original resources by utilizing
the genetics of active ingredients thus preventing the exploitation or damage to the
environment [4].
Cosmetics and make-up have been around for many centuries around the world applied
equivalently among all the genders. Anatomically modern humans have used various
substances to alter their appearance or adorn their features for at least 10,000 years and
possibly a lot longer. The etymology of the Greek word “Kosmeticos” means “related to
beautifying or decorative substance.” From a consumer point of view, cosmetics are
products applied locally on external body parts for beautifying mainly the complexion,
skin, nails, teeth, and hair [5]. The term cosmetics differs slightly throughout the world.
“The US Federal Food, Drug and Cosmetic Act defines cosmetics more precisely in two
ways 1) as products intended to be rubbed, poured, sprinkled, or sprayed, introduced into
or otherwise applied to the human body, or any part to cleanse or beautify or promote
attractiveness or alter the appearance (for instance, shampoos and lipstick) and 2) products
intended for use as a component of any such articles (the term soap is excluded)” [6].
Article 2 of the EU Cosmetics Regulation (Regulation (EC) No. 1223/2009) incorporating
the following definition of a cosmetic product: “A ‘cosmetic product’ shall mean any
substance or mixture intended to be placed in contact with the various external parts of the
human body (epidermis, hair system, nails, lips, and external genital organs) or with the teeth
and the mucous membranes of the oral cavity with a view exclusively or mainly to cleaning
them, perfuming them, changing their appearance and/or correcting body odors” [7].
The Indian Drugs and Cosmetics Act, 1940, and the Drugs and Cosmetic Rules 1948 defines
cosmetics almost similar to the US Federal Food, Drug and Cosmetic Act [8]. The word
cosmeceutical a hybrid category of the words “cosmetics and pharmaceutical,” was first
coined by Raymound Reed, a member of U.S. society of cosmetic chemist. Furthermore,
the term was popularized by Dr. Albert Kligman in 1984 describing it as a cosmetic with
pharmaceutical action [9]. However, the term is still not officially recognized in US, EU, or
India. Nutricosmetics is another closely related term used which conceptualize that orally
ingestible dietary products can act as nutritional supplements to care skin, nails, and hair
natural beauty [10].
Cosmetics can be synthesized artificially or naturally occurring and a combination of
both [11]. The basic ingredients in a majority of cosmetics include water, thickeners, mois-
turizers, emulsifiers, stabilizers of pH, colors, fragrances, preservatives, and other active
formulations specific to skin, hair, nails, or hygiene [12]. Cosmetics essentially require inter-
action and penetration to multilayers of skin and different cell types; hence, compounds
having properties like biosurfactants, antioxidants, antiaging, etc. produced by various
sources serve as the best replacement to chemical entities available in marker [5]. The cos-
metic industry is constantly seeking new natural products and has explored the untapped
bioresources in the past decade and is one of the frontiers in bioprospecting. Bioprospecting
in cosmetics has made leaps from utilizing plant seeds to fungi, from traditional and herbal
cosmetics to vegan and natural products made from seaweed and oats. A changing prefer-
ence in the cosmetics industry from synthetic to natural and biologically obtained sources
has undoubtedly changed the cosmetics market. Use of herbal and natural beauty products
reduces the chance of side effects or any other harm that may be caused by chemical-based
products. The natural genetic resources have an enormous capacity to produce novel
10.3 India’s Biodiversity and Its Traditional Knowledge/Medicine in Cosmetic 191
compounds for the medical and food industry. The undiscovered potential of our ecosystem
and diverse species has never-ending possibilities of biological value. The chapter attempts
to recapitulate the bioprospecting of plant biodiversity in cosmetic industry.
10.2 The Fury of Synthetic Chemicals

in Cosmetics on Health
Synthetic chemicals in cosmetics and skincare products often unleash their fury on users
causing several mild to severe health problems. With the increasing use of cosmetics and
the consequent exposure to the chemicals for a lengthy period and repeated frequency, the
adverse biological effects of these products are becoming more prevalent globally [13, 14].
Both men and women are being affected by these harmful chemicals. Although there exist
several stringent regulations and quality control tests for cosmetic manufacturing, these
standards are often not met, as evidenced by the harmful effects still occurring in the cos-
metic consumers [15]. The cosmetic industry is creative and always striving for improved
products, which is in the never-ending process of finding and using new ingredients. Many
of these new ingredients are not enlisted in the restriction’s list and may serve as the latest
potential allergens. Contrary to medicine manufacturing, there neither exists any specific
agency to critically assess the standards of cosmetics and skincare products nor any mar-
keting authorization is required [16]. Moreover, there is no analysis of the risk–benefit ratio
involved in the production and no guarantee of maintenance of consistent quality from one
batch of products to the other. It is, therefore, the practice of “cosmetovigilance,” a kind of
quality surveillance devoted to ensuring that the safety of cosmetics has been emerging in
public health sciences [17]. In the past nine years, almost 12% of global cosmetic users had
experienced toxic effects caused by cosmetics and skincare products [17, 18]. Thus, the
increasing use of cosmetic products is emerging as a public health concern. Table 10.1
enlists several public health risks caused by chemicals used commonly in a wide variety of
cosmetics.
10.3 India’s Biodiversity and Its Traditional Knowledge/

Medicine in Cosmetics
World Health Organization defines “Traditional medicine” as a collection of indigenous

and long-established medicinal practices seen all around the world, among various coun-
tries, cultures, and civilizations, which has its roots from the humankind’s knowledge and
experience. India’s rich biodiversity has been preserved and ethnically shared by people
practicing traditional medicine like Yoga, Homeopathy, Siddha, Amchi, Unani, and
Ayurveda in conjunction with modern medicine [19]. The recent few decades have seen an
increase to search for new natural products in drugs and cosmetics industry from the tradi-
tional medicine systems as they bear distinct similarities to contemporary pharmacognosy
along with experiential wisdom, holistic approach, better tolerance, and safety [20, 21].
Ethnobotany is again a study of traditional folklore knowledge and customs of mankind
Table 10.1 Chemical cosmetics and their toxic effects.
Causative chemical/s Chemical cosmetics Harmful effects
Dibutyl phthalate (DBP), Nail polish Allergic reactions and skin

formaldehyde, toluene irritation, headache, eye
(Toxic trio) irritation, dizziness. If ingested
acts as endocrine disruptor,
worsen infertility, causes liver
and kidney failure
Benzophenones, Sun-screening agents Irritant, allergic, phototoxic or
debenzoylmethanes photo-allergic dermatitis
Para-aminobenzoicacid (PABA) Sun-screening agents Irritant, allergic, phototoxic or
photo-allergic dermatitis
Cinnamates Sun-screening agents Irritant, allergic, phototoxic or
photo-allergic dermatitis
Para Phenylenediamine (PPD) Commercial hair dye Skin inflammation and irritation
(eczema), blisters and surface
oozing, contact dermatitis,
erythematous rash, carcinogenic
Hydroquinone (HQ) Skin lightening cream Severe skin redness, burning
sensation, ochronosis (gradual
darkening of the skin)
Coumarin Deodorants, anti- Potential carcinogen
perspirants, fragrances
Phethleugenol Deodorants, anti- Potential carcinogen
perspirants, fragrances
Phthalates Deodorants, Potential hormone disruptors
antiperspirants, fragrances
Hydrogen peroxide solutions Hair bleaching products Allergic contact dermatitis
(Type I and IV)
Ammonium persulfate Hair bleaching products Allergic contact dermatitis
(Type I and IV)
Butylated hydroxyanisole (BHA), Moisturizers, lipsticks Skin allergies, carcinogenic
Butylated Hydroxytoluene (BHT)
Parabens (methylparaben, Shampoo, toothpastes Skin irritation, contact
propylparaben, butylparaben, moisturizers, sunscreens, dermatitis, endocrine disruptors
and ethylparaben) face and body
washes, deodorants,
antiperspirants, shaving gels
Diazolidinyl Urea Child care products, skin Mutagenic and carcinogenic
care products, hair, nails, agent, allergic contact dermatitis
eye and face make-up
Imidazolidinyl urea Lotions, creams, shampoos, Mutagenic and carcinogenic
hair conditioners, agent, allergic contact dermatitis
deodorants
1,4-dioxane Toothpaste, mouthwash, Potential carcinogen, can trigger
shampoo breast, skin and liver cancer
10.3 India’s Biodiversity and Its Traditional Knowledge/Medicine in Cosmetic 193
Causative chemical/s Chemical cosmetics Harmful effects
Heavy metals [lead (Pb), Lipstick, eyeliner, eye Contact dermatitis, systemic
cadmium (Cd), nickel (Ni), shadow, whitening allergic dermatitis, circulatory
arsenic (As), mercury (Hg), toothpaste, nail color, face and peripheral nervous
antimony (Sb), cobalt (Co)] powder, hair cream, hair disorders can serve as potential
dyes (contaminants) carcinogen, pneumoconiosis,
emphysema, bronchitis, bone
fragility, kidney damage
Methylchloroisothiazolinone- Personal hygiene products Allergen, may cause
methylisothiazolinone (MCI-MI) hypersensitivity on long term
exposure
Methyldibromoglutaronitrile- Moisturizers Contact dermatitis
phenoxyethanol (MDBGN-PE)
Quaternium-15 Soaps, shampoos, hair Allergen, may cause systemic
conditioners, hair styling anaphylaxis
products, shaving creams,
moisturizers
Thimerosal Tattoo inks, mascara Respiratory and nervous system
toxicant
Glyceryl monothioglycolate Hair styling chemicals Allergen
(straightening, perming)
Myroxylon Pereirae (Balsam of Fragrances Allergen
Peru)
Cocamidopropyl betaine Shampoo, toothpaste, hand Allergen
soaps, shower gels, liquid
soaps, skin cleansers,
deodorants
Toluenesulfonamide- Nail lacquers, polishes Allergen
formaldehyde resin
Propyl gallate, octyl gallate, Moisturizing cream, Allergen
dodecyl gallate lipstick
concerning plants and its varied uses not just in medicine but also religious and other
anthropological uses [22]. Herbal products have been used all over the world either as
home remedies or on large-scale industrial beauty and health products with plant sources
as the main ingredient. Ayurveda, a well-documented traditional medicine of India, is
accepted by modern medicine and western countries for its rich and established history of
effectiveness. These findings are published in ancient scriptures like Charaka Samhita,
Sushruta Samhita, Ashtanga Hridayam, and Ashtanga Sangraha. It is still widely used in
rural areas as their foremost health care. Bioproducts include health foods and medicines,
botanicals, and supplements. In India, Ayurveda, Unani, and Siddha systems are based
on a holistic lifestyle that incorporates everything from diet to the drug, from physical to
spiritual healing of mind and body. Traditional and herbal medicine is also widely used
around the globe, such as traditional Chinese medicine and Kampo, Japanese traditional
medicine [23].
India’s biodiversity in plants provides sources not only to medicine and drugs but also
cosmetics, and hence incorporated in clinical practices backed by ages of results and ongo-
ing evidence of being safe and natural. India has four biodiversity hotspots, The western
Ghats, home to 6000 vascular plants with 2500 genus. Three thousand plants are endemic,
and most spices of black pepper and cardamom in the world are all believed to have origi-
nated in the Western Ghats. The Himalayas are another biodiversity hotspot, where 10,000
species of plants, a third of which are endemic and cannot be located anywhere else in the
world can be found. Similarly, the Indo-Burma Region and Sundaland are the other hot-
spots that are home to thousands of endemic species of fauna, half of which cannot be
found anywhere else in the world. Due to the rich biodiversity and established benefits of
traditional and herbal medicine, public interest in plant-based products has increased over
the years, leading to a surge in the development of plant-based nutraceuticals and cosmet-
ics. Thus, a multidisciplinary approach, including traditional knowledge-based bio-
prospecting, may offer promising leads in the cosmetic industry with natural and safe use.
10.3.1 Herbal Cosmetics

The plethora of plants and herbs, which have been traditionally used for ages in India for
healing, led to the development of modern drugs and are now making a comeback for
being a safe and effective substitute for chemical and synthetic medicine and drugs. Herbal
cosmetics have one or more herb and shrubs as base ingredients that provide cosmetic
benefits. Herbal cosmetics tend to suit all skin types and are available in a wide range and
at a lower price than synthetic cosmetics and cosmeceuticals. They are natural and are
mostly gentle on the skin and are cruelty-free. These features make herbal cosmetics a safer
and more attractive option for the new generation of more conscious buyers. Few examples
of the most popular and developed herbs and bioingredients of natural cosmetics are
shown in Table 10.2.
10.4 Use of Plant-Based Products in the Cosmetic Industry
Earlier botanicals were source of a small percentage of cosmetics. However, the consumers
worldwide are looking for cosmetic products devoid of any synthetic chemicals. Many fac-
tors attribute in interest in natural products research: First, unmet therapeutic needs; sec-
ond, the remarkable diversity of both chemical structures and biological activities of
naturally occurring secondary metabolites; third, the utility of bioactive natural products
as biochemical and molecular probes; fourth, the development of novel and sensitive tech-
niques to detect biologically active natural products; fifth, improved techniques to isolate,
purify, and structurally characterize these active constituents, and last, advances in solving
the demand for the supply of complex natural products.
Cosmetics typically contain around 15–50 ingredients. Many products differ from
company to company and vary with ingredients. However, most cosmetics contain certain
core components such as emulsifier, thickener, preservative, emollient, pH stabilizers,
fragrances, and colors. Emulsifiers are ingredients used to produce homogenous and even
texture usually to bind the water–oil mixtures in the cosmetics [25]. Some of the
10.4 Use of Plant-Based Products in the Cosmetic Industr 195
Table 10.2 Popular herbs and their ingredients in natural cosmetics [24].
Skin type Natural compound/ingredient Feature and benefit
Dry skin Coconut oil Liquid or solid

Directly applied on skin and hair as a
moisturizer; can be used as cooking oil,
Sunflower oil Smoothing properties and considered
non-comedogenic
Antiaging Golden Root (Rhodiola rosea) Increases resistance to physical stress and
treatment has antioxidant properties.
Carrot seed oil Many essential vitamins like Vit A,
promotes new skin cell formation,
antiwrinkle, and natural toner.
Ginkgo (Ginkgo biloba) Circulatory tonic, strengthening
capillaries, protects the nervous system,
and fights oxidation.
All-round Neem Antiseptic, reduce dark spots, antibacterial
skincare and
protection
Aloe Moisturizer, sunscreen, emollient
Green tea Antioxidant that can be consumed as a
drink or applied on the skin, lessen
inflammation, and cell damage protection
Calendula Topically used for acne, reducing
inflammation, controlling bleeding, and
soothing irritated tissue
Haircare Amla It contains vitamin C, tannins,
phosphorus, iron, and calcium that give
nutrition to the hair, prevents graying, and
stimulates thicker hair growth.
Kapoor, Heena, Amalaki, Used in moisturizers, shampoos, hair
Bhringraj, Hirda, Rosary pea, growth, darkening of hair, hair oil, and
Brahmi, Lemon and Shikakai preventing dandruff.
Essential Oils Eucalyptus oil, Rose oil, Copreservatives with antibacterial activity,
Citronella oil for fragrance, in hair care and skincare for
having sustained effects in the skin
commercially used natural emulsifiers are glyceryl monostearate (GMS) and polysorbate
20 derived from natural oils. Thickeners [26] are agents that help in increasing the viscosity
of the final cosmetic products. Some of the natural ingredients have both emulsifying and
thickening properties. The use of natural preservative from plants has both antioxidant and
antibacterial effect. Many essential and aromatic oils of plant origin exhibit preservative
properties [27]. Emollients are skin softeners by preventing dry skin formation [28].
Stabilizing agents maintain pH of the cometic preparation. Most of the stabilizers are of
microbial origin than plant source. The fragrances used in cosmetics usually have their
origin from a wide range of plants. Natural pigments of plant origin are gradually used in
Table 10.3 Core components of plant origin used in cosmetics.
Core component Plant name References
Emulsifier Helianthus annuus L. – sunflower oil [29–31]

Corylus americana Marshall – hazelnut oil
Acacia senegal – gum arabic
Cyamopsis tetragonoloba – guar gum
Elaeis guineensis – palm kernel oil [32]
Glycine max – soy oil
Cocos nucifera – coconut oil
Thickeners Gossypium arboretum – hydroxyethyl cellulose, [33]
Cyamopsis tetragonoloba – guar gum,
Preservatives Melaleuca alternifolia – tea tree oil [34, 35]
Thymus vulgaris – thyme oil
Emollient Olea europaea – olive oil, Cocos nucifera – coconut oil [32, 36]
pH stabilizers Triticum aestivum – hydrolyzed wheat protein [37]
Colors Curcuma longa – bright yellow [38]
Croccus sativa – yellowish orange
Beta vulgaris – red
Lithospermum – red
recent years. Many of the plant-based components are used commercially by many of the
noted brands all around the world. Some of the plant-based core components are listed in
Table 10.3.
Apart from the core ingredients, many other bioactive materials are added depending on
the type of cosmetic action required. This may include skin protection from harmful UV
irradiation, skin whitening, antiaging, hair care, acne-protection, anticellulite activity, and
hair coloring, etc. Some the plant bioactives used in the natural cosmetic industry are
enlisted in Table 10.4.
10.5 Green Cosmetics – Significance and Current Status

of the Global Market
The word “green” is interchangeably used with “organic,” “environment-friendly,” “natu-

ral,” “sustainable,” or “chemical-free.” Thus, “Green cosmetics” are those cosmetics which
are not merely plant-based but manufactured from plants which are cultivated or growing
naturally in organic and chemical-free condition. Green cosmetics “guarantees environ-
mental conservation all along the production line, consumer’s respect and utilization of
natural matter with superior ecological quality” (Eco cert 2003). They are devoid of geneti-
cally modified ingredients and thus aim to achieve environmental conservation, minimal
pollution, sustainable utilization of nonrenewable resources, and preservation species.
Table 10.4 Plant Bioactives and their mode of action in natural cosmetics.
Cosmetic
property Plant name Bioactives Action References
Skin Grape, Vitis sp. Proanthocyanidins or procyanidins High antioxidant activity, Skin whitening [39]
protection White mulberry, Morus Phenolic compounds Eg. gallic acid; Skin whitening property [40]
alba quercetin and the fatty acids e.g.
linoleic acid, palmitic acid;
Mulberroside F
Liquorice/Licorice, Glabridin, glabrene, isoliquiritigenin, Skin whitening property [41, 42]
Glycyrrhiza glabra licuraside, licochalcone a, liquiritin
and its isoform
Aloe vera Aloesin Skin whitening, photoprotection property [43]
Citrus sinensis Hesperidin (Flavonoid) Skin whitening [44]
C. paradisi Hesperidin (Flavonoid) Skin whitening [44]
Cranberry, Vaccinium Proanthocyanidins or procyanidins High antioxidant activity, skin whitening [45]
oxycoccos, Vaccinium
macrocarpon
Anti-aging Cofee, Coffea arabica Polyphenols, especially chlorogenic Improves pigmentation, fine lines, wrinkles, [46, 47]
acid, condensed proanthocyanidins,
quinic acid, and ferulic acid
Photo Moringa oleifera Rutin, quercetin, ellagic acid, Protects from UVA-induced oxidative stress, [48]
protection chlorogenic acid, ferulic acid UV-induced erythema, photoaging, prevent collagen
destruction, enhances expression of immune factors
Hair care Chinarose, Hibiscus Flavonoids, tannins, terpenoids, Hair growth promotion and antigraying activity [49]
activity rosasinensis saponins, and alkaloids
Clove basil, Ocimum α-Pinene, camphene, cis-ocimene, Enhance normal hair growth, promotes follicular [50]
gratissum β-ocimene, trans-caryophy-llene, proliferation in cyclophosphamide (used frequently
eugenol in chemotherapy) – induced hair loss, antimicrobial
activities
(Continued)
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Cosmetic
property Plant name Bioactives Action References
Anti-acne Bitter melon, Momordica Charantin (steroidal saponin), Antibacterial, anti-inflammatory efficacy, anti-acne [51]
charantia Momordicoside U, Geracrene activity, anti-psoriasis, anti- leprosy
Neem, Azadirachta Azadirachtin, nimbolinin, nimbin, Anti-inflammatory, anti-acne activity [52]
indica nimbidin, nimbidol, sodium
nimbinate, gedunin, salannin,
quercetin, ß-sitosterol
Eucalyptus globulus Gamma-terpinene (Monoterpene) Antibacterial, antifungal and anti-inflammatory [53]
activity, antiacne efficacy
Anti-cellulite Coffee, Cofffea robusta Caffeine Removes skin swellings and fatigue signs [54]
activity L., Coffea arabica L.
Coleus forskohlii Forskolin Dislodges localized fat [55]
Centella asiatica L. Asiaticoside Strengthens circulation, increases metabolism of [56]
proline and lysine
Horse chestnut, Aesculus Saponin Anti-inflammatory and vasoprotective actions [56]
hippocastanum L.
Ginkgo biloba L. Proanthocyanidins Prevent edema, anti-inflammatory activities [56]
Ivy, Hedera helix L. Saponin Have vasoconstrictor properties, prevents edema, [56]
improves body fluid drainage
Hair coloring Henna, Lawsonia Lawsone/hennotannic acid Red dye [57]
inermis, Lawsonia alba,
Lawsonia spinosa
Juglans cinerea, J. Regia, Naphthoquinones (Juglone and Yellow-brown dye [58, 59]
J. Nigra juglandin)
Indigofera tinctoria Indigotin Indigo dye [60]
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10.5 Green Cosmetics – Significance and Current Status of the Global Marke 199
Green or environment-friendly cosmetics are manufactured from organically grown plant

and fruit extracts and their concentrates [61].
The main reason for increasing consumer preferences toward these products is related to
an increase in health awareness and environmental consciousness [62]. Our skin offers the
first line of defense to our body in fighting an array of infections. It can wholly or partially
soak the applied products, which thus can reach to our bloodstream. About 60% of the used
cosmetics and skincare products reach our circulatory system. An average woman soaks up
almost 30 pounds of the ingredients present in moisturizers in 60 years [63]. Hence, using
chemical-free, green cosmetics is a safer avenue.
Moreover, the ability to perform physiological functions of the skin decreases with aging.
Plant products can offer a solution in the fight against aging by performing hydration, anti-
oxidant, antimicrobial, and UV-protection activity, reducing inflammation, irritation, and
many other things. Some of the examples are coconut and argan oil, calendula, green tea,
pomegranate, crocin occurring in crocus and gardenia flowers, soy, and feverfew [64].
The chemicals used in cosmetics also take a toll on the environment [65]. Examples are
some preservatives (parabens, triclosan), UV-shields, plastics, and heavy metals [66]. The
potential of environmentally safe green cosmetics toward solving this problem is also a
reason for their gradually increasing popularity [67]. However, there is still a considerable
lack of awareness among customers about these products [68], which requires unique
marketing strategies. Strategies undertaken for Green marketing highlight a new edge of
socioeconomic and environmental responsibility adopted by organizations [69].
The Indian market has also seen a surge in such Green companies in the past few decades.
Brands like Khadi naturals, Ayur, Jovees, Himalaya, and Lotus herbals, Shahnaz Hussain,
Biotique, Aroma essentials, Fab India, are successful ventures so far [70].
10.5.1 Sustainable Development Goals (Economic, Ecological Benefits)

in Cosmetic Industry – How Bioprospecting and Green Cosmetics Can Help?
Intelligent innovations serve as the keystone business strategy driving not only economic
growth but also plays a prominent role in solving an array of social and environmental
issues. Sustainability related intelligent innovations or sustainable innovations, also called
GreenTech solutions, act at production, service, strategy, process design level to achieve a
win–win situation for both producer and consumer. The use of synthetic chemicals in the
cosmetic industry causes several adverse effects, e.g., chemical abuse leading to mild to
severe health issues (Table 10.1), environmental pollution, and unsustainable use of natu-
ral resources. The harmful effects of synthetic chemicals in cosmetics are raising health
concerns among customers globally. Researchers have reported an increasing trend of
consumer awareness toward sustainable cosmetic products in recent decades due to
health issues caused by UV rays and customers becoming aware of the benefits of natural
products [71–73].
Hence, several research efforts have been focused on the conceptualization and develop-
ment of sustainably innovated green cosmetic products and their marketing models [73].
These products are characterized by the consumption of less water, material, and energy
during production, least or zero environmental pollution [74, 75]. For reduction in environ-
mental pollution caused by cosmetics and body care products, the use of an array of higher
plants, macro, and micro algae-derived bioactive natural components, agro wastes, greener
extraction methods is being envisaged [76–78]. Volatiles and nonvolatiles extracted from
plants, mainly herbs, and spices serve as the primary sources of higher plant-derived bioac-
tive ingredients [77]. Some examples of bioactive ingredients include gallotannins, caffeic
acid, and that of agrowaste is the husk of quinoa (Chenopodium quinoa), which serves as a
skin exfoliant. Consideration toward the materials used for packaging of the finished cos-
metic is also of utmost importance. The use of eco-friendly products like paper, glass, wood
is gradually increasing in cosmetic packaging.
“The United Nations defined 17 Sustainable Development Goals (SDGs) covers a range
of environmental, economic, and societal problems required for sustainable, innovative
actions.” According to Reuters, the global cosmetic sector was valued to be 507.75 billion
USD in 2018 and predicted to reach 758.45 billion USD by 2025. Hence, the activities
undertaken in this sector can contribute directly toward achieving certain SDGs if imple-
mented sustainably. Production of green cosmetics by using organically cultivated plant
products, use of environment-friendly packaging, low energy, low water-intensive produc-
tion techniques are such approaches that can directly contribute toward reducing the car-
bon footprint. Organically cultivated plants use natural fertilizer and are pesticide-free.
Environment-friendly packaging is biodegradable and hence will contribute toward lesser
or zero solid waste production. Hence, such actions can aid in achieving responsible pro-
duction (SDG12) and climate action (SDG13). The use of natural bioactive compounds,
ecofriendly packaging in cosmetics, and body care products will ensure customer health
while ensuring economic benefits. These actions will contribute toward achieving good
health and well-being (SDG3).
10.6 Ethical and Legal Implications of Bioprospecting

and Cosmetics
Bioprospecting is defined as search for identification, collection, extracting, and screening

of biological resources mainly of plants, animals, microorganisms, cells, or genes, for com-
mercial purposes, such as for pharmaceutical products, cosmetics, dyes, and foods.
Bioprospecting is hence governed by laws and has ethical implications and is connected
and communicated by local populations, governments, other persons in the scientific com-
munity, and the international public.
From the first step of discovery of novel natural compounds, the process and activities of
bioprospecting have direct implications to the environment, habitat, and natural surround-
ings and the indigenous community of that place. The search for bioresources and or tradi-
tional knowledge has gained tremendous momentum and has led to biopiracy, denial of
rights, and exploitation of natural resources. Hence, both national and international laws
as well as various private and regional measures have been put in place to regulate access
to genetic resources and benefit-sharing.
There are various legal issues related to bioprospecting. Most prominent among them is
the protection and conservation of traditional knowledge of biodiverse and indigenous
lands, which gets exploited by developed countries. Other legal issues concerning bio-
prospecting revolve around pharmaceutical and other industry research based on
10.6 Ethical and Legal Implications of Bioprospecting and Cosmetic 201
traditional medicine and knowledge of the indigenous communities; Biopiracy, which is

unauthorized and plagiarized use of biological resources or traditional knowledge or its
patenting and lastly unequal benefit sharing between resources or knowledge provider and
technology developer [79].
Apart from the laws pertaining bioprospecting, the ethical and legal guidelines applica-
ble to cosmetics should also be taken into consideration. Bioprospecting of novel cosmetic
compounds in given biota by the use of tools like bioinformatics and cheminformatics can
also lead to quicker results with new botanical-based cosmetics. However, one major
drawback would be the side effects of the use of such cosmetics in a long run. But the
traditional knowledge has the advantage of the above limitation because they have obser-
vations over a long period of many generations. The future cosmetics can thus be based on
time tested use of traditional herbal cosmetics developed by using advanced technologies
bringing together the best use of knowledge, resources, science, and technology in an
amicable manner.
10.6.1 International Laws Regulating Bioprospecting

1) Convention on Biological Diversity (CBD) was formed in 1992 to stop the loss of biodi-
versity and to regulate the inconsistency between the resource and knowledge giver and
the technology and product developer [80]. The CBD acknowledges the state’s sovereign
rights upon its bioresources and builds the structure for equal access for the same. It
recognizes the efforts and rights of indigenous communities for their knowledge and
long traditional practices of sustainable use and conservation of the biological resources,
all the while protecting and providing equal share and benefits leading from the use of
those biological resources and traditional knowledge [79].
2) Rio Earth Summit formed the Statement of Forest Principles, which also oversees grants
of equal share to indigenous communities for providing any traditional knowledge or
resources from their environment and its ultimate use [81].
3) “Declaration on the Rights of Indigenous Peoples provides rights of traditional medi-
cines (protecting plants, animals, and minerals) and health practices of Indigenous
communities.” It also supports the recognition of complete ownership, protection, and
control of their traditional and intellectual property. This article clearly outlines the full
rights of indigenous people to protect and create technologies and products and to con-
trol their cultural properties, which includes medicine, genetic resources, literature,
humans and seeds, and their knowledge of flora and fauna [82].
4) Nagoya Protocol dictates the user of biological resources and traditional knowledge, to
follow the benefit sharing and access protocols and regulations that are set as prerequi-
sites of those resource countries. Bioprospectors must adhere to mutually agreed to con-
ditions between the resource giver and user when trying to utilize their bioresources
along with following the regulations of equal access and sharing. The Nagoya Protocol
is relevant public international law, which makes it mandatory for associations which
use and benefit (bioprospectors) from biological and genetic resources. The protocol
helps the traditional knowledge to follow and adhere to jurisdictions and local Access
and Benefit Sharing (ABS) governing laws of the provider country and, when appropri-
ate, agree to ABS conditions with genuine providers of bioresources and knowledge [83].
10.6.2 Indian Law Regulating Bioprospecting

India is a hub for aromatic and medicinal plants, and its rich and diverse bio-history makes
it extremely unique (from too hot and dry to cold regions with plenty of species variations)
and an important country for bioprospecting. Thus, it requires special care and attention
toward the safety, preservation, and conservation and sustainable utilization of the biologi-
cal and traditional resources. The Biological Diversity Act 2002 (BD Act) and the Biological
Diversity Rules 2004 (BD Rules) protect Indian biological and traditional resources, regu-
lated by the center through the National Biodiversity Authority (NBA). The BD act dele-
gate’s responsibilities and influence the State Biodiversity Board and Biodiversity
management committees at the local level [80].
As mentioned, India is a significant source for bioprospecting, considering that 80% of
the medicinal plants patented in the US are of Indian origin, and 50% of the drugs reported
in the British Pharmacopoeia have Western Himalayas medicinal plant origins. The
Biodiversity Act and rule emphasis on Biosurvey and Bio Utilization and form the frame-
work for safeguarding and providing approvals for administering bioprospecting in India.
Among that is obtaining a beforehand approval from the NBA for bioprospecting activities
such as intellectual property rights for any research or innovation based on biological
resources or traditional knowledge from India; for collecting or procuring any biological
resource originating in India or any traditional knowledge of India for any research or com-
mercial use such as drugs, industrial enzymes, food flavors and extracts, fragrance, cosmet-
ics, emulsifiers, colors, and genes used for improving crops and livestock through genetic
intervention. It also covers the transfer of traditional knowledge or biological resources and
biosurvey or bioutilization. With all these regulations and monitoring, the NBA must pro-
tect the terms and conditions of all approvals and secure equal sharing of benefits between
the user and provider of the biological resources and traditional knowledge.
Another essential article of the BD act and rules is to provide the prior declaration and
information of any Indian citizen or corporate body registered in India to the state biodiver-
sity board before beginning any bioprospecting activity like biosurvey or procure biological
resource for any commercial or research utilization [79].
10.6.3 Access and Benefit Sharing (ABS)

Access and benefit sharing (ABS) is the manner of accessing genetic resources between
provider and user and the agreement among them for equal and fair sharing of the result-
ant benefits of the resource. CBD lays out rules that oversee the benefit sharing and access
of bioresources and traditional knowledge that promote environment-friendly access of
genetic resources and their purposes and to also protect the resultant benefits of their use,
which should be fairly and equally shared between the provider and user.
Two key features in ensuring ABS are the prior informed consent (PIC), which is pro-
vided by the central governing body (NBA in India) in a legal and institutional framework
and the mutually agreed terms (MAT) of fair and equal distribution of benefits between
provider and user [80].
For centuries local communities and indigenous people have been using and benefiting
and developing biological resources and lived off of their local biodiversity by using them
10.7 Laws Regulating Cosmetic 203
from medicine, shelter, food, agriculture, clothing, and animal husbandry. Hence, tradi-
tional knowledge refers to practices and innovations and complete knowledge of local
communities and indigenous people related to bioresources and genetic materials, which
gets adapted and changed and developed based on needs and environment over centuries
and continues to be passed down to new generations [81].
Traditional knowledge, including genetic resources, is relevant to indigenous and local
communities (ILCs), which depend on these bioresources for their day to day activities and
livelihood. The bioprospector or user benefits from traditional knowledge by seeking per-
mission and accessing these resources to develop commercial and new products as well as
for research in both commercial and academic fields. Competent National Authorities
(CNAs) generally oversee the negotiations and agreements and validity between users and
providers of traditional knowledge and maintain a balance in the bioprospecting area [82].
Bonn Guidelines – the CBD adapted Bonn Guidelines in 2002 to support governments in
regulating ABS in their countries. It assists countries in developing and enforcing ABS
effectively to make sure that both users and providers of bioresource share the benefits
equally and fairly [81].
10.6.4 World Intellectual Property Organization (WIPO)

World Intellectual Property Organization (WIPO) – formed in 1967 as a global frame-
work that promotes and protects Intellectual property (IP) policy under the United
Nationals with 193 members. It is a forum to shape international IP rights and protects IP
across borders and resolves conflicts. Through WIPO IP knowledge can be shared and can
be used as a reference for world IP policies [84].
10.6.5 Intergovernmental Committee on Intellectual Property and Genetic

Resources, Traditional Knowledge, and Folklore (IGC)
Intergovernmental Committee on Intellectual Property and Genetic Resources,
Traditional Knowledge, and Folklore (IGC) was further formed in 2000 as a forum for
WIPO associate states to examine IP concerns pertaining to accessing, sharing, and protect-
ing bioresources and traditional knowledge. Formal negotiations are held by IGC to reach
agreements regarding international laws that can establish adequate protection of biore-
sources and traditional and cultural practices and knowledge. Motivation to protect IP was
taken from growing awareness of folklore creativity belonging to local people and is part of
indigenous community identity and should hence be protected as intellectual property and
should prevent misuse or exploitation by growing technologies [85].
10.7 Laws Regulating Cosmetics
Every country has laws governing access to provide safe and effective cosmetics. Cosmetic
laws are regulated by Central Drugs Standards Control Organization (CDSCO) headed by
drugs controller general of India and Bureau of Indian Standards (BIS) [86]. The manu-
facturer of cosmetics has to obtain licensing authority from the respective state
governments and manufacture the product under the strict guidelines. The United States
(U.S.) and European Union (EU) share similar goals of warranting the safety of cosmet-
ics for consumers through critical and proven science-based regulation. The means by
which each regulates the safety of cosmetics are quite similar. EU also has a Cosmetic
ingredient Review that reviews and declares a given constituent of a cosmetic to be safe
or not safe.
Proof of safety of cosmetic product, especially each ingredient, has to be provided by the
manufacturer. Unlike pharmaceutical drugs, no pre-market approval is needed for cosmet-
ics except color additives, active ingredients, sunscreen, and preservatives. The International
Fragrance Association (IFRA) also establishes safety standards for fragrance materials.
Safety of the cosmetic products is tested based on toxicity and irritancy potential, the rules
which are similar to that of screening drugs for treatment of skin diseases. However, the
strict guidelines for testing depend on the ingredients used in cosmetics. The cosmetics
once manufactured have to abide by the labeling, import and export rules, and advertising
rules of the country of origin [5].
10.8 Role of Biotechnology in Bioprospecting

and Cosmetics
Although organic or green cosmetics are on a rise, they still remain a small part of the
overall cosmetics industry. The main reason would be faster depletion of the natural
plants than the time required for the plants to grow. Another reason is that the active
ingredient which gives the magical effects still remains confined to certain rarer and
endangered species. Hence, there is a simultaneous need for faster cultivation of such
rarer plants without compromising on the plant properties. Advances in biotechnology
and use of tissue culture of plants to produce secondary metabolites can be exploited for
commercial processing of even rare plants and the metabolites they produce. Examples
for such commercially available products are Shikonine a red pigment used especially for
lipsticks and nail color [87] is produced from Lithospermum erythrorhizon. Arbutin is a
whitening ingredient produced from Catharanthus roseus [88]. Both these ingredients
are commercially produced via tissue culture by Mitsui Petrochemical Industries, Japan.
However, the use of plant cell culture or their metabolite derived cosmetic active ingre-
dients is still in its infant stage [89].
White biotechnology or industrial biotechnology, a component of “green chemistry,”
plays a key role in producing the cosmetics in the present world. The use of renewable
substrates and biocatalyst results in an environmentally friendly valuable cosmetic prod-
uct. This not only reduces the use of animal killing thereby protecting the animals from
being extinct. Blue biotechnology exploits the ocean and sea resources to generate products
of industrial interest. There is rise in the use of materials, to generate natural cosmetics
with better efficacy and minimum side effects.
Ideally, a government and industry partnership should focus on multidisciplinary
research. The research joins the forces starting from bioprospecting of plant from its natu-
ral habitat. This begins with the consultation of local people for their ethnic knowledge of
plants for cosmetic uses. Once a right plant source with potential benefits is selected,
Reference 205
further study to know the natural products chemistry, the underlying molecular and cel-
lular biology, using the combined knowledge of biochemistry, synthetic, and analytical
chemistry, and pharmacology which ends with a consumer product profitable to all.
Box 10.1 A Good and Successful Initiative

The International Cooperative Biodiversity Groups (ICBG) Panama, 1998, was started to
establish shared benefits of Panama’s bioresources with the country as a provider of
genetic resources. This has resulted in benefits from drug discovery projects, scientific
infrastructures, training of scientists, and forming many research programs. One of the
objectives is to include local researchers in the center of commercially viable programs
that use local biodiversity for research. The ICBG has led to a higher incentive for conser-
vation helping advance the Coiba National Park to its UNESCO world heritage tag [83].
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211
11
Therapeutic Lead Secondary Metabolites Production Using

Plant In Vitro Cultures
Vikas Srivastava1, Aksar Ali Chowdhary1#, Skalzang Lhamo1#, Sonal Mishra2,
and Shakti Mehrotra3
1
Department of Botany, Central University of Jammu, Samba, Jammu and Kashmir, India
2
School of Biotechnology, University of Jammu, Jammu, Jammu and Kashmir, India
3
Department of Biotechnology, Institute of Engineering and Technology, Lucknow, Uttar Pradesh, India
11.1 Introduction
Plants being sessile in nature face adverse environment conditions and manage them by
stimulating various physiological, biochemical and molecular processes which in turn alter
the ion homeostasis, gene expression and also endogenous level of several metabolites [1, 2].
The plant metabolites have been identified as the basis for their nutritional and medicinal
properties, and therefore plants are used by human being since ancient time for such pur-
poses [3–5]. The metabolites are the product of metabolism, and based on their utility in
governing plant life, it can be categorized into primary (amino acids, fats, carbohydrate,
etc.) or secondary (terpene, alkaloid, phenolics, etc.) metabolites. However, there are no
clear cut criteria, which distinguish between the primary and secondary metabolism. For
example, both diterpenes (C20) and triterpenes (C30) represent primary and secondary
metabolites (SMs). Similarly, proline is considered as the primary metabolite, whereas C6
analog (pipecolic acid) is considered as an alkaloid [6, 7].
The significance of SMs has not only considered them as an effective stress mitigation
entity for plant itself but also as an effective molecule to alleviate many human ailments.
The research on phytochemistry, biochemistry and bio-prospection (biological activity test-
ing) has revealed the establishment of many SMs with authorized medicinal values, which
have also been qualified for commercialization in terms of medicines in various health care
systems. Production and accumulation of SMs in plants tissues are associated with a vari-
ety of accessory functions, but primarily it is for the defence against bacteria, fungi, herbi-
vores and as signals in plants [8]. SMs are highly inducible in response to stresses [9] and
include active molecule responsible for pharmacological or biological activity. Further, in
#
Equal Contribution

212 11 Therapeutic Lead Secondary Metabolites Production Using Plant In Vitro Cultures
the last 50 years due to their medicinal properties, research on plant SMs has increased
significantly [10]. The enormous opportunity offered by SMs leads to the distortion of the
natures’ sustainability through direct harvesting of medicinal plants from their natural
habitat and therefore the alternative production system is highly desired.
Plant in vitro cultures though emerged as a system of plant culturing under controlled
environment condition since a century ago. But, with the advancement of research in this
area, the focus has slightly changed and now the researchers’ effort to offer some value
application out of it. Since previous half century or so, the focus on raising plant in vitro
cultures of medicinal plants involves the analysis of their potential to produce the metabo-
lite of interest. The range of plant in vitro cultures, viz., shoot, adventitious root, callus and
cell suspension and hairy root cultures (HRCs) are explored for this purpose. Based on
growth and the way to manage (easy handling) the in vitro cultures, callus and cell suspen-
sion culture and HRCs are emerged as the imperative choice. Further, based on genetic and
biochemical stability along with culture cost criteria, HRCs [11, 12] currently supersede the
entire range of in vitro cultures. Besides the production of SMs, nowadays, plant in vitro
cultures are also used for many other biotechnological interventions such as elicitation,
metabolic engineering, phytoremediation, molecular farming, plant–microbe interaction,
bioreactor up-scaling, etc. [11–16]. In spite of all types of plant in vitro cultures, irrespective
of their pace of utility today, have been explored sometimes for SMs production with phar-
maceutical value, which is largely the subject of the current chapter. The below text will
highlight some of the excellent approaches to enhance the metabolite production using
plant in vitro cultures to further meet the desired production. Further, the emphasis will
also be on some of the instances of utilization of plant in vitro cultures for therapeutically
important plant metabolites.
11.2 Secondary Metabolites and Pharmaceutical Significance
The SMs are also known as plant constituents, natural products or phytochemicals and
have medicinal properties [17]. These are produced by almost all plants, and plant has
genetic information for the biosynthesis of these metabolites. Further, on the basis of their
biosynthetic pathways and precursor, they are classified into three main categories as ter-
penes (or terpenoid, terpene derivatives), phenolics and alkaloids [7, 17, 18], and each cat-
egory represents pharmaceutical lead molecules (Table 11.1). The terpenoids (both primary
and SMs) and alkaloids are synthesized from C5 precursor isopentenyl diphosphate and
amino acids, respectively. However, the phenolics biosynthesis involves either shikimic
acid pathway or the malonate/acetate pathway [7].
Some SMs such as terpenes and phenolic compounds are almost produced by major line-
age of land plants, whereas alkaloids are restricted to a particular taxon, but they may also
occur in other groups due to convergence [19]. These metabolites also serve as the basis for
chemotaxonomy [20]. For instance, the plants have been demarcated into groups with spe-
cific metabolites such as tropane alkaloid bearing plants (Atropa, Hyoscyamus, Scopolia,
etc.), terpene indole alkaloid bearing plants (Catharanthus, Rauwolfia etc.), benzyl isoqui-
noline alkaloid bearing plants (Papaver), etc. In plants, the functions of SMs are not crucial
for vital processes such as growth, development and reproduction [21]. However, they
11.2 Secondary Metabolites and Pharmaceutical Significanc 213
Table 11.1 Major classes of plant secondary metabolites (SMs) and biological activity of some of
the relevant metabolites produced through plant in vitro system.
Major class
of SMs Metabolite Plant system Biological activities
Terpenoids Artemisinin Artemisia annua L. Antimalarial and Anticancerous

Astragaloside Astragalus Cardioprotective, antitumor,
membranaceus antihypertensive, antiinflammatory,
antidiabetic, neuroprotective, etc
Tanshinone Salvia miltiorrhiza Used for the treatment of
cardiovascular disease
Eclalbatin Eclipta alba L. Antioxidant
Abietane Cephalotaxus fortune Antitumor
Alkaloids Codeine Papaver somniferum Antitussive and analgesic
Morphine P. somniferum Narcotic and analgesic
Atropine Hyoscyamus niger Anticholinergic
Caffeine Coffea arabica Stimulant
Quinine Cinchona officinalis Antimalarial
Scopolamine & Hyoscyamus Anticholinergic
Hyoscyamine reticulatus L.
Reserpine and Rauwolfia serpentina Antihypertensive and
Ajmalicine Antiarrhythmic
Paclitaxel Corylus avellana Anticancerous
Vinblastine Catharanthus roseus Antileukemic
Phenolics Reseveratrol Vitis vinifera Anticancerous, anti-cardiovascular
and antineurodegenerative
Rosmarinic acid Rosmarinus officinalis L. Antioxidant
Caffeic acid R. officinalis L. Antioxidant
Daidzein Glycine max Anticancerous
Genistein G. max Anticancerous
protect plants from any harmful situation imposed by the environment [21–24]. Further,
the significance of SMs as therapeutics has also been identified in many studies [25].
The SMs are widely presented as an imperative molecule of high industrial significance
and function as therapeutic, flavour, fragrance, nutraceutical, etc. [26]. Among them, the
pharmaceutical significance of SMs is primarily targeted in many medicinal systems, and
many commercial products utilizing these metabolites are available in the market. The
current understanding of them suggested their role in curing many human diseases as
mentioned in Table 11.2. Some of the important medicinal values they offer include
their utility as anticancer, antidiabetic, antidepressant, antiinflammatory, antiviral, anti
oxidant, anticholinergic, antimalarial, antimicrobial, hypotensive, hepatoprotective, neu-
roprotective, etc. For instance, the metabolites like catharanthine, camptothecin,
podophyllotoxin are known for their contribution in cancer therapy. Gymnemic acid and
Table 11.2 Some examples of pharmaceutical lead molecules produced through plant in vitro
cultures.
Pharmaceutical value Secondary metabolites
Anticancerous Aloe-emodin, Andrographolide, Artemisinin, Camptothecin, Emodin,

Ginsenoside, Glycyrrhizin, Paclitaxel, Plumbagin, Podophyllotoxin,
Resveratrol, Rutamarin, Vinblastine, Vincristine
Anticholinergic Atropine, Hyoscyamine, Scopolamine
Antidepressent Chrysophanol, Harmine
Antidiabetic Gymnemic acid, Serpentine
Antihypertensive Ajmalicine, Reserpine
Antiinflammatory Aloe-emodin, Aloin, Glycyrrhizin, Hypericin, Kaempferol, Verbascoside,
Withanolides
Antimalarial Artemisinin, Quinine
Antioxidant Arbutin, Daidzin, Ginsenoside, Kaempferol, Plumbagin
Antiviral Aloe-emodin, Hypericin, Glycyrrhizin, Rosmarinic acid
Hepatoprotective Glycyrrhizin, Picroside I
Neuroprotective Ginsenoside, Withanolide A
Sedative Codeine
serpentine are known for the antidiabetic function. The reserpine and ajmalicine are good
hypo-tensive metabolites. Picroside I and Withaferin A are hepatoprotective and neuropro-
tective, respectively.
11.3 Plant In Vitro Cultures and Strategies for Secondary

Metabolite Production
Though being a mimic of in vivo plant, it is obvious that in vitro cultures may generate the
biochemical reactions, similar to the parent plant, which is also true for SMs production.
Therefore, many investigations during generation/induction of in vitro culture and subse-
quent maintenance are accompanied with secondary metabolite analysis. Successes of the
in vitro production of various SMs of high medicinal value are reported such as scopola-
mine, reserpine, camptothecin, withanolides, serpentine, catharanthine, etc. (Table 11.2.
Section 11.4). But, as independent system, these cultures also offer the route to further
enhance the metabolite content, the knowledge of which can also be translated at bench
scale. Some of the approaches that meet higher production of SMs are mentioned in the
below text.
11.3.1 Precursor Feeding

This approach is used to supply the immediate or distant precursors of the targeted biosyn-
thetic pathway. The genesis is the presumption that the targeted pathway is available, but
11.3 Plant In Vitro Cultures and Strategies for Secondary Metabolite Production 215
due to scarcity of their immediate or distant precursors, the desired production of metabo-
lite of interest could not be achieved. Precursor feeding is one of the types of biotransfor-
mation, where the efforts are done to make the substrate (precursor) available to obtain
desired product (SMs), the other two are co-culture and exogenous/non-native molecule
biotransformation [16, 27, 28]. Chao and Lan [29] have reported the production of scopola-
mine using 6-hydroxyhyoscyamine as biotransformation substrate in in vitro cultures of
Anisodus tanguticus. The exogenous supplementation of tropic acid, tropinone and tro-
panol as precursors lead to increased production of hyoscyamine and scopolamine in
Atropa belladonna suspension cultures [30]. Anitha and Ranjitha [31] have reported the
stimulation of reserpine biosynthesis in the callus cultures of Rauwolfia tetraphylla L. using
tryptophan as precursor. In Solenostemon scutellarioides in vitro culture, improved ros-
marinic acid was noticed after feeding of phenylalanine and l-tyrosine [32]. Besides, the
exogenous molecule biotransformation and co-culture approach also provide ways to
develop molecule with therapeutic significance [16]. For instance, in an attempt of bio-
transformation of betuligenol in A. belladonna HRCs, the bioactive molecule raspberry
ketone (antiobese) and betuloside (hepatoprotective) are generated [33]. The co-culture of
Atropa belladonna hairy root with its shooty teratomas results in hyoscyamine production
by one system (HRCs) which is translocated by the medium to the system (shooty tera-
toma) that convert hyoscyamine into scopolamine [34].
11.3.2 Metabolic Engineering

The production of SM requires a specific biosynthetic pathway. The substrate (precur-
sor) enters into the pathway and leads to the production of desired metabolites. Besides,
as the metabolic pathways are mostly complex in nature, some diversions are also pos-
sible that may divert the pathway, thereby reducing the expected outcome. The meta-
bolic engineering (genetic engineering to regulate metabolism) offer the opportunity to
strengthen the biochemical activity through over-expression of genes responsible for
the production of desired metabolite or through suppression of any possible diversion of
the pathway of interest [15]. For instance, the expression of Catharanthus roseus
tryptophan decarboxylase (CrTDC) gene leads to enhanced accumulation of reserpine
and ajmalicine in transgenic HRCs of R. serpentina [35]. Sun and Peebles [36] have
reported increase alkaloid (ajmalicine, catharanthine, tabersonine, etc.) production in
Catharanthus roseus HRCs overexpressed with ORCA3 and Strictosidine glucosidase
(SGD). The expression of AaPMT (Putrescine methyltransferase) and AaTRI (Tropinone
reductase I) in Anisodus acutangulus HRCs led to enhanced biosynthesis of tropane alka-
loids [37]. Enhanced scopolamine production (17mg/L) which is 100 times more than
the control clones was also reported in transgenic HRCs of H. muticus overexpressed
with H6H (Hyoscyamine 6β-hydroxylase) [38]. Similarly, the overexpression of PMT and
H6H simultaneously led to the improvement of tropane alkaloids in transgenic A. bel-
ladonna HRCs [39]. The expression of Arabidopsis thaliana squalene synthase gene
(AtSQS1) in W. somnifera transformed HRCs demonstrated increased withaferin A con-
tent [40]. Besides, nowadays using heterologous expression approaches, the desired SMs
have also been produced in non-native (plant with no report of desired SM production)
plant or microbial cultures.
11.3.3 Elicitation
It is evident that the environmental cues may influence the plant growth and develop-
ment; this is also true for the metabolism including secondary metabolism. Under such
conditions (biotic or abiotic stress or media/nutrient stress), the additional SMs thus
produced provide additional advantage to the plant and thus the plants better manage
the stress impacts. This feature of induction of secondary metabolism leading to the pro-
duction of desired metabolites is being used to enhance the production profile of plant
in vitro cultures by application of certain physical, chemical or biological factors, an
application known as elicitation. The approach has been widely used in in vitro cultures
and offer enough opportunity to optimize factors, which can dramatically increase the
metabolite content [13, 15, 41]. In Hypericum perforatum cell suspensions, the elicitation
with MeJA (methyl jasmonate) exhibited stimulation of flavonoid production, which was
2.7 times as compared to control [42]. Similarly, elicitation with salicyclic acid enhanced
production of chrysophanol and aloe-emodin more than 5–13 and 10–11 fold as com-
pared to control in the adventitious root culture of Aloe vera [43]. The elicitation effect of
Piriformospora indica on withaferin A biosynthesis was reported in cell suspension cul-
tures of Withania somnifera [44]. The improved gymnemic acid accumulation was also
achieved through endophytic fungi in the cell suspension cultures of Gymnema sylves-
tre [45]. Besides, the elicitation-based metabolite enhancement is also reported in
HRCs [15]. For instance, Kai et al. [46] have reported the improved accumulation of
tropane alklaoids by 1.51 (with ethanol), 1.13 (with methyl jasmonate) and 1.08 (with
Ag+) times in Anisodus acutangulus HRCs. In Catharanthus roseus HRCs, the improve-
ment of ajmalicine content was noticed after elicitation with optimized mixture of elici-
tor composed of jasmonic acid, methyl jasmonate and KCl [47]. Zaheer et al. [48] have
reported the enhanced daidzin production in Psoralea corylifolia L. HRCs after elicitation
with jasmonic acid and acetyl salicylic acid.
11.3.4 Bioreactor Up-scaling

This should be the ideal interventions, when everything is set (in vitro cultures are produc-
ing maximum production) and one need to up-scale the entire process. Bioreactor is a
physical instrument used for the proportionate increase in the culture volume, which is
also proportional to the metabolites it contains. Several successful attempts for plant in vitro
culture-based SMs production utilizing various type of bioreactor are reported [49, 50]. For
instance, in the cultivated shoot cultures of Artemisia annua L. in bioreactors (a modified
airlift bioreactor, a multi-plate radius-flow bioreactor and an ultrasonic nutrient mist bio-
reactor), artemisinin production was reported. Out of three bioreactors used, the nutrient
mist bioreactor exhibited 1.4 and 3.3 fold higher production as compared to multi-plate
radius-flow bioreactor and the modified airlift bioreactor, respectively [51]. In R. serpentina
HRCs, the comparison was made in 11-week old HRCs grown in shake flask and bioreactor,
which suggested an increase in the dry matter and reserpine content in later [52]. Baque
et al. [53] have reviewed the utilization of bioreactor for the production of biomass and
useful compounds from adventitious roots of many high value medicinal plants (Morinda
citrifolia, Echinacea purpurea and E.angustifolia, Hypericum perforatum and Panax ginseng).
11.4 Exemplification of the Utilization of Different Types of Plant In Vitro Cultures for SMs Production 217
Besides, the mentioned interventions, the combinatorial approaches utilizing two or more
strategies of them further provide better results.
11.4 Exemplification of the Utilization of Different Types

of Plant In Vitro Cultures for SMs Production
The plant in vitro cultures have been presented as an important system for the production
of plant SMs. Based on the site of in planta biosynthesis of SMs, the different types of
in vitro cultures are used for this purpose (Table 11.3). For instance, if the SMs of interest
are synthesized in natural roots, then the in vitro alternative would be adventitious roots or
hairy roots. Similarly, if the choice of SMs belongs to aerial plant parts, the in vitro alterna-
tive would be either shoot or callus/cell suspension cultures. However, this does not make
an error-free categorization and several exceptions do exist. The below text describe some
of the instance of success in the therapeutic lead SMs production through different types of
in vitro cultures.
11.4.1 Shoot Culture

The exploitation of shoot cultures was reported for the production of many pharmaceutical
lead compounds such as artemisinin, scopolamine, kaempferol, digitoxin and digoxin,
cocaine, vindoline, withanolide, etc. The shoot cultures of Artemisia annua were reported
to produce artemisinin [51, 75]. Khanam et al. [76] have demonstrated the presence of
hyoscymaine and scopolamine in the shoot cultures of Duboisia myoporoides R. Br. In
Scopolia parviflora Nakai (a rare medicinal plant native to Korea), the shoot cultures were
generated from adventitious shoots of rhizome [77]. The analysis of tropane alkaloids
(hyoscyamine and scopolamine) was also studied, which demonstrated their occurrence in
root, stem and leaves of in vitro propagated plants [77]. Another tropane alkaloid cocaine
was reported in the shoot cultures of Erythroxylum coca var. coca, though its level was 50%
as compared to the parent plant. Further, maximum accumulation was achieved in the
leaves of shoot cultures [78].
The Catharanthus roseus is a well-known plant producing natural antineoplastic drugs
(vinblastine and vincristine). Due to the complexity and spatiotemporal regulation of ter-
penoid indole alkaloids (TIAs) biosynthetic pathway responsible to their biosynthesis, the
callus culture, cell suspension and hairy roots of C. roseus do not provide adequate system
to produce them [79]. Yet, the multiple shoot cultures are explored to accomplish TIA bio-
synthetic pathway. The multiple shoot cultures of C. roseus (Madagascar periwinkle)
induced directly from seedlings in the presence of 1.0mg/l benzyladenine, additionally it
also composed of an unorganised tissue. The HPLC assay demonstrated the presence of
vindoline and catharanthine in the shoot (especially leaf tissue), whereas ajmalicine was
reported in the unorganized tissue. Comparison with parent demonstrated several fold
higher catharathine content in leaf tissue; however, the vindoline content was comparable
with the parent [80]. Miura et al. [81] have studied the formation of vinblastine in C. roseus
multiple shoot cultures. Satdive et al. [82] have reported the production of ajmalicine in the
Table 11.3 Few examples of production of secondary metabolites using plant in vitro cultures.
S. No Plant System In vitro culture Family Class of SMs Secondary metabolite References
1 Alstonia scholaris Callus culture Apocynaceae Alkaloid Echitamine Jeet et al. [54]
Picrinine
Tubotaiwine
2 Anisodus luridus Hairy root culture Solanaceae Alkaloid Scopolamine and hyoscyamine Qin et al. [55]
3 Artemisia annua L. Cell suspension culture Asteraceae Sesquiterpene Artemisinin Zebarjadi
et al. [56]
4 A. annua L. Hairy root culture Asteraceae Sesquiterpene Artemisinin Ahlawat
et al. [57]
5 Astragalus membranaceus Hairy root culture Leguminosae Triterpene Astragaloside Jiao et al. [58]
saponin
6 Catharanthus roseus Callus culture Apocynaceae Alkaloid Vinblastine and vincristine McGehee
et al. [59]
7 Calotropis gigantea Hairy root culture Asclepiadaceae Steroid Cardenolides Sun et al. [60]
8 Corylus avellana Cell suspension culture Betulaceae Alkaloid Paclitaxel Salehi et al. [61]
9 Datura metel Hairy root culture Solanaceae Alkaloid Atropine Shakeran
et al. [62]
10 Eclipta alba L. Callus culture Asteraceae Coumestan Wedelolactone and Khurshid
demethylwedelol actone et al. [63]
Triterpene Eclalbatin
saponin
11 Hyoscyamus reticulatus L. Hairy root culture Solanaceae Alkaloid Hyoscyamine, scopolamine Moharrami
et al. [64]
12 Ophiorrhiza mungos var. Callus and cell suspension Rubiaceae Alkaloid Camptothecin Krishnan
angustifolia culture et al. [65]
0005092149.INDD 218 06-03-2021 19:02:41

13 Papaver orientale L Hairy root culture Papaveraceae Alkaloid Morphine, thebaine Hashemi
and codeine et al. [66]
14 Rauwolfia serpentina Hairy root culture Apocynaceae Alkaloid Ajmaline Srivastava
Ajmalicine et al. [67]
15 R. serpentina Transgenic hairy roots Apocynaceae Alkaloid Reserpine Mehrotra

