New Horizons in Natural Compound Research 0443152322, 9780443152320

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New Horizons in Natural

COMPOUND RESEARCH

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Progress in Biochemistry and Biotechnology

New Horizons in Natural

COMPOUND RESEARCH

Edited by

SURYA NANDAN MEENA VINOD NANDRE KISAN KODAM RAM SWAROOP MEENA

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2023 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Notices

Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-443-15232-0 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

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Contents

List of contributors Biographies Preface

1. Natural compounds for health and environment: past, present, and future

xiii xxi xxiii

1

Shreeram Suresh Joglekar, Yogini Soman and Anup Atul Kale 1. Introduction 2. Vital natural compounds 3. Future of natural compounds 4. Conclusions References

2. Recent advances in extraction of natural compounds

1 3 10 11 12

17

Mahesh S. Majik and Umesh B. Gawas 1. Introduction 2. Extraction methodologies and their applications 3. Concluding remarks Acknowledgments References

3. Cutting edge approaches for natural product purification

17 18 29 29 29

35

Aiyshwaryalakshmi and Joyita Sarkar 1. Introduction 2. Extraction 3. Pre-isolation 4. Isolation 5. Purification 6. A typical approach for isolation of anthocyanin monomers from red cabbage 7. Conclusion Acknowledgment References

35 36 38 39 41 42 43 43 44

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Contents

4. Mass spectrometry-based metabolomics for high-throughput natural products screening and compound discovery: an emerging trend

47

Jisun H.J. Lee and Deepak M. Kasote 1. Introduction 2. MS-based metabolomics 3. Applications of MS-based metabolomics in NP research 4. Conclusions References

5. Green synthesis of natural compounds

47 48 50 52 52

55

Aafaq Tantray, Nitin Rode, Lina Khandare and Santosh Terdale 1. Introduction 2. Conclusion Acknowledgment References Further reading

6. Diversity of chemical skeletons: a practical strategy to benefit

55 71 71 71 73

75

Gayatri D. Kotkar, Abhijit D. Shetgaonkar and Santosh G. Tilve 1. Introduction 2. Structural diversity in natural products 3. Semisynthetic or modified NPs 4. Synthetic approaches for building chemical skeletons 5. Conclusion References

7. Modern approaches for mining of novel compounds from the microbes

75 76 119 126 129 130

133

Savita Girawale, Surya Nandan Meena and Kisan M. Kodam 1. Introduction 2. Traditional methods and techniques used for mining of novel compounds along with their limitations 3. Modern use of databases in hunting new compounds 4. Mining for novel compounds using genomic databases 5. Proteomics approach for the mining for NCs 6. Metabolomics and mass spectrometry approach in the discovery of NCs 7. Conclusion and future prospects

133 134 136 136 139 141 142

Contents

Acknowledgments References

8. Informatics and computational methods in natural product drug discovery

142 143

147

Heena Shoket and Monika Pandita 1. Introduction 2. Evolution of bioinformatics concept: a new vision 3. High-throughput virtual screening Author contributions Conflict of interest statement References

9. Compound synergy in natural crude extract: a novel concept in drug formulation

147 155 158 163 163 163

167

Vivek T. Humne and Mahendra N. Lokhande 1. Introduction 2. What is the synergy effect? 3. Factors responsible for the synergetic effect 4. Synergistic effect on diabetes 5. Synergistic effect on antimicrobial activity 6. Synergistic effect of natural drugs on breast cancer cell 7. Conclusion References

10. Small molecules vs biologics

167 168 168 170 172 174 176 177

179

Preethi Poduval, Sonia Parsekar and Surya Nandan Meena 1. Introduction Acknowledgment References Further reading

11. Introduction to enzymes and organocatalysis

179 196 197 199

201

G.D. Ametefe, O.O. Ajani, E.E.J. Iweala and S.N. Chinedu 1. Definition and classifications of enzymes 2. Introduction to organocatalysis 3. Conclusion Suggestion References

201 215 220 220 220

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12. Natural products for the prevention and management of nephrolithiasis

225

Farah F. Al-Mamoori 1. Introduction 2. Prospects for nephrolithiasis management and improvements in natural product approaches References

13. Enzymatic preparation, purification, and therapeutic applications of marine oligosaccharides

225 226 229

231

Md Imran, Hetika Kotecha, Elaine Da Costa, Devika R. Jadhav and Sanjeev C. Ghadi 1. Introduction 2. Marine polysaccharides and their oligosaccharides 3. Enzymatic preparation of bioactive oligosaccharides from complex polysaccharides 4. Purification of oligosaccharides 5. Application of oligosaccharides in biomedicine 6. Conclusion and future prospects Acknowledgments References Further reading

14. Marine microalgae: an emerging source of pharmaceuticals and bioactive compounds

231 233 234 235 237 244 244 244 250

251

M.K. Anusree, K. Manasa Leela, M. Sreehari, Subhisha Raj, Arathi Sreenikethanam and Amit K. Bajhaiya 1. Introduction 2. Bioactive compounds from microalgae 3. Applications of bioactive compounds 4. Microalgae as a source of pharmaceuticals 5. Enhancement of algal metabolites 6. Conclusion References

15. Natural compounds in chemopreventive foods for prevention and management of non-communicable diseases

251 252 256 257 260 260 260

267

Asmita N. Bambole, Surya Nandan Meena, Vinod S. Nandre and Kisan M. Kodam Abbreviations 1. Introduction 2. Cause of civilization diseases 3. How oxidative stress cause NCDs and antioxidants can prevent NCDs 4. What are chemopreventive foods?

267 267 268 269 275

Contents

5. Antioxidants in chemopreventive foods to prevent or control civilization diseases 6. Challenges or loopholes in chemoprevention strategy 7. Conclusion and future perspective Acknowledgment References

16. Insect metabolome: New paradigm of novel metabolites discovery and its potential applications

275 279 284 285 285

293

Yogita P. Patil, Sharada D. Mohite, Ashok P. Giri and Rakesh S. Joshi 1. Introduction 2. Specialized methods for insect metabolome analysis 3. Insect metabolome: diversity and spatio-temporal dynamics 4. Uniqueness of insect metabolome 5. Insect-associated metabolome 6. Potential application of insect metabolites 7. Conclusion and future prospects Acknowledgment References Further reading

17. New targets for old drugs: drug repurposing approach for accelerating the drug discovery engine with minimum financial inputs

293 294 296 297 299 302 308 309 309 314

315

Navanath M. Kumbhar, M.A. Aparna, Snehal K. Nimal, Pallavi Shewale, Sagar Barale and Rajesh Gacche 1. Introduction to drug repurposing: beyond the “old wine in new bottle” 2. Experimental and computational approaches for drug repurposing 3. Repurposing the drugs for effective cancer management 4. Repurposing for cardiovascular diseases 5. Repurposing for neurodegenerative disorders 6. Drug repurposing in diabetes 7. Repurposing for viral diseases 8. Repurposing for microbial diseases 9. Conclusion References

18. Modern role of essential oils in drug discovery and medicinal products

315 317 317 324 325 329 331 333 339 339

351

Varsha Kumari, Priyanka Kumawat, Surya Nandan Meena, Shyam Singh Rajput, Ramesh Saini, Sharda Choudhary, Bhuri Singh, S.B. Yeri, D.K. Gothwal, Radheshyam Sharma, Poonam Kumari and Sarfraz Ahmad 1. Introduction 2. Methods of extraction of essential oils (EOs)

351 353

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3. Medicinal plants as a source of essential oils 4. Essential oils as a source of medicine and drug discovery 5. Conclusions References

19. Cyclodextrins (CDs) derived from natural source as an essential component in biopharmaceutics

353 357 358 358

361

Bharat Shinde, Priyanka Khot, Dadasaheb Patil, Pooja Doshi, Manish Gautam and Sunil Gairola 1. Introduction 2. Application of cyclodextrins in biopharmaceuticals 3. Regulatory aspects of CDs 4. Conclusion References

20. Natural compound-based scaffold to design in vitro disease systems

361 365 368 368 369

373

Chirag Varshney, Brijesh Kumar and Swapnil C. Kamble Abbreviations 1. Introduction 2. 2D cell culture system 3. 3D system 4. Scaffold for 3D cell culturing 5. Types of scaffolds 6. Conclusion 7. Future perspective Acknowledgment References

21. Natural compounds as pesticides, emerging trends, prospects, and challenges

373 373 374 375 376 376 385 385 386 386

391

Puja Gupta, Mohd Shahnawaz, Vasudeo Zambare, Naresh Kumar and Amanpreet Thakur 1. Introduction 2. Different sources for biopesticides 3. Mode of action of different biopesticides 4. Challenges 5. Prospects 6. Conclusions Acknowledgments References

391 392 398 405 406 407 408 408

Contents

22. Natural compounds as insecticidesda novel understanding

415

Gulzar A. Rather, Madhu Raina and Sakshi Saini 1. Introduction 2. Natural plant compounds as insecticides 3. Conclusion Acknowledgment References

23. Nanoformulations of natural compounds for herbicide and agri-food application

415 419 422 423 423

427

Rajashri Satvekar, Yogita Chavan, Akshyakumar Sahoo and Vinod S. Nandre 1. Introduction 2. Nanotechnologies in agriculture 3. Nanotechnologies in food science 4. Summary and future prospect Declaration of competing interest References

24. Natural compounds for bioremediation and biodegradation of pesticides

427 429 434 437 440 440

445

Mudasir Ahmad Dar, Mohd Shahnawaz, Khalid Hussain, Puja Gupta, Mohd Yaseen Sirwal, Beenish Sadaqat, Sehrish Gazal, Romana Akhtar, Sarita Parihar, Daochen Zhu, Charles Oluwaseun Adetunji, Tahira Fardos, Jyoti Parihar, Osemwegie Osarenkhoe Omorefosa, Rongrong Xie and Jianzhong Sun 1. Introduction 2. Pesticides and their impacts on sustainability 3. Concerns related to pesticide pollution 4. Bioremediation of pesticides 5. Types of pesticide bioremediion based on the type of microbes/enzymes 6. Conclusions and future perspectives Acknowledgments References

25. Role of natural compounds in metal removing strategies

445 448 449 453 461 474 475 475

489

Harshada Sowani, Mithil Mahale, Vinod S. Nandre, Surya Nandan Meena, Kisan M. Kodam, Mohan Kulkarni and Smita Zinjarde 1. Introduction 2. Natural compounds used in heavy metals removal

489 492

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3. Conclusions References

26. Policies, regulatory requirements, and risks in natural product research

504 505

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Priyanka Khot, Swapnil Jadhav, Dinesh Chaudhari and Kisan M. Kodam 1. Introduction 2. Protection of new plant variety 3. Natural products and international protection 4. Natural resources and associated traditional knowledge 5. Bioprospecting and biopiracy 6. Conclusion References Index

511 514 514 516 517 518 519 521

List of contributors

Charles Oluwaseun Adetunji Department of Microbiology, Edo State University Uzaiure, Edo State, Iyamho, Nigeria Sarfraz Ahmad S.K.N. Agriculture University, Jobner, Rajasthan, India Aiyshwaryalakshmi Institute of Chemical Technology Marathwada Campus, Jalna, Maharashtra, India O.O. Ajani Department of Chemistry, Covenant University, Ota, Ogun State, Nigeria Romana Akhtar Department of Zoology, Government Degree College Kishtwar, Kishtwar, Jammu and Kashmir, India Farah F. Al-Mamoori Department of Pharmaceutical Sciences, Faculty of Pharmacy, Zarqa University, Zarqa, Jordan G.D. Ametefe Department of Biochemistry, Covenant University, Ota, Ogun State, Nigeria M.K. Anusree Algal Biotechnology Lab, Department of Microbiology, School of Life Sciences, Central University of Tamil Nadu, Thiruvarur, Tamil Nadu, India M.A. Aparna Department of Biotechnology, Savitribai Phule Pune University, Pune, Maharashtra, India Amit K. Bajhaiya Algal Biotechnology Lab, Department of Microbiology, School of Life Sciences, Central University of Tamil Nadu, Thiruvarur, Tamil Nadu, India Asmita N. Bambole Department of Chemistry, Division of Biochemistry, Savitribai Phule Pune University, Pune, Maharashtra, India Sagar Barale Department of Microbiology, Shivaji University, Kolhapur, Maharashtra, India Dinesh Chaudhari The College of Military Engineering, Pune, Maharashtra, India Yogita Chavan MIT-School of Food Technology, MIT-ADT University, Pune, Maharashtra, India S.N. Chinedu Department of Biochemistry, Covenant University, Ota, Ogun State, Nigeria

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Sharda Choudhary National Research Centre on Seed Spices, Ajmer, Rajasthan, India Elaine Da Costa School of Biological Sciences and Biotechnology, Goa University, Taleigao Plateau, Goa, India Mudasir Ahmad Dar Biofuel’s Institute, School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, China Pooja Doshi Department of Chemistry, Division of Biochemistry, Savitribai Phule Pune University, Pune, Maharashtra, India Tahira Fardos Department of Botany, Government College for Women, Jammu, Jammu and Kashmir, India Rajesh Gacche Department of Biotechnology, Savitribai Phule Pune University, Pune, Maharashtra, India Sunil Gairola Serum Institute of India Pvt. Ltd, Pune, Maharashtra, India Manish Gautam Serum Institute of India Pvt. Ltd, Pune, Maharashtra, India Umesh B. Gawas Department of Chemistry, Government College of Arts, Science and Commerce, Khandola, Marcela, Goa, India; Department of Chemistry, Dnyanprassarak Mandal’s College and Research Centre, Assagao, Goa, India Sehrish Gazal Department of Environmental Science, Government Degree College Kishtwar, Kishtwar, Jammu and Kashmir, India Sanjeev C. Ghadi School of Biological Sciences and Biotechnology, Goa University, Taleigao Plateau, Goa, India Savita Girawale Department of Chemistry, Savitribai Phule Pune University, Pune, Maharashtra, India Ashok P. Giri Biochemical Sciences Division, CSIR-National Chemical Laboratory, Pune, Maharashtra, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India D.K. Gothwal S.K.N. Agriculture University, Jobner, Rajasthan, India Puja Gupta Department of Life Sciences, RIMT University, Mandi Gobindgarh, Punjab, India Vivek T. Humne Department of Chemistry, Shri R.R. Lahoti Science College, Amravati, Maharashtra, India

List of contributors

Khalid Hussain Department of Botany, University of Kashmir, Srinagar, Jammu and Kashmir, India Md Imran School of Biological Sciences and Biotechnology, Goa University, Taleigao Plateau, Goa, India E.E.J. Iweala Department of Biochemistry, Covenant University, Ota, Ogun State, Nigeria Swapnil Jadhav Subhadra Educational Society, Pune, Maharashtra, India Devika R. Jadhav School of Biological Sciences and Biotechnology, Goa University, Taleigao Plateau, Goa, India Shreeram Suresh Joglekar School of Biology, MIT World Peace University, Pune, Maharashtra, India Rakesh S. Joshi Biochemical Sciences Division, CSIR-National Chemical Laboratory, Pune, Maharashtra, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India Anup Atul Kale School of Biology, MIT World Peace University, Pune, Maharashtra, India Swapnil C. Kamble Department of Technology, Savitribai Phule Pune University, Pune, Maharashtra, India Deepak M. Kasote Plants for Human Health Institute, North Carolina State University, Kannapolis, NC, United States Lina Khandare Department of Physics, Savitribai Phule Pune University, Pune, Maharashtra, India Priyanka Khot Department of Chemistry, Division of Biochemistry, Savitribai Phule Pune University, Pune, Maharashtra, India Kisan M. Kodam Department of Chemistry, Division of Biochemistry, Savitribai Phule Pune University, Pune, Maharashtra, India Hetika Kotecha School of Biological Sciences and Biotechnology, Goa University, Taleigao Plateau, Goa, India Gayatri D. Kotkar School of Chemical Sciences, Goa University, Taleigao, Goa, India Mohan Kulkarni Department of Chemistry, Division of Biochemistry, Savitribai Phule Pune University, Pune, Maharashtra, India Brijesh Kumar School of Bio-Medical Engineering, Indian Institute of Technology, Banaras Hindu University, Varanasi, India

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Naresh Kumar RIMT University, Mandi Gobindgarh, Punjab, India Varsha Kumari S.K.N. Agriculture University, Jobner, Rajasthan, India Poonam Kumari S.K.N. Agriculture University, Jobner, Rajasthan, India Priyanka Kumawat S.K.N. Agriculture University, Jobner, Rajasthan, India Navanath M. Kumbhar Department of Biotechnology, Savitribai Phule Pune University, Pune, Maharashtra, India Jisun H.J. Lee Department of Plant Science and Technology, Chung-Ang University, Anseong, South Korea Mahendra N. Lokhande Department of Chemistry, Avvaiyar Government College for Women, Karaikal, Pondicherry, India Mithil Mahale Department of Chemistry, Division of Biochemistry, Savitribai Phule Pune University, Pune, Maharashtra, India Mahesh S. Majik Goa State Higher Education Council, Directorate of Higher Education, Porvorim, Goa, India; Department of Chemistry, Government College of Arts, Science and Commerce, Khandola, Marcela, Goa, India K. Manasa Leela Algal Biotechnology Lab, Department of Microbiology, School of Life Sciences, Central University of Tamil Nadu, Thiruvarur, Tamil Nadu, India Surya Nandan Meena Department of Chemistry, Division of Biochemistry, Savitribai Phule Pune University, Pune, Maharashtra, India Sharada D. Mohite Biochemical Sciences Division, CSIR-National Chemical Laboratory, Pune, Maharashtra, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India Vinod S. Nandre Department of Chemistry, Division of Biochemistry, Savitribai Phule Pune University, Pune, Maharashtra, India Snehal K. Nimal Department of Biotechnology, Savitribai Phule Pune University, Pune, Maharashtra, India Osemwegie Osarenkhoe Omorefosa Department of Food Science and Microbiology, Landmark University, Omuaran, Kwara, Nigeria Monika Pandita School of Biotechnology, Shri Mata Vaishno Devi University, Katra, Jammu and Kashmir, India

List of contributors

Jyoti Parihar Department of Pedagogy of Bioscience, Government College of Education, Jammu, Jammu and Kashmir, India Sarita Parihar Department of Economics, Government Degree College Kishtwar, Kishtwar, Jammu and Kashmir, India Sonia Parsekar Department of Biotechnology, Dhempe College of Arts and Science, Miramar, Goa, India Yogita P. Patil Biochemical Sciences Division, CSIR-National Chemical Laboratory, Pune, Maharashtra, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India Dadasaheb Patil Serum Institute of India Pvt. Ltd, Pune, Maharashtra, India Preethi Poduval Department of Biotechnology, Dhempe College of Arts and Science, Miramar, Goa, India Madhu Raina Department of Botany, University of Jammu, Jammu, Jammu and Kashmir, India Subhisha Raj Algal Biotechnology Lab, Department of Microbiology, School of Life Sciences, Central University of Tamil Nadu, Thiruvarur, Tamil Nadu, India Shyam Singh Rajput S.K.N. Agriculture University, Jobner, Rajasthan, India Gulzar A. Rather Agricultural Research Organization, Rishon, Israel Nitin Rode Department of Chemistry, Savitribai Phule Pune University, Pune, Maharashtra, India Beenish Sadaqat Biofuel’s Institute, School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, China Akshyakumar Sahoo Department of Technology, Shivaji University, Kolhapur, Maharashtra, India Sakshi Saini Department of Botany, University of Jammu, Jammu, Jammu and Kashmir, India Ramesh Saini Deaprtment of Crop Science, Konkuk University, Seoul, Korea Joyita Sarkar Institute of Chemical Technology Marathwada Campus, Jalna, Maharashtra, India Rajashri Satvekar Department of Technology, Shivaji University, Kolhapur, Maharashtra, India

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Mohd Shahnawaz Biofuel’s Institute, School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, China; Department of Botany, University of Ladakh, Kargil Campus, Khumbathang, Kargil, Ladakh UT, India; Department of Botany, Government College for Women, Jammu, Jammu and Kashmir, India Radheshyam Sharma Jawaharlal Nehru Krishi Vishwa Vidhalya, Jabalpur, Madhya Pradesh, India Abhijit D. Shetgaonkar Dnyanprassarak Mandal’s College and Research Center, Assagao, Mapusa, Goa, India Pallavi Shewale Department of Biotechnology, Savitribai Phule Pune University, Pune, Maharashtra, India Bharat Shinde Serum Institute of India Pvt. Ltd, Pune, Maharashtra, India Heena Shoket School of Biotechnology, Shri Mata Vaishno Devi University, Katra, Jammu and Kashmir, India Bhuri Singh Agriculture University, Kota, Kota, Rajasthan, India Mohd Yaseen Sirwal Department of Chemistry, Government Degree College Kishtwar, Kishtwar, Jammu and Kashmir, India Yogini Soman In-vitro Toxicity and Mutagenicity, Intox Private Limited, Pune, Maharashtra, India Harshada Sowani Department of Chemistry, Division of Biochemistry, Savitribai Phule Pune University, Pune, Maharashtra, India M. Sreehari Algal Biotechnology Lab, Department of Microbiology, School of Life Sciences, Central University of Tamil Nadu, Thiruvarur, Tamil Nadu, India Arathi Sreenikethanam Algal Biotechnology Lab, Department of Microbiology, School of Life Sciences, Central University of Tamil Nadu, Thiruvarur, Tamil Nadu, India Jianzhong Sun Biofuel’s Institute, School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, China Aafaq Tantray Department of Chemistry, Savitribai Phule Pune University, Pune, Maharashtra, India Santosh Terdale Department of Chemistry, Savitribai Phule Pune University, Pune, Maharashtra, India Amanpreet Thakur RIMT University, Mandi Gobindgarh, Punjab, India

List of contributors

Santosh G. Tilve School of Chemical Sciences, Goa University, Taleigao, Goa, India Chirag Varshney Department of Technology, Savitribai Phule Pune University, Pune, Maharashtra, India Rongrong Xie Biofuel’s Institute, School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, China S.B. Yeri University of Agricultural Sciences, Raichur, Karnataka, India Vasudeo Zambare R&D Department, Om Biotechnologies, Nashik, Maharashtra, India; University Teknologi Malaysia, Skudai, Johor, Malaysia Daochen Zhu Biofuel’s Institute, School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, China Smita Zinjarde Department of Biotechnology (with jointly merged Institute of Bioinformatics and Biotechnology), Savitribai Phule Pune University, Pune, Maharashtra, India

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Biographies

Dr. Surya Nandan Meena is currently a DSK PDF at the Department of Chemistry, Savitribai Phule Pune University, Pune, India. Earlier Dr. Meena has secured the National Postdoctoral Fellowship through the Department of Science and Technology (DST), India, and worked at the National Institute of Oceanography, Goa for 2 years. He has completed his MSc (Agri) Plant Biotechnology from the University of Agriculture Sciences, Dharwad, India, and PhD in Biotechnology from the Department of Biotechnology, Goa University, Goa, India, through research fellowship of the Department of Biotechnology (DBT). Dr. Meena has more than 5 years of postdoctoral research experience in natural products chemistry which resulted in more than 10 peer-reviewed research papers in international journals, three book chapters, and editors of two books in Elsevier. Dr. Meena has presented his research work in different national and international conferences. Dr. Vinod Nandre is working as a Postdoctoral Fellow in the Department of Chemistry, Savitribai Phule Pune University, Pune, India. His postdoctoral work is based on silica extraction from e-waste and rice husk. He has obtained his PhD from Savitribai Phule Pune University, Pune, on the topic of arsenic metabolism in prokaryotic system and arsenic removal from waste. He has 21 international publications and 1 patent based on phosphate removal from industrial waste sample. He also has two book chapters to his credit. He has teaching experience in medical biochemistry and industrial experience.

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Dr. Kisan M. Kodam is a Professor of Biochemistry at the Department of Chemistry, Savitribai Phule Pune University, Pune, India. Dr. Kodam’s research interests are focused on environmental biotechnology. In particular, his research team is working on biodegradation of organic pollutants, arsenic, and metal bioremediation. He has 27 years of teaching and research experiences and he has supervised 16 students for their doctoral studies and mentored 5 postdoctoral fellows. He has published around 100 research publications and 4 book chapters. His citations are 2704 with an h-index of 29. He is also working on biotransformation reactions for synthesis of natural products and cytotoxicity analysis. He has reported two novel bacterial species, namely Alishewanella solinquinati and Microvirga indica isolated from dyes and metal-contaminated soils, respectively. His research work has been presented in many international conferences. He has been awarded as Fellow of Maharashtra Academy of Sciences, India, for his valuable contributions in the field of environmental biotechnology. Dr. Ram Swaroop Meena is an Agronomist working in the Department of Agronomy, Institute of Agricultural Sciences, Banaras Hindu University (BHU), Varanasi, India. Dr. Meena has been awarded Raman Research Fellowship by the Ministry of Education, Government of India. He has completed his postdoctoral research on soil carbon sequestration under Padma Shri Prof. Rattan Lal, World Food Prize Laureate, Columbus, USA. Dr. Meena has completed 10 external funded projects including, MHRD, ICAR, DST, etc., and published more than 130 international research and review papers in peer-reviewed reputed journals, and has an h-index of 52 and citations over 8000 with a total impact factor of 268.35. He has published 4 books at the national level and another 20 books (Springer, Elsevier, etc.) at the international level, and contributed to the books with 15 chapters at the national and 70 at the international level.

Preface The “Chemical biology” spans a wide range and includes a variety of science disciplines. This book entitled New Horizons in Natural Product Research describes recent progress in various fields in natural product research, such as pharmaceutical, agriculture, and environment. It highlights the new advancement in isolation, purification, and identification of natural compounds (NCs) from various sources. Twenty-six chapters make up this book, and each one was written by a highly qualified expert in their particular field of study. The first section of the book comprises of “Natural compounds for health and environment: past, present, and future” chapter which covers the overview of the NCs and their importance and major breakthroughs in the NCs discovery. Further chapters in this section are cutting-edge approaches for NC purifications, emerging trends in NC screening, and identifications that describe the latest techniques or methods used for NCs isolation, purification, and characterization. The chapter titled “Diversity of chemical skeletons: Different strategies for its benefit” is a key chapter of this book, the authors have described the reason for the diversity of the compounds and stressed their importance in biological activities. The second section of the book covers the latest innovations in techniques, methods, and scientific knowledge about the drug discovery from the NCs. This part includes some latest trending chapters such as “Compound Synergy in Natural Crude Extract: A Novel Concept in Drug Formulation”; “Green Synthesis of Natural Compounds”; “Role of Natural Compounds in Chemoprevention Foods”; and “Natural Compounds for Animal Free Disease Scaffold Model.” The editors are certain that these topics, which are highlighted in a book for the first time in depth, will be of great interest to a large readership. Third section of the book dedicated to the agriculture and environment covers broad topics such as “Natural Compounds as Insecticides: A Novel Understanding”; “Natural Compounds as Pesticides: Emerging Trends, Prospects, and Challenges”; “Natural Compounds as Herbicides and Nano-formulations”; “Role of Natural Compounds in Metal Removing Strategies”; and “Natural Compounds for Bioremediation and Biodegradation of Pesticides.” The contents of this section covered all the major environmental and agricultural concerns while emphasizing the role of NCs. The authors have raised the concerns in these areas and also provided the solutions to these issues through the most recent scientific findings, approaches, and methodologies connected to NCs. This section is followed by a chapter on intellectual property rights and NCs.