Ajmalicine et al. [35]
16 R. serpentina Callus, leaf. Root, and stem Apocynaceae Alkaloid Reserpine Zafar et al. [68]
culture Ajmalicine
17 Rosmarinus officinalis L. Callus culture Lamiaceae Phenolic Rosmarinic acid Coskun
and caffeic acid et al. [69]
18 Salvia miltiorrhiza Transgenic hairy roots Lamiaceae Diterpenes Tanshinone Hao et al. [70]
19 Solanum khasianum Hairy root culture Solanaceae Alkaloid Solasodine and α-solanine Srivastava
et al. [67]
20 Tinospora cordifolia Callus culture Menispermaceae Alkaloid Berberine Pillai et al. [71]
21 Vitis vinifera Hairy root culture Vitaceae Phenolic Reseveratrol Hosseini
et al. [72]
22 Withania somnifera Transformed Hairy Root Solanaceae Steroid Withaferin A Yousefian
Culture et al. [40]
23 W. somnifera Adventitious root culture Solanaceae Steroid Withanolides, Withaferin A Rangaraju
et al. [73]
24 W. somnifera Hairy Root Culture Solanaceae Steroid Withanolide A, withaferin A Sivanandhan
and withanone et al. [74]
0005092149.INDD 219 06-03-2021 19:02:41

multiple shoot cultures of C. roseus. In a study of precursor feeding, Sharma et al. [79] have
demonstrated the feeding of tryptamine and tryptophan was found effective, with better
results in former.
The production of hypericin and pseudohypericin was achieved in the shoot cultures of
Hypericum hirsutum and H. maculatum, which was highly stimulated by salicylic acid elic-
itor [83]. The multiple shoot cultures of Rauvolfia serpentina were reported to produce
3-epi-α-yohimbine [84]. The production of pharmaceutical important compounds such as
casticin, rutin, neochlorogenic and p-coumaric acids was achieved in Vitex agnus-castus
agitated shoot cultures [85]. The effect of plant growth regulators (PGR) on shoot develop-
ment and biologically active indolizidine alkaloids (securinine, and allosecurinine) were
assessed in Phyllanthus glaucus (Euphorbiaceae) in vitro shoot cultures. Among tested
PGR, the highest number of shoot per explant was achieved on MS medium supplemented
with IBA 0.5 mg/L and BAP 1.0 mg/L, and the optimal rooting response was obtained in
MS + IBA 1.0 mg/L [86]. In the stationary liquid phase, the shoot cultures of Ruta graveo-
lens L. were demonstrated to produce psoralen, bergapten, xanthotoxin, isopimpinellin
(linear furanocoumarins), rutamarin (linear dihydrofuranocoumarin), kokusaginine and
skimmianine (furanoquinoline alkaloids) [87]. Kilby et al. [88] have reported the produc-
tion of quinoline alkaloid by juvenile shoot cultures of Cinchona ledgeriana. The produc-
tion of two withanolides (Withaferin A and Withanolide D) was investigated in the multiple
shoot cultures of Withania somnifera [89]. Quinoline alkaloids (camptothecin and 9-meth-
oxycamptothecin) production from shoot cultures of Nothapodytes foetida was reported by
Roja and Heble [90]. Gopalakrishnan and Shankar [91] have reported the Camptotheca
alkaloids (Camptothecin) from multiple shoot cultures of O. decumbens (0.056% dry
weight). Remarkable production of cardiotonic glycosides digitoxin and digoxin were repo-
retd in the shoot culture of Digitalis purpurea [92, 93].
11.4.2 Adventitious Root Culture

Adventitious root culture is another important choice for secondary metabolite production
and used successfully in many plant species for the production of many metabolites of high
pharmaceutical, nutraceutical and industrial values. It usually requires auxin (IBA, NAA,
IAA, etc.) supplementation to the basal salt solution for the induction and maintenance as
reported in the adventitious roots of many medicinal plants [94]. Production of several
important SMs of pharmaceutical significance was carried out using the adventitious root
cultures of diverse medicinal plants, viz., Withanolide (Withania somnifera, [95]; [96],
Silymarin (Silybum marianum, [97], Scopolamine (Scopolia parviflora, [98], Psoralen
(Psoralea coryfolia, [99]), Podophyllotoxin (Podophyllum hexandrum, [100]), Gingenoside
(Panax ginseng, [101]), Rosmarinic acid (Orthisipon stamineus, [102]), Hypericin,
(Hypericum perforatum, [103]), Andrographolide (Andrographis paniculata, [104]), etc.
The improvement of SMs production was also observed after adoption of elicitation and
bioreactor-based methods [105].
11.4.3 Callus and Cell Suspension Culture

The callus and cell suspension systems are simple, cost-effective method for secondary
metabolite production. The callus culture represents a mass of mostly undifferentiated
11.5 Conclusio 221
cells grown on semi-solid medium; however, the suspension cultures are free cell or small
group of cells grown in the form of suspension in continuously agitated liquid culture
medium. These cultures provide an alternative option to whole plant cultivation for the
production of SMs of pharmaceutical interest [106]. For instance, the accumulation of
reserpine in NaCl-treated calli was reported in R. tetraphylla [31]. Zafar et al. [107] have
reported the improvement of reserpine content in the R. serpentina callus after alumium
chloride elicitation. The callus and adventitious roots were developed from leaf segments
of shoot cultures of Cephalis ipecacuanha A. Richard in phytohormone-supplemented MS
media. The content of emetic alkaloids (emetine and cephaeline) was analysed by HPLC in
calli, root and root suspension cultures, which demonstrated their presence; however, in
calli, emetic alkaloids were present in traces [108]. Similarly, the cell suspension cultures
of medicinal plants are largely utilized for production of various therapeutic metabolites.
Some of relevant examples of such important metabolites produced through cell suspen-
sion cultures are ajmalicine (C. roseus, [109]), artemisinin (A. annua, [110]), camptothecin
(C. acuminata, [111]), crocin (Crocus sativus, [112]), forskolin (Coleus forskohlii, [113]),
gymnemic acid (G. sylvestre, [114]), hypericin (Hypericum perforatum, [115]), reserpine
(R. serpentina, [116]), etc.
11.4.4 Hairy Root Cultures

Currently, the HRCs are considered as one of the effective method for the production of
imperative SMs and also offer several other allied applications in biotechnology [11, 12, 15,
117]. In HRCs, the amalgamation of several other biotechnological interventions such as
elicitation [13], metabolic engineering [14], precursor feeding [16], bioreactor up-
scaling [49], ploidy alteration [15] are further reported to enhance the production profile of
HRCs. The HRCs have also proved to be very effective source for long-term production of
pharmaceutical lead molecules because of its genetic and biochemical stability and very
low induction and maintenance associated cost. Using HRCs, the important pharmaceuti-
cal lead molecules produced are very rich in number, some of imperative examples are
Andrographolide (A. paniculata, [118]), Artemisinin (A. annua, [57]), Ajmalicine
(C. roseus, [47]), Atropine (Datura metel, [62]), Podophyllotoxin (Linum album, [119]),
Reserpine (R. serpentina, [35]), withanolide (W. somnifera, [120]), Dopamine (Portulaca
oleracea, [121]), Camptothecin (Ophiorrhiza pumila, [122]), Tanshinone (Salvia miltior-
rhiza, [123]), etc.
11.5 Conclusion
In a nut shell, the plant in vitro cultures are widely used for the production of a variety of
SMs having therapeutic values for the treatment of many serious human ailments, viz.,
cancer, diabetes, hypertension, etc. The diverse biotechnological interventions have also
utilized to make the optimum yield of metabolite of interest, and up-scaling process
through mediation of bioreactor is also well documented. The journey so far is very excit-
ing, which provide sustainable and long-lasting alternative method for the production of
therapeutic lead plant natural metabolites that will be available for generous supply to
meet the desired pharmaceutical and commercial demand.
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231
12
Plant Diversity and Ethnobotanical Knowledge of Spices

and Condiments
Thakku R. Ramkumar1 and Subbiah Karuppusamy2
1
Agronomy Department, IFAS, University of Florida, Gainesville, FL, USA
2
Department of Botany, Botanical Research Center, The Madura College, Madurai, Tamil Nadu, India
12.1 Introduction
Spices and condiments are used for flavoring the food and beverages. They may be defined
variously that “Spices are plant parts like seed, bark, berries, buds, floral parts, fruits, ker-
nels, leaves, rhizome, latex etc., which impart strong flavor or aroma to food or drinks” [1].
These aromatic vegetable products are used in the whole, broken, or in ground form to
season food rather than for nutrients [2]. The meaning of spice derived from Latin term
spec, the noun alluding to appearance or kind. Traditional medicinal practices are using
herbs including spices, in Indian subcontinent and Chinese proximities dating back to at
least 4000 bce. In popular culture, spices usage was also connected with magic, religion,
tradition, and cultural rituals. Condiments on the other hand are usually a combination of
herbs and spices blended in a liquid form. Condiments are prepared food, mainly preserved
or fermented and used in invariable quantities depending on diner’s taste [3]. However,
International Organization for Standardization [4] defines spices and condiments, do not
show any clear-cut differences, and can be clubbed together under one term “spices and
condiments,” whichever used for flavoring and seasoning, including meat preparation,
baking, confectioning, and other food processing and preserving.
India is bestowed with rich ethnic and cultural diversity. There are about 440 ethnic
communities in this region [5]. India is also one of the mega biodiversity countries that has
the one of the world’s richest plant diversity reservoir supporting about 50% of India’s bio-
diversity [6]. Most of the ethnic communities residing in hilly tracts and slopes form small,
isolated remote villages. The ethnic people mostly depend on natural resources from the
nearby forest for their food, livelihood, and ailments. They are repository of indigenous
knowledge system belonging to agriculture, food, medicine, etc. As they have very good
knowledge of their natural resources, the local crops, wild plants, ethnic vegetables, and
indigenous fruits are mainly used in their local diet for food [7]. They use varieties of plants

232 12 Plant Diversity and Ethnobotanical Knowledge of Spices and Condiments
in ethnic food for flavoring, seasoning, coloring, and food preservation as well. About
60 varieties of spices are grown in India [8], which are well-documented from ancient
period onward [9].
The important spices traditionally traded throughout the world are mostly products of
tropical environments. The major exceptions to this group are the capsicums (chilli, paprika)
and coriander, which are grown over a much wider range of tropical and nontropical envi-
ronments as well. Production of spices and essential oils in these wet and humid environ-
ments brings special difficulties for crop and product management. Drying the crop to
ensure a stable stored product is of particular importance, and in wet humid environments,
this creates the need for efficient and effective drying systems. In the passage of time, the
uses of the spices have steadily increased; in culinary art, beverages, confectionery, liquors
and cordials, perfumery, cosmetic industry, and even notably as medicines. Today, the prin-
cipal uses of spices in medicine lie with their adjuvant and a lleviative qualities [10].
Spices are even more important today all around the world for improving taste and clamor
of food items. Most of the spices are rich sources of essential oils. Essential oils are volatile
liquid products of steam or water distillation of plant parts such as leaves, stems, bark, seeds,
fruits, roots, and plant exudates. An essential oil may contain up to several hundred chemi-
cal compounds, and this complex mixture of compounds gives the oil its characteristic fra-
grance and flavor. Usually spices are used directly or indirectly in several industries such as
cosmetics, perfumery, medicine, and pharmaceutical. The main properties of spices are
antioxidants, antimicrobial, antibiotic, and preservatives. Spices are also used for coloring
the foodstuff [11]. Many leafy spices are sometimes used as “herbal” teas for improving
general health. Apart from enhancing the taste and flavor of food, spices have been widely
believed to exert digestive stimulant action. A few medicinal properties of spices such as
tonic, carminative, stomachic, diuretic, and antispasmodic have long been recognized [12].
12.2 Habitat and Diversity of Major Spices and Condiments

in India
India is historically known as the “The home of spices.” India has produced the largest
quantity spices and condiments and also consumed and exported highest level significantly
when compared to other countries. The diverse agroclimatic regions across the subconti-
nent offer tropical, subtropical, and temperate conditions, suitable to grow almost all such
species [13]. Among the 109 spices listed by International Organization for
Standardization [4], India grows about 60 species and Indian spices flavor foods in over 130
countries of the world [14]. In India, at least 52 spices are being cultivated including tree
spices, seed spices, and herbal spices. Most of these spices can be grown alone or in combi-
nation with other crops as a system. The choice of crops depends on the physiography,
topography, soil, climate, and the market demands. Major spices, namely black pepper,
cardamom, vanilla, ginger, turmeric; tree spices nutmeg, clove, cinnamon, allspice (dried
unripe berry of Pimenta dioica) and garcinia; and seed spices, coriander, fennel, mustard
are ideal crops for inter or mixed cropping [15]. Some of the important Indian spices and
condiments with scientific name, family name, useful part, common name, distribution in
Indian states, and their cultivation status are summarized family-wise in Table 12.1.
Phenotypic images of selected spice plants also provided in Figure 12.1.
Table 12.1 Diversity and distribution of spices and condiments in India.
Family Botanical name Part(s) used Common name(s) Distribution Cultivation status
Acoraceae Acorus calamus L. Rhizome Sweet flag, myrtie flag, calamus, Throughout India Cultivated
flag root
Amaryllidaceae Allium ascalonicum L. Bulb Shallot Assam, Tripura Cultivated
Amaryllidaceae Allium cepa L. Bulb Onion Throughout India Cultivated
Amaryllidaceae Allium cepa var. aggregatum Bulb Potato onion Assam, Maharashtra Cultivated
Amaryllidaceae Allium fistulosum L. Bulb, leaves Japanese bunching onion, stony North eastern India Cultivated
leek
Amaryllidaceae Allium porrum L. Bulb, Leaves Leek, winter leek Northeastern States Cultivated
Amaryllidaceae Allium sativum L. Bulb Garlic Throughout India Cultivated
Amaryllidaceae Allium tuberosum Rottler. Bulb Indian leek Assam Cultivated
Anacardiaceae Buchanania lanzan Spreng. Seed Cuddapah almond Andhra Pradesh, Tamil Nadu Wild
Anacardiaceae Mangifera indica L. Immature fruit Mango Throughout India Cultivated
Anacardiaceae Schinus molle L. Fruit Californian pepper tree Karnataka, Kerala, Tamil Nadu Wild
Anacardiaceae Schinus terebinthifolia Fruit Brazilien pepper tree Maharashtra Cultivated
Radde.
Annonaceae Xylopia aethiopica (Dunal) Fruit Negro pepper Arunachal Pradesh, Assam Cultivated
A. Rich.
Apiaceae Anethum graveolens L. Leaves Dill Andhra Pradesh, Madhya Cultivated
Pradesh, Kerala, Tamil Nadu
Apiaceae Anethum sowa Kurz. Fruit Indian Dill Rajasthan, Gujarat, Odisha Cultivated
Apiaceae Angelica archangelica L. Fruit, petiole, Garden angelica Assam, Meghalaya, Tripura Cultivated
root
Apiaceae Anthriscus cerasifolium (L.) Leaf Chervil Assam, Arunachal Pradesh Wild
Hoffm.
(Continued)
0005092150.INDD 233 06-03-2021 19:06:05

Apiaceae Apium graveolens L. Seeds, root, Celery Assam, Nagaland, Maharashtra, Cultivated
leaves Gujarat
Apiaceae Bunium persicum (Boiss) Fruit, bulb Black caraway Rajasthan, Assam, West Bengal Cultivated
Fedtsch.
Apiaceae Carum bulbocastanum L. Fruit, Bulb Black caraway Assam, Tripura, Meghalaya Wild
Apiaceae Coriandrum sativum L. Leaves and Coriander Andhra Pradesh, Madhya Cultivated
fruits Pradesh Telangana, Tamil Nadu
Apiaceae Carum copticum L. Fruits Ajowan caraway, blond caraway Tamil Nadu, Kerala, Karnataka Cultivated
Apiaceae Cuminum cyminum L. Fruits Cumin Throughout India Cultivated
Apiaceae Eryngium foetidum L. Fruits Elephant pepper Kerala, Arunachal Pradesh Wild
Apiaceae Ferula asa foetida L. Rhizome Asafoetida Rajasthan Cultivated
exudates
Apiaceae Foeniculum vulgare Miller. Seeds Fennel Kerala, Madhya Pradesh Cultivated
Apiaceae Levisticum officinale Koch. Leaves Lovage Northern India Cultivated
Apiaceae Pimpinella anisum L. Fruits Aniseed Assam, Tripura, Rajasthan, Cultivated
Gujarat, Odisha
Asteraceae Artemisia dracunculus L. Leaves Tarragon Arunachal Pradesh Wild
Brassicaceae Armoracia rusticana Gaertn. Root Horse radish Assam, Arunachal Pradesh Cultivated
Brassicaceae Brassica juncea (L.) Czernj. Seeds Mustard Andhra Pradesh, Bihar, Madhya Cultivated
et Cosson. Pradesh, Uttar Pradesh
Brassicaceae Brassica nigra (L.) Koch. Seeds Black mustard Bihar, Uttar Pradesh Cultivated
Brassicaceae Brassica rapa (L.) Hanelt. Seed Field mustard Uttar Pradesh, Haryana, Cultivated
Chhattisgarh
Brassicaceae Sinapis alba L. Seeds White mustard Northern India Cultivated
Caesalpiniaceae Tamarindus indica L. Fruit Tamarind Throughout India Cultivated
0005092150.INDD 234 06-03-2021 19:06:05

Capparidaceae Capparis spinosa L. Flower bud Caper bud Tamil Nadu Wild, rarely
cultivated
Cleomaceae Cleome viscosa L. Seeds Jakhiya Throughout in India Wild
Clusiaceae Garcinia gummi-gutta (L.) Fruit Malabar tamarind, camboge Kerala, Karnataka Cultivated and
Robson. wild
Clusiaceae Garcinia indica (Thouars) Fruit Kokam Kerala Wild
Choisy.
Cupressaceae Juniperus communis L. Fruit Juniper berry (technically seed Assam, Meghalaya Cultivated
cone)
Fabaceae Abrus precatorius L. Root Indian liquorice, rosary pea Throughout India Wild
Fabaceae Glycyrrhiza glabra L. Root Liquorice Odisha, Andhra Pradesh, Tamil Cultivated
Nadu
Fabaceae Trigonella foenum-graecum Leaves, seeds Fenugreek Throughout India Cultivated
L.
Illiciaceae Illicium verum Hook.f. Fruit Star anise Sikkim, Meghalaya Cultivated
Iridaceae Crocus sativus L. Anther and Saffron Jammu and Kashmir Cultivated
filament
Lamiaceae Hyssopus officinalis L. Leaves Hysopp Northeastern India Cultivated
Lamiaceae Lippia graveolens Kunth. Leaves, Mexican oregano Northern India Cultivated
terminal shoot
Lamiaceae Melissa officinalis L. Leaves Balm, Lemon balm Kerala, Maharashtra, Karnataka Cultivated
Lamiaceae Mentha arvensis L. Leaves Mint, field mint Throughout India Wild
Lamiaceae Mentha citrata L. Leaves Bergamol Northeastern India Cultivated
Lamiaceae Mentha piperita L. Leaves Pepper mint Throughout India Cultivated
(Continued)
0005092150.INDD 235 06-03-2021 19:06:05

Lamiaceae Mentha spicata L. Leaves Spear mint, garden mint Throughout India Cultivated
Lamiaceae Ocimum bascilicum L. Leaves Sweet basil Throughout India Cultivated
Lamiaceae Ocimum sanctum L. Leaves Holy basil Throughout India Cultivated
Lamiaceae Origanum hortensis L. Leafy shoots Marjoram Andhra Pradesh, Tamil Nadu Cultivated
Lamiaceae Origanum vulgare L. Leaves Origano Throughout India Cultivated
Lamiaceae Petroselinum crispum Leaves Parsley Kerala, Karnataka, Maharashtra Cultivated
(Miller) Nyman.
Lamiaceae Rosmarinus officinalis L. Leaves Rosemary Kerala, Tamil Nadu, Cultivated
Maharashtra, Assam
Lamiaceae Salvia officinalis L. Leaves Garden sage, Dalmatian sage Kerala, Tamil Nadu, Cultivated
Maharashtra
Lamiaceae Satureja hortensis L. Leaves Summer savory Northern India Cultivated
Lamiaceae Thymus vulgaris L. Leaves Thyme Throughout India Cultivated
Lauraceae Cinnamomum burmanii Bark Indonesian Cassia Meghalaya, West Bengal Cultivated
Blume.
Lauraceae Cinnamomum cassia L. Bark Cinnamon cassia Kerala Cultivated
Lauraceae Cinnamomum loureiroi Bark Vietnamese cassia bark Kerala, Karnataka Cultivated
Nees.
Lauraceae Cinnamomum tamala Nees. Leaves Indian cassia bark Kerala, Karnataka, Tamil Nadu Cultivated
Lauraceae Cinnamomum verum Blume. Leaves Sri Lankan cinnamon, common Kerala, Tamil Nadu Wild
cinnamon
Lauraceae Laurus nobilis L. Leaves Bay leaf, true laurel Meghalaya, Nagaland Cultivated
Lauraceae Phoebe cooperiana Kanjlal& Leaves Assamese mekhali Arunachal Pradesh, Assam Wild
Das
Malvaceae Bombax ceiba L. Fruit Maratimokka, cotton tree Kerala, Tamil Nadu Wild
0005092150.INDD 236 06-03-2021 19:06:05

Moringaceae Moringa pterygosperma Leaves Drumstick leaf Throughout India Cultivated

Gaertn.
Myristicaceae Myristica argenteaWarb. Kernal and Papuan nutmeg Gujarat, Odisha, Assam Cultivated
mace
Myristicaceae Myristica fragrans Houtt. Kernal and Nut meg Throughout India Cultivated
mace
Myrtaceae Syzygium aromaticum Flower bud Clove Kerala, Tamil Nadu Cultivated
Thunb.
Myrtaceae Pimenta dioica (L.) Merr. Leaves Allspice Tamil Nadu Cultivated
Myrtaceae Pimenta racemosa (Miller) J. Leaves West Indian bay Arunachal Pradesh Cultivated
Moore.
Orchidaceae Vanilla planifolia Andrews. Fruit Vanilla Kerala, Tamil Nadu Cultivated
Orchidaceae Vanilla tahitensis J. Moore. Fruit Tahitian vanilla West Bengal, Assam Cultivated
Orchidaceae Vanilla pompona Schiede. Fruit Pompona vanilla Arunachal Pradesh, Nagaland Cultivated
Pandanaceae Pandanus amaryllifolius Leaves Pandanwangi Maharashtra Wild
Roxb.
Papaveraceae Papaver somniferum L. Seeds Poppy, blue maw Jammu and Kashmir, Sikkim Cultivated
Parmeliaceae Parmotrema perlatum Thallus Kalpasi, black stone flower, Northeastern India Wild
(Huds.) Choisy. lichens
Pedaliaceae Sesamum indicum L. Seeds Sesame Throughout India Cultivated
Piperaceae Piper guineense Schum. Fruits West African pepper Assam, Arunachal Pradesh Cultivated
Piperaceae Piper longum L. Fruits Long pepper Kerala, Tamil Nadu Cultivated
Piperaceae Piper mullesua Buch.-Ham. Fruits Wild pepper Maharashtra, Karnataka, Wild
ex D.Don. Kerala, Tamil Nadu
Piperaceae Piper nigrum L. Fruits Black pepper Kerala, Tamil Nadu Cultivated
(Continued)
0005092150.INDD 237 06-03-2021 19:06:05

Poaceae Cymbopogon citratus (DC.) Leaves Lemon grass, West Indian lemon Throughout India Wild
Stapf. grass
Poaceae Cymbopogon nardus L. Leaves Sri Lankan citronella Throughout India Wild
Punicaceae Punica granatum L. Seeds Pomegranate Throughout India Cultivated
Ranunculaceae Nigella damascena L. Seeds Damas black cumin Rajasthan Cultivated
Ranunculaceae Nigella sativa L. Seeds Black cumin Jammu and Kashmir, Rajasthan Cultivated
Rutaceae Murraya koenigii (L.) Leaves Curry leaf Throughout India Cultivated
Sprengel.
Rutaceae Zanthoxylum armatum DC. Fruit Mountain prickle pepper Arunachal Pradesh Wild
Rutaceae Zanthoxylum bungee Planch. Fruit Chinese prickly ash pepper Arunachal Pradesh Wild
Rutaceae Zanthoxylum Fruit Chinese pepper Arunachal Pradesh, Wild
acanthopodium DC.
Rutaceae Zanthoxylum oxyphyllum Fruit Wild thorn pepper Arunachal Pradesh Wild
Edgew.
Rutaceae ZanthoxylumrhetsaDC. Fruit Small thorn pepper Karnataka, Odisha, Kerala, Wild
Arunachal Pradesh
Solanaceae Capsicum annuum L. Fruit Wild chilli, capsicum Throughout India Cultivated
Solanaceae Capsicum frutescens L. Fruit Bird eye chilli Throughout India Cultivated
Zigiberaceae Kaempferia galanga L. Rhizome Galanga Kerala Cultivated
Zingiberaceae Aframomum angustifolium Fruit, seeds Madagascar cardamom Assam, Meghalaya, Tripura Cultivated
Schum.
Zingiberaceae Aframomum hanburyi Fruit, seeds Cameroon cardamom Assam, Tripura, Nagaland, Cultivated
Schum. Maharashtra
Zingiberaceae Aframomum corrorim Engl. Fruit, Seeds Korarima cardamom Assam, Meghalaya, Nagaland Cultivated
0005092150.INDD 238 06-03-2021 19:06:05

Zingiberaceae Aframomum melegueta Fruit, seeds Grain of paradise, guinea grains Assam, Manipur, Mizoram, Cultivated
Schum. Nagaland
Zingiberaceae Alpinia calcarata Roscoe. Rhizome Galanga Southern states, Assam Wild
Zingiberaceae Alpinia galanga (L.) Willd. Rhizome Greater galangal Throughout India Cultivated
Zingiberaceae Alpinia officinarum Hance. Rhizome Lesser galangal Assam, Nagaland, Tripura Cultivated
Zingiberaceae Amomum aromaticum Roxb. Fruit, seeds Bengal cardamom Throughout India Cultivated
Zingiberaceae Amomum kepulaga Fruit, seeds Round cardamom, chester Arunachal Pradesh Cultivated
Sparague. cardamom, Siamese cardamom
Zingiberaceae Amomum subulatum Roxb. Fruit Large cardamom Arunachal Pradesh, Kerala, Cultivated
Sikkim, Tamil Nadu
Zingiberaceae Curcuma amada Roxb. Rhizome Mango ginger Arunachal, Maharashtra, Kerala Cultivated
Zingiberaceae Curcuma longa L. Rhizome Turmeric Telangana, Tamil Nadu Cultivated
Zingiberaceae Curcuma zedoaria Roscoe. Rhizome Zedoary Kerala Cultivated
Zingiberaceae Elettaria cardamomum (L.) Fruits Cardamom, Green cardamom, Kerala, Karnataka, Tamil Nadu Cultivated
Maton. small cardamom
Zingiberaceae Zingiber montanum Rhizome Wild ginger, mountain ginger Arunachal Pradesh Wild
(Koenig.) Link.
Zingiberaceae Zingiber officinale Roscoe. Rhizome Ginger Throughout India Cultivated
0005092150.INDD 239 06-03-2021 19:06:05

a b c
d e f
g h i
j k
Figure 12.1 Selected Indian spice and condiment plants. (a) Alipnia calcarata, (b) Cinnamomum
zeylanicum, (c) Costus speciosus, (d) Curucuma neilgherrensis, (e) Elettaria cardamomum, (f) Melissa
officinalis, (g) Pimenta dioica, (h) Piper longum, (i) Piper nigrum, (j) Piper mileusa, (k) Zanthoxylum
armatum. Source: Santosh Kumar Upadhyay.
Ethnobotanical evidences indicate that the use of turmeric in India has begun in ancient
time in connection with Goddess Sakthi worship of divine mother and later as a commodity
of trade as coloring material and as a condiment [16]. The genus originated in
12.3 Ethnobotanical Context of Spices and Condiments in Indi 241
Indo-Malayan region and has spread from Southeast Asia to tropical West Africa and East
Africa and later introduced to Caribbean islands and to Americas. Turmeric is popular
among rice-eating people of South Asia, Southeast Asia, and Indochina (continental por-
tion of Southeast Asia) as a spice and condiment. Turmeric is the main cash crop in the
tribal dominated districts of Kandhamal, Gajapati, Ganjam, Mayurbhanj, and Koraput in
Orissa since time immemorial [17].
The cardamoms are the capsules of dried fruits in different genera of the Zingiberaceae
family, primarily Elettaria, Amomum, and Aframomum. Among them, Elettaria cardamo-
mum (cardamom/green cardamom/small cardamom) is the most important and is grown
predominantly in southern India [18]. The false cardamom, large cardamom, and black
cardamom from the allied genus Amomum are native to Nepal, Sikkim, Bengal, and
Southeast Asian countries. African cardamom, which is botanically known as Aframomum
danielli, is native to Southeast Africa especially in Tanzania, Cameroon, Madagascar, and
Guinea. Small cardamom is extensively cultivated in Nepal and Sikkim and to a limited
extent with the large cardamom (Amomum subulatum). However, international trade is
now limited to Asian countries as far as small and large cardamoms are concerned because
of high prices. Worldwide, cardamom is recognized as the “queen of spices” for its pleasant
aroma and taste and is the third most expensive spice after saffron and vanilla [19, 20].
Spices produced from tree crops are called “Tree spices.” Tamil Nadu, Kerala, Andhra
Pradesh, Maharashtra, and Karnataka are the major states cultivating tree spices in larger
areas of India. There are 17 tree spices, commonly grown in India, which are clove, nut-
meg, cinnamon, tamarind, garcinia, curry leaf, and allspice [12]. The commercially impor-
tant products have been obtained from whole spice, ground spice oil, and oleoresin. The
Western Ghats belt of Kanyakumari and Nilgiris districts of Tamil Nadu and in a few areas
in Karnataka and Kerala are cultivating the clove crop. Nutmeg cultivation is more com-
mon in Kerala and limited areas of Tamil Nadu and Karnataka. All spice was cultivated
rarely in the gardens of Kerala, Tamil Nadu, Karnataka, West Bengal, and Odisha [21].
In India, Kerala is highly suitable for the cultivation of spices. Important spices culti-
vated are black pepper, cardamom, ginger, turmeric, clove, garcinia, nutmeg, and mace.
Out of this 95% of the total production is contributed by pepper, cardamom, ginger, and
turmeric. Pepper and cardamom are cultivated in the high ranges, while ginger and tur-
meric predominate in plains. The major production centers are Idukki and Wayanad
districts [12].
12.3 Ethnobotanical Context of Spices and Condiments

in India
The earliest literary record in India on spices is the Rig Veda (at least or before 4000 bce),
one of the ancient Hindu scriptures, listing more than a thousand healing plants including
spices and condiments. Thus, the story of Indian spices dates back to at least 6000 years
into the past. In the modem world, major trade is related to eating, and the spices provide
the major thrust – traditionally a country of agriculture [15, 22]. Domestication of turmeric
is probably much earlier to Indus valley civilization [23]. In the current scenario of ever-
increasing human stress, ethnomedicinal plants having antioxidant properties are thought
to be extremely fruitful to relieve stress and improve health and eventually economy. Many
Indian spice plants have been investigated for their beneficial use as antioxidants or source
of antioxidants using presently available experimental techniques [24]. This plant-based
traditional knowledge has become a recognized tool in the search for new sources of drugs
and nutraceuticals [25].
Tribals of Eastern Ghats in Andhra Pradesh make use of some rare plants for their
condiments and culinary purposes that are specific to that region, including dried fruits
of Tacca leontopetaloides and tubers of Decalepis hamiltonii, which are rare spices [26].
Arunachal Pradesh was inhabited by Adi, Apatani, Nyishi, Galo, Khampti, Khowa,
Mishmi, Idu, Taroan, Momba, Sherdukpen, Singpho, Hrusso, Tagin, Khamba [27]. Some
of the plant species are recorded in tribal belts of Arunachal Pradesh as new source of
spice plants such as Acmella paniculata, Dendrocalamus hamiltonii, Etlingera lingui-
formis, Gynura prcoumbens, Hydrocotyle sibthorpioides, Magnolia oblonga, Oenanthe
javanica, Paederia foetida, Perilla frutescens, Persicaria nepalensis, Phyllostachys bambu-
soides, and Spilanthes acmella. Some ethnic tribal communities of Assam, namely
Ahom, Deori, Mishing, Sonowal-kachari use 51 species as spices and condiments,
including Garcinia pedunculata, Hibiscus sabdariffa, Houttuynia cordata,
Neocinnamomum caudatum and Peperomia pellucida, which are new spice species [28].
Tribes in Maharashtra such as Pawara, Bhil, Mavachi, Kokani, and Tadavi use 26 com-
mon spices and added a new spice species of Guizotia abyssinica as used in food
flavoring [29].
Owing to their aromatic properties, three species, namely Artemisia scoparia (seeds,
flowers, and leaves), Carum carvi (fruits, seeds), and Murraya koenigii (leaves), are added
to pulses and vegetables as condiment and spice in Jammu and Kashmir region. Ar. sco-
paria is a locally grown wild spice [30]. The Tangkhul people of Manipur region are mainly
dependent on the forest, as forest plants are gathered for food, medicinal, spices, fuel, etc.
They use at least 30 plant species as spice belonging to 17 genera of 8 families. Several plant
species used by them are recorded as new spice species such as Allium hookeri, Allium
tuberosum, Costus speciosus, Curcuma cassia, Elshotzia blanda, Elshotzia communis,
Hedychium coronarium, Hedychium marginatum, Persicaria posumbu, Zingiber cassumu-
nar and Zingiber zerumbet [31]. Babu et al. [8] assessed the status of traditional practices of
farmers and women, in particular for storage of spices and condiments in Odisha especially
for ginger (Zingiber officinale), turmeric (Curcuma longa), chilli (Capsicum annuum),
onion (Allium cepa), garlic (Allium sativum), and coriander (Coriandrum sativum).
Observations revealed that a large number of farmers still practice the traditional storage
system. Ginger and turmeric are stored in pit method, heap method and in situ method
while chilli, onion, and garlic are stored in mesh bags and hanging method. In traditional
method of storage, farmers primarily rely on traditional practices using local resources [8].
The Sikkim state was inhibited by various ethnic tribes such as Lepcha, Bhutis, Limboo,
and Nepalese [32]. The tribes live mostly on hilltops and on slopes, forming small and iso-
lated villages. It is important to save this traditional knowledge of biological heritage and
explore new resources. Traditional and ethnic knowledge has played a significant role in
the discovery of novel ideas about conservation of natural resources. Spices have good anti-
oxidant and preservative properties as well as antimicrobial and antibiotic properties, and
therefore, are also used for medicinal purposes, 14 such species are used by tribes of
12.4 Major Spices and Condiments in Indi 243
Sikkim [7]. Spices and condiments used by traditional communities of Tripura were also
record [33]. In West Bengal, local communities using 32 spices distributed in 27 families
including some wild plants such as Zanthoxylum armatum were recorded [34].
Western Ghats are tribal-dominated areas in southern India where Kadar, Malasars,
Mudhuvan, Kurumbans, Kaatunaickens, Thodar, Kanis, and Paliyars reside. Still they fol-
low traditional way of life with close association of forest vegetation for their livelihood
materials including food. Several local spice and condiments species are recorded as they
are used for preservation, flavoring, and coloring material of the food stuffs. Some of the
plants such as Costus speciosus, Garcinia gummi-gutta, Curcuma neilgherrensis, Syzygium
palghatense, Curculigo orchioides, Cleome monophylla are exclusively used as spice plants
in this region [35]. In Kerala and Tamil Nadu, crushed cardamom capsules are boiled with
tea and water to impart a pleasant aroma to tea, which is popularly called “Elakkai tea,”
and which has been used to relieve tiredness and depression. Cardamom capsules contain
significant concentration of β-carotene [36]. In traditional medicine, consumption of car-
damom daily with a table spoon of honey improves the eyesight [37]. However, some peo-
ple believe that excessive uses of cardamom capsules may cause impotency [20]. A total of
11 spices out of 109 ISO approved spices are most frequently used in South Indian cuisines,
particularly in Kerala and Tamil Nadu [38].
12.4 Major Spices and Condiments in India
12.4.1 Black Pepper

Piper nigrum is a perennial climbing vine grown for its berries. The matured and dried ber-
ries are used in flavoring and medicinal purposes. India is a main producer, consumer, and
exporter of black pepper in the world. Annually it produces 17,563 tonnes of black pepper
worth Rs. 150.95 crore that were exported to various other countries mainly from Kerala
and Karnataka and Tamil Nadu [11]. It is used in different medicines from very ancient
periods. Long pepper (Piper longum) is a slender aromatic climber whose spike is widely
used in Siddha, Ayurvedic, and Unani systems of medicines particularly for diseases of
respiratory tract. Pipalarishta, Pippalyasava, Panchakola, Pippalayadilauha, and
Lavanabhaskarchuran, which include contributions from both P. nigrum and P. longum,
are common ayurvedic preparations [10]. Combination of sukku (dried Z. officinale) milagu
(dried fruits of P. nigrum), and thippili (dried fruits of P. longum), generally referred as
sukku-milagu-thippili, is a common home medicine for cold in South India, especially
Tamil Nadu.
12.4.2 Capsicums
Capsicums are dried and processed fruits of annual peppers (Ca. annuum var. annuum;
Capsicum chinense; Capsicum frutescens) commonly referred as chilli or chili pepper or
Chile pepper or bell pepper. A rainfall of 600–1250 mm is desirable. Rainfall is needed over
the growing season but is not needed as the fruits ripen. Heavy rain during flowering
adversely affects pollination and wetness at ripening encourages fungal spoilage. Capsicums
flourish in warm sunny conditions, require 3–5 months with a temperature range of
18–30 °C; growth retards at below 5 °C, and frost kills the plants at any growth stage. A
seedbed temperature of 20–28 °C is optimum for germination. Red chili powder, a common
spice, is dried and powdered red chilli fruits. Being the hottest part of the chili, the powder
is exceptionally strong and used in small quantities. Originating in the Americas, chillies
were then introduced to India by the Portuguese and since then became an integral part of
Indian cooking. Chillies are used as powder, crushed, and in its whole form in various
South Indian curries.
12.4.3 Cinnamomum
Cinnamomum verum bark is widely used in aromatic rice preparations such as Biriyani and
Pulao in India. It is a sweet-tasting spice with a warm and woody aroma. These properties
make it a great ingredient to be used in cakes and desserts. Cinnamomum bark powder is
highly favored for flavoring processed drinks in western hemisphere. Apart from adding
flavor to food, cinnamon also has various health benefits; thought to help prevent cancer
and lower blood pressure. It is predominantly grown along the Western Ghats of Kerala
and Tamil Nadu [39]. While the origins of this spice can be traced back to India, it is also
native to Sri Lanka. Ci. verum is an evergreen tree of Lauraceae family whose bark and
leaves are strongly aromatic. It is useful in bronchitis, asthma, cephalalgia odontalgia, car-
diac diseases, diarrhea, uropathy, nausea and vomiting, flatulence, fever, halitosis, and skin
protection [40].
12.4.4 Coriander
Coriandrum sativum, is a member of the parsley family, and its seeds are oval-shaped,
ridged, and turn from bright green to beige when ripe. This spice tastes sweet and tangy,
with a slightly citrusy flavor. This omnipresent spice is probably the oldest in the world and
is widely grown in the states of Rajasthan, Madhya Pradesh, and Tamil Nadu. Coriander
seeds are also used as an alternative to salt. Seeds and leaves contain essential oils ranging
from 0.1 to 1.0%, which is used for preparation of soaps of pleasant odor and good leather-
ing property. The dried fruit powder is used to flavor foods, such as pickles, sauces, and
confectionery. The fresh leaves are added in almost all-sauce-like preparations in Tamil
Nadu. The distilled essential oils from the fruits are used in perfumes, soaps, candy, choco-
lates, tobacco, meat products, baked food, canned foods, liquors and alcoholic beverages,
and also in pharmaceutical preparations.
12.4.5 Cumin
Cuminum cyminum L., another Apiaceae member, is used to add a smoky note and a robust
aroma to most Indian curries and vegetables. Fried in its dry form or roasted before use,
cumin seed is usually the first spice added while cooking Indian dishes. It is also dry roasted
and converted to powder before being added to dishes such as pudding and buttermilk. It
is used to flavor rice, stuffed vegetables, many savory dishes, and curries. Since it burns
easily and can become overpowering, it is used sparingly. Cuminol or cuminaldehyde is the
12.4 Major Spices and Condiments in Indi 245
typical volatile principle present in the cumin seeds, which is the reason for characteristic
aroma of the cumin. The cumin seeds are used for flavoring vegetables, pickles, soups,
sausages, cheese and for seasoning breads, cakes, and biscuits. The seeds are also used for
the preparation of traditional and homeopathy medicines prescribed for several common
ailments. The seeds have major properties such as carminative, stimulant, astringent, and
emmenagogue.
12.4.6 Cardamom
Elettaria cardamomum (green/small cardamom) is the concerned species, a tall growing
perennial herb from Zingiberaceae. The fruits borne on panicles at the base of the plants
have trilocular capsule with 15–20 seeds. The natural altitudinal growing range is between
750 and 1500 m while the most productive cultivated zone is 1000–1200 m. The annual
rainfall required is usually 2500–4000 mm in the monsoon belt. A temperature range of
10–35 °C occurs over the production areas with a lower limit of about 17 °C, and an opti-
mum temperature between 22 and 24 °C is favored. Cardamom grows naturally in shade
but will produce good yields in only partial shade if well-watered. It is an indigenous spice
of the land of the Malabar Coast in India. This is the third most expensive spice in the
world, mainly because it is hand-harvested and requires a lot of manual work [41]. While
the green cardamom has a mild and light eucalyptus tone to it, the black/large cardamom
(A. subulatum) is spicy, smoky, and generally used only for its seeds. The most common use
for cardamom is to enhance the flavor of tea and puddings [11].
12.4.7 Fennel
Foeniculum vulgare is an annual crop from Apiaceae, which is commonly cultivated in
frost-free area of Rajasthan and Gujarat. The fennels are aromatic fruits and used in most
kinds of Indian foods. Fennel seeds for their fragrant odor and pleasant aromatic taste due
to the presence of aromatic volatile oil principles are widely used in soaps, pickles, meat
dishes, sauces, pastries, confectioneries, etc. Besides fruits, the tender leaves are used in
salads. The seeds are used in several Allopathic, Siddha, and Ayurvedic medicines, which
are administered in diseases such as cholera, piles, gripping, constipation, dysentery, and
diarrhea.
12.4.8 Ginger
Zingiber officinale is an herbaceous perennial from Zingiberaceae, the rhizome of which is
used as a spice, and it is also one important spice native of South and Southeast Asia. It is
recommended as a medicine in Siddha and Ayurveda for curing liver complaints, flatu-
lence, anemia, rheumatism, piles, and jaundice. Kerala and Meghalaya are major ginger-
growing states in India. India exported 7,250 tonnes of ginger to the value of the Rs. 40.755
cores annually [11]. Dried ginger in locally called as sukku, and the decoction is the first
home remedy for illness in much of southern parts of the country. Sukku decoction is also
taken with milk and jaggery (a locally processed sugar), also a traditional greeting drink
for guests.
12.4.9 Mustard Seed

In Indian cooking, brown mustard (Brassica juncea L.) seeds are more commonly used
than the black mustard (Brassica nigra L.) seeds. They belong to Brassicaceae. These seeds
can be fried whole in order to flavor oil that is then used for cooking raw food. This favored
oil can also be used as a garnish. While the seeds are native to Rome, the earliest reference
to their use is in stories of Buddha, where it refers the seeds to justify death as a natural
phenomenon [42]. Rapeseed cultivation is confined only to northern India because of late
maturity and shattering of pods owing to high temperature prevailing during harvest in
February–March.
12.4.10 Nutmeg
Myristica fragrans of Myristicaceae is the common nutmeg, while Myristica argentea, the
papuan nutmeg also is referred as nutmeg. Nutmeg is a perennial tree, reaching up to 20 m
in height. The useful part of the nutmeg is kernel of the seed, while mace is the net-like
crimson-colored leathery outer growth (aril) covering the shell of the seed. Nutmeg pickle
is one of the favorites in southern parts of India. The tree requires an optimal growing tem-
perature between 20 and 30 °C and the annual rainfall should be between1500 and
2500 mm. The common nutmeg cultivating areas of India are Kerala and parts of Tamil
Nadu [11]. The seed and mace of nutmeg, M. fragrans are useful in vitiated conditions of
kapa and vata, inflammations, cephalalgia, helminthiasis, dyspepsia, flatulence, cough,
asthma, diarrhea, vomiting, ulcer, hepatopathy, skin diseases, cardiac disorders, fever, and
generally debility. Thriphaladichoorna, Karppooradichoorna, Athisaragrahanichoorna,
jeerakadichoorna, etc., in Siddha and Ayurveda contain nutmeg kernel as well as mace.
12.4.11 Saffron
Crocus sativus is the most expensive spice in the world. It is cultivated predominantly in the
regions of Jammu and Kashmir and to a lesser extent in Himachal Pradesh and Ladakh
regions. Saffron is derived from the stigma of crocus flowers. Saffron is believed to be more
valuable than gold. The most striking feature of this spice is its pungent, honey-like aroma.
The deeper the color of saffron, the purer it is. It is often used after being soaked in water
or milk, which softens its strong aroma and taste. The glycosides crocin and picrocrocin are
the coloring principles and the bitter substance of saffron. Saffron is an important ingredi-
ent of the Ayurvedic and Unani systems of medicine in India other than food additives and
coloring agent. The major properties of saffron are nervous stimulant, helping in urinary,
digestive, and uterine troubles.
12.4.12 Turmeric
Curcuma longa, another spice belonging to the ginger family, turmeric is probably the most
commonly used spice in India. Turmeric was predominantly used as a dye and in Siddha
medicine for thousands of years. Derived from the roots of C. longa, a leafy plant native to
India, turmeric has an earthy consistency and a warm aroma and taste. Mainly used for its
12.5 Importance of Indian Spice 247
flavor and color, turmeric also has antiseptic qualities and is therefore used for its health
benefits as well. Turmeric forms an integral part of the rituals, ceremonies, and cuisine.
Due to the strong antiseptic properties, turmeric has been used as a remedy for all kinds of
poisonous affections, ulcers, and wounds. It gives good complexion to the skin. The rhi-
zomes of mango ginger (Curcuma amada) are useful in vitiated conditions of pitta, ano-
rexia, dyspepsia, flatulence, colic, bruises, wounds, chronic ulcers, skin diseases, pruritus,
fever, constipations, strangury, hiccough, cough, bronchitis, sprains, gout, halitosis, otalgia,
and inflammations [16]. C. aromatica is another Zingiberaceous perennial tuberous herb
with aromatic yellow rhizome, which is internally creamy in color and the fresh root has a
camphoraceous odor. Rhizomes are used in combination with astringents and aromatics
for bruises, sprains, hiccough, bronchitis, cough, leukoderma, and skin eruptions.
Maximum area under turmeric is in Andhra Pradesh, followed by Maharashtra, Tamil
Nadu, Odisha, Karnataka, and Kerala [23].
12.4.13 Vanilla
Vanilla belongs to the member of Orchidaceae with potential economic values due to its
unique flavor and pleasant aroma of its fruits. Vanillin is an aromatic substance obtained
from the unripe fruits of Vanilla planifolia. This plant is native to South-Eastern Mexico
and Central America. Vanilla tahitensis (Tahitian vanilla) and Vanilla pompon are also cul-
tivated in some parts of the South Pacific Islands for vanillin extraction. Vanilla was intro-
duced in India during 1990s for covering an area of 90 hectares. At present, it is widely
cultivated in Kerala and Tamil Nadu as an intercrop of coconut, areca nut, and black pep-
per plantations.
12.5 Importance of Indian Spices
Indian spices possess varieties of phytochemicals, for example, in curry leaves:

α-Amorphene, γ-Eudesmol, β-Pinene, α-Terpinolene, Limonene, (Z)-β-Ocimene. Terpenes,
Sesquiterpenes, Eucalyptol, Terpinyl acetate are found in bay leaves. In hemp seeds,
Cannabinol, Tetrahydrocannabinol, Cannabidiol, Tetrahydrocannabivarin, Cannabivarin,
Cannabichromene are found. Black pepper seeds contain Piperine, Guineensine,
Piperamide, and PiperoleinB [43]. Some pharmaceutically important compounds are
Eugenol (allspice, cinnamon, cassia, clove), Piperine (black pepper), Gingerol (ginger),
Myristin (nutmeg), Curcumin (turmeric), and Vanillin (vanilla) (Table 12.2).
These are phytochemical compounds that are accumulated in the usable spice and con-
diments and are essential for the quality of the spices [44]. Almost all the spices and condi-
ments are aromatic in nature, and it is by their respective volatile phytochemical usually
essential oils with composition of several terpenoids and oleoresins. The essential oils and
oleoresins help to impart specific aroma to the food, act as antioxidants, are active against
microbes, preserve from chemical deterioration, and increase shelf-life of the food. They
act as natural preservative. Quality of the spice is determined by the quality of respective
phytochemicals present in them. Indian spices and condiments possess quality aromatic
chemicals with superior quality [45]. Flavonoids and essential oils present in spice and
Table 12.2 Important flavor compounds in selected Indian spices and condiments.
Spice Important flavor compound(s)
Allspice Eugenol, 𝛽-caryophyllene

Anise (E)-Anethole, Methyl chavicol
Sweet basil Methyl chavicol, Linalool, Methyl eugenol
Bay laurel 1,8-Cineole
Black pepper Piperine, S-3-Carene, 𝛽-caryophyllene
Caraway d-Carvone, Carvone derivatives
Cardamom α-Terpinyl acetate, 1-8-Cineole, Linalool
Chilli Capsaicin, Dihydrocapsaicin
Cinnamon cassia Cinnamaldehyde, Eugenol
Clove Eugenol, Eugenyl acetate
Coriander d-Linalool, C10-C14-2-alkenals
Cumin Cuminaldehyde, p-1,3-Mentha-dienal
Dill d-Carvone
Fennel (E)-Anethole, Fenchone
Ginger Gingerol, Shogaol, Neral, Geranial
Mace α-Pinene, Sabinene, 1-Terpinen-4-ol
Marjoram e- and t-Sabinene hydrates, 1-Terpinen-4-ol
Mustard Allyl isothiocyanate
Nutmeg Sabinene, α-Pinene, Myristicin
Oregano Carvacrol, Thymol
Origanum Thymol, Carvacrol
Parsley Apiole
Peppermint 1-menthol, Menthone, Menthofuran
Rosemary Verbenone, 1-8-cineole, Camphor, linanool
Saffron Safranal
Sage, Clary Salvial-4 (14)-en-1-one, Linalool
Savory Carvacrol
Spear mint 1-Carvone, Carvone derivatives
Tarragon Methyl chavicol, Anethole
Thyme Thymol, Carvacrol
Turmeric Turmerone, Zingiberene, 1,8-Cineole
Vanilla Vanillin, p-OH-Benzyl-methyl ether
condiments act against a range of human illness including cancer, cardiovascular disease,
nervous diseases, and digestive problems [46]. Gingerol in ginger is also an intestinal
stimulant and promoter of the bioactivity of drugs. Capsaicin in chilli is an effective coun-
terirritant used in both pharmaceuticals and cosmetics. Fenugreek, onion, and garlic help
12.6 Spice Plantation and Cultivation in Indi 249
to lower cholesterol levels. A number of spices have also been identified as having antimi-
crobial properties. All these compounds are rich in Indian spices [47].
Selection of germplasm for cultivation is an important step for maintaining the quality of
spice species. The classic sage is Salvia officinalis but widely traded Salvia triloba and Salvia
tomentosa are commercial sages. Similarly, thyme is usually referred to as Thymus vulgaris,
but most thymes traded are a mixture of Thymus capitatus, Thymus serpyllum, and T. vul-
garis. The well-known turmeric is botanically defined as C. longa, but there are many sub-
species such as Alleppy turmeric and Cuddapah turmeric used to market with varied
qualities. The qualities of spices determined the trade niche. Hence, several spices are gath-
ered from their natural habitat where mixing of their coped one and wild varieties occurred
in that location [48].
Currently, biomedical efforts are focused on their scientific merits, to provide science-
based evidence for the traditional uses and to develop either functional foods or nutraceu-
ticals. The Indian traditional medical systems use turmeric for wound healing, rheumatic
disorders, gastrointestinal symptoms, deworming, rhinitis, and as a cosmetic. Studies in
India have explored its anti-inflammatory, cholekinetic, and antioxidant potentials with
the recent investigations focusing on its preventive effect on pre-carcinogenic, anti-
inflammatory, and anti-atherosclerotic effects in biological systems. Both turmeric and
curcumin were found to increase detoxifying enzymes, prevent DNA damage, improve
DNA repair, decrease mutations rate and tumor formation, and exhibit antioxidative
potential [49]. Recently several molecular targets have been identified for therapeutic or
preventive effects of turmeric. Fenugreek seeds, a rich source of soluble fiber used in
Indian cuisine, reduce blood glucose and lipids and can be used as a food adjuvant in dia-
betes. Similarly garlic, onions, and ginger have been found to modulate favorably the
process of anti-carcinogenesis [50].
12.6 Spice Plantation and Cultivation in India
India produces 75 varieties of spices of the 109 listed by ISO and is also the world’s largest
producer. According to the Spices Board of India, every state of India is cultivating at
least 10 varieties of spice plants, which are utilized in domestic use and export. The black
pepper production in Kerala (94.1%) and followed by Karnataka (3.6%) contributed major
production in India [3]. India is particularly suitable for cultivation of many spices due to
existing various soil-related parameters such as soil moisture, water content, soil tem-
perature, and soil nutrients, environmental parameters such as climate and
rainfall [51].
As a result of diverse agroclimatic conditions, India produces more than 20 seed
spices. Cumin, coriander, dill seeds, fenugreek, and fennel are the major seed spices
cultivated in the country. Different states are known for different spices, but seed spices
are mostly grown in Rajasthan and Gujarat with more than 80% contribution. [52]. India
has been producing 7.07 million tonnes of spices, coming from 3.52 mha area during
2015–2016. Seed spices play a crucial role in Indian spice economy contributing 50.31
and 21.30% area and production share to nation’s total spices. Individually chillies,
cumin, coriander, garlic, and fenugreek are the largest grown spices in the India with
23.05, 22.79, 17.48, 8.39, and 6.42% area share to total spice and 21.88, 7.25, 8.06, 23.07,
and 3.56 % production share to total spices production, respectively [53]. Tamil Nadu
state occupies the second position in both production and area under turmeric among all
the states in India after Odisha [54]. Small cardamom is cultivated in the states of Kerala,
Karnataka, and Tami Nadu in India about 50,000 hectares intercropping with black pep-
per plantations [55]. In India, cardamom is being cultivated majorly in Cardamom hill
range of Western Ghats covering an area of 1050 km2 designated as Cardamom hill
reserves [41].
Mustard cultivation is also being extended to nontraditional areas of southern states such
as Karnataka, Tamil Nadu, and Andhra Pradesh. It is cultivated in 26 states in the northern
and eastern plains of the country, about 6.8 mha is occupied under these crops. Rajasthan
is the giant mustard-growing state and alone contributes 43% of the total mustard seed
production in India. The cultivation of brown sarson, which once dominated the entire
rapeseed–mustard-growing region is now shadowed by Indian mustard (B. juncea). There
are two different ecotypes of brown sarson (Brassica rapa var. brown sarson) and lotni (self-
incompatible). The lotni is predominantly cultivated in colder regions of the country par-
ticularly in Kashmir and Himachal valley. Yellow sarson (Brassica rapa var yellow sarson)
is now mainly grown in Assam, Bihar, north-eastern states, Odisha, eastern Uttar Pradesh,
and West Bengal [56].
A recent analysis showed that growth and instability are there in spice cultivation in
India. For example, chilli registered a higher production and yield growth in Andhra
Pradesh, Karnataka, and at all India level. The area growth was 1.32% per annum in Andhra
Pradesh and negative in Karnataka (−1.05%), Maharashtra (−1.85%), and at all India level
(−0.06%). However, West Bengal registered a positive and significant growth in area, pro-
duction, and yield of chilli during 2008–2009. Black pepper has registered a substantial
growth in both area and production in Karnataka and Kerala states and also at all India
level. Interestingly, yield growth was found to be negative in Karnataka. Turmeric produc-
tion growth was found to be the highest in Andhra Pradesh (7.58%) and about 4–5% in
Karnataka, Odisha, and at the national level, while it is Odisha that registered highest
growth in turmeric yield [57].
12.7 Cultivation Technology of Caper Bud in India
Capers (Capparis spinosa) of commerce are immature flower buds, which have been pick-
led in vinegar or preserved in granular salt, and it is a new spice crop for India. Semi-
mature fruits (caperberries) and young shoots with small leaves may also be pickled for use
as a condiment. Capers are said to reduce flatulence and to be antirheumatic in effect. In
Ayurvedic system, capers are recorded as hepatic stimulants and protectors, improving
liver function. Capers have reported uses for arteriosclerosis, as diuretics, kidney disinfect-
ants, vermifuges, and tonics. Infusions and decoctions from caper root bark have been tra-
ditionally used for dropsy, anemia, arthritis, and gout. Capers contain considerable
amounts of the antioxidant bioflavonoid rutin [58]. Capers probably originated from dry
regions in west or central Asia. Known to be used millennia, capers were mentioned by
Dioscorides as being a marketable product of the ancient Greeks. In India, the plant is
12.8 Export of Indian Spice 251
growing in wild almost in all the states of dry and arid regions. Indians are not aware of the
use of caper buds as spice except some local medicinal uses [59]. Recently the plant is
under cultivation in Tamil Nadu after recognizing its trade importance.
Ecological conditions are needed for the caper plants in dry heat and intense sunlight
that make the preferred environment for caper plants. Plants are productive in zones hav-
ing 350 mm annual precipitation (precipitating mostly in winter and spring months) and
easily survive summer temperatures higher than 37 °C (100 °F). Caper plants are small
shrubs and may reach about 1 m upright. However, uncultivated caper plants are more
often seen hanging, draped, and sprawling as they scramble over soil and rocks (Figure 12.2a
and b). The caper’s vegetative canopy covers soil surfaces, which help to conserve soil water
reserves. Leaf stipules may be transformed into spines. Flowers are born on year-old
branches. The cultivated variety is thornless and developed through in vitro technique by
selecting somaclonal variants. In Tamil Nadu, the stem cuttings were procured from the
Argentina and the variety is known as “inermis.”
Caper plants propagated usually through the stem cuttings. Cuttings are collected in
February, March, or April. Basal stem portions, greater than 1 cm diameter and 8 cm in
length with 6–10 buds, are used. A dip in IBA solution of 1.5–3.0 ppm is recommended
(15 seconds). A 70% rooting percentage would be considered good. Transplanting is car-
ried out during the wet winter and spring periods, and first-year plants are mulched with
stones. In India, plants are spaced 1–1.5 m apart (depending on the roughness of the
topography; about 2000 plants per hectare). A full yield is expected from second or three
year. Plants are pruned back in winter to remove dead wood and water sprouts. Pruning is
crucial to high yield. Heavy branch pruning is necessary, as flower buds arise on one-year-
old branches. Three-year-old plants will yield 1–3 kg of caper flower buds per plant. The
unopened flower buds should be picked on a dry days. Harvesting is carried out regularly
throughout the growing season. Caper flower buds (Figure 12.2c) are collected by hand
mechanically about every 8–12 days, resulting in 9–12 harvest times per season. Immature
flower buds are preserved either in vinegar or under layers of salt in a jar (Figure 12.2d).
Raw capers are bland flavored and need to be cured to develop their piquant flavor.
Mechanized screens are used to sort out the various-sized capers after being hand-picked
from the cultivation. After processing the dried flower buds are preserved in air-tight
packs and are ready for exports.
12.8 Export of Indian Spices
Spices trade is a big business from time immemorial. Spices from India and far Eastern
Asia were in demand from ancient times. Caravan carried them from India and China to
the ports of the Mediterranean Sea or the Persian Gulf and then to the marketplaces of
Athens, Rome, and other cities, where they were sold at exorbitant Prices. When the
Mongols and Turks cut off overland trade routes from Asia, the European demand for
spices was a major factor in motivating a search for new trade routes around Africa and
across the Atlantic and Pacific oceans. The high price obtainable for spices was partially
responsible for the bitter rivalry of European powers for the control of spice-producing
areas and of trade routes. Even after adequate supplies of spices were found and means of
c d
Figure 12.2 Caper bud form in Tamil Nadu. (a) Farm field, (b) Capparis spinosa – caper plant,
(c) fresh caper buds, (d) processed Caper buds in US market (Whole Foods Market, Gainesville, FL, dt.
24th May 2020). Source: Santosh Kumar Upadhyay.
transportation made available, the cost long remained very high in Europe and America.
This was largely because of the transportation costs, expenses incident to attempts to retain
monopoly of markets and to limit crops in order to secure high prices. The Indian spices are
having high demand in the global market due to their geographically indicative quality
principles [3].
12.8 Export of Indian Spice 253
India as “the land of spices” plays a significant role in the global spices as quality spices
come from Kerala. Some of the spices such as chillies, coriander seeds, cumin, fenugreek,
fennels are coming from other parts of India. At present, India produces around 2.75 mil-
lion tonnes of different spices valued at approximately 4.2 billion US$ and holds the pre-
mier position in the world spices market. Climatic diversity of India is from tropical to
subtropical with varied temperature gradient (45–0 °C), suitable for growing several spice
crops in this country. In almost all of the 28 states and eight union territories of India, at
least one spice is grown in abundance. About 4,00,000 tonnes equivalent to $1.5 billion
worth of spices are traded worldwide annually [12]. In 2009–2010, the export of spices from
India has been 502750 tonnes valued Rs. 5560 crores [60]. Though every state of the coun-
try grows at least a few spice crops, the states of peninsular India and bordering region,
Kerala, Andhra Pradesh, Odisha, Gujarat, Maharashtra, West Bengal, Karnataka, Tamil
Nadu, and Madhya Pradesh are the major states in spice production. Odisha grows several
spices such as chillies, ginger, turmeric, coriander, garlic in the area of 2.37 lakh ha with a
production of 2.17 lakh tonnes. Turmeric and ginger are two most important spice crops
grown in Odisha and more than 50% of these crop growers are tribals [8].
Initially, the trade of export was mostly confined with black peeper, cumin, and car-
damom, but later covered a number of spices. Out of 109 spices listed by ISO, about 75
are produced by India. The average export touches around 550 thousand tonnes of spices
annually. Major share is contributed by chilly, ginger, cumin, and turmeric in terms of
volume. Other important products are cardamom, black pepper, and nutmeg. In terms
of value, mint and mint products stand first followed by chilly and oleoresins. In the
past 10 years, the Indian spice exports increased substantially in terms of volume and
value [61].
Among the agricultural commodities, total export contributes around 7% and spices
exports contribute around 3% in India [11]. In world trade of spices, India is at number
three with 8.8% of the share. The major spices exported by India are chillies (40%), turmeric
(10%), cumin (10%), coriander (9.5%), fenugreek (4.2%), black pepper (4%), and others con-
stitute up to 19%. Though these spices provide innumerable benefits, they should be used
sparingly [3]. India is a leading producer, consumer, and exporter of black pepper in the
world. Andhra Pradesh leads in chilly and turmeric production in the country with 49 and
57%. In coriander, cumin, and fenugreek production, Rajasthan stands as the largest pro-
ducer in the country with 63, 56, and 87% of domestic production [62].
Turmeric of India has tremendous demand in the market of foreign countries. India was
the largest producer (4.35 lakh tonnes) and exporter (24900 tonnes) of turmeric during
1996–1997. The yellow coloring chemical ‘Curcumin’ is gaining wider use in the food
industry, pharmaceuticals, in preservatives, and in health and body care. The Alleppey
Finger Turmeric (AFT) with a curcumin content of more than 5.5% is in great demand
abroad. India exports turmeric to the foreign countries such as UAE, Iran, Japan,
Bangladesh, and South Africa. India exported about 6580 tonnes of turmeric worth Rs.
2296 crores during2009–2010. Though, growth and instability fluctuated in area, produc-
tion, productivity, and export of major seed spices, namely cumin, coriander, and fenu-
greek, grown along with total spices from 1985 to 2015 [53].
The main export centers in India are Spice Board, Cochin, Kerala; Agricultural and
Processed Food Products Export Development Authority, New Delhi; All India Spices
Exporters Forum (AISEF), Cochin, Kerala; Directorate of Marketing and Inspection,

Faridabad, Haryana.
12.9 Conservation Efforts Against Selected Uncultivated

Wild Spices and Condiments
India has a good amount of diversity in spices such as black pepper, cardamom, ginger,
turmeric, cinnamon, tamarind and, garcinia. The other important spices relevant to India
are coriander, fennel, fenugreek, cumin, nutmeg, clove, and vanilla. Apart from these com-
mon spices and condiments, some of the plants and plant products are used for preserva-
tion and flavoring of foods by traditional knowledge or folklore system. These spice or
condiment plants are available locally and use of the plants in such a way for purpose for
spices in addition to medicinal and local therapeutic applications. India is a land of spice,
where several spice genera originated in the diversity hotspots of either The Himalayas or
The Western Ghats. Considering gingers (Zingiberaceae), reports indicate that around
eight tuber-bearing, one stoloniferous, and 16 non-tuberous species of gingers are found in
The Western Ghats and most of the genera are indigenous to India [63].
Nearly 25% of spice and condiment plants are growing in wild and collected from local
vegetation. Several local spices are wild relatives of the known valuable spice, for example,
Allium, Alpinia, Cinnamomum, Curcuma, Hedychium, Zanthoxylum, etc. Wild germ-
plasms of cultivated spice plants and wild relatives of the usable spice species are much
important for the improvement and development of quality spice varieties [62]. Many
research centers are actively engaged in collection, cultivation, and agronomic develop-
ment of wild relatives of the usable spice plants. Collection and conservation of valuable
wild spice species in Indian region have become imperative due to increasing interest in
herbal spices for health care across the globe. India alone contributes 140 species and con-
diments plant species used locally as well export to other countries from which 60 spices
are used in industries for making drugs [27, 28].
12.10 Institutions and Organization Dedicated

for Research and Development in Spices and Condiments
in India
The Indian Institute of Spices Research (IISR), Kozhikode (Calicut), a constituent body of
Indian Council of Agricultural Research (ICAR), is a major institute devoted to research on
spices and condiments. Regional station of the Central Plantation Crops Research Institute
(CPCRI), Kasaragod, engaged in research on spices since 1976. A National Research Centre
for Spices was established in 1986 with its headquarters at Kozhikode, Kerala, by merging
the erstwhile Regional Station of CPCRI at Kozhikode and Cardamom Research Centre at
Appangala, Karnataka. Realizing the importance of Spices Research in India, this Research
Centre was upgraded to Indian Institute of Spices Research in 1995. The National Research
Centre on Seed Spices, at Ajmer, Rajasthan, is an apex center of ICAR working on
12.11 Recent Researches on Spices and Condiment 255
improvement of seed spices and betterment of their stakeholders since its inception in
2000. Government of India has notified the formation of 10 spice development agencies
(SDAS) in the main spice-growing regions for the overall development of spices grown in
the region. These agencies will be chaired by the Chief Secretary of the concerned State
Government and consist of members from Ministry of Commerce and Industry, the State/
Central Agriculture/Horticulture Ministry, other related Central/State organizations, Agri
university, Member of Spices Board from the region, and various stakeholders of the indus-
try, namely growers, traders, and exporters of spices. Indian government has also formed
the “Saffron Production and Export Development Agency” (SPEDA) for the overall devel-
opment in the saffron industry in the state of Jammu and Kashmir in 2015. SPEDA acts as
a subordinate agency under Ministry of Commerce.
Other institutions and research organizations in India for development of spice and con-
diments crops are Indian Cardamom Research in Myladumpara, Kerala; Jawaharlal Nehru
Tropical Botanical Garden and Research Institute, Palode, Kerala; Cardamom Research
Centre (ICAR), Appangala, Karnataka; Central Institute of Medicinal and Aromatic Plants
(CSIR), Lucknow, Uttar Pradesh; Central Food Technological Research Institute (CSIR),
Mysore, Karnataka; Several state- and central-funded agriculture, science, and technical
universities are also engaged in spices research, with focus on localized species concerned
with localized problems and development.
12.11 Recent Researches on Spices and Condiments
Plants used as spices and condiments are usually aromatic and pungent. Most of the spice
plants have been reported that they owe these properties to the presence of varying types of
essential oils. Several studies indicated that the rich presence of essential oils and oleores-
ins determines the aromatic, flavoring, coloring, and pungent properties of spices and con-
diments [2] (Table 12.2). Indian spices are “part and parcel” of traditional health care for
thousands of years. Over the couple of decades, largely due to the growth in popularity of
complementary and alternative medicine, spices have regained attention due to their phys-
iological and functional benefits. By applying modern research methods to traditional rem-
edies, it is possible to discover what made these spices such effective ailment
treatments [64].
Low productivity in the spice sector is one of the serious problems facing the Indian spice
industry, resulting in low competitiveness in the international markets. The rapid disap-
pearance of some indigenous varieties of spices due to mixing of planting material results
in loss of genetic purity; examples are varieties contributing to the production of Cochin
ginger (namely Kuruppampady, Ellackal), Alleppey finger turmeric (namely Elanji), and
Byadagichilli [65]. Major challenges facing seed spices production in India are low produc-
tivity, lack of high-yielding varieties. Such challenges should be overcome by developing
fertilizer-responsive varieties and developing pests and disease resistance. Introduction of
germplasm of spices from their centers of diversity both in general and for specific charac-
ters will help in broadening the genetic base and for attempting meaningful crop improve-
ment programs [66]. India being the primary and secondary center of diversity for many
spices, the genetic variability in major spices such as black pepper, cardamom, cumin,
coriander, nigella can be addresses by assessing diverse germplasm and integrating benefi-
cial agronomic trait into cultivable varieties. Genetic resource enhancement, its evaluation,
and valuation for effective use to meet the challenges of biotic and abiotic stresses to sus-
tain the impact of climate change are also the need of the hour besides yield quality and
nutritional value.
12.12 Conclusion and Future Perspectives

Spices are rich source for phytochemicals having precise health benefits. Spices are used
individually or in combination as food adjuncts to impart flavor, color, and aroma.
Traditional knowledge prevailing in countries such as India has shown the medicinal prop-
erties of many spices for treating wounds, cough and cold, fever, hyperglycemic, and hyper-
lipidemic conditions. Some of the important bioactive compounds of spices that are shown
to possess medicinal value include curcumin from turmeric, capsaicin from red pepper,
piperine from black pepper, eugenol from cloves, allyl sulfides from garlic and onion. These
compounds are shown to possess antioxidant, anti-inflammatory, antimicrobial, hypolipi-
demic, and anti-lithogenic activities and pro-anti-cancer properties as well. There is need
to increase the productivity to fulfill the domestic requirement and for export of valued
spices and condiments. It has been observed that technological interventions such as rhi-
zome treatment, soil application of biocontrol agents, manures, fertilizers, micronutrients,
crop rotation, mulching, and correct form of plant protection measures increase the yield
performance. Reintroducing the germplasm from the centers of diversity in crops such as
paprika, nutmeg, clove, allspice, and vanilla will improve our genetic stocks for future uti-
lization. Popular spices such as lavender, anise, dill, oregano, marjoram etc. need to be
introduced to help in diversifying Indian spices production. Introduction of spices species
to nontraditional areas must be done in order to conserve the germplasm. To recognize the
climate-dependent high-yielding spice varieties, new improved spice crops for Indian cli-
mates are to be developed through modern research tools. Introduction of suitable modern
hybrids, considering the market demand and more emphasis on utilization of exotic wild
species in breeding programs, is to be considered. To enhance the productivity of spices and
condiments, integration of advanced farming practices with novel technologies and cou-
pling it with ecofriendly traditional production technologies among the farming commu-
nity is the need of the hour.
Acknowledgments
No funding has been received to conduct this work.
Authors’ Contribution
SK and TRR designed the work. SK did major contributions, compiled the information, and
written the manuscript. TRR did minor contributions and formatted the manuscript.
Reference 257
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261
13
Plants as Source of Essential Oils and Perfumery

Applications
Monica Butnariu
Chemistry & Biochemistry Discipline, Banat’s University of Agricultural Sciences and Veterinary Medicine “King Michael I of
Romania” from Timisoara, Timis, Romania
13.1 Background
The plants are a valuable source of secondary metabolites (SMs), which due to their
biological activity are widely used in the food, pharmaceutical, and cosmetology industries.
SMs are derivatives of primary metabolites (PMs) obtained as a result of methylation,
hydroxylation, glycosylation, and other biochemical reactions. Although they are very
important, MS have a much more limited distribution in nature compared to PMs and are
not essential for the functioning of the whole plant. They are not continuously synthesized
and are often produced during the non-growth phase of cells.
Among the SMs can be mentioned phenols, phenylpropanoids, flavonoids, essential oils
or volatile oils (EOs), terpenes, alkaloids, steroids, lignins, resins, etc. Over time, views on
the role of SM have changed significantly. This hypothesis was put forward by A. Kossel in
1891, a German physiologist, considered to be the first to belong to the concept of SMs [1].
According to another definition, SMs have the role of storage (accumulation).
Subsequently, it was assumed that all SMs are in fact PMs, because the important processes
in which SMs participate are not yet known. The starting point for the development of this
idea was the discovery of the shikimic path of biosynthesis of phenolic compounds.
Another determinant of SMs derived from the protective role of secondary compounds
against various abiotic and biotic stressors. Thus, the definition of SMs has changed over
the years.
The most acceptable definition of SMs is that they are naturally occurring substances
and play an explicit role in the internal economy of the organism that produces them [2].
The functions of the SMs are diametrically different from those of the PMs, and their
main function is to maintain the fundamental processes in plant cells and the survival of
the species in unfavorable environmental conditions. The delimitation between primary
and secondary metabolism is not absolute, as many of the intermediate PMs play similar
roles in SMs. Thus, some amino acids, such as 3-β-alanine and γ-aminobutyric acid, are

262 13 Plants as Source of Essential Oils and Perfumery Applications
certainly SMs, while sterols are essential structural compounds of many organisms and
should therefore be considered PMs. Therefore, the overlap of the biological functions of
several components of metabolism creates a complex interrelationship between primary
and secondary metabolism. A more precise presentation for the description of SMs is the
one proposed by Luckner. According to him, the following particularities are characteristic
for SMs: the diversity of chemical structures; limited distribution in plants; formation of
enzymes encoded by special genetic material; strict control of SMs biosynthesis by
regulating the amount of enzymes and their activity; compartmentalization of enzymes,
precursors, intermediates, and products involved in biosynthesis, storage, and breakdown;
the expression of the secondary metabolism as an aspect of the cellular specialization or of
the cells formed de novo individualized by the integration in the processes of differentiation
and development of the producing organs; the lack of importance of SMs for the synthesized
cell itself, but essential for the whole organism. Plant PMs and SMs of economic interest
have several common characteristics: most of them are non-protein biochemical
compounds, and they can be extracted from plant material by steam distillation or by
extraction with organic or inorganic solvents. Over the last 20–30 years, SMs analysis has
advanced greatly.
The use of modern analytical techniques by researchers, such as chromatography,
electrophoresis, the labeled isotope method, and enzymology, allowed them to elucidate
the exact structural formulas and biosynthesis pathways of SMs. The SMs are a diverse
class of compounds produced by plants, which perform many important functions. Unlike
PMs, these compounds are not essential for the basic metabolism of the plant but still
perform significant functions that allow the plant to adapt and thrive in its living
environment. Because plants do not have certain fundamental characteristics that we
encounter in humans and animals, they must have the means to meet additional challenges.
All plants taken together produce over 100 000 different varieties of secondary compounds
that serve a variety of purposes.
The main categories of SMs in EOs are terpenes and terpenoids, phenols, and alkaloids.
The compounds are grouped into one of these categories based on their chemical structure.
Terpenes are volatile organic compounds, which are based on a 5-carbon hydrocarbon,
known as isoprene. The smallest and most volatile compounds are monoterpenes, which
are biosynthesized by joining two isoprene molecules. Larger and less volatile compounds
are biosynthesized by joining three or more isoprene molecules. The monoterpenes are
followed by sesquiterpenes, which are formed by the union of three isoprene molecules.
Terpenes are SMs that give the plant its organoleptic characteristics (aroma and taste) and
which make up most of the EOs produced by aromatic plants [3].
13.2 Biochemistry of Essential Oils
13.2.1 The Physiological Mechanism of Biosynthesis of Essential Oils

EOs have a still unknown biological role. It accumulates in various plant organs, but
especially in petals, seeds, roots, bark, and wood. These substances are mainly composed of
terpene hydrocarbons. Rose petal oil contains an acyclic terpene called citronellol.
d-limonene and l-limonene are found in orange peels, lemons, cumin, and celery. In the
13.2 Biochemistry of Essential Oil 263
mint leaves, there are menthol, pine, lemon, in those of basil (ocimen methyl chavicol),
and wormwood thion + phellandrene. The scent of chrysanthemum flowers has the
following chemical compounds: tetracolsan hexacosan; in lavender, there is lavandulol,
ethylamyl ketone linalool, butyrate of isoamyl; chamomile contains a mixture of acetylene
and furfuryl paraffins [4].
EOs produced by glandular cells accumulates, as in all Lamiaceae species, between the
outer wall of glandular cells and the cuticle. This leads to the detachment of the cuticle
from the glandular cells and the emergence of a common storage space for EOs.
When the subcuticular space is filled with the secretion product, the glandular brushes,
entering the post-secretory phase, degenerate. Only a slight enlargement of the subcuticular
space was observed in the capped brushes, in which case the secreted material accumulates
in the lumen of the glandular cell. At maturity, the multicellular secretory gland has a large
subcuticular space. This cavity will be filled with EOs at the beginning. The cell space that
thus arises increases as the oil is secreted and released into this cavity. The secretions pass
through the plasma membrane and the wall of the secretory cells and accumulate in this
cavity to accumulate the secretion products. When the cuticle ruptures, the secretion
products are released. In secretory brushes with a multicellular gland, this subcuticular
space exceeds in height the glandular cells themselves. The EOs also exhibit optically active
properties due to the content of compounds with asymmetric carbon atoms. Among these
compounds are linalool, camphor, pinene, borneol, etc. Each of these compounds is
characterized by its own angle of rotation of the plane of polarization, and the value of the
angle of rotation of the volatile oil is dependent on the values of the angles of rotation of
each component, as well as the percentage content of these compounds in the mixture.
Although EOs contain optically active compounds, the value of the angle of rotation for
each oil falls within relatively well-defined limits and can serve to establish the identity of
the EOs compounds. EOs are biosynthesized in various organs or tissues of plants, including
leaves and stems (e.g. basil, bay, eucalyptus, fennel, lemon grass, mints, oregano, parsley,
pine, rosemary, sage, spicebush, tarragon, and wintergreen), seeds (e.g. anise, almond,
cumin, and celery), berries (e.g. allspice and juniper), bark (e.g. cinnamon and sassafras),
fruit peel or rind (e.g. citron, grapefruit, lemon, lime and orange), roots (e.g. valerian),
rhizomes (e.g. ginger), and flowers (e.g. chamomile, clove, geranium, jasmine, lavender,
orange, and rose) [5].
13.2.2 The Role of Terpenes in Plants

The two main functions are protection against inspections and herbivores, as well as
protection against high temperatures. Plants react by producing terpenes in areas affected
by the action of insects and herbivores, and these terpenes acting as bitter compounds or,
in some cases, even as pesticides that repel or “attract” (phenolic in nature [e.g. menthol
from Mentha piperita], volatile alkaloids [e.g. Valerotropes from Valeriana off.], resins and
gums [e.g. Humulus lupulus]). Plants in turn have evolved rather interesting strategies to
“attract.”
They include flowers that produce differently colored, often hairy “nectar guides” on
their petals (e.g. Iris); plants that produce ultraviolet pigments that insects see as “bulls-
eyes”; various colored petals and/or sepals whose flavonoid and anthocyanin pigments
attract specific pollinators; flowers that open only at night when moth-type pollinators are
active in flight (as with yucca flowers visited by hawkmoths); flowers that produce a rotten
meat smell (due to indoles, skatole, or amines) that attract flies or beetles, as in the case of
skunk cabbage and other aroids; flowers that produce pheromones/plant hormones (e.g.
ethylene, methyl jasmonate, and methyl salicylate). The flowers produce essential oils as
olfactory cues that “attract specific” because of their highly evolved sensing systems.
Monoterpenes (2 isoprene units, so 10 carbon atoms), which are more volatile, dominate
the inflorescences (e.g. limonene and menthone from the youngest leaves of peppermint),
can acting as a good spatial repellent. Sesquiterpenes (C15-terpenoids built from three
isoprene units), which are more bitter, are more abundant on the leaves, acting against
herbivores and can good contact repellent (e.g. α-bisabolene in black pepper [Piper nigrum]
and the candeia tree [Vanillosmopsis erythropappa] and β-caryophyllene in ylang ylang
[Cananga odorata], yomogin [Artemisia princeps Pamp.], and nerolidol in genus Tanacetum
[Compositae]). When plants notice an increase in temperature, they begin to synthesize
more terpenes and thus, at higher temperatures, day or night, more terpenes are released.
Terpenes evaporate at high temperatures, producing air currents that cool the plant and
decrease perspiration, preventing the plant from drying out. In the case of hemp, terpenes
are exuded in the resin and give it a sticky and viscous appearance that will catch and
immobilize some insects, thus acting as a protection against insects and high temperatures.
Thus, it is easy to notice that the hemp plant smells stronger during the first hours of the
morning than during the hottest part of the day, when a larger amount of terpenes
evaporates.
This is why it is recommended that mature plants should be harvested in the early hours
of the morning in order to have a maximum production of EOs. An important feature of
EOs and their compounds is hydrophobicity, which allows them to affect the lipid structure
of the bacterial cell membrane and increase their permeability, with cells losing ions and
other cellular components. The chemical structure of the individual compounds of EOs
affects their specific mode of antibacterial action. The importance of presence of the
hydroxyl group in phenolic compounds such as carvacrol and thymol has been
confirmed [6].
13.2.3 The Prevalence Essential Oils in Plants

EOs are relatively widespread in the plant kingdom, with some families being very rich in
such substances. Among these families, more representative is Pinaceae, Labiatae,
Umbelliferae, Myrtaceae, Lauraceae, Rutaceae, Caryophylaceae, Compositae,and
Zingiberaceae. Plants containing EOs are commonly called aromatic plants or hetero-
oleaginous plants. Aromatic plants contain EOs in amounts from hundreds of percent to
20–25% relative to dry mass. For example, in the inflorescences of white acacia and lily of
the valley, there is about 0.05% EOs, in the rose flowers 0.06–0.20%, in the inflorescences of
lavender 1.0–2.0%, in the fruits of fennel 4.0–6.0%, in star anise fruits up to 11.5%, and in
the flower buds of the clove tree up to 22%. About 2500 species of aromatic plants are
known in the world, of which more than 40% grow in the tropics.
However, of the total number of aromatic plants, only about 200 are of industrial
importance. The others are not recovered either due to a low content of EOs, or the
unsatisfactory quality of the volatile oil they contain. EOs are spread unevenly in the plant’s
body, usually concentrating in one or more of the organs. For example, in the rose, the
organ in which EOS accumulates is the flower, in mint – leaves and inflorescences, in
vetiver – in roots, in bay and eucalyptus – in the leaves and young shoots.
Usually, only the parts of the plant where the content of EOs is maximum is used in
industry. Sometimes, the raw material may contain parts of the plant (ballast) in which
practically no EOs are contained (mint, geranium, lavender stems, etc., are the ballast in
the process of extracting the respective EOs). The content of EOs in this type of raw material
depends a lot on the ratio between the parts rich in EOs and ballast.
The quantity and quality of volatile oil in the plant vary depending on a number of
endogenous (species, life cycle phase, etc.) and exogenous factors (temperature and
humidity of the air and soil, light intensity, etc.). In the body of the aromatic plant, EOs are
contained in the free and/or fixed state (in the form of glycosides).
EOs in the free state are located in such structures as: isolated secretory cells – idioblasts
(family Lauraceae); secretory organs (schizogenous oil glands from the secretory pockets –
characteristic of the family Rutaceae, Myrtaceae); secretory canals (Pinaceae, Umbelliferae);
secretory hairs (Lamiaceae, Compositae). The form in which EOs are found in the plant raw
material influences the storage method and its processing technology. EOs are common
aromatic compounds from different parts of plants (flowers, buds, seeds, branches, bark,
wood, fruit, or roots). They can be obtained by fermentation or extraction, but the most
commercially used method is hydrodistillation. It is estimated that over 3000 EOs are
known so far, of which about 300 are of practical importance in the perfume industry [7].
EOs contain a mixture of compounds that are chemically derived from terpenes or their
oxygenated compounds. The widest use of EOs in the European Union is in the food
(spices), cosmetics, and pharmaceutical industries. Terpenes are volatile organic
compounds, which are based on a hydrocarbon with five carbon atoms, known as isoprene.
The smallest and most volatile compounds are monoterpenes, which are biosynthesized
by joining two isoprene molecules. Larger and less volatile compounds are biosynthesized
by joining three or more isoprene molecules. The monoterpenes are followed by
sesquiterpenes, which are formed by the union of three isoprene molecules. Terpenes are
SMs that give the plant its organoleptic characteristics (aroma and taste) and which make
up most of the EOs produced by aromatic plants. The ratio of monoterpenes to sesquiterpenes
in leaves and flowers has been shown to be different. This is due to the predominance of
sessile trichomes in the leaves, which are much more specialized in the synthesis of
terpenes, while capitate trichomes are much more abundant in flowers and are specialized
in the synthesis of volatile terpenes (monoterpenes and sesquiterpenes). Normally, the
proportion of terpenes in the plant is less than 1%, reaching of up to 10% of the resin
composition [8].
13.2.4 Paths of Biosynthesis of Volatile Compounds in Plants

13.2.4.1 Metabolic Cycles Involved in the Biosynthesis of Different Groups
of Secondary Metabolites
SM biosynthesis involves a relatively small number of chemical compounds, which are
subsequently modified by various cycles into an unlimited number of secondary substances.
There are four cycles involved in the biosynthesis of SMs, each being responsible for the
biosynthesis of different classes of compounds: the shikimic cycle: simple phenols,
hydroxycinnamic acids, phenylpropene, phenylpropanoids, flavonoids, lignins, coumarins,
naphthoquinones, anthraquinones, tannins; mevalonic cycle and methylerythritol-
phosphate cycle: terpenes; acetate cycle: alkaloids. At the same time, according to their
biosynthetic origin, SMs are classified into several classes, including the class of
phenylpropanoids, terpenes, alkaloids, etc. These cycles are carried out with the involvement
of the essential derivatives of acetyl-CoA intermediates, shikimic acid, mevalonic acid, and
methylerythritol-phosphate, respectively [9].
13.2.4.2 Metabolic Cycles of Biosynthesis of Phenolic Compounds

13.2.4.2.1 The Shikimic Ccle of Biosynthesis of Phenolic Compounds The shikimic cycle takes
place through the cyclization of some pseudo aromatic intermediate compounds
(2-phosphoenol-pyruvic acid and 4-phosphoerythrosis), which lead to the formation of the
structure of the benzoic ring.
The shikimic cycle includes a key intermediate, namely shikimic acid. It is the main
precursor of the biosynthesis of aromatic amino acids, such as l-phenylalanine, l-tyrosine,
and l-tryptophan, as well as other compounds such as alkaloids, phenols, and
phenylpropanoids. That cycle is common for the biosynthesis of aromatic acids in bacteria,
fungi, yeasts, microorganisms, and plants, but not for the animal kingdom. This cycle is
essential because it is involved in the generation of structural blocks for the biosynthesis of
proteins, vitamins, and electron-carrying compounds, such as co-factors and quinones.
Shikimic acid was first isolated from the fruits of a species native to Japan Illicium
religiosum Sieb. It is currently the raw material for the synthesis of antiviral oseltamivir,
used to treat avian influenza. The main source of extraction of shikimic acid is aniseed
fruits (Illicium verum Hook F.). The precursors of the synthesis of shikimic acid in the
respective cycle-2-phosphoenol-pyruvic acid are formed from 3-phosphoglyceric acid, and
4-phosphatitrose is formed by cleavage of the condensation product between
6-phosphofructose and 3-phosphoglyceraldehyde. Thus, upon condensation of the two
precursors, 3-deoxy-d-arabino-2-heptulosonic-7-phosphate (DAHP) is formed [10].
The compound is a combination of carbohydrate residues, which although have a linear
chain, the carbon atoms are oriented in space according to a pseudo cyclic structure, which
allows the subsequent cyclization of the chain. The cyclization process takes place in
several stages, through oxidation and reduction reactions, with the formation of
3-dehydroquinic acid, subsequently entrained in a series of transformations, with the
formation of cyclohexane derivatives. In the next step, the dehydration of 3-dehydroquinic
acid takes place with the formation of 3-dehydroschemic acid. The latter, under the
influence of the oxidative-reducing enzyme shikimat-dehydrogenase, is converted to
shikimic acid.
Shikimic acid, unlike aromatic compounds, contains only a double bond. Subsequently,
as a result of a series of biochemical conversions, shikimic acid is transformed into horismic
acid, another important intermediate, which already has two double bonds. At that stage,
the branching of the shikimic cycle takes place. One of the biosynthetic pathways based on
horismic acid leads to the formation of l-tryptophan (and other indole derivatives), the
second pathway to the formation of l-phenylalanine and l-tyrosine. It is with the second
biosynthetic pathway that subsequent transformations are associated, which ultimately

lead to the formation of phenolic compounds in plant cells and tissues. This is the end
point of the shikimic cycle, being the source of these amino acids, it is also one of the
elements of primary cellular metabolism. Secondary specific transformations lead to
phenol biosynthesis [11].
13.2.4.2.2 The Metabolic Cycle of Phenylpropanoid Biosynthesis l-phenylalanine and

l-tyrosine are important precursors of many SM in higher plants, and the enzymes
responsible for redirecting these essential MPs in secondary metabolic cycles are the
enzymes l-phenylalanine ammoniacylase (PAL) and tyrosine decarboxylase (TirDC).
These precursors form the basic units of C6C3 phenylpropanoids (FPs), which are found in
many natural products, such as cinnamic acid, coumarins, lignins, and flavonoids.
Due to the commercial interest, regarding the use of FPs in pharmacological and other
industrial fields, their functions and biosynthesis cycle have been intensively studied in
many plant species. In the general biosynthesis scheme of FPs, the aromatic amino acid-l-
phenylalanine derivative of the shikimic cycle is subsequently subjected to biochemical
transformations, which consists in its deamination in the reaction catalyzed by the enzyme
PAL (EC 4.3.1.5) in trans-cinnamic acid. The latter is exposed to the para-hydroxylation
process with cinnamate-4-hydroxylase (C4H, EC 1.14.13.11), obtaining 4-hydroxycinnamic
acid (p-coumaric acid). P-coumaric acid is the first, and from a biogenetic point of view, the
simplest phenolic compound, being also the primary compound in the biosynthesis of
other plant phenolic compounds. Following the enzymatic transformations of p-coumaric
acid in plant cells, the biosynthesis of derivatives of different classes of phenolic compounds
takes place. By oxidative reduction of the side chain of p-coumaric acid, acetophenones,
phenylacetic acids, and phenylcarbonic acids are formed. The recovery of its side chain,
together with the subsequent dimerization or polymerization of the reconstituted product,
leads to the formation of lignin and phenolic resins-lignin type [12].
13.2.4.2.3 Metabolic Biosynthesis Cycles of Terpenes Terpenes, also called terpenoids or

isoprenes, are the largest class of plant metabolites reported to date, which includes
compounds with a wide structural variety.
They are produced by all living organisms. To date, more than 45 000 plant terpenes have
been identified and this number is constantly growing. Of the tens or thousands of plant
terpenes, only a few can be considered MPs, considered essential for the functioning of the
whole plant and are therefore common to all plant species. These include molecules
involved in the processes of respiration (ubiquinones), photosynthesis (chlorophylls,
carotenoids, tocopherols, phylloquinones, and plastoquinones), regulating the growth and
development of the plant (brassinosteroids, cytokinins, gibberellins and abscisic acid),
strigol.
The rest of the terpenes are specialized metabolites, whose biosynthesis is usually limited
to certain plant families or even certain species. Terpenes usually participate in protecting
plants against herbivores and pathogens, in attracting pollinators and dispersing seeds and
as allelochemicals, which influence competition between plant species [13].
A large number of terpene metabolites have commercial value and are used as pigments,
flavors, polymers, or drugs. Typical terpene structures contain carbon skeletons represented
by (C5)n units and are classified as hemiterpenes (C5), monoterpenes (C10), sesquiterpenes
(C15), diterpenes (C20), sesterterpenes (C25), triterpenes (C30), and tetraterpenes (C40).
According to the isoprene rule, all terpene compounds derive from two common precursors
containing two isoprene units bound head-to-tail, namely from isopentenyl pyrophosphate
(PPI) and dimethylallyl pyrophosphate (DMAPP) (allylic isomer of PPI). In plants, two
independent cycles are responsible for the biosynthesis of isoprene units: the mevalonic
acid cycle (MVA) and the methylerythritol-phosphate cycle (MEP) according Figure 13.1.
Abbreviations: PPP, pentose phosphate pathway; EDP, Entner-Doudoroff pathway; G3P,
glyceraldehyde-3-phosphate; DXP, deoxy-d-xylulose 5-phosphate; MEP, 2-C-methyl-d-
erythritol-4-phosphate; HMBPP, 4-hydroxy-3-methyl-but-2-enyl pyrophosphate; MVA,
mevalonate pathway (mevalonic acid); HMG, 3-hydroxy-3-methylglutaryl; IPP, isopentenyl
pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GPP, geranyl diphosphate; FPP,
farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; IDP, isopentenyl diphosphate;
FDP, farnesyl diphosphate; GAP, glyceraldehyde-3-phosphate; GGDP, geranylgeranyl
diphosphate; HMG-CoA, 3-hydroxy-3-methylglutaryl CoA; IDP, isopentenyl diphosphate.
The normal biosynthesis of terpenes in plants depends essentially on the flow of
precursors released by the nucleus of the isoprene metabolic cycles and, consequently, on
the dynamic regulation of these cycles. The MVA cycle offers predominantly biosynthesis
precursors of sesquiterpenes, triterpenes, and brassinosteroids. Respectively, in the
biosynthesis of hemiterpenes, monoterpenes, diterpenes, tetraterpenes, carotenoids and
their degradation products-cytokinins, gibberellins, chlorophyll, tocopherols, and
plastoquinones participate in the MEP cycle derivatives. This cycle is considered exclusively
plastid, because the set of enzymes involved in SM biosynthesis exists only in plastids, the
Glycolysis Ieucine, valine

and isoleucine
PPP metabolism
DXP pathway
G3P DXP MEP HMBPP
EDP
MVA pathway
Pyruvate Acetyl-CoA Acetoacetyl-CoA HMG MVA IPP DMAPP
C5 C5
Fatty acid
metabolism
Diterepenoids
Phytoene GGPP
biosynthesis (C20)
Carotenoids
biosynthesis (C40)
Sequalene FPP GPP
Triterpenoids Sesquiterpenoids Monoterpenoids

biosynthesis (C30) biosynthesis (C15) biosynthesis (C10)
Figure 13.1 Schematic representation of a biosynthesis of terpenes in plants.

13.3 The Metabolism Terpene 269
data being based on experimental research and predictions of their subcellular location.
Unlike the previous one, the subcellular location of the MVA cycle is not as clear.
Historically, this cycle has been referred to as the cytosolic cycle, and new claims suggest
that the MVA cycle is distributed between the cytosol, the endoplasmic reticulum, and
peroxisomes [14].
13.3 The Metabolism Terpenes

The metabolism of terpenes synthesized in the mevalonate cycle has as initial product
acetyl CoA. In this cycle, monoterpenes, sesquiterpenes, diterpenes, triterpenes, and
tetraterpenes are synthesized, among which we can mention: linalool, limonene,
gibberellin, abscisic acid, ubiquinone, carotenoid pigments, etc. The biosynthesis of these
substances is genetically coordinated, by coding the synthesis of enzymes that catalyze the
reactions in this cycle: phytoen-desaturase, phytoen-synthase, hydroxymethylglutaryl-CoA
reductase, geranylgeranyl-pyrophosphate synthase, lycopene cyclase, and capsanthin-
capsorubin synthase.
In the first stage of terpene biosynthesis, the formation of 3-hydroxy-3-methylglutaryl-
CoA takes place, from three molecules of acetyl CoA, a reaction catalyzed by an enzyme
that cofactor with iron and quinone. In the second step, catalyzed by hydroxymethyl-
glutaryl CoA reductase, mevalonate is formed. By decarboxylation and phosphorylation,
mevalonate results in isopentyl pyrophosphate (C5), from which geranyl pyrophosphate
(C10), farnesyl pyrophosphate (C15), and geranylgeranyl pyrophosphate (C20) are formed.
These substances are precursors for different terpenes [15].
Thus, monoterpenes are formed from geranyl pyrophosphate: linalool and limonene.
Sesquiterpenes and squalene are formed from farnesyl pyrophosphate. From geranylgeranyl-
pyrophosphate are synthesized diterpenes, kauren which is the precursor of gibberellins
and abscisic acid and phytoene, which is the precursor of carotenoid pigments. Locating at
the cellular level, the various stages of the terpene synthesis process is difficult. Isopentyl
pyrophosphate can be biosynthesized in all cellular structures where terpenes are
synthesized.
The specific site for the synthesis of monoterpenes is plastids. The synthesis of farnesyl
pyrophosphate and derived sesquiterpenes, as well as triterpenes including phytosterols,
takes place in the cytoplasm and in the endoplasmic reticulum. The biosynthesis of
diterpenes takes place in plastids, that is, where the activity of geranylgeranyl-pyrophosphate
synthase has been identified. The activity of entkauren synthase, which catalyzes the
synthesis of entkauren, the precursor of cytokinins, has been identified in chloroplasts.
Also, in chloroplasts, carotenoid pigments and tocopherols are biosynthesized, and
ubiquinone is biosynthesized in mitochondria and microsomes.
Terpene biosynthesis takes place in all plant cells. In the case of species that synthesize
large amounts of terpenes, their synthesis takes place in specialized cells such as resinous
channels in pine leaves or isolated resinous cells in Thuja.
In the case of angiosperm plants, monoterpenes are synthesized in glandular hairs on
Mentha leaves, in flower petals or pistils (e.g. linalool), and latex in latex. Numerous
terpenes with 10 or 15 carbon atoms, with a high degree of volatility, are known as EOs and
give the characteristic aroma to some plant organs: flowers, fruits, seeds, etc. [16].
Sterols are made up of five isoprene units, such as cholesterol, sitosterol, stigmasterol,
and campesterol, and are found in the composition of plasma membranes having a role in
regulating their permeability. Some isoprenoid compounds secreted by plant roots are toxic
to the roots of other plants, being considered allelopathic substances. The shikimic acid
cycle has as substrate erythroso-4-phosphate, produced in the pentosophosphate cycle. The
result is phenols, lignins, anthocyanins, and phenolic amino acids such as tryptophan from
which auxin is biosynthesized. It is estimated that under normal conditions about 20% of
the fixed carbon of plants is used in the shikimat cycle. In this cycle, phosphoenolpyruvate
and erythroso-4-phosphate are condensed into a compound with seven carbon atoms: 3
deoxy-d-arabino-heptulosonate-7-phosphate (DHAP), which results in chorismate, as
follows:
DAHP 3 dihydroquinate 3 dihydroshikimat shikimat chorismat.
Chorismate is the starting compound for three cycles: of the first results in phenylalanine,
lignins, and flavonoids, of the second tryptophan, auxins, glucosinolates, phytoalexins, and
alkaloids, and of the third tyrosine and melanin. The key enzyme in the shikimic acid cycle
is phenylalanammonium lyase (PAL), which is bound to the membrane of the endoplasmic
reticulum, chloroplasts, mitochondria, and plasma membranes. Mechanical damage and
pathogen attack induce the formation of mRNA encoding the synthesis of DAHP synthase,
stimulating the shikimat cycle. The enzymes that catalyse the reactions in this cycle are
synthesized in the ribosomes in the cytoplasm. The activity of these enzymes has been
shown in chloroplasts and its identification in the cytoplasm is uncertain [17].
Some of the amino acids that form in this cycle are in turn precursors of other substances.
Tryptophan is the precursor of the growth hormone auxin, phenylalanine is the precursor
of flavonoid pigments and lignins, tyrosine is the precursor of ubiquinone, an electron
transporter in the process of respiration. In the shikimic acid cycle, which takes place in the
endoplasmic reticulum and in the cytoplasm, phenolic acids are also biosynthesized:
p-coumaric, cinnamic, caffeic, ferulic, chlorogenic, as well as galotanins. Phenolic
substances can stimulate or inhibit the action of hormones, inhibit the synthesis of ATP in
mitochondria, as well as the activity of enzymes, or cytoplasmic currents in the cells of
absorbent hairs.
Some phenolic substances (ferulic, lunular, chlorogenic acid, and catechins) have an
inhibitory effect on seed germination. By oxidizing substances such as tyrosine from the
tubers of Solanum tuberosum, dopamine from bananas, phenolic acids from apples, in the
presence of phenolase black melanin is formed, which gives the characteristic color for
mechanically damaged or senescent fruits. Some phenols are allelopathic substances:
juglone produced by Juglans regia, salicylic acid produced by Quercus falcata, and ferulic
acid produced by Adenostoma etc. Cinnamic acid derivatives have been identified in
vacuoles and chloroplasts. Enzymes involved in the biosynthesis of flavonoid substances:
chalcone-flavone isomerase and flavonoid hydrolase have been identified in the
endoplasmic reticulum and chloroplasts, and flavonoid substances have been identified in
vacuoles, extraplasmatic space, and chloroplasts. The biosynthesis of flavonoid substances
is genetically coordinated [18].
The synthesis of anthocyanins can be performed in any plant cell, being located in the
cytoplasm for monomers, dimers, and trimers and in vesicles, improperly called
13.3 The Metabolism Terpene 271
anthocyanoplasts (intensely pigmented organelles), for the final products. The anthocyanins
formed are transported from these vesicles, in vacuole, through a process of pinocytosis,
and the membranes of the vesicles can be incorporated into the tonoplast. Organic acids
result from the Krebs cycle, and phenolic substances from the shikimic acid cycle.
Alkaloids are nitrogen heterocyclic substances, found in over 13 000 species. Most plant
alkaloids come from amines or amino acids and only some come from isoprenoid precursors
in which nitrogen is incorporated into a late stage of the biosynthetic cycle. This is the case
with solanine (glycoalkaloid) from Solanum tuberosum and tomatine and its aglycone,
tomatidine from Lycopersicon esculentum. Alkaloids have been identified in vacuoles,
chloroplasts, and extraplasmatic space. Enzymes involved in the synthesis of these
substances have been identified in endoplasmic reticulum membranes, plasmalemma, and
tonoplast.
Among alkaloids, nicotine has the best studied biosynthetic cycle [19].
The primary compounds are arginine and ornithine which are decarboxylated and
metabolized to the putrescein (C4H12N2) conjugate form. Nicotine biosynthesis takes place
in small vesicles, which come from the endoplasmic reticulum or Golgi bodies and which
contain the enzymes involved in this process. The membrane of these vesicles is permeable
to tertiary compounds that can be synthesized at other sites. The formed quaternary
compounds are released into the vacuole, following its fusion with vesicles. Cyanogenic
glycosides such as amygdalin (C20H27NO11) and prunasin (C14H17NO6) have been identified
in cell vacuoles, and enzymes involved in the synthesis of cyanogenic glycosides have been
identified in the membrane of the endoplasmic reticulum. Cyanogenic glycosides have
been identified in over 1000 species, 500 genera, and 100 plant families. Of these substances,
the best known are amygdalin and prunasin from Rosaceae and sambunigrin from
Caprifoliaceae.
Biogenic amines are widespread in plants both as simple amines (primary, secondary,
and tertiary) and as amines with different functional groups (alcoholic, phenolic, carboxylic,
etc.). Among the amines, putrescine is important, which is formed from ornithine (Pisum,
Nicotiana), cadaverine is formed from lysine (Lupinus and Pisum), tryptamine is formed
from tyrosine (Hordeum and Lolium), dopamine is formed from dihydroxyphenyl alanine.
Putrescein, spermine, and spermidine interact with nucleic acids and may be involved in
protein biosynthesis. Putrescein, cadaverine, spermine, and spermidine in a concentration
of 10−4–10−6 M stimulate the growth process, and dopamine is the precursor for the
formation of melanoid compounds in bananas. Volatile substances that impart fruit flavor
are intermediate compounds of metabolism: alcohols, aldehydes, ketones, esters, ethers,
etc., substances with a high degree of volatility [4].
13.3.1 Metabolic Cycle of Mevalonic Acid Biosynthesis

The first step in this cycle is the formation of coenzyme A-(S)-3-hydroxy-3-methylglutaryl
(HMG-CoA) by the condensation of acetyl-CoA with acetoacetyl-CoA.
The next step involves the irreversible enzymatic reduction of HMG-CoA with hydrogen
from nicotinamide-adenine-dinucleotide-phosphate (2 × NADPH) to obtain mevalonic
acid (MVA). Subsequently MVA undergoes two successive phosphorylations with adenosine
triphosphate (2 × ATP) to form 5-pyrophosphate.
The next step already involves the trans-elimination of the hydroxyltertiary group and
the carboxyl group of 5-pyrophosphate, with the formation of PPI. This transformation is
in balance with the formation of DMAPP. The key enzymes involved in the MVA cycle are
acetoacetyl-CoA thiolase (AACT), HMG-CoA synthetase (HMGS), HMG-CoA reductase
(HMGR), and mevalonate diphosphate decarboxylase (MPDC or MVD) [20].
13.3.2 Metabolic Cycle of Methylerythritol Phosphate Biosynthesis

Plasticid isoprenoids are derived from PPIs via the 2-C-methyl-d-erythritol 4-phosphate
(MEP) ring. The first stage of this cycle involves the condensation of pyruvic acid
(2-hydroxypropanoic acid) and d-glyceraldehyde 3-phosphate. The initial reaction is
catalyzed by 1-deoxy-d-xylulose 5-phosphate (DXP) synthetase (DXS) to produce DXP. The
second step is intramolecular rearrangement and reduction of DXP using the enzyme
1-deoxy-d-xylulose 5-phosphate reductisomerase (DXR) leading to the formation of
2-C-methyl-d-erythritol 4-P (MEP). It, in turn, continues to undergo a series of reactions to
form PPIs.
The next step is the stereospecific and reversible isomerization of the PPI double bond
that leads to the formation of DMAPP. This step is very important because it generates
reactive pyrophosphate alkyl, which helps to combine two isoprene units to form geranyl
pyrophosphate (GPP and C10). Farnesyl diphosphate (FPP and C15) is derived from two
units of PPI and one unit of DMAPP, geranylgeranyl diphosphate (GGPP and C20) is from
three PPIs and one DMAPP. The biosynthesis of monoterpenes uses GPP as a substrate to
synthesize monoterpenes with linear and cyclic structure; the biosynthesis of sesquiterpenes
uses FPP, and of diterpenes-GGPP. The key enzymes involved in the MEP cycle are 1-deoxy-
d-xylulose 5-phosphate synthetase (DXS), 1-deoxy-d-xylulose reductoisomerase
5-phosphate (DXR), and (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase
(HDR) [21].
13.4 The Role of Essential Oils and the Specificity of Their

Accumulation in Plants
Although only 20–30% of higher plants have been investigated so far, tens of thousands of
SMs have already been isolated, and their structures have been determined by mass
spectrometry (MS), nuclear magnetic resonance (1H NMR, 13C NMR), or single crystal
X-ray diffraction. The role of SM has long been overlooked but is now widely recognized.
They are considered non-essential for sustaining life, but crucial for the survival of the
producing organism. EOs are often accumulated by plants in smaller quantities than MP,
about 1–3% of the dry mass, and can be identified only at a certain stage of growth and
development of the species or within only one species, which makes the extraction and
purification their difficult.
Although EOs have been known for hundreds of years and have been used as dyes (e.g.
indigo, and shikonin), flavorings (vanillin, capsaicin, and mustard oil), flavors (rose oil,
lavender oil, and other EOs), stimulants (caffeine, nicotine, and ephedrine), hallucinogens
(morphine, cocaine, scopolamine, and tetrahydrocannabinol), insecticides (nicotine,
13.4 The Role of Essential Oils and the Specificity of Their Accumulation in Plant 273
piperine, pyrethrin, and rotenone), human and vertebrate toxins (coniine, strychnine,
aconitine, colchicine, and glycoside), and therapeutic agents (atropine, quinine, and
codeine), their supposed biological functions were a contradictory issue. In natural
ecosystems, there is a regulation of population numbers, a natural control, in which the
internal factors of the respective populations (endogenous factors) intervene, as well as the
external factors (exogenous). In turn, exogenous factors can be abiotic and biotic. Among
the abiotic factors, the most relevant are temperature, UV radiation (affects phenols and
phenylalanine derivatives), soil composition (affects SMs containing nitrogen). Some EOs,
for example flavonoids, are essential compounds that absorb UV rays and also show
protective functions against those rays, thus preventing the photodegradation of
chlorophylls, or even the destruction of the structural integrity of chloroplasts. However,
biotic effects include plant pathogens, such as viruses and bacteria, as well as herbivorous
animals. During the periods of stress, caused by the attack of microorganisms, the
biosynthesis of SM is activated [22].
Among them were described SM with antibiotic, antifungal, antiviral action, which are
able to protect plants from pathogens (phytoalexins). They are also known the EOs with
anti-germinative properties, such as monoterpenes, in the following order of inhibitory
power: geraniol > carvone > borneol > β-citronellol > α-terpineol > camphor > menthol > m
entone > limonene > citral. At the same time, camphor also shows toxic (allelopathic)
effects for plants, which have an important role in the interaction with pollinators and the
seed coat.
In addition, plants also have mechanisms for responding to stressors, similar to the
animal’s immune system. They are based on the action of salicylic and jasmonic acid. Their
biosynthesis seems without direct significance for the synthesizing cell but can be decisive
for the development and functioning of the whole organism. Some SMs determine the taste
and aroma of fruits and vegetables, and ethers, esters, terpenes, etc., through their pleasant
smell, and they have an important role in plant pollination. Pigments, which are also
products of secondary metabolism, play an important role in redox reactions. They
determine the color of flowers, fruits, vegetables, and all plant organs and play an important
role in the pollination process [23].
Phenylpropanoids (FPs) are one of the most important groups of natural compounds.
They can be found in a wide variety of very valuable herbs as well Echinacea purpurea,
Rhodiola rosea, Silybum marianum, Melissa officinalis etc. FPs play an essential role in
plant integrity and have a protective effect against biotic and abiotic stresses. They are
divided into several major classes, such as coumarins, flavonoids, hydroxycinnamic acids,
and phenylpropenes. FPs have antioxidant activity and have beneficial effects on human
health. FPs are also known for their antibacterial, antifungal, and anti-inflammatory
properties.
Terpenes, being synthesized by all living organisms, represent the most numerous class
of natural compounds. They are an essential component of human nutrition, and many of
these substances are economically important as pharmaceuticals, aromatics, and potential
biofuels of the latest generation. In the pharmaceutical industry, terpenes have a wide
spectrum of use, being used both as excipients to improve skin penetration and as active
medicinal principles with pharmacological properties anticancer, antimicrobial, antifungal,
antiviral, antihyperglycemic, anti-inflammatory, etc. [24].
Data from the literature suggest that most of the materials studied describe the analgesic
profile of monoterpenes and sesquiterpenes, alone or in combination with other
biochemical components such as EOs extracted from medicinal plants. Examples of
biologically derived drugs currently marketed as terpene products are artemisinin
(sesquiterpenoid), taxol (diterpenoid), and vincristine (monoterpenoid). Hydroxycinnamic
acids (chlorogenic, ferulic, caffeic, etc.) and other aromatic hydroxy acids (gallic and
salicylic acids, etc.) are the most common phenolic acids in higher plants and act as
biogenetic precursors in the biosynthesis of other plant phenolic compounds. Both acids
and their derivatives are powerful antioxidants, which possess pronounced antiradical
properties in in vitro; tests. The pronounced collagenic effect of ferulic, caffeic, and
chlorogenic acids was established. All caffeic, chlorogenic, ferulic, coumaric, and other
hydroxycinnamic acids are essential in the prevention and treatment of obesity, diabetes,
and associated disorders, improve kidney function, stimulate antitoxic function of the liver,
and have an antimicrobial and antimalignant effect.
The high content of hydroxycinnamic acids and their derivatives is characteristic of the
species Echinacea purpurea, Rhodiola rosea, whose plant materials are widely used in
medicine to correct immune system disorders. Therefore, the evaluation of the mechanisms
involved in the accumulation of SM with beneficial effect for the improvement of health,
as well as the highlighting of those exogenous factors with stimulating effect of active
principles, represents a scientific issue of significant social and economic importance [25].
EOs are complex liquid mixtures consisting mainly of terpene and terpenoid
hydrocarbons, extracted from parts of hetero-oil plants (by hydrodistillation, maceration,
enfleurage, pressing, etc.) and used in the composition of perfumery, cosmetics, food, etc.
in order to change their smell and/or taste. EOs are a by-product of plant metabolism that
has a characteristic odor. The role of these compounds in the body of the plant is not well
defined, but it is known that some substances behave as attractants (attract pollinating
insects), others on the contrary-repellents-keep away potentially harmful organisms to the
plant (show antifungal effects, herbicides, insecticides, etc.).
Also, these compounds, evaporating in hot weather, partially reduce the temperature of
the plant. Some of them serve as intermediates in the biosynthesis of complex products
necessary for the plant organism or participate directly in various biochemical processes.
In the general monograph “Aetherolea” from the European Pharmacopoeia, tenth edition,
EOs are defined as mixtures of volatile and lipophilic substances, with aromatic odor,
belonging to different classes of organic compounds (especially terpenes and their
oxygenated derivatives).
Named “oils,” these mixtures were obtained due to their resemblance to the appearance
of fatty oils. The full name “EOs” is appropriate, as it expresses one of the most characteristic
properties of these compounds, namely the increased volatility at normal temperature.
Due to this property, they differ from fatty oils, which under normal conditions are
virtually non-volatile. Other names, such as essential oil and essential oil, are less
characteristic because neither these oils are composed of essential (and/or ester) compounds
nor is the name essential. EOs contain different classes of chemical compounds, of which
terpene compounds and terpenoids predominate [26].
Usually, non-terpenic compounds are found in smaller quantities, among them aromatic
and aliphatic compounds (aldehydes, alcohols, amines, etc.) can be highlighted. In total,
up to 500 compounds can be contained in an EOs, and their number vary depending on the
plant from which the EOs were extracted. Thus, EOs from geranium and rock rose contain
about 300 compounds, and those obtained from rose, bergamot, orange ofabout 500
compounds each.
Of this set of compounds, one or several are contained in high concentrations influencing
the odor character of the volatile oil.
For example, in coriander EOs, the basic component is tertiary alcohol-linalool (38%); in
rose EOs alcohols predominate: phenylethyl, citronellol, geraniol, and nerol (summary up
to 95%); in lavender EOs-linalool (51%) and linalyl acetate (35%).
The class of terpene compounds in EOs predominate acyclic terpenes: monoterpenes
(myrcene and ocimen), sesquiterpenes (farnesen); cyclic terpenes: monocyclic
monoterpenes (limonene, terpinolines, and terpenes) and bicyclic monoterpenes (pinens,
camphene, and hull); monocyclic sesquiterpenes (bisabolen), bicyclic sesquiterpenes
(cadinen), etc. Oxygenated derivatives predominate in the class of terpenoids: terpenic
alcohols (linalool and geraniol), terpenic aldehydes (citral and citronellal), and terpenic
esters (linalylacetat and geranylbutyrat). Among the compounds of non-terpenic nature
are phenols (anethole and eugenol), organic acids (acetic acid and benzoic acid), aliphatic
alcohols and aldehydes, amines, thiocompounds, heterocyclic compounds, etc. [27].
EOs together with synthetic odor compounds are the basic raw material in the production
of perfumery, cosmetics, but also food flavors.
Thus, EOs of sage, eucalyptus, sandalwood, and geranium are applied in various antiacne
products due to their antiseptic properties. Many of these compounds are used as raw
materials in various organic syntheses, for example, linalool in the synthesis of vitamin E,
myrcene in the synthesis of cyclohexenes applied in perfumery, carvone in the synthesis of
bravellin (antimalarial action), tetrahydrocannabinoids (anti-analgesic action, etc.), and
some EOs (mint, lemon, rose, coriander, etc.) are used as taste and odor correctors in
pharmaceuticals.
Terpenoids are one of the most common classes of natural compounds.
The aromas of many flowers and fruits are due to the mixtures of volatile compounds
that they emit. These are often called terpenes.
Their molecule consists of 5n carbon atoms where n is a natural number. Many terpenes
are used as food flavorings (clove extract, mint, etc.), as perfumes (rose, lavender, etc.), or
as solvents (turpentine). Hydrocarbons included in the category of terpenoids have the
general formula (C5H8)n, where n is a natural number greater than or equal to 2.
For this reason, they were considered isoprene oligomers in which the binding of
monomers is performed in positions 1, 4 (head = tail).
Natural EOs are composed together with the mentioned hydrocarbons and a series of
their functional derivatives containing oxygen (alcohols, carbonyl compounds, etc.). The
most common classification of terpenoids is made according to the value of n in the
mentioned molecular formula. In reality, plants do not synthesize terpenoid compounds
from isoprene.
The source of these units is acetic acid (precursor) which with coenzyme A leads to
acetyl-coenzyme A and then by trimolecular condensation to hydroxy-b-methyl-glutaryl-
coenzyme A, which is reduced fermentatively to mevalonic acid, a compound which then
transforms in isopentenyl pyrophosphate, the key element in terpenoid synthesis.
Examining the polycyclic structures of terpenes, it is obvious that they can be detailed by
conformation and configuration formulas that approximate more correctly the molecules
of these compounds, e.g. (−) menthol 2 has a chair configuration with equatorially oriented
substituents.
Volatile terpenes are low molecular weight molecules derived from isopentenyl
diphosphate (PPI) and play an important role in direct and indirect defense, being also
used as a signal for neighboring plants. Table 13.1 shows the provenance, components, and
physiological action of several plant species representative for the content of EOs.
Plants constantly secrete small amounts of volatile terpenes, but in some cases, such as
the attack of herbivorous insects, terpenes are secreted in larger quantities in response to
the negative action of biotic factors.
Table 13.1 Components of essential oils from some plant’s species.
Chemical structure/origin Oil Plants species Components
CHO3 Anise Fruit of anise Anethol (80–90%)

(Pimppinella
Anetol anisum)
CH2 CH CH2
CH3
Bergamot Orange peel (−) Linalool acetate,

(Citrus nerol, terpineol, (−)
OH aurantum, pinene, (−) camphen,
Nerol Bergamia (+) lemon
subspecies)
H3C CH3
Caraway Fruits Carum Carvone (50–60%),

CH3 carvi (+) limonen,
O dihydrocarbon,
H 3C CH2
CH3
CH2OH
Lemon Cymbopogon Citral (70–85%),
grass flexuosus methylheptenone,
nerol, farnesol,
H3C CH3
geraniol, dipentene
Chemical structure/origin Oil Plants species Components
COOH
Cinnamic acid
Lavender Flowers Linolyl acetate
Lavendula (30–60%), linolyl
officinalis butyrate, geraniol,
coumarin, cineole
O O
Coumarin
CH3
H H
Valerian Odolean Butyric acid, valerian
(Valeriana acid, (−) camphene,
officinalis) (−) pinene, (−)
H3C CH3 borneol, bornyl
H valerianate
COOH
H3C CH3 CH
CH3 3CH
CH2 3
Fennel Seed of fennel (50–60%), (+) pine,

Camphen α-Pinen anethole (Foeniculum camphen, dipentene,
vulgare)
CH3 CH3
O
Geranial OH Neroli Citrus 27% (pine, camphen,
Geraniol (orange bigaradiarisso dipenten), (−)
H3C CH3 H3C CH3 blossom) linalool 30%, geraniol,
nerol, anthranilic
acid methyl ester 16%
Source: Santosh Kumar Upadhyay.
Terpenes are an important source of olefinic compounds (alkenes) that are involved in
the formation of phytotoxic products.
Volatile terpenes combine with nitrogen oxides and form ozone-type photo-oxidants,
thus increasing the stress around the plant.
The synthesis of terpenes is very energy-consuming, and in essence, the cost of producing
volatile terpenes is higher than that of any primary or SMs.
Terpenes are generally accumulated in plants at the level of specialized secretory
structures: glandular hairs (Lamiaceae, Asteraceae), secretory pockets and cavities
(Fabaceae, Rutaceae), secretory canals (Pinaceae, Apiaceae), laticifers (Euphorbiaceae,
Asteraceae), and idioblasts (Magnoliacs, Lauraceae) [28].
The compound that contributes to the scent depends on the plant species, and it can be
seen from Table 13.2.
Table 13.2 The compound that contributes to the scent some spices plant.
Common name of the

plant species The compound/compounds responsible for odors
Carnations Eugenol/beta-caryophyllene, and benzoic acid derivatives