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The book New Horizons in Natural Compound Research focuses on research and practical techniques in several disciplines, encouraging young scientists to pursue unique research while also encouraging them to generate strong research ideas. The book will also help scholars understand what has been done and what still needs to be done in the fields of natural product research, technology intervention, and sustainability. Since text and visuals are equally important to us, earnest attempts have been made to use flow charts, tables, and figures to support the text’s clarifications and explanations. In order to increase the researchers’ sense of self-motivation, it is written in a simple and succinct manner. Graduate and postgraduate students can use this book to look into more research opportunities in NCs. The teachers and researchers from diverse life sciences institutes across the world will undoubtedly benefit from the most recent knowledge on the numerous research disciplines and approaches in natural product research. The editors are grateful to all the contributors for their active participation and prompt response, without their contributions the book would not have been possible. The editors express their sincere gratitude to Dr. Deepak, Dr. Imran, Dr. Aditi, Dr. Neeraj, Dr. Chaaya, Dr. Leena, Dr. Namdev, Dr. Virendra, Dr. Omkar, Dr. Deeksha, and Dr. Sagar, for their timely support, valuable inputs, and critical reviews. We would like to thank Elsevier book publication team for their support. Lastly, we would like to thank Prof. and Head, Department of Chemistry, and authorities of Savitribai Phule Pune University, Pune, India, for their encouragement and support in this endeavor. Surya Nandan Meena Vinod Nandre Kisan Kodam Ram Swaroop Meena

CHAPTER 1

Natural compounds for health and environment: past, present, and future Shreeram Suresh Joglekar1, Yogini Soman2 and Anup Atul Kale1 1

School of Biology, MIT World Peace University, Pune, Maharashtra, India; 2In-vitro Toxicity and Mutagenicity, Intox Private Limited, Pune, Maharashtra, India

1. Introduction 1.1 Natural compounds in history The most reliable source of medicine leads has consistently been natural chemicals (secondary metabolites) [1e4]. They have, however, recently seen a minor decline in interest in medication research and development efforts [1]. However, compared to conventional combinatorial chemistry, natural molecules continue to exhibit amazing structural diversity, opening the possibility of discovering new low molecular weight lead compounds. Numerous prospective natural lead compounds are yet to be uncovered because around 10% of the biodiversity from the biosphere has been assessed for possible effective biological activity. Thus, the challenge is figuring out how to admittance this natural chemical variation [3]. Even today, two essential oils that have been utilized since Mesopotamia (2600 B.C.) are Cupressus sempervirens (Cypress) and Commiphora species (Myrrh), which are used for a variety of diseases including coughs, colds, and inflammation [3]. About 700 plant-based remedies, including gargles, tablets, infusions, and ointments, are listed in the Egyptian medical record known as the Ebers Papyrus (dated to 2900 B.C.). Provable records of the uses of natural compounds can be found in the Chinese different medicine repositories [3]. Greeks had knowledge of the collecting, storage, and application of medicinal plants in the first century AD, and the majority of natural scientists at that time focused on the study of medicinal herbs. From the ancient time, cloisters in the majority of European nations kept their knowledge about natural compound, while those in the Arab world preserved their Greco-Roman knowledge [3]. Since Avicenna was a Persian pharmacist, he made substantial contributions to the fields of pharmacy and medicine through books like The Canon of Medicine [3]. The first persons to own and operate pharmacies privately were the Arabs (8th century). 1.2 Medicinal plants Many of the bioactive natural compounds that have been employed as medicines throughout history by conventional drugs, treatments, and oils are still unknown. Through different experimentation for hundreds of years while looking for meals that New Horizons in Natural Compound Research ISBN 978-0-443-15232-0, https://doi.org/10.1016/B978-0-443-15232-0.00017-5

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may be used to treat illnesses, including palatability tests or premature deaths, man has developed most of the knowledge about the benefits of medicinal plant natural products [5,6]. For instance, Indian tribes in southern California used the plant Salvia that thrives throughout the southwest part of US and northwest of Mexico to assist with birthing. It was thought that male newborn babies grew up to be the healthiest and strongest members of their communities and were immune to all respiratory illnesses for life [6]. Camel’s thorn, i.e., Alhagi maurorum Medik, produces a delicious, sticky substance from its stems and leaves on hot days. Ayurvedic practitioners have documented and asserted that the plant can aid in the variety of treatments. This gummy sap, known as “manna,” is primarily made up of melezitose, sucrose, and inverted sugar [7]. The Israelites also used it to treat violent diarrhea by boiling the roots and ingesting the extract while Konkani people used it to treat asthma [7]. The herb Ligusticum scoticum Linnaeus consumed raw on a daily basis will help in the disease prevention [8]; the root was also employed as a sedative in the Faeroe Islands an aphrodisiac, and a remedy for flatulence [9,10]. The deadly nightshade, Atropa belladonna, distributed on all continents of the world. It was effectively excluded from folk medicine compilations due to its wellknown poisonousness, and it looked to have gained acceptance as being risky to experiment with [11]. 1.3 Medicinal natural compounds from other sources Piptoporus betulinus, one of the fungi, produces the charcoal which is used as an antiseptic and disinfectant [12]. The part of P. betulinus used to control bleeding; they also created incredibly comfortable corn pads [13]. Agaricus campestris Linnaeux ex Fries, sometimes known as the field mushroom, is another illustration. It may be found in the Caribbean, as well as the northern and southern temperate zones. For the treatment of throat cancer, A. campestris was reportedly cooked in milk [14]. Early on, in the 17th and 18th centuries, lichens were valued significantly higher than oriental spices and were employed as colors. Although there are nonlichen-based pharmaceuticals on the commercial level now, their folkloric uses are well known [15]. Lichens have been utilized as a source of basic materials for medicines, cosmetics, and fragrances since ancient China and Egypt [16]. Usnea dillenius ex Adanson, for instance, is still offered pharma compass an active component in antidandruff soap and shampoos and is used to cure sore eyes. It was once used to treat conditions of the scalp. The U. subfloridana was used as a lotion after being cooked with butter and tobacco [11]. The Parmelia omphalodes, widely distributed in the group of islands in the North Atlantic Ocean, which is used to produce dye of brown colors. It was traditionally used to cure burns and cuts, as well as to treat painful sores under the chin, by sprinkling it on stockings before a voyage to avoid foot inflammation [17,18]. Comparatively, there aren’t many documented uses of the marine environment in con ventional medicine. However, Chondrus crispus and Mastocarpus stellatus were used to

Natural compounds for health and environment: past, present, and future

produce infusion as a traditional remedy against flu like symptoms including chest diseases was well known. Burns and kidney issues were also treated by boiling the algae in milk or water [19,20]. Additionally, it has been discovered that consuming of Porphyra umbilicalis algal juice for 3 weeks is beneficial against different malignancies, especially against mammary gland carcinoma cancer [21]. P. umbilicalis has also been used to cure dyspepsia. It was additionally cooked and given to cows in the spring to help them pass gas [9,22]. 1.4 Different class of metabolites as natural compounds All living things require the creation and decomposition of different biomolecules, and this process is known as primary metabolism. The substances engaged in these metabolic pathways are collectively known as primary metabolites [23]. The process through a living system produces substances referred to as secondary metabolites (Natural Compounds) is known as “secondary metabolism” and is frequently found to be interspecific and intraspecific to an organism [24]. The synthesis of secondary metabolites occurs because of an organism adapting to its microenvironment or as a potential safeguard against predators to ensure the survival of the organism. Secondary metabolites are typically not necessary for an organism’s growth, development, or reproduction. Secondary metabolite biosynthesis is generated from the basic biochemical reactions inside the cells which required for cell survival which generates intermediates molecules that finally lead to the development of secondary metabolites [23]. Despite there being a finite number of building components, secondary metabolites can form indefinitely. The most crucial components utilized in the manufacture of secondary metabolites are the precursor compounds like shikimic acid, mevalonic acid, and acetyl coenzyme A (acetyl-CoA). They take part in a variety of biosynthetic pathways involving a wide range of processes and biological reactions, such as Schiff base creation, alkylation, decarboxylation, aldol synthesis, and Claisen synthesis [23]. According to one theory, small biomolecules like different amino acids, acetate and shikimic acid, and intermediates forms “shunt metabolites” that have followed an alternative biochemical route, resulting in secondary metabolite biosynthesis [25]. These biosynthetic pathways change by either natural or unnatural factors to help the organism adapt or live longer [25]. These natural compounds are created by an endless number of terrestrial and marine creatures, and their distinctive biosynthesis gives rise to their distinctive chemical structures, which exhibit a wide range of biological functions.

2. Vital natural compounds Most early medicines were based on traditional medicinal practices, which were then followed by clinical, pharmacological, and chemical research [26]. The anti-inflammatory drug acetylsalicylic acid known as aspirin extracted from the bark of the willow tree Salix alba as a salicin that is probably the most renowned and recognized case to date [27]. The

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separation of numerous alkaloids from Papaver somniferum (opium poppy), e.g., morphine, an economically main medicine, was first described in 1803 (Table 1.1). The crude morphine obtained from the plant P. somniferum was heated in acetic anhydride to produce heroin (diacetylmorphine), which was further easily converted to codeine as an effective painkiller. Historically, poppy extracts employed for medical purposes, whereas the Arabs described opium as addicting [26]. Digitalis purpurea L. (foxglove) was first discovered in the 10th century, which contains the active ingredient digitoxin, a glycoside, was discovered to improve cardiac conduction and its contractibility. As a result, other drugs were gradually phased out in favor of digitoxin in the treatment of congestive cardiac failure, but they could have protracted detrimental effects on the heart [27]. The antimalarial medication quinine, isolated from the Cinchona succirubra and approved by the US FDA in 2004, had been used for millennia to treat mainly malaria and other ailments like fever, gastric problems, and throat infections, and malignancy. When the British initiated widespread cultivation of the plant in the mid-1800s, formal usage of the bark to cure malaria was established [27]. Pilocarpine, discovered in Pilocarpus jaborandi, is an amino acid derived alkaloid that has been applied in medicine for more than a century to treat types of glaucoma. The oral formulation of pilocarpine was used to treat dry mouth condition, during the radiation therapy for head and neck malignancies, by stimulating sweat glands to assess salt and chloride concentrations [28]. 2.1 Natural compounds from fungi From the beginning of time, several fungus species have been a part of human existence. They were used for eating, making alcohol, treating illnesses, and for cultural purposes. Fungi have explored for additional synthesis of enzymes, biopesticides, medicinally significant bioactive chemicals, and enormous production of various antibiotics as microbiology has progressed [54]. The penicillin is indubitably one of the most renowned natural products isolated from the fungus Penicillium notatum by Fleming in 1929 [55]. Thus, the 1945 Nobel Prize was awarded to Chain and Florey (together with Fleming) for the in vivo testing of penicillin [56]. It was necessary to use a countercurrent extractive separation process with high yields. Following its commercialization in the early 1940s, this finding led to the reisolation and clinical testing of synthesized novel penicillin’s, which ultimately altered drug discovery research [57e59]. Between 1942 and 1944, the first clinical data with the publication on penicillin G, there was a huge effort to develop novel antibiotics from microbes and bioactive natural compounds across the globe [29,60]. Even until 1968, when it was determined that all-natural lactams had been discovered, one of the traditional methods for finding beta lactams was still in use. Nonetheless, with the introduction of novel testing methods in the 1970s, the synthesis of bacterial strains supersensitive to lactams, activity for lactamase inhibition, and preciseness for sulfur containing

Natural compounds for health and environment: past, present, and future

Table 1.1 Summary of the natural compounds. S. No.

Natural product

Source

References

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Acetylsalicylic acid Salicin Morphine Digitoxin Quinine Pilocarpine Penicillin Norcardicin Imipenem Aztreonam Vancomycin Erythromycin Betulinic acid Bevirimat (PA-457) Ganoderic acid b Amrubicin hydrochloride Doxorubicin Torreyanic acid Paclitaxel (Taxol) Baccatin III Ingenol 3-O-angelate PG490-88 Combretastatin A-4 phosphate Calanolide A Calanolide B Prostratin Arteether Artemisinin Grandisine A Grandisine B Apomorphine Tubocaurarine Plitidepsin ET743 Spisulosine Cryptophycin Polycavernoside-A 4-Acetoxydictylolactone Dictyolide A Dictyolide B Nordictyolide Crenuladial

Plant Plant Plant Plant Plant Plant Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Fungi Plant Plant Plant Plant Plant Plant Plant Plant Plant Plant Plant Plant Plant Plant Marine Marine Marine Marine Marine Marine Marine Marine Marine Marine

[27] [27] [27] [27] [27] [28] [21,26,29,30] [31] [31] [31] [26] [1,23,26] [13,32e34] [35] [36] [23,26] [23,26] [37] [38] [23] [39,40] [41,42] [43,44] [45e47] [45e47] [45e47] [3,23] [3,23] [48] [48] [49] [23] [50e53] [53,79,80] [81] [85] [79,89] [90] [90] [90] [90] [90]

environment environment environment environment algae algae algae algae algae algae

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metabolites help in the development of novel antibiotic structural classes was made possible, e.g., norcardicins, carbapenems, and monobactams, by taking these antibiotic isolations into account. In addition to the glycylcyclines, a novel class of broadspectrum antibiotics, there are currently nine lactams, e.g., two cephalosporins, six carbapenems, and one venom, undergoing clinical studies or through drug registration [30]. The phyla Basidiomycota and Ascomycota consist of enormous group of woodrotting fungi known as polypores, which are a significant source of the biologically active substances utilized in medications. There are more than 20,000 species of basidiomycetes, with 500 Aphyllophorales described among them [31]. A significant amount of antibacterial activity was present in about 75% of the polypore fungi under study, indicating that they might be a promising source for the development of novel antibiotics. Many substances have been shown to have multidimensional effect of variety of illness, e.g., potent anticancer agent, anti-inflammatory, antiviral, cytotoxic, cardiovascular, and immunestimulant [61]. The uniqueness of fungal cells lies into its peculiar characteristics that can survive inside different plant tissues like intercellular spaces of plant stems, petioles, roots, etc. throughout their life cycle without displaying any symptoms of disease or infection [62e64]. These fungi are collectively referred to as endophytes, and they provide some of the most significant natural chemicals for the pharmaceutical industry [3]. Vancomycin, a glycopeptide-based antibiotic, was first discovered by Edmund Kornfeld in 1953 and produced via the growth of Amycolatopsis orientalis. This culture is mostly active against diverse group of bacteria including Gram-positive and Gram-negative bacteria, mycobacteria, and fungi. It is used to treat severe infections and against susceptible organisms in patients who are penicillin sensitive [26]. The primary macrolide antibiotic, erythromycin, was found in Saccharopolyspora erythraea and was composed of propionate molecules that were then joined to form a 14-membered macrocyclic unit. Erythromycin is used to treat mild to moderate upper and lower respiratory tract infections and has a broad spectrum of activity against gram positive cocci and bacilli [23]. Currently in clinical development are three semisynthetic ketolide derivatives of erythromycin, cethromycin (ABT-773, Restanza TM), EP-420 (by Enanta Pharmaceuticals), and BAL-19403 (by Basilea). Viral diseases that are fatal are caused by viruses, which are unicellular proteinaceous entities. There are very few naturally occurring substances or synthetically produced counterparts from fungus that have antiviral activities [65]. For example, betulinic acid, compound derived from Betula pubescens plant, was initially found to be a poor inhibitor of topoisomerase I and HIV replication [32,66]. Phase I studies are testing betulinic acid as a cancer chemopreventive medication [33]. Bevirimat (PA-457), which is being studied in Phase II preclinical trials, was obtained from the herb Syzygium claviflorum and is believed to interfere with the last stage of HIV protein synthesis [34]. Ganoderic acid, isolated from Ganoderma lucidum fruiting bodies and spores, had substantial anti-HIV-1 protease activity with an IC50 value of 20 M [35].

Natural compounds for health and environment: past, present, and future

In 2002, amrubicin hydrochloride, related to Anthracyclin group compound extracted from Streptomyces peucetius. Doxorubicin is a powerful chemotherapy drug for treating a variety of lymphomas and sarcomas [23,26]. Torreyanic acid was isolated from an endangered tree Torreya taxifolia [36] and evaluated in numerous cancer cell lines, where it was found to have increased potency/cytotoxicity in cell lines susceptible to protein kinase C, triggering by apoptosis [37]. 2.2 Natural compounds from plants Since ancient times, the medicinal properties of plants have been well-documented. Over millions of years, they have evolved and adapted to different microbial systems and environment producing distinctive secondary metabolites with a wide range of structural characteristics. Due to their ethnopharmacological characteristics, they have been used as a significant source of drugs for the development of new drugs; therefore, around 80% of population still utilizes plant-based traditional medicines for basic healthcare [67,68]. But of 122 plant-derived medicines, more than 80% were linked to their original ethnopharmacological purpose [69]. Research into medicinal plants as potential drugs has increased as a result of traditional medicine knowledge (complementary or alternative herbal products), and some naturally occurring compounds that have become wellknown pharmaceuticals have been isolated. Taxol or Paclitaxel, derived from the tree Taxus brevifolia, is the most frequently used breast cancer medication (Pacific Yew). The US Department of Agriculture first collected the bark in 1962 as part of their exploratory plant screening program at the National Cancer Institute [70]. The course of treatment may require for 2 g of the drug, and it takes around three mature trees that are at least 100 years old to produce 1 gr of Taxol. Taxol is now synthesized to meet the 100e200 kg per year (or 50,000 treatments) demand that exists [23]. Interestingly, Taxol received the multiple FDA approvals for diverse purposes in 1992 [71]. Low amounts of Taxol can be discovered in natural sources, but its synthesis has been successfully completed (despite being challenging and expensive) [38]. T. brevifolia and its derivatives, which are significantly more plentiful and accessible in Baccatin III, are examples of chemical counterparts that can be successfully transformed into Taxol [23]. Ingenol and 3-O-angelate, a derivative of the polyhydroxy diterpenoid ingenol produced from the sap of Euphorbia peplus, are further instances of antitumor substances that are now being tested in clinical studies for the treatment of skin cancer [50,72]. PG49088, a semisynthetic counterpart of triptolide known as 14-succinyl triptolide sodium salt, derived from Tripterygium wilfordii that is utilized in China for autoimmune and inflammatory disorders [39,40]. Moreover, phase II clinical investigations are currently being conducted on the stilbene derivative combretastatin A-4 phosphate, which is produced from the South African bush willow Combretum caffrum [65]. By acting as an antiangiogenic agent, this substance causes vascular blockages in tumors.

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The National Cancer Institute (NCI) and other organizations were compelled by the AIDS pandemic of the 1980s to look into natural compounds as potential therapeutic possibilities for the treatment of AIDS. Over 60,000 plant and marine organism extracts evaluated against HIV-1 infected white blood cells. The calanolide class of chemicals, which were isolated from Homalanthus nutans and Calonphyllum species and are known as prostratin and calanolide A and B, respectively, is the most significant result of these investigations [41,42]. However, Sarawak Medichem Pharmaceuticals licensed and tested Calanolide A for Phase II clinical studies, but no further drug development announcements have been made. The AIDS Research Alliance in Los Angeles, California, carried out phase-I human clinical trials of prostratin in 2010. In 2000, both artemotil and the artemisinin which was discovered from the plant Artemisia annua became recognized antimalarial drugs. However, this herb was originally used by Chinese to treat fevers and chills. As antimalarial medications, different artemisinin derivatives are in various stages of clinical development in Europe [3,23]. Currently, a synthetic trioxolane based on the 28 pharmacophore is used with piperaquine to treat malaria [43]. The indole alkaloids grandisine A and grandisine B, also known as isoquinuclidinone and indolizidine, are found in the leaves of the Australian rainforest tree Elaeocarpus grandis, as opposed to Grandisine A, which has a distinct tetracyclic skeleton structure. Both of these substances could be employed as analgesic leads because of their affinity for the human opioid receptor [44]. The alkaloid galantamine hydrobromide, which is derived from the plant Galanthus nivalis, has long been used to treat Alzheimer’s disease [73,74]. Apomorphine, a morphine derivative extracted from poppy (P. somniferum), works as a potent dopamine agonist as well as a fast-acting D1 and D2 dopamine receptor agonist to treat Parkinson’s disease [45]. Curare is a South American Indian arrow poison that is manufactured in the Amazon and Orinoco rain forests. In order to reduce the need for deep anesthesia during surgery, the active component tubocaurarine, which was extracted from the climber species Chondrodendron tomentosum (Menispermaceae), is used as a muscle relaxant. Due to tubocurarine’s dearth on the market, a number of synthetic analogues have been developed and are now preferred over the original substance. 2.3 Natural compounds from the marine environment Plants have arisen as a novel source of bioactive natural chemicals, despite the long history of the marine environment producing distinctive structural entities. Pharmaceutical companies started to realize that the ocean, which makes up 70% of the planet’s surface, would have a distinctive biodiversity and would be a source of new drug prospects [4]. The advent of contemporary snorkeling made it possible to explore the marine environment’s rich diversity of various organisms. The worldwide marine pharmaceutical market currently consists of a vast array of marine substances that have been researched and investigated for use in preclinical trials. In addition, there are now three FDA-

Natural compounds for health and environment: past, present, and future

approved medications and 13 compounds (or their derivatives) in various phases of clinical development [47]. Recently, a peptide-based medication called ziconotide, also known as Prialt, was isolated in a tropical cone snail and obtained a license to treat pain in a number of pathological disorders. The recently identified peptide plitidepsin, which is present in the Mediterranean tunicate Aplidium albicans, is currently being studied in Phase II clinical trials for the treatment of various tumors [47,73]. Ecteinascidin, however, was only isolated in very small quantities from the ascidian Ecteinascidia turbinata [49,74]. Due to the need for larger quantities for advanced preclinical and clinical testing, E. turbinata was produced on a large scale by aquaculture in open ponds, but semisynthesis has been well established [75,76]. Thus, Ecteinascidin, also known as Trabectedin, was given a license by the European Union in October 2007 as the first marine anticancer drug and is currently finishing up important Phase-III trials in the United States for approval. The saltwater clam Spisula polynyma, from which the peptide medicine Spisulosine was developed, showed notable selectivity against solid tumor cells [50e52]. In the middle of the 1990s, cryptophycin was chosen for clinical trials. These trials advanced to phase-II before being stopped due to toxicity and ineffectiveness [53]. 2.4 Natural compounds from marine algae More than 30,000 species of algae exist in the world, which helps to keep the biosphere’s oxygen levels stable. Algae also serve as a rich supply of structurally distinct natural substances as well as food for people and marine animals as well as a source of medication and biofertilizers [77]. Diverse terpenoids with oxygen and nitrogen heterocycles, phenazine group derivatives, and brominated heterocyclic groups, which are responsible for a variety of biological functions, have been identified from marine algae [78]. Numerous algae species have been shown to possess antibacterial and antifungal activities [79]. Seafood poisoning is caused by the substance Polycavernoside-A poisonous glycoside, which was isolated from the alga Polycaverosa tsudai. A brown alga called Dictyota dichotoma produced a wide range of chemicals, including 4-acetoxydictylolactone, diterpenes, dictyolides A and B, and nordictyolide, which has anticancer properties. Dilophus ligatus generates a substance called crenuladial, which has been found to have antibacterial effects against Aeromonas hydrophyla, Micrococcus luteus, and Staphylococcus aureus [80,81]. Researchers have been searching for secondary metabolites in the red algae genus Laurencia (Rhodophyta) since the 1970s. Halogenated sesquiterpenes are produced by Laurencia, while diterpenes are the most common secondary metabolites. Additionally, this genus is exceptional in that it produces C15-acetogenins and the chamigrenes class of compounds, which are halogenated terpenes, allowing it to grow in a variety of geographical zones [82,83]. With the development of synthetic chemical pesticides during the past 50 years, agriculture productivity has improved [84]. Additionally, the search for novel pesticides has become important because to the significant growth in resistance to existing control agents. According to reports, between 1984 and 1990, varied insect resistance

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to synthetic chemical pesticides grew by 13% [85,86]. Because of this, a lot of study has focused on extracting insecticidal substances from marine algae. This has led to the isolation of over 40 active components [86]. Examples of natural pesticides include the isolation of cyclic polyhalogenated monoterpenes from the red alga Plocamium cartilagineum that are effective against the leafhopper Macrosteles fascifrons, for example, 1-(2-E-chlorovinyl)-2,4,5-trichloro-1,5-dimethylcyclohexane. All of these have been shown to have considerable insecticidal effects against mosquito larva, including Laurepinnacin and (Z)-Laureatin, an acetylenic cyclic ether produced from the red alga Laurencia pinnata.[87] 2.5 Natural compounds from marine sponges Because Porifera sp. lacks an organ system and a stalk, they must rely on a constant flow of water to obtain food, gases, and secretory bodies. All sponges serve as current feeders or filters and have weaker physical defense against their foes. Spongouridine and spongothymidine, which Bergmann isolated from the Caribbean sponge Cryptotheca crypta in the early 1950s [88], are still regarded as two of the most important discoveries to have come from marine sources. These substances were shown to exhibit antiviral activity, and their structural analogues were later converted into the therapeutic anticancer medication cytosine arabinoside, which was later converted into the potent anticancer chemical adenosine arabinoside [89]. This was an important discovery since it will open up new avenues for future research into the development of potent antileukemia agents based on nucleoside chemistry [65].

3. Future of natural compounds Omics is a new field of study in biology that aims to characterize all macromolecules and cellular components that contribute to cell survival. Examples of omics techniques include transcriptomics, proteomics, glycomics, and fluxomics. There is no one analytical method in the developing field of metabolomics that can characterize all low-molecularweight metabolites of a specific organism. The investigation of thousands of minute molecules (metabolites) in each biological sample is made possible by this developing field, which blends analytical chemistry, biology, and cutting-edge informatics. The best analytical platforms include mass spectrometry coupled with liquid, gas, or capillary chromatography, as well as nuclear magnetic resonance spectroscopy. Primary and secondary metabolites in tissues are extracted using particular extraction techniques that were created to effectively remove all metabolites in their original form before analysis in the solvents used. There is no one analytical platform and approach that can be used to evaluate all metabolites simultaneously because metabolite extracts show a huge chemical variety of metabolites. Multiple separation chemistries are necessary for the most thorough analysis [79].

Natural compounds for health and environment: past, present, and future

Numerous compounds can now be precisely and thoroughly evaluated in high-end instrumentation facilities using sophisticated informatics techniques thanks to developments in analytical tools. With the help of various algorithms that eliminate or filter background noise and, most crucially, data peak integration with massive data sets using various statistical analysis techniques, these technologies can extract information from the generated data. The biggest obstacle to metabolomics progress is the inability to identify the chemical make-up of detected signals. Even today, 60%e80% of all detected compounds are still unidentified [78,79], and the metabolomic community has taken on various projects to address this problem by building huge mass spectral and NMR spectrum libraries. Some of these unknown compounds, or secondary metabolites, might be unrecognized natural product sources. The terms “fingerprinting,” “footprinting,” “profiling,” and “target analysis” are frequently used in this subject. The goal of fingerprinting is to take a “snapshot” of the organism; however, the signals utilized to do so are highly technique-dependent and may not always be able to detect or identify particular metabolites. Whether a metabolite is well-known or brand-new, signals must be able to be linked to a specific metabolite. Target analysis [90] goal is to locate and measure a specific metabolite of interest. There aren’t many studies in the scientific literature that investigate the use of metabolomics in conjunction with conventional natural product chemistry techniques to identify novel bioactive natural chemicals. Research on plants has taken up the majority of these [90]. It is still challenging to find bioactive natural chemicals from plants because of their enormous chemical diversity and complexity.

4. Conclusions In conclusion, there are various benefits to merging natural product discovery techniques with metabolomics technologies. To start, by increasing the number of identifications in metabolomics data, we might be able to develop novel frameworks for evaluating biological activity for the many illnesses that are being considered. The high-throughput chemical characterization operations for a variety of natural resource species will benefit from the emerging novel technique in metabolomics science. Second, chemists must create various libraries that will aid in the development of enormous databases for natural compounds in the near future by increasing biological interpretation of metabolomic data for various natural compounds based on mass and NMR spectra. Increased detection and resolution of small molecule compounds in biological samples containing primary and secondary metabolites were made possible by advances in instrumentation science and related sophisticated separation techniques coupled with sensitive detectors. These developments will undoubtedly be used to advance natural product chemistry discover to identify effective new drug candidates that will aid in the maintenance of human welfare and help in the relief from various diseases.

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References [1] Mishra BB, Tiwari VK. Natural compounds: an evolving role in future drug discovery. Eur J 2011;46: 4769e807. [2] Rey-Ladino J, Ross AG, Cripps AW, Mcmanus DP, Quinn R. Natural products and the search for novel vaccine adjuvants. Vaccine 2011;29:6464e71. [3] Cragg GM, Newman DJ, Biodiversity. A continuing source of novel drug leads. Pure Appl Chem 2005;77:7e24. [4] Marderosian AD, Beutler JA. The review of natural compounds, Seattle, WA, USA. 2002. [5] Hicks S. Desert plants and people. San Antonio, TX, USA: Naylor Co; 1966. [6] Duke JA, Duke PAK, Cellier JLD. Duke’s handbook of medicinal plants of the bible. Boca Raton, FL, USA: CRC Press Taylor and Francis Group; 2008. [7] Dillenius JJ, Innys J. Synopsis methodica stirpium britannicarum. p. 482. [8] Svabo JC. Indberetninger fra en Reise I Færøe 1781 og 1782. Copenhagen, Denmark: Selskabet til Udgivelse af Færøske Kildeskrifter og Studier; 1959. [9] Beith M. Healing threads: traditional medicines of the Highlands and Islands. Edinburgh: Polygon; 1999. [10] Allen DE, Hatfield G. Medicinal plants in folk tradition: an ethnobotany of Britain and Ireland. Cambridge, UK: Timber Press; 2004. [11] Hatfield G. Country remedies: traditional East Anglian plant remedies in the twentieth century. Woodbridge, UK: Boydell Press; 2005. [12] M€ uller K. Pharmaceutically relevant metabolites from lichens. Appl Microbiol Biotechnol 2001;56:9. [13] Cameron J. The Gaelic names of plants (Scottish, Irish and Mnax) collected and artanged in scientific order, with notes on their etymology, uses, plant superstitions, etc., among the Celts, with Copious Gaelic, English and Scientific Indices. Glasgow, Scotland, UK: John Mackay; 1900. [14] Moloney MF. Irish, ethno-botany and the evolution of medicine in Ireland, Dublin, Ireland. 1919. [15] Vickery R. A dictionary of plant-lore. Oxford, UK: Oxford University Press; 1995. [16] Borlase W, Books. The natural history of Cornwall, Oxford, UK.  Folk medical beliefs and practices in the Aran Islands. Ireland: Galway; 1983. [17] Heithir RO. [18] Dewick PM. Medicinal natural compounds: a biosynthentic approach. West Sussex: John Wiley and Son; 2002. p. 520. [19] Maplestone RA, Stone MJ, Williams DH. The evolutionary role of secondary metabolitesda review. Gene 1992;115:151e7. [20] Colegate SM, Molyneux RJ. Bioactive natural compounds: detection, isolation and structure determination. Boca Raton, FL, USA: CRC Press; 2008. [21] Butler MS. The role of natural product in chemistry in drug discovery. J Nat Prod 2004;67:2141e53. [22] Aniszewski T. Alkaloids-secrets of life. In: Alkaloid Chemistry. Elsevier Science; 2007. p. 334. [23] Mann J. The elusive magic bullet: the search for the perfect drug. New York, NY, USA: Oxford University Press; 1999. [24] Abraham EP, Chain E, Fletcher CM. Further observations on penicillin. Lancet 1941;16:177e89. [25] Lax E. The Mold in Dr. Florey’s coat: the story of the penicillin miracle. New York, NY, USA: John Macrae/Henry Hol; 2004. [26] Kinghorn D, Pan L, Fletcher JN, Chai H. The relevance of higher plants in lead compound discovery programs. J Nat Prod 2011;74:1539e55. [27] Alder AL. The history of penicillin production. New York, NY, USA: American Institute of Chemical Engineers; 1970. [28] Williams JD. b-lactamases and b-lactamase inhibitor. Int. J. Antimicrob. Agents 1999;12:2e7. [29] Li R P Johnson, Porco JA. Total synthesis of the quinine epoxide dimer (þ)-torreyanic acid: application of a biomimetic oxidation/electrocyclization/Diels-Alder dimerization cascade. J Am Chem Soc 2003;125:5059e106. [30] Fellows L, Scofield A. Chemical diversity in plants. In: Intellectual property rights and biodiversity conservationdan interdisciplinary analysis of the values of medicinal plants. University Press; 1995.