Violets Ionones
Lilies (e)-Beta-ocimene and linalool, eucalyptol (also referred to as 1,8-cineole),
(E)-ocimene, 8-oxolinalool, benzyl methyl ether, indole, lilac aldehyde, lilac
alcohol, and hydroquinone dimethyl ether
Hyacinth Hyacinth, ocimenol, cinnamon, and ethyl 2-methoxybenzoate
Chrysanthemums Alpha-pinene, eucalyptol, camphor, and borneol, chrysanthenone and
chrysanthenyl acetate, beta-caryophyllene
Lilacs (e)-Beta-ocimene, benzyl methyl ether
Roses (−)-Cis-rose oxide, beta-damascenone, geraniol, nerol, (−)-citronellol,
farnesol, and linalool
Lily 1,8-Cineole, benzaldehyde, methyl benzoate, ethyl benzoate, creosol, and
isoeugenol
Coffee Linalool, nerol and geraniol (as major constituents 40% total),
epoxygeraniols and epoxynerols
Jasmine (−)-Jasmine lactone, (Z)-jasmone, (−)- and (−)-epi-methyl jasmonate
Lavender (−)-(R)-linalool, (−)-(R)-lavandulol, 1-octen-3-yl acetate
Lemon Geranial + neral, (+)-limonene
Lemongrass Geranial, neral, myrcene, neomenthol, linalyl acetate, (Z)-beta-ocimene
Lily-of-the-valley Floral-rosy-citrusy notes: citronellol, citronellyl acetate, geraniol, nerol,
geranyl acetate, geranial+benzyl acetate, neral, benzyl acohol, phenethyl
alcohol, phenylacetonitrile, farnesol and 2,3-dihydrofarnesol, green-grassy
notes: (Z)-3-hexenal and (E)-2-hexenal, (Z)-3-hexenyl acetate, (Z)-3-hexen-
1-ol, green pea and galbanum-like notes: 2-isobutyl-3-methoxypyrazine and
2-isopropyl-3-methoxypyrazine, fatty, waxy, aldehydic notes: octanal,
nonanal, decanal and fruity, raspberry notes: beta-ionone
Magnolia (+)-Verbenone, isopinocamphone, (Z)-jasmone. Magnolia (+)-verbenone,
isopinocamphone, (Z)-jasmone
Mandarin Methyl N-methyl-anthranilate, linalool, trans-4,5-epoxy-(E)-2-decenal,
alpha-sinensal and (E,Z)-2,6-dodecadienal
Mango Delta-3-carene, limonene, terpinolene, (E)-beta-ionone, a-phellandrene,
(E,Z)-2,6-nonadienal, ethyl 2-methylpropanoate, (E)-2-nonenal, ethyl
butanoate, methyl benzoate, decanal and 2,5-dimethyl-4-methoxy-3[2H]-
furanone (mesifurane)
Neroli (orange (+)-Linalool, (+)-(E)-nerolidol and (E)(E)-farnesol, methyl anthranilate,
flower) indole, phenylacetonitrile and 1-nitro-2-phenylethane
Petunia Benzaldehyde, phenylacetaldehyde, isoeugenol, methyl benzoate, phenethyl
alcohol, benzyl benzoate
Peppermint (−)-Menthol, (−)-menthyl acetate, (−)-menthone and (+)-menthofurane
Pink pepper Alpha-cadinol
Common name of the

plant species The compound/compounds responsible for odors
Pine Alpha- and beta-pinene and two exotic macrolides from maritime pine,
longifolene from long-leaved pine, and the two derivates isolongifolene and
isolongifolanone
Raspberry Raspberry ketone, (R)-(+)-(E)-alfa-ionone, mesifurane, beta-damascenone
Robinia 2-Aminobenzaldehyde and 3(Z)-hexen-1-ol, 3(Z)-hexen-1-ol
Rosemary (+)-Borneol, (+)-bornyl acetate, (+)-camphor, (+)-alpha-pinene,
(+)-verbenone and 1,8-cineole
Saffron Safranal and 2-hydroxy-4,4,6-trimethyl-2,5-cyclohexadien-1-one
Tobacco Beta-damascenone, megastigmatrienone, oxo-edulan, 4-oxo-beta-ionone
Thyme Thymol, carvacrol
Vanilla Vanillin and a vanilla vitispirane
Patchouli (−)-Patchoulol and norpatchoulenol
Peach Gamma-decalactone and delta-decalactone
Pear Ethyl 2(E),4(Z)-decadienoate
Turmeric Ar-turmerone, turmerone and curcumin
Yuzu Branched aldehydes, yuzu lactone and 6-methyl-5-hepten-2-ol
Ylang-Ylang p-Cresyl methyl ether, benzyl acetate, methyl salicylate, methyl benzoate
and cinnamyl acetate
Both the effective synthesis of volatile terpenes and the formation of these specialized
structures in their accumulation are very expensive for organisms. Terpenes serve as signals
that can induce systemic defensive responses, in the unattacked areas of the plant, but also,
through the air, can reach the neighboring plants where they can induce defensive
responses to possible attacks. It is the volatile nature of terpenes that gives them the ability
to behave as the highly efficient signaling molecules. Mircene is found in hops, geraniol has
a “rose”-like (the scent of geraniums and roses) in rose flowers, limonene (the major scent
of citrus flowers and fruits) in cumin, nerol (orange blossom), menthol in mint, camphor
in Laurus camphor wood and wormwood, borneol in lavender, etc.
The origin and some physical properties of terpenoids are presented in Table 13.3.
The quality of EOs depends very much on the concentration of the basic components.
But this criterion is not a determining one because some EOs that correspond to all physico-
chemical quality parameters may receive a low grade from the perfumer due to the content
of unpleasant-smelling compounds, which exceed the allowable limits.
As such compounds serve: for rose EOs-organic acids with low molecular weight; for
lavender and coriander EOs-camphor; for EOs of mint and geranium-mint, etc. Also, the
odor nuances of an EOs are influenced by a series of compounds that are contained in low
concentrations (tenths or hundreds of percent). Thus, the presence or absence of rosenoxid,
methylenugenol, eugenol, and acetic aldehyde influences the smell of volatile rose oil;
menthylacetat and mentofuran – the smell of volatile peppermint oil, etc.
Table 13.3 Provenance and some physical properties of terpenoids.
Terpenoid Provenance (essential oil)
Ocimen Basil
Citronelol (R = CH2OH) (−) Of rose oil
Citronelal (R = CHO) Essential oils of geranium and rose
4 Geraniol (R = CH2OH) Rose oil, geranium, animal tissues
Geranial (R = CHO) (citral Lemon oil
a)
Nerol (R = CH2OH) Rose oil, bitter orange blossom oil
Neral (R = CHO) (citral b) Cis, trans, in lemon peel
Limonen (+) In lemon peel (−) In turpentine (fir needles) (±) it is called
dipentan
Terpineol Peppermint oil
Menthol Peppermint oil
Pulegonă Peppermint oil
Alpha-pinene Turpentine oil
Beta-pinene Turpentine oil
Camphor From camphor tree wood, from wormwood
Thujone Thuja oil
Farnesol From linden, rose, citrus oil, pearl
Nerolidol Nerol oil
Juvenile hormone III In insects
G-bisabolene Lemon and bergamot oil
Abscisic (+) acid Plant growth hormone
Caryophyllene Clove oil
Drimenol Drimys winteri tree (America)
Geranyl-geraniol Conifer resin
Geranyl-linalool Conifer resin
Phytol Component of chlorophyll and vitamins K and E
Vitamin A Growth factor in milk, butter, eggs, fruits
Manool Yellow pine oil
Sclareol Sage oil, incense
Abietic acid Conifer resin
Pimaric acid Conifer resin
squalene In the shark liver
Ambreina In gray amber (sperm whale)
Cucurbitacina In cucumbers, it gives a bitter taste
13.5 Essential Oils from Plants in Perfum 281
The quality parameters of EOs include odor, color, taste, acidity index, relative density,
refractive index, iodine index, peroxide index, esterification index, saponification index,
and the content of specific individual compounds of certain oils (content of linalool,
menthol, charcoal, etc.). For a qualitative EOs, these parameters must correspond to those
indicated in the corresponding normative documentation. EOs are complex liquid mixtures
consisting of volatile organic compounds of plant origin. The composition of EOs is
dominated by terpenic compounds and terpenoids (monoterpenes, sesquiterpenes,
monoterpenic alcohols, etc.).
Classes of aliphatic compounds (alcohols, aldehydes, aliphatic esters, etc.), aromatics,
macrocyclics, and their derivatives (amines, organic sulfides, heterocyclic compounds,
etc.) are contained in small quantities [29].
13.5 Essential Oils from Plants in Perfume
Perfume is a mixture of aromatic EOs or aromatic compounds, fixatives and solvents, that
used to give the human body, animals, food, objects, and living spaces a pleasant smell. The
types of perfume reflect the concentration of aromatic compounds in a solvent, in which
the fine perfume is usually ethanol or a mixture of water and ethanol. Various sources
differ considerably in the definitions of perfume types. The intensity and longevity of a
perfume are based on the concentration, intensity, and longevity of the aromatic
compounds, or perfume EOs used. As the percentage of aromatic compounds increases, so
does the intensity and longevity of the perfume. Specific terms are used to describe the
approximate concentration of a perfume with the percentage of perfume oil in the volume
of the final product.
The precise formulas of commercial perfumes are kept secret. Even if they were widely
published, they would be dominated by ingredients and fragrances so complex that they
would not be very useful in providing a guide to the general consumer in describing the
experience of a perfume. However, perfume connoisseurs can become extremely skilled at
identifying the components and origins of perfumes in the same way as wine experts.
The most practical way to start describing a perfume depends on the elements of the
perfume notes of the perfume or “family” it belongs to, all affecting the overall impression
of a perfume from the first application to the last persistent suggestion of the perfume.
The traces of the perfume left behind by a person wearing perfume are called his “sillage,”
after the French word for “wake,” like the traces left by a boat in the water. The perfume is
described in a musical metaphor as having three sets of notes, which makes the harmony
of the perfume harmonious.
The EOs (aroma compounds) produced by plants can be systematized by functional
groups. These groups involve alcohols (e.g. eugenol, furaneol, hexanol, and menthol),
aldehydes (e.g. acetaldehyde [pungent], benzaldehyde [marzipan, almond], cinnamaldehyde
[cinnamon], citral [lemon oil and lemon grass], furfural [burnt oats], hexanal [green and
grassy], nonanal, octanal, and vanillin [vanilla]), amines (e.g. skatole and indole), esters
(e.g. lutein fatty acid esters from marigold), ethers (nerolin = methyl β-naphthyl ether),
terpenes (e.g. caryophyllene, citronellol in rose, geraniol, linalool in many flower species,
nerol, and β-ionone).
The notes unfold over time, with the immediate impression of the top note leading to
deeper middle notes, and the base notes gradually appear as the final stage [30].
The smells like, tobacco flavors (which are similar to cigarettes), fruit flavors (peach,
blueberry, etc.), menthol flavors, sweet flavors (chocolate, candy, etc.), and other flavors
(black tea, coffee, wine, etc.) are carefully created by knowing the process of evaporation of
the perfume and the three categories listed below:
●● Top notes: they are also called top notes. Perfumes that are perceived immediately when
applying a perfume. Top notes consist of small, light molecules that evaporate quickly.
They form the initial impression of a person’s perfume and are therefore very important
in selling a perfume, e.g. of top notes include mint (rich in menthol, menthone,
menthofuran, 1,8-cineole, menthyl acetate, etc.), lavender (rich in linalool, perillyl
alcohol, linalyl acetate, camphor, limonene, tannins, etc.), and coriander (rich in linalool,
limonene, camphor, geraniol, etc.).
●● Middle notes: also called heart notes. The scent of a perfume that appears just before the
top note dissipates. Compounds with middle notes form the “heart” or main body of a
perfume and act to mask the often-unpleasant initial impression of the base notes, which
become more pleasant over time, e.g. of middle notes include sea water (oceanic, salty/
seawater vibe, idea is smell “breezy,” “outdoorsy,”) sandalwood (sesquiterpenic alcohols/
tricyclic α-santalol/β-santalol, etc.), and jasmine (ester benzyl acetate, nerolidol, cedrol,
jasmone, etc.).
●● Base notes: the smell of a perfume that appears close to the departure of the middle notes.
Base and middle notes are together the main theme of a perfume. The base notes bring
depth and solidity to a perfume. Compounds in this class of flavors are usually rich and
“deep” and are usually not perceived until 30 minutes after application, e.g. of base notes
include tobacco/tobacco smoke (e.g. cyclohexane, ionone, theaspirone, safanal,
cyclocitral etc.), amber (dry woody amber ambergris musk sweet, etc.), and musk
(Angelica archangelica, Abelmoschus moschatus produce musky-smelling macrocyclic
lactone compounds, etc.).
The perfumes in the top and middle notes are influenced by the base notes; conversely,
the smells of base notes will be altered by the types of scented materials used as middle
notes. Manufacturers who publish perfume notes typically do so with perfume components
presented as a perfume pyramid, using imaginative and abstract terms for the listed
components. The various aroma constituents are produced in tobacco leaf via oxidative
carotenoid degradation, according Table 13.4
Grouping perfumes can never be completely objective or definitive. Many perfumes
contain aspects of different families. Even a fragrance designated as a “single flower” will
have subtle hints of other aromatics. There are almost no real unique perfumes made from
a single aromatic material [31]. Table 13.5 lists some of the chemical compounds in
essential oils and their characteristic aroma in alphabetical order.
According to the fragrance classification chart using the disk of aromas, there are five
main families Floral, Oriental, Woody, Fougère aromatic, and Fresh, the first four in
classical terminology and the last in the modern oceanic category. Each of them is divided
into subgroups and arranged around a wheel. In order to be able to describe the notion of
smell, they were conceived to odor descriptor corresponding to flavor index.
Table 13.4 Aroma/smell/odor characteristics of tobacco carotenoid derivatives.
Name Aroma/smell/odor characteristics Structure
H3C CH3 O
CH3
3-Oxo-alpha-Ionone Sweet, floral
O
CH3
H3C CH3 O
CH3
4-Oxo-beta-ionone Sweet rich like virginia tobacco
CH3
O
H3C CH3 O
Alpha-Ionone Woody balsamic, violet-raspberry in dilution CH3
CH3
H3C CH3
CHO
Beta-Cyclocitral Green, grassy hay like odor
CH3
O
H3C CH3
Beta-Damascenone Fruity, floral with apple, plum-raisen, tea, CH3
rose, tobacco note
CH3
O
H3C CH3
Beta-Damascone Fruity (apple-citrus), tea-like with slight CH3
minty note
CH3
H3C CH3 O
Beta-Ionone Woody, violet, fruity; woody-raspberry on CH3
dilution
CH3
H3C CH3
Dihydroactinodiolide Weak, slightly cooling

O O
CH3
H3C CH3
Oxo-Edulan I Oriental tobacco like

O O CH3
CH3
(Continued)
Name Aroma/smell/odor characteristics Structure
H3C CH3
Oxo-Edulan II Oriental tobacco like

O O CH3
CH3
H3C CH3
CHO
Safanal Saffron, green, sweet, hay-like
CH3
H3C CH3
Theaspirone Tea like O CH3

O
CH3
Table 13.5 Chemical components of essential oils and aroma/smell/odor characteristic.
Chemical components The aroma/smell/odor characteristic
6-Methyl heptan-2-one Green, herbaceous, weak oily

Benzyl acetate Floral-fruity, fresh-sweet-notes
Benzyl alcohol Faint aromatic, weak floral
Benzyl benzoate Sweet-balsamic, faint-floral
Benzyl salicylate Faint-sweet, weak herbaceous-medicinal
Chavicol Sweet-phenolic, anise-like, green-minty-notes
cis-3-Hexenol Green, fresh-grass
cis-3-Hexenyl acetate Intense green, weak fruity, pungent
cis-3-Hexenyl benzoate Green-herbal, weak woody
cis-Jasmone Intense jasmine-like, warm-floral, weak fruity
cis-Linalool oxide Fresh-floral, citric-notes
cis-Methyl jasmonate Floral-sweet, herbaceous
Eugenol Spicy, strong clove-cardamon-notes
Geraniol Rose-notes, sweet-floral, weak fruity
Geranyl linalool Floral, rose-geraniol-lavender-notes, weak fruity
Indole Unpleasant-fecal, musty-cadaverous-floral in dilution
Isoeugenol Spicy, intense clove-like, sweet and woody undertones
Isophytol Weak floral-balsamic
Linalool Fresh-floral, sweet-fruity-woody, lavender-notes
Methyl anthranilate Fruity, grape-like
Chemical components The aroma/smell/odor characteristic
Methyl benzoate Fragrant-fruity

Methyl linoleate Fatty-oily
Methyl oleate Fatty-oily
Methyl palmitate Fatty-oily
Methyl salicylate Fragrant-minty, sweet-spicy, wintergreen-notes
Nerol Floral, weak rose-notes, sweet-fruity
p-Cresol Medicinal-aromatic
Phenylethyl acetate Sweet-fruity, rose-honey-notes
Phenylethyl alcohol Floral, rose-like, fragrant-honey-notes
Phytol Weak floral-balsamic, sticky side-notes
Phytyl acetate Faint-floral-balsamic
trans-Jasmone Floral-jasmine-like, warm-fruity, weak spicy
trans-Linalool oxide Fresh-floral, weak sweet-citric
trans-Nerolidol Floral-fruity, rose-apple- and green-citrus-notes, woody-waxy
α-(E,E)-Farnesene Weak floral
α-Terpineol Fragrant-floral, weak fruity with lilac-notes
δ-Jasmine lactone Jasmine-like, warm-floral
Perfume compounds in perfumes will degrade or decompose if improperly stored in the

presence of heat, light, oxygen, and foreign organic matter. Proper preservation of perfumes
involves keeping them away from heat sources and storing them where they will not be
exposed to light. An open bottle will keep its aroma intact for several years, as long as it is
well preserved. However, the presence of oxygen in the space of the glass head and
environmental factors will change the smell of the perfume in the long run.
Fragrances are best stored when stored in airtight aluminum bottles or in their original
packaging when not in use and refrigerated at relatively low temperatures: between 3 and
7 °C (37–45 °F). Although it is difficult to completely remove oxygen from the headspace of
a stored perfume bottle, opting for spray dispensers instead of “open” rollers and bottles
will minimize oxygen exposure. Sprays also have the advantage of isolating the perfume
inside a bottle and preventing it from mixing with dust, skin, and detritus, which would
degrade and alter the quality of a perfume.
The family classification is a starting point for describing a perfume, but it does not fully
characterize it. E.g., Roses spp. come in two types of perfume extracts: as an essential or
absolute oil (deeper and sweeter than its oil counterpart). Rose was the test of time due to
its ability to combine perfectly with other floral notes, wood and citrus. Rose extracts
contain hundreds of molecules, which explains why its scent is so rich and multifaceted.
Rose perfume extracts have notes of citrus (lemongrass), green, fruity (peach, plum,
wine), spicy (cloves), amber, and sweet veneers, all in one fragrance. Pelargonium graveolens
(green, accentuated, and herbaceous scent) meaning strong smell, geranium is often
confused with “the other” rose (but geranium has an aromatic quality [similar to lavender],
which makes it smell more “masculine”), but with a less powdery and more lemony,
herbaceous aroma with a soft but strong green scent. Known molecules found in geranium
oil are citronellol, nerol, geraniol, and linalool. Nerol is found in lemongrass, contributing
to the smell of lemony geraniums, and geraniol is one of the primary components in rose
oil, contributing to the smell of rose geraniums [32].
Among the compounds contained in a relatively large number of EOs can be highlighted:
13.5.1 Linalool (3,7-dimethylocta-1,6-dien-3-ol), C10H18O

Smell and taste: it has a floral scent, free of terpenic and camphor shades, if it is pure enough,
with fresh, woody, and citrus shades. The taste is spicy, similar to coriander EOs. Distribution
in nature: it has been identified in over 200 EOs of different origins (leaves, flowers, wood,
and fruit). Important sources: for the dextro form – coriander oil (60–70%); for the form
levo – HoSho oil (80%), linaloe (80%), bois de rose (80–90%), ylang-ylang and bergamot
(linalool 7–20%, linalyl acetate 20–40%). Field of use: it is used as an odorant compound in
perfumery compositions and cosmetics. It serves as a raw material in the synthesis of other
odorous compounds (such as citral, linalylacetate, etc.) and some biologically active
compounds (vitamin E, cyclohexylgeranilacetic acid [Cygerolum]), etc. [33].
13.5.2 Camphor (1,7,7-trimethylbicyclo [2.2.1] heptan-2-one), C10H16O

Physico-chemical properties: chemical compound of the class of bicyclic terpenes. It has two
asymmetric carbon atoms in positions C1 and C4, but only two enantiomeric forms (+) and
(−) are possible. It is slightly soluble in water (0.12%), well soluble in ethanol, ether, chloroform,
acetone, acetic acid, benzene. Smell and taste: after the smell, camphor can be considered as a
prototype, and it shows a warm, mentholated smell, almost radiant, but a little tenacious. The
taste is fresh, bitter-hot, and cool. Spread in nature: it has been identified in over 180 EOs. It is
the main component of Cinnamomum camphora camphor tree oil in China. From this oil,
camphor is separated by crystallization. Field of use: it is used as a component of perfumery
compositions; as a plasticizer in the production of plastics; in medicine it is applied as an
antiseptic, antirheumatoid, and analeptic remedy; as a raw material for the synthesis of other
valuable compounds (such as 3-bromocamphor, borneol) etc. [34].
13.5.3 Cedrol (1S, 2R, 5S, 7R, 8R)-(2,6,6,8-tetramethyltricyclo [5.3.1.01,5]

undecan-8-ol or cedran-8-ol), C15H26O
Physico-chemical properties: chemical compound of the class of sesquiterpene alcohols. It is
in the form of colorless crystals, well soluble in benzyl benzoate, moderately soluble in glycols
and mineral oils, insoluble in water. It contains five asymmetric carbon atoms. Smell and taste:
it manifests a smell of moderate intensity, similar to the smell of cedar wood. It has weak and
woody taste. Distribution in nature: it is contained in EOs from some conifers (cedar [Cedrus
atlantica], juniper [Juniperus virginiana], cypress [Cupressus sempervirens] etc.), some species
of oregano, etc. Field of use: it is used as an odorant and flavoring compound or in the synthesis
of methyl ether and cedrilacetat, which have the same applications [35].
13.5.4 Eugenol (2-methoxy-4-allylphenol; 1-hydroxy-2-methoxy-4-

allylbenzene), C10H12O2
Physico-chemical properties: chemical compound of the phenol class. It appears as a
colorless to slightly yellowish liquid. Smell and taste: it manifests a strong clove smell,
characterized as hot, spicy, hot, less hot, and spicy than clove oil. It is hot and spicy taste.
Distribution in nature: It contained in many EOs, including: EOs from cloves (85%), basil
(60–70%), cinnamon leaves (even over 90%), in smaller quantities in obligatory EOs, ylang-
ylang, Citronella, Sassafras, etc. Field of use: It is used as an odorant compound; as a raw
material in the synthesis of other odorous compounds, such as dihydroeugenol, isoheugenol,
etc. Esters are a class of chemical compounds derived from inorganic or organic acids in
which at least one hydroxyl (−OH) group is substituted by the −O-alkyl group. In nature,
the most common odorant aliphatic esters consist of the residues of formic, acetic,
propionic, butyric acids, and the residues of methyl, ethyl, butyl, isoamyl alcohols, etc.
Carboxylic acids and their esters are contained in various EOs, in fruits, flowers, fermented
foods (cheeses, etc.). Uses: esters, due to their specific odor, represent the most numerous
class of odorous and flavoring compounds. It is used in perfumery, but especially in the
food industry to make food flavor compositions (food essences), which give the products
aromas of flowers, fruits (the largest group), and berries [36].
13.5.5 Citral (3,7-dimethyl-2,6-octadien-1-al), C10H16O

Belongs to the class of monoterpenic aldehydes, it is a weak-yellow liquid with a strong
lemon smell, with a bitter-bitter taste. It is a mixture of two geometric isomers: the trans-
form (geranial or citral A) (55–70%) and the cis-form (neral or citral B) (35–45%).
Distribution in nature: citral is found in various EOs, in large quantities in EOs of various
species of Cymbopogon or Lemongrass (50–95%) and in EOs of Litsea cubeba (plant native
to China, Indonesia) (60–80%). In smaller quantities, it is contained in EOs from: lemon
( 2%), eucalyptus, verbena (widespread in tropical and subtropical regions), ginger,
rhubarb, etc. Uses: due to the pronounced smell of lemon, it is applied as an odorant or
flavoring agent in cosmetics and food, and it is rarely used in perfumery (due to the
tendency of polymerization and oxidation). It is added to chewing gum, pastries, sweets,
ice cream, and various drinks. The concentration of citral in these products varies in the
range of 9–170 ppm. To give the smell of lemon and verbena, it is added in the composition
of perfumes, soaps, detergents, creams, and cosmetic lotions. Citral is also used as an
intermediate in the synthesis of vitamin A, ionone, and methil ionone [37].
13.5.6 Vanillin (4-hydroxy-3-methoxybenzaldehyde) C8H8O3

Vanillin comes in the form of white aciform crystals with a pleasant smell, vanilla, and
sweet taste. In general, the toxicity of vanillin is relatively low (LD50 [rat, oral
administration] = 1580 mg kg−1; LD50 [rabbits, dermal administration] = 5010 mg kg−1),
but it can still induce dermal reactions in people allergic to compounds such as acid
benzoic, Peruvian balm, cinnamon, and cloves. Natural vanillin is extracted from fermented
vanilla pods. Three species of vanilla are known to be produced to obtain natural vanillin:
Vanilla planifolia A., Vanilla pompona S., and Vanilla tahitensis M. Uses: vanillin is used as
a flavoring in various foods: in confectionery (ice cream [0.005–0.03%], chocolate
[0.03–0.08%]-the ice cream and chocolate industry together consumes about 75% of all
vanillin used as a flavoring), in confectionery (0.03–0.1%), in dairy products, and in various
beverages. Vanillin is also used as an odorous compound in perfumery but also to mask the
unpleasant smell and taste of medicines, animal feed and cleaning products. It is also used
in the production of biologically active compounds. Other fields of application of vanillin
are: in the composition of antifoams, in galvanostegy (gives gloss to zinc-coated surfaces),
attractive in some insecticides, catalyst in the polymerization reaction of methyl
methacrylate, yeast inhibitor in food, etc. [38].
13.5.7 Syringe Aldehyde (4-hydroxy-3,5-dimethoxybenzaldehyde)

C9H10O4
It comes in the form of colorless crystals soluble in ethanol (and other polar organic
solvents), chloroform, etc. In nature, it is found in spruce and maple wood; it is also formed
in alcoholic beverages kept in oak pots (the content of the compound increasing during the
maturation of the drink, gives a spicy, smoky aroma with woody notes). At the same time,
the syringe aldehyde together with vanillin participates in the chemical communication of
some insect species. Uses: in the literature, it can be found studies that show properties:
antioxidants, anti andfungals, moderate hypoglycemic antimalarials (in studies in rats),
anticancer (due to inhibition of nitrosamines or antiproliferatives in colon cancer) for
syringe aldehyde [39].
Some researcher, conducting research on a species of beetle (Acanthoscelides obtectus),
identified insecticidal properties for syringe aldehyde. It is used as a flavoring compound in
various alcoholic beverages, giving them an aroma reminiscent of chocolate, grapes, smoke,
and wood. There are thousands of types of mono-, sesqui-, di-, tri-, and tetra-terpenic
compounds (which differ from each other in molecular mass), and many of them have
been studied extensively. These compounds are found in nature and in most plants.
Many monoterpenes are known for their strong aromas and high degree of volatility
through which they attract insects to the flowers of plants for pollination [40].
Myrcene or beta-myrcene is a linear carbohydrate monoterpene and is the main
component of wild thyme EOs, which comprises 40% myrcene. Myrtle is also found in high
concentrations in other plants such as hops, mangoes, and citronella. Pinene is the common
name for two isomeric bicyclic monoterpenoids, alpha-pinene, and beta-pinene, which are
the main components of the pine resin that gives it its name, and of the resin of other
conifers, being also the most widespread terpene in nature. In fact, the pine is not only
found in the plant kingdom, the two compounds being part of the insect’s communication
system and also acting as an insecticide. Alpha-pinene is an acetylcholinesterase inhibitor.
Limonene is a cyclic carbohydrate and the main component of lemon EOs, hence its name,
and other citrus EOs. Limonene is also the second most widespread terpene in nature and
is an intermediate in other terpene biosynthesis processes. In contrast to pine, limonene is
not found in insects, but it still has some insecticidal effects. Limonene is widely used in the
food and pharmaceutical industry as a flavoring. Linalool is a linear monoterpenic alcohol,
resulting from the main substances of lavender EOs, but we also find it in many other
13.6 Conclusions and Remark 289
plants. It is widely used as a flavoring in cleaning and hygiene products, as an intermediate

in the chemical industry and as an insecticide against flies and beetles, but is not a broad-
spectrum insecticide. Lavender EOs relieve skin burns and can even reduce the need for
morphine when inhaled by patients with postoperative treatment. These effects are
attributed to linalool because it is the main component of lavender EOs, because after
ingestion, other substances, such as monoterpene linalyl acetate, hydrolyze into linalool.
Eucalyptol, also known as 1,8-cineole, is a monoterpenic ester that is almost entirely
eucalyptus oil, hence its name, but is also widespread in the plant kingdom. It acts as an
insecticide, although it is produced by certain species of orchids to attract bees. Eucalyptol
is used as a food additive to add a certain flavor. Caryophyllene is the generic name for a
mixture of three compounds: alpha-caryophyllene or humulene, beta-caryophyllene,
which is the main component of black pepper EOs and caryophyllene oxide, the result of
oxidation of the rhubarb and eucalyptus plant [41].
The EOs isolated from rose, mint, lemon, and lavender contain numerous oxygenated
monoterpenes, aliphatic, and aromatic compounds that give these oils their unique
pleasant scent. Other non-aromatic terpenes and higher molecular weight terpenes can act
as protective compounds, giving the plant a more bitter taste, a pungent aroma or a sticky
texture, to repel potentially harmful predators. Another protective feature of terpenes is
hydrophobicity (the water repellent characteristic), which allows them to easily pass
through the membrane of invading cells. When crossing a cell membrane, these compounds
can increase the fluidity of the membrane, so that the cell no longer has the ability to
maintain a balanced internal environment. Because cell survival depends heavily on the
balance of the internal environment, this can cause apoptosis (cell death) [42].
Although terpenes often do not endanger the lives of large organisms (such as animals
and humans), they can be effective against many environmental threats.
13.6 Conclusions and Remarks
EOs consist largely of monoterpenes and a variable proportion of sesquiterpenes. These

proportions, together with the extraction performance, will be mainly affected by the
drying degree of the plant, when it is processed for the extraction of EOs. In fact, the
performance of EOs extraction by steam distillation of fresh plant is less than 1%, with a
composition of 80–90% monoterpenes and 10–20% sesquiterpenes. However, the extraction
performance will be around 0.1% in the case of the dried plant and its composition will be
lower in monoterpenes, reaching instead up to 50% sesquiterpenes, due to the fact that
monoterpenes are very volatile and evaporate quickly during the process of drying plants.
Some sesquiterpenes remain in the plant even after 15 minutes of decarboxylation at
120 °C. This is the case of caryophyllene, which has the characteristic aroma of moist earth
of ripe or boiled hemp. Also, the evaporation of monoterpenes during the drying process is
responsible for the transformation of the aroma from that of a fresh plant to the aroma of a
well-dried plant, although the change in taste comes from the degradation of chlorophyll.
Thus, fresh plants have menthol, citrus, fruity aromas, etc., which fade when the plants are
dry. However, terpenes are not only responsible for the aroma of plants, but they also have
an important biological and therapeutic activity. It has been proven that essential plant oils
have therapeutic properties and are the pharmacological basis of aromatherapy. These pure
oils and terpenes can also be used as flavorings in the food industry, being nontoxic
compounds. The therapeutic properties will depend specifically on the respective terpene.
Plants need more attention in order to complete and complete studies on immunity, because
acting at this level can ensure the food needs of the population on Earth. It is likely that the
slow progress in the study of plant immunity can be attributed to the impossibility of knowing
the mechanisms at the cellular level due to the lack of technology to allow this. In recent years,
science and technology have advanced almost simultaneously, which gives us hope that
perhaps in the coming years the food needed for the population of our planet can be provided.
Nutritional support must provide all the necessities of human development, both quantitatively
and qualitatively, and this poses quite serious problems. However, due to the fact that we have
clear data about some plant immune mechanisms and the fact that at certain levels it has been
possible to manipulate plants in order to reduce the activity of pathogens, we can hope that in
the future, plant diseases will no longer be an economic catastrophe.
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293
14
Bioprospection of Plants for Essential Mineral

Micronutrients
Nikita Bisht1,2 and Puneet Singh Chauhan1,2
1
CSIR‐National Botanical Research Institute (CSIR‐NBRI), Lucknow, India
2
14.1 Introduction
As a result of the green revolution, hunger/energy deficits have seen steady improvement
in developing countries. According to the Food and Agriculture Organization (FAO), the
undernourishment in the global population has declined from 18.6% in 1990–1992 to 10.9%
in 2014–2016. But still, one in nine people worldwide suffers from hunger [1]. Regardless
of the improvement, more than two billion people suffer from deficiency of micronutri-
ents, also recognized as hidden hunger [2]. Micronutrients, as the name suggests, are the
compounds that are required in small amounts to aid the growth, development, and main-
tenance of the body and are classified into two groups, i.e. vitamins and minerals [3].
Micronutrients are essential to human health and are significant for human body functions
[4]. Despite their small requirement, micronutrient deficiencies, in particular of mineral
micronutrients such as iron (Fe) and zinc (Zn), are common in developing countries, as
well as in developed countries [5]. Micronutrient shortages thus create major public health
problems worldwide, posing nutritional and serious health effects to approximately two
billion people and causing 25 000 children to die every day [6, 7].
14.2 Plants as a Source of Mineral Micronutrients
The mineral elements that living beings require come into the food chain via plants.
Therefore, mineral element concentrations in the edible plant tissues are essential for
human nutrition. Up to two‐thirds of the global population are estimated to be at risk of
deficiencies in one or more important minerals with deficiencies of Fe and Zn being the
most frequent [8, 9]. People require mineral elements for their well‐being, and plants are
the dietary source of most of these elements [10]. The diets widely eaten worldwide include
a variety of plant products such as grains and vegetables. The combination of different food

294 14 Bioprospection of Plants for Essential Mineral Micronutrients
ingredients and culinary practices such as heating, sprouting, fermentation, etc. can there-
fore have a significant impact on the availability of micronutrients from the plant‐based
diet [5]. However, edible plant tissues may contain low concentration of mineral elements
for different reasons, or some plant species inherently have low concentration of particular
mineral elements, for example, crops grown in areas with low phyto availability of minerals
[11–15], or those edible plant parts, such as fruits, seeds, and tubers are used that are natu-
rally low in mineral elements with restricted phloem mobility [8, 16].
14.3 Bioavailability of Micronutrients from Plants
Plants are packed with essential mineral micronutrients, but it is important to better under-
stand their bioavailability to derive appropriate amount of nutrition from them. In a plant‐
based diet, the key concept for achieving balanced nutrition is food synergy that is defined as
an additive or more than additive influences on health from dietary patterns, foods, and food
constituents [17]. Maintaining the bioavailability of micronutrients as a cornerstone and
developing intelligent food synergies have the benefit of being close to the population’s psyche
but involve efforts to build them as national missions [18]. Mineral micronutrients such as Fe
and Zn have poor bioavailability from plant foods that are also affected by various dietary com-
ponents including both inhibitors and enhancers of absorption. Phytic acid, tannins, dietary
fibers, and calcium are among the most active inhibitors, while organic acids are known to
help absorb Fe. Zn bioavailability from food grains has also been reported to be similarly influ-
enced by the dietary factors [5]. Table 14.1 summarizes the micronutrient content of different
food groups derived from plants based on the food composition database [18, 19].
14.3.1 Bioavailability of Fe and Zn

Nonheme Fe is the most important form of Fe present in plant foods. The absorption of
nonheme Fe is inhibited by the phytic acid found in whole grains, lentils, and nuts.
Moreover, in tea, coffee, red wines, and a number of cereals, vegetables, and spices, poly-
phenols impede Fe absorption [18, 20]. In India, a study was conducted on previous reports
to estimate Fe absorption and was found to be 5% for adult male and 10% for adult female
[21, 22] and was 12–15% low compared to the suggested Western diets [21, 22]. Another
report on Fe bioavailability documented that the bioavailability of Fe in the cereals and
Table 14.1 Recommended dietary limits and dietary micronutrient content (per 100 mg) for adults.
Plant‐derived foods
Micronutrient AR (M/F) RDA Vegetables Fruits Cereals, millets Pulses, legumes Nuts, oilseeds
Iron (mg) 6/8.1 17/21 0.99 1.4 3.78 5.57 5.4

Zinc (mg) 9.4/6.8 12/10 0.34 0.3 1.71 3.31 5.18
AR (M/F) = average requirement (Male/Female); RDA = recommended dietary allowances.

14.3 Bioavailability of Micronutrients from Plant 295
millets ranged from 7.1% in pearl millets to 15% in rice [5, 23]. However, in the same study
authors reported that in another study bioavailability of Fe differed greatly and suggested
that the differences in the values could be due to regional differences in the food grains
examined and the procedure employed for determination [5, 23].
Plant foods such as whole grains, legumes, nuts, and seeds are rich source of Zn, but they
also contain high amounts of phytic acids (inhibitor of Zn absorption) due to which only
30–35% absorption of Zn takes place [24]. Thus, as compared to the nonvegetarian foods that
are high in bioavailable Zn, the bioavailability of Zn from vegetarian foods is lower [24, 25].
Moreover, when the bioaccessibility of Fe and Zn was determined, the bioaccessibility of Zn
from food grains was higher than that of Fe, and the difference in pulses was more promi-
nent. While Fe’s bioaccessibility from cereals ranged from 4% in sorghum to 8% in rice, Zn’s
bioaccessibility ranged from 5.5% in sorghum to 21% in rice. The availability of micronutri-
ents from the diets was very poor, ranging between 3.3 and 4.4% for Fe, and between 7.8 and
8.7% for Zn. The low quality of minerals was due to the presence of high phytate and dietary
fiber content in the diet [26]. Given comparable concentrations of Fe and Zn in rice and fin-
ger millet–based meals, the bioaccessibility of both of these minerals in finger millet–based
meal was lower, and this was due to the higher tannin content of the finger millet, which was
found to be the only difference in the two meals [27]. Furthermore, when the availability of
Fe and Zn from 60 vegetarian diets eaten by infants, teenagers, adults, and older adults was
evaluated in vitro, the abundance of these minerals was found to be very low in terms of diets,
ranging from 3.3 to 4.4% for Fe and from 7.8 to 8.7% for Zn. [28].
14.3.2 Impact of Food Processing on Micronutrient Bioavailability from Plant Foods

It is generally observed that most of the plant‐based food that is consumed normally under-
goes some form of processing that leads to alterations in the food matrix and inherent com-
ponents of foods. Hence, food processing techniques such as heat processing, sprouting,
fermentation, etc. affect nutrient bioavailability from the plant‐based foods (Figure 14.1).
Organic acids
Heat processing Amino acids
β-carotene-rich
Germination Micronutrient bioavailability vegetables
Malting Fermentation
Sulfur-rich
compounds
Figure 14.1 Different food processing techniques that influence nutrient bioavailability from the
plant‐based foods.
14.4 Manipulating Plant Micronutrients
The concentration of mineral elements in crops can be increased by the careful application
of mineral fertilizers and/or by the use of genotypes with a higher mineral content. Besides,
mineral element bioavailability may also be increased by crop husbandry, breeding, or
genetic engineering [8]. For ensuring sufficient dietary intake of essential mineral nutrients,
scientists have turned with renewed interest for the research and manipulation of plants. A
major focus is to identify and isolate the genes needed to synthesize and accumulate a target
compound in order to induce the desired dietary shift by increasing its levels in staple crops.
A critical point for all micronutrient research, however, is that unlike macronutrients that
can account for up to 30% of a tissue’s dry weight, individual micronutrients are generally
much less than 0.1% of a tissue’s dry weight, and thus it is technically feasible to increase the
micronutrient levels [29]. Moreover, due consideration should be given to identifying target
compounds, their efficacy, and whether excessive dietary consumption may have unin-
tended negative health consequences before attempting to alter nutrient components in
food crops. Before addressing the potential of manipulating plant micronutrient content
through emerging technologies, it is important to emphasize that there are more conven-
tional approaches and that both separately and in conjunction with emerging technologies
should be followed. Modern farming and breeding programs have aimed mainly to increase
productivity and yields over the past 50 years, a task that will remain a major concern in
supplying the caloric intake required by the world’s increasing population. Nevertheless, the
micronutrient composition and crop density are equally important but largely overlooked in
breeding programs. In the rare cases where the content of micronutrients was assessed,
significant genotypical variations were observed. Such variability can and will be used to
grow nutritionally improved cultivars and to help establish the genetic and physiological
basis for differences in nutrients [29]. Plant scientists have started to use genomic tools and
the DNA technology’s ability to research all areas of plant biology. The established biochem-
ical knowledge for individual pathway steps, well‐defined protein motifs common to differ-
ent reaction mechanisms, and the presence or absence of subcellular targeting information
in the primary amino acid sequence are combined for the identification of gene(s) by bioin-
formatics. These techniques complement biochemical and genetic approaches and can eas-
ily be integrated to add new dimensions to complex plant pathways elucidation.
14.4.1 Improving Bioavailability of Micronutrients from Plant Foods

Improving the micronutrient content of crop products through biotechnology is a promis-
ing technique for the worldwide fight against micronutrient malnutrition. Modifying the
nutritional composition of plant foods is a critical global health issue, as essential nutri-
tional requirements for most part of the world’s population remain unfulfilled. Throughout
most of the developing countries, there are large numbers of people on simple diets that
consist mainly of a few staple foods (cassava, wheat, rice, maize, etc.) that are poor sources
of several important micronutrients [29]. To fix mineral micronutrient shortages in human
populations, plant scientists are working to establish methods for the application of fertiliz-
ers and/or to encourage plant breeding techniques to increase concentrations/bioavailabil-
ity of mineral elements in plant products [2, 8, 13, 14, 30]. These approaches to
14.4 Manipulating Plant Micronutrient 297
Agronomic biofortification
Approaches
for Biofortification based on conventional
biofortification plant breeding
Biofortification based plant breeding

using genetic engineering
Figure 14.2 Different approaches that are used for the biofortification of mineral micronutrients
in agricultural crops.
biofortification are classified according to genetic engineering as agronomic, conventional

plant breeding, and plant breeding (Figure 14.2).
Agronomic biofortification through the application of fertilizers temporarily enriches
micronutrients. This approach helps to increase micronutrients that can be ingested
directly by the plant, such as Zn, but less so for micronutrients that are synthesized in the
plant and cannot be absorbed directly [31]. In the light of sustainable economic growth and
environmental health, various authors have recently examined the agronomic approaches
for growing mineral nutrient concentrations in edible portions of major crop plants [2, 8,
13, 14, 30]. These included reviews of suitable methods, requirements for infrastructure,
and practical benefits of agronomic biofortification of edible crops with Fe and Zn for eco-
nomic sustainability, food production, and human health [8, 13, 32]. Conventional plant
breeding recognizes and establishes high‐mineral parent lines and crosses and segregates
the generations to grow plants with the required nutrient and agronomic characteristics
[30, 33]. Biofortification by genetic engineering aims to do the same and was primarily used
in crops where the target nutrient naturally does not exist at the necessary levels [2].
Researchers have also tried to investigate genetic variation in mineral concentrations, the
interaction between the environment and the genotype, and breeding potential for
increased mineral element concentrations in the produce [8, 14, 30]. Previous studies pro-
vided a detailed overview of genetic factors influencing concentrations of mineral elements
in edible tissues of popular crops and also established research under the Harvest Plus
program to increase concentrations of Fe and Zn in dietary staples [8, 33].
14.4.2 Metabolic Engineering of Micronutrients in Crop Plants

Metabolic engineering can be used, as in molecular breeding, to produce crops with
increased micronutrient content, each with its advantages and disadvantages. Metabolic
engineering uses genetic modification by engineering metabolic pathways to enhance nutri-
tional value in crop plants. This strategy involves modulating endogenous metabolic path-
ways or introducing one or the introduction of one or more heterologous performers to
enhance the production of a target compound, decrease the amount of unwanted molecules,
or alter the flux to accumulate a more bioavailable, stable, and active compound [31].
However, this requires detailed knowledge of the endogenous metabolic pathways involved
in the regulation. Only then a successful engineering strategy can be developed for the over-
expression/downregulation of key enzymes gene(s) responsible to enhance micronutrient
biosynthesis/accumulation without affecting crop development and yield.
Fe and Zn are essential elements for many of the human metabolic processes [34, 35].
The biofortification of crop with these micronutrients involves many processes to be
orchestrated, such from taking up by the roots to retaining in the edible parts. From the
rhizosphere, Fe and Zn are absorbed via the root epidermis, taken to the xylem, and trans-
ferred to different tissues throughout the plant where they can be stored. The transgenic
strategy for increasing the content of Fe and Zn in crops is to primarily improve the absorp-
tion and effectiveness of Fe and Zn by modulating transporter gene(s) expression [31, 36]
and reducing antinutritional factors such as phytic acid [37]. The coexpression of
LACTOFERRIN (Fe‐chelating glycoprotein) and FERRITIN further enhances the content
of Fe in crops [38–41]. The simultaneous expression of NAS (nicotianamine synthase) and
Ferritin increases not only the content of Zn but also iron content in crops [42–44]. In
transgenic rice, it has been documented that the combined expressions of four genes, NAS,
FERRITIN, PSY, and CrtI, increase the Fe, Zn, and β‐carotene contents significantly [45].
Therefore, a number of biofortified crops such as rice, maize, and wheat with enriched
contents of Fe and Zn were produced to counter global human mineral deficiencies [46].
While the metabolic engineering has made tremendous progress in the field of crop bioforti-
fication, many challenges still remain [47]. The main issue is lack of knowledge of an organ-
ism’s metabolic pathways and key regulators. Though at present the sequencing of plant
genomes has become easy, but the lack of annotations for effective gene functions makes it
difficult to determine the composition of genes that encode key enzymes in different metabolic
pathways. The constitutive metabolite synthesis is likely to trigger abnormal cell development
and plant growth. Metabolic processes may at the same time be affected by feedback regulation.
Nevertheless, the expression of transgenes expressed in specific tissues must increase in order
to achieve metabolite synthesis and accumulation in specific tissues and to avoid adverse effects
on normal plant development [48]. Furthermore, most of the metabolic pathways require
numerous regulatory factors and enzymes. The application of high‐efficiency multigene expres-
sion vector systems (TGS II system) and the CRISPR gene editing tool would enable the expres-
sion and regulation of upstream and downstream genes of whole metabolic pathways in more
versatile and precise ways [49–51]. Thus, with the advances in metabolic engineering technol-
ogy, and deep understanding of the metabolic pathways, synthetic metabolic engineering can
then accomplish more accurate reconstruction and regulation of complex multistage metabolic
networks, producing more novel biofortified crop varieties.
14.5 Microbes in the Biofortification of Micronutrients in Crops
The concept of hidden hunger is well documented in the last two decades [52]. Different
strategies have been used to grow biofortified crop varieties with improved micronutrient
bioavailability, such as conventional and molecular plant breeding or the use of chemical
supplements. The role of microorganisms in enhancing nutrient content in crops has also
Reference 299
been documented [53–55]. There are vast amounts of Fe and Zn found in the earth’s crust,
but are not available to plants because they are present in the form of insoluble salts. Intrinsic
plant‐based strategies such as the production of organic acids or phytosiderophores/chela-
tor secretions not always deliver sufficient micronutrients to plants [56–58]. The use of
microorganisms to help the crop take up and translocate Zn and Fe more efficiently and
effectively is a promising option that needs to be effectively integrated into agricultural or
breeding approaches [58]. The mobilization of micronutrients by microorganisms high-
lighted the importance of (i) rhizospheric soil acidification, (ii) phenolic secretion stimula-
tion, (iii) root morphology and architecture modifications, (iv) reduction of phytates, and (v)
upregulation of transporters. The formulation(s) of these microbes can be explored as seed
priming or soil dressing options for the biofortification of Zn and Fe [59].
14.6 Conclusions
Plants have been important for human health and wellbeing since ancient times and were
used as a source of mineral micronutrients. But low concentration of mineral micronutri-
ents and presence of their inhibitors in plants is the main problem. However, in recent
years, advancement in the field of plant research have created new opportunities to extract
maximum advantage from the plants. Plants are now considered as a rich resource for the
production of mineral micro nutrients. With time as the genomic, transcriptomic, prot-
eomic and metabolic data has rapidly escalated, the number of target molecules/pathways
related to accumulation of micronutrients has also increased. Given that micronutrient
nutrition is of global concern, it is imperative that the synergies between scientists, policy
makers and educators concentrate on developing multi‐pronged, environmentally friendly
solutions and incorporating microbial options into the mainstream of integrated agricul-
tural practices. Thus, convergence of all the above‐mentioned factors with the potential to
provide good quantities of mineral micronutrients from plants.
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15
Algal Biomass
A Natural Resource of High-Value Biomolecules
Dinesh Kumar Yadav, Ananya Singh, Variyata Agrawal, and Neelam Yadav
Molecular Biology and Genetic Engineering Laboratory, Department of Botany, University of Allahabad, Prayagraj, India
15.1 Introduction
Algae (microalgae and macroalgae) the extremely diverse group of photoautotrophs have
almost ubiquitous inhabitation preferentially in aquatic environments. They successfully
dwell in highly acidic and frozen soils, rocks, snow, in/on plants and animals as symbionts,
hot spring, volcanic waters, and arid conditions [1]. They represent ecologically imperative
group of organisms.
All microscopic unicellular prokaryotic (Cyanophytes) and eukaryotic (other algae
phyla), and microscopic multicellular algae are commonly known as microalgae, whereas
macroscopic, multicellular algae are called as macroalgae, are found in fresh and marine
waters, and frequently referred as seaweeds. Algae constitute a fundamental part of aquatic
food chains in various ecosystems. Microalgae contribute up to 80% of the biomass and
primary productivity in marine ecosystems and perform >40% of global photosynthesis [2].
Representing their enormous ability to sequester carbon dioxide through photosynthesis
and produce a huge biomass resource.
Algae have been classified based on their photosynthetic pigments, reserve food materi-
als, and mode of reproduction. The additional criteria considered for their classification
include morphological and cytological features, chemical composition of cell wall, and spe-
cific gene sequences along with photosynthetic pigments [3]. The latest seven kingdoms
classification has divided living organisms into Archaebacteria, Eubacteria, Protozoa,
Chromista, Fungi, Plantae, and Animalia. Seven-kingdom classification has distributed
algae into eight phyla under four kingdoms [4]. Majority of algae have been clustered into
seven phyla under domain Eukaryota. The Heterokontophyta lineage has c. 15 000 identi-
fied and about 10 million anonymous species remain. The bacillariophytes or diatoms are
predominant form in phytoplankton populations and largest group of biomass producers
on the Earth [5]. Rhodophtya lineage has ~5000 identified and ~15 000 new unidentified

304 15 Algal Biomass
species. Chlorophytes have ~16 000 known and ~100 000 undescribed extant species. The
members of four minor lineages (Dinophyceae, Euglenophyceae, Cryptophyceae, and
Glaucophyceae) are very ambivalent and under revision [2]. The domain Prokaryota has a
single algal phylum, Cyanobacteria that represent another highly abundant group of algae
next to Heterokontophyta and Chlorophyta [6, 7].
Algae are one of the fastest-growing life forms with short multiplication time, which may
be due to their enormous strain diversity showing remarkable tolerance to seasonality lead-
ing to >3% greater annual photon-to-biomass conversion efficiencies [8]. Based on these
properties, algae offer tremendous natural resource for commercial exploitation of high-
value molecules like proteins, carbohydrates, lipids, triglycerides, pigments, polyphenols,
phytosterols, hormones, vitamins, minerals for food and health, aquaculture feed, animal
feed, dyes, cosmetics, and others. With the increasing awareness for advantages of using
natural products over the synthetic ones, their utilization is expanding especially in food
and pharmaceutical industries. Additionally, algae can be cultivated on nonagricultural
landmass or wastewater bodies without pesticide so quality of food or produce is not com-
promised and atmospheric CO2 sequestration reduces environmental issues. Improvement
in bioprocessing strategies for biomolecules extraction, bioconversion (chemical catalysis
maceration, maturation, and supercritical liquid extraction), and algal biotechnology has
led to unexceptional utilization algal bio-resource to meet current food, energy, and envi-
ronmental challenges [9–11]. This chapter focuses on algal bio-resources under four major
categories, i.e., carbon dioxide capture and sequestration, high-value biomolecules, biofuel
production, and feed for livestock and aquaculture (Figure 15.1).
15.2 Carbon Dioxide Capture and Sequestration
Carbon dioxide is the principal control button that governs the Earth’s temperature. So,
menace caused by global warming is worsening due to increasing atmospheric CO2 con-
centration by anthropogenic activities. The global emission of CO2 has increased from 2 to
36 billion tons in 115 years. The rate of increase in atmospheric CO2 concentration in
2018 was recorded as 1.231 ppm year−1. The rate CO2 emission increased by eightfold in
2018 from that of 1960 [12]. At this rate of increment, CO2 is the major component
augmenting in total global warming, and climate change that may lead to substantial bio-
logical extinctions.
Algae have being considerably recognized as most productive biological systems for CO2
capture and biomass generation. Microalgae have ~10 fold higher photosynthetic effi-
ciency compared to land plants [13]. Tsai et al. [14] reported that algae can absorb
159 mg l−1 day−1 CO2 at a consumption efficiency of 93% in high rate ponds (lab-scale algal
niche), compared to continuous stirred tank reactor (178 mg−1 l−1 day−1 and 96%). The
bicarbonate pumps arrayed in plasma- and chloroplast outer membrane of eukaryotic
algae transport bicarbonate ions into the cells with ~90% efficiency in open water bod-
ies [15]. The concentrated bicarbonate in the chloroplast is dehydrated spontaneously or
by carbonic anhydrase, releasing CO2 for photosynthesis and producing algal biomass.
Some dominant algal species suitable for CO2 capture are Anabaena, Oscillatoria, Lyngbya,
Spirulina, Chlorella, Chlamydomonas, Monoraphidium, Nannochloropsis, Oedogonium,
Sunlight Sunlight
Carbon dioxide capture

through photosynthesis
Pigments
Polyphenols
PUFAs
Biogas Polysterols
Biomethanol Microbial fuel cell Carbohydrates
Hormones
Bioethanol Proteins
High-value
Biofuel Minerals
biomolecules
Biodiesel production
Algal
biomass Vitamins
Syngas
Pharmaceuticals
Biochar
Cosmetics
Aquaculture feed Livestock feed

Wastewater
treatment
Figure 15.1 High-value natural products obtained from algal resources.