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[31] Farnsworth NR, Akerele RO, Bingel AS, Soejarto DD, Guo Z. Medicinal plants in therapy. Bull WHO 1985;63:965e81. [32] Kiviharju TM, Lecane PS, Sellers RG, Peehl DM. Antiproliferative and proapoptic of triptolide (PG490), a natural product entering clinical trials, on primary cultures of human prostatic epithelial cells. Clin Cancer Res 2002;8:2666e74. [33] Fidler JM, Li K, Chung C. PG490-88, a derivative of triptolide, casues tumor regression and sensitizes tumors to chemotherapy. Mol Cancer Ther 2003;2:855e62. [34] Gustafson KR, Cardellin JH, Mcmahon JBI, Gulakowski RJ, Ishitoya J, Szallasi Z, Lewin NE, Blumberg PM, Weislow OS, Beutler JA. A nonpromoting phorbol from the Samoan medicinal plant Homalanthus nutans inhibits cell killing by HIV-1. J Med Chem 1992;35:1978e86. [35] Cox PA. Saving the ethnopharmacological heritage of Samoa. Pharm Biol 2001;39:33e40. [36] Davidson RN, Boer M, Ritmeijer K. Paromomycin. Trans R Soc Trop Med Hyg 2009;103: 653e60. [37] Carroll AR, Arumugan G, Quinn RJ, Redburn J, Guymer G, Grimshaw P, Grandisine A and B. novel indolizidine alkaloids with -opioid receptor binding affinity from the leaves of the human Australian rainforest tree Elaeocarpus grandis. J Org Chem 2005;70:1889e92. [38] Henríquez R, Faircloth G, Cuevas C. ET-743, Yondelis), aplidin, and kahalalide F. In: Anticancer agents from natural products. vol. 743; 2005. p. 215. [39] Manzanares CC, Garcia-Nieto R, Marco E, Gago F. Advances in the chemistry and pharmacology of ecteinascidins, a promising new class of anticancer agents. Curr Med Chem 2001. [40] Cuevas A, Francesch A. Development of Yondelis (trabectedin, ET-743). A semisynthetic process solves the supply problem. Nat Prod Rep 2009;26:322e37. [41] Cuadros R, Garcini EMD, Wandosell F, Faircloth G, Fernandez-Sousa JM, Avila J. The marine compound spisulosine, an inhibitor of cell proliferation, promotes the disassembly of actin stress fibers. Cancer Lett; 2000. p. 23. [42] Salcedo M, Cuevas C, Otero G, Sanchez-Puelles JM, Fernandez-Sousa JM, Avila J, Wandosell F. The marine antitmor compound ES 285 activates EGD receptors. Clin Cancer Res 2003;9. [43] Salcedo M, Cuevas C, Sanchez-Puelles JM, Otero G, Sousa JMF, Avila J, Wandosell F. A novel antitumoral compound, interacts with EDG receptors. In: Proceedings of American Association for cancer research 94th meeting; 2003. p. 11e4. [44] Trimurtulu I Ohtani, Patterson GML, Moore RE, Corbett TH, Valeriote FA, Demchik L. Total structures of cryptophycins, potent antitumor depsipeptides from the bluegreen alga Nostoc sp. strain GSV 224. J Am Chem Soc 1994;116:4729e37. [45] Faulkner DJ. Marine natural products. Nat Prod Rep 1988;20:269e309. [46] Baslow MN. Marine pharmacology; a study of toxins and other biologically active substances of marine origin. Baltimore, MD, USA: Williams & Wilkins Co; 1969. [47] Faulkner DJ. Marine natural products. J Nat Prod Rep 2002;19:1e48. [48] San-Martin A, Negrete R, Rovirosa J. Insecticide and acaricide activities of polyhalogenated monoterpenes from Chilean Plocamium cartilagineum. Phytochemistry 1991;30:2165e9. [49] Georghiou P. Managing resistance to agrochemicals: from fundamental research to practical strategies. ACS Symp Ser 421 Am Chem Soc 1990:18e41. [50] Wright AE, Forleo DA, Gunawardana GP, Gunasekera SP, Koehn FE, Mcconnell OJ. Antitumor tetrahydroisoquinoline alkaloids from the colonial ascidian Ecteinascidia turbinate. J Org Chem 1990;55: 4508e12. [51] Mcconnell O, Longley RE, Koehn FE, Gullo VP, editors. Butterworth-Heinemann, The discovery of natural products with therapeutic potential, Boston, MA, USA; 1994. [52] Roessner U, Beckles DM. Metabolite measurements. In: Plant metabolic networks. Heidelberg, Germany: Springer; 2009. [53] Roessner U, Nahid A, Hunter A, Bellgard M. Metabolomicsdthe combination of analytical chemistry, biology and informatics. In: Comprehensive biotechnology. vol. 1. Springer; 2011. p. 447e59. [54] Buss AD, Waigh RD. Antiparasitic drugs. In: Wolff ME, editor. Burger’s medicinal chemistry and drug discovery, vol. 1. Wiley-Interscience; 1995. p. 1021e8.

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[55] Zjawiony JK. Biologically active compounds from aphyllophorales (polypore) fungi, J. Nat, K.D. Gwinn, C.L. Schardl, A. Friburg, Southern regional information exchange group (SRIEG-37) on the tall fescue endophyte. J Prod Agric 1992;5:189e90. [56] Petrini O. Taxonomy of endophytic fungi of aerial plant tissues. In: Fokkema NJ, van den Heuvel J, editors. Microbiology of phyllosphere. Cambridge University Press; 1986. p. 175e87. [57] Kashiwada Y, Hashimoto F, Cosentino LM. Betulinic acid and dihydrobetulinic acid derivatives as potent HIV agents. J Med Chem 1996;39:1016e7. [58] Yogeeswari P, Sriram D. Betulinic acid and its derivatives: a review on their biological properties. Curr Med Chem 2005;12:763e71. [59] Min S, Nakamura N, Miyashiro H, Bae KW, Hattori M. Triterpenes from the spores of gano- derma lucidum and their inhibitory activity against HIV-1 protease. Chem Pharm Bull J 2000. [60] Lee GA, Lobkovsky E, Clardy JC. Torreyanic acid: a selectively cytotoxic quinone dimer from the endophytic fungus Pestalotiopsis microspora. J Org Chem 1996;61:3232e3. [61] Fabricant S, Farnsworth NR. The value of plants used in traditional medicine for drug discovery. Environ Health Perspect 2001;109:69e75. [62] Cragg M. Paclitaxel (taxol): a success story with valuable lessons for natural product drug discovery and development. Med Res Rev 1998;18:315e31. [63] Cseke LJ, Kirakosyan A, Kaufmann PB, Warber SL, Duke JA, Brielmann HL. Natural compounds from plants. CRC, Taylor and Francis; 2006. p. 640. [64] Nicolaou KC, Yang Z, Liu JJ, Ueno H, Nantermet PG, Guy RK, Claiborne CF, Renaud J, Couladouros EA, Paulvannan K, Sorensen EJ. Total synthesis of taxol. Nature 1994;367:630e4. [65] Kedei N, Lundberg DJ, Toth A, Welburn P, Garfield SH, Blumberg PM. Characterization of the interaction of ingenol 3-angelate with protein kinase C. Cancer Res 2004;64:3243e55. [66] Ogbourne SM, Suhrbier A, Jones B. Antitumour activity of ingenol 3-angelate: plasma membrane and mitochondrial disruption and necrotic cell death. Cancer Res 2004;64:2833e9. [67] Deleu D, Hanssens Y, Northway MG. Subcutaneous apomorphine: an evidence-based review of its use in Parkinson’s disease. Drugs Aging 2004;21:687e709. [68] Marris E. Marine natural products: drugs from the deep. Nature 2006;443:904e5. [69] Alejandro M, Glaser KB, Cuevas C, Jacobs RS, Kem W, Little RD, Mcintosh JM, Newman DJ, Potts BC, Shuster DE. The odyssey of marine pharmaceuticals: a current pipeline perspective. Trends Pharm Sci 2010;31:255e65. [70] Rinehart KL, Lithgow-Bertelloni AM. Novel antiviral and cytotoxic agent, dehydrodidemnin B. PCT Int Pat Appl 1991. [71] Urdiales JL, Morata P, Castro IND, Sanchez-Jimenez F. Anti-proliferative effect of dehydrodidemnin B (DDB), a depsipeptide isolated from Mediterranean tunicates. Cancer Lett 1996;102:31e7. [72] Rinehart KL, Holt TG, Fregeau NL, Stroh JG, Keifer PA, Sun F, Li LH, Martin DG. Ecteinascidins 729, 743, 745, 759A, 759B, and 770: potent antitumor agents from the Caribbean tunicate Ecteinascidia turbinata. J Org Chem 1990;55:4512e5. [73] Kimura N Kamada, T Yoshikazu. Fifteen chamigrane derivatives from the red alga Laurencia nidifica. Bull Chem Soc Jpn 1999;72:289e92. [74] Duke SO, Menn JJ, Plimmer JR. Pest control with enhanced environmental safety. ACS Symp Ser 1993;514. [75] Sayed E, Dunbar KA, Perry DC, Wilkins TL, Hamann SP, Greenplate MT, Wideman JT. Marine natural products as prototype insecticidal agents. J Agric Food Chem 1997. [76] Watanabe K Umeda, M Miyakado. Isolation and identification of three insecticidal principles from the red alga Laurencia nipponica Yamada. Agric Biol Chem 1989;53:2513e5. [77] Rochfort S. Metabolomics reviewed: a new “Omics” platform technology for systems biology and implications for natural products research. J Nat Prod 2005;68:1813e20. [78] Roessner U, Beckles DM. Metabolite Measurements. In: Schwender J, editor. Plant Metabolic Networks. New York, NY: Springer; 2009. https://doi.org/10.1007/978-0-387-78745-9_3. [79] Roessner U, Nahid A, Hunter A, Bellgard M. Metabolomics-The combination of analytical chemistry, biology and informatics. In: Comprehensive Biotechnology. 1. Springer; 2011. p. 447e59. [80] Faulkner DJ. Marine natural products. J. Nat. Prod. Rep 2002;19:1e48.

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[81] Tringali C, Oriente G, Piattelli M, Geraci C, Nicolosi G, Breitmaier E. Crenuladial, an antimicrobial diterpenoid from the brown alga Dilophus ligulatus. Can. J. Chem 1988;66:2799e802. [82] San-Martin A, Negrete R, Rovirosa J. Insecticide and acaricide activities of polyhalogenated monoterpenes from Chilean Plocamium cartilagineum. Phytochemistry 1991;30:2165e9. [83] Kimura J, Kamada N, Yoshikazu T. Fifteen chamigrane derivatives from the red alga Laurencia nidifica. Bull. Chem. Soc. Jpn 1999;72:289e92. [84] Duke SO, Menn JJ, Plimmer JR. Pest Control with Enhanced Environmental Safety. ACS Symposium Series 1993;514. [85] Georghiou GP. In Managing Resistance to Agrochemicals: From Fundamental Research to Practical Strategies. ACS Symp. Ser. 421. Am. Chem. Soc 1990:18e41. [86] Sayed E., Dunbar K. A., Perry D. C., Wilkins T. L., Hamann S. P., Greenplate M. T., et al. Marine natural products as prototype insecticidal agents. J. Agric. Food Chem. [87] Watanabe K, Umeda K, Miyakado M. Isolation and identification of three insecticidal principles from the red alga Laurencia nipponica Yamada. Agric. Biol. Chem 1989;53:2513e5. [88] Mcconnell O, Longley RE, Koehn FE, Gullo VP. In: Butterworth-Heinemann, editor. The Discovery of Natural Products with Therapeutic Potential; 1994. Boston, MA, USA. [89] A. E. Wright, D. A. Forleo, G. P. Gunawardana, S. P. Gunasekera, F. E. Koehn, O. J. Mcconnell [90] Rochfort S. Metabolomics reviewed: A new “Omics” platform technology for systems biology and implications for natural products research. J. Nat. Prod 2005;68:1813e20.

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CHAPTER 2

Recent advances in extraction of natural compounds Mahesh S. Majik1, 2 and Umesh B. Gawas2, 3 1

Goa State Higher Education Council, Directorate of Higher Education, Porvorim, Goa, India; 2Department of Chemistry, Government College of Arts, Science and Commerce, Khandola, Marcela, Goa, India; 3Department of Chemistry, Dnyanprassarak Mandal’s College and Research Centre, Assagao, Goa, India

1. Introduction The negative impact of synthetic substances on health is one of the major concerns in this modern era. As consequence, pharmaceutical, food, and cosmetic industries are focusing more on natural products as the source of new chemical entities, and hence natural products have gained tremendous attention for their commercialization in the form of useful products [1]. Plants are the richest source of diverse chemicals, which are used not only as the traditional systems of medicines but also as modern medicines, food supplements, nutraceuticals, and as a source of lead molecules for synthetic drugs [2]. Natural sources such as plants, animals, microbes, etc. produce chemicals in the form of a complex mixture containing alkaloids, amino acids, glycosides, terpenoids, flavonoids, lignans, etc. To use them for the benefit of the human being either in the form of drugs or value-added chemicals, there is a need to separate these mixtures into individual components and to know their quality. The first step in the chemical value addition of any natural biomass involves the extraction of chemical entities using various technique from simple traditional extraction to the most advanced modern extraction methods, followed by purification and subsequent characterization to establish the complete chemical identity of a natural product. The steps involved in isolating natural products from natural sources are depicted in Fig. 2.1. Extraction is a process, which involves separation of chemical components from the natural biomass using a solvent or mixture of two solvents by following the standard procedures. The soluble secondary metabolites from natural sources are extracted in the organic solvent leaving behind the insoluble cellular marc of natural biomass. Moreover, extraction methods are useful in various ways such as in the preparation of a sample for biological testing, qualitative and quantitative analysis of bioactive compounds, and so on [3]. The extraction process is embedded with several issues which include (i) the stabilities of extractives and the solvent, (ii) the formation of artifact and chemical conversion during the extraction process, and (iii) properties such as purity, viscosity, and the toxicity of extraction solvent [4]. In recent years, extraction techniques have been continuously developed prompting the search for more efficient and environmentally friendly New Horizons in Natural Compound Research ISBN 978-0-443-15232-0, https://doi.org/10.1016/B978-0-443-15232-0.00010-2

© 2023 Elsevier Inc. All rights reserved.

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Figure 2.1 Outline of steps involved in obtaining natural compounds from various samples highlighting the importance of extraction techniques, which is a crucial step of the chemical analysis process.

extraction techniques [5] which involve (i) shortening operation times and less laborious, (ii) minimum usage of organic solvent, (iii) improving the efficiency of extraction, and (iv) cost-effective protocol. In order to overcome the shortcomings of the traditional solvent extraction process, several extraction approaches have been developed during the last two decades. These include ultrasonic-assisted extraction, microwave-assisted extraction, supercritical fluid extraction, accelerated solvent extraction, and enzyme-assisted extraction [6]. This chapter covers the highlights of various extraction techniques used in the separation of natural products along with their evolution over the period. Moreover, these green extraction methods are discussed with emphasis on the practical issues, the important parameters influencing their performance, and the advantages associated with them. Also, the applications of the various extraction methods in natural product chemistry are discussed with the help of representative examples.

2. Extraction methodologies and their applications 2.1 Classical solvent extraction The extraction methodologies for isolation of natural products from the diverse biomass have observed tremendous progress over the last several decades; however, the traditional solvent extraction method was also extensively used [7]. The broad range of bioactive compounds existing in natural resources must be effectively separated and recovered before subjecting it for purification and use. The traditional protocol of extraction involves two steps: (i) the biomass to be extracted is soaked in a suitable solvent and (ii) the soluble chemical constituent present in the biomass moves into the extraction

Recent advances in extraction of natural compounds

solvent by either the mass transfer actions of diffusion or permeation process. The classical extraction method relied on the choice of chemical nature of solvents and the use of heat and/or agitation and still survived in with the advancement in technology due to its simple operating conditions [8]. Examples of this method include the following: (i) Heat reflux extraction: The infusion and decoction are the two methods under this category wherein infusion involves pouring boiled water or solvent over the sample, allowing it to stand for 15 min with periodic stirring, and finally filtration to give an extract. Whereas in the decoction method, the sample materials to be extracted are boiled along with a specified volume of solvent followed by cooling and filtration to give an extract [9]. (ii) Soaking extraction: The maceration is the simplest method under this category. The sample to be extracted is soaked in the solvent in a corked conical flask for a period of one week, followed by filtration and evaporation of solvent to give an extract [10]. It is extensively used for the extraction of thermally labile components. The other method is percolation wherein the powdered sample is soaked in a suitable solvent for about 1e2 days. The solvent is decanted and the fresh solvent is added and kept for another 1e2 days for percolation. The procedure is repeated to get maximum yield, and the evaporation of solvent from combined filtrate gives crude extract. (iii) Soxhlet extraction: This method is used extensively for the isolation of chemical components from different types of biomass wherein the desired compound is sparingly soluble in the given solvent. This method involves the wrapping of sample powder in a thimble which is made up of filter paper and placed in the holder of the glass assembly of the Soxhlet apparatus [11]. The Soxhlet apparatus is fitted over a distillation flask and a condenser, and refluxed for several hours wherein the extract is collected into a distillation flask along with solvent. The evaporation of solvent gives the crude extract which is used for further analysis. These modes usually have drawbacks in terms of the long operation time, high sample size, and consume more volumes of organic solvents. One of the major limitations of Soxhlet extraction is the variation in the quality of final products as it involves heating for evaporation of the solvent and hence thermally labile components of the sample lose their identity. This has led to the further advancement in the extraction methodologies providing an efficient yield of extractions along with many more other benefits. 2.2 Ultrasound-assisted extraction (UAE) The development of green extraction methods for isolation of natural products has been demonstrated with numerous examples in the literature [12]. The utilization of ultrasound technology in the extraction process has tremendous scope in order to achieve

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the objective of sustainable green chemistry. In 2017, Chemat et al. reviewed the ultrasound assisted extraction techniques and their application in the food and pharmaceutical industries [13]. The sound waves with frequencies in the range of 20 kHz are capable of producing mechanical vibration in solid, liquid, and gas samples. These waves involve expansion and compression cycles during their travel through the sample. The expansion process is capable of creating bubbles in a liquid produces negative pressure and thus pulls the molecules apart, whereas the compression cycle pushes them together. The bubbles form, grow, and finally collapse producing a liquid jet which have a strong impact on the solid surface facilitating the movement of molecules from the solid phase to the liquid phase in an efficient manner. Through this mechanism, ultrasound in extraction is responsible for disrupting biological cell walls, which ultimately releases the contents into the extracting solvent [14]. Using ultrasound technology, extraction can be completed in the fastest way with added advantages in terms of high reproducibility with the reduction in the consumption of organic solvent and energy. The use of ultrasound-assisted extraction (UAE) is specifically recommended for thermally labile compounds which may be chemically altered under refluxing conditions prevail in the Soxhlet extraction conditions [15]. The versatility of UAE in natural product chemistry is highlighted in Table 2.1. 2.3 Microwave-assisted extraction (MAE) The conventional extraction strategies involve tedious procedures utilizing more volumes of organic solvents with limited applicability to thermally sensitive compounds. This has triggered the use of microwaves in the solvent extraction of bioactive compounds from diverse samples such as marine/terrestrial flora and fauna, environmental samples, geological materials, etc. [24]. The heating of the sample is done using microwave radiations of frequency in the range 0.3e300 GHz. Microwave radiations consist of electric and magnetic field that are perpendicular to each other. Microwaves can easily penetrate biological samples and interact with the polar molecules present in the sample to be extracted. The electric field causes heating in a sample via dual mechanisms consisting of dipolar rotation and ionic conduction. In ionic conduction, the changing electric field leads to the migration of ions which in turn is responsible for producing collision of ions with each other and with solvent molecules [25]. Similarly, polar molecules in samples migrate due to electromagnetic fields leading to the generation of heat. This unique heating mechanism of microwave radiation is capable of reducing the extraction time almost by 30 min compared to the heating process of the Soxhlet method. Through this mechanism, the components present in the sample absorb microwave energy based on their dielectric properties and the nature of the extracting solvent (e.g., nonpolar solvent heptane to polar solvent water) [26]. However, in certain cases, mixture of solvents was used to get enhanced selectivity in extraction. MAE system consists of two types:

Recent advances in extraction of natural compounds

Table 2.1 UAE used to obtain NPs from various sources. S. No.

Sources (marine or terrestrial)

Bioactive compound

1.

A. platensis

Proteins

2.

A. nodosum

FSPs, total soluble carbohydrates

Phenolic compounds

3.

L. hyperborean

Laminarin

Phenolic compounds 4.

A. nodosum

Phenolic compounds Laminarin

5.

Nanochloropsis sp.

Free fatty acids

6.

H. banksii

7.

E. cava

Phenolic compounds Phenolic compounds

8.

G. pusillum

9.

G. gracilis

Phycobiliproteins (R-PE and R-PC) R-PE

Optimal extraction condition (solvent/ enzyme/temp)

Room temperature, 60 min, W 0.1 M HCl, room temperature, 5 min, 20 kHz, 500 W, sonification amplitude 0.1 M HCl, room temperature, 5 min, 20 kHz, 500 W, 50% sonication amplitude 0.1 M HCl, room temperature, 15 min, 20 kHz Room temperature, 15 min, 20 kHz, 500 W Room temperature, 15 min, 20 kHz, 500 W 0.1 M HCl, room temperature, 15 min, 20 kHz 98% ethanol, 69.6 C, 5 min, 20 kHz, 500 W, 50% sonification amplitude 70% ethanol, 30 C, 60 min, 150 W 50% methanol, 30 C 12 h, 40 kHz, 200 W 0.1 M PBS, 30 C, 10 min, 120 mm sonication amplitude 0.26 M PBS; 10 min, 45 kHz, 400 W

Yield of extraction

References

84%

[16]

95.4 mg F/100 g DM/ 2573 mg G eq./100 g DM 2340.5 mg GA eq./100 g DM

[17]

6.2%

[18]

0.4%

0.16%

5.8%

7%

[19]

23.1 mg/g DM

[20]

6.2 g/100 g DM

[21]

0.16 mg/g DM/ 0.11 mg/g DM 1.6 mg/g DM

[22]

[23]

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focused or open vessel system and closed vessel system. In an open vessel system, the condenser is fixed on top of the extraction vessel to avoid the loss of volatile components under microwave conditions. It is preferentially employed for thermally stable compounds. In a closed vessel system, the extraction is performed at elevated temperature and pressure. However, the open vessel or atmosphere pressure microwave system can be even more effective than closed vessel method. In 2012, Delazar et al. outlined the principles of microwave-assisted extraction (MAE) methods along with the extracting protocols [27]. Further development of this technique has led to more advanced vacuum-MAE, nitrogen protected-MAE, ultrasonic-MAE etc. The details of these modified MAE have appeared in the form of a review article by Chan et al. [28]. MAE has been widely employed for the extraction of different types of samples which are summarized in Table 2.2. 2.4 Ionic liquid-assisted extraction (ILAE) Extraction of natural products in their pure form is of great interest, and ionic liquids are gaining growing interest for the extraction and separation of bioactive compounds from biomass matrices [40]. Ionic liquids are the salt type chemical compounds with low melting temperature and exist in the liquid state at temperatures below 100 C. These salts are ionic in nature and hence serve as very good solvent for both inorganic and organic compounds. It can be employed for the extraction of simple molecules to complex polymers as well as biomolecules. Consequently, the ionic liquids have replaced the organic solvents in the liquid-liquid extraction process over the years. For example, the solubility of ionic liquid in water largely depends on a type of counter anion, which can be easily modified by making the variation in the hydrophobic alkyl substituent of the cationic site [41]. The solubility in water is increased in the presence of inorganic anions such as halide, tetrafluoroborate, thiocyanate, sulfonic, trifluoroacetate, and nitrate; however, biphasic systems formed in the presence of organic anions like hexafluorophosphate or bis (trifluoromethylsulfonyl) imide anions and so on impart hydrophobicity to the ionic liquid. The common ionic liquid is consisting of an organic cation of phosphorus or nitrogen containing skeleton along with its organic or inorganic anion. The major five classes of ionic liquid include ammonium, phosphonium, imidazolium, pyridinium, and pyrrolidinium, which are widely used in extraction and separation processes in analytical chemistry (Fig. 2.2) [42]. The applications of UAE and MAE in the extraction and isolation of natural products from different bio-mass is discussed in Sections 2.1 and 2.2. Furthermore, the combination of these techniques in presence of ionic liquid as solvent has gained growing attention and hence its application has culminated in the form of several review articles in the literature [43]. Although ionic liquids have been extensively explored for various purposes, but the data on biodegradability and environmental impact is scarce which leaves enormous scope

Table 2.2 MAE used to obtain natural products from various sources. S. No.

Sources (marine or terrestrial)

Optimal extraction condition (solvent/ enzyme/temp)

Bioactive compound

1. 2.

A. platensis A. nodosum

Proteins FSPs, total soluble carbohydrates Phenolic compounds

3.

Vicia faba

Vicine

4.

Vitis vinifera

Terpenes

5. 6.

Mentha piperita Capsicum annuum

Volatile oil Carotenoids

7.

Ginseng

Ginsenoside

8.

Zingiber officinale

Zingiberene

9.

Melilotus officinalis

Coumarin

10.

Tobacco

Solanesol

11.

Artemisia annua

Artimisinin

12.

Liquorice root

Glycyrrhizic acid

13.

Lochroma gesnerioides

Withanolides

W, 1000 W, 3 min 0.1 M HCl, 5 min, 1000 W, 245 MHz 0.1 M HCl, 5 min, 600 W, 2450 MHz Methanol: water (1:1), 30 s, 1140 W Dichloromethane, 475 W, 90 C 10 min Hexane, alkanes Ethanol, acetone, dioxanel, 50 W, 120 s. Methanol, 300 W, 72.2 C, 2450 MHz, 30 s. Microwave absorption media (ICP, GP, ACP), 85 W, 100 C, 30 min Ethanol 50%, 50 C, 5 min Methanol, 700 W, 60 C, 12 min Water: acetonitrile (40: 60 v/v), 700 W, 50 C, 40 min Ethanol 40%e60%, 85e90 C, 5 min Methanol, 25 W, 40 s.

References

[29] [30]

[31] [32]

[33]

[34]

[35] [36] [37]

[38] [39]

Organic or inorganic cations R1 N+ R2 R R4 3

R1 N

N+ R

+

N+ R pyridinium

2

imidazolinium

ammonium

N R2 R1 pyrrolidinium

R2

R1 P+ R4

R3

phosphonium

Organic or inorganic anions X

-

PF6

BF 4

ROSO 3

PhSO 3

CH3 SO3

Figure 2.2 Common ionic liquid cation and anions used in liquid-liquid extraction.

24

New Horizons in Natural Compound Research

for research in this area [44]. The various applications of ionic liquids in obtaining natural compounds are summarized in Table 2.3. 2.5 Supercritical fluid extraction (SFE) For the last several decades, one of the most common methods for performing analytical separations was based on liquid-liquid extraction using nonpolar solvents like n-hexane or other chlorinated organic solvents. The use of supercritical fluids in separation chemistry is reported in the late 1970s; however, it was not been explored to its full potential [52]. Supercritical fluid extraction (SFE) is a process of isolation wherein natural products/bioactive components are extracted from organic biomass using supercritical fluid as an extracting solvent. The physicochemical characteristics of the supercritical fluids including the dissolving power can be controlled easily by varying either pressure or temperature without crossing phase boundaries, thus making them good solvent for extraction process. Supercritical CO2 is the most common solvent used in various extraction processes, since it can be employed at near ambient critical temperature (31 C) and thus more suitable for the extraction of bioactive compounds that are susceptible to degradation at a higher temperature. Moreover, the solvent strength of supercritical CO2 can be altered by varying temperature, pressure, and by the use of cosolvents. This property of supercritical CO2 makes it perfectly useful in various industries like food, aromas, essential oils, and nutraceuticals [53]. During the extraction process using supercritical fluid as the solvent, the biomass to be extracted is loaded into the extraction vessel equipped with temperature controller and pressure valves at the inlet and outlet in order to maintain optimum extraction conditions. The extraction vessel is pressurized with the fluid using a pump whereby the supercritical fluid along with dissolved components get transported into the separators [54]. The solvating power of fluid can be controlled by adjusting the pressure and the extract is collected at the lower part of the separator using a valve. SFE with supercritical CO2 is a method of choice for the extraction of thermally labile compounds, wherein removal of solvent is done simply by reducing the pressure to obtain the solvent free extract. This greener extraction technique is widely utilized for diverse applications like the removal of fat from food, vitamin E enrichment from natural sources, removal of alcohol from wine and beer, and so on. However, the extraction of polar compounds requires the use of polar solvents like Freon-22 or nitrous oxide [55]. Supercritical CO2 is widely used in this technique; however, polar compounds are difficult to extract in supercritical CO2 due to their limited solubility. Moreover, the solubility of the polar compounds in the supercritical CO2 solvent can be enhanced by the addition of polar solvents such as methanol, ethanol, acetonitrile, acetone, water, diethyl ether, dichloromethane, etc. [56]. The use of supercritical fluid in the extraction of natural products listed in Table 2.4.

Recent advances in extraction of natural compounds

Table 2.3 Ionic liquid used to obtain natural products from various sources. Sr. No.

Sources (marine or terrestrial)

1.

Apocynum venetum

Hyperoside, isoquercitrin

2.

Nelumbo nucifera

Phenolic alkaloids

3.

Polygonum cuspidatum

trans-Resveratrol

4.

Psidium guajava

Gallic acid, ellagic acid, quercetin

5.

Smilax china

trans-Resveratrol, quercetin

6.

Flos Lonicerae Japonicae

Caffeoylquinic acids

7.

Iris tectorum

Iristectorin A, iristectorin B, and tectoridin

8.

Saururus chinensis and Flossophorae

Rutin

Bioactive compound

Optimal extraction condition (solvent/ enzyme/temp)

1-butyl-3methylimidazolium tetrafluoroborate 1-alkyl-3methylimidazolium bromide, 1-alkyl-3methylimidazolium chloride, 1-alkyl-3methylimidazolium tetrafluoroborate 1-butyl-3methylimidazolium bromide 1-alkyl-3methylimidazolium chloride, bromide and other anions 1-alkyl-3methylimidazolium chloride, bromide and other anions N-butyl-Nmethylimidazolium bromide (water) N-butyl-Nmethylimidazolium tetrafluoroborate, 1hexyl-3methylimidazolium bromide and 1octyl-3-methyl imidazolium bromide (water) N-butyl-Nmethylimidazolium bromide, N-butylNmethylimidazolium chloride, N-butylNmethylimidazolium tetrafluoroborate (water)

References

[45]

[46]

[47]

[48]

[49]

[50]

[51]

25

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New Horizons in Natural Compound Research

Table 2.4 Supercritical fluid extraction to obtain natural products from various sources.

S no.

Sources (marine or terrestrial)

Bioactive compound

1.

Fermented orange pomace (Citrus sinensis)

Phenolic compounds

2.

Annatto seed (Bixa Orellana)

Geranylgeraniol, tocotrienols

3.

Chokeberry pomace (Aronia melanocarpa (Michx.)) Turmeric rhizomes (Curcuma longa L.)

Phenolic compounds

4.

Curcumin

5.

Strawberry leaves

Phenolic compounds

6.

Coriandrum sativum L. and Ocimum basilicum L. Tomato peels

Linalool

7.

Lycopene and b-carotene

Optimal extraction condition (solvent/ enzyme/temp)

Yield of extraction

References

Ethanol, ethanol: water (9:1), 15, 25 and 35 MPa, 40, 50 and 60 C Supercritical CO2 (density 290 kg/m3), 60 C, 10 MPa Ethanol (80% m/m), 35 C, 10 MPa

18e22 mg/g dry extract

[57]

39  9%

[58]

1.52 g per 100 g of pomace

[59]

Supercritical CO2, ethanol (at 25 C), water (at 60 C), 400 bar Supercritical CO2, 35 e60 C, 15 e30 MPa 40 C, 100, 300 bar

23.4%

[60]

e

[61]

782 mg/g

[62]

Supercritical CO2, 50 e80 C, 300 e500 bar, 105 min

32%e60% for lycopene and from 28% to 58% for b-carotene

[63]

Recent advances in extraction of natural compounds

Table 2.4 Supercritical fluid extraction to obtain natural products from various sources.dcont’d

S no.

Sources (marine or terrestrial)

Bioactive compound

8.

Flower of Fritillaria thunbergii

Alkaloids, peimine, peiminine

9.

Piper auritum and Porophyllum ruderale Salvia ocinalis L. leaves

Essential oil

10.

Oxygenated monoterpenes, a-humulene, viridiflorol and manool

Optimal extraction condition (solvent/ enzyme/temp)

Yield of extraction

Ethanol: water (80% e100% v/v) cosolvent, 50 e70 C, 35 MPa, 1.5e3.5 h 40, 50 C, 10.34, 17.24 MPa

Total alkaloids: 0.9 mg/g, peimine (0.7 mg/g), peiminine (0.07 mg/g) 2.37e3.09 g oil/100 g dry material

15 MPa or 20 MPa

0.242% e7.361%

References

[64]

[65]

[66]

2.6 Enzyme-assisted extraction (EAE) Enzymes are well known as biocatalysts which are derived from microbes (bacteria, fungi), animal organs, vegetable extracts, and fruits. The basic principles of enzymeassisted extraction (EAE) involve catalytic hydrolysis reaction via disruption of plant cell wall under optimum experimental conditions which then releases the intracellular components into the extraction medium. The mechanism involves binding of plant cells of a biomass with an active site on the enzyme which causes change in the shape of the enzyme to fit the substrate in the active sites. Consequently, the active components present in the cells are released in the extraction medium [67]. Although enzymatic extraction looks to be simple, its effective utilization in application requires knowledge of various parameters such as biochemical composition, mechanism of action, and optimal operational conditions [68]. Additionally, enzymes are highly specific and hence the controlled temperature condition is maintained for the extraction of thermos-sensitive compounds such as perfumery agents, flavors, volatile oil, etc. Among the various types of enzymes, carbohydrate hydrolyzing enzymes are preferentially used during EAE techniques due to their effectiveness over other enzymes [69]. The selective examples of extraction of natural products using EAE are given in Table 2.5.

27

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New Horizons in Natural Compound Research

Table 2.5 Enzyme assisted extraction to obtain natural products from various sources. S. No.

Sources (marine or terrestrial)

Bioactive compound

Enzyme used in the extraction

Yield of extraction

1.

Rosemary leaves

Total phenolic content

Increased extraction efficiency by 30% compared to conventional extraction

[70]

2.

Ulmus pumila L. barks

Polyphenols

w16 mg/g

[71]

3.

Grape marc seeds Concord grape juice Longan (Dimocarpus longan Lour.) Bay leaves (Laurus nobilis L.)

Phenolics

Disodium phosphatecitric acid buffer at pH 5, proteolytic (alcalase 2.4L FG) and pectinolytic (bioprep 3000L) enzyme Cellulase, pectinase and b-glucosidase Pectinase

w20 mg/g

[72]

e

[73]

8.55%

[74]

Cellulase, hemicellulose and xylanase

92.5%

[75]

Viscozyme L, pectinase and cellulase Pectinex

Max. 280 mg/ 100 g

[76]

w7 mg/g

[77]

4. 5.

6.

7.

Capsicum annuum

8.

Crocos sativus

Polyphenols Polysaccharide a-terpenyl acetate, methyl eugenol, linalool, apinene, sabinene and b-pinene Carotenoid

Anthocyanins

Cellulase and pectinase Cellulase

References

2.7 Accelerated or pressurized-solvent extraction (PSE) Accelerated solvent extraction is a solid-liquid type extraction which is carried out at elevated temperatures, i.e., in the range of 50e200 C and at a pressures in the range of 10e15 MPa. It is also known as pressurized solvent extraction (PSE) [78]. In this method, the extraction is carried out under pressure to maintain the solvent in liquid state at high temperature below its critical temperature. The use of high temperature enhances

Recent advances in extraction of natural compounds

the diffusivity of the solvent and high pressure allows the extraction cells to be filled faster, thus forcing the liquid into extraction medium [79]. The efficiency of extraction can be improved further by employing the sequential extraction using a solvent of different polarities. Accelerated solvent extraction is the best alternative method for supercritical fluid for the extraction of polar compounds from natural biomass [80]. The amount of solvent and time required for extraction have shown a dramatic decrease in the accelerated solvent extraction method as compared to the traditional extraction. This technique is extensively utilized for the extraction of stable organic pollutants from environmental samples at high temperature [81].

3. Concluding remarks Solid-liquid extraction is widely used for the early isolation/purification of natural products from various natural bio-mass such as plants, animals, and microorganisms. The traditional extraction techniques like Soxhlet extraction, maceration, and percolation were popular over decades. However, these traditional extraction methods involve laborious procedures and utilize more quantities of organic solvents making them environmentally hazardous. In recent years, unconventional extraction techniques like UAE, MAE, SFE, and pressurized solvent extraction have emerged as efficient methods for the extraction of analytes from various natural samples. These greener and more cost-effective methods have ample scope to extend their applicability for various types of samples. Interestingly, the combination of these extraction techniques is more helpful in the separation of complex mixtures of analytes. We believe that the scientific and technological advances discussed in this chapter will provide a strong basis for the investigation and application of newer/existing extraction techniques to isolate natural compounds for commercialization and various applications.

Acknowledgments Authors are thankful to the Directorate of Higher Education, Government of Goa, India for research funding wide Grant No. 9/328/2016-17/SPSE-PP/DHE/3607 dated 17/06/2019.

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CHAPTER 3

Cutting edge approaches for natural product purification Aiyshwaryalakshmi and Joyita Sarkar Institute of Chemical Technology Marathwada Campus, Jalna, Maharashtra, India

1. Introduction Natural products are chemical substances possessing distinctive pharmacological or bioactive properties. Isolated and purified from natural sources like plants and/or animals, natural products provide wide scope to strategize solutions for health-related issues. They furnish raw and starting materials for various industries such as pharmaceutical, cosmetic, flavor, dietary supplements, etc. Plants mainly synthesize such molecules as secondary metabolites to combat adverse environmental conditions, aromas, colors as well as toxicity. Earlier, medicines derived from plants and animals were used to treat diseases and as source for various drugs [1e3]. The paradigm shifted and instead of traditional strategies, modern medicines gained tremendous popularity for treatment of pathological conditions [4e6]. Over the past few decades although, natural medicinal plants are gaining ground for health promotion and treatment of diseases as well as pharmaceutical drugs are now being approved based on natural products or their derivatives [7,8]. However, the use of natural products for use in therapeutic purposes faces certain challenges that mainly include lack of standardized techniques for isolation, extraction and/or purification, batch-to-batch variation in product quality and quantity, lack of insight in mechanism of drug action, etc. [9]. All natural products exist in the form of complex mixtures with other compounds. The product of interest must be extracted, isolated, and purified from this complex mixture. Isolation of products from natural sources is the most crucial, demanding, and tedious step in the process of natural product purification. Starting with extraction, a series of different techniques are employed to obtain pure natural product. In this chapter, we provide an overview of standardized techniques that are currently being employed for isolation, extraction, and purification of natural products. The general approach and techniques for purification of desired natural product from raw material is depicted in the schematic presented in Fig. 3.1.

New Horizons in Natural Compound Research ISBN 978-0-443-15232-0, https://doi.org/10.1016/B978-0-443-15232-0.00011-4

© 2023 Elsevier Inc. All rights reserved.

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Figure 3.1 General approach and techniques employed in purification of natural product. CCC, counter current chromatography; GAS, gas antisolvent; HPLC, high pressure liquid chromatography; HSCCC, high-speed counter current chromatography; LPLC, low pressure liquid chromatography; SAS, supercritical antisolvent; SFC, supercritical fluid chromatography.

2. Extraction Extraction is the primary step for obtaining a crude extract of the desired natural product from the source. The most extensively used method is solvent extraction [10]. Extraction of natural products from the source constitute customary stages of penetration of solvent into the solid natural source, dissolution of natural compound in the solvent, diffusion of the compound, and collection of extracted compounds [11]. Some of the conventional extraction methods are listed in Table 3.1. They usually use large amount of organic solvents and have lengthy extraction time [12]. Currently employed techniques are less cumbersome as they exhibit higher selectivity toward a particular product and consume less solvent. One of the most advanced cutting-

Cutting edge approaches for natural product purification

Table 3.1 Some common conventional extraction methods. Extraction method

Description

Solvent extraction

Transfer of component from one solvent to another based on its solubility Partially soluble product of interest transferred from solid source to solvent at its boiling temperature Assisting technique for liquid-liquid and solid-liquid extraction by creating vortex Powdered source immersed in solvent at room temperature Assisting technique for liquid-liquid and solid-liquid extraction using centrifugal force Releasing product of interest by crushing source at room temperature without generating heat

Soxhlet extraction Vortexing Maceration Centrifugation Cold pressing

edge techniques is supercritical fluid extraction (SFE) which uses a supercritical fluid as the extraction solvent that has solubility like liquid with diffusivity like gas [12]. Their solvating properties drastically change at their critical pressure and temperature that allows for enhanced dissolution of many natural products. Supercritical carbon dioxide (S-CO2) is the most commonly used supercritical fluid owing to its intriguing features such as low critical temperature (30.98 C) and low polarity that helps in extraction of thermally labile and nonpolar components [13]. Apart from this, S-CO2 is also inert, inexpensive, and shows no toxic effects [14]. In pressurized liquid extraction (PLE), high pressure is applied to mediate the extraction that maintains the liquid state of solvents beyond their boiling point. This imparts enhanced penetration, dissolution, and diffusion rates. Extraction time is considerably lowered and the solvent is efficiently reused. PLE is commonly used in the extraction of saponins, flavonoids, and essential oils [15]. Though a highly competent method, PLE is still disputed for its ability to extract thermally labile components [16]. Microwave assisted extraction (MAE) uses microwave to generate heat that interacts with both polar and nonpolar components of the source matrix. The interaction is basically mediated by dipole rotation and ionic conduction. The heat and mass transfer in the same direction facilitates extraction and augment yield while using lesser solvent. The major advantage of MAE is however its solvent-free extraction technique that allows for efficient extraction of volatile components [17]. Enzyme-assisted extraction (EAE) is mainly advantageous for releasing the product of interest out of cell and its organelles. Commonly used enzymes are a-amylase, cellulase, and pectinase, that hydrolytically act on different polysaccharides of cell wall, which is a difficult plant cell component to breakdown [18,19]. Pulsed electric field (PEF) uses short pulses of electricity to break open cell membrane that drastically increases the mass transfer and efficiently releases

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the product of interest. PEF extraction method is valuable for thermolabile compounds. The efficiency of extraction can be tuned by pulse time, field strength, etc. [20]. Finally, ultrasonic-assisted extraction (UAE) employs ultrasonic waves to create cavitation in solvent that increases its diffusion, heat transfer and solubility of solute. Since it allows for extraction in lowered temperature, it is an excellent technique for extraction of thermolabile components. It also consumes lesser solvent and therefore is a green technique [14].

3. Pre-isolation The crude extract from the source contains myriad of cellular and noncellular components and impurities and therefore cannot be directly subjected to isolation techniques and must undergo pre-isolation, clarification or enrichment step. Pre-isolation basically is intended to capture and concentrate the product of interest from its crude extract. There are three major techniques that are used for pre-isolation of desired product from crude extract: (i) Solvent partitioning/extraction: As already mentioned in Table 3.1, this method relies on transfer of component from one solvent to another based on its solubility. Basically, two immiscible solvents (with varying polarities) are mixed with the extract. When the solvents separate, the compounds in the extract also separates according to their partition coefficients [21]. (ii) Adsorption enrichment: Macroporous resins are used adsorption of desired components based on their affinity toward the resin. The compound interacts with the matrix with noncovalent interactions like hydrophobic, electrostatic, hydrogen bonds, etc. Other than specific interactions, properties like molecular weight, size, shape, etc. can also be manipulated. Many natural products like phenolics, saponins, flavonoids, alkaloids, and coumarins has been enriched using adsorption techniques [10,11,22,23]. For hydrophobic compounds, the extracellular fraction is passed through a hydrophobic adsorption resin. Such resin binds hydrophobic components that can be later eluted using a more nonpolar solvent like methanol, acetone, ethyl acetate, butanol, etc. depending on the polarity of product of interest. The efficiency of the process can be tailored by selecting resin having higher affinity toward the product and using gradient elution methods in which the polarity of the eluting solvent is decreased gradually in a step-wise or continuous manner. If the component is acidic/basic or charged at particular pH, then anion or cation exchange resin could be employed. Components bound to such resins can be eluted out by altering ionic strength and pH of the solvent. In some cases, the product of interest can be rendered hydrophobic upon neutralization by pH adjustments. In such cases, after neutralization a hydrophobic resin can be used to capture and concentrate the product [24e28].

Cutting edge approaches for natural product purification

(iii) Membrane separation: In this case, a selectively permeable membrane is used to filter the crude extract. Usually, cross flow of the extract tangential to the membrane surface is employed to minimize deposition of insoluble fraction on membrane and clogging of membrane pores. Based on their ability of membrane to filter different components, membrane separation techniques are classified as microfiltration (filters suspended solids and bacteria), ultrafiltration (filters viruses), nanofiltration (filters multivalent ions), and reverse osmosis (filters monovalent ions) [29,30]. Extracellular products are simpler to capture as they are secreted out of the cell. Capture of such extracellular components depend on their chemical nature and a appropriate method can be chosen accordingly. Isolation of intracellular components could be a bit more complicated as the product is associated with biomass, i.e., cellular components. In such cases, conventional technique like solvent extraction followed by concentration by evaporation using thin film or rotary evaporator could be used. Finally, the component is subjected to resin-based capture for further concentration. The major limitation of these techniques is however the use of large volume of organic solvents. Further, emulsion formation and/or flocculation of the desired component can occur that could be difficult to deal with. Therefore, integrated techniques that omit the pre-isolation and clarification steps are high on demand. One such technique is expanded-bed adsorption chromatography that uses a single pass of extract for isolation and purification of the desired products. In this technique, the densely packed resin is expanded by upward flow of solvent and then the crude extract is subjected to it. However, the technique has been successfully applied for purification of few extracellular components like peptides, proteins, and few antibiotics [21].

4. Isolation Isolation aims to obtain a natural compound in adequate quantities to ascertain its chemical structure as well as physicochemical, biological, and bioactive properties. Solid phase extraction (SPE) techniques have been extensively used for isolation of natural products. SPE exploits the capacity of solid stationary matrix (chromatography column) to adsorb analytes from liquid mobile phase based on the properties of the matrix and analyte. SPE techniques exhibit high recovery rate that makes it advantageous over other techniques [31]. SPE can be classified as ion exchange SPE and adsorption SPE. Ion exchange SPE can be further divided into cation exchange (negatively charged matrix, positively charged analyte) and anion exchange (positively charged matrix, negatively charged analyte) [32]. Adsorption SPE can further be categorized as normal and reverse phase based on matrix characteristics and retention time of the components on the matrix. Normal phase SPE employs polar matrix and nonpolar solvent; the least polar component elutes first. In case of reverse phase SPE, the least polar substance has highest retention time on

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the column; polar mobile phase and nonpolar stationary phase are used in this case. Most polar components come out first as eluate in reverse phase. Before devising a protocol for isolation of natural product, the properties of the compound of interest like polarity, charge, acid/base characteristics, molecular size, solubility, etc. must be taken under consideration. It is much simpler to isolate a known compound of interest as the physicochemical characteristics of such compound is discernible. However, in case of isolation of unknown entities, it is more complicated to strategize a protocol and qualitative analysis to unveil the properties of the unknown compound, which needs to be done before proceeding to isolation. The qualitative analysis mainly includes different biochemical tests to confirm presence of a group of chemical entities like flavonoids, phenolics, alkaloids, etc. and subsequent analytical thin-layer or high-performance liquid chromatography (HPLC) profiling. Apart from this, the physical and chemical nature of the crude extract can also be taken under consideration to strategize an isolation protocol. Some of such characteristics are listed below [33]: (i) Polarity: The hydrophobic/hydrophilic properties of the compound extracted from source can be determined by the polarity of solvent used for extraction. For example, more hydrophobic fraction of the raw material will be extracted with solvents of decreasing polarity viz. water, methanol, ethanol, acetonitrile, dimethylsulphoxide, ethylacetate, chloroform, dichloromethane, xylene, and hexane. Further, the polarity of the extract can also be modulated by first drying it, followed by redissolution in different solvents. (ii) Charge: The information on the charge present on the molecule is particularly important to determine the type of ion exchange SPE required. The charge could be positive or negative and can be deduced by using ion-exchangers. (iii) Acidic/basic properties and pH stability: The acid-base properties of the compounds can easily be discerned by partitioning in aqueous solvent in acidic, neutral and basic pH. Carrying out the analysis in a wide range of pH will also provide information about the stability of the compounds in different pHs. (iv) Molecular size: The presence of macromolecules like proteins or polysaccharides in the extract can be determined by carrying dialysis using a membrane with appropriate molecular cut-off. Usually, the secondary metabolites being smaller in size will pass through the membrane while macromolecules will be retained in the dialysis tube. (v) Thermal stability: Thermal stability of the components of the extract can be revealed by heating the extract in water bath at a specific temperature for a particular time period, for example at 90 C for 10 min. This analysis is advantageous as many of the natural products are heat-labile and gets degraded at higher temperatures leading to loss of bioactivity. However, such an analysis will be inconsequential if the initial extraction method has employed a high temperature.

Cutting edge approaches for natural product purification

5. Purification Even after enrichment and isolation, the product is bound to be contaminated with other compounds with similar properties as well as impurities. This renders the purification techniques an essential step to obtain highly pure and stable compound of interest with high specific activity. Though many efficient techniques for purification have been developed, none of them is powerful enough to purify all natural products. Usually, a combination of two or more techniques are employed for appropriate purification of the compound of interest. One of the most widely used purification techniques is chromatography, that exploits the binding capacity of analyte in mobile phase to stationary phase as well as retention time of the analyte on stationary phase [34]. Separation is effectuated by repeated sorption and desorption on the stationary phase when analyte moves along with the mobile phase. However, binding relies majorly on the stronger interaction of analyte with stationary phase as compared to mobile phase. Some of the highly efficient chromatography techniques used for purification are described as follows: (i) Low-pressure liquid chromatography (LPLC): This chromatographic technique uses low pressure to impel mobile phase through stationary phase. The instrumentation usually does not use pump and uses gravity for the movement of mobile phase. The stationary phase is composed of particles of size 40e200 mm packed into a column while a wide variety of solvents can be used in mobile phase. It provides comparatively lower resolution and requires long time. Commonly used separation technique that uses LPLC is molecular sieve chromatography that separates components according to their molecular size [35]. (ii) High-performance liquid chromatography (HPLC): As compared to LPLC, high pressure is used to force solvent through stationary matrix in HPLC. The different matrices employed for HPLC are same as that of SPE as described in Section 4. The pressure is imparted by a pump that introduces the solvent into the column at a constant speed. The particle size of stationary phase is around 3e10 mm and the pump pressure renders smooth movement of mobile phase through the densely packed column. Further, the small particle size provides high surface area ensuring higher resolution of separation [21]. (iii) High-speed counter-current chromatography (HSCCC): HSCCC relies on the partition of analyte in a two-phase solvent system composed of two immiscible liquids. There is no solid stationary matrix in this case. This liquid-liquid chromatographic technique is advantageous because of its high recovery rate of introduced sample as there is no irreversible binding of analyte with the stationary phase. Further, the risk of loss of activity of the compound of interest is lower [36,37]. (iv) pH-zone-refining counter-current chromatography: This technique is an upgraded form of HSCCC that uses an acid or base as retainer in the stationary liquid phase and a base or acid eluter in mobile phase, respectively. The analytes

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bind and get eluted as per their acid dissociation constant and polarity. The major advantage of the technique is its capacity for high sample loading [36]. (v) Supercritical fluid chromatography (SFC): SFC uses a supercritical fluid (liquid or gas above its critical points) as mobile phase that imparts high and fast mass transfer due to increased diffusivity and lowered viscosity. Two types of SFC are currently used for separation viz. packed-column SFC (pSFC) and capillary SFC (cSFC). pSFC uses stationary phase particles packed in macro-columns whereas cSFC uses micro-columns that enables it to be used for high throughput applications [38]. (vi) Membrane-based techniques: Though majorly used for pre-isolation and enrichment, some membrane-based techniques like micro- and ultrafiltration are also used for purification of natural compounds by separating impurities. (vii) Selective precipitation using supercritical/gas antisolvent (SAS/GAS) techniques: In this technique a supercritical fluid/gas expanded liquid is formed that drastically reduces the solvation power of the liquid [39]. The compound of interest is dissolved in the liquid that is injected into supercritical fluid/gas. Due to miscibility of the two, large volumetric expansion takes place, thereby precipitating the solute, i.e., the compound of interest. The precipitation can be modulated by changing the pressure, temperature, solvent-supercriticlal fluid/gas ratio as well as concentration of extract. Apart from the chromatographic techniques, crystallization is also used for purification, which is basically the precipitation of analyte in a supersaturated solution. After the formation of supersaturated solution, nucleation starts which is the starting point of crystal formation. The crystal grows by deposition of the analyte on the nucleation point.

6. A typical approach for isolation of anthocyanin monomers from red cabbage Anthocyanins are water soluble phenolic pigments which are excellent natural food colors responsible for red, blue and purple colors. Apart from this, anthocyanins also possess antioxidant and anticancer properties rendering them pharmaceutically valuable. Red cabbage (Brassica oleracea L.) is an excellent source of anthocyanins. Chen et al. have devised a highly efficient protocol for isolation of anthocyanin monomers for red cabbage that is discussed as follows [40]: (i) Extraction: Homogenized red cabbage, in 50:50 methanol:water, containing 1% formic acid was subjected to UAE in an ultrasonic bath for 1 h at room temperature followed by rotary evaporation at 40 C. (ii) Enrichment and isolation: For enrichment and isolation of the anthocyanin content, adsorption SPE was used with XDA-8 macro-mesh resin column and two different mobile phases i.e., 1% formic acid in water and 80:20 methanol:water with 3% formic acid followed by drying by rotary evaporation.

Cutting edge approaches for natural product purification

(iii) Purification: Crude anthocyanin obtained by enrichment and isolation was subjected to HPLC with following parameters: (a) Stationary phase: C18 column (1.9  25 cm, 10 mm) (b) Sample load: 50 mg crude anthocyanin (c) Mobile phase: Phase A (3:97; formic acid:water); Phase B (3:97; water:formic acid). Elution carried out with 10%e35% B, 0e15 min; 35%e45% B, 15e35 min; 45%e100% B, 35e55 min; 100% B, 45e55 min. (d) Flow rate: 15 mL/min (e) Detectors: UV/visible Using this approach, 10 major anthocyanin monomers were isolated from red cabbage. The structural identification of the anthocyanins was performed by mass spectroscopic analysis. Approximately 99% pure anthocyanins were obtained.

7. Conclusion Natural products have a great potential to be pharmaceutical candidates but are limited by their purity as the desired product exist with myriad of other chemical entities and impurities. Therefore, it is necessary to carefully strategize a purification method of the product of interest. The approach can be broadly classified into extraction, isolation, and purification steps. As compared to conventional extraction methods, modern methods are green, fast, and compatible with heat-labile components thereby retaining their bioactive properties. Extraction is followed by isolation techniques that lead to enrichment of the desired component. Mainly solid phase extraction is employed. For purification, advanced chromatographic techniques and crystallization is used. A lot of techniques exist for extraction, isolation, and purification that enable it to design a suitable purification protocol depending on the physicochemical properties of desired product. A combination of different techniques can also be used for enhancing purification. Though advances have been made toward obtaining pure natural products, still there are scope of improvement. Greener yet inexpensive techniques are required to completely eradicate usage of harsh organic solvents to render the process more environment friendly. Further, batch-to-batch variation in quality and quantity of natural products also needs to be tackled. In conclusion, multiple techniques presently exist for purification of natural products, and environmentally safe approaches with efficient yield could be devised by tuning the parameters of existing techniques as well as combining different techniques.

Acknowledgment JS would like to acknowledge Science Engineering and Research Board, Government of India, Start-up Research Grant (File No. SRG/2021/000305) for funding her research.

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CHAPTER 4

Mass spectrometry-based metabolomics for high-throughput natural products screening and compound discovery: an emerging trend Jisun H.J. Lee1 and Deepak M. Kasote2 1

Department of Plant Science and Technology, Chung-Ang University, Anseong, South Korea; 2Plants for Human Health Institute, North Carolina State University, Kannapolis, NC, United States

1. Introduction The chemical compounds with biological activities obtained from natural sources, such as plants, microbes, algae, insects, and animals are generally called natural products (NPs). Plants are the main source of NP compounds, followed by marine organisms, microbes, fungi and lichens, and animals [1]. Thousands of NP-based compounds have been reported with a range of bioactivities, and records of them are found in around 98 accessible databases [2]. In the Dictionary of NPs (DNP), over 250,000 NP compounds have been reported so far [3]. NPs are the main source of medicines. Nearly 34% of total medicines approved by the US Food and Drug Administration (FDA) in the years 1981e2010 were NPs or direct derivatives of NPs [4]. In classical NP research, screening, purification, and identification of bioactive compound(s) of interest in crude extracts or fractions is a challenging and tedious process due to the complex biological matrices [5]. This requires high-throughput techniques, such as mass spectroscopy (MS) and nuclear magnetic spectroscopy (NMR). NMR is used for structural elucidation of novel natural compounds, considering additional data from ultraviolet spectroscopy and mass spectrometry [4]. Conversely, liquid chromatography (LC) or gas chromatography (GC) in conjunction with MS methods are used qualitative and quantitative analyses [6]. However, this classical approach is time-consuming and may have limited applications in the rapid and comprehensive screening of natural compounds in the complex biological matrices, including the identification of marker compounds. Unlike this, the metabolomics approach can be a valuable tool in NP research, especially in the rapid screening and quantification of natural compounds, and identification of (bio)marker compounds from the complex matrix. New Horizons in Natural Compound Research ISBN 978-0-443-15232-0, https://doi.org/10.1016/B978-0-443-15232-0.00020-5

© 2023 Elsevier Inc. All rights reserved.

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Thus far, NMR- and MS-based metabolomics approaches have been effectively used in NP research. Both of these approaches have their own merits and demerits, especially in terms of feasibility and sensitivity of analytical tools. For example, NMR is noncompound destructive technique that requires minimal sample handling. However, it is less sensitive and predominantly detect the most abundant metabolites. Conversely, mass spectrometry can measure metabolites at very low concentrations [6]. Considering the dominance of the MS-based metabolomics approach in NP research, in this chapter, the fundamentals of MS-based metabolomics and its applications in NP research have only been discussed.

2. MS-based metabolomics Metabolomics is defined as the comprehensive measurement of all metabolites, including low-molecular-weight molecules in a biological specimen [7]. In general, MS-based metabolomics analyses, analytical techniques such as LC-MS or GC-MS are used along with biostatistical tools for the identification or (semi) quantitative estimation of metabolites in biological samples, including interpretation of the spectral information [8]. Based on study objectives, metabolomic analyses are categorized as targeted or untargeted. The general workflow used in MS-based metabolomics in NP research is shown in Fig. 4.1. Sample preparation, mass spectrometric measurement, and data analysis are the three main steps in MS-based metabolomics. 2.1 Targeted MS-based metabolomics In targeted MS-based metabolomics, analysis of specific metabolites, especially (semi) quantitatively, is carried out using hyphenated mass spectrometry. In NP research, this approach is predominantly used for the quantitation of known and candidate biomarkers, including their validation. Tandem mass spectrometry (MS/MS) coupled with GC or LC technology is mainly used in MS-based targeted metabolomics owing to its high sensitivity, speed, and throughput [9]. However, a comparatively small number of metabolites, mainly annotated ones, can be analyzed using targeted approaches [10]. 2.2 Untargeted MS-based metabolomics Untargeted metabolomics also referred to as comprehensive or global metabolomics that comprises the detection of thousands of molecular features of metabolites. Generally, high-resolution mass spectrometry (HR-MS) is used in untargeted MS-metabolomics. Examples of HR-MS instruments are time-of-flight (TOF), Fourier transform ion cyclotron resonance (FT-ICR), and Orbitrap-MS. HR-MS can achieve mass accuracy less than 5 to 0.1 parts per million (ppm), and a mass resolution higher than 10,000 to 1,000,000 full width at half maximum (FWHM) [11]. The molecular features of crude

Mass spectrometry-based metabolomics for high-throughput natural products screening and compound discovery

Figure 4.1 The general workflow used in mass spectrometry-based metabolomics (targeted or untargeted) in NP research.

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extract or fraction are captured after separation on the column or by introducing samples directly into the mass spectrometer; this method is called direct infusion mass spectrometry (DIMS). In NP research, the untargeted metabolomics approach is used for metabolic fingerprinting or profiling and (bio)marker discovery studies. However, there are several technical challenges in MS-based untargeted metabolomics that limits its widespread adoption, mainly related to the robust and rapid compound identification, including comprehensive coverage of metabolome [12]. A huge amount of spectral data is generated in untargeted MS-based metabolomics, which needs to be statistically processed to infer resultant spectral information. In metabolomics, data is initially processed (data processing steps such as alignment, bucketing, normalization, and scaling are performed), and later on, analyzed by univariate and multivariate techniques. In univariate methodologies, only one variable (in LC-based metabolomics, usually one spectral feature out of a panel of many measured) is analyzed at a time between two or more groups to assess group-wise differences and verify the normality of its distribution [13e15]. For univariate analysis, tests like t-test, analysis of variance (ANOVA) volcano plot, and fold change are routinely performed [14]. However, the univariate analysis does not provide systemic information, especially information about co-relation among multiple variables. In contrast, multivariate analysis enables the understanding and interpretation of metabolomic data, including association among metabolites [14]. Multivariate data analysis tests are classified into two categories: (1) Unsupervised methods: principal component analysis (PCA), hierarchical clustering analysis (HCA)eDendrogram, Heatmap, and (2) Supervised methods such as partial least squares (PLS), partial least squares-discriminant analysis (PLS-DA), orthogonal partial least squares (OPLS), and orthogonal partial least squares-discriminant analysis (OPLS-DA). Among these, PCA and PLS-DA are most preferred techniques [14]. There are several web-based tools such as MetaboAnalyst, XCMS, MZmine 2, GNPS, OpenNMS, and MS-DIAL currently available for MS-based metabolomics data analysis, and feature annotation [16].