Microspora, Scenedesmus, etc. About 1.6–2.0 g of CO2 is sequestered for each gram of algal
biomass produced [16]. Complete harvesting and processing of the algal biomass are more
efficient than terrestrial biomass production systems. It suggests that algae can accumu-
late considerable amount of carbon within short period of time and could be the best
substitute to biologically sequester CO2 to mitigate global warming. Flue gases released
from fossil-fuel power plants typically contain high CO2 concentrations (10–20%) blended
with biologically significant concentrations of nitrous- and sulfuroxides. Douskova
et al. [17] reported that use of flue gases as fertilizer in algal ponds increases biomass pro-
ductivity by 30% in comparison to direct injection of an equivalent amount of pure CO2.
High CO2 fixation efficiency of ~99% can be accomplished under optimal conditions with
as short as two seconds of retention time of flue gases [18]. An algal pond of 3600 acres
with the biomass productivity rate of 20 g dry weight m−2 day−1 can efficiently fix 80% of
CO2 in flue gas exhaust during day from a typical 200 MWh fossil-fuelled power plant.
However, the efficiency of CO2 mitigation by algae can be altered by pond chemistry,
physiological condition of algae, temperature, and available nutrition.
The exploitation of algae for CO2 sequestration offers several other advantages. Algae can
be cultivated in open water bodies as well as in closed photobioreactors. The later system
offers high productivity due to stringent control over physiochemical growth parameters,
sterile conditions, and easy operations. Airlift bioreactors are provided with light period at
regular intervals, controlled stress to cells, and even distribution of nutrients resulting in
higher energy-to-mass transfer [19]. The huge revivable algal biomass is a rich source of
high-value biomolecules and renewable green energy.
The CO2 capture solely may not have the greatest impact on the accretion of greenhouse
gases unless it is effectively sequestered over geological time intervals preventing its re-
entry into carbon cycle. Multiple strategies may be adopted for algal carbon sequestration.
It can be permanently deep buried in a geologic formation where ~7% of algal dry weight is
not available for natural biochemical cycles. The disadvantage of burying algal biomass can
be overcome by depositing only after the selective extraction of hydrocarbon or neutral
lipid fraction, i.e. triacylglycerols (TAG), containing 75% carbon and accounting for more
than 60% of the total dry weight. Additionally, burial of TAGs is safer as it prevents risk of
escape of gaseous CO2 from the geologic deposits. Carbon dioxide can also be chemically
converted into stable solid or liquid carbonate salts as construction material. Although this
approach has potentially lower risks of carbon escape, but is energy inefficient. Another
method of geological storage for mitigated carbon in algal biomass is to transform it into
biocharby pyrolysis at elevated temperature. Biochar contains >90% carbon and can endure
for centuries under soil [20].
15.3 Algae in High-Value Biomolecules Production

Algal biomass represents a sustainable and cost-effective source of diverse essential nutri-
ent supplements to meet the growing needs of undernourished population of developing
countries. A large number of high-value, bioactive, novel compounds from algae have been
recognized as viable source in nutraceuticals (protein, lipids carbohydrates, vitamins, and
other important metabolites) with key role in nutritional food security of the world
(Table 15.1).
15.3 Algae in High-Value Biomolecules Productio 307
Table 15.1 Major high-value health biomolecules in microalgae [6, 7].
High-value health products Algae Applications
Food Chlorella, Spirulina, Odontella auriata, Nutraceuticals

Tetraselmis chuii, Aphanizomenon
flosaquae, Nostoc, Ascophyllum sacrum,
Spirogyra, Oedogonium, Caulerpa,
Hydroclathrus, Enteromorpha, Ulva,
Monostroma, Codium, Hizikia, Gracilaria,
Cladosiphon, Sargassum, Laminaria,
Porphyra, Gelidiella, Halymenia, Hypnea,
Laurencia, Macrocystis, Ascophyllum,
Laminaria, Undaria pinnatifida, Porphyra,
Gelidium, Palmaria palmata, Chondrus
crispus
Feed Chlorella, Arthrospira platensis, Pavlova, Livestock feed,
Tetraselmis, Isochrysis, Chaetoceros, aquaculture feed,
Phaeodactylum, Thalassiosira, heterocystous
Nannochloropsis, Skeletonema, Porphyra, cyanobacteria used as
Kappaphycus, Gracilaria, Undaria, biofertilizers
Laminaria, Hizikia fusiforme,
Porphyridium valderianum, Hypnea
cervicornis, Cryptonemia crenulata
Proteins, peptides Arthrospira platensis, Chlorella vulgaris, Nutraceuticals
and amino acids Chlorella ellipsiodea, Dunaliella salina, Cosmeceuticals,
Nannochloropsis oculata, Porphyridium Pharmaceuticals,
cruentum, Haematococcus pluvialis,
Scenedesmus
PUFAs Nannochloropsis oculata, Chlorella Food, Nutraceuticals,
vulgaris, Botryococcus braunii, Pharmaceuticals
Crypthecodinium cohnii, Ulkenia,
Scenedesmus obliquus, Schizochytrium,
Labyrinthula, Isochrysis galbana,
Thraustochytrium, Phaeodactylum
tricornutum, Nannochloropsis,
Porphyridium cruentum, Monodus
subterraneus, Pavlova salina, Chaetoceros
calcitrans, Isochrysis galbana
Carbohydrates Tetraselmis, Isochrysis galbana, Food, Nutraceuticals,
Porphyridium cruentum, Odontella aurita, Pharmaceuticals
Porphyridium purpureum, Chlorella sp.,
Rhodella reticulate, Chlorella
stigmatophora, Dunaliella salina,
Haematococcus pluvialis, Nannochloropsis,
Diacronema vlkianum, Pavlova lutheria,
Phaeodactylum tricornutum, Scenedesmus
dimorphus, Anabena cylindrica, Spirilina
platensis, Ankistrodesmus angustus,
Aphanizomenon flos-aquae, Laminaria
hyperborea, Laminaria digitata, Laminaria
japonica, Ascophyllum nodosum,
Macrocystis pyrifera
(Continued)
Pigments chlorophyll All algae, Chlorella vulgaris, Food, Colouringagents,

Monoraphidium, Aphanizomenon Nutraceuticals,
flos-aquae, Gloeothece membranacea Pharmaceuticals,
Cosmeceuticals,
Carotenoids Dunaliella salina, Dunaliella bardawil, Food, Colouring agents,
Dunaliella tertiolecta, Chlorella Nutraceuticals,
zofingiensis, Chlorococcum, Scenedesmus Pharmaceuticals,
almeriensis, Isoehrysis galbana, Cosmeceuticals,
Haematococcus pluvalis, Codium fragile,
Synechococcus, Nanocloropsis gaditana,
Gracilaria chilensis, Porphyridium
cruentum, Phordium autumnale,
Anabaena cylindrica, Undaria pinnatifida,
Spirulina platensis, Nostoc, Porphyridium
marinum, Rhodella reticulata, Galdieria,
Aphanizomenon flosaquae, Porphyra
haitanensis
Phycobilliproteins Arthrospira platensis, Pseudanabaena Fluorescent dyes used as
mucicola Porphyridium marinum, fluorophores in
Rhodella reticulata, Galdieria, Research &diagnostics
Aphanizomenon flosaquae, Porphyra
haitanensis, Griffithsia pacifica,
Kappaphycus alvarezii
Vitamins and carotenoids Food, Nutraceuticals,
Pro-vitamin A Pharmaceuticals,
α-Carotene Chlorella, Tetraselmis suecica Cosmeceuticals
β-Carotene Dunaliella salina, Dunaliella bardawil,
Dunaliella tertiolecta, Haematococcus
pluvialis, Scenedesmus almeriensis,
Codium fragile, Synechococcus,
Nanocloropsis gaditana, Gracilaria
chilensis, Porphyridium cruentum,
Phordium autumnale, Anabaena
cylindrica, Undaria pinnatifida, Ulva
lactuca, Chlorella, Spirulina, Tetraselmis
suecica, Isoehrysis galbana
Vitamin B complex Dunaliella salina, Chlorella stigmatophora,
Chaetoceros gracilis, Thalassiosira
pseudonana, Pavlova lutheri, Nannochloris
atomus, Eisenia arborea, Nannochloropsis
oculata, Anabaena cylindrica,
Pleurochrysis carterae, Undaria
pinnatifida, Ulva lactuca
Vitamin C Porphyra umbilicalis, Porphyridium
cruentum, Himanthalia elongata, Gracilaria
changii, Tetraselmis suecica, Isoehrysis
galbana, Eisenia arborea, Dunaliella
tertiolecta, Chlorella stigmatophora, Eisenia
arborea, Ulva lactuca
Pro-vitamin D Pediastrum, Scenedesmus, Crucigenia,

Coelastrum, Chlorella, Cosmarium,
Navicula, Cyclotella, Gomphosphania,
Oscillatoria
Vitamin E Porphyridum cruentum, Tetraselmis
suecica, Isoehrysis galbana, Dunaliella
tertiolecta, Dunaliella salina, Chlorella
stigmatophora, Nannochloropsis oculata,
Macrocystis pyrifera, Haslea ostrearia,
Chaetoceros calcitrans
Vitamin K1 Anabaena cylindrical
Polyphenols Phaeodactylum tricornutum,
Nannochloropsis gaditana, Nannochloris
sp., Haslea ostrearia, Dunaliella tertiolecta,
Ankistrodesmus, Spirogyra, Caespitella
pascheri, Tetraselmis suecica, Chlorella,
Euglena cantabrica, Tolypothrix,
Chlamydomonas, Synechocystis, Dunaliella
salina, Nostoc sp., Nostoc commune,
Leptolyngbya protospira, Anabaena,
Nodularia spumigena, Arthrospira
platensis, Phormidiochaete
Phytosterols (Sitosterol, Pavlova lutheri, Tetraselmis, Isochrysis
Stigmasterol, galbana, Phaeodactylum tricornutum,
Brassicasterol) Chattonella antique, Dunaliella tertiolecta,
Dunaliella salina, Nannochloropsis,
Chrysoderma, Chrysomeris,
Chrysowaernella, Giraudyopsis,
Peyssonnelia, Chlorella vulgaris,
Glaucocystis nostochinearum
Phytohormones Nannochloropsis oceanica
Mineral nutrients Food, Nutraceuticals
Inorganic salts Fucus vesiculosus, Tetraselmis suecica,
Isochrysis galbana, Dunaliella tertiolecta,
Chlorella stigmatophora, Undaria
pinnatifida, Chondrus crispus, Porphyra
tenera, Anabena cylindrica, Spirilina
platensis
Iodine Alaria esculenta, Palmaria palmata, Ulva
intestinalis, Ulva lactuca, Laminaria
digitata, Saccharina japonica, Undaria
pinnatifida
Iron Ulva lactuca, Sargassum, Porphyra,
Gracilariopsis, Pyropia yezoensis
Use of seaweed and microalgae as food greatly depends upon the heterogeneity in
iomass composition that can be varied by strain selection and altered growth conditions.
b
Algae tremendously modulate their biochemical composition by synthesis and accumula-
tion of secondary metabolites in response to variation in their biochemical niche. These
features of algae have been exploited to develop metabolic imbalance-based strategies to
divert algal cell to preferably synthesize desired metabolites [21]. The metabolic imbal-
ance-based strategies involve stressing procedures such as high salt, light intensity and pH,
unfavorable temperatures, or altered concentrations of metal salts.
15.3.1 Proteins, Peptides, and Amino Acids

Algae are unorthodox and protein rich source of protein containing balanced amount of
essential and nonessential amino acids as compared to animal and plant protein sources
like meat, egg, milk, or legumes [22]. Most of the microalgae (Arthrospira platensis,
Chlorella vulgaris, Nannochloropsis oculata, Porphyridium cruentum, Haematococcus plu-
vialis, Scenedesmus, etc.) have proteins, i.e. ~50% their total dry weight, that can be scaled
up to 70% by selecting suitable algal species and changing the physicochemical growth
conditions [23]. Microalgal proteins are primarily used in the formulation of nutraceuticals
or functional foods [24]. It is assumed that by the year 2054, 50% of protein market will be
replaced by unconventional protein sources like algae or insects. In spite of having bal-
anced amino acid profile, algal proteins have reduced digestibility in comparison to con-
ventional sources [25] which varies from 50 to 82% species to species. The lower digestibility
of algal proteins is attributed to thicker, more rigid, and high cellulose content in the cell
wall. Their digestibility can be improved up to 78–84% on average by treating the cell with
disruption processes [26, 27].
Furthermore, algal proteins are widely used in the skin-related cosmetic industry
because of their elegant biological activities [28]. For example, oligopeptide extracted
from Chlorella vulgaris has been used in the formulation of Dermochlorella DG® for skin
rejuvenation. Application of Dermochlorella DG on skin induces collagen fiber produc-
tion that increases the compactness of skin, lessens wrinkles and stretch marks, and
increases vasculature [29]. The Arthrospira oligopeptide containing formulation is used as
an antiaging ointment [30]. Micosporin-like amino acid containing formulation has anti-
oxidant and photo-protective properties and is used in sunscreen lotion [31]. Three to
twenty residue-long oligopeptides obtained after enzymatic hydrolysis of microalgal
protein soup have shown antibacterial, antioxidant, antihypertensive, anticancer, and
anti-inflammatory properties which can be exploited in pharmaceuticals for health bene-
fits [32]. In vitro enzymatic hydrolysate of protein soup from Chlorella ellipsiodea gener-
ated a unique, antioxidant hexapeptide (LNGDVW) which scavenged peroxy
l,1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl-free radicals. Also, under in vitro
condition this purified hexapeptide protected monkey kidney cells from hydrochloride-
induced apoptosis and necrosis [33]. The same lysate generated another unique tetrapep-
tide (VDGY) with an antihypertensive effect, i.e. angiotensin I-converting enzyme (ACE),
inhibitory activity and systolic blood pressure-lowering, in mice on oral administra-
tion [34]. Various peptides purified from Chlorella vulgaris and Spirulina platensis hydro-
lysate showed antihypersensitive effect [35], and Dunaliella salina peptides exhibited
cytotoxic effects [36]. These hydrolysate-originated peptides with 3 to >10 kDa molecular
weight also exhibited significant antimicrobial activity against Escherichia coli, Helicobacter
pylori, and Staphylococcus aureus. Smaller peptide of <3 kDa from Dunaliella salina
hydrolysate showed antiproliferative activity even at lower concentrations. Studies have
shown that enzymatic hydrolysate of microalgal biomass is safe for use in food additives
nutraceuticals [37].
15.3.2 Polyunsaturated Fatty Acids (PUFAs)

Lipid is a distinct group of chemical compounds composed of predominantly nonpolar
carbon–carbon or carbon–hydrogen bonds. It is an integral component of cell membrane
and is crucial for the maintenance of structural and functional integrity of cell and organ-
ism. Lipids in most of organism can be broadly classified as storage lipids (primarily triglyc-
erides) used for energy generation; and structural lipids (phospholipids, glycolipids, and
sterols) are major constituent of cellular membranes. Specialized lipids serve a variety of
cellular functions like cofactors (vitamin K), pigments (carotene, retinal pigment), deter-
gents (bile juice), hormones (sex hormones, vitamin-D derivatives), transporters (dolich-
ols), and different cellular signaling molecules (eicosanoids, phosphatidylinositol
derivatives).
Fatty acids, a type of storage lipids, can be further categorized depending on the number
of carbon–carbon (C–C) double bonds (unsaturated hydrocarbon) in their carbon chain as
saturated, monounsaturated (MUFA), or polyunsaturated fatty acids (PUFAs). PUFAs have
a long hydrocarbon chain possessing more than one double bond and have significant roles
in metabolism. PUFAs can be further categorized into omega-3 and omega-6 (n–3 and n–6
families) fatty acids, depending on the position of first C–C double bond present in fatty
acid chain with 18 or more carbon atoms present with respect to the terminal methyl end
of the of PUFA molecule.
PUFAs include eicosapentaenoic acid (EPA), arachidonic acid (AA), γ-linolenic acid
(GLA), and docosahexaenoic acid (DHA), as precursors for synthesis of a range of fatty
acids like cofactors, pigments, hormones, and transporters. These fatty acids function as
signaling molecules, usually of 20- or 22-CPUFA, i.e. very long-chain PUFAs (VLCPUFAs),
and are essential for proper growth and development of human. Humans are deficient of
endogenous synthesize PUFAs longer than 18-C [32], while fishes and terrestrial plants do
not anabolize it de novo, but only accumulate EPA and DHA. Microalgae have emerged as
promising exogenous dietary sources of long-chain PUFAs of ω3 (EPA and DHA) and ω6
(GLA and AA) families. The fatty acid profile and quantity of PUFAs in fishes are naturally
low and they amass it by feeding on PUFA-rich microalgae. The fatty acid profile and con-
tent varies with algal species, growth conditions, and seasonal variations [38]. Various
attempts have been made to understand role of environmental changes in lipid profile. It
has been shown that factors such as nitrogen starvation, heterotrophic nutrition condi-
tions, and high irradiation-induced elevated levels of reactive oxygen species; activated
lipid, and DHA accumulation by ~57 and ~43% respectively, in microalgal biomass [39].
Although, stress factors increase lipid accumulation but significantly reduce algal growth
rates and biomass production. Paes et al. [40] proposed a two-phase culture cultivation
procedure to mitigate the deleterious effects of exposure to stresses, where Nannochloropsis
oculata culture was initially cultivated under optimum conditions for maximum growth of
algal biomass. Then biomass was exposed to nutrient deficient or excess irradiations for
lipid accumulation. This showed similar biomass yields both under nitrogen supplemented
and starved conditions. However, cell volume of nitrogen starved cells increased due
26–34% increased lipid accumulation of total dry mass.
The fish oil extracted from salmon and codfishes is key dietary sources of EPA and
DHA. These oils have limited usage due to their low oxidative stability, unpalatable taste
and smell, and nonvegetarian origin. As a result, EPA and DHA of microalgal origin have
wider acceptability and market values. However, production of algal fatty acids at industry
scale is limited to DHA, whereas that at laboratory scale is limited to EPA [11].
Schizochytrium, Crypthecodinium, and Ulkenia are microalgae that yield high amounts of
DHA, i.e. >40% of total fatty acids [10]. Other common PUFA-producing microalgae are
Labyrinthula, Thraustochytrium, Phaeodactylum tricornutum, Nannochloropsis,
Porphyridium cruentum, Monodus subterraneus, Pavlova salina, Chaetoceros calcitrans,
Isochrysis galbana, and Crypthecodinium cohnii [31] (Table 15.1).
15.3.3 Polysaccharides
Simple saccharides are primary by-products of photosynthesis in oxygenic photoauto-
trophs. Polysaccharides are glycosidic bond-linked hydrophilic sugar polymers and com-
plex biomolecules used in numerous biochemical processes. Based on their biochemical
role, polysaccharides can be categorized as energy-providing storage polysaccharides like
starch; structural polysaccharides that make up the cell wall, and polysaccharides associated
in cellular signaling as recognition molecules [41]. Five types of species-specific storage
polysaccharides (starch, floridean starch, glycogen, chrysolaminarin, and paramylon) are
found in microalgae and blue-green algae. Cyanobacteria exclusively store carbohydrates
in the form of glycogen, sucrose, or glucosylglycerol [42]. The algal starch, floridean starch,
and glycogen are polyglucans having α-1,4-/α-1,6-type of glycosidic linkages in distinctive
proportions. Glucose residues in monomer unit of chrysolaminarin starch are β-1,3-/β-1,6-
linked, whereas in paramylon are only β-1,3-linked. The starch granules are accumulated
in chloroplasts; chrysolaminarin in vacuoles, whereas floridean starch, paramylon, and
glycogen in cytoplasm [43]. Different types of algal polysaccharides vary in degree of
polymerization, branching, and topology within the cell, imparting high degree of struc-
tural and compositional diversity.
The storage and structural polysaccharides have great industrial applications in food,
pharmaceutical, and cosmetics industries. Microalgal species like Pavlovalutheri,
Porphyridium cruentum, Odontella aurita, etc. can accumulate carbohydrates by >50% of
total dry mass [25]; however, polysaccharide accumulation largely depends upon the geno-
type and growth conditions. Storage polysaccharides have been largely exploited in biofuel
industry as starting material (in Section 15.5).
Alginates are complex polysaccharides made of linear copolymers of β-1-4-linked
d-mannuronic acid and β-1-4-linked l-guluronic acid monomers. They are found in brown
algae and considered as dietary fibers due to resistance to digestion in human gastrointes-
tinal tract. Consumption of algae like Codium rediae, Rhodella reticulate, and Gracilaria
containing 23–64% alginate of total dry weight supplement high amounts of dietary fiber
but have nonnutritive physiological effects [44]. Fermentation of such dietary fibers in
intestine by inhabitant microflora produces short-chain fatty acids like acetic acid, propi-
onic acid, and butyric acids which foster both gut epithelia and exert a probiotic effect on
its microbial consortia [45]. Hydrogels formed by alginates in the presence of divalent cati-
ons are used in wound healing, drug delivery, and tissue engineering.
Heavily sulfated heteroglycans, ulvans are obtained from members of Ulvales. Ulvans are
unusually hydrophilic that possess anionic property and structural similarities with animal
glucosaminoglycan regulators and other lectins. Ulvan polysaccharides show antibacterial,
antiviral, antioxidant, anticoagulant, antihyperlipidemic, antitumor, and immunomodula-
tory properties [46].
Carrageenans are heavily sulfated galactans formed by disaccharide polymeric units of
3-linked β-d-galactopyranose or 4-linked α-d-galactopyranose or 4-linked 3,6-anhydro-α-
d-galactopyranose. Primary sources of carrageenans are red algae orders Gelidiales,
Gigartinales, and Gracilariales. They exhibit antiviral activities against herpes simplex,
dengue-2, rabies virus, herpes zoster, and vesicular stomatitis virus. On the other hand,
partially depolymerized carrageenan extracts show anticancer and immunostimulatory
activities [46]. Carrageenan supplemented diet act as prebiotics in poultry and rat feeds [47,
48]. Polysaccharides from Porphyridium cruentum show antiproliferative activity [49].
Even carrageenans of low molecular mass have potential harmful effects. There high doses
in feed caused ulceration in animals and modified cytokine profile innormal human
colonic mucosal epithelial cell line culture [50]. Oral administration of λ-κ carrageenan to
mice increased their blood glucose, and carrageenan-induced insulin tolerance might lead
to diabetes [51].
Laminarin is a sulfated polysaccharide of phaeophytes and chemically made of β-1,3-d-
glucan with β-1,6- branching with different reducing ends having either mannitol or glu-
cose residues. The dietary effects of laminarins (β-1,3-d-glucan) depend on complexity of
its primary structure. Oral doses of M-series β-1,3-d-glucan have antitumor and hepatopro-
tective effects [52, 53]. Fucoidans are sulfated fucose-containing polysaccharides found in
edible phaeophytes like Cladosiphon okamuranus, Undaria pinnatifida, Saccharina japon-
ica, etc., and used as antiaging biomolecules in cosmetics [54].
15.3.4 Pigments
Pigments are fundamental, essential, and one of the most studied biomolecules present in
thylakoid membrane of chloroplast that selectively absorb visible spectrum of sunlight.
Pigments are widely used as colorants, food additives, vitamin precursors, in pharmaceuti-
cals as antioxidants, anticarcinogenic, antihypertensive, anti-inflammatory molecules, and
feed of livestock and aquaculture [6, 10]. Algae produce pigments according to their geno-
type that are grouped as chlorophylls, carotenoids, and phycobiliproteins (PBPs), which
impart green-, yellow/orange- and, red/blue-colored appearance to algae, respectively, as
per their abundance.
15.3.4.1 Chlorophylls
Chlorophylls (a, b, and c) are lipid-soluble green pigments essentially present in virtually
all photoautotrophs. Use of natural pigments and chlorophylls as colorants in
nutraceutical, pharmaceutical, and cosmetics industry is growing rapidly due to intense

green pigmentation and health and environmental concerns associated with synthetic
dyes. Sodium and cupric derivatives of chlorophylls (chlorophyllin) are popularly used as
food or beverage additives for rich green color [29]. Chlorophyll and its derivatives like
phaeophytin, pyrophaeophytin, and chlorophyllin are strong protectants against mutagens
like alkylating agents, heterocyclic amines, polycyclic aromatic hydrocarbons, aflatoxins,
etc., and used as therapeutic biomolecules. Combinations of chlorophylls have shown
strong antioxidant activity, increased glutathione S-transferase content, reduced
cytochrome P450 enzyme activity, reduced cell differentiation, induced cell arrest, and pro-
grammed cell death [55, 56]. Chlorophyll content is dependent on the genotype and growth
conditions. Low light intensity, red/green light spectrum, elevated temperature, and nitro-
gen and phosphorus-rich medium can significantly increase chlorophyll content.
Chlorophyll c is found only in brown algae, and reports on its health benefits are elusive.
15.3.4.2 Carotenoids
Carotenoids form a family of isoprenoid structured lipophilic yellow to orange-red
accessory light-harvesting pigments. These are found in certain bacteria, fungi, microalgae,
and plants. Carotenoids harbor the membranes of chloroplasts, mitochondria, and endo-
plasmic reticulum. Sometimes are present in chloroplast matrix as plastoglobulin, or cyto-
plasmic lipid globules. Carotenoids are very important dietary components with therapeutic
and cosmetic uses as precursor of vitamin A. Natural β-carotene is exclusive source of 9-cis
β-carotene which is a strong quencher of oxygen-free radicals and is reported to improve
retinal and visual dysfunctions in humans and animals [57]. Additionally, they are very
strong antioxidants because of their free radical quenching properties and protect organ-
isms from oxidative stresses. The β-carotene has been shown prevent advancement of ath-
erosclerosis (plaque formation in arteries) in humans [58]. Oral doses of β-carotene prevent
UV-induced erythema in humans [59]. There is an increasing demand for algae as source
of carotenoids as they possess a variety of carotenoids and also microalgae have enormous
capacity to produce them high amounts, i.e. ~0.1–0.2% of total dry mass [9]. However, in
green microalgae, it can be increased up to 14% by exposure of algal cells to high irradiance,
elevated temperature, high salinity, and reduced nutrition [60]. More than 600 different
natural carotenoids have been reported and categorized as oxygen-free hydrocarbon carot-
enoids, carotenes, oxygenated derivatives of carotenes, and xanthophylls. Green microal-
gae produce a huge range carotenes (β-carotene, lycopene) and xanthophylls (astaxanthin,
antheraxanthin, violaxanthin, lutein, zeaxanthin, neoxanthin, lutein, loroxanthin, can-
thaxanthin). Caretenoids of diatoms and brown algae are diatoxanthine, diadinoxanthin,
and fucoxanthin [61]. The β-carotene or provitamin A is first commercially produced
microalgal biomolecule used as an additive in pharmaceutical products and as colorant in
food products like cheese, butter, and margarine [30, 62]. Common β-carotene producing
microalgae are Dunaliella bardawil, D. tertiolecta, and Scenedesmus almeriensis [7].
Dunaliella salina and Haematococcus pluvialis are richest commercial producers of two
most desirable caretenoids β-carotene and astaxanthin, respectively [61]. D. salina pro-
duces ~14% carotenoids of its total biomass and up to 98.5% β-carotene of its total carote-
noid content [62]. Phordium autmnale produces 24 different all-trans-carotenoids with
β-carotene as a major component [63].
Astaxanthin, a red xanthophyll pigment, is the second most desirable carotenoid and its
microalgal sources include Chlorella zofingiensis, Scenedesmus almeriensis, and
Chlorococcum. A freshwater green microalga, Haematococcus pluvalis, produces up to 7%
carotenoids of its total dry mass and ~81% astaxanthin of total carotenoid content [62] and
is a promising species for industrial scale astaxanthinproduction. Astaxanthin is a popular
aquaculture feed that elicits pinkish-red color to the flesh of salmon, shrimp, lobsters, and
shellfish [10, 29, 64]. Also, it is most potent antioxidant which is ~10 fold more active than
other carotenoids and protects from oxidative stresses, cancer, diabetes, ophthalmic, and
neurodegenerative diseases [65]. Consumption of natural astaxanthin reduces inflamma-
tory reactions, improves health of patients with cardiovascular issues, prevents lipid per-
oxidation in heavy smokers [66], and increases serum high-density lipid and
adiponectin [67].
Lutein and zeaxanthin (yellow carotenoids) are essential pigments in human retina and
are obtained from microalgae. They protect photoreceptor cells against blue light-induced
damage to lens and retinal cells [68]. Their regular dietary consumption protects from cata-
ract, diabetic retinopathy, and age-related retinal degeneration by free radicals [69].
Common lutein-producing genotypes are Chlorella zofingiensis, Chlorella protothecoides,
Neospongiococcus gelatinosum, Scenedesmus almeriensis, Chlorococcum citriforme,
Muriellopsis, etc. [29]. Zeaxanthin producing strains are Scenedesmus almeriensis and
Nannochloropsis oculata.
Other carotenoids are lycopene (red), violaxanthin (orange), canthaxanthin (reddish-
orange), and fucoxanthin (olive green). Violaxanthin from Dunaliella tertiolecta and
Chlorella ellipsoidea is anti-inflammatory, antiproliferative and anticancerous [70].
Lycopene is used in formulation of sunscreen and antiaging products in cosmetic industry.
Fucoxanthin from brown algae and some marine diatomsis used as a strong antioxidant,
anti-inflammatory, anticancerous, antidiabetic, tanning, and neuroprotective in pharma-
ceutical and cosmeceuticals.
15.3.4.3 Phycobilliproteins (PBPs)

PBPs functioning as accessory photosynthetic pigments are found in members of
Cyanophyta, Rhodophyta, Cryptophyta, and Glaucophyta. PBPs are disc-shaped multi-
subunit hydrophilic protein complexes with covalently linked linear tetra-pyrrole
groups (bilins). They absorb light between 470 and 660 nm and transfer it to photosyn-
thesis reaction center thereby enhance the photosynthetic efficiency. Based on spectral
properties PBPs are sub-grouped asphycocyanin (PC), phycoerythrin (PE), allophyco-
cyanin (APC), and phycoerythrocyanin (PEC). PBP’s composition and content may
vary with genotype and growth conditions and may reach up to 13% of total dry bio-
mass. Being natural PBPs are extensively used in food, pharmaceutical, and cosmetic
industries. In food industries, PBPs are used to make chewing gums, popsicles, wasabi,
confectionaries, beverages, and dairy products [30]. Physiologically active PBPs are
widely used as strong antioxidants, antiviral, anticarcinogenic, immunity booster, anti-
inflammatory, hepatoprotective, and neuroprotective in pharmaceuticals and in cos-
meceuticals for perfumes and eye makeup powder. Phycocyanin, a blue fluorescent dye
from Spirulina, is used to prepare fluorescent-labeled probes for various research
activities [30].
15.3.5 Vitamins
Vitamins are vital micronutrients that are precursors of many important enzyme cofactors,
and antioxidants essentially required for normal metabolic processes. As majority of vita-
mins are neither synthesized de novo nor stored in body, so their regular dietary intake is
needed. Microalgae are an excellent source of almost all vitamins (Table 15.1) such as pro-
vitamin A (α- and β-carotene), vitamin B complex (B1, B2, B3, B5, B6, B8, B9, and B12),
vitamins C, D, E, and K1 [71]. The quality and quantity of vitamins in microalgae are largely
dependent on their genotype and physicochemical growth phase, culture conditions, and
seasonal variations [32]. Fabregas and Herrero [72] reported that Tetraselmis suecica,
Isoehrysis galbana, Dunaliella tertiolecta, and Chlorella stigmatophora produce high vita-
min contents analogous to traditional sources. D. tertiolecta produces Provitamin A, vita-
mins B12, B2, E, and β-carotene. T. suecica is rich in vitamin B1, B3, B5, B6 and vitamin C,
whereas Chlorella contains high levels of vitamin B7 and five times more B12 than fruits and
vegetables. So, microalgal supplemented vegan foods can fulfil vitamin B12 requirements,
viz., 1 g of Anabaena cylindrica powder supplements 64% of adult’s vitamin B12 require-
ment [71]. Seaweeds are rich source of water- (vitamins B1, B2, B12, and C) and fat-soluble
(pro-vitamin A, β-carotene, and vitamin E) vitamins. Also they contain other vitamins of
B-complex (B3, B6, B9, and H) but in lesser amount. Sea vegetables like laver (Porphyra
umbilicalis), sea spaghetti (Himanthalia elongata), and Gracilaria changii have vitamin C
contents comparable to tomatoes and lettuce. Vitamin C content in a brown alga, Eisenia
arborea is ~35 mg/100 g dry weight which is fairly close to mandarin oranges. G. chilensis
and Codium fragile contain more β-carotene than carrots [73]. Common kelp, Macrocystis
pyrifera, contains more of vitamin E (tocopherol) than soybean and sunflower oil.
Although microalgae are rich in vitamins, their bioavailability depends upon algal geno-
types, seasons, and growth conditions [32]. Thus require high-throughput screening of
promising algal genotypes, implementation of appropriate strategies for harvesting, drying,
and processing of algal biomass to protect heat-labile vitamins.
15.3.6 Polyphenols
Polyphenols or phenolic compounds form a large group of secondary metabolites produced
by plants and microorganisms and have gained attention as an important class of natural
antioxidants. They possess antiallergic, antimicrobial, anticancer, anti-inflammatory, anti-
diabetes, antiaging activities. Polyphenols extracted from Nannochloropsis and Spirulina
exhibitantifungal and antimycotoxigenic activities. Primary sources of polyphenols are
fruits, vegetables, and beverages. Majority of phenolic compounds are derivatives of the
aromatic amino acid metabolism. Polyphenols are the aromatic compounds consisting of
one or more phenyl ring bearing one or more hydroxyl group that makes them polar. Based
on their chemistry, polyphenols have been categorized into phenolic acids (hydroxycin-
namic acids, hydroxy benzoic acids), flavonoids (anthocyanins flavonols, flavones, fla-
vanonols, flavanones), isoflavonoids (coumestans, isoflavones), lignans, stilbenes, and
phenolic polymers (hydrolyzable tannins, proanthocyanidins-condensed tannins) [74].
Phlorotannins are oligomers of phloroglucinol units and predominantly found in cell walls
of phaeophycean genera. Halogenation of phlorotannins increases their structural
complexities. Polyphenols protect organisms from oxidative damages caused by various

environmental stresses and their profile and amounts are dependent on algal species and
their growth conditions. Seaweeds (green-, red-, and brown-algae) are the primary source
of polyphenols [75], however, microalgae (Dunaliella tertiolecta, Haslea ostrearia
Phaeodactylum tricornutum, Ankistrodesmus, Caespitella pascheri, Spirogyra, Euglena can-
tabrica) and cyanophytes (Nostoc spp., Nostoc commune, Leptolyngbya protospira Nodularia
spumigena, Arthrospira platensis, Phormidiochaete spp.) are also reported as their
sources [76]. Polyphenol contents in Haematococcus pluvialis, Tetraselmis, Neochloris oleo-
abundans, Arthrospira platensis, and Chlorella vulgaris ranges from 54 to 375 mg Gallic acid
Equivalent (GAE)/100 g dry weight is almost same as that from classical sources [77].
Microalgae have distinct flavonoid compositions from higher plants and a rich source of
catechins and flavonols. Phaeophyceae genera have higher phlorotannin content than
other algal groups. Eckols, a phlorotannin, isolated from brown alga, Ecknonia cava has
antiadipogenic and neuroprotective effects, and Eisenia bicyclis has high free radical scav-
enging content that inhibits melanin formation [78, 79]. Bromophenols, commonly found
in all algal groups, have significant pharmaceutical values due to their antioxidative, anti-
microbial, anticarcinogenic, antithrombotic, and antidiabetic activities [80]. Thus, suitable
methods producing significant amounts of polyphenols should be studied in detail.
15.3.7 Phytosterols
Sterols are ubiquitous amphipathic steroid alcohols, a form of lipid that are essential com-
ponent of eukaryotic cellular membrane and regulate its fluidity, stability, and permeabil-
ity by interacting with other membrane phospholipids and proteins [32]. Sterols function
as hormones or their precursors and neurotransmitters. Sterols of plants, animals, and
microorganisms are termed as phytosterols, zoosterols, and mycosterols, respectively.
Sterols are triterpenes derived from isoprenoid biosynthesis with tetracyclic cyclopenta-α-
phenanthrene (rings A, B, C, and D) including a long elastic aliphatic side chain attached
to C-17 of ring D [81]. Most of the phytosterols have double bond-linked C-5/C-6, and
methyl groups attached to C-10 and C-13. Also, aliphatic side chain length, position of
double bond, availability of alkyl group, saturation condition, and stereochemistry of C-24
alkyl side chain of phytosterols have functional importance. Cholesterol is the primary
zoosterol and about 200 structurally and functionally similar phytosterols are reported
among which β-sitosterol is most abundant. Other common phytosterols are campesterol
and stigmasterol. Phytosterols can lower the low-density lipids in blood and promote car-
diovascular health, hence, have commercial values as nutraceuticals and pharmaceuticals.
Additionally, they have antiatherogenicity, anticancer, anti-inflammatory, antioxidative
activities and used for curing neural disorders, like Alzheimer’s disease and autoimmune
encephalomyelitis [6].
Microalgal phytosterols have 24-ethylcholesterolas dominant form with an extra alkyl
group in side chain [32]. Extremely diverse algal phytosterols consist of cholesterol, fucos-
terol, isofucosterol, clionosterol, dihydroxysterols, etc., and are biosynthesized by meva-
lonate or methyl-d-erythritol 4-phosphate pathways. Algal strains and physicochemical
growth conditions can modify phytosterol diversity and content. Microalgae may produce
5.1% phytosterols (brassicasterol, sitosterol, and stigmasterol) of their total biomass.
Phytosterol-producing microalgae are Pavlova lutheri, Tetraselmis, Isochrysis galbana,

Phaeodactylum tricornutum, Chattonella antique, Dunaliella tertiolecta, Dunaliella salina,
Nannochloropsis, Chrysoderma, Chrysomeris, Chrysowaernella, Giraudyopsis, Peyssonnelia,
Glaucocystis nostochinearum, Chlorella vulgaris, etc. [81].
15.3.8 Phytohormones
Phytohormones are endogenously produced low molecular weight trace signal molecules
that regulate diverse cellular processes in plants. Phytohormones like auxins, gibberellins
(GAs), cytokinins (CKs), abscisic acid (ABA), ethylene (ET), salicylic acid (SA), jasmonic
acid (JA), and brassinosteroids (BRs) function synergistically to initiate appropriate signal-
ing network in response to various stimuli and maintain cellular homeostasis. Lu and
Xu [82] have reported various essential and bioactive phytohormones (auxin, GA, ABA,
CK, and ET) from diverse microalgal lineages. Despite controversial functional signifi-
cance of algal phytohormones and related signaling pathways, endogenous ABA and CK in
Nannochloropsis oceanica have shown physiological effects similar to higher plants [82].
Although, microalgal phytohormone profiles are similar to higher plants, but their nature
and abundance, and biosynthetic intermediates seem to vary among species. Comprehensive
understanding of phytohormones pathways in microalgae can be utilized to increase algal
biomass, production of desired secondary metabolites, and many other biomolecules of
industrial use.
15.3.9 Minerals
Mineral nutrients include essential inorganic dietary elements in addition to carbon,
hydrogen, oxygen, and nitrogen needed for normal metabolism and growth of organisms.
Important dietary elements are calcium, magnesium, phosphorus, copper, iron, manga-
nese, molybdenum, potassium, sodium, iodine, and zinc. Based on their quantity stored in
body minerals are classified into major minerals and trace minerals. Major minerals are
recycled and stored in large quantities, viz. calcium, sodium, potassium, magnesium, phos-
phorus, chloride, and sulfur. However, trace minerals are equally vital, but required in very
small amounts, viz. manganese, zinc, copper, iron, molybdenum, chromium, iodine, fluo-
ride, and selenium. Algae are excellent source of mineral supplements such as potassium,
iron, magnesium, calcium, and iodine are frequently used as sea vegetables and in aquacul-
ture. Marine algae like Fucus vesiculosus, Tetraselmis suecica, Isochrysis galbana, Dunaliella
tertiolecta, Chlorella stigmatophora, Undaria pinnatifida, Chondrus crispus, Porphyra ten-
era, etc. contain a significant amount of inorganic ions such as phosphorus, calcium,
sodium, potassium, iron, magnesium, zinc, cobalt, and copper. They particularly have high
concentration of Cl− and Na+ possibly due to their noteworthy osmotic potential [83, 84].
Algae have high bioadsorptive and bioaccumulative capacities leading to higher mineral
content than terrestrial plants. Ash content of specific seaweeds may reach up to 40% of dry
weight whereas mineral content in spinach can be 20% of dry weight [85]. However, the
concentration of minerals may vary with genotypes, seasons, and geographic locations.
Seaweeds like Alaria esculenta, Palmaria palmata, Laminaria, Saccharina japonica,
Undaria pinnatifida, Ulva intestinalis, etc. are excellent sources of iodine. Iodine content is
15.4 Algae in Biofuel Production/Generatio 319
altered during preparation and storage as many iodine compounds are water-soluble or
may vaporize under humid conditions [86]. Macroalgae like Gracilariopsis, Sargassum,
Ulva, and Porphyra from the Venezuelan sea contain a potentially rich source of iron suit-
able for human consumption [46]. Thus, edible brown and red algae can be consumed as
sea vegetables to supplement essential dietary mineral nutrition.
15.4 Algae in Biofuel Production/Generation

Steep population growth has driven enormous global industrialization where most of the
energy demand is met with nonrenewable fossil fuels like petroleum oil, coal, and natural
gas. Thus, conventional fossil fuel reserves are exhausting rapidly and global energy
demand is anticipated to inflate by 1% per year by 2040 [87]. Environmental issues associ-
ated with fossil fuels and inflating prices have led the soaring energy sector to quest for
alternative inexpensive, renewable, and green energy resources to ensure energy security.
Solar, hydroelectric, wind, and biomass has represented potential and dynamic genesis of
renewable energy. Biomass from agriculture and forestry has been used as feedstock for the
production of biofuels like bioethanol, biodiesel, and biogases [88]. Exhaustion of agricul-
ture and forestry biomass feedstock has serious concerns as their combustion expands car-
bon imbalance and their inappropriate harvesting has led to other crisis, viz. deforestation,
soil erosion, and loss of biodiversity due to their slow growth rate.
Algal biomass has emerged an alternate feedstock for biofuel production over food-crop
and non-food crop biomasses due to high growth rate, lipid, and carbohydrate contents as
compared to terrestrial plants. Various biofuel products like biodiesel, bioethanol, biogas,
biohydrogen, bio-oil, syngas, and biochar can be obtained from algal biomass according to
the species type, growth conditions, and conversion processes. Members of Cyanophyceae,
Chlorophyceae, and Pyrrophyceae contain higher amount of fatty acids and are most suit-
able for biofuel production [89], whereas those of Phaeophyceae are rich in carbohydrates
and most suitable feedstock for bioethanol production [90]. Various algal biomass transfor-
mation pathways for biofuel production are summarized in Figure 15.2. Technologies for
algal biomass conversion into biofuel involve four broad ways, i.e. thermochemical, chemi-
cal, biochemical, photosynthetic microbial fuel cell.
15.4.1 Thermochemical Conversion

In process of thermochemical conversion algal biomass is disintegrated followed by its
chemical transformation into biofuels in the presence or absence of oxygen at different
temperatures using combustion, gasification, pyrolysis, or hydrothermal liquefaction
methods [91]. Direct combustion involves thermal conversion of biomass at >800 °C tem-
peratures in excessive presence of an oxidant (typically oxygen) in a steam turbine or boiler
to generate heat. Primary by-products produced are CO2 and water vapor. Drying of algal
biomass is preceded by its direct combustion for better efficiency. Gasification is the ther-
mal conversion of algal biomass under-regulated oxygen at ~1000 °C into synthesis gas or
syngas, which is primarily a mixture of hydrogen, carbon monoxide, and methane. Syngas
can be directly used as fuel or can be further fractionated in a range of fuels, chemical
Conversion routes Conversion methods Products
Electricity, heat
Combustion
mechanical power
Gross biomass
Thermochemical conversion Liquefaction Bio-oil
Gasification Syngas
Syngas
Pyrolysis
bio-oil char
Lipids
Chemical conversion Transesterification Biodiesel
Algal biomass
Photobiological Hydrogen gas
Ethanol
Sugars
Biochemical conversion Fermentation
Hydrogen gas
Anaerobic digestion Biogas
Photosynthesis
Microbial fuel cells Photo microbial fuel cell Bioelectricity
Figure 15.2 Different algal biomass modification pathways for biofuel production [7, 88].
intermediates, and liquid products. Pyrolysis is the thermal conversion of biomass by heat-
ing it under anoxic conditions producing charcoal (solid), bio-oil (liquid), and syngas [92].
Pyrolytic thermal conversion can be conventional (biomass heating rate is 0.1–1 K s−1 for a
long resident time of 45–550 s), fast (10–200 K s−1 for a short resident time of 0.5–10 s), and
flash (>1000 K s−1 for a very short residence time of <0.5 s). Pyrolysis is the most preferred
thermal conversion procedure due to high ash content in algal biomass. Depending upon
the biomass feedstock, fast pyrolysis may yield 60–75% of bio-oil, 15–25% char, and 10–20%
noncondensed gases. The flash pyrolysis procedure can transform biomass into crude oil
with ~70% efficiency. Crude bio-oil produced through pyrolysis can be used as biofuel in
engines and turbines. In hydrothermal liquefaction, the algal biomass slurry is deoxygen-
ated with alkali solution at more than 400 °C and 2900 psi pressure in the absence of oxygen.
Several compounds such as high viscosity bio-oil (~73%), gas (~20%), and char (0.5%) can
be collected from algal biomass [93]. Average bio-oil yield with the catalytic hydrothermal
liquefaction is nearly twice (63%) than with noncatalytic aqueous liquefaction [91].
15.4.2 Chemical Conversion by Transesterification

High lipid contents of algal biomass allow its chemical conversion in biofuel. Heterotrophic
algal cells contain significantly high amount of total and neutral lipids than the photoauto-
trophics [94]. Although, lipid content of microalgae depends upon its ecological niche and
species, they have significantly high lipid content (50–86% total dry weight) as compared to
macroalgae except chlorophytes. Microalgal cells exhibit extensive lipid accumulation
under high light intensity or nutritional (N or P) stresses [95]. High lipid content of algal
cells can be converted into high efficiency biodiesel by the process of chemical
transesterification.
Transesterification is an acid or base-catalysed chemical exchange of organic alkyl group
of an ester with the organic alkyl group of an alcohol. It is utilized for the production of
biodiesel by chemical reaction between algal fatty acids or triglycerides and alcohol in the
presence of an acid, base, or lipase as a catalyst. Transesterification converts algal fatty
acids or triglycerides to fatty acids methyl ester (FAME) or biodiesel and glycerol using
alcohol in the presence of catalyst (Figure 15.3).
Although any alkyl alcohol (methyl, ethyl, propyl, or butyl, and amyl alcohol) can be
used for transesterification but, methanol, and ethanol are preferred at commercial scale
due to their physicochemical advantages and low cost [96]. This reaction highly depends
on the molar ratio of reactants, alcohol, and catalyst used thus regulating the fluidity of
biodiesel enabling its direct use in engines by blending with petroleum diesel [97].
CH2-O-CO-R1 R1-COOCH3 CH2-OH

Catalyst
CH2-O-CO-R2 + 3CH3OH R2-COOCH3 + CH2-OH
CH2-O-CO-R3 R3-COOCH3 CH2-OH
Fatty acid or TGA Methyl alcohol FAME Glycerol

(Biodiesel)
Figure 15.3 Transesterification of algal fatty acid (TGA) to glycerol and biodiesel.
High-density glycerol produced as a by-product can be routinely drawn out to maintain

chemical equilibrium and be utilized in cosmetic and pharmaceutical industries.
Transesterification process is of two types, i.e. conventional extractive and direct or in
situ transesterification. Conventional extractive transesterification is a multistep process
involving controlled desiccation of biomass, fatty acid extraction, transesterification, and
biodiesel refining. Despite its higher production cost and processing time, it yields a refined
biofuel excellent for high-speed diesel engines [98]. Direct or in situ transesterification is a
single-step method where untreated wet algal biomass is directly delivered into the reac-
tion chamber for transesterification without extraction of fatty acids. It uses excess volumes
of alcohol to tolerate water present in the biomass, facilitates fatty acid extraction, and
serves as esterification reagent. This method has greater yield, low cost, and reduced waste
compared to the conventional transesterification. Catalyst-free direct supercritical methyl
alcohol transesterification of wet biomass can significantly improve the quality of
biodiesel [99].
15.4.3 Biochemical Conversion

Biochemical conversion technologies of biofuel production rely on the presence of energy
reserve carbohydrates in algal feedstock. These energy reserve polysaccharides differ from
species to species and are different from structural polysaccharide of the cell wall. Their
subcellular location and degree of polymerization vary with algal species. Most common
carbohydrates stored in algal biomass are starch, floridean starch, chrysolaminarin, para-
mylon, and glycogen [25]. Microalgae have variable carbohydrate content like Porphyridium
cruentum and Chlamydomonas have up to 57 and 17% carbohydrate reserves of total dry
weight, respectively [42]. Algal polysaccharide content can be modified by altered growth
conditions. Algal biomass can be directly used as feedstock in biochemical conversion
pathways of biofuel generation without any pre-treatment. Theses pathways involve
hydrolysis of energy reserve polysaccharide in algal biomass by fermenting bacteria into
simple sugars through anaerobic digestion which can then be fermented into biofuels viz.
biogas, bioalcohols, or biohydrogen.
Biogas is produced from delipidized, polysaccharide-rich wet algal biomass in consecu-
tive stages of hydrolysis, fermentation (acidogenesis), acetogenesis, and methanogenesis.
During hydrolysis, the wet biomass is broken into soluble sugars and amino acids by bacte-
rial decomposition. In the next step, fermentable sugars are oxidized into alcohols, lactate,
acetic acids, volatile fatty acids, and carbonic acid by acidogenic bacteria under anaerobic
conditions. The fermented products are further oxidized into acetic acid substrates by ace-
togenesis. Methanogenic bacteria convert acetic acid into biogas (methane and CO2) at
final step of conversion (Figure 15.4).
The efficiency of anaerobic digestion is significantly impaired by C:N ratio of in feed-
stock, temperature, pH, and rate of feedstock input in the plant. Low C:N ratio is due to
high protein content in feedstock that leads to ammonia accumulation that inhibits bacte-
rial degradation reactions. Depleted content of lipid and protein in feedstock biomass have
shown better yield of biogas [100]. Co-digestion of waste papers, use of halophilic bacteria,
and microwave pretreatment of biomass can significantly improve the biogas
production [101–103].
Complex carbohydrates, lipids

and proteins
STEP 1 Hydrolysis
Simple sugars, fatty acids,

and amino acids
Acidogenesis
STEP 2
(Fermentation)
Volatile fatty acids

(acetic acid, propionic acid, butyric acid, and valeric acid)
STEP 3 Acetoogenesis
Hydrogen gas,
Acetate
carbon dioxide gas
STEP 4 Methanoogenesis
Biogas
CH4, CO2
Figure 15.4 Biochemical conversion of algal biomass for biogas production.
Contrary to terrestrial plant biomass, algal biomass has low lignin and hemicellulose and
high starch and cellulose content making it better choice for ethanol production. Algae like
Spirogyra, Chlorococcum vulgaris, Prymnesium parvum, Gracilaria, Gelidium amansii,
Laminaria, Sargassum, Nizimuddinia zanardini, Ulva pertusa, Eucheuma cottonii, Alaria
crassifolia, etc. have been widely used for bioethanol synthesis [104, 105]. Saccharification
of polysaccharide-rich algal biomass with hot water, acidic and enzymatic hydrolysis, and
liquefaction is essential for efficient fermentation. Saccharification and fermentation can
also be performed together by using amylase-producing strain of yeast to reduce the cost of
bioethanol production. Saccharomyces cerevisiae and Zymomonas mobilis are widely used
to perform fermentation under anaerobic conditions during bioethanol production. Since
fermentation of mannitol takes place in the presence of oxygen, so the use of S. cerevisiae
and Z. mobilis is not possible. A facultative anaerobic bacterium, Zymobacter palmae, is
used for fermentation of mannitol-rich algal biomass. Major advantage of commercial
bioethanol production is that it can be readily used as automobile fuel or blended with fos-
sil fuel petroleum. Nonetheless, certain features of bioethanol like corrosiveness, low vapor
pressure, flame luminosity, and energy density make it inferior fuel than diesel and petrol.
Instead, butanol is considered as a better transportation fuel due to its higher energy den-
sity, and it has more potential for integration with petroleum fuel. Algae like Ulva lactuca
and Saccharina (Phaeophyceae) provide promising biomass for butanol production [106].
Development of technologies for use of hydrogen gas as a fuel has leaped since last dec-
ade. Unlike fossil fuels that produce greenhouse and other toxic gases on combustion,
hydrogen gas as fuel has unique features like high energy content, extremely low density,
and production of water as a by-product on burning have made it the cleanest and most
efficient fuel. Microalgae can synthesize biohydrogen during photosynthesis with high
photon conversion efficiency. The ability of Scenedesmus obliquus to produce hydrogen gas
through fermentative and photochemical processes [107] has opened the possibilities to
use algae to assimilate the solar energy into clean fuel. Algae being oxygenic photosyn-
thetic organism produce hydrogen through biochemical processes like biophotolysis and
photofermentation. During the light reaction of photosynthesis, photolysis of water mole-
cules generates hydrogen ions, electron, and molecular oxygen. Consequently, in the
absence of oxygen, Fe-hydrogenase enzyme in microalgal cell converts hydrogen ions into
biological hydrogen gas [108]. However, co-produced molecular oxygen tends to inhibit the
activity of hydrogenase enzyme and impedes biohydrogen production. Such repression of
Fe-hydrogenase activity by oxygen can be protected by exposing the algal culture to anaero-
bic conditions [109] which leads to a “two-step photosynthesis and biohydrogen produc-
tion” system. First aerobic stage of such systems is composed of culturing of
photosynthetically active algae, while during the second anaerobic stage, sulfur deficiency
is stimulated in culture by biohydrogen production [110]. The sulfur deprivation in culture
leads to gradual degradation of a sulfolipid in the thylakoid membrane exclusively associ-
ated with photosystem II and partially inactivates it, while photosystem I remains active.
Thus, sulfur-deprived cells lower the oxygen production while steady respiratory oxygen
consumption creates an anoxygenic environment in the algal cell. In this physiological
condition, Calvin cycle stops the generation of NADP+ and cell keeps releasing the electron
to prevent oxidative damage. Some algae produce hydrogenase enzymes in such conditions
to release the reductive pressure in the cell and produce hydrogen by accepting electron
from ferredoxin [111]. Chlamydomonas reinhardtii and Chlorella sorokiniana are potential
source of biohydrogen fuel production under a sulfur-deprived condition.
15.4.4 Photosynthetic Microbial Fuel Cell (MFC)

Energy demands have led to the development of alternative renewable energy sources in
form of microbial fuel cell that can directly generate electricity in single or multiple
steps [112, 113]. MFCs are bioelectrical equipment that directly harvest electrical energy
generated during the anaerobic respiration of certain microbes grown on organic feed-
stocks. MFC is widely used as a bio-electrochemical system in wastewater management,
sustainable electricity production, and regeneration of valuable reduced reaction end prod-
ucts in biochemical reactions.
In a hydrogen fuel cell, hydrogen gas is oxidized into protons and electrons at anode
while oxygen is reduced to water at cathode in presence of catalyst. The structure of MFCs
is comparable to hydrogen fuel cells in which protons flow from anode to cathode chamber
through an electrically insulated proton-exchange membrane while electrons flow in a
similar direction through an electric wire. In a MFC, anaerobic bacteria oxidize the organic
feedstock in anode compartment and transfer the electrons to an electrode instead of an
electron-acceptor molecule. The dissimilatory metal-reducing or anodophilic bacteria
(Geobacter metallireducens, Geothrix fermentans, Shewanella, etc.), can make a biofilm on
the surface of metal electrode (electron acceptor) in anode chamber and reduce it with the
15.5 Algae in Additional Application 325
help of periplasmic c-type cytochrome protein as metal reductase [114–116]. Major

constraint of typical MFC is the requirement of continuous aeration with oxygen as a
potential terminal electron acceptor at cathode, which is an energetically intensive proce-
dure in a real-world application.
The limitation of cathode aeration was eliminated by the assimilation of algal photosyn-
thesis with MFCs, hence known as photo MFCs. In a photo MFC, bacterial respiration
takes place in anaerobic anode chamber which is electrochemically coupled with an algal
oxygenic photosynthesis cathode chamber as a potential terminal electron acceptor.
Unicellular microalgae are widely used in photo MFCs for electricity generation. Thus,
algal photo MFCs can efficiently treat wastewater, generate decent electricity, and fix CO2
simultaneously [117].
Dominant anodophilic bacterial consortia for photo-MFCs are composed of Shewanella
oneidensis, Geobacter sulferreducens, Cytophaga xylanolytica, Cytophaga, Pseudomonas aer-
uginosa, Thiomonas perometabolis, Dechloromonas, Desulfovibrio, etc. whereas algal spe-
cies commonly used in cathode chamber are Spirulina platensis, Anabaena cylindrical,
Synechococcus leopoliensis, Scenedesmus obliquus, Chlorella vulgaris, Chlorella pyrenoidosa,
Desmodesmus, Phormidium, Chlamydomonas reinhardtii, Laminaria saccharina, etc.
Photo-MFC with a combination of Geobacter sulferreducens suspension in acetate as
anolyte and Chlamydomonas reinhardtii is shown to generate power output of
mW m−2 [118]. Thus, photo-MFCs propose a cost-effective and eco-friendly technology for
electricity generation and wastewater management.
15.5 Algae in Additional Applications
15.5.1 Algae as Livestock Feed and Nutrition

Aquaculture, livestock animals, and poultry farm must be intensified in order to meet the
growing needs of milk, meat, eggs, wool, and hides for increasing population. Productivity
of reared animals greatly depends upon the quality of their feed so insufficient quantity or
nutritionally incomplete agricultural products as feed lead to suboptimal production of
meat, milk, and eggs. Algae serve as an excellent nutrition-rich alternative feed for a variety
of animals. Algal supplemented feeds are more nutritive and rich in proteins, carbohy-
drates, PUFAs, carotenoids, essential vitamins, and mineral than conventional ones. These
can boost immunity of ruminants and protect animals and fishes from diseases and lead to
production of healthy animal products. At present, >30% of global algal production is used
to feed reared animals, and >50% of global Arthrospira platensis production is used to pre-
pare such feeds. Genetically divergent Australian sheep fed on Arthospira-fortified feed
significantly gained body mass, growth, and body contour [119]. Algal feed increase ω-3
fatty acid content in milk without harming its lipid yield [120]. Laminaria digitate supple-
mented feed increased pig weight by 10% [121].
Chicken meat and eggs are among major sources of protein globally and algae serve as
nutrition supplement for poultry. Algal biomass up to 10% of total feed weight is used as a
partial replacement of traditional protein supplement in poultry feed. Chicken fed on algal
biomass-supplemented feed exhibited bright yellow colour of egg yolk, skin, and shanks
with 10% less cholesterol due to higher carotenoid content infeed [122]. Due to high carot-
enoids, use of excessive algal feeds for extended periods cause yellowing and change in
texture of meat, so are not liked in some parts of the world, but it is not reported to cause
toxicity in animal during feeding trails.
15.5.2 Algae as Feed in Aquaculture

Aquaculture, an important sector in animal husbandry, is a vital source of protein. Various
microalgal species (Chlorella, Arthrospira platensis, Pavlova, Tetraselmis, Isochrysis,
Chaetoceros, Phaeodactylum, Thalassiosira, Skeletonema, Porphyra, Kappaphycus,
Nannochloropsis, etc.) are natural food for herbivores like rotifers, brine shrimps, copep-
ods, and other crustacean that are directly or indirectly fed by fishes and secondary con-
sumers in aquatic food chains. PUFAs (AA, EPA, and DHA) are essential for fecundity, egg
quality, spawning, larvae hatching rates, growth and development, and pigmentation of
fishes [123, 124]. Animals having limited ability of de novo synthesis of PUFA shave high
demand of PUFA and its derivatives from fishes as food and nutraceuticals. The
carotenoid-rich algal feeds that enhance the pigmentation of reared shrimps, salmonids,
lobsters, red sea breams, etc., are an ideal alternative source of balanced nutrition for the
blue revolution.
15.5.3 Algae as Bio-Fertilizer

Algal biomass of cyanobacteria and green algae is crucial in building organic component of
soil on decomposition in an agro-ecosystem. Secretion of exopolysaccharides and other
phytohormones promotes growth of crop, microflora, and fauna. Inoculation of fields with
cyanobacteria and green algae augments microbial activities in soil and rhizosphere,
increases plant growth promoters, viz., vitamin B12, indole-3-acetic acid, indole-3-propi-
onic acid, or 3-methyl indole, and improves crop yield [125]. Inoculation of crop fields with
heterocystous cyanobacteria (Nostoc, Aulosira, Tolypothrix, Scytonema, Anabaena variabi-
lis, etc.) can fix atmospheric nitrogen, i.e. up to 40 kg of nitrogen per hectare and thereby
significantly reduce the cost of crop production. Whereas non-heterocystous cyanophytes
(Plectonema boryanum, Oscillaoria, Lyngbya) and green algae enrich soil by secreting insol-
uble salts. Thus, microalgae are used as biofertilizers and to maintain soil fertility.
15.6 Conclusion and Future Prospects
Algae have tremendous potential to produce a variety of high-value biomolecules.