3. Applications of MS-based metabolomics in NP research 3.1 Fingerprinting and profiling of NPs MS-based fingerprinting or profiling is a quick mass singles capturing technique, mainly used in the untargeted approach. The LC-orGC-MS and DIMS are the techniques, usually used to obtain MS-based fingerprints or profiles of metabolites. In NP research, metabolites signatures or fingerprints of crude extracts and fractions are routinely captured to understand the chemical pattern and purity of isolates. Moreover, MS-based metabolomics approach has also been successfully used in NP bioprospecting studies, especially for

Mass spectrometry-based metabolomics for high-throughput natural products screening and compound discovery

comparing changes in fingerprints or profiles of metabolites in response to exposure and metabolism [17]. 3.2 NPs de-replication and drug discovery In NP research, the term “de-replication” refers to the fast identification of known metabolites from complex mixtures. This process is fully embedded with targeted and untargeted metabolomics studies [18]. De-replication studies are based on a comparison of data obtained from chromatographic and spectroscopic techniques, like GC-MS, and LC-MS [19]. In a chemical de-replication study, the MS-based metabolomics approach has been used to group bacteria according to the chemical diversity of their produced metabolites [20]. Numerous commercial MS-based databases such as MarinLit and NAPRALERT are available that can assist in the de-replication process, and also reduce the time taken for the structure elucidation of known compounds [21]. In addition, the MS-based metabolomics approach is used for the discovery of novel NPs and in drug development. With this approach, discovery and testing of new drugs can be enhanced [22]. Moreover, metabolomics studies can be valuable in reducing drug failures that are associated to toxicity in early-phase trials or drug withdrawals in late-phase trials [23]. 3.3 Bioactivity assessment, metabolism, and biomarker discovery of NPs In herbal medicine research such as Chinese Traditional Medicine (CTM), MS-based metabolomics has been considerably used in screening and identifying potential bioactive components from herb drugs, including evaluating therapeutic effects of CTM and its products [24]. Metabolomics is also found to be a useful tool to understand the mode of action, xenobiotic metabolism, and toxic side effects of NPs [25]. LC-MS-based metabolomics techniques have advantages, especially in handling large datasets [26]. MS-based metabolomics, both targeted and untargeted, is found to be an evolutionary technology in rapid biomarker identification, quantification, and validation. In NP research, biomarker discovery is a crucial event of study, which is usually undertaken to get information on bioactive metabolites and their metabolic products and to identify drug-responsive metabolite(s) under in vitro and in vivo conditions. 3.4 Quality control of NPs The chemical composition of sources of NPs is highly complex and variable. Genetic, environmental, and processing factors have a remarkable impact on the quality of NPs [27]. Hence, quality control of NP is challenging. etabolomics can be a powerful tool for the quality assessment of NPs. MS-based targeted and untargeted metabolomics techniques have been used to monitor changes in the quality of NPs and the content of quality control marker metabolites.

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4. Conclusions MS-based metabolomics has been emerging as a valuable and indispensable tool in accelerating NP research. Recent advances in analytical aspects of mass spectrometry and metabolomics data processing tools are making MS-based metabolomics a fast, highly sensitive, and more robust platform in NP research. Currently, MS-based metabolomics has been widely used in both NP profiling, quality control, and NP drug discovery. The brief information provided in this chapter related to the fundamental aspects of MS-based metabolomics and its application in NP research can be a preliminary means for readers and researchers in this field.

References [1] Lautie E, Russo O, Ducrot P, Boutin JA. Unraveling plant natural chemical diversity for drug discovery purposes. Front Pharmacol 2020;11:397. [2] Sorokina M, Steinbeck C. Review on natural products databases: where to find data in 2020. J Cheminf 2020;12(1):1e51. [3] Wolfender J-L, Nuzillard J-M, Van Der Hooft JJ, Renault J-H, Bertrand S. Accelerating metabolite identification in natural product research: toward an ideal combination of liquid chromatographye high-resolution tandem mass spectrometry and NMR profiling, in silico databases, and chemometrics. Anal Chem 2018;91(1):704e42. [4] Harvey AL, Edrada-Ebel R, Quinn RJ. The re-emergence of natural products for drug discovery in the genomics era. Nat Rev Drug Discov 2015;14(2):111e29. [5] Atanasov AG, Zotchev SB, Dirsch VM, Supuran CT. Natural products in drug discovery: advances and opportunities. Nat Rev Drug Discov 2021;20(3):200e16. [6] Marshall DD, Powers R. Beyond the paradigm: combining mass spectrometry and nuclear magnetic resonance for metabolomics. Prog Nucl Magn Reson Spectrosc 2017;100:1e16. [7] Clish CB. Metabolomics: an emerging but powerful tool for precision medicine. Mol Case Stud 2015; 1(1):a000588. [8] Beale DJ, Pinu FR, Kouremenos KA, Poojary MM, Narayana VK, Boughton BA, et al. Review of recent developments in GCeMS approaches to metabolomics-based research. Metabolomics 2018; 14(11):1e31. [9] Ren J-L, Zhang A-H, Kong L, Wang X-J. Advances in mass spectrometry-based metabolomics for investigation of metabolites. RSC Adv 2018;8(40):22335e50. [10] Zhang X, Quinn K, Cruickshank-Quinn C, Reisdorph R, Reisdorph N. The application of ion mobility mass spectrometry to metabolomics. Curr Opin Chem Biol 2018;42:60e6. [11] Ghaste M, Mistrik R, Shulaev V. Applications of fourier transform ion cyclotron resonance (FT-ICR) and orbitrap based high resolution mass spectrometry in metabolomics and lipidomics. Int J Mol Sci 2016;17(6):816. [12] Cui L, Lu H, Lee YH. Challenges and emergent solutions for LC-MS/MS based untargeted metabolomics in diseases. Mass Spectrom Rev 2018;37(6):772e92. [13] Bartel J, Krumsiek J, Theis FJ. Statistical methods for the analysis of high-throughput metabolomics data. Comput Struct Biotechnol J 2013;4(5):e201301009. [14] Mattoli L, Gianni M, Burico M. Mass spectrometry-based metabolomic analysis as a tool for quality control of natural complex products. Mass Spectrom Rev 2022;3:e21773. [15] Saccenti E, Hoefsloot HC, Smilde AK, Westerhuis JA, Hendriks MM. Reflections on univariate and multivariate analysis of metabolomics data. Metabolomics 2014;10(3):361e74. [16] Witte TE, Overy DP. Untargeted metabolomic profiling of fungal species populations. In: Proteomics in systems biology. Springer; 2022. p. 349e65.

Mass spectrometry-based metabolomics for high-throughput natural products screening and compound discovery

[17] Wolfender J-L, Marti G, Ferreira Queiroz E. Advances in techniques for profiling crude extracts and for the rapid identification of natural products: dereplication, quality control and metabolomics. Curr Org Chem 2010;14(16):1808e32. [18] Hubert J, Nuzillard J-M, Renault J-H. Dereplication strategies in natural product research: how many tools and methodologies behind the same concept? Phytochem Rev 2017;16(1):55e95. [19] Carnevale Neto F, Pilon AC, Selegato DM, Freire RT, Gu H, Raftery D, et al. Dereplication of natural products using GC-TOF mass spectrometry: improved metabolite identification by spectral deconvolution ratio analysis. Front Mol Biosci 2016;3:59. [20] Forner D, Berrue F, Correa H, Duncan K, Kerr RG. Chemical dereplication of marine actinomycetes by liquid chromatographyehigh resolution mass spectrometry profiling and statistical analysis. Anal Chim Acta 2013;805:70e9. [21] Dias DA, Urban S, Roessner U. A historical overview of natural products in drug discovery. Metabolites 2012;2(2):303e36. [22] Cuperlovic-Culf M, Culf A. Applied metabolomics in drug discovery. Expet Opin Drug Discov 2016; 11(8):759e70. [23] Wishart DS. Emerging applications of metabolomics in drug discovery and precision medicine. Nat Rev Drug Discov 2016;15(7):473e84. [24] Hu C, Xu G. Metabolomics and traditional Chinese medicine. TrAC, Trends Anal Chem 2014;61: 207e14. [25] Yuliana ND, Khatib A, Choi YH, Verpoorte R. Metabolomics for bioactivity assessment of natural products. Phytother Res 2011;25(2):157e69. [26] Chen C, Gonzalez FJ, Idle JR. LC-MS-based metabolomics in drug metabolism. Drug Metab Rev 2007;39(2e3):581e97. [27] Yuliana ND, Jahangir M, Verpoorte R, Choi YH. Metabolomics for the rapid dereplication of bioactive compounds from natural sources. Phytochem Rev 2013;12(2):293e304.

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CHAPTER 5

Green synthesis of natural compounds Aafaq Tantray1, Nitin Rode1, Lina Khandare2 and Santosh Terdale1 1

Department of Chemistry, Savitribai Phule Pune University, Pune, Maharashtra, India; 2Department of Physics, Savitribai Phule Pune University, Pune, Maharashtra, India

1. Introduction Designing and creating chemical products in a way that minimizes or completely avoids the use of hazardous materials is known as “green chemistry.” This process involves working with the various components of a chemical product, from its design to its final disposal. The principles, created by Paul Anastas and John Warner, were designed to help scientists develop effective strategies to reduce the harmful effects of chemical synthesis and the environmental impact of their byproducts. One of the purposes is to improve the understanding of the various steps involved in chemical synthesis. A key part of this approach is to control pollution by means of nontoxic catalysts using safer catalysts, solvents, and appropriate reaction conditions to increase atom economy and energy efficiency [1e3] (Fig. 5.1). 1.1 Principles of green chemistry [2] Green chemistry encompasses a broad range of principles, as illustrated below (Fig. 5.1): 1. Waste prevention The chemical process designed to prevent waste formation can be used to minimize the amount of waste that can be generated by a synthesis method. It is advisable to focus on preventing the waste generation from first step, instead of treating it after its formation. The amount of waste that can be produced by a process should be kept as much as possible to a minimum level. An environmental factor E is a metric that measures the amount of waste that can be generated by a process. Continued

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2. Atom economy Atom economy is also useful when analyzing the efficiency of a synthesis. To maximize the atom economy, the various materials used in a chemical process must be incorporated into the final product. The yield of the product obtained from a process measures the atom economy. This can be done through the careful design of synthetic methods so as to minimize the formation of other products. This is why it is generally preferred to follow the processes that can increase the atom economy. 3. Less hazardous chemical synthesis The use of less-toxic materials in synthetic methods should be preferred in order to reduce their adverse effects on the environment and human health. However, this practice is not advised in the production of hazardous chemicals.

4. Designing safer chemicals The design of chemical products should ensure their safe nature and effectiveness with reduced toxicity. This principle is closely related to the previous one. The manufacturers of industrial, medical, and other products should aim to produce products that are both environmentally safe and effective in their action. The various factors that are involved in formulating the chemicals and can affect the environment and human bodies in various ways must also be considered. The knowledge of such factors can help to avoid/reduce the production of toxic chemicals. 5. Safer solvents and auxiliaries If possible, the use of auxiliary materials such as solvents and extractants should be avoided. These substances are sometimes required in chemical processes to facilitate a reaction, but can be of concern due to various inherent hazards, such as volatility and flammability. Although the use of such auxiliary materials is not always unavoidable, their use should be kept to a minimum level, should be employed only to reduce the energy requirements of the reaction, and should be recycled if possible.

Green synthesis of natural compounds

6. Design for energy efficiency To minimize the energy requirements of a synthesis process, it is generally advised to perform reactions at ambient pressure and temperature. It will allow for the creation of a chemical product with minimal energy usage. The energy efficiency of the reaction depends upon selecting the right reaction design and associated environment of reaction. 7. Use of renewable feedstocks This principle is focused on the use of petroleumbased chemicals that are mainly used in various industrial chemical processes. However, these materials are not sustainable and are depleting over the time. To get rid of the problem, the raw materials required in the process can be converted from renewable sources such as natural resources. 8. Reduce derivatives In chemical synthesis, protecting groups are commonly used to prevent the modification of certain groups in a molecule during a reaction. However, this process can also lead to waste generation. These steps require additional reagents and increase the waste produced by a reaction. In some reactions, the use of enzymes has been considered an alternative to the use of protecting groups. These enzymes are highly specific and can be used to target specific regions of a molecule. 9. Catalysis Instead of using a stoichiometric amount of reagents, choose a catalyst that can increase the selectivity, minimize waste formation, and can be recycled many times in a given reaction. It can also help to reduce energy consumption and reaction time. In certain reactions, the use of catalysts can lead to a higher atom economy.

Continued

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10. Design for degradation Ideally, chemicals should be designed so that they can break down into harmless products after they have fulfilled their purpose. However, persistent organic pollutants can remain in the environment even after they have been used. These chemicals, such as those used for the treatment of pesticides, are typically only broken down by the water or ultraviolet light. To avoid these chemicals remaining in the environment after their use, they should be replaced with safer alternatives. 11. Real-time pollution prevention A chemical reaction can be monitored in real-time which can help to prevent the release of harmful substances into the environment due to the unexpected reaction happens. This process can help identify the cause of the incident and keep harmful substances out of the environment.

12. Safer chemistry for accident prevention Although working with chemicals can be hazardous, to minimize the risk of accidents, choose and develop safer procedures. Before implementing these procedures, workers must be aware of the possible risks. It is also possible to reduce the risk of exposure to these products by carefully managing the hazards. This principle is linked to other principles that deal with the handling of hazardous substances.

1.2 The future of green chemistry The concept of green chemistry is a new approach to designing safer chemicals and processes. The improvements can still be made in various chemical processes. It aims to minimize the negative effects of chemicals on the environment and human health. For instance, many of the chemicals we use come from processes that don’t meet the principles of green chemistry. Despite the many advantages of this approach, many of these products still end up as waste products. Many challenges go into meeting the principles of multiple processes, but it can also help drive discoveries and improve the quality of life for everyone. In the coming years, many more procedures will be adapted to these principles.

Green synthesis of natural compounds

Figure 5.1 Principles of green chemistry.

Since the 12 principles of green chemistry have been elaborated in detail in this chapter, the focus of this chapter is on developing green methodologies for the synthesis of natural compounds [3]. An organic chemical component found in nature that is formed by a living organism is known as natural product. Natural products have also been synthesized by chemical synthesis (both semi-synthesis and total synthesis) and have played an important role in the advancement of organic chemistry [4]. The study of natural products was also important since it extended to commercial purposes such as cosmetics, dietary supplements, and food made from natural sources without added ingredients. In addition to this, natural products possess so many properties that make them beneficial for the development of drugs [5].

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1.3 General synthetic approaches to green chemistry 1.3.1 Microwave synthesis Microwave synthesis is the process of applying microwave radiation (MW) to a chemical reaction. The high-frequency electric fields are used to heat a substance containing charged particles or conducting ions in substances. In 1986, the first reports about the use of MW radiation for organic synthesis were published. Since then, its use has become an essential part of various synthetic organic chemistry processes such as solvent-free and solvent-mediated multicomponent reactions, coupling reactions, condensation reactions, nanoparticle synthesis, etc. The main advantages of using MW radiation for organic reactions are simple, efficient, and eco-friendly processes. It is having the ability to accelerate the process and provide selectivities that are not possible with conventional heating. In addition, the effects of the MW radiation on the reaction can be controlled by the chemist [6e8]. Shabalala Nhlanhla Gracious et al. have reported the green method of naturally occurring chromene derivatives under UV irradiation conditions (Scheme 5.1) [9]. Scheme 5.2 shows the synthesis by Shivaputra A. Patil et al. of naturally occurring chromene derivatives using phenols, aldehydes, and malononitrile using the microwave method in a single step [10]. Islam H. El Azab et al. (Scheme 5.3) have reported the conventional synthetic routes as well as microwave-assisted, for the synthesis of 6-OH-4-methyl-2H-Chromene-2one depicted in Scheme (1Be1E) (Table 5.1) [11].

Scheme 5.1 Green method of naturally occurring chromene derivatives.

Scheme 5.2 Chromene derivatives using phenols, aldehydes, and malononitrile using the microwave method.

Green synthesis of natural compounds

Scheme 5.3 Conventional synthetic routes as well as microwave-assisted, for the synthesis of 6-OH-4methyl-2H-chromene-2-one. Method 1: Microwave-assisted synthesis: AcOH, DMF, 120  C, 8e10 min; Method 2: Conventional synthesis: AcOH, DMF, reflux, 4e7 h; Method 3: Conventional synthesis: Anhydrous EtOH, stirring at r.t. for 1 h, then refluxes for 2 h at 60  C Table 5.1 Comparison between microwave-assisted synthesis (green synthesis) and conventional synthesis of products (1Be1E) in terms of time and % yield [10]. Time

Yield (%)

Compounds

Microwave (min)

Conventional (h)

Microwave

Conventional

1B 1C 1D 1E

8 e e 9

5 1 1 4

97 96 e 97

85 75 80 85

Scheme 5.3. Method 1: Microwave-assisted synthesis: AcOH, DMF, 120 C, 8e10 min; Method 2: Conventional synthesis: DMF, CH3COOH, reflux, 4e7 h; Method 3: EtOH, string 1 h, refluxes, 120 min at 60 C. 1.4 Ultrasound supported green synthesis [1] Sonochemistry is a powerful technique that involves the use of ultrasound waves to accelerate the reactions in organic compounds synthesis. The ultrasonic range is composed of various frequencies, ranging from 20 kHz to 10 MHz. Sonochemistry is a sustainable technique that can be used to boost the chemical properties of compounds. It can also be used to alter the reactivity of the compounds.

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The significant uses of ultrasound in chemical synthesis are as follows. Majority of reactions/synthesis reported are conducted at room temperature unless otherwise specified. The reactions carried out on exposure to ultrasound is represented by symbol. The use of ultrasound instead of harmful chemicals and high energy consumption makes this method of organic synthesis a classic contribution to the field of green chemistry. Additionally, with the exception of high-power ultrasonic horns, the cost of the apparatus is often modest for a straightforward ultrasonic cleaning bath (frequencies 20e40 kHz) [12,13]. The carbon-carbon bond formation serves as the cornerstone for building the carbon skeleton of organic molecules. As such, it is central to the chemical sciences and is regarded as the most important conversion in organic synthesis, including the total synthesis of natural products or compounds with significant pharmacological value. This section focuses on the carbon-carbon bond-forming processes that happen in diverse biologically active natural compounds when they are exposed to ultrasound as a clean energy source [9,14e16]. The entire synthesis of numerous complex natural compounds serves as evidence that the Barbier-type reaction is a powerful tool for forming a carbon-carbon bond. 1.5 Total synthesis of natural product psymberin using ultrasonic irradiation Ye and co-workers in 2019 reported a convergent, stereo controlled complete synthesis of natural product namely psymberin (E) from known aldehyde derivative (A) involving Barbier-type reaction under ultrasonic irradiation as a nonpolluting tool. The researchers are attracted to psymberin (E) due to its complex structure possessing stereochemistry, 5,S,8S,9S,11R,13R,15S,16R, and 17R, and also fascinating biological activity such as antitumor activity. During this Barbier-type reaction, ultrasonic sonication is crucial in creating a new CeC bond. Thus, under the influence of freshly activated zinc powder and ultrasound sonication, the known starting aldehyde (A) interacted with prenyl bromide (B) to produce a separable mixture of (C) and (D) in 92% yield (Scheme 5.4). Finally, using ultra sonication as a green tool, a highly convergent technique was used to finish the whole synthesis of the bioactive natural substance psymberin [1]. 1.6 Total synthesis of natural products such as ()-geigerin, ()-geigerin acetate, and ()-6-deoxygeigerin [1,18] Cycloaddition methodologies [19] have become more significant in the field of complete synthesis of complex natural compounds such as ()-geigerin (J), ()-geigerin acetate (K), and ()-6-deoxygeigerin (L), because of their high atom efficiency. Popular cycloaddition processes like the DielseAlder and Paterno Buchi reactions, etc. are 100% atom efficient, and they play a significant part in the green synthesis of bioactive natural

Green synthesis of natural compounds

Scheme 5.4 Synthesis of natural product psymberin using ultrasonic irradiation from known aldehyde.

compounds. Depres et al. used a tropylium cation (F) as the starting point for the successful stereocontrolled synthesis of natural compounds (J, K, and L) by cycloaddition process and using ultrasonic irradiation (Scheme 5.5) [18]. 1.7 Grinding technique The use of a grinding procedure is thought to be a crucial methodology for carrying out synthetic reactions with a high percentage yield of product and without the use of solvents. A mortar and pestle or a high-speed vibrating mill can be used to grind the reactants for a chemical reaction. The chemical reaction progresses because of collisions between the interacting molecules. As a result, the molecules collide, resulting in the production of the final product. In comparison, to traditional heating approaches, the reaction takes less time to complete [7,20] (Fig. 5.2). The different kinds of organic reactions that can be carried out utilizing the grinding technique in solvent-free environment at room temperature are shown in Fig. 5.3. J-T et al. have reported the use of amino sulphonic acid for the synthesis of barbituric acid by treating aromatic aldehydes with pyrimidine trione through a grinding technique (Scheme 5.6) [21]. An important class of naturally occurring chemical compounds is flavones and chromenes [22]. These substances have reportedly been shown to possess a range of pharmacological properties, such as anticancer and anti-HIV. D. Sharma et al. have reported the

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Scheme 5.5 Total syntheses of ()-geigerin (J), ()-geigerin acetate (K), and ()-6-deoxygeigerin (L) by ultrasound-assisted method.

Figure 5.2 The chemical reaction by grinding technique.

eco-friendly method for the synthesis of flavones and 2-styrylchromenes without the use of any organic solvents employing a grinding technique [23]. (Scheme 5.7 and Table 5.2). 1.8 Ball milling Ball milling is a mechanical process that is frequently used to combine materials and grind powders into extremely small particles. It has found extensive use in the industry across

Green synthesis of natural compounds

Figure 5.3 Reactions carried out using the grinding technique.

Scheme 5.6 Use of amino sulphonic acid for the synthesis of barbituric acid by treating aromatic aldehydes with pyrimidine trione through a grinding technique.

Scheme 5.7 Synthesis of flavone and chromene.

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Table 5.2 Synthesis of flavones and 2-styrylchromenes [23]. Compound

R1

R2

R3

Time (min)

Yield (%)

aa ab ac ad ae af ba bb bc bd be

H H H H OCH3 OCH3 H H H H OCH3

H CH3 H CH3 H H H CH3 H CH3 H

H H OCH3 OCH3 OCH3 H H H OCH3 OCH3 H

10 10 10 10 10 10 15 15 15 15 15

80 85 90 95 90 80 75 80 95 85 90

the globe as a cost- and environmental-friendly method. This method is used for the preparation of different kinds of heterocyclic compounds [24,25] (Fig. 5.4). Yu et al. have reported the synthesis of 2,4-diphenyl tetrahydroquinolines in good yield by using the ball milling technique via Diels Alder cycloaddition reaction in presence of FeCl3 (Scheme 5.8) [24]. For the synthesis of these kinds of naturally occurring heterocyclic compounds, this method provides a very effective and environmentally

Figure 5.4 Reactions performed by ball milling method [26].

Green synthesis of natural compounds

Scheme 5.8 Synthesis of 2,4-diphenyl tetrahydroquinolines in good yield by using the ball milling technique via Diels Alder cycloaddition reaction in presence of FeCl3.

friendly replacement for conventional techniques. The benefits of this approach include the absence of solvents, ease of accessibility, faster reaction times, and non-toxic catalysts. 1.9 The biological-based green chemistry methods It is difficult to maintain and improve efficiency when transitioning from synthetic to sustainable processes. In this sense, biocatalysis, which uses enzymes [27] or entire microorganisms, offers a valuable option. Due to the quick development of the organisms, simplicity of scaling up, environment-friendly, solvent-free catalysis, and increase in selectivity, microbial biotransformation is regarded as a potent technique. 1. Biosynthesis of quinoline alkaloids. Gapar et al. have prepared quinolone alkaloid natural products by enzymatic biosynthesis. In this synthesis, first tryptophan is converted into the metabolite 3hydroxyanthranilic acid by a series of enzyme processes to produce quinoline alkaloids. Quinoline alkaloid is created by the condensation of 3-hydroxyanthranilic acid and malonyl-SCoA, followed by cyclization (Scheme 5.9) [28].

Scheme 5.9 Tryptophan is converted into the metabolite 3-hydroxyanthranilic acid by a series of enzyme processes to produce quinoline alkaloids.

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2. STR variant was used to achieve the chemoenzymatic synthesis of (R)-harmicine, a natural alkaloid with anti-Leishmania effects that was isolated from the plant Kopsia griffithii. Tryptamine and methyl 4-oxobutanoate are cyclized by the enzyme, producing a (R)-isomer precursor that can be further reduced with LiAlH4. In two incremental steps, a yield of 62% was attained, with an enantiomeric excess of 98% (Scheme 5.10). In comparison to previously published approaches, this study presents a quicker and more creative synthetic route to harmicine. It also shows that it is possible to produce target molecules with excellent stereocontrol by combining chemical and biological catalysts [29]. 3. Enzymatic synthesis of natural product Acutumine. Kim et al. [30] recently created a discovery of a halogenase in plants. The T-cell cytotoxic tetracyclic chloroalkaloideAcutumine, which belongs to the Menispermaceae family, is terminally chlorinated by the enzyme (Scheme 5.11) [29,31]. 1.10 Microorganism synthesis of natural product Acutumine 1.10.1 Chalcones transformation using bacteria Chalcones, also known as (E)-1,3-diphenylpropene-1-ones, are one of the most important groups of natural compounds found in plants. They are flavonoid compounds with two aromatic rings connected by a three-carbon, unsaturated carbonyl bridge (Scheme 5.12) [32].

Scheme 5.10 Tryptamine and methyl 4-oxobutanoate are cyclized by the enzyme, producing a (R)isomer precursor that can be further reduced with LiAlH4.

Scheme 5.11 The T-cell cytotoxic tetracyclic chloroalkaloideAcutumine synthesis.

Green synthesis of natural compounds

Scheme 5.12 Chalcones ((E)-1,3-diphenylpropene-1-ones) with two aromatic rings connected by a three-carbon, -unsaturated carbonyl bridge.

1.10.2 Chalcones transformation using green solvents The work described by Moemeni et al. reports the use of a green solvent for the synthesis of heterocyclic spiroindeno quinoxaline indolizidine ring systems from the 1,3-dipolar cycloaddition of substituted chalcones with phenylenediamines, ninhydrin, and pipecolinic acid (Scheme 5.13). Although the reaction showed to be highly regio- and diastereoselective, it is a slowdown transition (reaction time 20 h) with yields between 67%e82%. Indolizidine rings and tetracyclic indenoquinoxalines, two very important nitrogen heterocyclic groups that exhibit both biological and pharmaceutical properties, are synthesised as a result of the reaction, providing a unique structure that presents a great opportunity for the creation of a variety of biologically active compounds [32]. 1.11 Ionic liquids are used as green catalysts Tantray et al. [33] have reported the green catalyst ionic liquid for one-pot synthesis of Dihydropyrimidine derivatives under solvent-free conditions at 120 C.The benefits of this method are the absence of any harmful organic solvents and toxic catalysts, the need for only a tiny amount of catalyst, faster reaction times, simple workup procedures, and the solvent-free condition (Scheme 5.14).

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Scheme 5.13 Synthesis of heterocyclic spiroindeno quinoxaline indolizidine ring systems from the 1,3-dipolar cycloaddition of substituted chalcones with phenylenediamines, ninhydrin and pipecolinic acid.

Scheme 5.14 Synthesis of dihydropyrimidine derivatives under solvent-free conditions at 120 C using green catalyst ionic liquid.

Rode et al. [34] described the use of ionic liquid namely proline hydrogen sulfate as a green catalyst for the synthesis of Dihydropyrimidine derivatives under solvent-free conditions (Scheme 5.15).

Green synthesis of natural compounds

Scheme 5.15 Synthesis of dihydropyrimidine derivatives under solvent-free conditions using ionic liquid as catalyst.

2. Conclusion The design of sustainable products and systems involves the systematic combination of multiple principles of green chemistry. The principles are a set of methodologies that can be used to achieve this goal. This can help to achieve the ultimate goal of sustainability, which is a benefit to society and the environment while reducing the harmful effects. In this chapter, we have presented the green methodologies for the synthesis of natural compounds and also highlight the application of these green tools in the total synthesis of bioactive natural products. The green techniques such as microwave, ultrasonic, grinding, and milling process are done using environmentally friendly and sustainable methods. Due to the toxicity and volatility of the majority of organic solvents, these strategies are used to make the reactions less harmful to the environment. Furthermore, biological methods like the use of enzymes, and bacteria have also been studied for the synthesis of natural products. Additionally, the use of a green solvent such as ionic liquids for the synthesis of naturally occurring heterocyclic compounds has been described. Being more eco-friendly processes, some of the most common factors that contribute to this include the absence of organic solvent, enhanced atom economy, minimal waste production, use of catalyst, and energy efficient processes that supports the development of green chemistry.

Acknowledgment The authors are grateful to Savitribai Phule Pune University, Pune.

References [1] Majhi S. Applications of ultrasound in total synthesis of bioactive natural products: a promising green tool. Ultrason Sonochem 2021;77(July):105665. https://doi.org/10.1016/j.ultsonch.2021.105665. [2] Anastas PT, Zimmerman JB. Design through the 12 principles of green engineering. IEEE Eng Manag Rev 2007;35(3):16.

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[3] Rana KK, Rana S. Fundamentals, representative applications and future perspectives of green chemistry: a short review. OALib 2014;01(05):1e16. [4] Kumar V, Kaur K, Gupta GK, Sharma AK. Pyrazole containing natural products: synthetic preview and biological significance. Eur J Med Chem [Internet] 2013;69:735e53. https://doi.org/10.1016/ j.ejmech.2013.08.053. [5] Osbourn AE, Lanzotti V. Plant-derived natural products: synthesis, function, and application. In: Plant-derived natural products: synthesis, function, and application; 2009. p. 1e597. [6] Gawande MB, Shelke SN, Zboril R, Varma RS. Microwave-assisted chemistry: synthetic applications for rapid assembly of nanomaterials and organics. Acc Chem Res 2014;47(4):1338e48. [7] Sahoo BM, Banik BK, Pa J. Microwave-assisted green chemistry approach. In: Advances in microwave chemistry; 2019. p. 475e508. [8] Nayak J, Devi C, Vidyapeeth L. Microwave assisted synthesis: a green chemistry approach. Int Res J Pharmaceut Appl Sci 2016;3(5):278e85. [9] Gracious SN, Kerru N, Maddila S, van Zyl WE, Jonnalagadda SB. Facile one-pot green synthesis of 2amino-4H-benzo[g]chromenes in aqueous ethanol under ultrasound irradiation. Synth Commun 2020;50(13):1960e71. https://doi.org/10.1080/00397911.2020.1761393. [10] Patil SA, Patil SA, Patil R. Microwave-assisted synthesis of chromenes: biological and chemical importance. Future Med Chem 2015;7(7):893e909. [11] Azab IHE, Youssef MM, Amin MA. Microwave-assisted synthesis of novel 2H-chromene derivatives bearing phenylthiazolidinones and their biological activity assessment. Molecules 2014;19(12): 19648e64. [12] Mohamed MA. Sonochemistry (applications of ultrasound in chemical synthesis and reactions): a review Part III. J Pharm Res Int 2019;53:1e19. [13] Bouzina A, eddine AN, Berredjem M. Ultrasound assisted green synthesis of a-hydroxyphosphonates under solvent-free conditions. Res Chem Intermed 2016;42(6):5993e6002. [14] Abrams DJ, Provencher PA, Sorensen EJ. Recent applications of C-H functionalization in complex natural product synthesis. Chem Soc Rev 2018;47(23):8925e67. [15] Bariwal J, Van Der Eycken E. C-N bond forming cross-coupling reactions: an overview. Chem Soc Rev 2013;42(24):9283e303. [16] Cho SH, Kim JY, Kwak J, Chang S. Recent advances in the transition metal-catalyzed twofold oxidative CeH bond activation strategy for CeC and CeN bond formation. Chem Soc Rev 2011;40(10): 5068e83. [17] Majhi S. Applications of ultrasound in total synthesis of bioactive natural products: a promising green tool. 2020 Ultrason Sonochem 2021;77:105665. https://doi.org/10.1016/j.ultsonch.2021.105665. Available from. [18] Carret S, Depres JP. Access to guaianolides: highly efficient stereocontrolled total synthesis of ()-geigerin. Angew Chemie Int Ed. 2007;46(36):6870e3. [19] Ylijoki KEO, Stryker JM. Cycloaddition reactions in organic and natural product synthesis. Chem Rev 2013;113(3):2244e66. [20] Sahoo BM, Banik BK. Solvent-less reactions: green and sustainable approaches in medicinal chemistry [Internet]. In: Green approaches in medicinal chemistry for sustainable drug design; 2020. p. 523e48. https://doi.org/10.1016/B978-0-12-817592-7.00014-9. Elsevier Inc. [21] Li JT, Dai HG, Liu D, Li TS. Efficient method for synthesis of the derivatives of 5-arylidene barbituric acid catalyzed by aminosulfonic acid with grinding Synth. Commun Now 2006;36(4e6):789e94. [22] Ahmed SK, Praveen A. A Novel Synthesis and antimicrobial activity of flavanone using environmental friendly catalyst H[bimBF4]. Res J Pharm Biol Chem Sci 2010;1:809e15. [23] Sharma D, Makrandi JK. A green synthesis of 2-phenyl/2-styrylchromones under solvent-free conditions using grinding technique. Green Chem Lett Rev 2009;2(3):157e9. [24] Tan YJ, Zhang Z, Wang FJ, Wu HH, Li QH. Mechanochemical milling promoted solvent-free imino Diels-Alder reaction catalyzed by FeCl3: diastereoselective synthesis of cis-2,4-diphenyl-1,2,3,4tetrahydroquinolines. RSC Adv 2014;4(67):35635e8.

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[25] El-Sayed TH, Aboelnaga A, El-Atawy MA, Hagar M. Ball milling promoted N-heterocycles synthesis. Molecules 2018;23(6). [26] Stolle A, Szuppa T, Leonhardt SES, Ondruschka B. Ball milling in organic synthesis: solutions and challenges. Chem Soc Rev 2011;40(5):2317e29. [27] Suman C, Evan OR, Joshua BP, Jessica AY, Alison RHN. Chemoenzymatic total synthesis of natural products. Acc Chem Res 2021;54(6):1374e84. [28] Diaz G, Miranda IL, Diaz MAN. Quinolines, isoquinolines, angustureine, and congeneric alkaloidsd occurrence, chemistry, and biological activity. In: Rao V, Rao L, editors. PhytochemicalsdIsolation, characterisation and role in human health. Brazil: Intech; 2015. p. 142e62. [29] Chubatsu Nunes HH, Nguyen TD, Dang TTT. Chemoenzymatic synthesis of natural products using plant biocatalysts. Curr Opin Green Sustain Chem 2022;35:100627. Available from: https://doi.org/ 10.1016/j.cogsc.2022.100627. [30] Kim CY, Mitchell AJ, Glinkerman CM, Li FS, Pluskal T, Weng JK. The chloroalkaloid ( )-acutumine is biosynthesized via a Fe(II)- and 2-oxoglutarate-dependent halogenase in Menispermaceae plants. Nat Commun 2020;11(1):1e7. [31] Li F, Zhang X, Renata H. Enzymatic CeH functionalizations for natural product synthesis. Curr Opin Chem Biol [Internet] 2019;49:25e32. Available from: https://doi.org/10.1016/j.cbpa.2018. 09.004. [32] Rosa GP, Seca AML, Barreto MDC, Pinto DCGA. Chalcone: a valuable scaffold upgrading by green methods. ACS Sustain Chem Eng 2017;5(9):7467e80. [33] Tantray AA, Rode NR, Terdale SS. Synthesis, molecular docking, and in silico ADME studies of dihydropyrimidine derivatives using tetrabutylphosphonium methanesulphonate ionic liquid as a catalyst under solvent-free conditions. Can J Chem 2022;100(6):447e57. [34] Rode N, Tantray A, Shelar A, Patil R, Terdale S. Amino acid ionic liquid-catalyzed synthesis, antiLeishmania activity, molecular docking, molecular dynamic simulation, and ADME study of 3,4-dihydropyrimidin-2(1H)-(thio)ones. Synth Commun 2021;52(2):1e15. https://doi.org/10.1080/ 00397911.2021.2010757.

Further reading [1] Kharissova OV, Kharisov BI, Gonzalez CMO, Mendez YP, L opez I. Greener synthesis of chemical compounds and materialsvol. 6. Royal Society Open Science; 2019. p. 1e41. [2] Arends I, Sheldon R, Hanefeld U. 1. Introduction. Green Chem Catal 2007:1e48. [3] Ganesh KN, Zhang D, Miller SJ, Rossen K, Chirik PJ, Kozlowski MC, et al. Green chemistry: a framework for a sustainable future. Org Process Res Dev 2021;25(7):1455e9.

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CHAPTER 6

Diversity of chemical skeletons: a practical strategy to benefit Gayatri D. Kotkar1, Abhijit D. Shetgaonkar2 and Santosh G. Tilve1 1

School of Chemical Sciences, Goa University, Taleigao, Goa, India; 2Dnyanprassarak Mandal’s College and Research Center, Assagao, Mapusa, Goa, India

1. Introduction Since ancient times, human civilization has grappled with diseases. The remedies employed were animal products, inorganic materials, and herbs. Medicinal herbs are sources of phytochemicals, which function as defense chemicals against diseases, fungus, insects, and herbivorous animals. Efforts to isolate active ingredients (chemicals), characterize them, and investigate their effects on biological targets resulted in modern medicinal chemistry. With the advent of knowledge regarding the active sites of a protein for a particular ailment, the structure of the phytochemical became relevant. Plants produce thousands of chemicals, of which very few hit the market as drugs. The search for an active component involves testing the biological activity of the plant extract as the first step. Further success depends upon the bio-guided fractionation approach. Subsequent development of compound testing led to high throughput screening (HTS). This demanded a large number of chemicals in pure form, which were difficult to obtain from nature (natural products), though considerable development of extraction and purification methodologies has taken place. The high demand for chemicals from pharma companies led to the development of combinatorial synthesis, where libraries of compounds were prepared by performing simple reactions. Huge libraries of compounds prepared by combinatorial synthesis are now available. However, no significant bioactive compounds could be realized by this approach due to an inherent limitation of this technique of combinatorial chemistry. Similar types of compounds were prepared by this mode of synthesis, which lacked the diversity required for effective interaction with the active site of the biological target. Recent efforts to overcome the limitations of this technique have been successful to some extent in providing lead compounds. The drug discovery program got more streamlined with the aid of a computer, because of its effective use in silico studies. This helps to do rapid structure-activity relationship studies (SAR). However, the limitation of available chemical entities vis-
a-vis bio testing facilities remains a bottleneck. As a result, natural products (NPs) continued to be the primary source of supply for drug-like molecules. Diversity-oriented synthesis is a recent offshoot to cater to the needs of the pharma industry. Also, multicomponent New Horizons in Natural Compound Research ISBN 978-0-443-15232-0, https://doi.org/10.1016/B978-0-443-15232-0.00023-0

© 2023 Elsevier Inc. All rights reserved.

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synthesis, though with some limitations, produces libraries of compounds for biological evaluation. The interaction of a drug with a binding site depends on the shape of the molecule and its electrophilic potential with the target protein. Binding kinetic properties, desolvation, and conformational changes are also important. As a result, structural motif diversity has remained an essential component of medicinal chemistry.

2. Structural diversity in natural products A broad diversity of chemical skeletons is observed in natural products. This diversity of skeletons has made a tremendous contribution to chemical biology and drug development. Plants have evolved over time to manufacture natural products to target macromolecules within the system. Because the structural domains of human targets are similar to those of plant targets, natural products bind to human targets. Natural products are the result of biological evolution formed by the maximum number of interactions with their biological targets, due to which NPs contain a vast structural diversity. This structural diversity originates from the biogenesis of how the enzymes in plants generate these molecules. More than 360,000 NPs are available from different plant sources. In the following sections, we will see the complex structural diversity of natural products through different classifications. 2.1 Terpenes The class of naturally occurring organic compounds biosynthesized from 5-carbon unit isoprene is called terpenoids or terpenes. This is the largest group of natural products, constituting about 60% of all known NPs. Terpenoids with the greatest structural diversity are the most biologically active and are employed in the treatment of various diseases. For example, Taxol and its derivatives are employed as anticancer agents due to their property of inhibiting human cancer cells. Similarly, Artemisinin and its related analogues are used as antimalarial agents. Terpenes are also used as aroma chemicals due to their nice flavors and fragrances. There is a common metabolic pathway for terpenes, which depend-s on the number of carbon atoms (usually multiples of five), display a variety of isomeric carbon skeletons and stereochemical configurations. The formation of a dimethylallyl pyrophosphate (DMAPP) intermediate followed by three synthase pathways classifies the different classes of terpenes [1,2] (Fig. 6.1). This section covers the vast structural canvas of terpenes isolated from natural sources, which showcases the diversity of this class of compounds. 2.1.1 Monoterpenes Monoterpenes constitute an important and diversified group of compounds of natural origin and are classified based on the total number of isoprene units, i.e., they contain C-10 carbon atoms with two isoprene units in them. Further, based on their structural arrangement, these are classified as acyclic and cyclic. Because of their low molecular

Diversity of chemical skeletons: a practical strategy to benefit

Figure 6.1 Biosynthetic pathway of different class of terpenes.

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weight, many are available in the form of essential oils. Geraniol, pinene, and camphor show good biological properties. Myrtenal is a bicyclic monoterpene found mostly in plants like cumin, pepper, mint, and eucalyptus. It shows good antioxidant, anticancer, antidiabetic, immunostimulant, and cyclooxygenase inhibitory activity. Borneol is a monoterpene available in both optical forms, Dextro (D-) and Levo (L-). Borneol and its derivatives are mostly used in the production of perfumes, cosmetic manufacturing, agriculture, the wood industry, and pharmaceuticals. It displays antimicrobial, antiinflammatory, and antiviral activity. Monophenolic monoterpene thymol has pharmaceutical and food preservative applications. Fig. 6.2 depicts the diversity displayed by monoterpenes by means of various physical and chemical modifications of the common precursor, geranyl pyrophosphate (GPP) [3]. Further functional diversity in these monoterpenes is seen in their oxidation products like sabinene hydrate, menthone, etc. 2.1.2 Sesquiterpenes Sesquiterpenes are a terpene class of compounds composed of three isoprene units. The molecular formula associated with this class of compounds is often C15H24. Similar to monoterpenoids, they are structurally classified as acyclic and cyclic. The biochemical modification of farnesyl diphosphate resulted in the diversity of sesquiterpenoids’ chemical skeleton [4] (Fig. 6.3). The reported compounds of this class mostly act as pheromones and juvenile hormones, especially from the plants. The lactone ring of sesquiterpene exhibits a variety of biological activities such as analgesic, insect deterrent, antifungal, antibacterial, and so on. Michael addition of amino functionality of proteins to the a-methylene-g-lactone group of the lactone is responsible for its reactivity. The 1,2,4-trioxane heterocycle of Artemisinin is very potent against cancerous cells. An extract from Artemisia argyi contains a high amount of arteminolides A-D [5] which are effective against leukemia and colon cancer. Other lactones like yomogin and tehranolide show good efficacy against cancer cells. Dehydroleucodine [6] has a cytoprotective anti-inflammatory and gastrointestinal effect by inhibiting the release of histamine and serotonin by intestinal mastocytes. Also, it acts as a wide-spectrum antiparasitic agent and is effective against Plasmodium, Schistosoma, Taxoplasma, Trichomonas vaginalis, etc. Secotanapartholides A & B were found to be active [7] agents against bacterial cells, possessing an inhibitory action against Bacteroides fragilis, Clostridium perfringens, and Staphylococcus aureus. Derivatives of artemisinin also possess antiviral effects. They are active against hepatitis B and C viruses, a range of human herpes viruses, influenza virus A and bovine viral diarrhea virus [8]. The guaianolide structure of sesquiterpene lactones possesses antifungal potency (Fig. 6.4).

Diversity of chemical skeletons: a practical strategy to benefit

Figure 6.2 Structural diversity of monoterpenes through GPP precursor.

2.1.3 Diterpenes Diterpenes are a structurally diversified class of natural C-20 compounds comprising four isoprene units. These are structurally classified as linear, bicyclic, tricyclic, tetracyclic, pentacyclic, or macrocyclic diterpenes contingent on their skeletal essential (Fig. 6.5). The diversity in chemical skeleton of this class is found both in Plantae kingdom and

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Figure 6.3 Diversity of skeleton of sesquiterpenes.

Diversity of chemical skeletons: a practical strategy to benefit

Figure 6.4 Important chemical skeleton of sesquiterpenes possessing bioactivities.

marine sources. The plant-based compounds possess biological activities and therapeutic uses (Fig. 6.6). A number of diterpenes have interesting insect chemistry, while several other soft corals rich in diterpenes are extremely lethal to fish. Not many applications are reported of linear or acyclic diterpenes obtained from terrestrial plants. In marine species, the extract of the alga Bifurcaria bifurcate, which contains acyclic diterpenoids possesses antimicrobial, antimitotic, cytotoxic, and antifouling activity. Labdane and clerodane skeletons isolated from plants, bacteria, fungi, and marine organisms, profoundly show antibacterial, antitumoral, hypolipidemic, and smooth muscle relaxing activity. The labdane diterpene is a key constituent of the aromatic oleoresin of balsam fir, Abies balsamea, and is consumed in the perfume industry. Asmarine A & B, which are isolated from the clerodane class of diterpenes, displayed anti-proliferative action against many human cancer cell lines. Also, clerocidine [9], a natural antibiotic, shows anticancer and antimicrobial activities. Forskoline [10] is a tricyclic diterpene isolated from the Coleus forskohlii plant. It has an effect on cardiovascular ailments and possesses broncho-spasmolytic action. Totarol [11], a compound obtained from the totara tree, and salvinorin A [12], isolated from the Mexican plant salvia divinorum, both exhibit antibiotic and hallucinogenic activity. Tetracyclic compounds are well studied for their biological activities, particularly. The

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Figure 6.5 Diversity of diterpenes with precursor GGPP.

tetracyclic diterpenes of Stevia and Ginko. These molecules, containing ester and glucosidase linkages, have natural sweetening properties [13]. Ginkogolides [14] isolated from Ginkgo biloba have traditionally been shown to be effective against peripheral and cerebrovascular disease. Taxol [15], also known as paclitaxel, extracted from Taxus brevefolia, has gained great attention for possessing anticancer action against some tumor cell lines. In clinical trials of bladder hyperiflexia and diabetic neuropathy, the Resiniferatoxica [16]

Diversity of chemical skeletons: a practical strategy to benefit

Figure 6.6 Diterpenes possessing various biological activities.

is a potent drug-able molecule that is effective over vanilloid receptors. Another potent natural product, Pepluanin A [17] showed two times higher drug-like properties than cyclosporine A by decreasing the resistance of carcinogenic cell lines to the cytotoxic drug daunomycin. 2.1.4 Triterpenes Triterpenes are metabolites of s-isoprene units and are the most significant group of phytochemicals (Fig. 6.7). They are found in various plants with the wax-coating of various fruits, flowers, etc. They are classified according to the number of rings found in the structure as acyclic, monocyclic, bicyclic, tricyclic, tetracyclic, pentacyclic, and hexacyclic. Among all, the pentacyclic skeleton shows the most applications. Traditionally, triterpenes have been used in several countries as analgesics, anti-inflammatory drugs, antipyretics, hepatoprotectives, cardiotonics, sedatives, tonics, etc. Asiaticoside [18] is a triterpenoid glycoside obtained from Centella asiatica. It is active as a psychoactive drug, an anti-inflammatory and a neuroprotective agent. Astragaloside IV [19e22] is another cycloartane-type skeleton that helps in wound healing in the skin and has other epithelial pharmacological effects. An ethanolic extract of Panax ginseng leaves

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Figure 6.7 Natural diversity of triterpenes.

Diversity of chemical skeletons: a practical strategy to benefit

contains another potential molecule called ginsenoside Rd, which acts as a healer in laserinduced wounds. Lupeol belongs to the Celastraceae botanical family. It possesses many bioactivities, such as anti-inflammatory, antioxidant, mitogen-activated protein kinase (MAPK) activation, and actin cytoskeleton remodeling. Oleanolic acid [23] is a bioactive molecule isolated from 120 different plant sources, including Anredera diffusa. It has cardioprotective, anti-atherosclerotic, anti-inflammatory, and immunomodulatory properties, It is also useful against allergic asthma, gastric ulcers and hypersensitivity. Another natural pentacyclic triterpenoid, ursolic acid, also shows diverse biological activities (Fig. 6.8). 2.1.5 Tetraterpenes Tetraterpenes are a class of terpenes with C-40 carbon atoms and conjugated double bonds. These are found in higher plants, cyanobacteria, algae, fungi, and nonphotosynthetic prokaryotes. They are also referred to as carotenoids, which impart color to the leaves, stems, flowers, fruits, shellfish, bird feathers, cosmetics, etc. These pigments have earned attention because of their physiological and environmental roles as well as industrial and pharma applications. Astaxanthin [24] is isolated from the fresh water microalga Haematococcus pluvialis, and the yeast fungus Xanthophyllomyces dendrorhous. It is a keto-carotenoid with various applications in the dye industry and possesses good anti-obesity, antioxidant, and other therapeutic properties. Fucoxanthin, isolated from Fucus, Dictyota, and Laminaria, also showed its efficacy in the above inhibitory

Figure 6.8 Biologically active molecules from triterpene class.

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action. Similarly, other chemical skeletons isolated from various plant and microorganism sources, such as fucoxanthinol, halocynthiaxanthin, carotenoid, b-carotene, lutein, zeaxanthin, chantaxanthin, siphonaxanthin, etc., have shown their potency in various biological activities [25] (Fig. 6.9).

Figure 6.9 Natural skeletons obtained from tetraterpene precursor GGPP.

Diversity of chemical skeletons: a practical strategy to benefit

2.1.6 Polyterpenes The category of terpenes with more than 40 carbon units is known as polyterpenes. They are widely used as CO-tackifiers in many quality hot melt and pressure-sensitive adhesives. In paints, varnishes, detergents, sealants, caulks, and rubber compounds, they are also used as performance modifiers, homogenizing agents, and adhesion promoters. Due to their light color, mild smell, and exceptional resistance to oxidation and discoloration, styrenated terpenes make a superb choice for high-quality adhesives. Excellent peel adherence to a variety of substrates is produced by the polymer’s mix of aromatic and aliphatic groups. 2.2 Alkaloids Alkaloids are a type of secondary metabolite with important displaying varied biological activities, such as analgesic, muscle relaxant, antioxidant, and so forth. These are utilized to benefit humanity and have been shown to be useful for some life-threatening diseases. Certain alkaloids have been demonstrated to cause suffocation, paralysis, and, in severe cases, patient death. A large variety of alkaloid extraction and estimation methods have been developed, making it easier for researchers to improve on previous approaches. Alkaloids are classified based on the heterocycle present in the system. The following sections describe the alkaloids based on ring size and the number of nitrogen atoms. 2.2.1 Pyrrole and pyrrolidine alkaloids Pyrrole and pyrrolidine ring-containing alkaloids are biologically active molecules having diverse effects in different biological systems. They disply a variety of biological activities, such as antipsychotic, b-adrenergic antagonist, etc. Similarly, pyrrolidine-based natural alkaloids like (S)-nicotine (from tobacco), scalusamides A (from the cultured broth of Penicillium citrinum), (R)-bgugaine (from Arisarum vulgane), 1,4-dideoxy-1,4-iminoD-ribitol (from Morus alba), and aegyptolidine A (from Aspergillus aegyptiacus) are found effective in treating different ailments [26,27] (Fig. 6.10). 2.2.2 Indole (pyrrole ring fused to benzene) Indole-based molecules present in different naturally occurring systems have shown significant therapeutic effects [28,29]. The naturally occurring amino acid tryptophan is an essential amino acid. The indole skeleton in nature shows diversified chemical structures originating from different precursors and pathways (Fig. 6.11). Many naturally occurring indole alkaloids like vincristine, vinblastine, vinorelbine, ajmaline, etc. have gained FDA approval for the respective therapeutic applications that they display. Indoles isolated from Catharanthus roseus have proven to be potent against carcinogenic states, including leukemia, lymphoma, melanoma, and so on. Allylated indole diketopiperazine (DKP) alkaloid, Okaramine S, obtained from Aspergillus taichungensis, and Jerantinine A, display cytotoxic activity. Reserpine, isolated from Rauwolfia serpentine, is used to

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Figure 6.10 Natural pyrrole and pyrrolidine alkaloids.

reduce high blood pressure. It is also found to be useful for treating people with schizophrenia. A few selected natural indole molecules are listed in Table 6.1, which shows potency against different cancerous cell lines. Strictamine, isolated from Alstonia scholaris, an indole alkaloid belonging to the Apocynaceae family, shows antimalarial, antijaundice and gastrointestinal properties. The plant extract of this is used in traditional medicine. The deethylibophyllidine alkaloid isolated from Aspidosperma sp., family Apocynaceae, inhibits the growth of plasmodium, leishmania, and Trypanossoma sp.

Diversity of chemical skeletons: a practical strategy to benefit

Figure 6.11 Indole-based natural products obtained from various sources.

2.2.3 Piperidine alkaloids Piperidine (Hexahydropyridine) is an organic heterocycle which is a key motif in depicting a variety of biological activities. L-Lysine amino acid is a precursor for the biosynthesis of it. The abundance of this amino acid in nature makes it adept of producing a large quantity of piperidine natural products. These NPs obtained from various sources possess several biological properties and act as anticancer, anti-inflammatory, and antipsychotic agents. Piperidine alkaloids are found in plants of the Piperaceae family. Several piperidine alkaloids isolated from herbs exhibit antiproliferative and anti-metastatic effects. Fig. 6.12 depicts the versatility of the piperidine skeletons.

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Table 6.1 Naturally occurring indole containing bioactive alkaloids. Drug-like molecule

Source

Biological activity

Drymaritin Caulerpin Arvelexin Harmaline Dragmacidins Trigonoliimines A-c Eudistomins Cycexpansamine A & B Methylaplysinopsin 6-Bromoaplysinopsin Rauvolfine C Physostigmine Gelliusine A

Drymaria diandra Caulerpa racemosa Isatis indigotica Optiorrhiza nicobarica Halicortex Trigonostemon Lii Ascidians Penicillium Aplysinopsis Reticulata Smenospongia aurea Rauwolfia reflexa Physostigma Venenosum Coledonian sponge Gellius sp.

Anti-HIV Bovine viral diarrhea virus Influenza virus A Anti-HSV-1 activity Antiviral activity Anti-HIV-1 activity Against RNA viruses Anti-inflammatory MAOa inhibition Affinity 5HT2A and 5HT2C Anticholinergic activity Anticholinergic activity Anti-migraine activity

a

MAO, monoamine oxidase.

Figure 6.12 Naturally occurring NPs containing piperidine as core structure.

2.2.4 Quinoline alkaloids (benzene ring fused at 2,3-position of pyridine ring) The quinoline alkaloid is another important class of nitrogen-based heterocycle showing a broad range of biological activities. The first isolated quinolone-based alkaloid, quinine, puts forward a new research topic. Since then, a wide range of new findings on such NPs and their applications have been studied and documented. The most important

Diversity of chemical skeletons: a practical strategy to benefit

quinolone alkaloid in anticancer treatment appears to be camptothecin obtained from the tree Camptotheca acuminate. Numerous alkaloids from these classes have also been reported to have antifungal, antiparasitic, insecticidal and anti-inflammatory properties. A few of the naturally occurring alkaloids are shown in Fig. 6.13. Evodiamine, extracted from Evodia rutaecarpa, is a quinolone alkaloid that has shown its efficacy by inducing apoptosis or cell cycle arrest, further preventing metastasis and angiogenesis. 2.2.5 Isoquinoline alkaloids (benzene ring fused at 3,4-position of pyridine ring) Isoquinoline alkaloids are an important family of heterocyclic compounds present in various plants. They occur mainly in families like Papaveraceae, Berberidaceae, and Ranunculaceae. They possess very good biological properties. Primarily derived from phenylalanine and tyrosine, they are obtained from dopamine by condensing with aldehyde or ketone. They are divided into two classes: simple isoquinolines and benzylisoquinolines. Berberine, an alkaloid found in Coptidis rhizome and Cortex phellodendri, shows good pharmacological activity. Recently, it has shown noteworthy amelioration of Ab25e35-induced apoptosis. Cedrin, obtained from Cedrus deodara, displayed a significant effect against AD by ameliorating mitochondrial dysfunction and apoptosis in pheochromocytoma (PC12) cells (Fig. 6.14). Cularines, isocularines and isoquinoline alkaloids are obtained from the genera Cearatocapnos, Corydalis, Dicentra, and Sarcocapnos (Papaveraceae). Salsolidine was obtained from Arthrocnemum glaucum. Recently, the alkaloid tamynine was obtained from Murraya paniculata.

Figure 6.13 Naturally occurring quinoline alkaloids.

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Figure 6.14 Naturally occurring isoquinoline alkaloids.

2.2.6 Aporphine alkaloids Aporphine alkaloids have an isoquinoline core base structure and broadly exist in nature. They are a class of Nelumbo nucifera alkaloids and are isolated from Nelumbo nucifera. They are annonaceae, menispermaceae, papaveraceae, ranunculaceae, lauraceae, monimiceae, magnoliaceae, berberidaceae, aristolochiaceae, leguminoseae, liliaceae, nymphaeaceae, piperaceae, symplocaceae, sabiaceae, and fumariaceae. Many structural analogues of aporphine have been isolated and tested for their bioactivity efficacy [30,103] (Fig. 6.15). Aporphine alkaloids show their selective efficacy as a-adrenoreceptor blocking agents. Nantenine alkaloids of the aporphine type display potency in antiserotonergic activity. Boldine is an example that shows its potency in preventing lipid peroxidation and the deactivation of cytochrome. Similarly, many other pharmacological activities are being exhibited by this class of alkaloids such as antiplatelet effect, anti-neurological effects, immunoregulatory activity, antivirus, anthelmintics, antifungal, antimalarial, antileishmanial, anti-inflammatory, antipyretic, cytoprotective effects and so on [30e32] (Fig. 6.16). 2.2.7 Indolizidine alkaloids Indolizidine alkaloids are chemical skeletons with two fused rings, one five-membered and another six-membered, with a common nitrogen atom. The unusual biosynthesis of amino acid pipecolic acid and subsequent transformation results in a diverse set of indolozidine-based chemical skeletons (Fig. 6.17). From the literature, it is noticed that the tylophorines, lycorines, and securinines, which contain multicyclic structures,

Diversity of chemical skeletons: a practical strategy to benefit

Figure 6.15 Aporphine alkaloids diversity.

Figure 6.16 Aporphine analogues showing biological variabilities.

show better potency against their respective biological targets. Further molecules possessing sterioisomeric centers enhance the activity. Securine-like molecules are not well known for their biological activity. Slaframine and swainsonine, two biologically active alkaloids isolated from R. leguminicola, are best known to pass through the blood-brain barrier and can cause neurological defects. Other indolizidine alkaloids with stereocenters have also demonstrated relative activities [33] (Fig. 6.18).

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Figure 6.17 Diversity of indolizidine NPs from different source.

Figure 6.18 Important indolizidine scaffolds possessing various biological activity.

Diversity of chemical skeletons: a practical strategy to benefit

2.2.8 Pyrrolizidine alkaloids Unlike indolizidine, the pyrrolizidine chemical skeleton is composed of two fivemembered fused rings with a bicyclic N-atom nucleus. These are derived from ornithines that are distributed in plants of certain taxa and are also found in insects that uptake them for defense against predators. This type of alkaloid occurs as tertiary bases or as their Noxides (PANO). Retronecine, heliotridine, otonecine, and platynecine have the necine base structure of pyrrolizidine alkaloids (Fig. 6.19). This alkaloid class also has a range of activities, including anti-HIV and acetylcholinesterase inhibitors. Along with the beneficial biological activity, these compounds show some toxicity due to bioactivation when administered. Therefore, only a few FDA approved drugs are being reported [34] from this class of alkaloids (Fig. 6.20). 2.2.9 Tropane A skeletal pattern with 8-azabicyclo [3.2.1] separates tropane alkaloids. Tropanecontaining molecules are abundant in the Apocynaceae, Brassicaceae, Faboideae, Moraceae, Olacaceae, and Proteaceae (Fig. 6.21). Tropane chemistry research has paved the way for structurally distinct synthetic drugs (e.g., local anesthetics) [35]. 2.2.10 Purines Purine is another heterocyclic alkaloid containing a fused pyrimidine ring with an imidazole ring. They exhibit anti-viral, antimicrobial, phosphodiesterase inhibitory,

Figure 6.19 Naturally occurring pyrrolizidines NPs.

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Figure 6.20 Biologically active natural pyrrolizidines.

Figure 6.21 Tropane moiety containing NPs from different sources of natural plants.