Microalgae offer a potential resource of renewable, clean fuels like biohydrogen, biogas,
syngas, bioethanol, biodiesel, and photo-electricity to address current environmental
imbalance and increased carbon foot printing due to fossil fuel combustion. Due to their
remarkable ability to utilize carbon emitted from combustion as a source to recycle it in the
form of biomass at an impressive rate algae helps to reduce global warming. Also, they have
the potential to assimilate pollutants into biomass as nutrients and are greatly useful in
wastewater management. Being a rich source of protein, carbohydrate, PUFAs, vitamins,
Reference 327
and minerals, they help to meet global demands of healthy food. As per WHO recommen-
dations on the intake of PUFA by infants and adults has increased its demand. However,
the major PUFA demand is met with DHA obtained from fish oil of fishes feeding of micro-
algae producing it. Similarly, coloring agents, β-carotene, and astaxanthin are used as fish
food and growing awareness of nutritional food will also drive their demand as health sup-
plement molecules. At present, algal products and algae are gaining popularity and greater
acceptability as a source of proteins, vitamins, minerals, polyphenols, and other value-
added health supplements will improve their commercial production. Algae are rich source
of pharmaceutical and cosmeceutical molecules and could drive their specific molecule-
targeted commercial cultivation.
Although algae offer an array of value-added molecules, many challenges impede their
exhaustive exploitation. A large gap exists between perceived commercial capabilities of
various algal species and actual abilities of industrial processes. Evolution from niche mar-
ket to the extraction of high-value algal products requires extensive research and improve-
ment in methods to expand yields to gain multiple compounds from a single strategy. The
extraction of bioactive algal compounds is greatly challenged by the cost of production, i.e.
cultivation system, maintenance, limited productivity, and bio-refining techniques.
Production and hyper-accumulation of single high-value molecule require identification
and selection of appropriate algal strain, desired genetic engineering, and development of
strain-specific culture cultivation strategies. Additionally, efforts are needed to understand
the mechanisms of high-value molecule production and devise high-density culture
production, dehydration, gentle methods of cell wall disruption, and suitable recovery pro-
cedures without altering its biochemical nature.
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335
16
Plant Bioprospecting for Biopesticides and Bioinsecticides

Aradhana Lucky Hans and Sangeeta Saxena
Department of Biotechnology, Babasaheb Bhimrao Ambedkar University, Lucknow, India
16.1 Introduction
Agriculture holds an exceptional position in the growth of a country, especially developing

ones. The major setback it faces is from the attack of various pests causing harm to crops.
Loss due to harmful pest causes severe economic losses of agricultural crops and commodi-
ties. This threat to economic loss in the quality of agricultural crops has paved a way to
major insights in the field of research and new discoveries of pest-controlling agents.
Therefore, one of the crucial components of agricultural management is efficient manage-
ment of these pests. They can be managed and controlled by the application of various
pesticides in the different ways and at the different time points.
Over the years, chemical pesticides have made a great contribution to the fight against
several pests and diseases. However, the repetitive and indiscriminate use of these pesti-
cides has developed insecticide resistance in many major pests causing resurgence of minor
pests, which in turn resulted in irreparable damage to crops. A new range of pests had even
more and wider range of pesticide application, making the situation even more worse.
Harmful effects, such as soil and water contamination and dramatic increase of the harm-
ful residues, cause harm to both environment and human health. The use of chemical
insecticides in crop pest control programs around the world had caused tremendous dam-
age to the environment, pest resurgence, pest resistance to insecticides, and lethal effects
on nontarget organisms [1].
It is evident that population rise has been a continuous process and to meet the needs,
agriculture also has been pacing up at faster rate. The improvement in the yield and pro-
duction of agriculture can be easily credited to the application of pesticides, therefore
improving production in agriculture is most certain. The indiscriminate use of pesticides
over the years, particularly in developing countries, has caused enough damage in various
spheres. Overlooking the safety measures and recommended usage has led to many health-
related problems. The risk is not only on humans but on the surrounding environment and
the nontarget flora and fauna that are exposed to these harmful chemicals. The residues

336 16 Plant Bioprospecting for Biopesticides and Bioinsecticides
linger in the air, water, and soil and persist for a long duration causing more damage to the
environment. According to World Heath Organizations (WHO), about 25 million cases of
acute occupational pesticide poisoning and 20 000 deaths occurred globally per year. This
adverse effect of these pesticides is a matter of great concern, leading to awareness about
safety of food and environment. A search for alternatives to these nonjudiciously used
chemicals is catching up pace toward a more greener and cleaner alternative. In recent
decades, there has been a heap change in the viewpoint, causing some changes in the use
of conventional chemical pesticides [2]. Pertaining to the high risk with the use of chemi-
cal pesticides, integrated pest management (IPM) programs have brought out application
of pesticides with more efficient and judicious manner. Eventually these changes have sig-
nificantly reduced pesticide use and improved pest management practices. As a result,
there is an increasing demand for less toxic or environmentally safe pesticides that would
pose less risk to application as well as consumption. The need of the hour is to look for
alternatives that are less adverse and possess lesser health risk and be more environment-
friendly. Moving back from chemical toward natural resource would be more apt owing to
immeasurable diversity of molecules present in the nature. To look for an alternative that
would be ecologically better and environmentally safe, biopesticides seem to be the choic-
est option. Also, chemical pesticides are more expensive in comparison to biopesticides.
Plants are regarded as one of the most diverse and richest resources of various important
bioactive natural products. Since ancient times, the use of plants by the natives of various
parts of the world as pesticidal has been well utilized. Innumerable active compounds have
been identified from plants and still the source has not exhausted its prospective. Hence,
this interminable reservoir of bioactive compounds should be screened, searched, and
secured for more applications. Biopesticides conceptualized natural enemy or predatory
organism or their products that would include plants, microbial products, or by-products as
they were capable of managing or reducing pests. There is an obvious urge for the impetus
in developing biopesticides as they have the potential to protect plants from pest infesta-
tion. In search for novel bioactive substances from botanicals, screening of diverse plants
can be of much utility. This process of discovering and further commercialization of new
products based on biological resource is known as bioprospecting. Nature has been an
unending reservoir wherein one can unleash plant compounds that can be helpful in con-
trolling various pests. Biological resource comparatively has higher diversity than the
chemical compounds, holding more potential for novel compounds for agricultural appli-
cation [3]. In this chapter, the present scenario of plant-based biopesticides, advantages,
and future prospects will be discussed.
16.2 Current Scenario in India

Due to its rich biodiversity, India offers plenty of scope in terms of sources for natural biologi-
cal control organisms as well as natural plant-based pesticides. Traditional knowledge avail-
able with the highly diverse indigenous communities in India may provide valuable clues for
developing newer and effective biopesticides. Owing to its biological background biopesti-
cides are eco-friendly, easily biodegradable, and safer alternative to chemical pesticides.
Although the production is not as par chemical pesticide, yet the demand is pacing up the
production every passing year. Presently, biopesticides may represent approximately 4.2% of
16.3 Plants-Based Active Compound 337
Table 16.1 List of biopesticides from their resource plants and target pests.
Plant product used

as biopesticide Target pests Plants
Limonene and Fleas, aphids, and mites also kill fire ants, several Citrus fruits
Linalool types of flies, paper wasps, and house crickets
Neem/ A variety of sucking and chewing insect Azadirachta indica
Azadirachtin
Pyrethrum/ Ants, aphids, roaches, fleas, flies, and ticks Chrysanthemum cineraria
Pyrethrins folium
Rotenone Leaf-feeding insects, such as aphids, certain Genus Lonchocarpus
beetles (asparagus beetle, bean leaf beetle, majorly from Lonchocarpus
Colorado potato beetle, cucumber beetle, flea utilis and Lonchocarpus
beetle, strawberry leaf beetle, and others) and urucu
caterpillars, as well as fleas and lice on animals
Ryania Caterpillars (European corn borer, corn earworm, Ryana speciosa
and others) and thrips
Sabadilla Squash bugs, harlequin bugs, thrips, caterpillars, South American lily
leaf hoppers, and stink bugs Schoenocaulon officinale
Nicotine Caterpillars chewing pests Nicotiana tabacum
Acetogenins Lepidopterans and Colorado potato beetle Annona spp., including A.
(Leptinotarsa decemlineata) squamosa and A. muricate
Capsaicinoids Lepidopterans and Hemiptera insects Capsicum spp.
the total pesticides market in India. There are more than 6000 species that are screened of
which 2500 plant species were found to possess biologically active compounds against vari-
ous kinds of pests. The important plant families containing the biopesticide plants are
Apocynaceae, Asteraceae, Euphorbiaceae, Fabaceae, Meliaceae (maximum), Myrtaceae,
Ranunculaceae, and Rosaceae [4]. The higher demand for organic, chemical-free food has
augmented the demand of eco-friendly chemicals isolated from different parts of plants
(Table. 16.1). They can be from leaves, barks, roots, fruits, flowers, seeds, or seed kernels. In
nature, the higher plants have the ability to synthesize and produce various kinds of second-
ary metabolites that are naturally avoided by insects. Some are able to interfere in the insect
life cycle, which are known as semiochemicals. Majorly four types of botanical products are
used for insect control (neem, pyrethrum, rotenone, and essential oils); few others such as
ryania, sabadilla, acetogenins, capsaicinoids, and nicotine have limited use. Additionally,
plant oils and extracts also seem to have effect on insects that have even lesser use.
16.3 Plants-Based Active Compounds
16.3.1 Azadirachtin
The plant Neem or Azadirachta indica is the most extensively used plant for insect pest
management. Several farmers in Indian villages collect the neem seeds to prepare crude
seed kernel extract for pest control. Various reports highlight that neem-related products
do not leave any toxic residues on application on plants. Neem oil is said to be effective
against mites, soft-bodied insects, and phytopathogens. The compound Azadirachtin is
said to have low toxicity toward mammals and pollinators [5] according to Environmental
Protection Agency. This compound is isolated from the kernels and used in various biofor-
mulations of biopesticides. Effect of azadirachtin on insects can be in two major ways. It
blocks the synthesis and release of ecdysteroids (molting hormones), leading to incom-
plete ecdysis in young insects and sterility in adult female insects. The other effect seems
to be its antifeedant property inhibiting pest attack. These excellent effects of neem on
pest definitely make neem to the millennium paradigm toward the development of biope-
sticides. The reason is there is a plethora of neem-based commercial products Neemmark,
Wellgrow, Azatin, Bio-neem, Nimbin, Neemark, to name a few, used effectively through-
out the world.
16.3.2 Pyrethrins
Pyrethrum is obtained from dries flowers of Chrysanthemum cineraria folium and has
been and is globally used as a biopesticide [6]. Pyrethrins are insecticidal chemicals
extracted from the dried pyrethrum flower. The flower heads are processed into a powder
to make dust. This dust can be used directly or infused into water to make a spray.
Technical-grade pyrethrum, the resin used in formulating commercial insecticides, typi-
cally contains pyrethrin from a range of 20–25% [7]. Effect of pyrethrins on insects is
characterized by rapid knockdown particularly in flying insects and hyperactivity and
convulsions in other insects. The mode of action is quite similar to commercial chemical
pesticides such as DDT (Dichloro-diphenyl-trichloroethane) and other organochloride-
based insecticides, effecting mostly the neurotoxic action potential blocking voltage-gated
sodium channels in nerve axons. Pyrethrins are less toxic and easily break down in sun-
light as they are liable to the UV rays, making them safer alternatives to chemical
pesticides.
16.3.3 Rotenone
Rotenone is prepared from the plant species belonging to the genus Lonchocarpus majorly
from Lonchocarpus utilis and Lonchocarpus urucu. A cube resin, root extract is the major
component consisting of active ingredients rotenone, deguelin, and tephrosin. Rotenone is
also used as blends along with pyrethrins and approved as organic insecticides. Rotenone
is an important insecticide extracted from various other leguminous plants. It is an effec-
tive insecticide, it blocks the electron transport chain and prevents energy production act-
ing as mitochondrial toxin. To be effective it must be ingested. It is considered similar to
DDT but is much lesser toxic in its formulated products. In insects, rotenone exerts its toxic
effects primarily on nerve and muscle cells, causing rapid cessation of feeding. Death
occurs several hours to a few days after exposure. The disruption energy metabolism and
the subsequent loss of ATP result in a slowly developing toxicity, and the effects of all these
compounds include inactivity, paralysis, and death.
16.3 Plants-Based Active Compound 339
16.3.4 Sabadilla
Sabadilla is derived from the seeds of South American lily Schoenocaulon officinale. They
have active ingredients as celandine-type alkaloids and toxic to mammals, but in commer-
cial preparations, the active ingredients is at very low concentration, diluting its toxic effect.
These alkaloids are similar to pyrethrins and are used on citrus crops and avocado in
organic farming.
The mode of action of these alkaloids affects nerve cell membrane action, causing loss of
nerve action potential, causing paralysis and death [6].
16.3.5 Ryania
These botanical insecticides are derived from stem of Ryana speciosa. It contains an alka-
loid ryanodine that interferes with calcium release in muscle tissue. It is a slow-acting
abdominal toxin. The effect exerted by it is not rapid, rather delayed as insects do not imme-
diately stop feeding after ingesting it. Although not much has been studied about its mode
of action yet, it is said to be effective in hot weather. It is found to be toxic against citrus
thrips and fruit moths [8].
16.3.6 Nicotine
Another alkaloid is nicotine, which is obtained from the leaves of tobacco plant or Nicotiana
tabacum and few related species. Nicotine, along with nornicotine and anabasine, is a syn-
aptic toxin that mimics the neurotransmitter acetylcholine. They cause similar effects as
that of organophosphate and carbamate insecticides. It can be absorbed dermally and is
toxic to humans; therefore, the use has seen a decline. It is a fast-acting killer for soft-
bodied insects but not to all chewing pests. It mimics acetylcholine, neurotransmitter
causing uncontrolled nerve firing. It is said that nicotine is fairly selective and affects only
certain types of insects [9].
16.3.7 Acetogenins
Acetogenins have been traditionally prepared from Annona spp., including A. squamosa
and A. muricate, which are used as botanical pesticides. These are source of fruit juice in
some parts of Asia. The major acetogenins are obtained from the seeds of A. squamosa [8].
These compounds show toxicity as slow-acting gut poison, mostly effective against chew-
ing pest, especially Lepidopteran pest and Colorado potato beetle (Leptinotarsa decemline-
ata). The mode of action of acetogenins is similar to rotenone. They work in blocking
energy production in mitochondria.
16.3.8 Capsaicinoids
Peppers are very abundant in nature and found around easily. They are extremely pungent
and hot in nature. The burning sensation in chilli owes to the presence of capsaicinoids
found only in Capsicum spp. The main components of capsaicinoids are capsacin,
dihydrocapsaicin, nordihydrocapsaicin, etc. Various reports have been indicating the
broad-spectrum activity of capsaicin against insects such as Myzus persicae, Bemicia tabaci,
Sitophilus zeamais, Alfalfa weevil, to name a few [10–11]. The mode of action is majorly by
impairing the nervous system of insects or dysfunction of Na or K-gated channels.
16.3.9 Essential Oils

In addition to these, there are many plants that contain essential oil, which is a complex
mixture of volatile compounds accumulated in seeds, flowers, and leaves. These are vola-
tile in nature, complex secondary metabolites having a strong odor, and lipophilic in
nature [12]. These compounds interfere with physiological, biochemical, metabolic func-
tions of insects. Therefore, plant essential oil compounds are considered to be alternative
insect-controlling compounds and act as biopesticides. They have a wide range of quality
that is sufficient to manage insects and pests well. They have the ability to delay develop-
ment activities such as adult emergence and fertility, deterrent effect on oviposition [13],
arrestant and repellent action [14]. They have antifeedant and larvicidal activity [15].
The volatile components of essential oils are classified into four main groups: terpenes,
benzene derivatives, hydrocarbons, and other miscellaneous compounds. Monoterpenoids
are the most representative molecules consisting 90% of essential oils having diverse func-
tion. They have acyclic alcohols (linalool, citronellol, geraniol), cyclic alcohols (menthol,
terpineol, isopulegol), bicyclic alcohols (borneol, verbenol), phenol (thymol, carvacrol),
ketones (carvone, menthone, thujone), aldehydes (citronellal, citral), acids (chrysanthemic
acid), and oxides (cineole) [16]. Linalool has been studied and found to have effect on the
nervous system, affecting ion transport and release of acetylcholine esterase in insects [16].
The mode of action of most monoterpenes is by causing a drastic reduction in the number
of intact mitochondria and Golgi bodies, impairing respiration and photosynthesis, and
decreasing cell membrane permeability [17].
The essential oils of Ocimum basilicum contain active compounds juvocimenes, which
are an analogue of juvenile hormones of insects. Matricaria recutita contains precocenes,
which stimulate the production of juvenile hormone that suppresses the growth of insects
during molting. Additionally, some aromatic plants such as coriander, marigold have
strong odor, which act act insect repellents and act in managing the harmful pests.
Therefore, an analytical approach in selecting right biomolecules for the production of
biopesticides having broader range of activity would be a preferable alternative for eco-
friendly pest management. From unravelling new and better molecules to utilizing them
into useful application in the field is the actual need of the hour. Mere pilling up of numer-
ous novel compounds would not be ideal, rather disseminating into biochemicals will be
highly appreciated.
16.4 Advantages and Future Prospects of Bioinsecticides
Advantages attributed to biopesticide usage over chemically synthesized products are

many (Figure 16.1). They are supposedly less toxic, more selective in combating unwanted
biological targets, possess higher efficiency at lower concentrations, decompose more
16.4 Advantages and Future Prospects of Bioinsecticide 341
Botanical pesticides Chemical pesticides
Weekly to monthly
Hourly application at
Application application at pre
preharvest interval
harvest interval
Repellent, growth,
morphological and Toxic, repellent, non-
Mode of physiological selective harms non-
action interference, selectively target organisms too
toxic target pests
Attaining Efficacy on repeated Efficacy on fewer

efficacy application application
Degradation Takes few days to Takes almost years to

degrade degrade
Environmentally safe, Accumulation in

Outcome safe on heat environment, health
sustainable agriculture risks, loss of
biodiversity
Figure 16.1 Advantages of botanical pesticides over chemical pesticides. Source: Adapted from
Lengai et al. [18] Licence no. 4870610831544.
quickly, reducing adverse environmental effects. They can be used as an active component
of IPM and reduce the use of conventional chemical pesticides while crop yields remain
high. To use biopesticides effectively, however, one needs to know a great deal about man-
aging pests.
In IPM-related programs, biopesticides are needed to be the preferred component in
implementation of resistance management programs. The agrochemical industry is shift-
ing from chemical to botanicals owing to its great implications. The demand for chemical-
free food is increasing, and so is the awareness. The consumers are now more willing to pay
beyond the regular prices for foods produced organically without the use of chemicals.
Adaptation toward botanical pesticides from the more prominent chemical pesticides is
gradual.
Although the scenario is fast changing, yet a lot has to be changed in the coming years to
get a higher impact in modern agriculture. Resistance risk analysis is a key requirement, as
efficacy demonstration of pesticide active substances and their formulations is mandatory.
This kind of resistance risk assessment requires examination of the inherent risk (associ-
ated with the government authorities of the product and the pest) and the agronomic risk
(influenced by the crop, the geographic area, and the use pattern). Therefore, resistance
risk analysis is a complex issue, and the basis for risk assessment requires a clear willing-
ness on the stakeholders to cooperate in order to maintain a sustainable, viable, and safe
agricultural environment.
The actual benefits of biopesticides can be best realized in developing countries such as
India, where farmers are not always able to afford chemical insecticides. The ancient and
traditional use of plants and plant derivatives for protection of stored products is estab-
lished well in our country. Although synthetic insecticides might be affordable to growers
(e.g. through government subsidies), yet limited literacy and a lack of protective equipment
result in thousands of accidental poisonings annually. The lack of judicious use of pesti-
cides and the health and environmental risk it possesses can be overcome by the use of
biopesticides. No security measure is taken care of while applying chemical pesticides; this
kind of risk will be curbed by botanicals.
The area under organic cultivation (crops) in India is estimated to be around 100 000
hectare. Besides, there are lakhs of hectare of forest area being certified as organic.
Furthermore, some states such as Uttaranchal and Sikkim have taken botanicals on a larger
scale and declared their states as organic. The future is in need of making a shift from syn-
thetic to natural source to minimize the adversity of chemicals. The nature has immense
treasure instead of damaging approach; care should be taken to utilize and coexist benefit-
ting the crop and its growers.
16.5 Conclusions
To acquire knowledge is good, but to apply the same is even better. The same stands true for
biopesticides. Bioprospecting for novel molecules having varied properties should be car-
ried out. But, perhaps it is time to converge the attention of the research community toward
the development and application of known botanicals rather than screen more plants and
isolate further novel bioactive substances that satisfy our curiosity but are unlikely to be of
much utility [19].
The development of the biopesticide industries has to adopt a strategic, comprehensive
and more forward, and applicable approach. With the growing population and the growing
need of population, there will be greater need of crops and other products. The biopesticide
industry has to pace up to stand par with the chemical industry. For this, the policymakers,
government authorities, and the consumers demand has to work in sync. These together
will boost up the importance and ease of trade will make it more available in the market for
easy application. The increasing concern of consumers and government on food safety has
led growers to explore new environmentally friendly methods to replace, or at least supple-
ment, the current chemical-based practices. The use of biopesticides has emerged as prom-
ising alternative to chemical pesticides. Biopesticides have a precious role to play in the
future of the IPM strategies. It is evident that the path would not be as easy although we
have a wealth of knowledge and resource, but application still remains at a nascent stage.
The efficacy of biopesticides in managing insect pests is sure very effective, but the mat-
ter that needs to be pondered upon is their limited source. Though nature has immense
treasure of these important botanicals, yet, the supply is limited, since a large number of
source plants are needed for extraction of desired active compounds. And the supply in
Reference 343
nature or natural habitat is marginal and would exhaust if used extensively. Therefore,
commercial agriculture of such plants would raise income and keep the supply of required
raw material at pace. To scale up the produce, landscapes that are not under direct cultiva-
tion can be utilized.
The process of extraction and processing of the biopesticides using appropriate methods
should be worked upon also considering cost cutting during production. An efficient pro-
duction with fluent distribution up to the smallhold farmers will see the real success of
biopesticides. A better marketing strategy for biopesticides will also help in better circula-
tion in the much upscale pesticide market. Also, a regular education or awareness program
to farmers and supplier would boost up the botanicals immensely. All of such measures
will surely help biopesticide to amplify its application in the market.
Apart from all these things, there are some areas that to my concern seem to be impor-
tant to be pondered upon. Generally, discovering something hidden in nature and report-
ing seems to be satisfying, yet, how much is it valued or accepted is a critical point of
discussion. A far more acceptable approach that also is rewarding is proving one’s hypoth-
esis; publication-wise too this seems better. Reporting a potent botanical compound and
moving onto its application in the fields is a long process. Yet, there are many compounds
successfully being applied in fields, such as Pyrethrin and Azadirachtin, etc. Therefore, the
dawn of biopesticides is decent, but to see it a daylight, more effort with applicable focus
is needed.
A
cknowledgment
ALH and SS would like to acknowledge the funding from DST NPDF-SERB.
R
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345
17
Plant Biomass to Bioenergy

Mrinalini Srivastava and Debasis Chakrabarty
Molecular Biology and Biotechnology Division, Tissue Culture and Transformation Lab, CSIR- National Botanical Research
Institute, Lucknow, India
17.1 Introduction
We are living in the age where continuous use of fossil fuels as energy resources enhances the
pollution speedily and instigates serious health issues. As a result, there is high demand for
alternative energy production ways. Whole scientific community across the world is trying to
discover and promote renewable energies, which share the load and in some extent replace
the conventional energy resources. Among alternative energy sources, plant biomass emerges
as vital source of bioenergy and highly recommended by the European Union [1]. Renewable
biomass sources can be converted to fuels and are a logical choice to replace oil [2]. From
ancient times, biomass is considered as a main source of energy for humanity, and now there
is a time for strong transition from old to modern source of energy [3].
Moreover, to overcome the crisis of environmental deterioration due to the impact of fos-
sil fuels, maximum utilization of biofuels especially in transportation sector is obligatory.
Plant-based biofuels are the most abundant source of renewable fuels, involved in the pro-
duction of small (ethanol and butanol as gasoline additives) and long-chain hydrocarbons
(for diesel additives or as jet fuels). Another advantage associated with biofuels is continu-
ous cycling of carbon rather than being released in the environment as in case of fossil
petroleum and natural gas [4].
Global warming is today’s major problem, and CO2 release can be minimized to a large
extent with the use of biomass as an energy source instead of traditional one. Oil and natu-
ral gas are not infinite assets, definitely they all have end, and we cannot imagine the situ-
ation without these primary sources of fossil fuel until we have develop a backup plan. In
these contexts, very high emphasis is needed to explore new sources of energy [5].
India generates approximately 450–500 million tons of biomass every year, and 32% of all
the primary energy use in the country comes from biomass [6]. The composition of bio-
mass has large diversity, for example, it may compose of cellulose, hemicellulose and

346 17 Plant Biomass to Bioenergy
lignin; such variations are responsible for different chemical properties of biomass. Besides
biofuel, biomass can also be utilized in the production of electricity and heat [7].
The Government of India has shown an enthusiastic interest toward renewable energy
and aims to initiate a project regarding 175 GW renewable power installed capacity by the
end of 2022. India is a country of huge biodiversity and considered as one of the biggest
economies with a growing population. The demand for electricity is rapidly growing year
by year due to more development in industrial sectors, increase in population, more urban-
ization, economic growth, and more electrification in rural areas. According to the Ministry
of New and Renewable Energy (“MNRE”), more than 70% of the population of rural India
directly or indirectly depends upon biomass-based energy, and it is very fortunate that bio-
energy is a well-known form of energy in rural India due to easy availability of agricultural
residues such as straw, etc. [8].
According to an estimate, 82% of total crude oil imports are needed to feed the domestic
consumption demand; it exhibits how India is depended and affected by the unpredicted
price hike in International market. Hence, it is very sensible and necessary to use and
divert our potential in the production of bioenergy [8].
On the other side, continuous increase in world’s population evokes us to explore new
form of energy rather than depend on old sources and strengthen our country as
“Aatmnirbhar Bharat.” Data also suggested world population projected to increase further
to nine billion by 2050 [9]. The MNRE ministry has started a project to reinforce the bio-
mass-based industries up to year 2020 [10].
India generates 150 000 tons of municipal solid waste per day, use of this waste as bio-
mass in power generation is the demand of time, and it can solve two major issues of the
country: one is alternative power supply and other is waste management [11]. So, consider-
ing all these studies, we write this chapter and discussed about plant biomass, bioenergy,
its conversion, and some other related major issues.
17.2 Plant Biomass
Plant biomass is considered as a renewable source of energy, basically it is an organic mate-

rial that comes from plants. Biomass is used for various purposes as in electricity genera-
tion, in heat production, and in biofuel production. Biomass belongs to many categories
such as wood, forest residue, garbage, and agricultural waste, etc.
It accommodates stored energy from the sun from the process known as photosynthesis,
and the chemical energy is released as heat after burning of biomass. Biomass can be
burned through two ways either directly (wood and wood processing wastes burned to
produce heat and to generate electricity) or indirectly (agricultural crops and waste materi-
als burned as a fuel); in latter case, it is converted into liquid biofuels [12].
Several types of biomass, such as corn, wood chips, and garbage, are used in the produc-
tion of electricity. Biomass can be converted into liquid fuels called biofuels that can be
helpful for cars, trucks, and tractors. Leftover food products such as vegetable oils can cre-
ate biodiesel, while corn, sugarcane, and other plants can be fermented to produce
ethanol [13].
17.3 Bioenerg 347
Plant biomass is a feedstock to generate sustainable fuels and that fuels have potential to
replace petroleum products. Biological fermentation of plant biomass is an assured method
for liquid fuel production, but there are many challenges to overcome. Bioconversion of
lignocellulose requires an appropriate deconstruction of the plant biomass and transforma-
tion of sugar into the desire fermentation products.
Thermal and chemical treatment of biomass and addition of enzymes for the hydrolysis
raises the process economic costs. In this respect, CBP (consolidated bioprocessing) is
emerging as promising transformative options, it combines all biological steps into one
unit, and enzyme synthesis, hydrolysis, and fermentation steps are occurring in a single
reactor. CBP is a cheaper and valuable option for cellulosic biofuel production. Clostridium
thermocellum is well known for its role in degradation and utilization of cellulose, and it is
also used as in CBP as biocatalyst for the synthesis of cellulosic ethanol [14].
Plants efficiently convert photons into electrons, in this regard new solar panels are
developed with 30–36% efficiency. According to PV Magazine, current solar panels are
more efficient in comparison to previous one. The electrons from the plants should be cap-
tured before its conversion into sugar. Once the electricity is captured from the plant, the
electricity can be used for many purposes. Some electrical engineers at University of
Washington designed a circuit that can convert the natural energy into valuable electric-
ity [15]. Earlier in 2007, another Dutch research group at Wageningen University patented
the process of collecting plant power; today, a Netherlands-based company named Plant-e
holds this patent [16].
17.2.1 Types of Biomass (Source: [17])

●● Wood from natural forests and woodlands
●● Forest plants
●● Forest residues
●● Agricultural waste, for example, stover, straw, green agricultural wastes, and cane trash
●● Agro-industrial wastes such as sugarcane bagasse and rice husk
●● Industrial waste material, such as black liquor from paper manufacturing
●● Sewage
●● Food processing wastes
●● Municipal solid wastes
17.3 Bioenergy
Bioenergy holds a remarkable position in the energy economy share about 9.5% of total
primary energy supply and 70% of total renewable energy [18]. Usually bioenergy gener-
ates heat in buildings and industry, but it is expected that bioenergy would be involved in
the 3% of electricity production and approximately 4% of transport energy till 2023 [19].
Extension of bioenergy relies on agricultural policies worldwide, there is a continuous
increase observed in contribution of bioenergy of 40–55 EJ/year. A hike is estimated from
200 to 400 EJ during this century, and it makes biomass a significant option for energy
supply rather than mineral oil [20]. There should be an awareness program regarding
utility of biomass in power generation and in other sectors.
The concept of biomass energy is very simple and understandable. Plants take carbon
dioxide from the air and perform photosynthesis; when plant dies, much of the carbon is
again released into the atmosphere as carbon dioxide. According to Samuel Stevenson, a
policy analyst at the Renewable Energy Association in London, “When we use biomass as
an energy source, we are intercepting this carbon cycle, using that stored energy produc-
tively rather than it just being released into nature [21].”
Energy obtained from biomass has some advantages as it is free of fluctuation and does
not require storage. Although biomass-generated energy has some challenges as biomass is
not available throughout the year. So, there is a need to store biomass beyond the harvest-
ing period [11].
Bioenergy accounts for 13–14% of the total energy consumption; presently among all
renewable energy sources, bioenergy is the largest energy source and accounts for approxi-
mately second/third of the renewable energy mix. Bioenergy is a complex energy network,
a range of steps, and various feedstock involved in the generation of energy from the
biomass [22].
On the global platform, forestry sector is the biggest contributor to the bioenergy mix.
Forestry products such as charcoal, pellets, fuelwood, and wood chips are responsible for
more than 85% of biomass, which is used for energy purposes. Wood fuel (1.9 billion m3) is
the primary products, i.e. used for bioenergy production; mainly wood fuel is used for tra-
ditional cooking and heating in countries such as Asia and Africa [23].
17.4 Biomass Conversion into Bioenergy
There are some challenges to overcome when we want to shift from fossil fuel to biomass
fuels. The heavy solid biomass has to be converted into liquid and gaseous fuels to make
transportation convenient, and it can be done through two processes: one is biochemical
and another one is thermochemical [24].
In regard to biomass conversion into bioenergy, combustion, pyrolysis, and anaerobic
digestion are well-established technologies and already used at commercial level but lique-
faction, gasification, hydrolysis, fermentation, and fractionation are in queue for future
implementation [25]. Mainly three types of energy are generated through conversion of
biomass, i.e. (i) thermal energy, (ii) electrical energy, (iii) transportation fuel energy.
Thermal energy is released by the transformation of woody biomass, it provides heat,
which is needed for cooking. Steam generated in combustion can be used for domestic as
well as industrial processes and can also be used in the production of electricity and
donated as electrical energy. Synthesis gas and biogas forms in other conversion technolo-
gies such as pyrolysis, gasification, and anaerobic digestion are also suitable for electricity
production. Transportation fuel is the energy obtained in self-propulsion motors from bio-
fuels. These are also known as “second generation biofuels” and originated from lignocel-
lulosic type of biomass. These biofuels are obtained through thermochemical and
biochemical conversion processes. Cultivation of appropriate “energy crops” especially for
bioenergy is actually the need of the hour [25].
17.5 The Concept of Biomass Energy (Source: [27]) 349
Second-generation biofuels are different from first-generation biofuels in their origin.

These are generated through lignocellulosic materials such as jatropha, cassava, switch-
grass, wood, and straw and some other biomass residues. First-generation biofuels are that
derived from edible food crops, i.e. sugarcane, wheat, barley, corn, potato, soybean, sun-
flower, and coconut [26].
17.4.1 Cogeneration
Cogeneration is a term used for simultaneous production of more than one form of energy
via using one type of fuel. Cogeneration has more potential growth in comparison to gen-
eration of energy alone because it produces both heat and electricity.
17.5 The Concept of Biomass Energy (Source: [27])
Chemically biomass is composed of carbon and hydrogen elements, and in the transforma-
tion of biomass to bioenergy, the bound energy of these compounds is released. The energy
can be release through two main kinds of conversion.
17.5.1 Thermochemical Conversion

This type of conversion is performed at high temperature; bond breaking and reforming of
organic matter take place into biochar (solid), synthesis gas, and highly oxygenated bio-oil
(liquid). Thermochemical conversion is divided into three subtypes. These are gasification,
pyrolysis, and liquefaction. Mainly three factors decide which type of conversion is going
to happen, i.e. nature, quantity of biomass, and the preferred type of energy. It was reported
that thermal conversion has some advantages as it can produce energy from plastics,
requires short processing time and reduced water usage, etc. Besides, thermochemical con-
version is independent of environmental conditions [26].
17.5.1.1 Direct Combustion

It is the simplest and very economical; energy is released by burning the material through
direct heat.
17.5.1.2 Pyrolysis
Pyrolysis is the temperature-based degradation of biomass by heat but in the absence of
oxygen. Biomass is heated to a temperature between 400 and 750 °C, and end products are
gas, fuel oil, and charcoal.
17.5.1.3 Gasification
Biomass is used in the production of methane through heating or anaerobic digestion.
Syngas, a mixture of carbon monoxide and hydrogen, can be derived from biomass.
Methane is the major constituent of natural gas, and decomposition of organic waste gen-
erates gas; this gas contains approximately 50% methane.
17.5.2 Biochemical Conversion

Biochemical conversion involves yeast or specialized bacteria to convert biomass or waste
into beneficial energy. The subtypes are anaerobic digestion, alcoholic fermentation, and
photobiological techniques, which produce different biofuels [26].
17.5.2.1 Anaerobic Digestion

Biomass such as manure, waterwaste (sewage), and food processing waste are mixed with
water and added into a digester tank without air. This kind of digestion converts organic
matter to a mixture of methane. Landfill gas is produced by anaerobic digestion of buried
garbage in landfills.
17.5.2.2 Alcohol Fermentation

Feedstocks such as barley, wheat, potatoes, and waste paper, sawdust, and straw containing
sugar, starch, or cellulose are used in fermentation. Alcohol is produced by converting
starch into sugar, and then fermentation of sugar produces alcohol after that alcohol and
water mixture are separated through distillation.
17.5.2.3 Hydrogen Production from Biomass

A serious issue is associated with biomass utilization, i.e. its low efficiency of utilizing bio-
mass. In this regard, conversion of biomass into hydrogen is a very well approach, and
hydrogen production plays a very significant role in the development of hydrogen economy.
Thermochemical process such as gasification or pyrolysis and biological process such as
fermentation or biophotolysis can be practically applied to produce hydrogen [27].
17.6 Use of Biofuel in Transportation
Involvement of biofuel in transport is exceeded day by day and reached up to 6% on a yearly

basis in 2019, and 3% annual production growth is expected over the next five years. There
is a strong need of policymaking and remodeling in this sector to reduce the cost, and it will
enhance the biofuel consumption and initiate its use in aviation and marine transport. In
2019, electricity generation from biomass increased by over 5%, and recent positive policy
and market developments in emerging economies indicate an optimistic outlook for bioen-
ergy, supporting its “on track” status [28].
17.7 Production of Biogas and Biomethane from Biomass
Biogas is a combination of methane, CO2, and small quantities of other gases, and it can be
used to generate power and fulfill heating and cooking demand. Biogas and biomethane
can originate from a variety of feedstocks/biomass such as crop residues, animal manure,
municipal solid waste, wastewater, but these two are dissimilar products and used in sepa-
rate activities. Biomethane can be directly produced through gasification of forestry resi-
dues. Biogas provides a sustainable way to supply community energy demands, especially
in area that requires more electricity or the area where power supply through national grids
17.8 Generation of Biofue 351
is not easily reachable. Biomethane can be prepared from biogas via removing the CO2 and
other impurities. Replacement of solid biomass with biogas for cooking purposes is improv-
ing health in developing countries. According to some reports, biogas provides a way
toward clean cooking to 200 million people by 2040 [29].
17.8 Generation of Biofuel
As present Indian government is giving more emphasis to make in India program, biofuel
from biomass is gelling very well in it, and it also gives right direction toward some more
government schemes such as Swachh Bharat Abhiyan, skill development, doubling of
farmer’s income, employment generation, etc.
Recently, some national policies on biofuels are finalized and approved by the Union
Cabinet; hopefully these policies will provide a massive force to the small growing bioen-
ergy sector in India. A policy about usage and production of ethanol from damaged farm
products and food grains has cleared the way to utilize the agricultural waste for the gen-
eration of bio-power. This policy also has provision to convert waste such as plastic as well
as municipal solid waste to fuel. It will certainly help India to achieve the target of 10 GW
of biomass power by 2022. The policies provide a practical and acknowledgeable support
for the bioenergy sector in India [11].
Production of plant-based fuels definitely decreases our dependence on fossil fuels.
Besides, many other advantages are also associated with flora fuels as these fuels release
very less quantity of greenhouse gases such as carbon dioxide, etc. Biofuels are sustainable,
and energy companies mix them with gasoline. In contrast to coal, oil, or natural gases,
biofuels are renewable at some levels.
Biofuels are divided into two major headings, bioalcohol and biodiesel. The bioalcohol as
ethanol is made from corn and some other plants because of breakdown of starch via yeast
and bacteria. On the other hand, biodiesel is manufactured in refineries by using the oil
crops such as soybeans. Vegetable oils are treated with alcohol for conversion into biodiesel
[30]. According to the study, any plant material can be converted to biofuel, as waste prod-
ucts such as sawdust corn stalks, and corn kernels. Research is continuously going on to
explore plants that can produce fuel with very less requirement of marginal land and little
or no irrigation or no need of fertilizer. Some invasive species can also fulfill the need as
feedstock for biofuel plants [31].
17.8.1 Bioethanol
Bioethanol can be produced by two ways: one is through sugar fermentation process and
another one is by chemical process through the reaction of ethylene with steam. Corn,
wheat, maize, willow, waste straw, and popular trees, reed canary grass, sawdust, cord
grasses, artichoke, Jerusalem sorghum plants, and Miscanthus are mainly use for ethanol
production. Ethanol is biodegradable, causes lesser toxicity and pollution, and it is a high
octane fuel. Bioethanol has huge importance over conventional fuels. This fuel is manufac-
tured from the renewable sources such as plants, by creating low pollution, it will be help-
ful in improving the air quality. Rural economy also gets benefited due to more need of
necessary crops for bioethanol production.
Plant cell wall is made up of cellulose, hemicellulose, and lignin, these are complicated
mixture of carbohydrate and create problem during burning of biomass. Ethanol is pro-
duced through the hydrolysis and sugar fermentation of biomass. Biomass is treated with
acids or enzymes, and it degrades the polymers into monomers. The cellulose and the
hemicellulose are broken down into sugar and fermented into ethanol. The lignin is usu-
ally used as a fuel for the ethanol production [30].
17.8.2 Biodiesel
Biodiesel is manufactured through chemical reaction by the reaction of vegetable oil with
an alcohol such as methanol or ethanol. The chemical reaction that produces biodiesel is
called “transesterification.” Oils and fats are categorized into the ester family. Biodiesel can
be made from any usual source such as canola oil, soybean oil, pork lard, beef tallow, and
also from some exotic oils, for example, walnut oil or avocado oil. Although waste/used oil
can also be used to make biodiesel, but due to the presence of some contaminants, these
oils should be filtered before use in reaction.
Ethanol is quite expensive; that’s why methanol is the ultimate choice for biodiesel pro-
duction although it is very toxic in comparison to ethanol. Quality masseurs for biodiesel
are determined by the American Society for Testing and Materials (ASTM) specification
D6751. Fuel testing and quality check are expensive, but there is no alternative, and it is the
most reliable method to supply the best-quality product to consumers [31].
A very interesting study was conducted by Mao et al. [32]; they make bibliometric analy-
sis by collecting data from 9514 studies with the help of searching key words “Biomass
energy” and “Environment.” Data was collected from the year 1998 to 2017, and many
fascinating topics on biomass utilization and bioenergy production were covered in this
analytical paper. List of some important biofuel crops and next generation biofuel crops are
given in the Tables 17.1 and 17.2 [34] and [35].
17.9 Advanced Technologies in the Area of Bioenergy
Various research proposals are funded by the several agencies in public as well as in private
sectors to promote, establish, and advance renewable energy, especially bioenergy. Among
them, some of the advanced biomass-related technologies are named here. Many of them
are already used at commercial level and some are ready to be commercialized [33].
●● Advanced plantation systems for some unexplored or newly explored plants.
●● Development of advanced biorefinery system for uninterrupted production of products
such as biodiesel.
●● Modification in anaerobic treatment methods to induce higher destruction of pathogens
in wastewaters to achieve more biogas yields along with high production rates.
●● Development of close-coupled biomass gasification–combustion systems to generate hot
water and steam for the utilization in commercial buildings and schools.
●● Strengthen the biomass gasification processes to meet the high-efficiency production of
medium-energy content fuel gas and power.
17.10 Conclusio 353
●● Use of genetically engineered and efficient microbes, which can convert pentose and
hexose sugars from cellulosic biomass and further convert it into ethanol through
fermentation.
●● Use of zero-emissions waste biomass-combustion systems for joint association of dis-
posal-energy recovery and recycling.
●● Selection of catalysts for thermochemical conversion especially for gasification of bio-
mass to produce gases at higher yield.
●● In case of pyrolysis process, emphasis is given to short residence time to produce chemi-
cals and liquid fuels from appropriate biomass.
17.10 Conclusion
The present chapter aims to collect the information about plant biomass, bioenergy, and its
conversion. We have concluded from this chapter how biomass-based energy is needed at
present time, it can deal with several issues at a single time as pollution, waste manage-
ment, oil crisis, etc. It can also generate employment and strengthen the economy as well.
Biomass holds significant potential to overcome the current energy demand and have
strong ability to compensate the excessive dependence on fossil fuels. Due to continuous
depletion in fossil fuel resources, biomass-based energy along with other biotechnological
approaches would become focus of future research.
Table 17.1 Important biofuel crops.
S. no Plant Uses
1 Corn Corn is the major crop in the world, used for ethanol-based biofuels production.
2 Rapeseed/ It’s an important biodiesel crop, Rapeseed oil has been used in cooking. Canola
Canola gains special importance due to presence of low erucic acid content, and it makes
it healthier for animals as well as humans to eat.
3 Sugarcane Sugarcane is also used in bioethanol production, and it is six times cheaper than
corn. It is exclusively used in Brazil.
4 Palm oil Palm oil is obtained from the fruit of palm trees, and it is among the energy-
efficient biodiesel fuels. Palm-oil-based biodiesel causes lesser pollution in
comparison to gasoline. Palm oil is helpful in boosting the economies of Malaysia
and Indonesia.
5 Jatropha This crop is a master player in the biofuel market. Presently, India is the world’s
largest Jatropha producer. The main benefit associated with this crop is
requirement of very normal agricultural land, and in this way it will be helpful for
rural farmers. Jatropha plants can also live on land that suffered from the drought
and pests for approximately up to 50 years. Oil is extracted from the seeds
although seed cases and vegetable matter are also good source of biomass fuel.
6 Soybeans Soybeans exclusively used for biodiesel production in the United States. Motor
vehicles, buses, and heavy equipment can run on pure soybean-based biodiesel
or, blending is also done with more traditional diesel fuels. According to The
National Academy of Sciences, soybean diesel yields more energy than corn
ethanol.
Source: Ref. [34].

Table 17.2 Next-generation biofuel crops.
S. no. Name of plant Uses
1 Hemp Hemp plant can produce oil equivalent to soybean.

2 Switchgrass This plant has potential to replace corn as a feedstock for ethanol
production.
3 Carrizo Cane It is native in Europe and utilized at the commercial scale in Europe
and produces almost highest biomass per acre than any other plant.
4 Algae Algae produce up to 200 times more oil per acre in comparison to
soybean. Several world largest companies have invested hundreds of
millions of dollars for scaling up the algal fuel production.
Source: Ref. [35].
Acknowledgment
Chapter manuscript no. CSIR-NBRI_MS/2021/01/04.
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357
18
Bioenergy Crops as an Alternate Energy Resource

Garima Pathak1 and Shivanand Suresh Dudhagi2
1
B.D. College – A Constituent Unit of Patiliputra University, Patna, India
2
CSIR-National Botanical Research Institute, Lucknow, India
18.1 Introduction
A significant challenge in the twenty-first century is to provide adequate energy to the ris-
ing world population, which needs more and more energy per person. Today, this energy
supply is based predominantly on fossil energy carriers which adversely affect the climate
and degrade the natural resources by emitting greenhouses gases, carbon dioxide, and
nitrogen oxide. Nuclear fission-generated electricity requires huge infrastructure and has
adverse effects on the environment and human health [1]. The use of fossil fuels is linked
to long-term environmental impacts, which can contribute to land degradation and fertile
soil desertification [1]. For instance, coal emits greenhouse gasses such as carbon dioxide,
particulate soot, and sulfur-containing compounds which lead to soil acidification. Now
concern is rising about how such rising demands can continue to be met by finite and
slowly depleting resources. There is definitely a need for renewable energy sources. Several
countries changed their energy fulfillment goals from non-renewables to renewables.
There are only few energy sources which are renewable and having lesser impact on the
environmental. One such potential alternative with long-term positive future results is the
use of “bioenergy crops” for generating energy [2]. Energy from bioenergy crops is drawn
from plant and animal biomass [3]. Bioenergy crops reduce carbon dioxide, reduce green-
house gas emissions, increase soil carbon, reduce soil erosion, increase transpiration, and
provide heat and electricity [4–6]. The bioenergy crops also help in remediation of heavy
metals from the contaminated soil [7]. Large-scale bioenergy cultivation could also have a
positive impact on the wildlife. Bioenergy concept is very popular mango scientific com-
munity because of its renewability and eco-friendly nature. However, the worldwide mar-
ket has more conventional use of bioenergy crops as food, which raises food safety issues
for energy use. Bioenergy plants also compete with food crops for agricultural land, water
resources, and nutrient needs. Another negative impact linked to the use of bioenergy

358 18 Bioenergy Crops as an Alternate Energy Resource
crops involves the destruction of wildlife habitats and increased dispersion of invasive
plant species [8]. Different types of bioenergy crops and their features are described in this
chapter.
18.2 Classification of Bioenergy Crops
The concept of “traditional biofuel” was introduced to overcome the environmental and
associated issues. Traditional biofuels were derived from cultivated vegetables. Their use in
bioenergy is debatable because of issues relating to food security. Bioenergy crops are
screened based on specific characteristics such as oil yields, oil quality, and mitigation of
global climate change. Traditional bioenergy crops could improve the production of food
and fodder, with the additional advantage of mitigating global climate change [9]. Bioenergy
crops are mainly classified into five groups namely, first-generation, second-generation,
and third-generation, energy crops dedicated, and halophytes (Figure 18.1), and compo-
nents of various types of bioenergy crops are listed in Table 18.1.
18.2.1 First-Generation Bioenergy Crops

Biofuel generation programs have been initiated with the first-generation bioenergy crops
(FGECs). These crops are also a common local or global food source. FGECs such as sweet
sorghum, corn, sugarcane, oil palm, and rapeseed were initially used for biofuel prepara-
tion [10]. However, bioenergy crops of first generation have limited capacity to replace petrol
oil products due to higher production costs [11–14]. Such limitations were overcome by the
use of lingo-cellulosic materials from crop residues in the fuel extraction phase in the second-
generation bioenergy processing model [15]. Some common FGECs are discussed below.
First-generation
bioenergy crops
Second-
Halophytes generation
bioenergy crops
Bioenergy
crops
Third-
Dedicated
generation
bioenergy crops
bioenergy crops
Figure 18.1 Types of bioenergy crops.

18.2 Classification of Bioenergy Crop 359
Table 18.1 Constituents of various types of bioenergy crops.
First-generation Sweet sorghum, corn, sugarcane, oilseed rape, linseed, field mustard, hemp,
bioenergy crop sunflower, safflower, castor oil, olive, palm, coconut, and groundnut
Second- Switchgrass, reed canary grass, alfalfa, Napier grass, and Bermuda grass
generation
bioenergy crop
Third- Boreal plants, crassulacean acid metabolism (CAM) plants, eucalyptus, and
generation microalgae
bioenergy crop
Dedicated Cellulosic plants (eucalyptus, poplar, willow, birch, etc.), perennial grasses
bioenergy crop (giant reed, reed canary grass, switchgrass, elephant grass, etc.), non-edible
oil crops (castorbean, physic nut, oil radish, pongamia, etc.), and oil plants
(Jatropha curcas, Pistacia chinensis, Sapium sebiferum and Vernicia fordii)
Halophytes Acacia, Eucalyptus, Casuarina, Melaleuca, Prosopis, Rhizophora, and Tamarix
18.2.1.1 Sugarcane
Sugarcane (Saccharum officinarum L.) is the largest sugar-producing plant perennial plant
that grows year-round and adapted to warm temperate or tropical climates. Therefore, sug-
arcane feedstock remains available year-round at comparatively lower costs compared to
other bioenergy crops [3]. Sugarcane is primarily produced to obtain sugar. The sugarcane
juice contains a high proportion of sucrose, which is a substratum for biofuel. Several
breeding projects are underway to boost the germplasm of the sugarcane to increase the
production of sucrose and cellulosic biomass. Commercial bioethanol is derived from the
molasses, a sugar industry by-product.
18.2.1.2 Corn
Corn (Zea mays) is an effective crop of feed due to high grain yield and better accumulation
of starch in grains [16]. Corn has high content of volatile compounds and simple process-
ing method makes it a preferred crop for bioconversion. Corn is used in the manufacture of
ethanol in the United States and elsewhere. The key drawback of maize feedstock, how-
ever, is its predominant use in many countries as a staple food. The use of corn in the pro-
duction of bioenergy fuel could increase food prices worldwide, contributing to hunger and
famine. In order to overcome this issue, sweet corn variety was produced in the corn kernel
endosperm by spontaneous recessive mutations in genes that regulate conversion of sugars
to starch. Using dual-purpose and photosynthetically efficient sweet corn hybrids could
benefit farmers by contributing to energy generation without affecting the environment
and food supply [17, 18].
18.2.1.3 Sweet Sorghum

There are several varieties of sweet sorghum (Sorghum bicolour L.) which have high con-
tent of sugar. Sweet sorghum accumulates a significant quantity of fermented sugar in
stems to produce greater biomass. The plant needs less fertilizer and therefore is easily
grown on marginal lands. Agronomic characteristics of sorghum include high resistance to
drought and C4 photosynthesis. Limited research attempts have been made to classify the
genetic and molecular features of sorghum compared to crops such as maize and sugar-
cane. Sweet sorghum is a used as model bioenergy crop for understanding the complex
genomes of other bioenergy crops such as corn, sugarcane, miscanthus, and switch-
grass [19]. Sweet sorghum comprises high amounts of sugar in stems, and hence higher
activity of sugar metabolic enzymes detected during stem development [20]. The sorghum
crop has good nitrogen use efficiency and accumulates higher amounts of sugar in the stem
during drought [21, 22]. Crops of sorghum and sweet sorghum could be cross-breed to
improve crop productivity, and the desirable attributes could be detected by genetic map-
ping [23, 24].
18.2.1.4 Oil Crops

Oil crops consists of oilseed rape, linseed, field mustard, hemp, sunflower, safflower, castor
oil, olive, palm, coconut, and groundnuts. Vegetable oils could be refined to generate biofu-
els for transport or used directly as fuel for heating [25].
18.2.2 Second-Generation Bioenergy Crops

Second-generation bioenergy crops (SGECs) include perennial forage crops (switchgrass,
reed canary grass, alfalfa, Napier grass, and Bermuda grass) [26, 27]. The second-generation
bioenergy generation takes the more pragmatic approach of using crop remains as feed-
stock. SGECs are more efficient than FGECs, as SGECs utilized cellulosic biomass for
biofuel generation and the biofuel is non-oxygenated and in pure form [27]. SGECs reduce
many environmental problems and require very less cost of production of biofuels. Biofuels
from SGECs are thermo-chemically or biochemically derived from lingo-cellulosic crop
wastes [14, 28]. Second-generation biofuel’s main components are annual grain crops and
annual biomass crops [4]. The SGECs require less processing and generate high energy
with reduced emissions of greenhouse gases compared to FGEC. Growing SGECs generate
important biomass for bioenergy generation [29]. The sugarcane industry has an enormous
potential as a SGEC because the remains of sugarcane stalks (bagasse) are currently being
burned in sugarcane factories to produce steam and electricity. Bagasse is mainly com-
posed of cellulose, which mainly contained a linear chain of thousands of β(1 → 4)-linked
d-glucose units. Upon bacterial fermentation, bagasse releases cellulose residues that
could be used to produce bioenergy using the latest technologies [30]. However, the second-
generation ethanol produced from sugarcane has not yet been commercialized due to
the lower rate of bagasse to sugar conversion. But certain countries like Brazil have met
their energy requirements for bioethanol produced from sugarcane. The following are
some of the principal bioenergy crops of the second generation.
18.2.2.1 Switchgrass
Switchgrass (Panicum virgatum L.) is a warm-season, perennial, C4 grass which is grown
on marginal and erosive lands. This crop needs less nutrients and water for growth, making
it an environmentally friendly crop for large-scale production of biofuels [31, 32].
Switchgrass, however, has a slow setting time that takes about two years for complete
encroachment [31]. This plant has not grabbed much attention from researchers, particu-
larly in the plant breeding sector [33]. Consequently, the germplasm of most switchgrass
cultivars is not far from the native genomes. Depending on genetic composition, few types
of switchgrass from natural populations are non-differentiable. Switchgrass, therefore,
holds immense potential for genetic advancement in effective development of biomass.
18.2.2.2 Miscanthus
The genus Miscanthus contains 14–20 species of tall, perennial grasses, native to Asia, grown
as ornamental plants [34]. The morphology of the plant constrains its use as crop forage. The
plant is the main feedstock of herbaceous biomass in Europe. The Miscanthus plant per-
forms C4 photosynthesis, has a high fixation rate of carbon dioxide, and requires less water
and nitrogen than the C3 plants [35]. This grass is considered a dedicated energy crop due to
its rapid growth, disease resistance, high productivity, and 10–15 years of comparatively
longer plant life [36]. Miscanthus biomass yield was 33% higher than switchgrass [37]. One
prime example of the genus Miscanthus is M. Giantiseus L. This needs 87% less land to gen-
erate equivalent biomass to prairie species [34]. The disadvantage of growing Miscanthus
crops includes longer propagation duration of two to three years for rhizome cuttings, exces-
sive irrigation, and energy consumption during greenhouse propagation.
18.2.2.3 Alfalfa
Alfalfa (Medicago sativa L.) is North America’s oldest forage crop [38]. The Alfalfa stems are
fibrous and burnt for electricity production in the gasification cycle. The leaves are enriched
with protein [39]. The plant is a feedstock for the production of biofuels and also an excellent
feed for animals [40]. Alfalfa has higher concentrations of polysaccharide and lignin in stem
cell walls which lead to higher yields of stem dry matter and theoretical yields of ethanol [41].
18.2.2.4 Reed Canary Grass

Reed canary grass (Phalaris arundinacea L.) is a North American C3 grass. It is tall-growing
perennial grass that is efficient in the recycling of internal nitrogen from shoots to
roots. Several characteristics of canary reed grass are common to switchgrass such as slow
growth and low yield. It is a native species of wetlands [42]. The grass yields relatively
higher biomass and therefore could produce enough biofuel [43].
18.2.2.5 Other Plants

Because of the related benefits, several other plants often contribute to bioenergy. For exam-
ple, a tall, perennial, and tropical grass, called Napier grass (Pennisetum purpureum Schumach)
is preferred bioenergy crop due to the ease of establishment, persistent, and drought tolerance
ability. The grass is full of taste and nutritious [44]. Napier grass’s ability as a bioenergy crop
was recognized by its low-lignin content and higher biomass yield per acre [[45]]. The Napier
grass biomass has higher levels of volatile matter, carbon content, and lower levels of ash,
nitrogen, and sulfur [46]. Reportedly, Napier grass’s simultaneous saccharification and fer-
mentation (SSF) yielded 74.1% ethanol. Bermuda grass (Cynodon dactylon L.) is an another
plant used in bioenergy. It is a highly diverse perennial grass, short-lived, and mostly used as a
warm-season forage. Because of its innovative existence and resistance to salinity, Bermuda
grass acts as a soil binder in riverbanks or sea shore sand dams. It is a precious crop in irrigated
lands [47]. Other possible perennial grass feedstocks are Eastern gamagrass (Tripsacum dacty-
loides L.) and prairie cordgrass (Spartina pectinata Link) [48].
18.2.3 Third-Generation Bioenergy Crops

Third-generation bioenergy crops (TGECs) include boreal plant, CAM plants, eucalyptus,
and microalgas. CAM and boreal plants are the feedstock for direct cellulosic biomass fer-
mentation [49, 50]. Thermo-conversion process is used to generate bioenergy from euca-
lyptus [14, 51]. Some species of microalgae could be potential feedstock for biodiesel
production. The success of TGECs as a reliable source of biofuel depends on the efficient
metabolism of cellulolytic bacteria during the fuel conversion process. Cellulose is broken
down into water and carbon dioxide in the aerobic system. However, cellulose degrades to
CH4 and H2 in anaerobic systems. Newer methodologies such as genomics, biodiversity
studies, system biology, and metabolic engineering improve biofuel yields. TGECs are
being introduced to develop a renewable and non-polluting energy source that could
reduce global climate change [52–54].
18.2.3.1 Boreal Plants

Perennial grasses such as Phleum pratense and Phalaris arundinacea are examples of boreal
plant species. Under boreal conditions, perennial grass is a major producer of herbaceous
biomass. Boreal plants could be easily cultivated, harvested, stored, and used for the pro-
duction of CH4. Plants are tolerant of most phyto-pathogenic diseases, such as drought and
frost. Boreal plants can withstand cold winters and grow on low-nutrition soils [55]. Few
boreal plants such as Ananas comosus, Opuntia ficus-indica, Agave sisalana, and Agave
tequilana are commonly used for bioenergy production [56].
18.2.3.2 Crassulacean Acid Metabolism (CAM) Plants

Plants with CAM are well adapted to photosynthesis. These plants have potential to absorb
carbon dioxide at night. In arid habitats, CAM plants improve water efficiency and carbon
assimilation. CAM plants are drought tolerant and used as bioenergy crops [57]. The CAM
plants have three to six times higher water use efficiency than the C3 and C4 plants. CAM
plants such as cardoon are multifunctional bioenergy crops. These plants are used for the
production of solid and liquid biofuels [58, 59].
18.2.3.3 Eucalyptus
Eucalyptus (Eucalyptus sp.) is a native Australian plant. The plant grows faster with indefi-
nite growth and has a large genetic resource base. The plant is resistant to drought, fire,
insects, acid soils, low fertile soils, and other harsh conditions. Eucalyptus is cultivated in
tropical countries due to faster growth and higher yields (70 m3 ha−1 year−1). The rotation
period of the plant is as short as five years. Only four species and their hybrids (E. grandis,
E. urophylla, E. camaldulensis, and E. globulus) contribute 80% of lantations worldwide. Of
these four species, E. globulus is a widely adapted plant that is used in breeding programs
due to faster growth. Eucalyptus oil extracted by thermo-conversion from plant parts holds
enormous potential in the production of biofuel and bioenergy [14, 60].
18.2.3.4 Agave
Agave (Agave sp.) is a monocot plant native to hot, arid regions of Mexico. A plant species,
Agave tequilana, is used for the production of tequila. Agave nectar is used as an alternative
sugar for cooking. The plant grows in arid regions and has thick, fleshy leaves that end at a
sharp point. Agave uses the CAM path for photosynthesis. It opens up stomata for CO2
uptake during the night, causing less water loss during transpiration. The plant is used to
make alcoholic beverages, sweeteners, and fibers. Agave is preferred feedstock for biofuels
as it has minimal water requirements, is easily grown in wastelands, and does not compete
with food crop feedstocks [61].
18.2.3.5 Microalgae
Microalgae are an important feedstock for the production of biodiesel, bioethanol, bio-
methane, and bio-hydrogen [62]. Photosynthetically, they are more efficient than terres-
trial plants. Microalgae reduce greenhouse gas emissions by absorbing carbon dioxide
released by plants. They produce vast biomass in a short period of time through efficient
photosynthesis [50]. Microalgae minimize atmospheric carbon dioxide by sequestrating it.
Compared to conventional biofuel-producing crops, microalgae biofuels have less impact
on the environment and food supply in the world [49, 50, 63]. Microalgae have a very high
potential to mitigate global climate change [49] as they have an efficient conversion rate of
photons to photosynthates. In addition, they could be harvested all year round [64].
Microalgae provide nontoxic and biodegradable biofuels. Several programs are underway
to improve the rate of biofuel production by enhancing the efficiency of strains through
genetic engineering. Compared to other bioenergy crops, microalgae-derived fuel is consid-
ered greener due to higher conversion rates to biofuels.
18.2.4 Dedicated Bioenergy Crops

Examples of dedicated energy crops are perennial herbaceous and woody plant species. To
produce biomass, they require lesser biological, chemical, or physical treatments. Such
crops are considered environmentally friendly and would help mitigate global climate
change [28, 65]. Such crops may solve numerous environmental problems by reducing
salinity, carbon sequestration, enriching biodiversity, and improving the quality of soil and
water [53, 66]. Cellulosic plants (Eucalyptus, Poplar, Willow, Birch, etc.), perennial grasses
(giant reed, canary reed grass, switchgrass, elephant grass, etc.), non-edible oil crops (cas-
tor bean, physic nut, oil radish, pongamia, etc.), and oil plants (Jatropha curcas, Pistacia
chinensis, Sapium sebiferum and Vernicia fordii) are the devoted bioenergy crops. These
crops have a shorter life cycle and may therefore be harvested many times a year with long
harvesting periods [67, 68]. Short rotation coppice (SRC) is among the most promising
bioenergy-dedicated crop [69]. Countries such as Sweden and the United Kingdom are
leaders in the comprehensive cultivation of dedicated bioenergy crops [70].
18.2.5 Halophytes
Halophytes are unique plants growing in sandy, marshy, and semi-deserted soils. They also
inhabit coastal area, mangrove swamps, and estuaries [71]. The plants grow and propagate
better at higher concentrations of salt [72]. Halophytes help to sequestrate carbon and
rehabilitate degraded land, stabilizing habitats by providing the ecological niches required
to mitigate climate change. In addition, they protect the associated flora and fauna from the
environment and pathogens [73]. Frost-sensitive eucalyptus species and the frost-tolerant
populus species are the most likely to survive under saline conditions [60]. Halophytes are
easily established in salt-degraded soils and could also phyto-remediate soils contaminated
with heavy metals [74, 75]. Dicot halophytes have been shown to be more tolerant to saline
than monocots [76]. Halophytes may be used for food, medicine, and ornamental landscap-
ing purposes. They also protect the environment by promoting wildlife [75, 77]. Halophytes
of the genera Acacia, Eucalyptus, Casuarina, Melaleuca, Prosopis, Rhizophora, and Tamarix
are commonly used in the production of biofuels. Perennial halophyte (Kosteletzkya penta-
carpos) seeds have been shown to be used for the production of biodiesel [78]. Halophytes
have a high rate of biofuel conversion efficiency because of high percentage of secondary
metabolites [79].
18.3 Characteristics of Bioenergy Crops
Bioenergy crops can protect the environment in many ways [80]. Because of their perennial
nature, they are resistant to disease and pest [55]. Bioenergy plans have advanced pheno-
typic, architectural, biochemical, and physiological characteristics that are desirable for
biofuel production. In addition, cultivars of bioenergy crops are resistant to biotic and abi-
otic stresses that grows faster than other crops. Furthermore, crops with bioenergy need
fewer biological, chemical, or physical pretreatment, thereby reducing the costs involved in
processing biomass. New high-yielding energy crop varieties need to be introduced to meet
energy needs that could be accomplished through large screening of productive botanical
plants around the globe.
18.3.1 Physiological and Ecological Traits

Bioenergy plants store thermo-chemical and solar energy in a number of biochemical
forms. These plants need a variety of physiological and ecological features to maximize
radiation absorption, water efficiency, nutrient utilization, and environmental sustainabil-
ity [31, 67]. These physiological features include efficient nutrient cycling, low nutrient
requirement, carbon sequestration, low plant group competition, long canopy duration,
efficient C4 or CAM photosynthetic pathway, and effective light capture. All these physio-
logical features help plants to increase over-ground biomass during the growing season [66,
81]. Eco-physiological features of perennial short rotation coppice and lignocellulose grass
germplasm show great diversity [12, 82]. Bioenergy crops have vegetative storage organs to
store food reserves for longer periods of time. Vegetative storage structures are reported to
reduce environmental stress and minimize metabolic loss [14]. The ratio of carbon and
nitrogen is the deciding factor in generating bioenergy from plant biomass. Higher C:N
ratio of bioenergy crops yields more bioenergy in the form of methane [83].
18.3.2 Agronomic and Metabolic Traits

Bioenergy crops need low energy for establishment, are well suited to marginal lands, and
have higher biomass content. Such plants are reducing global warming and minimizing the
effects of global climate change. The bioenergy crop should carry features of long canopy
18.4 Genetic Improvement of Bioenergy Crop 365
length, perennial production, sterility, less dry matter to reproductive structures, and less
moisture content at harvest, according to agronomic characteristics. Miscanthus spp., a per-
ennial C4 grass, retains most of these agronomic characteristics [81, 84, 85]. The metabolic
architecture of the dedicated energy crop reduces “plant-to-plant” and “weed” competi-
tion. The plant metabolic improvement also decreases the interception of radiation,
increases the efficiency of water usage, and accelerates drying on the field. These plants are
simple, dense with upright stem branching, and waterlogging-resistant.
18.3.3 Biochemical Composition and Caloric Content

The biochemical composition of carbohydrates, proteins, lipids, and organic acids varies
between plants. Their use in the bioenergy field is based on biochemical composition being
unique. Bioenergy crops are a good source of energy, keep low cost of production, and
reduce greenhouse gas emissions [86]. In terms of calorific value, the plant bioenergy is
calculated, which is characterized as the expression of released heat value and energy con-
tent during the burning of material in air. In terms of calorific value, each type of bioenergy
plant has its own merits and demerits. For instance, more energy is obtained from poplar
plants than switchgrass and canary grass, whereas canary grass emits more greenhouse
gasses compared to switchgrass and hybrid poplar [87, 88]. Plant growth energy and crop
suitability issues are critical and related to bioenergy and food production [10]. Improving
the biochemical composition and structure of bioenergy crops increases its caloric value,
generating higher energy per ton of biomass [89]. Accumulated plant biomass is not pro-
portional to the energy absorbed during photosynthesis, as the magnitude of the accumu-
lated chemical forms varies in their energy densities. This difference depends on the species
and the plant’s stage of development. Carbohydrate generation is a valuable feature in bio-
energy crops. Carbon hydrates are used for the production of biofuel in the fermentation
process. Cellulose crops have more potential in the generation of bioenergy since their
degradation releases a large number of glucose units. Higher yields of biofuel from cellu-
losic crops correspond to lower greenhouse gas emissions per hectare and per unit of bio-
fuel produced compared to FGECs [12].
18.4 Genetic Improvement of Bioenergy Crops
Plants are commonly grown for food and feed. Traditional genetic modifications in breeding
techniques have helped to develop plant varieties with the desired morphological, pheno-
typic, and biochemical characteristics [90, 91]. The main focus of these efforts is on improv-
ing crop productivity and quality. In addition, food crops could be modified for bioenergy
generation by genotype alteration to produce more starch and a higher C:N ratio. Such mod-
ification could alter the pathway of lignin biosynthesis for better pre-processing via cellulose
and cellulose expression. Bioenergy crop characteristics can be improved by identifying
natural variations and genetic alterations in the production of transgenic plants [92, 93].
Genetically engineered bioenergy crops have greater adaptability to unfavorable conditions
and higher growth rates and caloric value. A high degree of similarity is present between the
grass genomes and poplar species. The transfer of gene function in such species into more
genetically recalcitrant grass plants, such as switchgrass, Miscanthus, and short rotation
coppice, could be encouraged. Because of easy propagation and faster growth in short rota-
tion coppice cycles with lesser fertilizer requirement, Willow was established as a promising
biomass crop. Willow plants need to be kept free of pests and diseases for better yield [94].
18.5 Environmental Impacts of Bioenergy Crops

Bioenergy crops provide numerous environmental and human benefits. The positive envi-
ronmental impacts of bioenergy crops can be measured through the review of the sustain-
ability indicators [95], risk–vulnerability–reliability assessment [96] and absolute impact
measurement or percentage change with baseline comparison [97, 98]. Different environ-
mental impacts of the production of bioenergy crops are shown in Figure 18.2 and well
described below.
18.5.1 Soil Quality

Prevalent cropping systems and crop characteristics affect soil quality by affecting nutrient
supply, availability of organic matter, soil structure, and pH. Miscanthus, switch-grass, and
other fiber crops, for example, are mild to nutrient requirements, while giant reeds and
Soil quality
Biodiversity Phytoremediation
Environmental
impact of
bioenergy crops
Water and Carbon

minerals sequestration
Figure 18.2 Impacts of bioenergy crops on environment.