Diversity of chemical skeletons: a practical strategy to benefit

antitumor, and antiprotozoal activities. Purine analogues substituted with dialkyl esters perform better in viral cell studies. O6-alkylguanine derivatives of purine have antitubercular, antiulcer, antimicrobial, cardiotonic, antiviral properties and antineoplastic, antitumor properties via induction of cancer mutations and cell death [36]. A few examples of this class are listed below, showing the structural variations (Fig. 6.22). 2.3 Phenolic compounds Polyphenols are plant-based natural products with an extensive range of intricate structures (Fig. 6.23). These are categorized as phenolic acids and phenolic alcohols. Polyphenols contain a phenolic ring as the basic monomer, but depending upon the number of phenol rings and chemical structure, they are further classified as phenolic acids, flavonoids, stilbenes, phenolic alcohols, and lignans. Polyphenols are highly active ingredients in the human body’s metabolism and are preventive scaffolds against medical complications. Polyphenols are mostly found in plant-based foods, and appear to have antioxidant properties [37]. 2.3.1 Phenolic acids This is a naturally occurring class of phenolic secondary metabolites. These molecules contain one carboxylic acid unit joined to the phenol ring. Therefore, all the skeletons containing this core structure are termed phenolic acids. They are classified as

Figure 6.22 Chemical skeleton diversity of purine in nature.

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Figure 6.23 Biogenesis of polyphenols.

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Figure 6.23 Cont’d

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hydroxybenzoic and hydroxycinnamic acids. The formation of phenolic acids follows the classical shikimic pathway via the formation of phenyl alanine into a diverse class of phenolic acids. All these chemical skeletons have a wide variety of applications as pharmaceutical agents, food preservatives, nutraceuticals, animal husbandry products, skin care products, etc. Phenolic acids partially work as signaling molecules in plant metabolism. They also act as defensive soldiers for plants. Phenolic acids such as hydroxybenzoic and hydrocinnamic acids preferably show antioxidant properties. They enhance the nutritional quality of food items. Due to the rate-limiting efficacy of ROS produced, they have been effective as antidiabetic agents. These molecules have also shown their efficacy as antimicrobial agents and in other therapeutic activities. Protocatechuic acid is a plant phenolic acid isolated from the stem bark of Boswellia dalzeilii and the leaves of Diospyros melasoxylon. They act as good antioxidants and anti-inflammatory agents. Caffeic acid, isolated from Eucalyptus globulus, barley grain, herb Dispsacus asperoides, fresh water fern Salvinia molesta, and mushroom Phellinus linteus, demonstrates potency in anticancer studies. p-Coumaric acid is a natural product found in Gnetum cleistostachyum. Gallic acid is present in plants such as Cynomorium coccinclum, Myriophyllum spicatum, and the blue-green alga Microcystis aeruginosa. Similarly, ferulic acid, sinapic acid, p-hydroxy benzoic acids, syringic acid, and gentistic acid are examples of isolated phenolic acids with diverse pharmaceutical effects [38] (Fig. 6.24). 2.3.2 Coumarins Coumarins (2H-1-benzopyran-2-one) have a benzene and pyrone ring system that is fused. They are found as secondary metabolites in plants, bacteria, and fungi. More than 1300 coumarin-skeletal chemical compounds have been isolated from approximately 150 different species. In the Plantae kingdom, coumarins are mostly isolated from 30 different families, such as Rutaceae, Oleaceae, Nyctaginuceae, Umbelliferae,

Figure 6.24 Naturally occurring phenolic acids.

Diversity of chemical skeletons: a practical strategy to benefit

Clusiaceae, Gluttiferae, and Apiaceae. Based on structural analogues, they are further classified into six sub-categories. They are coumarins, furano coumarins, dihydrofurano coumarins, pyrano coumarins, phenyl coumarins, and bicoumarins. All the above classes possess physicochemical properties and therapeutic applications. Esculetin is obtained from many plant species and is shown to have anti-inflammatory activity. Dicoumarol, isolated from sweet clover, exhibits anticoagulant activity. Coumarins with long-chain hydrocarbon substituents have antibacterial potential. For example, ammoresinol, found in Ferula ammoniacum and Ferula michaelii, showed good antibacterial properties. Another example of such is novobiocin, isolated from microbes, which shows similar activity. Osthole isolated from Angelica pubescens, Cnidium monnieri, and Peucedanum ostruthium exhibits a broad range of antifungal activity against important pathogens. Other isolated natural coumarins with therapeutic effects include (
) calanolide A/B, 300 -demethylchartreusin, scopoletin, xanthyletin, fraxin, methoxsalen, fraxidin, grandivittin and ayapin [39,40] (Fig. 6.25).

Figure 6.25 Bioactive natural coumarins.

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2.3.3 Flavonoids Flavonoids are a class of natural secondary metabolites with structurally diverse phytochemicals. These compounds are ubiquitous and known for their positive effects on health. They are key ingredients for nutraceutical, medicinal, and cosmetic purposes. They possess a wide range of bioactivities, such as antioxidant, anti-carcinogenics, etc. The use of flavonoids is not only restricted to human health but can also be effectively used in agricultural products. On the basis of plant source, they are extracted and further classified into subgroups. Chalcones, flavones, flavonols, and isoflavones are the most common subgroups. The collection of a specific type of flavone in a specific species is responsible for describing the color and aroma of flowers, reproduction, seedling development, and so on. Neoflavonoids are a group of polyphenolic compounds having a 4phenylchromen motif with no hydroxyl group at the second position. Calophylloide is the first neoflavonoid isolated from a Calophyllum inophyllum plant source. They are classified as Latifolin and Dalbergin based on the chemical skeleton and source from which they are obtained. Dalbergin analogues are isolated from D. nigra, D. odorifera, Pterocarpus santalinus, D. stevenonii, D. latifolia, and D. sissoo plants. Another class of diphenyl allyl neoflavonoids has been isolated from D. sissoo, D. nigra, Dalbergin louvelii, rosewood, D. odorifera, D. melanoxylon, D. inundata, D. riparia, P. Santalinus, Manchaerium Kuhlmannii, M. miscolobium, M. nictitans, Nepalese propolis, Pterolinus G., etc. [41,42] (Fig. 6.26). Flavonols or flavan-3-ols, are highly diversified 3-hydroxy analogues of flavones. These are chemical skeletons with two aromatic rings and one heterocyclic ring. These flavonols are mostly found in dietary plants. All the derivatives of flavonols show a range of biological activities, such as antitumor, anti-cancer, anti-diabetic, etc. Fig. 6.27 depicts a few of the beneficial flavanol subclasses isolated from various plants. Catechins are major constituents of tea-leaves that show potent antioxidant and physiological activities. They are basically isolated from Canellia sinensis (C. sinensis) and C. assumica. Kaempferol is another natural flavonol, isolated from pteridophyta, pinophyta, and angiosperm types of plants. Derivatives of kaempferol modulate apoptosis, angiogenesis, inflammation, and metastasis. Quercetin is a plant pigment, extracted from colored plants or food ingredients. They are well known for acting against blood sugar, cancer cells, heart disease, inflammation, etc. Mostly, they are good antioxidants. Myricetin is another example of a flavonol with high nutraceutical properties. Iterative studies of these compounds have proven that they act against Alzheimer’s and Parkinson’s diseases. Naringenin, a subclass of flavonols, is found in glycosidic forms. They are mostly found in citrus fruits, bergamot, tomatoes, and other fruits. Narigine derivatives have a variety of biological effects on human health, including promoting carbohydrate metabolism, acting as ROS scavengers, decreasing lipid peroxidation and protein carbonylation biomarkers [43].

Diversity of chemical skeletons: a practical strategy to benefit

Figure 6.26 Naturally occurring flavonoids.

Figure 6.27 Naturally occurring flavonols.

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The pigment in its glycosylated form, anthocyanin, is a colored water-soluble pigment belonging to the flavonoid family (Fig. 6.28). They are major constituents in red-to-purplish blue-colored leafy vegetables, fruits, and edible plants. Cyanidin-3-glucoside is the major component of these abiotic species, influenced by pH, light, temperature, and structure. They are well known as antioxidant, antimicrobial, and neurological health agents and are active against noncommunicable diseases. The most abundant anthocyanidins are cyaniding (50%), delphinidin (12%), pelargonidins (12%), peonidin (12%), malvidin (7%) and petunidin (7%). They are found in nutraceuticals and have traditionally been used as phytopharmaceuticals, appetite stimulants, choleretic agents, and so on [44]. Isoflavones are naturally occurring phytoestrogens, nonsteroidal phenolic plant compounds. These compounds are further classified into five subclasses such as genistein, daidzein, glycitein, biochanin A, and formononetin (Fig. 6.29). Liquiritigenin and naringenin are two precursors for the abovementioned formation of isoflavones. Equol and its metabolites are more bioactive and show predominately estrogenic and antioxidant properties. It also possesses anti-androgenic properties. Soyabean isoflavone supplements decrease

Figure 6.28 Naturally occurring flavonols as pigments.

Figure 6.29 Structures of naturally occurring isoflavones.

Diversity of chemical skeletons: a practical strategy to benefit

estrogenic activity and may show menopausal symptoms. Another isoflavone, genistein selectively antagonizes the catabolic effects of parathormone in the osteoblasts [45]. 2.3.4 Polyphenolic amides Polyphenolic amides are a class of polyphenols that have an amide group. They are classified into two subgroups; they are avenanthramides and capsaicinoids (Fig. 6.30). Avenanthramides are low molecular weight phenolic acid amides that are linked by an amide bond between anthranilic acid and hydroxycinnamic acid. Oats are the major source of these types of chemical skeletons. Approximately 40 different types of avenanthramides are present in oat grains and oat leaves. Capsaicinoids are a class of chemical skeletons actively present in chili peppers and belong to the genus Capsicum. They are classified as polyphonic amides rather than alkaloids due to the presence of a phenolic group and an amide moiety in the same structure. These are mostly found in chilli species. Both avenanthramides and capsaicinoids have anti-inflammatory and antioxidant properties [46,47]. 2.3.5 Stilbenes Stilbenes are chemical structures having a C6eC2eC6 framework with two isomeric forms. They are mostly found in botanical families such as Vitaceae, Leguminaceae, Gnetaceae, and Dipterocarpaceae. Over 459 natural stilbenes are identified from 45 botanical families and plants. Stilbene synthase is the crucial enzyme that is responsible for the diversified range of stilbene skeletons. The two isomeric forms of stilbene, (E)-stilbene and (Z)stilbenes, exist in different physical forms. Stilbenes are produced as defensive chemicals in response to infection in plants. For example, resveratrol and pinosylvin are two trans stilbenes acting as phytoalexins. They also possess pharmaceutical, therapeutic, and nutraceutical properties. They are well known for their biological properties. Combretastatin, isolated from Combretum caffrum bark, has shown good results as an anticancer agent. Resveratrol has also been investigated for antimicrobial activity. From the literature data, latitolol and 15 others showed significant anti-inflammatory activities. Hopeahainol A, isolated from Hopea lainanensin, shows anti-acetylcholinesterase activity. Rumexiod, derived from Rumex bucephalophorus, exhibited antidiabetic activity. Fig. 6.31 depicts the contribution of stilbenes as therapeutic agents with diverse bioactivities [48].

Figure 6.30 Polyphenolic amides.

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Figure 6.31 Chemical structures of naturally occurring bioactive stilbenes.

2.3.6 Tannins From ancient times to the present, tannins have been explored for their different variations and applications. They are present in all parts of the plant. These are classified into two types: hydrolysable tannins and condensed polyflavonoid tannins. Polyflavonoid tannins are much more stable. These condensed tannins are basically flavonoid oligomers. Other minor components present in the condensed type of tannins are flavon3-ol, flavan-3,4-diols, and other flavonoid analogues; carbohydrates; imino and amino acids. Hydrolysable tannins are simple compounds of gallic acid. They are further categorized based on the hydrolysis products that they form. The two subclasses are gallotannins and ellagi-tannins. Gallo-tannins are obtained from plants containing a polyol residue, whereas ellagi-tannins are found in berries, tea, and wine fermented in oak barrels. Tannins have much importance in the industrial sector. For example, as a bio-based material for the manufacture of leather from vegetable origins. Due to their adherence property, they serve as adhesives for wood products (especially in carpentry). Tannins are

Diversity of chemical skeletons: a practical strategy to benefit

also known for pharmaceutical and medical applications. Iterative studies showed them to be potential bactericides due to the irreversible process of protein activity. They also have positive anticancer properties. Acutissimin A is a bound flavonoid with ellagitannins, that has shown its effectiveness against apoptosis in comparison with the drug etoposide. The presence of naturally occurring tannins actually accounts for the characteristic change in taste in wine, beer, and fruit juices. Due to the many interesting facts about tannins, researchers began to exploit their use as antioxidant agents. The variety of applications of tannins on different platforms has been a hot-spot for researchers for future development [49,50]. 2.3.7 Lignans Natural phenolic compounds called lignans are derived from the phenylproponoid pathway. They are found in many dietary sources, such as vegetables, fruits, legumes, whole grain cereals, and oil seeds. Sesame and flax seeds contain a high concentration of lignans. They play a vital role in plant protection, nutraceuticals, and therapeutic use. They have the potential to act as remedies for diabetics, oxidation, cardiovascular disease, microbial infections, and inflammatory responses. The structure of lignans contains 9-carbons in a C6eC3 fused phenylpropane unit. This phenylpropane side unit is later seen with different substitutions, which further classifies lignans into eight subgroups. They are dibenzylbutane, aryltetralin, dibenzylbutyrolactone, dibenzylbutyrolactol, dibenzocyclooctadiene, arylnaphthalene, furan, and furofuran derivatives. Apart from these subclasses, they are diversified based on the presence and absence of oxygen [51,52] (Fig. 6.32). Phodophyllotoxin is mainly derived from the Podophyllum genus and is known for its potential anticancer properties [53]. 2.3.8 Lignins Lignin is the second largest biomass next to cellulose [54e56]. They are highly branched and naturally occurring long-chain polymers. Three common repeating units are p-coumaryl, coniferyl, and sinapyl alcohol. They are chemically active ingredients found in the environment, that are highly responsible for genetic alteration due to environmental factors. Lignin is a polymeric material, used as a fertilizer, herbicide, and in material development in the fields of bioplastics technology, nano-composite, and catalysis. Lignincarbohydrate-protein from Pimpinella anisun has shown good results for antiviral activity against HSV1 and 2 viruses. Lignophenol has been shown to be a potential antidiabetic agent due to its ability to inhibit the a-glycosidase enzyme. Lignin from sugarcane bagasse has been evaluated for antioxidant activity, and other functionalized chitosans seem to have improved antimicrobial properties. Lignin-based hydrogels and their derivatives are considered as scaffolds for tissue engineering applications. Lignosulfonate and lignin-based complexes are employed in drug delivery applications. Similarly, the lignin precursor sinapyl alcohol exhibits anti-inflammatory and anti-nociceptive properties.

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Figure 6.32 Chemical structures of naturally occurring bioactive lignans.

2.4 Polyketides Polyketides are a large group of natural reservoirs having COeCH2 repeated units. These large and diverse secondary metabolites were first discovered in 1893 by J. Norman Collie. Polyketides are globally produced by bacteria, fungi, plants, and certain marine organisms. For example, tetracycline is isolated from Streptomyces aureofaciens, lovastatin is isolated from Phomopsis vexans (bacteria), emodin from Rheum palmatum (plant), maitotoxin-1 from Gambierdiscus australes (protists), stegobinone from stegobium paniceum (insect), elysione from Elysia virdis (mollusks), etc. Polyketides are classified based on structure (as aromatics, macrolactones, macrolides, decalins, polyethers, and polyenes) and biosynthetic pathways [57] (as Type I PKS, Type II PKS, and Type III PKS). They share a range of applications in pharmaceutical platforms such as antibiotics, antifungals, cytostatics, anti-cholesteremics, etc., as shown in Fig. 6.33. 2.5 Carbohydrates Carbohydrates (CHOs) [58] are mainly recognized as dietary foods and are considered an important source of energy for various biological entities. Carbohydrates are the most abundant class of biomolecules. They are either found as free compounds or covalent glycoconjugates such as glycoproteins or glycopeptides, glycolipids, and several other glycosylated natural products. Carbohydrates are building units and are important in metabolism. They have been involved in notable biochemical events such as intercellular adhesion, molecular recognition, and signal transduction. Due to their crucial biological significance in life processes, they are visualized as new sources of drugs and therefore

Diversity of chemical skeletons: a practical strategy to benefit

Figure 6.33 Chemical structures of naturally occurring bioactive polyketides.

offer opportunities. Based on the number of saccharide units and the number of glycosidic linkages, carbohydrates can be classified as (1) sugars (monosaccharides and disaccharides, DP 1e2); (2) oligosaccharides (DP 3e9); and (3) polysaccharides (DP  10).

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Although carbohydrates have similar monomer compositions, they differ drastically with respect to physiological and biochemical behavior. For example, consumption of sucrose, which results in glucose and fructose formation, leads to different gastrointestinal and post-absorptive effects as compared with ingestion of glucose or fructose as a single source. Hence, it is important to understand its chemical structure. Further subclassification of the carbohydrates is given in Table 6.2. Carbohydrates acquire three important attributes: a high density of functional units, i.e., mainly hydroxyls; diversity of structural isomers, and ideal biocompatibility, as they are key ingredients in life processes. Due to this characteristic feature, carbohydrate moieties in glycosylated natural products increase the drug water solubility, decrease its toxicity, and improve the overall activity of the product. Many of these have gained gained popularity due of their documented biological activities, such as antioxidant, antimicrobial, antidiabetic, hypolipidemic, and immunomodulatory activities [59,60]. Thus, carbohydrates have medicinal importance, and one could consider them as functional handles toward the discovery of a new drug. 2.5.1 Sugars Monosaccharides [61] are the simpler, nonhydrolysable carbohydrates that are referred to as monosaccharides. These are the basic, single molecules that build the higher classes of carbohydrates. They are found both in ring and chain forms, and contain multiple Table 6.2 Classification of carbohydrates based on chemical structure. Category

Subclass

Common names

Sugars

• Monosaccharides • Disaccharides • Sugars alcohols/polyols

Oligosaccharides

• Non-digestible oligosaccharides • Starch • Maltodextrins

Polysaccharides

• Non-starch polysaccharides (NSP) • Resistant starch (RS)

• Glucose, galactose, fructose, mannose, arabinose, xylose, erythrose, and others. • Sucrose, lactose, isomaltulose, maltose, trehalose, and others. • Mannitol, lactitol, sorbitol, xylitol, erythritol • Raffinose, stachyose, fructooligosaccharides, arabinooligosaccharides, and others. • Amylose, amylopectin, and modified starches. • Glucose, maltose glucooligosaccharides • Cellulose, hemicellulose, pectin, hydrocolloids (Arabic gum, guar gum, others). • RS type 1,2,3, and 4

Diversity of chemical skeletons: a practical strategy to benefit

hydroxyl groups. The derivatives of these molecules impart diverse pharmacological effects, and the synthesis of them is widely studied. Based on the presence of a carbonyl functional group (i.e., ketone or aldehyde), they are classified as aldose and ketose. They are called as levorotary and dextrorotatory depending on which way the hydroxyl group is facing. They are also referred to as “optical isomers” of each other. Glucose, fructose, galactose, mannose, D-ribose, D-2-deoxyribose, and sorbose are examples of commonly studied molecules (Fig. 6.34). Disaccharides are sugar-based compounds that contain two monosaccharide units that are the same or different. The two monosaccharides are linked via a glycosidic linkage. Disaccharides with significant nutritional values include lactose, maltose, sucrose, isomaltose and trehalose (Fig. 6.35). For example, sucrose is a major component in fruits and vegetables and acts as a natural sweetener. Trehalose is mainly used as a food additive, and it’s mostly a constituent in mushrooms. Lactulose is a laxative. Unlike monosaccharides, disaccharides also possess pharmacological effects when bound to drug-able natural or synthetic molecules. The addition of sugars increases the pharmacological effects of the drug motif. 2.5.2 Oligosaccharides Oligosaccharides [61] are a well-studied subclass of carbohydrate natural products. They usually consist of 3e10 monosaccharide units linked covalently via glycosidic bonds. They differ structurally in terms of the number and type of monomeric sugar units (e.g., fructosyl, glucosyl, galactosyl, xylosyl), as well as the position and conformation of links between sugar moieties. They can exist in a linear or branched arrangement. Common naturally occurring oligosaccharides include raffinose, stachyose, and verbascose. Further, based on the type of monomeric or dimeric sugar moieties, they are further subclassified as fructo-oligosaccharides (FrOS), galacto-oligosaccharides (GOS), isomaltooligosaccharides (IMO), arabino-oligosaccharides (AOS), xylo-oligosaccharides (XOS),

Figure 6.34 Chemical structures of common natural monosaccharides.

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Figure 6.35 Chemical structures of common natural disaccharides.

lactulose, gentio-oligosaccharides, soybean oligosaccharides (SOS), lactuosucrose, and cyclodextrins (CD) [62e64]. Some of these oligosaccharides occur naturally in several legumes, cruciferous vegetables, and whole grains; others are synthesized from natural sugars or by enzymatic hydrolysis of polysaccharides. As illustrated in Table 6.3, we could also see some animal and microorganism derived oligosaccharides such as human milkderived galacto-oligosaccharides and algae derived marine oligosaccharides (ADMO). Oligosaccharides are soluble in water and are somewhat sweet. The sweetness decreases with increasing oligomeric chain length. Because humans lack the digestive enzyme galactosidase, we are unable to properly digest these carbohydrates, and as a result, oligosaccharides remain unhydrolyed and pass undigested into the lower gut. Nevertheless, they are primarily used as dietary supplements due to their nutritional properties. In recent years, oligosaccharides have been considered special due to their effect on the microflora of the gastrointestinal tract. Galacto-oligosaccharides and fructo-oligosaccharides are well known for their benefits to the host, promoting some interesting bacterial growth in the colon (e.g., lactobacilli, bifidobacteria), thereby improving host health. Hence, nowadays, they are termed as prebiotics. Oligosaccharides have low lipophilicity because of their large number of hydroxyl groups. Typically, the lower the lipophilicity of a drug, the inferior is its absorption profile when administered orally. Hence, pure oligosaccharide drugs are intended for curing gastrointestinal tract diseases where less absorption is necessary.

Table 6.3 Overview of chemical structure and source of common oligosaccharides. Type of oligosaccharides

Natural occurrence/synthetic

Fruits and vegetables onions, banana, garlic, etc.

Lactose derived

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Human milk

Stems and roots of gentian, gentiobiose derived

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Table 6.3 Overview of chemical structure and source of common oligosaccharides.dcont’d Natural occurrence/synthetic

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Type of oligosaccharides

Soy sauce and honey, enzymatic transformation of starch

Fermentation of sugar beet pulp

Algae derived

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Polyxylans hydrolysis

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Bioactive oligosaccharide natural products are secondary metabolites that have been discovered to target a range of biological processes, such as bacterial translation interference, disruption of cell wall biosynthesis, and inhibition of human a-amylase. They show potent biological activity against bacterial infections and type II diabetes. Oligosaccharide natural products mainly include aminoglycosides, steroidal glycosides, terpenoid glycosides, glycophospholipids, and phenolic glycosides. Acarviostatins [65] are naturally occurring aminoglycosides, also known as pseudooligosaccharides, isolated from cultures of S. coelicoflavus var. nankaiensis. Initially, six acarviostatins were identified and structurally characterized. Each acarviostatin consists of a pseudotrisaccharide core flanked by D-glucose units at the reducing and non-reducing ends [66,67] (Fig. 6.36). Among all, Aacarbose is the most basic acarviostatin among all acarviostatins, and is a powerful a-glucosidase inhibitor prescribed for type II diabetes. It was obtained from the fermentation broth of the Actinoplanes strain SE 50 and was the first commercially marketed a-glucosidase inhibitor under the trade name Glucobay. Acarviostatins have been found to be more potent a-amylase inhibitors than acarbose. Acarviostatin III03, for example, is 260 times more active than acarbose. It is important to understand here that a-amylase inhibition activity is caused by the combined effect of valienamine-based core and attached sugar residues. This is the reason why validamycin A, an antifungal drug, is not an inhibitor of a-amylase due to the absence of sugar units. Orthosomycins [68] are a class of antibiotics and naturally occurring phenolic glycosides. They have an orthoester connection between sugar residues. Based on the number of orthoester bonds and sugar residues, they are divided into two classes: Class I heptaand octasaccharides with two orthoester bonds and a dichloroisoeverninic acid ester appendage, such as Avilamycins, Everninomicins, Flambamycin, and so on. Hygromycin

Acarbose Acarviostatins II03 Acarviostatins II13 Acarviostatins III03

l 0 0 1 0

m 1 2 2 3

n 1 3 3 3

Figure 6.36 Generalized structure of naturally-occurring acarviostatin oligosaccharides.

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B and Destomycins are examples of Class II pseudotrisaccharides with one orthoester bond and an aminocyclitol. Everninomicins are isolated from Micromonospora carbonacea. Everninomicins [65] are octasaccharides having a dichloroisoeverninic acid. The majority of everninomicins have an orsellinic acid at the other end of the saccharide chain. Everninomicins have three unique oxidative features. A methylenedioxy bridge attached to ring F, orthoester linkages between rings C and D and rings G and H. L-evernitrose is a nitrosugar. Rings B, C, and sometimes D (D-evalose) are 2,6-dideoxy sugars, while ring E (4-O-methyl-D-fucose) is a 6-deoxygenated sugar. Ring F is 2,6-di-O-methyl-Dmannose, ring G is L-lyxose, and ring H is eurekanate (Fig. 6.37). Avilamycins [69] are heptasaccharides, isolated from Streptomyces viridochromogenes T€ u57. Structurally, they are alike to everninomicin but deficient in the nitrosugar unit. The orthosomycins act as bacterial translation inhibitors, differing in mechanistic pathway as compared to antibiotics currently in clinical use. Everninomicin A [70] was active against both Gramnegative and Gram-positive bacteria, whereas both everninomicin D and B were only active against Gram-positive bacteria. The absence of orsellinic acid appendage to eurekanate residue in everninomicin D and B is the reason. Additionally, reduction of nitro to amino eliminates Gram-positive action while considerably increasing Gram-negativity. Moenomycins [65,71] are naturally occurring, novel classes of phosphoglycolipids obtained from Streptomyces species. Moenomycin A is composed of three characteristic parts: pentasaccharide, phosphoglycerate, and a C-25 isoprenyl lipid chain. Most of the moenomycins contain a main tetrasaccharide linked to a lipid chain via phosphoglycerate moieties. The other variations are N-acetyl glucosamine or 6-deoxy chinovosamine as rings C and E or the C4 position of ring F (Fig. 6.38). The moenomycins are inhibitors of peptidoglycan glycosyltransferases. They have a potency of up to 1000 times higher than that of a gylcopeptide antibiotic, i.e., vancomycin.

Figure 6.37 Structure of naturally occurring orthosomycin oligosaccharides.

Diversity of chemical skeletons: a practical strategy to benefit

Figure 6.38 Structure of naturally occurring moenomycin A oligosaccharide.

Saponins [59] are a type of glycosylated secondary metabolite. Large number of saponins are identified and characterized using spectroscopic techniques. Saponins can be categorized as steroidal and triterpenoid glycosides according to their aglycones. For many years, due to their amphiphilic nature, they were used as natural surfactants in Africa. Additionally, they were used to kill diseased snails and avert the spread of schistosomiasis. Saponin extracts from ginseng, ivy quillaia, sarsaparilla, primula, and senega are employed as folk treatments. Steroidal glycoside, digoxin, is a well-known cardiac drug used to treat congestive heart failure (Fig. 6.39). Recently, digoxin showed anticancer activity. Also, several steroidal saponins such as dioscin, aginoside, etc. showed good antifungal activities. Microalgae are recognized as a source of oligosaccharides and polysaccharides. Algaederived marine oligosaccharides [72] are categorized into varied types according to their chemical diversity. Marine algae sourced oligosaccharides find potential application in functional foods (prebiotics), biomedicine, nutraceuticals, cosmetics, and environmental protection. Good solubility and little toxicity of chitosan oligosaccharides are important in medicinal applications for numerous biological activities such as antimicrobial, antiAlzheimer’s, antidiabetic, hypoglycaemic, anticoagulant, and hypocholesterolemic properties. Carrageenan oligosaccharide shows promising bio-activity, which includes cholesterol-lowering effects, antiviral, immunomodulatory, and antioxidant properties. 2.5.3 Polysaccharides In nature, polysaccharides exist in almost all living forms, including in the tissues of seeds, leaves, and stems of herbal plants, body fluids of animals, extracellular fluids, and cell walls of bacteria, yeast, and fungi. Polysaccharides [61] are complex biopolymers composed of

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Figure 6.39 Structure of naturally occurring saponins steroidal glycoside.

various monosaccharides such as hexoses, pentoses, and their acids. When repeating monomeric sugar units exceed 10 numbers, linked together through glycosidic bonds in a linear or branched structure, then they are known as polysaccharides (PSs). Polysaccharides can be classified in several ways, for example, chemical composition, structure, solubility, sources, and applications. Based on electric charge [73], PSs can be categorized as anionic polysaccharides (heparin, alginic acid, chondroitin sulfate, etc.); cationic polysaccharides (chitin, chitosan); and nonionic or neutral polysaccharides (dextran, starch, and cellulose). Based on of chemical composition [74], polysaccharides are broadly classified into two groups, namely homopolysaccharides (homo-PSs) and heteropolysaccharides (heteroPSs). A homopolysaccharide is defined to have only one type of monosaccharide repeating unit in the polymer chain, whereas a hetero-polysaccharide contains two or more distinct types of monosaccharide units. For example, cellulose and glycogen consist of glucose as a repeating sugar unit, whereas heparin is composed of a-L-idopyranosyluronic acid 2-sulfate and 2-deoxy-2-sulfoamino-a-D-glucopyranose 6-sulfate. Both homo-PSs and hetero-PSs may consist of homo or hetero linkages with reference to configuration and position of linkage. Thus, based on different glycoside linkages, polysaccharides can also be further classified as glycoproteins, glycolipids, and glycoconjugates. In both types of polysaccharides, the monosaccharide units can be linked in either a linear pattern or can branch out into complex formations. Furthermore, it should

Diversity of chemical skeletons: a practical strategy to benefit

also be noted that, for a polysaccharide to be considered acidic in nature, it should contain one or more types of acidic groups such as carboxyl, sulfuric, or phosphate. Earlier research suggested that the only PSs with medicinal properties are homopolysaccharides, particularly glucans [73,75,76]. However, hetero-PSs also exhibit interesting bioactivities. In fact, bioactivities of both homo-PSs and hetero-PSs are characteristics of their structure. Polysaccharides and oligosaccharides are the source of energy and building units of life and facilitate several biological signals. This led the researchers to develop polysaccharides for biomedical use such as therapeutic entities, drug carriers, and tissue scaffolds. Overall, due to diverse structures, polysaccharides result in distinctive functionalities and bioactivity. Further, based on the type, source, composition, and biological effects, recent advances in bioactive polysaccharides are summarized in Table 6.4. A few naturally occurring antibacterial polysaccharides were also isolated from natural resources. Saccharomicins [65] polysaccharides are one of its types. Till date, two kinds of saccharomicins are reported: saccharomicins A and B (Fig. 6.40). 2.6 Antibiotics Since the discovery of Penicillin from Penicillium notatum [77], microorganisms have become an imperative class to isolate diversified chemical compounds that have been utilized in medicine, agriculture, the food industry, and scientific research. Microbes account for 20% of all NPs and are used as antibiotic agents in pharmaceuticals. The most common classes of antibiotics that have been widely explored are penicillins, cephalosporins, carbapenems, tetracyclines, etc. (Fig. 6.41). Since microbes can produce an appreciable number of natural products, it is an emerging alternative to express biosynthetic genes from the original producers in microbial hosts, especially bacterial and fungi. Due to extensive growth and possible genetic alteration in bacterial cells, these organisms have been contributing more than fungi. More than 60% of current compounds are isolated from Taxomyces andreanae and Nodulisporium sulviforme. These include Actinomycin D, anthracycline (doxorubicis, and valrubicis), bleomycin, mitosanes, anthracenones, and enediynes. These compounds stimulate tubulin polymerization and interfere in normal microtubule breakdown.