18.5 Environmental Impacts of Bioenergy Crop 367
cardoons are heavily depleted. Supplementation of soil with proper nutrients is necessary to
ensure soil quality. In addition, nutrient supplementation needs to be carefully adjusted
with concentration. For example, sweet sorghum and potato crops require a relatively lower
concentration of phosphorus. Moderate concentrations of nitrogen and potassium are
needed by crops to prevent plant malnutrition. Lack of adequate nutrition decreases plant
biomass, and nutrient deficiency in the form of outward symptoms is evident. Sunflower,
giant reed, and cardoon exhibit deeper nitrogen deficits. Giant reed, cardoon, sugar beet,
sweet sorghum, canary grass, and wheat also reveal high deficiencies in potassium [99].
18.5.2 Water and Minerals

Cultivation of bioenergy crops could be water demanding to the point of compromising the
availability of natural water resources. The water requirements of the crop should therefore
be taken into account before planting bioenergy crops. The scarcity of water could hinder
the successful establishment of bioenergy crops as a biofuel resource. Careful selection of
bioenergy crops with tolerance to water stress is required in arid and semi-arid regions.
Some deep-rooted bioenergy crops are drought-tolerant and capable of efficient carbon
sequestration. However, such crops alter the dynamics of water and nutrients in soils to
negatively impact biodiversity [53]. Corn, sugarcane, and oil palm crops required plenty of
water for cultivation therefore well suited for growing in high-rainfall tropical areas [57].
Sugar beet, hemp, and potato also significantly affect water supplies [99]. Still, Miscanthus,
and Eucalyptus plants have a lower cumulative effect on water supplies. Bioenergy crops
have been known to influence minerals in the soil. The sorghum plant accumulates, for
example, Pb, Ni, and Cu in roots and shoots. Applying phosphorus and potassium to bioen-
ergy crop fields greatly decreases the loss of soil mineral content. Perennial crops are less
demanding for macronutrients, and their pattern of use of nutrients does not vary signifi-
cantly from annual crops. Eucalyptus and Willow plants impact mineral resources at lower
levels, while sweet sorghum and potato face higher risks of depletion of nutrients [99].
18.5.3 Carbon Sequestration

Carbon sequestration requires extracting CO2 from the environment through plant media-
tion. Bioenergy crops minimize atmospheric CO2 by means of a large accumulation of
biomass. The use of perennial crops could improve soil quality by increasing carbon
sequestration through high production of biomass and deep-rooted systems [100].
Bioenergy crops will now be used to sequester atmospheric CO2 and increase the produc-
tivity of biomass for generating bioenergy [101].
18.5.4 Phytoremediation
Phytoremediation means usage of plants for remediating polluted soil, sediments, and
groundwater by eliminating or degrading pollutants [102]. This technology is innovative
and cost-effective and maintains long-term applicability [103]. Bioenergy plant may
extract heavy metals from the soil to improve the quality of soil. The approach has an
added benefit of treating polluted site without digging [104, 105]. Phyto-stabilization and
phyto-extraction include the main phytoremediation methods used to remediate heavy

metal-contaminated property. Phyto-stabilization requires the use of growing root plants,
which reduces the bioavailability of stabilized metals in the substrate [106]. Phyto-
extraction involves the use of plants with the ability to accumulate heavy metals from
soils, sediments, and water. This way of handling metal-polluted land seems economically
feasible [107]. Phytoremediation is common to many plant genera. Effective phytoreme-
diation, however, requires the selection of appropriate plants. Plant selection depends on
its availability, adaptation to specific climate conditions, ability to extract heavy metals,
rate of production of biomass, and economic value [103]. A study on Sorghum bicolor for
the phytoremediation of heavy metals has shown that the plant is efficient in the absorp-
tion of metals due to high biomass. The plant accumulates a high concentration of metal
in its shoots. Sorghum plants were able to absorb metals such as Ni, Pb, and Zn effi-
ciently [108]. A major source of water pollution in agricultural land is widespread and
unrestricted use of fertilizer in the field. High levels of nitrate fertilizer are used in fields
to increase crop yields. The use of high nitrate fertilizers creates pollution of the surface
and groundwater by nitrates. Few bioenergy plants have the potential to remove soil or
water contaminants. Poplar plant is known to accumulate high nitrate levels from water
streams draining from agricultural lands [109]. This plant extracts nitrate from water bod-
ies and thereby decreases its concentration in polluted water [110]. Poplar is well suited
for growing in nitrate-rich soil through high- and low-affinity nitrate transporter pro-
teins [111]. Miscanthus crops are also used for phytoremediation [112, 113]. In phytoreme-
diation, the crop is favored because of its perennial existence, high productivity, better
growth rate, effective sequestration of CO2, higher efficiency in water use, and the ability
to protect soil erosion. However, the use of Miscanthus has been associated with the dis-
advantage of lower numbers of viable seeds for oil extraction [113, 114], making it unsuit-
able for biofuel extraction.
18.5.5 Biodiversity
Biodiversity defines the variety of species that exist on earth. This increases the efficiency
of the habitats, where each species contributes in its own way. Biodiversity preservation is
thus necessary for a healthy ecosystem. The biodiversity of nature is diminished by many
environmental factors, among which land conversions, deforestation, and grassland con-
versions contribute to great duration. Most such factors linked to the environment could be
regulated by growing crops with bioenergy. Bioenergy crops protect biodiversity by reduc-
ing emissions of greenhouse gases and mitigating global climate change [80]. Furthermore,
the blossoming period of biodiversity and other crops also increases the abundance and
diversity of birds or insects, particularly in the fields of sunflower [99, 115]. However,
growing annual crops reduces biodiversity due to short soil impacts and high growth
requirements. The development of biofuel-based lignocellulose systems that use a range of
feedstocks could increase the diversity of agricultural landscapes and increase arthropod-
mediated ecosystem services [116]. For example, perennial grasses with a high content of
lignocellulose reduce soil tillage and agrochemical usage, yield high above and below
ground biomass, favor soil microfauna, and provide shelter for invertebrates and birds [80,
117]. In contrast to perennial grasses, willow and poplar plants support more biodiversity
Reference 369
due to longer life cycles and habitat development for birds, vertebrates, and flora. The over-
all effect of these crops on biodiversity, however, may be marginal or not even posi-
tive [118–120]. Bioenergy plants such as eucalyptus do not support biodiversity because of
a more vigorous cultivation management.
18.6 Conclusion and Future Prospect

Plants expand by absorbing released CO2 during the combustion of biomass. No net CO2 is
produced by the use of crop biomass for energy generation, as the amount emitted during
use has been previously fixed during plant development. Using bioenergy crops for gener-
ating energy may help to make use of this alternative source of renewable energy.
Commercial development of bioenergy fuels could reduce our reliance on fossil fuel trans-
port using existing engine technology. Feedstock for bioenergy crops (cellulose or sugar,
starch plants) may play a major role in ethanol and biodiesel generation to improve the
rural economy, provide greater energy efficiency, and use environmentally degraded lands
in a sustainable way. A large-scale plantation of bioenergy crops may control these environ-
mental factors. Since bioenergy crops may alter the soil’s water and nutrient dynamics,
their pattern of water use should also be taken into account before field planting. A suitable
bioenergy crop should be recommended according to the land type.
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377
19
Marine Bioprospecting
Seaweeds for Industrial Molecules
Achintya Kumar Dolui1,2
1
Department of Lipid Science, CSIR-Central Food Technological Research Institute, Mysuru, Karnataka, India
2
Academy of Scientific and Innovative Research, Ghaziabad, Uttar Pradesh, India
19.1 Introduction
Seaweed is another term for marine algae, which is benthic in nature (since they reside on the
bottom of water bodies). They do not feature typical plant-like structures like root, stem, and
leaves and belong to the division of Thallophyta in the plant kingdom [1]. Seaweeds generally
lack a common ancestor and form a polyphyletic group as a result of their convergent
evolution. They usually refer to unicellular to multicellular and mostly macroscopic forms of
algae. Seaweeds are classified into three different groups, i.e. green algae (phylum: Chlorophyta,
classes: Bryopsidophyceae, Chlorophyceae, Dasycladophyceae, Prasinophyceae, and
Ulvophyceae, about 1200 species), brown algae (phylum: Ochrophyta, Classes: Phaeophyceae,
about 1750 species), and red algae (phylum: Rhodophyta, about 6000 species). This classifica-
tion is based on the types of pigment (composition) they possess, characteristics of their cell
walls, and the type of reserve polysaccharides [2, 3]. Sometimes tuft forming blue green algae
(Cyanobacteria) is also lumped together with the seaweeds. The presence of seawater (or at
least brackish water) is an absolute requirement for the seaweed to be physiologically func-
tional. Besides, the presence of sufficient light is needed to make the seaweeds photosyntheti-
cally active. From the ecological point of view, another prerequisite for seaweeds is a robust
attachment point. Thus, most of the seaweeds occupy the littoral zone, preferably or within
that zone on rocky shores than on sand or shingles. Some species of algae are living deepest
into the sea, where accessibility of sunlight is a major challenge. Seaweed like Sagassum is a
free-floating planktonic seaweeds (brown algae) thanks to their gas-filled sacs, which keep
them afloat in an acceptable depth. However, the free-floating seaweeds are subjected to major
challenges in the form of changing temperature, salinity, and periodic drying [2]. Considering
the plethora of benefits that seaweeds offer to humankind, the cultivation of seaweeds on a
commercial scale has been put into practice for biomass production. Unlike terrestrial agricul-
ture, there is no requirement for freshwater as well as arable land for its cultivation. Besides, it
does not require the application of fertilizer in most cases. From the industrial perspective,

378 19 Marine Bioprospecting
seaweed cultivation is an ever-expanding industry that is being exploited for the isolation of
pharmaceuticals, extraction of functional food ingredients, and the production of biofuels [4].
Globally, seaweeds cultivation is a lucrative industry which is dominated mainly by Asian
countries like China (47.9%) Indonesia (38.7%), the Philippines (4.7%), the Republic of Korea
(4.5%), Japan (1.3%), and Malaysia (0.7%) [5]. Seaweed is considered as superfoods and called
as sea vegetables, since it is one of the nutrient-dense food in the plant kingdom. Edible sea-
weeds are also a rich source of macronutrients and micronutrients, beyond the source of
essential nutrients, and also a reservoir of various health-promoting bioactive components.
Even though seaweeds make a huge contribution to the traditional human diets in oriental
countries, its inclusion into the Western diet was limited only to the coastal communities.
However, recently it has gained popularity in the Western population due to the impetus from
the health, food, and nutraceutical industry [6]. Since seaweeds are loaded with several health-
enhancing elements such as antioxidants, phycocolloids, carotenoids, soluble dietary fibers,
polyunsaturated fatty acids (PUFAs), phycobilins, polysaccharides, sterols, tocopherols, terpe-
nes, and phycocyanins, it has huge potential for the health supplement markets in the form of
value-added products or novel foods. Seaweeds can alleviate lifestyle-related disorders, such as
hyperglycemia, hypercholesterolemia, and hyperlipidemia [7]. Marine algae are also a rich
source of potential bioactive components of industrial interest. The list includes isolated poly-
saccharides (e.g. alginate, fucoidan), proteins (e.g. phycobiliproteins), polyphenols (e.g. phlo-
rotannins), carotenoids (e.g. fucoxanthin), and n-3 long-chain PUFAs (e.g. eicosapentaenoic
acid) [8]. Moreover, seaweeds produce a lot of secondary metabolites with substantial indus-
trial and therapeutic potential. These secondary metabolites exhibit potent biological activities
such as antimicrobial, antitumor, antidiabetic, anticoagulant, antioxidant, anti-inflammatory,
antiviral, antimalarial, anti-tubercular, anti-aging, antifouling, and antiprotozoal [9–11]. For
optimum exploitation of their full potential for human health and nutrition, there should be
well-established cultivation (aquaculture) practices along with good extraction protocol to
enrich and isolate the target bioactive compounds specifically. Also, the efficacy of the target
molecule for pharmaceutical and industrial applications should be validated with a proper
clinical assay, including a safety profile. Recent advancements in “omics” techniques (genom-
ics, metagenomics, and proteomics), along with molecular biology approaches such as combi-
natorial biosynthesis, synthetic biology, selection methods, expression systems, and
bioinformatics platform have also played a significant role and contributed substantially
toward the discovery of new drug leads with pharmaceutical significance from seaweeds [12,
13]. In this book chapter, I have discussed the various applications of seaweeds in human
health and nutrition (functional foods and nutraceuticals), industrial therapeutics and phar-
maceutical application, cosmeceuticals, and therapy to lifestyle-related disorders. I have also
highlighted various extraction techniques usually adopted for the isolation of seaweed com-
pounds. Finally, the market potential of seaweeds is also briefly discussed.
19.2 Seaweeds as Nutraceuticals and Functional Foods
Functional foods are a new category of food products that offer health-promoting extra-
nutritional benefits such as lowering the level of plasma cholesterol, antioxidant activities,
body weight regulation, and improved gut health. Since seaweeds are heterogeneous in
19.2 Seaweeds as Nutraceuticals and Functional Food 379
their composition, they have the potential to be used as functional foods. They have a
unique profile of bioavailable carbohydrates, protein content, soluble dietary fiber, and
higher levels of PUFA [14]. For example, phytosterol extracted from marine algae, espe-
cially from the brown algae, finds wider application as nutraceuticals since they are capa-
ble of lowering the cholesterol thereby reducing the risk of cardiovascular disease [15]. In
addition, there are pieces of evidence which provide the experimental proof of the potential
of seven phytosterols namely fucosterol, saringosterol, 24-hydroperoxy-24-vinyl-cholesterol,
29-hydroperoxy-stigmasta-5, 24(28)-dien-3β-ol, 24-methylene-cholesterol, 24-keto-
cholesterol, and 5α, 8α-epidioxyergosta-6, 22-dien-3β-ol from Sargassum fusiforme being
cholesterol-lowering agents in cell line study. The authors concluded that saringosterol was
more potent than other phytosterols in stimulating LXRα which in turn lower the level of
cholesterol [16]. In another study, it was reported that the lipid extracts of Nostoc commune
Vaucher seaweed lowered the expression level of genes that are involved in cholesterol
metabolism. The observed hypocholesterolemic effect was attributed to the campesterol,
sitosterol, and clionasterol of this alga [17]. However, increased phytosterol consumption is
reported to increase serum phytosterol levels. Thus, the consumption of phytosterol should
be avoided by phytosterolemic patients [18]. The United States has authorized the use of
oil, which is obtained from Schizochytium sp. as food ingredients since it is very high in
docosahexaenoic acid (22:6 ω–3), including very high levels of squalene and phytosterols.
It also has threefold less cholesterol than fish oil [14]. Although the lipid content in sea-
weeds is low, they have a unique fatty acid profile, which is different from terrestrial plants.
The predominant fatty acid present in seaweeds is ω–3 fatty acid [19]. The fatty acids usu-
ally have a linear chain with an even number of carbon atoms and one or two double
bonds. Red algae are rich in eicosapentaenoic acid (20:5 ω–3) arachidonic acid (20:4 ω–6),
whereas brown alga such as Wakame has a higher level of palmitic acids (16:0) and oleic
acids (18:1 ω–9). Green algae like U. pertusa have ample of hexadecatetraenoic acid (16:4
ω–3), and oleic and palmitic acids. Octadecatetraenoic acid (18:4 ω–3) is the predominant
fatty acid in Laminaria sp. and U. pinnatifida seaweeds, whereas the hexadecatetraenoic
acid (16:4 ω–3) is plentiful in Ulva sp. [20, 21]. Seaweeds are an excellent source of dietary
fibers. The predominant soluble fibers from seaweeds include alginate, carrageenan, and
agar. Besides, minor polysaccharides which feature fucoidans, xylans, and ulvans are also
found [22]. Algal species, such as Hypnea spp., Ulva lactuca, possess more soluble fiber
than terrestrial plants on a dry weight basis [23]. Alginate is one such polysaccharide which
constitutes 14%–40% of the dry mass of Laminaria sp. There are several advantages associ-
ated with the consumption of alginate-rich algae. It helps in decreased cholesterol uptake,
alters the colonial bacterial profile favorably, and absorbs toxin from the body [24]. Since
alginates are excellent metal-chelating agents, they are considered valuable nutraceuticals
as they can scavenge toxic and harmful elements in the human gut. In the scavenging pro-
cess, they may also reduce the concentration of essential elements that are di- or polyva-
lent. Further, algal polysaccharides act as satiety inducer and therefore are used as weight
control management agents [25]. Soluble fibers offer other health benefits such as improved
gut health through enhanced water-binding capacity as well as optimum nutrient absorp-
tion by decreasing the digestive transit time [26]. Higher intake of dietary fiber is associated
with a lower risk of lifestyle-related health complications such as cancers, diabetes, and
heart disease [27]. Virtually, all seaweeds are an excellent source of macro elements such
as Ca, Mg, Na, P, and K, as well as minor elements like Zn, I, and Mn, which are required
for growth, metabolism, and development [26]. It is reported that phosphorus and calcium
content of seaweeds surpasses that of potatoes, carrots, apples, and oranges. Green and
brown algae are a rich source of vitamin C. It varies anything between 50 and 300 mg/100 g
dry matter and comparable to that of parsley and peppers. The vitamin C content of red
algae varies from 10 to 80 mg/100 g of dry matter. Monostroma undulatum, green algae,
which are found in the coastal region of Southern Argentina coast, are very rich in vitamin
C, which is 159–455 mg/100 g of dry matter [14]. Seaweeds are also considered as an alter-
native and viable protein source for humans. Spirulina is one such alga that is considered a
superfood by the World Health Organization (WHO) and is consumed widely because it is
a rich source of protein. It offers several health benefits, such as renal protection, anti-
hyperlipidemia, anti-hypertension, and anti-hyperglycemic [28]. Moreover, it is also a rich
source of hypocholesterolemic γ-linoleic acid (GLA), B-vitamins, and free-radical scaveng-
ing phycobiliproteins [29]. Since spirulina is nutrient-rich and contains 180% more calcium
than milk, 670% more protein than tofu, 3100% more β-carotene than carrots, and 5100%
more iron than spinach, it has been sent to international space stations by NASA [30]. As
far as industrial applications concerned, lectins and phycobiliproteins are two bioactive
proteins which have been utilized by industry. Many patents have also been filed regarding
the health-enhancing bioactivities of phycobiliproteins as nutraceutical [31]. Some sea-
weeds (kelp, wakame nori, and Kombu) from japan is very high in iodine content. Thus,
dietary supplements containing iodine from kelp seaweed is very popular [32].
19.3 Seaweeds in the Alleviation of Lifestyle Disorders
There is literature that evidences the usage of fucoxanthin as potential food supplements
for managing obesity and diabetes in in vitro; studies as well as in animal models. For
instance, fucoxanthin is reported to inhibit α-amylase and α-glucosidase digestive enzymes.
As a result of this, lipid metabolism is modulated favorably by the downregulation of lipid
synthesis and upregulation of lipid hydrolysis [33]. In another trend, fucoxanthin supple-
mentation in animal study was correlated with a reduction of blood glucose and plasma
level as well as improvement of plasma lipid profile, which eventually reduce the risk of
insulin resistance [34]. Fucoxanthin was also found to have an antiobesogenic effect by
reducing visceral fat and BMI in human clinical trials spanning over four weeks [35].
Consumption of seaweeds and seaweed isolates rich in carotenoids and alginates was
found to exhibit a positive effect on satiety, appetite, blood glucose, and cholesterol
level [36]. Supplementation of diets with Undaria pinnatifida and Sacchariza polyschides
seaweeds in the human clinical trial had favorable glucose levels, reduced level of serum
triglyceride, and increased concentration of high-density lipoprotein in type 2 diabetes
persons [37]. Phlorotannins, fucoxanthin, polyphenolics, and polysaccharides have been
identified as the main seaweed components behind the mitigation of diabetes and its
related health complications. There are several pharmaceuticals such as Captopril,
Eplerenone, and the angiotensin-I converting enzyme (ACE) inhibitor available in the
market for the treatment of high blood pressure and heart disease. However, these com-
mercial drugs have some reported side effects, such as impaired renal function, dry cough,
19.5 Seaweed Is a Source of Anticoagulant Agen 381
and extremely low blood pressure [38]. Thus, a seaweed-derived functional molecule can
be an alternative to these commercial drugs. In epidemiological studies, it was found that
an inverse relationship exists between daily consumption of seaweeds and lower risk of
hypertension and cardiovascular disease [39]. Seaweed-derived bioactive peptides have the
potential to bind to the active site of the ACE and mitigate the high blood pressure. For
instance, in an in vitro; study, Gracilariopsis lemaneiformis peptides had potent ACE inhib-
itory activity [40].
19.4 Anti-Inflammatory Activity of Seaweeds
There are reports of seaweeds (marine algae) harboring compounds which are potent anti-
inflammatory. For example, methanolic extracts from the green seaweed Ulva conglobata
has been recognized as an anti-inflammatory in the cell (neuronal HT22 cells and micro-
glial BV2) line study [41]. Among the many algal bioactive compounds, carbohydrates are
reported to possess anti-inflammatory properties. For example, oligosaccharides derived
from alginate are shown to inhibit neuroinflammation. Similarly, laminarin, a polysaccha-
ride found in Laminaria species, was found to regulate neuroinflammation. A sulfated
polysaccharide fraction derived from the red seaweed Gracilaria cornea was demonstrated
to possess anti-inflammatory attributes and modulate the acute inflammatory process by
histamine inhibition, vascular permeability, and neutrophil migration [42]. In addition to
polysaccharides, lipids derived from algae also have potent anti-inflammatory properties.
PUFA, from microalgae, has multiple health benefits and reduces the risk of diseases such
as diabetes, arthritis, and obesity, mostly in the context of neuroinflammation. Sterols (cho-
lesterol in red algae, fucosterol in brown algae and isofucosterol, and clionasterol in green
algae) are another class of lipids that are proposed to alleviate neuroinflammation, since
they can cross the blood–brain barrier. However, there is limited literature regarding the
neuroprotective ability of algal sterols [43]. Lectins represent a group of glycoproteins.
They are functional biopeptides which are found in marine algae and possess anti-inflam-
matory activity. Further, phenolic and polyphenolic compounds such as phlorotannins
from marine algae have also gained much attention for their anti-inflammatory actions [44].
Carotenoids and terpenoids are an important class of isoprenoids that belong to marine
algae. Fucoxanthin is present in brown algae, β-carotene is predominantly present in green
microalgae. Carotenoids, in particular, fucoxanthin, has exhibited anti-inflammation and
anti-oxidative damage activity [41]. Brown seaweed, such as Sargassum, is reported to have
anti-inflammatory activity. Supplementation of diets with whole seaweed powder
(Sargassum hystrix) reported improving stress-induced liver inflammation conditions in
Wistar rats [45].
19.5 Seaweed Is a Source of Anticoagulant Agent
In the biomedical industry, heparin is the only predominant drug which is being used
extensively in the treatment of thromboembolic disorders. However, there are some long-
term side effects such as thrombocytopenia, a hemorrhagic effect associated with heparin.
This necessitates a hunt for an alternative source for the antithrombotic agent [46]. There
are reports of seaweeds polysaccharides being recognized for anticoagulant activity as their
usage does not involve any potential contamination from harmful prions or viruses associ-
ated with commercial heparins [47]. Seaweeds-derived polysaccharides do not pose any ill
effects toward cellular metabolism, and drugs derived from algal extracts are very much
affordable. From a pharmacological point of view, the mode of actions of these algal poly-
saccharides is dependent on the extent and position of the sulfate group present in the
polysaccharides, including their molecular weight. This property of algal polysaccharides
indicates that the length, shape, and density of negative charge of the molecule are the
prerequisite for the desirable anticoagulant activity. Literature evidence that phlorotannins
and fucoidans, sulfated polysaccharides from brown algae, have been identified as antico-
agulant agents [48]. Further, carrageenans from red algae and ulvans in green algae also
have an anticoagulant property that is proven in in vitro; model. Faggio et al. [47] evaluated
the anticoagulant activity of sulfated polysaccharides from Ulva fasciata (Chlorophyta) and
Agardhiella subulata (Rhodophyta) on human blood. Both the agents were shown to be
effective in prolonging coagulation time in PT and APTT assay [47]. In another study, two
sulfated polysaccharides, namely MP and SP, were extracted from Enteromorpha linza,
purified by ion-exchange and size exclusion chromatography, and characterized by fourier-
transform infrared analysis. These two molecules demonstrated potent anticoagulant activ-
ity in in vitro; system [49]. However, for the exploitation of the sulfated polysaccharides,
their efficacy, as well as safety profile, should be evaluated extensively and exclusively in
in vivo; model.
19.6 Anticancer Property of Seaweed
Cancer is one of the major causes of death around the world, and researchers are continu-
ously striving toward developing a new strategy or drugs to manage this pandemic in a
better way. Epidemiological studies support the health benefit of consumption of plant and
algal-derived foods with the reduction of risk of cancer. There are reports of certain algal
compounds being useful in the treatment of cancer because of the antioxidant property of
their compounds [50]. Bryopsis sp. is a green marine alga which holds a promising future
in the anticancer treatment since it contains compounds such as depsipeptides kahalalide
A and F that are reported to be potent drugs for tumors, AIDS, and lung cancers [51].
However, kahalalide A could not advance beyond the phase II trial because of its short
shelf life and lack of efficacy. Besides, it did not exhibit the expected response in patients.
Anyways, it is high cytotoxic agents that pave the way for the development of many syn-
thetic analogs of kahalalide A [52]. Bis-indolic amides such as chondriamide A and B are
derived from the red alga Chondria sp., and they have cytotoxicity. These two are potent
anticancer drug candidates/leads as they prevent the proliferation of human nasopharyn-
geal and colorectal cancer cells [53]. Algal polysaccharides such as fucoidans, laminarans,
alginic acids, carrageenans, along with other modified polysaccharides have also attracted
attention over the year for their capacity to ameliorate various forms of human cancer [54].
In addition to polysaccharide, terpene is another group of bioactive compounds, in
19.6 Anticancer Property of Seawee 383
particular from Chlorella sorokiniana and Chaetoceros calcitrans extracts, which have an
encouraging anticancer profile in comparison with commercially available drugs [55]. The
anticancer properties of different terpene compounds such as ursane-type triterpenoids,
lupane-type triterpenoids as well as known diterpenoids extracted from Acanthopanax tri-
foliatus were evaluated in the cell model. These compounds exhibited strong to moderate
anticancer activity against SF-268, MCF-7, HepG2, and NCIH460 cancer cells [56]. Further,
brominated cyclic diterpenes from Sphaerococcus coronopifolius, a red alga, (Rhodophyta
Phylum) has antitumor potential and cytotoxic activity against malignant cell lines which
are understudied, since the mechanism behind the observed effects are still unknown [52].
Liu et al. [57] reported that bis (2, 3-dibromo-4, 5-dihydroxybenzyl) ether, a bromophenol
compound from algae, is capable of inducing apoptosis of K562 cells by arresting cell cycle
at S phase[57]. The compounds such as lauren diterpenol, thyrsiferol, and caulerpin have
been reported to be involved in antitumor activity since they inhibit the transcription factor
HIF-1 [58]. Lee et al. [59] reported that phlorofucofuroeckol-A, which has been isolated
from brown alga Eisenia bicyclis, to be a potent antitumor compound as it inhibits Aldo-
keto reductase family 1 B10 AKR1B10, which is a therapeutic cancer target [59]. In another
instance, the compound sulfoquinovosyl diacylglycerol (SQDG), from red algae, exhibited
remarkable inhibition of telomerase enzyme, which is involved in the uncontrolled divi-
sion of malignant cells [60]. In a recent study, ethanolic extracts of Chaetomorpha sp.,
green algae, were used for the extraction of a novel anticancer molecule and tested in cell
culture (MDA-MB-231 breast cancer cell lines). It was found that the presence of antican-
cer property of this alga was primarily due to the antitumor chemicals like terpinol, oxi-
mes, and dichloracetic acid (DCA). In addition, these compounds also contain calcium,
silicon, along with other essential metals which make these molecules a novel target for
anticancer drug as well as a nutritional supplement [61]. Brentuximab vedotin (D)
(Adcetris®) is a drug of cyanobacteria origin which has got Food and Drug Administration
(FDA) approval for the treatment of Hodgkin’s lymphoma. There are other drug candi-
dates, mostly algal secondary metabolites, which are in phases of the clinical trial or in the
pipeline to be approved by the FDA [52]. Sometimes, structural characteristics are one of
the parameters which have to be considered when developing a therapeutic from marine
source, since in many cases the putative drug from different sources exhibits varied
response in vivo; [52]. For example, fucoidans showed promising activity on clinical trials
but exhibited varied responses when administered intraperitoneal, oral, or intravenous [62].
Algal-derived compounds are often administered along with therapeutic molecules as co-
adjuvant to improve the efficacy of the drugs. For example, when HepG2 cells were pre-
treated with fucoxanthin, a well-known pigment from brown algae, the efficiency of
cisplastin (well-known therapeutic used for chemotherapy) was improved [63]. In another
study, λ-carrageenan was conjugated with fluorouracil (5-FU), marketed as adrucil. As a
result of this, the antitumor activity was enhanced as well as the by-standing effect of
immunocompetence damage caused by 5-FU was fine-tuned or mitigated [64]. Most of the
identified compounds, such as polysaccharides, polyphenols, pigments, alkaloids, and ter-
penes, exert their inhibitory effects by mediating specific cellular processes [52]. For exam-
ple, laminarin, a storage polysaccharide from brown algae, is reported to induce apoptosis
on HT-29 colon cancer cells by arresting the cells in G2/M phase of the cell cycle [65].
19.7 Seaweeds as Antiviral Drugs and Mosquitocides
Seaweed-derived compound has also been used for the treatment of various vector-borne
diseases as well as mosquitocides. Among the mosquito-borne viral disease, dengue is one of
the diseases which outbreaks each year, in particular in India, and kills many people. Female
mosquitos that belong to Aedes aegypti and Aedes albopictus sp. are the common carrier of
this viral fever. In addition, it also transmits related viruses like Zika and Chikungunya [66].
The multiplication of the chikungunya virus starts with its attachment to the host cell sur-
face. Thus, inhibiting the binding of the virus will be a vital strategy to manage the virus.
Since the seaweeds polysaccharides can modify their cell surface properties, the use of algal
polysaccharides is touted as an effective approach to prevent this deadly viral disease. To this
end, sulfated polysaccharides are being exploited for the development of possible antiviral
agents. For example, ulvan is one such compound that contains 11% of sulfate and 6% of
uronic acids in its structure. It is extracted from Caulerpa cupressoidesis, a green alga found
in Brazil. Its antiviral potential was tested against DENV-1 in cell line study by Rodrigues
et al. [67]. Results are very promising with a satisfactory selective index (>714) and no cyto-
toxicity (CC50 > 1000 μg ml−1). Further, the analysis of sulfated polysaccharides by infrared
revealed a distinct pattern of molecular weight (8–100 kDa). In support of their observed
results, the authors pointed out that sulfate residue on C6 galactose residues was critical for
the observed inhibitory effect. Further, they mentioned the extent of sulfation (at least by
1.8 margins than the content of uronic acid) as well as they (<100 kDa) also have a secondary
role in the antiviral mechanism of ulvan [67]. Fucoidan is another sulfated polysaccharide
extracted from the brown algae Cladosiphon okamuranus. It has sulfated fucose and glucu-
ronic acid in a typical ratio of 6.1:1.0:2.9 (fucose, glucuronic acid, and sulfate, respec-
tively) [68]. This compound inhibited the activity of DENV-2 virus by 20% in cell line study
(BHK-21) with LC50of 4.7 μg ml−1. This compound also exhibited antiviral activity in a dose-
dependent manner [69]. When desulfated, the efficiency of inhibition was reduced markedly
(reduced to 1%), whereas fucoidan derivative wherein glucuronic acid was replaced by glu-
cose had no inhibition on DENV-2 virus. It was revealed that both glucuronic acid and sul-
fated fucose residues are crucial for the interaction with the viral glycoprotein present in the
envelope of DENV-2. Among the different serotypes (DENV1-, DENV-2, DENV3, and
DENV4) of the dengue virus, DENV-2 was found to be more vulnerable to fucoidan. DENV-2
and DENV3 had moderate susceptibility, whereas the DENV1 strain showed no vulnerability
to fucoidan. To explain this phenomenon, the authors analyzed the nucleotide sequence of
the four serotypes. It was revealed that amino acid residue located at 295 and 310 in their
protein sequence plays a vital role in the recognition of sulfated glycosaminoglycans of
fucoidan. Later, the antiviral activity of carrageenan and agaran (DL galactans) from
Gymnogongrus torulosus (Rhodophyta) against DENV-2 was demonstrated in in vitro; cell
line study. These novel series of DL galactans showed remarkable inhibitory activity with no
cytotoxicity effects [70]. There are also a few studies reported regarding the inhibitory effects
of seaweeds extracts against ZIKV virus [71, 72]. For instance, extracts from marine algae
such as Osmundaria obtusiloba, Kappaphycus alvarezii, and Caulerpa racemosa from
Brazilian coast were successfully tested against ZIKV. Inhibition of ZIKV replication was
more than 90% in a dose-dependent with LC50 value ranging from 1.38, 1.98, and 1.82 μg ml.
Among the three algae, Caulerpa racemosa was found to be more potent than other algae [72].
19.8 Use of Seaweeds in the Cosmeceutical Industr 385
SQDG, (1, 2-di-O-acyl-3-O-(6-deoxy-6-sulfo-α-d-glucopyranosyl)-sn-glycerol), a sulfolipid,

also exhibited antiviral activity against HSV-1 virus [71]. The mosquitocidal activity of sea-
weed-derived compounds has also been shown against mosquito, the carrier of most viral
disease. Yu et al. [73] reported that halogenated sesquiterpene (−)-elatol derived from
Laurencia dendroidea, a red algae typically found in Brazil, exhibited potent larvicidal effects
when tested against Aedes aegypti. The mortality rate was demonstrated to be very high
(>91% at 50 ppm). LC50 value was calculated to be 10.7 ppm, which was promising [73]. In the
same study, certain fatty acids such as palmitoleic, myristic, lauric, and capric acids isolated
from Cladophora glomerata were evaluated against Aedes triseriatus with LC50value varying
from 3 to 14 ppm. In another study, Salvador-Neto et al. [74] shown the herbicidal effect of
halogenated sesquiterpene, (+)-obtusol, and elatol derived from Laurencia dendroidea. The
mortality rate of elatol compound was found to be 30% at 10 ppm against A. aegypti within
24 hour, whereas the same concentration (10 ppm) of obtusol exhibited more potent larvi-
cidal activity (with a mortality rate of 90%). It was also revealed that the obtusol exhibited
dose-dependent larvicidal activity with LC50 of 3.5 ppm. On further probing, it was found that
larvae that were subjected to larvicide had their intestinal epithelium damaged. In their
chemical structure, these two compounds differ by one double bond and by an additional
bromine atom [74]. This might explain the differences in their activity profile.
19.8 Use of Seaweeds in the Cosmeceutical Industry
Recently, microalgae occupied a permanent place in the cosmeceutical industry and con-
solidated its presence in the markets. Cosmeceuticals are basically sold out as cosmetics
that contain biologically active ingredients. They are essential ingredients in a wide variety
of skincare formulation, mainly topical applications, and their intended use is limited to as
a beautifying agent for the enhancement of skin tone and color. For example, Arthrospira-
and Chlorella-derived extracts containing bioactive compounds are incorporated in face
and skincare products [75]. There are reports of anti-aging, skin-whitening, and anti-
pigmentation agents that are derived from marine algae [76]. Laminaria, Fucus, and
Chondrus are the algae that are predominantly being exploited in the cosmetics thanks to
their ability to rehydrate and nourish the skin [77]. Laminaran, a polysaccharide extracted
from brown algae Laminaria, is used as anticellulite cosmetics products due to its broad
range of bioactive properties [78]. Cellulite is basically not a health issue but a cosmetic
issue and can be alleviated by routine skincare regiments to improve the visual appearance
of skin. Fucoidan has found application in the anti-aging formulation as it enhances hydra-
tion and elasticity of cells by stimulating the production of heparin-growth factor (HGF),
which in turn helps in the growth of cells and tissues [79]. Sunscreen and skin-whitening
products is one segment that is steadily expanding because it plays a protective role from
the sunlight, sunburn, tanned skin, and pigmentation. Whitonyl®, a skin-whitening prod-
uct, commercialized by Silab is oligosaccharides derived from Palmaria palmata (red alga,
Rhodophyta). It alleviates the symptoms, such as deep wrinkles, loss of elasticity, sun
freckles, or brown spots, which are caused due to chronic exposure to the sun. These symp-
toms are localized in the face, arms, and hands in women who are exposed to sun and are
accentuated with age and with the extension of exposure [80]. Among polysaccharides,
alginic acid is the predominant polysaccharide in several species of brown algae. Alginic
acid is reported to inhibit scar formation and helps in the wound-healing process. Alginate,
along with collagen, is used in the clinical industry for repairing tissues [81]. One of the
important characteristics of alginate is the hydrogel formation capacity in the presence of
several cations. Thus, alginates are produced by the conversion of insoluble alginic acid
into soluble alginates in conjugation with salts of sodium or potassium [82]. At low pH,
they form gel-like structures. Alginates are used in a wide variety of gelling agents in drugs
and cosmeceuticals as emulsion stabilizers, protective colloids, as thickeners. They are also
used as a lotion, hand jellies, ointment bases, pomades, and hair products. They are also
used as skin products such as facial cream or beauty masks [83]. Carrageenans are another
class of polysaccharides that are found in carrageenophytes (Betaphycus gelatinum,
Chondrus crispus, Eucheuma denticulatum, Gigartina skottsbergii, Kappaphycus alvarezii,
Hypnea musciformis, Mastocarpus stellatus, Mazzaella laminaroides, and Sarcothalia
crispata), red algae. There are three major types of carrageenans, namely kappa (κ), iota (ι),
and lambda (λ). Iota and kappa carrageenans possess gelling property, whereas lambda
carrageenan is preferred as a thickening/viscosifier [84]. Carrageenans find application in
a wide array of personal hygiene and grooming products such as toothpaste, hair condi-
tioners, lotions, medicines, shampoos, deodorants, sunray filters, shaving creams, foams,
and sprays. As much as 20% of carrageenans are exploited in cosmetology and phar-
macy [82]. There is also evidence of seaweeds extract being useful in maintaining a slim
figure through gene regulation and protein expression [85].
19.9 Use of Seaweed as Contraceptive Agents
Three different varieties of red algae from coastal waters in Srilanka were evaluated for
post-coital contraceptive agents. Methanol and methylene chloride (1:1) extracts of two
varieties namely Gelidiella acerosa and Gracilaria corticata showed potent post-coital
contraceptive activity in female rats without showing any by-standing effects [86]. In
another study, Bhakuni et al. [87] demonstrated the anti-implantation activity of ethanol
extracts of G. edulis and G. corticata algae in the mouse model [87]. There is also a report of
100% inhibition of sperm motility by ethanolic extracts from G. edulis, which disrupts
plasma membrane in a similar fashion as spermicidal compounds [88]. Halimeda gracilis,
Indian seaweed, exhibited 100% inhibition of human spermatozoa at a dose of 10 mg ml−1
in just 20 seconds. EC50value was calculated to be 2.05 mg ml−1 in 20 seconds. It was revealed
that the plasma membrane of sperm was damaged due to the exposure to extracts of
Halimeda gracilis. Phytochemical analysis of the extracts of this seaweed showed the pres-
ence of secondary metabolites such as alkaloids and flavonoids and protein and sugar [89].
During the course of evolution, marine organisms acquired exceptional metabolic capac-
ity by virtue of their production of an array of secondary metabolites, which are quite
specific and potent in their actions. These secondary metabolites are produced by marine
organisms in defense of predation, in competition for space and food as well as to maintain
an ecological relationship in the marine ecosystem [90]. Here, in the table mentioned
below (Table 19.1), I have highlighted the different bioactive molecules present in the sea-
weeds along with their biological actions and applications.
19.9 Use of Seaweed as Contraceptive Agent 387
Table 19.1 Bioactive compounds from various seaweeds and their potential actions/applications.
Species Extracts/compound Application References
Rhodophyta (red algae)

Gelidiella acerosa, Gracilaria Methanolic extracts Contraceptive agents [86]
corticata
G. edulis, G. edulis Ethanolic extracts Male contraceptive [88]
Sphaerococcus coronopifolius Brominated cyclic Antitumor, [52, 91]
diterpenes anti-bacterial
Porphyra yezoensis SQDG Antitumor [60]
Laurencia dendroidea Halogenated Lavicidal [73]
sesquiterpene
Chondrus crispus, Eucheuma Carrageenan, Hydrogel, [84, 92, 93]
denticulatum, Kappaphycus source of zinc cosmoceuticals,
alvarezii lubricant, anticoagulant,
nutraceuticals
Agardhiella subulata Sulfated Anticoagulant [47]
polysaccharide
Palmaria palmata Extracts, source of Skin-lightening and [82, 94]
iron, and iodine skin-whitening agent,
anti-pigmentation
activity, health
supplements
(nutraceuticals)
Gelidium sp., Gracilaria sp. Agar Adhesives, [94]
suppositories, capsules,
textile printing/dyeing
Porphyra sp. Porphyrans Hypolipidemic, [95]
anticancer,
anti-inflammatory
Chlorophyta (green algae)
Ulva conglobata and Ulva Sulfated Anticoagulant [47, 96, 97]
reticulata, Ulva fasciata polysaccharide
Ulva conglobata Methanolic extracts Anti-inflamatory [41]
Bryopsis sp. Depsipeptides Anticancer [51]
kahalalide A and F
Chaetomorpha sp. Terpinol, oximes Anticancer [61]
and DCA
Caulerpa cupressoidesis Ulvan Anti-viral [67]
Dunaliella, Chlorella, Bio-ethanol, Biofuel/biodiesel, dye, [98, 99]
Chlamydomonas, Arthrospira, β-carotene, biogas vitamin C supplement
Sargassum, Spirulina,
Gracilaria, Prymnesium
parvum, Euglena gracilis and
Scenedesmus
Haematococcus pluvialis, Astaxanthin Antioxidant [100]
(Continued)
Species Extracts/compound Application References
Ochrophyta (brown algae)

Myagropsis myagroides Fucoxanthin Anti-inflamatory, [101]
antioxidative
Laminaria sp. (Laminaria Laminaran Cosmetics, anticellulite [78]
digitata, Laminaria
hyperborean)
Fucus vesiculosus, Eisenia Phlorotanins, Cosmetics, Alzheimer’s [102, 103,
bicyclis, Ecklonia cava Dieckol, Eckol, disease, MMP 104, 105]
Fucosterol, and inhibition, antidiabetic
8,8′-bieckol
Brown algae sp. Alginates Tissue engineering [65]
Ectocarpus siliculosus Fucostatin Anticancer [106]
Ecklonia cava, Eisenia Polyphenol Antioxidant, anti- [107]
arborea, Ecklonia stolinifera, inflammatory,
Eisenia bicyclis antidiabetic, antitumor,
antihypertensive,
anti-allergic
19.10 Extraction of Active Ingredients from Seaweed
The selection of the extraction method is the most critical step, which ultimately influences
the extraction yield of the target molecule. There is no single and well-established protocol
that ensures maximum recovery and compatible with most of the molecule with industrial
importance. Seaweeds extraction usually involves many steps, and during the process of
isolation, the seaweeds are often subjected to treatments such as exposure to solvents, high
temperature, extreme pH, and extended period of extraction. For each target compound,
the extraction method should be optimized and scaled up to a commercial scale. The end
products should be enriched in the active ingredients and devoids of any impurities to
maximize its efficiency. Here, we have discussed the different extraction methods usually
adopted for the extraction of bioactives from seaweeds.
19.10.1 Supercritical Fluid Extraction (SFE)

The supercritical fluid extraction (SFE) is an extraction technique which is adopted to extract
valuable component (target molecule) from a sample matrix using a suitable solvent.
Although a solid sample is the preferred matrix for this extraction process, liquid samples are
also amenable to this process. The solvent which acts as extractant has a major role for the
selective extraction of the molecule of interest. For example, phlorotannins such as phlore-
thols, fucols, or fucophlorethols are selectively extracted from Cystoseira abies-marina algae
by pure ethanol at 100 °C in subcritical state. Their chemical characterization was confirmed
by two-dimensional liquid chromatography (LC × LC-MS/MS) method [108]. Polyphenols of
Cystoseira tamariscifolia extracted with 100% methanol and chloroform exhibited more cyto-
toxic activity than water extract in the cell (leukemia, HL-60, PC3, and THP-1 cells) line study
19.10 Extraction of Active Ingredients from Seawee 389
with methanol extracts being seventeen times more potent [109]. Nowadays, green solvents
are preferred over organic solvents for safe and environmentally friendly extraction of bioac-
tive molecules from seaweeds. For example, 2-methyltetrahydrofuran (MTHF) is one such
green solvent that has been utilized for the successful extraction of carotenoids from Chlorella
vulgaris [110]. It is obtained from renewable and biodegradable sources (lignocellulosic bio-
mass), which can be recycled. It was also reported that ethanol and MTHF at 1:1 ratio and at
110 °C for 30 minutes yielded satisfactory carotenoids and can be an alternative to n-hexane.
There are pieces of evidence that suggest that a pretreatment method in the form of drying
the raw material is often helpful to recover maximum target compounds since it will enhance
the direct contact between the sample and the solvent. Moreover, the drying mode also influ-
ences the recovery of carotenoids. Maximum carotenoids were obtained by SFE method in
the optimized condition at 60 °C and 20–40 MPa with 23% water content of raw material from
Dunaliella salina [111]. More sophisticated techniques which allow integration of extraction,
identification as well as characterization of compounds into a single platform are also emerg-
ing. These platforms not only simplify the process of bioprospecting but also avoid possible
damage to the target molecule by minimizing the entire extraction process. For instance,
Abrahamsson et al. [112] developed an SFE-UV/Vis-ELSD equipment/platform. They dem-
onstrated its efficiency in the detection of ergosterol, chlorophyll A, carotenoids, and total
lipids from the algal extract obtained by SFE [112].
19.10.2 Ultrasound-Assisted Extraction (UAE)

Ultrasound-assisted extraction (UAE) of marine bioactive is another strategy that offers
several advantages such as simplicity, lower solvent consumption, and less extraction time.
Further, it can be operated at lower temperatures, which avoids the degradation of heat-
sensitive compounds. Moreover, it is affordable techniques that can be easily adapted to
any industrial scale [113]. Polysaccharides extracted from brown Ascophyllum nodosum
and Laminaria hyperborea algae by UAE method had better Laminaran yield than conven-
tional liquid–solid extraction and better antioxidant and antimicrobial profile [114]. UAE
has been exploited for the isolation of pigments such as fucoxanthin from brown marine
algae. Recovery of fucoxanthin to the tune of to 0.197 g/100 dry sample was achieved using
70% ethanol with high antioxidant activity [115]. UAE has also been utilized for the isola-
tion of protein from seaweeds. For instance, the extraction of protein from Palmaria pal-
mate was achieved by applying ultrasounds. Further, the crude protein isolates were treated
with papain to obtain hydrolyzed bioactive peptides. The recovered peptides had anti-ath-
erosclerosis activity and reduced blood pressure [116]. This report evidenced that the com-
bination of ultrasounds with proteolytic enzymes fine-tuned the overall recovery of
bioactive peptides, which ultimately is reflected in their activity profile.
19.10.3 Microwave-Assisted Extraction (MAE)

Microwave-assisted extraction (MAE) is an extraction technique that relies on microwave
energy to heat the solvents and increase the mass transfer of the solute embedded in the
sample into the solvents. The advantages of this method over soxhlet are manifold since it
drastically reduces the extraction time to 30–60 minutes. It is applied in the extraction of
organic compounds from plant and marine sources with better yield. However, the efficiency
of MAE can be optimized by manipulating several factors, such as the selection of solvent,
microwave power, temperature, and the solvent-to-solid ratio [117]. For example, microwave
power of 800 W and temperature of 40 °C for a period of 60 minutes was found to be critical
for optimal recovery (2.2 and 37.7 μg ml−1, respectively) of carotenoids and chlorophylls from
Ulva flexuosa [118]. The MAE has been successfully applied for obtaining phenolics from
brown seaweed Lessonia trabeculate. With an extraction time of 15 minutes, a satisfactory
yield of polyphenols to the tune of 74.13 GAE mg/100 g dry seaweed was achieved compared
with the conventional extraction method, which yields 49.80 GAE mg/100 g over a period of
four hours. The extracts also had inhibition activity on pancreatic lipase, α-glucosidase, α
amylase, and tyrosinase [119]. In addition, MAE of fucoxanthin from Undaria pinnatifida
was performed at 60 °C with a solid-to solvent ratio of 1:15 (g ml−1) for 10 minutes. With a
microwave power of 300 W, an optimal amount of fucoxanthin (109.3 mg/100 g dry weight)
was achieved [120]. However, it is not compatible with the thermostable compound and
sometimes cannot sustain the accuracy and activity of the target molecule.
19.10.4 Enzyme-Assisted Extraction (EAE) and EMEA

Enzyme-assisted extraction (EAE) of bioactive compounds from marine algae is another
promising strategy, since hydrolytic actions of enzymes render the cell disrupted and allow
enrichment of the bioactive compounds in the fractionations. Several brown algae species
such as Sargassum angustifolium, Sargassum boveanum, P. gymnospora, C. cervicornis,
Colpomenia sinuosa, Feldmannia irregularis, and Iyengaria stellate were subjected to carbo-
hydrase and proteases treatment for recovery of polyphenols, polysaccharides, and pro-
teins [121]. In another study, the extraction of ulvans from Ulva armoricana was enhanced
upon treatment with endo-protease. Further, the antiviral activity of the extracted ulvans
was established in this study [122]. It can be pointed out that the EAE is the method of
choice for the extraction of food grade and food compatible compounds for the purpose of
development of functional foods or nutraceuticals. It will increase the recovery rate and
enhance the biological attributes of the novel compounds. For example, EAE of Sargassum
muticum exhibited a better prebiotic and antioxidant potential than fructooligosaccharides.
In a similar way, EAE of Osmundea pinnatifida showed more radical scavenging activity,
and Codium tomentosum extracts exhibited potent α-glucosidase inhibition, which is a great
attribute for an antidiabetic compound [123]. For better results, enzymatic extraction is cou-
pled with MAE. Charoensiddhi et al. [124] applied enzymatic and microwave-assisted enzy-
matic extraction (MAEE) for higher recovery of phlorotannins as well as antioxidant
compounds from Ecklonia radiata. They reported that a short period of extraction of 30 min-
utes with MAEE yielded more phlorotanins than the enzymatic treatment alone at 24 hours.
MAEE enables high-performance recovery of the bioactive compound since the microwave
radiation, as well as hydrolytic actions of enzymes, act in synergy to disintegrate the cell wall
structure, which makes way for the release of the target compounds [124].
19.11 Market Potential of Seaweeds
According to the recent report released by Grand View Research, Inc., the global market vol-
ume of commercial seaweeds is predicted to reach USD 11.9 billion by 2027 and will grow at
a compound annual growth rate (CAGR) of 9.1%. In the Asian subcontinent, the market is
Reference 391
expected to witness a substantial growth at CAGR of 9.3% from 2020 to 2027, whereas, in
North America, the same market is estimated to witness a CAGR of 8.8% over the same fore-
cast period [125]. The main driving force that fuels this persistent growth of global seaweeds
market comprises of various factors such as diversified applications, acknowledgment of the
health benefits of seaweeds, the rapid increase in awareness among the consumers, and
aggressive promotions by manufacturers as well as increasing penetration levels in both
established and emerging markets. The leading players in the seaweeds market are always
trying to expand their business either by collaborating with or acquiring small-scale business
partners in order to increase their product portfolio. Some of the giant companies in the sea-
weeds market are Cargill, Inc., Roullier Group, E.I. DuPont Nemours and Company, Biostadt
India Ltd., and Compo GmbH and Co. Moreover, the environment-friendly regulation pro-
cess is expected to further augment the expansion of this market. The market segment
includes upstream producers, vendors, end users, several stakeholders, and government
organizations [125]. The market for the product derived from seaweeds is segmented into
green, red, and brown seaweeds. Among these, red and brown seaweed are largely exploited
in the food and pharma industry. These two industries are predicted to capture more than
70% market size of commercial seaweed market by 2025 [126]. Based on formulation, the
commercial seaweed market is bifurcated into liquid, powdered, and flakes. For example, the
seaweed hydrocolloid industry, which is segmented into agar, alginate, and carrageenan, is
steadily growing at the rate of 2–3% per year. The Asia Pacific region is the predominant mar-
ket known for the raw material and manufacturing process of this particular industry [127].
19.12 Conclusion
Seaweeds are boon to humankind as the potential benefits of seaweed consumption are
multilayered. Seaweeds offer an economical and sustainable source of health-enhancing
functional compounds in human nutrition and commercial value-added products. They
are also an incredible source of active ingredients and are exploited to full potential in
nutraceuticals, pharmaceuticals, cosmeceuticals, consumer products, and industrial thera-
peutics. Seaweeds are also a suitable and viable alternative source of drugs for several life-
threatening diseases (cancer and viral) and lifestyle-related diseases (cardiovascular,
diabetes, and hypertension).
References
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20
Bioprospection of Orchids and Appraisal of Their

Therapeutic Indications
Devina Ghai1, Jagdeep Verma2, Arshpreet Kaur1, Kranti Thakur3,
Sandip V. Pawar4, and Jaspreet K. Sembi1
1
2
Department of Botany, Government College, Rajgarh, Himachal Pradesh, India
3
Department of Botany, Shoolini Institute of Life Sciences and Business Management (SILB), Solan, Himachal Pradesh, India
4
University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, UT, India
20.1 Introduction
Bioprospecting or biodiversity prospecting refers to the systematic search for biochemical

and genetic information in nature in order to develop commercially important products for
pharmaceutical, agricultural, cosmetic, and other applications [1]. It is a purposeful explo-
ration, extraction, and evaluation of wild biological materials to develop products that are
valuable for the mankind in various important forms such as pharmaceuticals, agrochemi-
cals, nutritional supplements, cosmetics, flavorings, fragrances, biological controls, indus-
trial enzymes, molecular probes etc. According to Shaw [2], global biodiversity centers can
act as potential bioprospecting regions because of their unique bioresource richness.
A wide range of established industries (such as pharmaceuticals, manufacturing, and
agriculture) as well as a wide range of comparatively newer industries (such as aquacul-
ture, bioremediation, biomining, biomimetic engineering, and nanotechnology) are
actively engaged in bioprospection [3]. As the bioprospecting researches are fueled by tech-
nological developments, these remain confined to industries of the developed nations. It
has been seen in majority of cases that the industries exploit traditional knowledge accu-
mulated over centuries in developing and underdeveloped nations and give nothing or bare
minimum in return. The rights linked with revenue generation (manufacturing, refine-
ment, selling, etc.) remain reserved with the industry that produces the final product or
service produced at the end of the discovery and development chain. The controversy sur-
rounding the involvement of some multinational companies in developing weight loss
products based on the Hoodia cactus (Hoodia gordonii) used by the San tribe of the Kalahari
Desert for centuries as an appetite suppressant is an example of such problems [4]. Another
example is that of turmeric (powdered dry rhizome of Curcuma longa), a herb used in a
variety of culinary and therapeutic formulations since ages in India, where a patent granted

402 20 Bioprospection of Orchids and Appraisal of Their Therapeutic Indications
to researchers at University of Mississippi Medical Center for wound healing property of

turmeric was revoked by United States Patent and Trademark Office (USPTO) based on the
evidences produced by Council of Scientific and Industrial Research (CSIR), India [5], for
protection of traditional knowledge. Such a gap between traditional knowledge and prod-
uct development (leading to revenue generation) usually results in triggering debates over
indigenous claims to intellectual and cultural property and importance of ownership
rights. Convention on Biological Diversity (CBD), a multilateral treaty, attempts to bridge
these gaps by recognizing the sovereign rights of each country to control the access to the
biodiversity existing within its borders and ensuring benefit sharing and technology trans-
fer in exchange for access to bioresources [6].
Plants and their products have been utilized since long for quenching a vast variety of
human needs including food, clothing, shelter, and medicine. A large number of important
drugs have plant origin and many others are semisynthetic derivatives of such naturally
occurring products,and were discovered by using traditional knowledge of several ethnic
communities across the globe. The drugs such as atropine (Atropa belladonna), cocaine
(Erythroxylum coca), codeine and morphine (Papaver somniferum), colchicine (Colchicum
autumnale), digitoxin (Digitalis purpurea), ephedrine (Ephedra sinica), curcumin (Curcuma
longa), hyoscyamine (Hyoscyamus niger), methyl salicylate (Gaultheria procumbens), podo-
phyllotoxin (Podophyllum peltatum), prostratin (Homalanthus nutans), quinine (Cinchona
robusta), reserpine (Rauvolfia serpentina), scopolamine (Datura spp.), taxol (Taxus brevifo-
lia), theobromine (Theobroma cacao), vincristine (Catharanthus roseus), etc. are in use
throughout the world as therapeutics. Researchers have also stressed the importance of
marine fungi in producing several bioactive natural products with potential curative appli-
cations [7]. Microbial endophytes have yielded antibiotics, antivirals, anticancer, and anti-
diabetic agents, antioxidants, immunosuppressant, and insecticidal compounds [8, 9].
Microbes inhabiting varied habitats possess excellent bioprospecting potential in the dis-
covery of plant growth promoting substances and novel enzymes [10, 11]. Rapid sequenc-
ing and analysis of bacterial and fungal genomes have led to the discovery of certain gene
clusters in these organisms that potentially govern the biosynthesis of novel biologically
active compounds in them [12].
20.2 Orchids as a Bioprospecting Resource
Orchids represent one of the largest and highly evolved angiosperm families, the
Orchidaceae, and comprise plants possessing strikingly beautiful flowers of incredible
shapes, colors, and size range. Though popular as affluent ornamentals, orchids were first
discovered for their therapeutic properties. These plants find mention in various ancient
scriptures as curatives and still find a place of pride in traditional medicine throughout the
world [13–17]. According to Nugraha et al. [18], the vascular epiphytes including orchids
are important sources of therapeutic agents with diverse biological activities. Being one of
the largest plant families with nearly 28 500 species on record [19], Orchidaceae offers a
vast scope for varied researches including bioprospection. Their ability to have distributed
across almost every corner of the earth with variously adapted habits such as terrestrial,
epiphytic, lithophytic, and even subterranean makes these plants a valuable source of a
20.4 Therapeutics Indications of Orchids in Asian Regio 403
wide range phytochemicals. Their inherently slow-growing nature and unique associations
with symbiotic fungal partners for germination and growth in nature add to the uniqueness
of their biosynthetic pathways. The obligation to an insect for pollination ensures the pro-
duction of varied compounds to offer interesting rewards for the insect pollinator and add
on to the diverse array of phytochemicals being produced by the plant [20, 21]. All this
leads to the evolution of a variety of new compounds through activation of newer meta-
bolic pathways and thus providing tremendous resource for bioprospecting for therapeutic
indications.
20.3 Orchids as Curatives in Traditional India
In India, orchids find utility in local systems of medicine since the Vedic period [22–24].
The earliest reference to Indian orchids can be seen in “Charaka Samhita” wherein the
medicinal importance of some orchidaceous taxa is reported [25]. However, the first scien-
tific account of these plants appeared in Hortus Malabaricus, a 12-volume work by Van
Rheede published during 1678–1693, wherein the medicinal properties of some orchids
including Acampe praemorsa, Bulbophyllum sterile, Cleisostoma tenuifolium, Cymbidium
aloifolium, Dendrobium ovatum, Eulophia epidendraea, E. graminea, Liparis odorata,
Pholidota imbricata, Rhynchostylis retusa, Rhytionanthos rheedei, Seidenfia rheedii,
Taprobanea spathulata, etc. from peninsular region were provided [26]. In ancient Indian
literature, there are references to a group of eight plants, popularly known as “Astavarga,”
which were used for preparation of a number of rejuvenating herbal formulations, out of
these, four are orchids, i.e. Jivak (Malaxis muscifera), Rishbhaka (Crepidium acuminatum),
Riddhi (Habenaria intermedia) and Vriddhi (Platanthera edgeworthii) [27, 28]. Astavarga is
an important constituent of “Chyawanprash,” the popular immune booster Ayurvedic for-
mulation in India [29]. Some other botanicals including Ban-alu (Gastrodia falconeri),
Jewanti (Dendrobium spp., Flickingeria macraei), Salam/Salampanja/Hathpanja
(Dactylorhiza spp., Eulophia spp.), Shwethuli (Zeuxine strateumatica), Salabmisri (Eulophia
dabia), and Rasna (Acampe papillosa, Vanda tessellata) are used as aphrodisiacs, blood
purifiers, and general restorative tonics [30]. Rahamtulla et al. [31] provided ethnobotani-
cal aspects of 25 orchid species from the Darjeeling Himalaya (India). Over the years, there
are several reports on orchids curing a variety of human ailments [30, 32–43].
20.4 Therapeutics Indications of Orchids in Asian Region
It is believed that the Chinese were first to document their medicinal uses. Bletilla striata
and one Dendrobium species were described in “Materia Medica” of Shen-nung during
twenty-eighth century BCE. Shi-Hu, Tian-Ma, and Bai-Ji are three orchid-based therapeu-
tic formulations used in China [14]. The first one (Shi-Hu) is derived from Dendrobium
nobile and allied species and is valued as an important tonic because of its effectiveness in
lung, kidney, and stomach diseases, hyperglycemia, and diabetes [44–46]. “Tian-Ma” is
prepared from tubers of an achlorophyllous (mycoheterotrophic) orchid named Gastrodia
elata and finds use to treat headaches, migraine, epilepsy, high blood pressure,
rheumatism, fever, and nervous problems; till date, more than 50 chemical substances have
been isolated from this species [17, 45, 47]. Tubers of Bletilla striata are used for the prepa-
ration of third drug called “Bai-Ji,” which is used for curing tuberculosis and gastric ulcers,
boils, malaria, hemorrhage, inflammation, and malignant swellings [47]. Preparations
from Dactylorhiza hatagirea (dbang lag) provide sustenance for Tibetan monks (yogins)
practicing in remote caves [17]. Some orchids including Acriopsis javanica, Corymborchis
longiflora, Epidendrum bifidum, Eria pannea, Grammatophyllum scriptum, Nervilia arago-
ana, Tropidia curculigoides, etc. are used in some regions of Malaysia and Indonesia to cure
a number of problems such as postdelivery sickness in women, malaria, scabies and skin
lesions, intestinal tapeworms, and skin sores [48–50]. Orchids are famous by the name of
“Sunakhari,” “Sungava,” “Chandigava” in Nepal, and about 60 species find use as local
medicines in addition to energizers and aphrodisiacs [51, 52]. Vaidya [53] presented infor-
mation on medicinal properties of 130 Nepalese orchid species. Yonzone et al. [54] reported
74 orchid species used for various medicinal purposes in the Himalayan region. Different
species of Dendrobium, Vanda, Cymbidium, and many other genera are used in Indonesia,
Malaysia, Singapore, Vietnam, Sri Lanka, Thailand, Myanmar, Taiwan, Korea, and Japan
as tonic and to cure a variety of disorders [55–57]. Chauhan et al. [58] mentioned three
orchid species (Dactylorhiza hatagirea, Eulophia nuda, Vanda tessellata) in the list of plants
used for improving virility. Flickingeria nodosa, Dendrobium aqueum, and Pholidota pal-
lida have shown the antioxidant activity [59–61]. Minh et al. [62, 63] suggested the possible
use of root extracts of Phalaenopsis hybrids as a potential source of natural antioxidants.
Eating tubers of Geodorum sp. is linked with promoting longevity in Myanmar [17].
Hossain et al. [64] screened the bioactive phytochemicals in three epiphytic orchids (Luisia
zeylanica, Rhynchostylis retusa, Papilionanthe teres) of Bangladesh.
20.5 Evidences of Medicinal Uses of Orchids in Ethnic African Groups
Africans use traditional medicine as an important part of their cultural beliefs. Orchids
form an important part of their traditional medicine system because of their therapeutic
properties. People of Zulu community use an infusion of Ansellia humilis and Habenaria
foliosa as emetic. Some species of Eulophia are considered to prevent miscarriage and
relieve pain. Ansellia gigantea and A. humilis were used by Zulus as an aphrodisiac and as
antidote to bad dreams [65, 66]. Species such as Angraecum augustipetallum, Bulbophyllum
falcatum, B. maximum, B. pumilum, B. melinostachyum, Cyrtorchis arcuata, Diaphananthe
bidens, Eulophia cucullata, Graphorkis laurida, Habenaria procera, Liparis nervosa,
Polystachya cultriformis, etc. are still used for treatment of diabetes, skin infections, epi-
lepsy and fertility problems, arthritis, tuberculosis, and gastritis, paste of Ansellia africana
pseudobulbs is used as a contraceptive [67, 68]. According to Hutchings et al. [69] and
Chinsamy et al. [66], around 50 orchid species are informally traded and used in South
African traditional medicine especially by the Zulu community. In South Africa, “Iphamba”
refers to a group of 12 different orchids, namely Cyrtorchis arcuata, Diaphananthe millarii,
D. xanthopollinia, Eulophia ensata, E. ovalis, E. leontoglossa, Microcoelia exilis, Mystacidium
capense, M. venosum, Polystachya transvaalensis, Tridactyle bicaudata, and T. tridentate [66].
Tubers of some Disa, Habenaria, and Satyrium species are used as food in Malawi [70],
20.7 Remedial Uses of Orchids in American and Australian Culture 405
whereas the cooked root tubers of Eulophia cucullata are used as a poultice [71]. Infusion
prepared from the roots of Disa aconitoides is administered as an emetic for women (to
promote conception) and that prepared from the tubers of Eulophia clavicornis and
E. tenella to treat infertility [71, 72]. Ansellia africana, an African endemic, popularly
known as leopard orchid has been reported to be a species with high medicinal potential
and reserve of many important biomolecules [73]. Root infusion of A. africana is also given
to children for treatment of cough [71]. Chinsamy et al. [74] tested the anti-inflammatory,
antioxidant, anticholinesterase activity, and mutagenicity of seven orchid species that are
most commonly traded in herbal markets of South Africa and found that root extract of
A. africana to be the most effective. Vanilla flavor is the most important commercial pro-
duce of orchids in present time. Besides this, species of Vanilla (mainly Vanilla planifolia)
possess medicinal and aphrodisiac properties and cultural reliance [17, 75, 76].
20.6 Orchids as a Source of Restoratives in Europe
In Europe, orchids are thought to have many healing properties besides the ability to
enhance virility and potency. During the first century, Dioscorides in his book “De Materia
Medica” described nearly 500 medicinal plants including two terrestrial orchids. This book
highlighted the effectiveness of orchids as a determinant of sex of the offspring and pro-
moted the “Doctrine of Signatures” [14]. Some species of Ophrys, Orchis, Serapias, and
Dactylorhiza were used against alcoholic gastritis, and tuberous orchids were expected to
increase fertility in men [77]. An orchid-based nutritious drink, “Salep,” was sold at street
stalls in London [78], and its popularity declined only after introduction of tea and cof-
fee [17]. Salep was prepared from the tubers of around 35 orchids especially those belong-
ing to genus Orchis, Dactylorhiza, Neotinea, Ophrys, Himantoglossum, Serapias, Steveniella,
and Anacamptis [79, 80] and is rich in mucilage, sugar, starch, nitrogenous substance, and
traces of volatile oil and is considered as a valued tonic and aphrodisiac [48]. According to
Ghorbani et al. [81], wild orchids were traditionally collected for “Salep” in Iran for their
use in traditional medicine and ice-cream industry. Some species of Dactylorhiza are still
promoted as aphrodisiacs in many Asian countries, especially in Himalayan region.
20.7 Remedial Uses of Orchids in American and Australian Cultures
Vanilla, the spice orchid used since ancient times to flavor cocoa, finds mention in an Aztec
herbal of 1552 [14, 17] and was described to treat fevers, impotency, and rheumatism.
There are reports of Arethusa bulbosa roots being used to relieve toothache and hot juice of
the roasted fruits of Bulbophyllum vaginatum to treat earache [82]. Orchids such as
Goodyera pubescens (against mad dog bites), Bromheadia finlaysoniana (pain reliever),
Spathoglottis plicata (treatment of joint pains), Cypripedium spp. (as sedative, to treat anxi-
ety, fever, headache, nervous tension, etc.) are widely used in America [48, 83, 84]. Different
species of Cypripedium were used in North America by different ethnic groups for its seda-
tive and antispasmodic properties [84]. C. parviflorum is an important orchid employed to
treat hysteria and disorders of the nervous system by North American Indians [17]. In
Mexico, people of Mixtec and Triqui ethnicities use many native plants including orchids
as food and medicine [85]. According to Garcia et al. [86], the epiphytic orchids are known
by the name “ita ndeka” in the Mixtec language, and Prosthechea karwinskii (endemic to
Southern Mexico) are used to cure coughs, wounds and burns, diabetes, and miscarriage.
Orchids such as Bletia purpurea, Calanthe calanthoides, Catasetum integerrimum,
Cyrtopodium macrobulbon, Laelia autumnalis, L. speciosa, Myrmecophila tibicinis, and
Rhyncholaelia digbyana find mention in Mexican traditional medicine to heal wounds,
burns, and respiratory problems [13, 66, 87]. Asseleih et al. [88] listed 12 orchid species
(Epidendrum chlorocorymbos, Habenaria floribunda, Isochillus latibracteatus, I. major,
Mormodes maculata, Oestlundia luteorosea, Oncidium ascendens, Scaphyglottis fasciculata,
Sobralia macrantha, Spiranthes eriophora, Stanhopea oculata, Vanilla planifolia), which
are used for their ethnobotanical and pharmacological properties in Veracruz state
of Mexico.
Australians use the pseudobulbs of Cymbidium sp. for treatment of dysentery.
Dendrobium teratifolium and D. discolor were used to relieve pain and control ring-
worm [89]. Interestingly, the seed capsules of Selenipedium chica were occasionally used as
a substitute for Vanilla. Liparis reflexa is considered harmful for human health because of
its toxic properties [89]. Tubers and bulbs of some orchids (Gastrodia sesamoides,
Dendrobium speciosum, Caladenia spp.) are reported as emergency bush foods [78].
According to Teoh [17], at least 20 Australian species are edible, and only a few Cymbidium
and Dendrobium species are used as medicine.
20.8 Scientific Appraisal of Therapeutic Indications of Orchids
As evident from the preceding text, orchids are the backbone of the traditional medicine
systems across the globe. With the advent of medical science and technology, efforts have
been made to tap the immense potential as indicated by the crude ethnomedicinal evi-
dences, to develop products with sound scientific scaffolding for therapeutic use. This role
of orchids in traditional literature as restoratives and therapeutics has been the basis of
modern research to evaluate their potential as anticancer, antioxidant, antimicrobial,
immunomodulatory, antidiabetic, and anti-inflammatory agents (Figure 20.1; Table 20.1).
The following sections discuss in detail the various therapeutic indications in orchids:
20.8.1 Orchids as Potent Anticancer Agents

Cancer is a leading cause of death worldwide. Although several synthetic drugs are avail-
able for its treatment, none of them is completely effective and possesses many side effects
too. On the other hand, plant-based anticancer drugs have been proved to be effective and
safe for cancer treatment to some extent. Several reports elucidating the anticancer proper-
ties of the compounds extracted from orchids have been documented. The isolated biben-
zyl compounds from Dendrobium officinale and D. findlayanum displayed high cytotoxicity
against the growth of Hela human cervical cancer cell line [122, 145]. Moscatilin, a biben-
zyl derivative obtained from D. loddigesii showed an inhibitory effect toward human mela-
noma, esophageal cancer, and breast cancer [130–132]. Similarly, moscatilin isolated from
20.8 Scientific Appraisal of Therapeutic Indications of Orchid 407
Identification of orchids used in

traditional medicine
Sample collection and

extraction
Isolation and identification of

compounds
Polysaccharides Alkaloids
Bibenzyls Flavonols
Phenanthrenes
Stilbenes Flavonoids
Phenolics
Immunomodulatory Anticancer
Evaluation of
biological Antioxidant
Antimicrobial
activity
Antiinflammatory Antidiabetic
Therapeutic applications
Figure 20.1 An outline of bioprospecting in orchids and its therapeutic applications.
Table 20.1 Bioactive compounds derived from different orchid species.
Plant name Compound/Class Activity Reference
Anoectochilus Type II arabino-galactan (Polysaccharide) Immunomodulatory [90]

formosanus activity
Anoectochilus ARPP80 (Polysaccharide) Antioxidant activity [91]
roxburghii
Bletilla Blestriarene A, blestriarene B, blestriarene Antibacterial [92]
ochracea C (Phenathrenes) activity
4-Methoxyphenanthrene-2,7-diol Anti-inflammatory [93]
(Phenanthrene) activity
Bletilla striata BSP (Polysaccharide) Healing oral ulcers [94]
Coelonin (Dihydrophenanthrene) Anti-inflammatory [95]
activity
(Continued)
2,7-Dihydroxy-3,4- Antibacterial [96]

dimethoxyphenanthrene, 2,7-dihydroxy-4- activity
methoxy-9,10-dihydrophenanthrene,
2,7-dihydroxy-3,4-dimethoxy-9,10-
dihydrophenanthrene, shanciol C, shanciol
F, shanciol D blestriarene A (Stilbenoids)
Bletistrin F, bletistrin G, bletistrin J, Antibacterial [97]
bulbocol, shanciguol and shancigusin B activity
(Bibenzyl derivatives)
4,7,7′-trimethoxy-9′,10′-dihydro(1,3′- Antibacterial [98]
biphenanthrene)-2,2′,5′-triol, activity
4,7,4′-trimethoxy-9′,10′-dihydro(1,1′-
biphenanthrene)-2,2′,7′-triol;
4,7,3′,5′-tetramethoxy-9′,10′-dihydro(1,1′-
biphenanthrene)-2,2′,7′-triol,
4,8,4′,8′-tetramethoxy(1,1′-
biphenanthrene)-2,7,2′,7′-tetrol,
blestriarene C [4,4′-dimethoxy(1,1′-
biphenanthrene)-2,7,2′,7′-tetrol]
(Biphenanthrenes)
pFSP (Polysaccharides) Antioxidant activity [99]
BSP (Polysaccharides) Antioxidant activity [100]
BSP-1 (Polysaccharide) Immunomodulatory [101]
activity
BSPF2 (Polysaccharide) Immunomodulatory [102]
activity
Dihydropinosylvi, batatasin III, 3′-hydroxy- Anti- [103]
2-(4-hydroxybenzyl)-3,5-dimethoxy- neuroinflammatory
bibenzyl, gymconopin D and activity
5-[2-(3-methoxyphenyl)ethyl]-1,3-
benzenediol (Stilbenes)
Phochinenin K, bleformin F and Anti-inflammatory [104]
4,8,4′,8′-tetramethoxy-(1,1′- activity
biphenanthrene)-2,7,2′,7′-tetrol
Bulbophyllum Retusiusines B (Phenylpropanoid) Antifungal activity [105]
retusiusculum
Cremastra Coelonin, orchinol (Phenanthrenes) Antioxidant activity [106]
appendiculata
Cymbidium Cymbinodin-A (Phenathrenequinone) Anticancer activity [107]
finlaysonianum
Dendrobium DAP (Polysaccharide) Immunomodulatory [108]
aphyllum activity
Aphyllone B (Bibenzyl derivative) Antioxidant activity [109]
Dendrobium Moscatilin (Bibenzyl derivative) Anticancer activity [110]
aurantiacum
var. denneanum
Dendrobium n-Docosyl 4-hydroxy-trans-cinnamate Antidiabetic [111]

christyanum (Ester of cinnamic acid) property
Dendrobium Erianin (Bibenzyl) Inhibits diabetic [112]
chrysotoxum retinopathy
Dendrobium (+)-Homocrepidine A (Indolizidine) Anti-inflammatory [113]
crepidatum activity
(+)-Dendrocrepidamine A, Anti-inflammatory [114]
dendrocrepidamine B, (+)-homocrepidine activity
A (Octahydroindolizine-type alkaloids)
Dendrocrepine (Indolizidine alkaloid) Antidiabetic property [115]
Dendrobium 2,5-Dihydroxy-4-methoxy-phenanthrene Anti-inflammatory [116]
denneanum 2-O-β-d-glucopyranoside (Phenanthrene activity
glycosides), 5-methoxy-2,4,7,9S-
tetrahydroxy-9,10-dihydrophenanthrene
[9,10-dihydrophenanthrenes]
Dendrobium 5-Hydroxy-3-methoxy-flavone-7-O-(β-d- Antidiabetic [117]
devonianum apiosyl-(1-6))-β-d-glucoside (Flavonol property
glycoside)
DvP-1 (Polysaccharide) Immunomodulatory [118]
activity
DDP (Polysaccharide) Immunomodulatory [119]
activity
Dendrobium Gigantol (Bibenzyl compound) Anticancer activity [120]
draconis
Dendrobium Dendrofalconerol A (Bibenzyl) Anticancer activity [121]
falconeri
Dendrobium 4,4′-Dihydroxy-3,3′,5-trimethoxy bibenzyl Anticancer activity [122]
findlayanum (Bibenzyl)
Dendrobium Confusarin (Phenanthrene), 5-methoxy-7- Antidiabetic [123]
formosum hydroxy-9,10-dihydro-1,4-phenanth property
renequinone (Phenanthrenequinone)
Dendrobium DHP1A (Polysaccharide) Hepatoprotective [124]
huoshanense activity
GXG (galactoxyloglucan) (Polysaccharide) Protection of [125]
intestine
GXG (galactoxyloglucan) (Polysaccharide) Antidiabetic [126]
DHP-4A (Polysaccharide) Immunomodulatory [127]
activity
Dendrobium Dendrosinen B (Bibenzyl derivative) Inhibit pancreatic [128]
infundibulum lipase
Dendrobium Loddigesiinols G–J (Polyphenols) Antidiabetic [129]
loddigesii Moscatilin (Bibenzyl derivative) Anticancer activity [130–132]
(Continued)
Dendrobium Moscatilin (Bibenzyl) Protect retinal cells [133]

nobile from hypoxia/
ischemia
3′,4-Dihydroxy-3,5′-dimethoxybibenzyl, Antifungal activity [134]
3-hydroxy-5-methoxybibenzyl, batatasin
III, tristin, 3,3′,5-trihydroxybibenzyl
(Bibenzyl)
Nudol (Phenanthrene) Anticancer activity [135]
Dendronbibisline A, Dendronbibisline B Anticancer activity [136]
(dihydrophenanthrofurans)
Dendronbibisline C Dendronbibisline D
(Bisbibenzyl derivative)
Dendrobium 3,4-Dihydroxy-4′,5-dimethoxybibenzyl Antidiabetic [137]
officinale (Bibenzyl) property
DOP (Polysaccharide) Prevent lung [138]
inflammation
DOPS (Polysaccharide) Protective role in [139]
liver injury in acute
colitis
DOP (Polysaccharide) Treating [140]
constipation
Dendronan (Polysaccharide) Maintaining colonic [141]
health
DOP (Polysaccharide) Gastroprotective [142]
activity
DOPS (Polysaccharide) Neuroprotective [125]
activity
DOPW-1 and DOPW2 (Polysaccharide) Immunomodulatory [143]
activity
DOP-1-1 (Polysaccharide) Immunomodulatory [144]
activity
4,4′-Dihydroxy-3,5-dimethoxy bibenzyl Anticancer activity [145]
(Bibenzyl)
Rutin (Flavonoid) Antioxidant activity [146]
Dendrobium Dendroflorin (Phenolic) Antioxidant activity [147]
palpebre
Dendrobium Dendroparishiol (Bibenzyl- Anti-inflammatory [148]
parishii dihydrophenanthrene derivative) activity
Dendrobium 4′-Dihydroxy-3′,5-dimethoxybibenzyl Anticancer activity [149]
plicatile (Bibenzyl)
Dendrobium Dendroscabrol B (Bisbibenzyl) RF-3192C Antidiabetic [150]
scabrilingue (Dinaphthalenone) property
Dendrobium Dendrofalconerol A (Bisbibenzyl) Antidiabetic [151]
tortile property
Dendrobium DTP (Polysaccharide) Immunomodulatory [152]

tosaense activity
Eulophia 4-Methoxy-9,10-dihydro-2,7- Anti-inflammatory [153]
macrobulbon phenanthrenediol, 4-methoxy-2,7- activity
phenanthrenediol,
1,5-dimethoxy-2,7-phenanthrenediol,
1,5,7-trimethoxy-2,6-phenanthrenediol,
1-(4-hydroxybenzyl)-4,8-dimethoxy-2,7-
phenanthrenediol (Phenanthrene)
4-Methoxy-2,7-phenanthrenediol Antioxidant activity
Gastrodia elata GPs (Polysaccharides) Immunomodulatory [154]
activity
Bis(4-hydroxybenzyl)ether mono-β-l Antioxidant activity [155]
galactopyranoside
Gastrodin Antioxidant activity [156]
(4-hydroxyapatite-4-hydroxyapatite-
glucoside)
Gastrodinol (tetra-p-cresol substituted Antibacterial [157]
cyclopenta [a] naphthalene derivative) activity
Gavilea lutea Gavilein (Bibenzyl derivative) Antifungal activity [158]
Liparis regnieri Erianthridin, gigantol, hircinol, nudol, Antibacterial [159]
coelonin, moscatin (Phenanthrene activity
derivatives)
Paphiopedilum 3′-Hydroxy-2,6,5′-trimethoxystilbene, Anticancer activity [160]
callosum 3′-hydroxy-2,5′-dimethoxystilbene,
2,3′-dihydroxy-5′-methoxystilbene
(Stilbenes), galangin (Flavonoid)
Paphiopedilum 5,6-Dimethoxy-2-(3-hydroxy-5- Anticancer activity [161]
godefroyae methoxyphenyl)benzofuran (Stilbenes)
Pleione 2,5,2′,5′-Tetrahydroxy-3-methoxybibenzyl Anti- [162]
bulbocodioide and 2,5,2′,3′-tetrahydroxy-3- neuroinflammatory
methoxybibenzyl (Bibenzyl)
Spiranthes Spiranthesphenanthrene A Anticancer activity [163]
sinensis (Phenanthrene)
Vanda teres Eucomicacid (Auxin) and vandateroside II Antiaging activity [164]
(Glycopyranosyloxybenzyleucomate)
D. aurantiacum has also depicted its potential in the treatment of pancreatic cancer [110].
Likewise, the studies on D. draconis and D. falconeri also yielded bibenzyl compounds,
gigantol and dendrofalconerol A, which were cytotoxic against lung cancer cells [120, 121].
A phenanthrene derivative nudol, isolated from D. nobile, played a critical role in the inhi-
bition of osteosarcoma (U2OS) cell growth [135]. Spiranthes phenanthrene A from
Spiranthes sinensis inhibited the growth and migration of melanoma (B16−F10) cells [163].
Furthermore, the phenolic compounds isolated from Cymbidium finlaysonianum,
Paphiopedilum callosum, and P. godefroyae were found to be cytotoxic against human small
cell lung cancer (NCI-H187) cell lines [107, 160, 161]. So, in a nutshell, it can be said that
the metabolites of orchids have the potential to be used in cancer treatment after more
extensive investigations.
20.8.2 Immunomodulatory Activity in Orchids

The immune-enhancing potential of plant-derived compounds leads toward maintaining a
disease-free state in human beings. Both in vivo and in vitro; testing methods have been
employed for evaluating the immunomodulatory effects of various orchids. The polysac-
charides obtained from many species belonging to the genus Dendrobium have exhibited
noteworthy immunomodulatory activity due to their ability to promote the proliferation of
macrophages and lymphocytes, increase in the phagocytosis activity and upregulation of
nitric oxide and cytokine production, etc. These activities have been testified in the polysac-
charides of Dendrobium aphyllum, D. devonianum, D. huoshanense, D. officinale and D. tos-
aense [108, 118, 119, 127, 143, 144, 152]. Type II arabino-galactan, a polysaccharide purified
from Anoectochilus formosanus, stimulated the maturation of dendritic cells, which play a
crucial role in the induction of immune responses against pathogens [90]. Further, the
polysaccharides from Bletilla striata and Gastrodia elata improved the spleen and thymus
indices, which attributed toward their immune-enhancing potential [101, 154].
20.8.3 Orchids and Their Antioxidant Potential

As free radicals are the causal factors leading to many diseases such as cancer and cardiovas-
cular diseases. Therefore, the compounds having antioxidant activity can be utilized for the
treatment of such grave diseases. Many investigations have been undertaken so far to evalu-
ate the antioxidant potential of various orchids using both in vitro; and in vivo; methods. The
polysaccharides extracted from Anoectochilus roxburghii and Bletilla striata displayed remark-
able antioxidant effects [91, 99, 100]. Several phenolic compounds also possessed antioxidant
potential. For instance, Rutin (flavonoid), Aphyllone B (bibenzyl derivative), and 4-methoxy-
2,7-phenanthrenediol isolated from Dendrobium officinale, D. aphyllum, and Eulophia mac-
robulbon, respectively, showed significant DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical
scavenging activity, which is a standard test to evaluate the antioxidant potential [109, 146,
153]. Moreover, phenanthrenes such as coelonin and orchinol derived from Cremastra
appendiculata showed efficiency in both DPPH and ABTS (2,2′-azino-bis(3-ethylbenzothia-
zoline-6-sulfonic acid)) radical scavenging activities [106]. Furthermore, dendroflorin and
dendroparishiol isolated from D. palpebre and D. parishii exhibited antioxidant activity by
decreasing reactive oxygen species (ROS) in H2O2-treated cells and increasing the expression
of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPX),
and catalase (CAT) [147, 148]. The enhancement of the activity of these enzymes has also
been reported in a similar study conducted in Gastrodia elata [155].
20.8.4 Antimicrobial Studies in Orchids

The antimicrobial effects of the compounds extracted from orchids have been studied
against a diverse species of bacteria and fungi. The stilbenoids and the bibenzyl derivatives
isolated from the tubers of Bletilla striata showed significant antibacterial activity against
Staphylococcus aureus [96, 97]. In the earlier researches on Bletilla striata and Bletilla
ochracea, the derived biphenanthrenes and phenanthrenes have also been reported to pos-
sess inhibitory effects against gram-positive bacteria such as Staphylococcus aureus,
Staphylococcus epidermis, and Bacillus subtilis [92, 98]. In the same manner, the phenan-
threne derivatives (erianthridin, gigantol, hircinol, nudol, coelonin, and moscatin) in
Liparis regnieri displayed antibacterial activity against Streptococcus agalactiae and Bacillus
subtilis [159]. Furthermore, gavilein and retusiusines B isolated from Gavilea lutea and
Bulbophyllum retusiusculum, respectively, exhibited noteworthy antifungal activity against
Candida albicans [105, 158]. Additionally, broad-spectrum antifungal activity has been
shown by bibenzyl derivatives of Dendrobium nobile against Alternaria brassicicola,
Phytophthora parasitica var. nicotianae, Colletotrichum capsici, Bipolaris oryzae, Diaporthe
medusae nitschke, Ceratocystis paradoxa moreau, Exserohilum turcicum, Pestallozzia theae,
and Alternaria citri [134].
20.8.5 Orchids and Anti-inflammatory Activity

Lipopolysaccharide induces the production of nitric oxide synthase in macrophages, which
upon stimulation produces different inflammatory factors such as tumor necrosis factor-α,
interleukin-1β, etc. Different studies have been conducted to decrease inflammation with
the help of compounds derived from different orchid species by inhibiting nitric oxide pro-
duction. The stilbenes (dihydropinosylvi, batatasin III, 3′-hydroxy-2-(4-hydroxybenzyl)-
3,5-dimethoxy-bibenzyl, gymconopin D, and 5-[2-(3-methoxyphenyl)ethyl]-1,3-benzenediol)
isolated from Blettia striata were reported to have anti-neuroinflammatory activity [103].
Zhou et al. [104] studied the effect of phochinenin K, bleformin F, 4,8,4′,8′-tetramethoxy-
(1,1′-biphenanthrene)-2,7,2′,7′-tetrol on reducing nitric oxide in lipopolysaccharide-
induced BV-2 microglial cells. Bibenzyl derivative isolated from pseudobulbs of Pleione
bulbocodioides also exhibited anti-neuroinflammatory potential [162]. 2,5-Dihydroxy-4-
methoxy-phenanthrene 2-O-β-d-glucopyranoside and 5-methoxy-2,4,7,9S-tetrahydroxy-
9,10-dihydrophenanthrene of Dendrobium denneanum exert anti-inflammatory effects by
inhibiting MAPKs and nuclear factor κB pathways [116]. An indolizidine, (+)-homocrepi-
dine A, isolated from stems of D. crepidatum, inhibited accumulation of nitric oxide and
reduced inflammatory responses [113]. Similarly, a bibenzyl-dihydrophenanthrene deriva-
tive of D. parishii and phenanthrene derivatives of Eulophia macrobulbon could also invoke
anti-inflammatory responses [148, 153]. A dihydrophenanthrene, coelonin, identified from
ethanolic B. striata extract, and 4-methoxyphenanthrene-2,7-diol from B. ochracea have
remarkable anti-inflammatory properties [93, 95].
20.8.6 Antidiabetic Prospects in Orchids

Diabetes is a disease with defective glucose metabolism caused by reduced insulin
uptake or inefficient insulin production. Unmanaged diabetes could lead to several
health-related problems. Several studies have been conducted to evaluate the orchid
polysaccharides for their hypoglycemic potential. Noteworthy among these is a com-
parative study on various species of Dendrobium [165]. GXG (galactoxyloglucan), a
purified polysaccharide of D. huoshanense, prevents hyperglycemia in type 2 diabetes
in mice by improving insulin sensitivity [126]. Loddigesiinols G–J extracted from
stems of D. loddigesii and 3,4-Dihydroxy-4′,5-dimethoxybibenzyl from D. officinale

were used in treating type 2 diabetes [129, 137]. 5-Hydroxy-3-methoxy-flavone-7-O-
(β-d-apiosyl-(1-6))-β-d-glucoside, a flavanol derivative of D. devonianum, inhibited
the production of α-glucosidase, thereby reducing the blood sugar levels [117].
D. chrysotoxum–derived erianin could prevent diabetic retinopathy [112]. A phenol
from D. christyanum (n-Docosyl 4-hydroxy-trans-cinnamate) and a bisbenzyl (dendro-
falconerol A) from D. tortile exhibited high α-glucosidase inhibition showing their
antidiabetic potential [111, 151]. Similarly, confusarin and 5-methoxy-7-hydroxy-
9,10-dihydro-1,4-phenanthrenequinone of D. formosum also showed antidiabetic
activity [123]. These compounds can form the basis of future development of plant
based antidiabetic drugs.
20.8.7 Other Analeptic Properties in Orchids

There are several records of orchid-derived compounds exhibiting medicinal properties.
Dendrobium officinale polysaccharides could be suggested for treating chronic obstruc-
tive pulmonary disease (COPD), liver injury in acute colitis, and constipation [138–140].
Dendronan, a polysaccharide from D. officinale, was reported to be playing role in main-
tenance of colonic health in rat [141]. Moscatilin, a bibenzyl derivative from D. nobile,
was able to protect retinal cells from hypoxia/ischemia [133]. Bletilla striata polysaccha-
ride composed of mannose and glucose plays a role in healing oral ulcers [94]. Various
polysaccharides extracted from Dendrobium were reported to have multiple protective
roles such as DHP1A has hepatoprotective potential by decreasing the expression of sev-
eral inflammatory responses, DOP provided gastroprotective effect, and GXG (galactoxy-
loglucan) enhanced mucosal lining in intestinal areas [124, 142, 166]. Dendrosinen B, a
bibenzyl derivative of D. infundibulum, was found to inhibit pancreatic lipase [128].
Polysaccharides from D. officinale were proved to be protective against neurodegenera-
tive disease [125].
Also, orchids are being used as cosmetic agents. A detailed review by Hadi et al. [167]
emphasized upon the importance of antiaging properties of Vanda extract. Similarly, stim-
ulation of cytochrome c oxidase in Vanda teres by eucomic acid and vandateroside II pro-
vided antiaging effects [164].
20.9 Conclusions
Orchids are an immense source of active phytochemicals due to their varied life modes and
physiology and their wide adaptive potential. The traditional literature is dotted with
reports of their use as curatives and restoratives. However, to establish these plants as ther-
apeutics and to exploit their vast reserve of phytochemicals and produce pharmaceutically
active dosage forms, systemized observational studies need to be undertaken to create sci-
entific evidences and link the ethnobotanical knowledge to state-of-the-art research and
development in order to facilitate new drugs discovery.
Reference 415
Acknowledgments
JKS is grateful to CSIR for research support No. 38(1443)/17/EMR-II dated 25/5/17. SVP
acknowledges financial support from the UGC-Start up grant No.F.4-5(65-FRP)
(lv-cycle)/201 7 (BSR) under UGC Faculty Recharge Programme (GOI), New Delhi. DG is
grateful for Senior Research Fellowship (File No. 09/135(0809)/2018-EMR-I) by CSIR. AK
is grateful for DST INSPIRE (Provisional offer: DST/INSPIRE/03/2019/000643). The
authors are also grateful to Department of Science and Technology, Government of India,
for partial financial support under Promotion of University Research and Scientific
Excellence (PURSE) grant scheme.
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425
Index
a antifungal 6, 27, 32, 33, 56, 58, 61–63,

ABTS 110, 176, 412 109, 153, 154, 156, 162, 163, 166, 198,
agri‐biomass 131–144 273, 274, 408, 410, 411, 413
agriculture residues 144 anti‐inflammatory 3, 61, 63, 141, 154,
algal biotechnology 304 161, 198, 213, 214, 249, 256, 273, 310,
alkaloids 133, 154, 155, 158, 163–165, 313, 315–317, 378, 381, 387, 388,
183, 197, 211–213, 215, 217, 220, 405–411, 413
221, 261–263, 266, 270, 271, 339, 383, anti‐microbial 2, 6, 9, 101, 133, 153, 154,
386, 409 156, 160–166, 197, 199, 213, 232, 242,
α‐amylase inhibitors 16, 21, 24–26, 31, 33 249, 256, 273, 274, 311, 316, 317, 378,
amino acids 16, 21, 30, 32, 36, 105, 111, 389, 406, 412
113, 117, 160, 211, 212, 261, 266, 267, antioxidants xxi, 6, 9, 133, 141–143, 154,
270, 271, 296, 307, 310, 316, 322, 384 157, 161, 175–184, 190, 195, 197, 199,
amylases 21, 24–26, 100–102, 104, 105, 213, 214, 232, 241, 242, 247, 249, 250,
117, 118, 120, 143 256, 273, 274, 288, 310, 313–316, 378,
analeptic 286 382, 387–390, 402, 404–408, 410–412
analgesics 61–63, 73, 213, 273 arcelins 16, 24, 30, 31, 33
animal manure 131, 350 arsenic (As) 76, 79, 135, 193
anthelmintic 2, 6, 61, 162 artemisinin 62, 65, 162, 213, 214,
anticancer 2, 3, 6, 61–63, 106, 107, 110, 216–218, 221, 273
141, 154, 161, 273, 288, 310, 313, 316, asparaginase 100, 106
317, 382, 383, 387, 388, 402, astaxanthin 140, 314, 315, 327, 387
406, 408–411 asymptomatically 100
anti‐diabetic 5, 161, 213–214, 315, 317, atropine 61, 62, 213, 214, 218, 221,
378, 388, 390, 402, 406, 409, 410, 414 272, 402

426 Index
b cell suspension 212, 216–218, 220, 221

bacteria 16, 27, 33, 36, 55, 57–60, 80, 93, cellulases 100–102, 107–109, 117, 119,
99, 105, 106, 111, 114, 116, 117, 119, 134, 120, 134
143, 160–164, 166, 211, 266, 273, 314, cereal grains 179, 181
322, 324, 350, 351, 362, 412, 413 cereal straw 131
bioactive xxi, 3, 6, 7, 53, 55, 57, 58, 60, 62, chemical prospecting 3, 92
92, 93, 117, 133, 142, 143, 153, 156, 164, chitinases 16, 17, 20, 36, 109–110
175, 189, 194, 196–198, 200, 215, 256, chlorophylls 267, 268, 273, 280, 289,
306, 318, 327, 336, 342, 378, 380–382, 313–314, 390
385, 386, 388–390, 402, 404 chromatography 104, 105, 113, 114, 117,
bioenergy xxi, 10, 131, 132, 144, 262, 382, 388
345–353, 357–369 clustered regularly interspaced short
bioenergy crops 10, 357–369 palindromic repeats (CRISPR)
biofilm 116, 157–159, 165, 324 8, 298
biofuels 75, 78, 107, 132, 135, 273, 304, cocaine 217, 272, 402
312, 319, 321, 322, 345–348, 350, 351, condiments xxi, 9, 231, 232, 240–243, 247,
353, 354, 358–365, 367, 368, 378, 387 248, 250, 254–256
bioinsecticides 9 conservation 9, 53, 54, 64, 92, 94–96, 196,
biomass xxi, 7, 10, 73, 78–80, 107, 131, 200–202, 242, 254
132, 135–137, 140, 144, 216, 303–327, contraceptive 386, 387, 404
345–354, 357, 359–369, 377, 389 corn stover 131, 132, 135
bionic prospecting 3, 4, 92, 93 cosmaceuticals 4, 5
biopesticides xxi, 9, 16, 336–338, cosmetics xxi, 2, 4, 9, 54, 73, 91, 92, 99,
340–343 110, 140, 142, 189–191, 194–204, 232,
biopiracy 6, 55–56, 64, 93, 200, 201 248, 249, 265, 274, 275, 286, 287, 304,
bioprospecting xxi, 1–10, 15, 53–56, 60, 310, 312–315, 322, 385, 388, 401, 414
62–64, 91–93, 95, 96, 100, 101, 189–205, coumarins 156–158, 161, 162, 192, 266,
336, 342, 377–391, 401–403, 407 267, 273, 277
bioremediation xxi, 54, 57, 58, 79, 110, crassulacean acid metabolism
113, 115, 401 (CAM) 359, 362–364
biosensor 54, 60, 111 crop residues 131, 133, 350, 358
biosynthetic pathways 7, 53, 212, 214, cryptogams 1
215, 217, 266, 403 cyclotides 16, 20, 32–35
CYP450 58
c
canatoxins like proteins 33 d
carbon sequestration 306, 363, 364, 367 defensins 16, 32
carotenoids 133, 143, 154, 175, 182, deficiency 29, 56, 57, 293, 298, 324, 367
267–269, 282, 283, 308, 313–315, 325, disinfectant 73, 250
326, 378, 380, 389, 390 DNA 8, 16, 92, 93, 122, 158, 162, 163, 175,
cellobiohydrolase 108 249, 296
Index 427
e fodder 10, 94, 141, 358

ecological 1, 6, 16, 53, 92, 94–96, 115, 196, food xxi, 4, 5, 9, 10, 18, 32, 37, 56, 57, 60,
199–200, 251, 321, 363, 364, 377, 386 76, 77, 92–96, 99, 104, 107, 110, 114, 131,
ecological restoration 8, 91–96 133, 134, 138, 140, 141, 153, 161, 162, 175,
ecosystem biodiversity 91 176, 181–184, 189–191, 193, 200, 202, 231,
elicitation 212, 216, 220, 221 232, 242–247, 249, 253, 254, 256, 261, 265,
EMEA 390 274, 275, 281, 287–289, 293–297, 303, 304,
emollient 194–196 306–316, 326, 327, 336, 337, 341, 342, 346,
emulsifier 190, 194–196, 202 347, 349–351, 357–359, 363–365,
endophytes 7, 9, 56–58, 99–122, 402 378–380, 382, 383, 386, 390, 391, 402,
endophytic 7, 100, 101, 104, 106–119, 404, 406
216 food additives 4, 5, 92, 161, 175, 182–184,
entomotoxic 15–37 246, 288, 311, 313
enzyme‐assisted extraction (EAE) 390 Food and Drug Administration (FDA) 60,
enzymes 3, 4, 7, 9, 21, 24, 32, 33, 36, 54, 62, 63, 383
57, 59, 92, 93, 99–122, 134, 140, 156, 161, forests 54, 91, 96, 106, 231, 242, 243, 342,
163, 189, 202, 249, 262, 266–272, 298, 314, 346, 347
316, 324, 347, 352, 360, 380, 383, 389, 390, fossil fuels 132, 306, 319, 323, 326, 345,
401, 402, 412 348, 351, 353, 357, 369
essential oils 9, 162, 164, 183, 195, 232, fragrances 190, 192–195, 202, 204, 213,
244, 247, 255, 261–290, 337, 340 232, 281, 284, 401
ethnobiological 9 fruits 9, 102, 107, 113, 115, 133, 134, 136,
ethnobotanical 3, 231–256, 403, 406, 414 142, 143, 156, 160, 161, 175–181, 183,
ethnobotany 191 184, 199, 231–239, 241–245, 247, 250,
263–266, 269–271, 273, 275, 276, 279,
f 280, 282, 284, 287, 294, 316, 337, 339,
fatty acid 55, 63, 111, 141, 182, 197, 353, 405
282, 311–313, 319, 321, 322, 325, 378, fuels 94, 132, 135, 139, 141, 242, 319, 323,
379, 385 324, 326, 345–349, 351–354, 357–360,
fermentation 102–105, 107, 108, 110, 112, 362, 363, 369, 391
113, 117, 119, 132, 135, 139–141, 265, functional foods 184, 249, 310, 378–380,
294, 295, 313, 322, 323, 347, 348, 390
350–353, 360, 362, 365 fungi 27, 33, 55–60, 80, 99, 101, 104–117,
ferric ion reducing antioxidant power 119, 141, 143, 160, 162, 189, 190, 211,
(FRAP) 176–178, 181, 182 216, 266, 303, 314, 402, 412
fibre 94, 107, 115, 132, 133, 135, 138, fungus 2, 63, 106, 108, 110–114, 140
139, 141
flavonoids 133, 142, 143, 154, 156, 160, g
165, 183, 197, 216, 247, 261, 263, 266, 267, gardens 94, 233, 236, 241
270, 273, 316, 317, 386, 410–412 gene bank 95
fly ash (FA) 58, 75–79, 136, 137 gene prospecting 3–4, 92, 93
428 Index
genetic 1, 6–8, 16, 21, 57, 63, 80, 91–96, insecticides 6, 76, 94, 109, 272, 274, 287,
119, 138, 189, 190, 200–203, 212, 215, 288, 335, 338, 339, 342
221, 255, 256, 262, 296, 297, 327, insects 15–37, 57, 63, 76, 92, 109, 141,
360–363, 365–366, 401 263, 264, 274, 275, 280, 288, 310,
genetic biodiversity 91 337–340, 342, 362, 368, 403
Giberellic acid 57 integrated pest management (IPM) 336,
glucosinolates 133, 154, 175, 270 341, 342
green belt 77 intellectual property rights (IPR) 6, 53,
green cosmetics 196–200, 204 56, 202
h k
hairy root 215, 217, 219 Kosmeticos 190
halophytes 358, 359, 363, 364
heavy metal 57, 58, 60, 73, 74, 76–80, l
135–137, 140, 143, 144, 193, 199, 357, laccases 54, 100–102, 110–111, 118, 120
364, 367, 368 landfills 75, 77, 79, 131, 350
herbal cosmetics 190, 194, 201 lectins 6, 16–20, 24, 30, 33, 36, 313,
herbicidal 54, 385 380, 381
hexavalent 76, 80 legumes 16–18, 21, 24, 56, 57, 114, 183,
high‐performance liquid chromatography 294, 295, 310
(HPLC) 93, 217, 221 lipases 55, 100, 101, 103, 111–113, 120,
high‐value biomolecules 304, 140, 390, 409, 414
306–319, 326 lutein 133, 282, 314, 315
hirudin 4 lycopene 133, 134, 143, 175, 183, 269,
holobionts 9 314, 315
hyperaccumulators 74, 79, 80, 116
m
i marine 3, 4, 10, 55, 58, 60, 62, 63, 79, 108,
immunomodulatory 313, 406–412 116, 164, 303, 315, 318, 350, 377–391, 402
industrial application 9, 101, 111, 119, medicinal xxi, 4–6, 9, 27, 62, 104–106,
141, 312, 378, 380 108, 109, 140, 153–156, 175, 179, 181,
industrial molecules 10 183, 191, 202, 211–214, 216, 217, 220,
industrial waste 347 221, 231, 232, 242, 243, 251, 254, 256,
industry 3–6, 9, 54, 56–57, 59–64, 73, 273, 403–405, 414
75–80, 91, 96, 99–122, 133–135, 137, 140, medicinal plants 273, 405
144, 183, 189–191, 194–196, 199–200, medicine xxi, 2, 3, 5, 10, 53, 62, 63, 91, 93,
204, 232, 253–255, 261, 265, 273, 94, 105, 153, 154, 156, 163, 181, 183, 189,
287–289, 304, 310, 312, 314–315, 322, 191–194, 201, 203, 211, 231, 232, 243,
341–342, 346, 347, 359, 360, 378, 380, 245, 246, 255, 274, 286, 287, 364, 386,
381, 385–386, 391, 401, 405 402–404, 406
inhibitors 5, 6, 16, 21–26, 33, 61, 63, 135, metabolic engineering 212, 215, 221,
157, 158, 160, 162, 164–166, 287, 288, 297–298, 362
294, 295, 299, 380 metabolomic 27, 32–34, 63
Index 429
metagenomics xxiii, 4, 7, 122, 378 Penicillium 57, 62, 102, 103, 106, 108,
microalgae xxi, 9, 55, 80, 175, 180–183, 113, 120
303, 304, 310–312, 314–318, 321, 322, peptides 3, 16, 32–36, 58, 113, 160, 307,
324–327, 359, 362, 363, 381, 385 310, 311, 381, 389
microbial fuel cell (MFC) 319, 324–325 pesticidal 36, 54, 336
microorganisms 3, 4, 7–9, 57–59, 63, 91, Phanerogams 1
99, 100, 104, 105, 111, 113–115, 118, 119, pharmacological 6, 153–156, 212, 267,
131, 135, 143, 160–163, 166, 200, 266, 273, 289, 382, 406
273, 298, 299, 316, 317 phenolic acids 133, 141, 143, 182, 183,
mineral micronutrients xxi, 9, 270, 274, 316
293–299 phenols 58, 60, 110, 156, 165, 176, 177,
minerals xxi, 9, 57, 103, 133, 138, 139, 181, 261, 262, 265, 266, 270, 273, 286,
201, 286, 293–299, 304, 309, 318–319, 340, 414
325, 327, 348, 367 photosynthetic 156, 181, 303, 304, 315,
monoterpenes 162, 198, 262, 264, 319, 324, 364
265, 267–269, 272, 273, 275, 281, pH stabilizers 194, 196
287–289, 340 phytases 55, 101
morphological 1, 16, 303, 365 phytodetoxification 73
phytoextraction 73, 77–80, 368
n phytohemagglutinin 24, 30, 31
nanoparticles 59, 60, 73, 161 phytohormones 221, 309, 318, 326
natural resources 2, 3, 8, 53, 64, 92, 96, phytostabilization 73, 78, 79
131, 137, 153, 199, 200, 231, 241, 304, phytosterols 269, 304, 309, 317–318,
336, 357 379
nature xxi, 3, 6, 19, 22, 25, 26, 28, 31, 34, phytovolatalization 73
75–77, 92, 93, 96, 107, 113, 115, 142, 189, pigments 195, 204, 263, 267, 269, 270,
211, 212, 215, 247, 261, 263, 275, 284, 273, 303, 304, 308, 311, 313–319, 377,
286–288, 306, 318, 327, 336, 337, 339, 383, 389
340, 342, 343, 348, 349, 357, 364, 368, plant biodiversity 1–10, 15–37, 91–96,
377, 401, 403 153–166, 191
plant‐derived 4, 9, 62, 153, 154, 156,
o
164–166, 183, 184, 200, 412
omics 7–8, 378
pollutants 57, 58, 73, 77, 326, 367
orchidaceae 237, 247, 402
polyphenols 60, 133, 165, 175, 183, 189,
orchids 161, 288, 402–407, 412–414
197, 294, 304, 309, 316–317, 327, 378,
oxidative stress 175, 183, 184, 197,
380, 383, 388, 390, 409
314, 315
polysaccharides 101, 109, 116, 133,
p 141, 165, 175, 182, 184, 312–313,
paint additive 73 322, 323, 361, 377–387, 389,
paraffins 263 390, 407–414
pathogens 9, 15, 21, 24, 116, 141, 156, polyunsaturated 182, 311–312, 378
164, 165, 267, 270, 273, 290, 352, 363 precursor feeding 214–215, 220, 221
430 Index
preservative 73, 190, 194–196, 199, 204, sustainable xxi, 9, 53, 57, 64, 75, 77, 92,
232, 242, 247, 253 94–96, 100, 131, 132, 144, 189, 196,
proteinase inhibitors 16, 21–24, 33 199–202, 221, 297, 306, 324, 342, 347,
proteins 4, 5, 7, 8, 15–37, 56, 57, 92–94, 350, 351, 369, 391
101, 104, 108, 109, 112–114, 116, 120,
134, 139–141, 143, 156, 158, 160, 161,
t
tannins 56, 57, 154–157, 160–161, 165,
163, 165, 175, 189, 196, 266, 271, 296,
195, 197, 266, 282, 294, 295, 316
304, 306, 307, 310–311, 315, 317, 322,
terpene hydrocarbons 262
325–327, 361, 365, 368, 378–380, 384,
terpenes 159, 162–163, 211–212, 247,
386, 389, 390
261–275, 278, 279, 282, 286, 288–289,
q 340, 378, 382–383
quinones 160, 266, 269 Thallophyta 377
thickener 190, 194, 196, 386
r total phenolic content (TPC) 176–178,
reactive oxygen species (ROS) 9, 101, 175, 181, 182
311, 412 toxicity 18, 30, 32, 33, 76, 79, 80, 135, 156,
renewable resources 94, 144 181, 204, 287, 326, 338, 339, 351
ribosome‐inactivating proteins trace metals 73, 74
(RIPs) 16, 27–29 traditional knowledge 3, 4, 6, 194,
rice husk 131, 132, 135–139, 141, 347 200–203, 242, 254, 256, 336, 401, 402
root culture 216, 219, 220 transgenics 16, 18, 21, 24, 27, 30, 32, 33,
37, 56–58, 74, 93, 215, 219, 298, 365
s
Trichoderma 57, 108, 115, 119, 134
saponins 57, 143, 154, 175, 197, 198, 218
Trolox equivalent antioxidant capacity
seaweeds 10, 63, 190, 303, 310,
(TEAC) 176–178, 181, 182
316–318, 377–391
secondary metabolites (SMs) 1, 8, 9, 16,
u
21, 53, 57, 101, 117, 119, 133, 153, 154,
ultrasound‐assisted extraction
156, 160, 165, 194, 204, 211–221, 261,
(UAE) 253, 389
262, 265–268, 272–274, 278, 310, 316,
United Nation 91, 94, 140, 200, 203
318, 337, 340, 364, 378, 383, 386
shoot culture 216–221 v
slavic natives 155 vanilla 232, 237, 241, 247, 248, 254, 256,
species biodiversity 91 279, 282, 287, 405, 406
spices xxi, 9, 162, 194, 200, 231–256, 265, vegetables 9, 115, 133, 139, 140, 142, 143,
278, 294, 405 156, 160, 175–178, 181, 183, 231, 242,
sugarcane bagasse 102, 108, 117, 131, 244, 245, 273, 293, 294, 316, 318, 319,
134–138, 347 346, 351–353, 358, 360, 378
supercritical fluid extraction vinblastine 2, 5, 61, 62, 213, 214, 217, 218
(SFE) 388–389 vincristine 2, 5, 61, 62, 214, 217, 218,
superfood 378, 380 273, 402
Index 431
vitamins 133, 138, 139, 155, 195, 266, 293, xylanases 100, 101, 103, 115–116, 119,
304, 306, 308, 316, 325–327 121, 134, 140
volatile compounds 262, 265–268, 275,
y
340, 359
yeast 32, 102, 103, 109, 112, 140, 141, 266,
w 287, 323, 350, 351
wood preservatives 73
z
x zeaxanthin 54, 133, 314, 315
xanthophyll 314, 315

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Bioprospecting of Plant Biodiversity

for Industrial Molecules

Santosh Kumar Upadhyay

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Library of Congress Cataloging-in-Publication Data applied for

Set in 9.5/12.5pt STIXTwoText by Straive, Pondicherry, India

1 An Introduction to Plant Biodiversity and Bioprospecting 1

2 Entomotoxic Proteins from Plant Biodiversity to Control the

3 Bioprospecting of Natural Compounds for Industrial and Medical Applications:

4 Role of Plants in Phytoremediation of Industrial Waste 73

5 Ecological Restoration and Plant Biodiversity 91

6 Endophyte Enzymes and Their Applications in Industries 99

7 Resource Recovery from the Abundant Agri-biomass 131

8 Antimicrobial Products from Plant Biodiversity 153

8.3.1 Simple Phenols and Phenolic Acids 156

9 Functional Plants as Natural Sources of Dietary Antioxidants 175

10 Biodiversity and Importance of Plant Bioprospecting in Cosmetics 189

10.6.4 World Intellectual Property Organization (WIPO) 203

11 Therapeutic Lead Secondary Metabolites Production Using Plant

12 Plant Diversity and Ethnobotanical Knowledge of Spices and Condiments 231

12.5 Importance of Indian Spices 247

13 Plants as Source of Essential Oils and Perfumery Applications 261

14 Bioprospection of Plants for Essential Mineral Micronutrients 293

14.3 Bioavailability of Micronutrients from Plants 294

15 Algal Biomass: A Natural Resource of High-Value Biomolecules 303

16 Plant Bioprospecting for Biopesticides and Bioinsecticides 335

16.3.3 Rotenone 338

17 Plant Biomass to Bioenergy 345

18 Bioenergy Crops as an Alternate Energy Resource 357

18.2.1.2 Corn 359

19 Marine Bioprospecting: Seaweeds for Industrial Molecules 377

19.10.3 Microwave-Assisted Extraction (MAE) 389

20 Bioprospection of Orchids and Appraisal of Their Therapeutic Indications 401

Variyata Agrawal Fabrice Fekam Boyom

Puneet Singh Chauhan Institute of Urban Agriculture

Achintya Kumar Dolui

Ramya Krishnan School of Public Health

Aakanksha Kumar Shakti Mehrotra

Thakku R. Ramkumar Akanchha Shukla

Sangeeta Saxena Ananya Singh

Pankaj Srivastava Jagdeep Verma

Pankaj Kumar Verma

About the Editors

Dr. Santosh Kumar Upadhyay is

0005092138.INDD 24 06-08-2021 18:41:21

An Introduction to Plant Biodiversity and Bioprospecting

*Current: Accubits Technologies, Thiruvananthapuram, Kerala, India

Bioprospecting of Plant Biodiversity for Industrial Molecules, First Edition.

1.2.1 Chemical Prospecting

1.2.2 Gene Prospecting

1.2.3 Bionic Prospecting

1.3 ­Significance of Plants in Bioprospecting

1.4 ­Pros and Cons of Bioprospecting

Although bioprospecting has a huge list of advantages, it is not immune to certain

1.5 ­Recent Trends in Bioprospecting

1.6 ­Omics for Bioprospecting and in silico Bioprospecting

1.7 ­An Insight into the Book

Entomotoxic Proteins from Plant Biodiversity to Control

Bioprospecting of Plant Biodiversity for Industrial Molecules, First Edition.

1.3 Significance of Plants in Bioprospecting

1.4 Pros and Cons of Bioprospecting

1.5 Recent Trends in Bioprospecting

1.6 Omics for Bioprospecting and in silico Bioprospecting

1.7 An Insight into the Book

2.5 Ribosome-Inactivating Proteins (RIPs)

2.10 Ureases and Urease-Derived Encrypted Peptides

3.2 Why Bioprospecting Is Important

3.3 Major Sites for Bioprospecting

3.5 Biopiracy: An Unethical Bioprospecting

3.6 Bioprospecting Derived Products in Agriculture Industry

3.7 Bioprospecting Derived Products for Bioremediation

3.8 Bioprospecting for Nanoparticles Development

3.9 Bioprospecting Derived Products in Pharmaceutical Industry

3.10 Conclusion and Future Prospects

4.2 Different Toxic Materials from Industries