3. Semisynthetic or modified NPs Once a natural product shows some bioactivity, it is the job of organic chemistry to tweak the molecule to improve upon the activity. The tweaking of the molecule can be done by adding some groups or modifying the structure by removing some. For example, ester derivatives of naturally occurring geraniol are active in the selective inhibition of hCES2. Geranio-butanolide hybrid compounds showed excellent antibiofouling activity. Selective oxidation of geraniol leads to the potential antifungal agents [78e80] (Fig. 6.42).

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Table 6.4 Bioactive polysaccharides derived from different biotic sources. Chemical composition

Natural sources

Biological activity

Dietary fiber

Cellulose

b-(1 / 4) linked-Dglucopyranose, homo and linear chain Mannans, xylans, xyloglucans and b-glucans with mixed linkages

Fruit, vegetables, nuts, grains.

b-Glucans

b-(1 / 4) and b-(1 / 3)-D-glucose

Barley grains, oats

Resistant starch

a-(1 / 4) and/or (1 / 6) linked D-glucopyranosyl

Potatoes, rice, green bananas, legumes

Inulin

b-(1 / 2)-DFructofuranosyl

Wheat, onion, chicory root, garlic

Astragalus polysaccharides

Rhamnose, arabinose and glucose (molar ratio of 1: 6.25:17.86) (1 / 4)-linked homogalacturonan backbone Majorly glucose

Roots of Astragalus

Increase stool bulk and regulate bowel movement Antithrombotic activity, immunomodulating activity, antioxidant activity, cholesterol lowering effect, and regulate bowel movement Control blood glucose level and lipids; cholesterol reduction effect, lowering of hypertension Preclusion of colonic cancer, hypocholesterolemic and hypoglycemic effects, prebiotic Stimulate mineral absorption, hypolipidemic effects, prebiotic properties Immunomodulating activity

Hemicelluloses

Herbs

Ginseng polysaccharides

Polygonum multiflorum Thunb polysaccharides Chelidonium majus polysaccharide Reishi polysaccharide

Glucose, mannose and galactose (molar ratio 1: 4:5) Xylose, mannose, arabinose, rhamnose, glucose at the different ratios

Storage tissues of annual and perennial plants, legumes, fruit, and nuts.

Ginseng, the Panax ginseng roots

Anti-rotavirus activity

Polygonum multiflorum Thunb root Chelidonium majus

Antiglycation and antioxidant activity Antitumor immunostimulatory activity Stimulates the expression of inflammatory cytokines

Ganoderma lucidum

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Common name

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Category

Non-plant based

Repeating galactose units (D & L) and 3,6 anhydrogalactose

Red edible seaweeds

Alginate

Repeating units of 1,4linked b-mannuronate and 1,4-a-guluronate residues (1 / 2)-Linked Lrhamnose residues with sulfate groups b-Glucans and hetero-PS

Brown algae (Phaeophyceae)

Alternating units of (1 / 4)-linked Nacetylgalactosamine and (1 / 3)-linked glucuronic acid Repeated units of Nacetyl-2-amino-2deoxyd-glucose and 2amino-2-deoxy-Dglucose residues Repeated units of sulfonated hexuronic acid (1 / 4)glucosamine

Cartilage of animals

Sulfated rhamnan

Mushroom polysaccharides

Chondroitin sulfate

Chitin and chitosan

Heparin

Green algae, Monostroma latissimum Mushrooms

Antioxidant, anticoagulant, immunoregulatory effect, food additives Wound healing property, therapeutic agents, proteins delivery, and cell transplantation Anticoagulant activity

Antidiabetes, antiobesity, anticancer, and antibiotic properties Dietary supplement for treatment of osteoarthritis

Fungal mycelia and crab or shrimp shells

Drug delivery, wound healing

Porcine intestinal mucosa

Anticoagulation and binding affinity for growth factors

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Carrageenan

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Figure 6.40 Structure of naturally occurring saccharomicins polysaccharides.

Figure 6.41 Natural products as an antibiotic medicines.

The derivatives of thymol have shown different activities. Thymol-derived esters are active against E. coli bacteria. Thymol compounds having pyridine moieties display antimicrobial properties. Synthesized ether and aryl-azo-thymol derivatives have highly selective and potent antibacterial and antifungal activities [81e87] (Fig. 6.43).

Diversity of chemical skeletons: a practical strategy to benefit

Figure 6.42 Geraniol-motiff as drug agents.

Figure 6.43 Thymol-based derivatives possessing range of biological activity.

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1,2,4-Triazole compounds of myrtenal impart interesting bioactivities. Adamantane derivatives have been widely exploited as they are selective and potent against many biological targets. The ()-myrtenal-based 1,2,4- and 1,3,4-oxadiazole compounds demonstrate antiproliferative activity [88e92] (Fig. 6.44). The thio compounds of the pinene series exhibit high antiaggregation activity. A sulfide derivative of pinane shows hemocoagulation activity and thiosemcarbazone derivatives show anticancer activities [93,94] (Fig. 6.45). An imine derivative of camphor showed antitumoral activity by suppressing malignant tumor development. Hydrozone derivatives possess antimycobacterial activity against M. tuberculosis. Similarly, N-acylhydrazone derivatives of camphor show antiviral

Figure 6.44 Myrtenal-core structure analogues.

Figure 6.45 Pharmacological active molecules with pinene core structure.

Diversity of chemical skeletons: a practical strategy to benefit

activity against influenza virus. A-Truxillic acid derivatives were investigated for their analgesic activity and showed considerable analgesic activity. (þ)-Camphor-based sulfonamides with an N-heterocycle moiety were found to be superior filovirus inhibitors [95e97] (Fig. 6.46). The various heterocyclic ()-borneol derivatives with benzothiazole, benzoxaole, benzimidazole, etc., possess antiviral, antiulcer, and analgesic biological properties [98] (Fig. 6.47). Through combinatorial synthesis, the synthesis of paclitaxel C-7 derivatives has been reported. The aliphatic esters of this chemical library showed an effective cytotoxic effect. Similarly, rhamnospicamycin, a rhamnose analogue of spicamycin, a naturally occurring combinatorial library, has shown efficacious cytotoxicity toward human myeloma cells. A purine-based library from combinatorial methodology evolved effective protein kinase inhibitors. That is the selective binding mode of the 2,6,9-trisubstituted purine derivatives Roscovitine and Purvalanol A/B to the adenosine triphosphate binding site (CDK2), which is well complimented [99,100] (Fig. 6.48). Promysalin, a natural product, has the potential to inhibit the growth of the Gramnegative pathogen Pseudomonas aeruginosa (PA). Due to its effective contribution to antimicrobial activity and key structural features, 16-chemical libraries were built and screened as siderophores. Actinophyllic acid derivatives of an indole alkaloid that show high potential as anticancer agents, were synthesized by Diverted Total Synthesis

Figure 6.46 Camphor semi-synthetic analogs depicting various biological activity.

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Figure 6.47 Borneol-based biological active scaffolds.

Figure 6.48 Modified NPs as drugs.

(DTS) methodology. Notable examples, Epthilone B and Epoxomicin, which were successfully marked as drugs through this methodology [101] (Fig. 6.49). Phorbol esters and 1,2-diacyl-sn-glycerol (DAG) are two of the most important and effective ligands as anticancer and immunomodulatory analogs. Artemisinin is a potential antimalarial natural product, which on applying Function-Oriented Synthesis (FOS) formed a simplified analogue which showed equivalent potency. Similarly, another example is mevastatin, obtained by employing the FOS strategy on lipitor, a natural statin that is shown to possess better cardiovascular activity [102] (Fig. 6.50).

4. Synthetic approaches for building chemical skeletons The growing demand for active ingredients in the industry to meet human needs has made the design and development of new organic molecules imperative. The source of these chemical skeletons may be a direct natural product as seen above or may be

Diversity of chemical skeletons: a practical strategy to benefit

Figure 6.49 Drugs obtained via Diverted Total Synthesis (DTS) methodology.

Figure 6.50 Drugs derived from FOS route.

obtained by synthetic diversity. Due to the low abundance of natural products, an organic chemist needs to pin-up himself to design and synthesize target bioactive skeletons that are cost effective. The symbiotic relationship between organic chemistry and drug discovery has a history of significant milestones. Synthetic chemistry is the foundation of drug discovery. New developments or advanced capabilities in this area always lead to new discoveries and better drug entities on pharmaceutical platforms. Chemical synthesis is a set of skills to discover new drug molecules and is considered the heart of the entire drug discovery process. The design of drugs and their successful synthesis is a complicated process of hypothesis, conception, and synthesis. Different strategies have been employed to understand the development of the key to the lock-target. It is the most challenging task to find and design a specific synthetic target. Target-Oriented Synthesis (TOS) is the primitively used method to synthesize target molecules via retrosynthetic analysis. The

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TOS methodology has been effective over the years to date for getting diversified chemical skeletons. The effectiveness of HTS to screen a large number of chemical libraries has led to the development of combinatorial synthesis. Combinatorial synthesis creates large chemical libraries by combining different small molecules using the same synthetic method. A large chemical library created seems to have low significance when studying these molecules for pharmacological effects. To overcome the limitations of combinatorial synthesis, fragment-based drug discovery (FBDD) and parallel synthetic approaches have been developed. Furthermore, based on different criteria, these methodologies were further branched as different oriented synthesis to exploit effective lead optimization. The Diversity Oriented Synthesis (DOS) is another synthetic approach to build large chemical libraries. The aim is to design and synthesize structurally diverse compounds for different effects on various pharmacological properties. Further studies focused on biological functioning targets and then complimentary designing and synthesizing scaffolds were seen to be effective to some extent. This concept is referred to as Biology-Oriented Synthesis (BIOS) and was first introduced by Waldmann and coworkers. Another interesting approach by organic chemists to build drug able-analogues is seen in the DTS method. Furthermore, advanced intermediates formed by chemical synthesis are structurally studied using structure activity relations (SAR), physicochemical properties, and in vitro activity. The effective application and results of the DTS method led to a more general concept known as Function-Oriented Synthesis (FOS). The key idea employed in this strategy is to replace biologically active lead structures (a complex natural product) with simple scaffold designs having similar activity-defining structural features to those of the lead compound. To generate a diversity of chemical skeletons for various pharmaceutical applications, another exploited synthesis route is “Lead-Oriented Synthesis.” In this, the main principle is to find a lead entity based on lead-likeness physicochemical properties and structural considerations like chemical reactivity, electrophilic or redox active group alerts, favoring molecules with lower aromatic content, etc. Ketorolac [103] is a nonsteroidal anti-inflammatory drug that is used to treat moderate to severe pain. In a patient, this gives best results when administered with galactose as a prodrug. Similar to ketorolac, methotrexate (Fig. 6.51) is another medication used as a chemotherapeutic agent which is well coordinated with galactose. Chlorambucil’s good antitumor ability is proven due to the presence of 2-fluro-2-deoxyglucose. This glucose entity increases glycolysis in malignant cells and hampers the intake of glucose. Glycosylated paclitaxel showed much higher efficacy against cancer cells than paclitaxel. Another inorganic-organic motif, glucose-platimun conjugates (Glc-Pts), shows better effects against cancer cells.

Diversity of chemical skeletons: a practical strategy to benefit

Figure 6.51 Potent drugs derived from various synthetic approaches.

5. Conclusion Nature-derived products and their direct influence on human health have created several interesting research areas in the biomedical field. The developers of lead structures of industrial platforms have always been inspired and fascinated by the structural diversity of natural products. Through the drug discovery process, the screening of natural products as potential drugs through various biological activity studies has been carried out for several decades. As a result, effective results provided a new ray of hope for future

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improvements from time to time. As seen above, a large reservoir of natural products provides a wide range of molecules for future development. Their diverse functional properties also enhance research as lead compounds in different domains. It is also seen that due to the various changes in environmental factors and genetic drift, the naturally occurring natural products undergo structural variation, resulting in a new natural product. The intensive biological study of the core structure of natural products leads to the development of new drug-able molecules. The diversity of structures provided by nature and by synthetic organic chemists provides an ocean of opportunities for medicinal chemists to explore new skeletons as drug molecules.

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CHAPTER 7

Modern approaches for mining of novel compounds from the microbes Savita Girawale1, Surya Nandan Meena2 and Kisan M. Kodam2 1

Department of Chemistry, Savitribai Phule Pune University, Pune, Maharashtra, India; 2Department of Chemistry, Division of Biochemistry, Savitribai Phule Pune University, Pune, Maharashtra, India

1. Introduction Natural compounds (NCs) derived from plants, fungi, algae, and microorganisms have long been used as a source of novel drugs to treat a variety of human diseases. They differ from conventional synthetic compounds (SCs) in a few significant ways, such as they have more structural diversity, higher molecular masses (more carbon (sp3) and oxygen number), more hydrophilicity, and more hydrogen acceptors and donors [1]. These variations are beneficial because NCs with a higher degree of rigidity can be used in the creation of medications that target protein-protein interactions [2]. NCs have been essential in pharmaceutical and biotechnology industries, as a wide range of modern medications are made up of either naturally occurring molecules or derivatives of these. Since the ancient period, NCs obtained from living organisms including bacteria, fungi and plants have significant importance in treatment of different human diseases including cancer and in other therapeutic purposes such as cardiovascular diseases and multiple sclerosis [3e5]. More than half of the pharmaceutical drugs are now obtained from or inspired by NCs [3]. Some of the herbal constituents that are presently used as a drugs are codeine (Analgesic, Antitussive), morphine (Analgesic), quinine (Antimalaric), papaverine (Sympatholytic musculotropic) [6e8]. As per researcher, the unculturable microorganisms may serve as “untapped” sources for novel secondary metabolites. The diverse microbial taxa have the large potential to produce huge number of secondary metabolites with drug-like properties [9]. The bacteria are in high demand for the production of bioactive compounds because they are easy to grow, require less cultivation time [10] and are appropriate and robust platforms for the efficient production of recombinant proteins [11]. NCs are great source of cosmeceuticals (compounds possess cosmetic and pharmaceutical potential, known as cosmeceuticals). The NCs originating from different sources (plant, animal, and marine algal sources) have therapeutic and beautification potential based on its key ingredients. They are used in the treatment of various skin problems such as, infections, inflammation, and as a protectant of UV irradiation and pollution. The fatty acid obtained from plants and other natural sources have a huge market in the cosmetic industry [12]. NCs also play significant role as a “Nutraceutic,” the term is derived

New Horizons in Natural Compound Research ISBN 978-0-443-15232-0, https://doi.org/10.1016/B978-0-443-15232-0.00003-5

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from “nutrition” and “pharmaceutics.” These nutraceuticals are nutritional supplements which are applied for health purposes along with nutrition. The nutraceuticals have received high interest due to safety, potential nutritional and therapeutic effects. Some examples of NCs acting as nutraceutics are folic acid, omega-3, glucosamine, lutein, green tea and cod liver oil [13] Drug discovery slowed after the 1980s due to difficulties in identifying useful and novel drug components in microbes [14e16]. After the golden era, most of the pharmaceutical companies started developing small compounds libraries using combinatorial chemistry [17], for more bioactivity using high throughput screening strategies [18]. A key challenge in this area is to develop a method for the synthesis of chemically diverse and high-quality libraries that exhibit the desirable properties of natural products, such as diverse functionality, considerable skeletal diversity, a substantial number of chiral sp3 centers conferring conformational bias to the molecule, and good pharmacokinetic properties [19,20]. The NCs discovery once again caught the speed after the discovery of genome sequence of Streptomyces [21], wherein the sequence highlighted many silent biosynthetic gene clusters (BGCs) which upon the altered culture conditions express novel NCs. Nowadays, the necessities of the novel NCs are arising due to the acquired microbial resistance to the several available antibiotics as well as toxic side effects of certain medicines [22,23]. The new drugs required to treat emerging new diseases effectively. The COVID-19 pandemic has brought attention to the potential for the emergence of new diseases. New drugs can be developed to treat the causes or symptoms of diseases that have been well-known for a long time but were previously incurable like HIV/ ADIS, HBV, etc. [24]. New drugs are sometimes needed to treat different types of people, due to the various ways they are metabolized. The drugs may show various effects on males and females due to their different biology [25]. Current advances in genomics, synthetic biology, metabolic engineering, and bioand chemo-informatics have the potential to open up new avenues for novel NCs discovery from known and unknown bacterial species [26]. Using novel approaches, NCs discovery can be enhanced and focused on unexplored bioactive chemicals [27]. The present chapter deals with the detailed study of various modern approaches such as metabolomics, proteomics, and genomics (Fig. 7.1) with various bioinformatics tools in searching novel compounds in drug discovery.

2. Traditional methods and techniques used for mining of novel compounds along with their limitations Traditionally, bioactivity-guided isolation and extraction techniques are used to discover NCs. Since the methods are based on bioactivity screening, they had a higher success rate but also have limitation such as time consuming due to repeatedly isolating processes [28]. Conventional methods are labor intensive and time consuming that slow down the rate of NCs discovery. One of the reason for the reduction in the drug discovery rate in led

Modern approaches for mining of novel compounds from the microbes

Figure 7.1 Flowchart of modern approaches for hunting novel bioactive compound.

pharmaceutical company have seen due to the traditional screening methods. These screening processes involves library of extracts and non-compatible to the target-based assays [29,30]. Identification of bioactive compound is challenging and necessitates additional tools, such as dereplication tools, to prevent the rediscovery of compound. Sometimes, obtaining the sufficient biological material to isolate and define a bioactive NC can be difficult [31]. Furthermore, thorough identification and characterization of NCs using various analytical techniques like NMR, IR, LCMS, GCMS, etc. is expensive and necessitates high instrumentation expertise. Traditionally, natural compound extraction procedures based on bioactivity-guided protocols are carried out until a pure bioactive compound is isolated. The primary constraint is the difficulty in cultivating the organisms used to produce bioactive compounds or the factors that they do allow the organism to produce desired bioactive compounds outside of their natural habitat. These problems can be addressed by constructing innovative cultivating techniques, heterologous biosynthetic gene expression and induction can both improve NCs synthesis. Modern methods of natural product drug discovery are outperformed numerous issues must be resolved in order to isolate bioactive compounds from crude extracts, such as low levels of natural compounds, compounds that are already known, or the identification of those natural compounds that do not have drug-like properties. By developing techniques like

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dereplication, better extraction, and pre-fractionation steps, these issues can be resolved. It took a lot of time and effort to pinpoint the impacted molecular targets in order to determine the bioactive compound activity. These issues can be solved by the development of methods for accelerated elucidation of molecular modes of action; for example: (1) Nematic Protein Organization Technique (NPOT) [30], which identifies both therapeutic and toxic or side effect targets of drugs. The technique is label free and works in physiological conditions, directly on human tissue. In cases where the clinical relevance of genomics is uncertain, NPOT provides reliable toxicity profiling, identifies biomarkers, and provides predictors of isolate success [29,30]. (2) Cellular Thermal Shift Assay (CETSA) is an important method that confirms target engagement within a complex cellular environment by detecting variations in thermal stability of protein after binding with ligand [31]. (3) Drug Affinity Responsive Target Stability (DARTS) is a rapid and straightforward method that identifies the protein targets for small molecules [32]. (4) Stable Isotope Labeling with Amino acids in Cell culture and Pulse Proteolysis (SILAC-PP) is the method that uses the isotopic essential amino acids (L-lysine and Larginine) to replace the original amino acids for cell culture and passage for 8e10 generations followed by mass spectrometry for identification and quantification (by isotopic abundance difference) of protein [33]. (5) Thermal Proteome Profiling (TPP) approach is successfully utilized to identify targets and off-targets of drugs, to study the interactions between proteinemetabolite, proteineprotein, and to study posttranslational modifications [34]. (6) Similarity Ensemble Approach (SEA) is a promising approach that is applicable to drug-related studies and predicts the biological targets of a compound based on its resemblance to ligands annotated in a reference database such as ChEMBL [35].

3. Modern use of databases in hunting new compounds The databases make it possible to store, exchange, and compare data from various research projects, data types, and organisms. There are numerous databases available with the structural details of millions of chemical compounds, including ChemSpider (58 million), PubChem (83 million compounds), ChEMBL (2.1 million), ChemBank (1.2 million), ChEBI (440,000), and SciFinder (161 million). The databases like STRING (focusing the networks and interactions of proteins; https://string-db.org/), Top FIND (provides the information about the N- and C- terminal amino acid sequence of proteins; http:// clipserve.clip.ubc.ca/topfind/), Protein Data Bank (gives the information about proteins and nucleic acids), and many others are useful in proteomics study. The process of structural identification and re-isolation is ultimately eliminated by using this approach [36].

4. Mining for novel compounds using genomic databases Whole genome sequence of a microorganism is a gold mine for the identification of novel NCs. The microbial biosynthesis ability for secondary metabolites that remain

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silent/cryptic under laboratory conditions was revealed using a genome mining strategy. Activation of the silent biosynthetic gene clusters (BGCs) that decode for various metabolites could be a source for discovering novel NCs with potent activities that were previously unknown. Genome mining for silent BGCs can be carried out using a variety of bioinformatics tools that identify and characterize related gene clusters involved in NCs biosynthesis. The availability of a large number of bacterial genome sequences on various databases facilitate researchers to search for gene cluster that decodes for novel NCs. The various bioinformatics tools commonly used are NP (natural product) Searcher [37], SMURF (Secondary Metabolite Unique Regions Finder) [38], anti-SMASH (Antibiotics and Secondary Metabolite Analysis Shell) [39], CLUSEAN (Cluster Sequence Analyzer) [40], ClustScan (Cluster Scanner) [41], CASSIS (Cluster Assignment by Island of Sites) [42], and MIDDAS-M (Motif Independent De novo Detection Algorithm for Secondary Metabolite gene clusters) [43]. Since the year 2011, the anti-SMASH bioinformatics tool is gaining popularity among the researchers in genome mining studies. The anti-SMASH is most efficient and popular software that is widely used with additional key features and is helpful in the identification of secondary metabolite synthesizing gene clusters [44]. Furthermore, anti-SMASH is able to identify different biomolecules, small peptides, and other small compounds including beta-lactams, nucleosides, polyketide synthase (PKS), nonribosomal peptide synthetase (NRPS) or mixed PKS-NRPS, BGCs, siderophores, bacteriocins, terpenes, antibiotics, butyrolactones, melanin, and other metabolites [27]. Additionally, anti-SMASH provides the information about domain analysis and annotation related to PKS/NRPS, core structure prediction and substrate specificity, and comparative analysis of secondary metabolite, protein family, and gene clusters [45]. The general pathway for mining a novel NCs is described in Fig. 7.2. Mining microbial genomes for resistance genes is one method for specifically looking at antibiotic NCs. The molecular mechanisms of drug resistance, including enzyme-catalyzed antibiotic modifications, antibiotic target evasion, and active efflux of drugs from the cell, provide key information for new drug development and clinical use [46]. Since the necessary resistance genes frequently co-localize with the genes encoding the biosynthetic machinery for antibiotic production, they can be used as a guide to find potential antibiotics [47]. The gene clusters that code for proteins involved in biosynthesis, resistance, regulation, and transport are directly linked to antibiotics produced by microorganisms. Natural product genome mining for the rational discovery of novel chemical entities has emerged as a result of the capacity to link natural antibiotics to gene clusters and vice versa as well as the ongoing advancement of knowledge of biosynthetic machinery. Simultaneously, through the use of sophisticated sequencing technologies, DNA sequence data from a wide range of microbial genomes and environmental metagenomes has rapidly accumulated in public databases [48]. Thus, the resistance gene-based genome mining technique may be broadly applicable to genomics-driven natural product

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Figure 7.2 General pathway for mining a novel NCs using whole genome sequence of microbes.

discovery for the development of new antibacterial drug candidates based on the rapid increase in genome sequence data in public databases. Genome mining of NCs decoded by BGCs can be done in vivo using PCR techniques if the bacterial genome has not yet been sequenced. In this method, primers (specific short nucleotide sequences) bind complementary sequences of DNA and enable forward and reverse gene amplification using DNA polymerase. It is possible to locate the conserved region of biosynthesis cluster in vivo by designing a degenerated primer, which is a mixture of primers with similar sequences but different bases substituted at some points in its sequence [49]. This method was used by Ehrenreich et al. in cyanobacteria for detection of NRPS/PKS gene clusters and to compare them with the cytotoxicity of the strains [50]. This technique utilizes both molecular and culture-dependent screening methods to assess the biosynthetic potential. In recent study, by using PCR amplification sequencing approach with bioinformatics tool, the researcher detected four biosynthetic gene cultures encoded for NRPS, PKS-1, glycopeptide oxy b gene (Cytochrome P450 monooxygenase), and CYP specific polyene (cytochrome P450 hydroxylase). Detection was based on the presence of size of the PCR amplified DNA fragments. The various bioinformatics tools such as BLASTn, BLASTp, EMBOSS TRANSq, and MEGA 6.0 are used for functional analysis of the sequenced strains [51]. These findings demonstrate that the PCR-based genome screening method is an

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effective method for detecting potentially valuable Streptomyces. Bioinformatics analyses validate the presence of glycopeptide Oxyb, NRPS, and PKS-1 proteins, all of which play significant roles in antibiotic production pathways.

5. Proteomics approach for the mining for NCs Using the proteomics techniques or tools, it is easy to determine the relationship between NCs and BGCs in a microorganism. As mentioned earlier, many bacteria produce secondary metabolites through the large enzyme complexes known as polyketide synthases (PKSs), and nonribosomal peptide synthetase (NRPS) are encoded as specific clustered regions of the bacterial genomes, called biosynthetic gene clusters (BCGs). The proteome mining method reveals a link between genetic expression for the synthesis of NC in microorganisms under various cultivation conditions and levels of synthesized secondary metabolite or bioactivity of the compound of interest. Expression-based analysis is useful for detecting active BGCs with necessary modifications. The unexpressed/silent BGCs can be activated by varying the cultivation conditions, i.e., by changing the medium composition, culture pH, temperature, aeration, and container type, to induce the expression of silent BGCs and addition of inducers [52]. Several methods are available for the expression of silent BGCs including heterologous expression, promoter engineering, ribosome engineering, and transcriptional regulators engineering [53]. The detection of active BGCs are carried out by heterologous expression method. It mainly includes three steps: (1) Cloning of the target BGCs; (2) Engineering of the target BGCs; and (3) Transformation to the selected heterologous host [53]. By using affinity purification or mass spectrometry detection, these methods have been designed to specifically detect PKS and NRPS with essential phosphopantetheine modifications [54]. Many automated drug discovery approaches are based on testing a large number of compounds against disease targets; however, scientists developed a method that tested drug molecules against thousands of protein targets at the same time. As a result, proteome mining provides information on drug interactions with their targets [55]. Proteome mining study can be further classified on the basis of their mode of action. 5.1 Chemical biology: activity-based probes The multi-resolution of bioactive compounds is possible using targeted proteomics specially using chemical proteomics approaches. Chemical proteomics helps to identify the cellular targets for NCs. This method involves the immobilization of bioactive molecule of interest which is used to bind the proteins from targeted organism. The affinities of protein molecules for immobilized NCs can be used to determine whether a protein is a potential target or not. Proteins with a higher affinity are thought to be more potent targets. In proteomics research, the carrier protein domain is a frequently occurring

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component of the NC-synthetic machinery. It serves as a guideline for anchoring the biological synthesis of the PKS, NRPS, and fatty acid synthase (FAS) systems [56]. Carrier protein (CP) domains can be marked enzymatically in vitro by using CoA analogues and Phosphopantetheinyl transferases (PPTase) or in vivo by tagged CoA precursors [57]. As an alternative, activity-based protein profiling (ABPP) can be employed as a proteomics technique to detect enzymatic activity in intricate biological samples. In this case, the active site is marked with a covalent reporter probe, commonly a marked inhibitor that can be quantitatively detected by using protein gel electrophoresis, LC-MS/MS, and microarray systems [58]. PKS as well as NRPS biosynthetic systems make great targets for ABPP due to their highly modular properties. The acyltransferase (AT) and thioesterase (TE) domains have been targeted specifically by fluorescently labeled probes [59]. The use of LC-MS/MS in the initial screening steps can be avoided with ABPP while maintaining compatibility with online methods, making it appropriate for low-cost analysis of the NC biosynthetic potential of many strains and samples [60]. Matrix Assisted Laser Desorption Ionization (MALDI) mass spectrometry techniques are also used for the identification of small peptides and other molecules. In this mass spectrometry, MALDI is an ion source, and time-of-flight (TOF) analyzer is the mass analyzer. This technique is relatively easy to operate with high sensitivity, mass accuracy, and high resolution. It is commonly used in proteomics as peptide mass fingerprinting to identify proteins [61]. In this method, peptides are obtained by digesting proteins sample with sequence-specific protease enzyme like trypsin. The generated peptides are further analyzed by MALDI-TOF mass spectrometry for mass determination. The obtained masses can be compared to a database of theoretical peptide masses from a given organism using the same sequence-specific protease [62]. 5.2 Direct proteomic analysis of biosynthetic enzymes The biosynthetic machinery in NRPS and PKS systems is typically very large, with the synthetases consisting of multiple domains to form a molecular assembly line [63]. The mechanism is based on the covalent binding of NC scaffold to carrier protein (CP) domains by means of a thioester bond (phosphodiester linkage) to the 40 -phosphopantetheine (PPant) arm, a posttranslational alteration on a serine amino acid residue in the CP active site. This PPant alteration can be easily isolated from the peptide utilizing collision-induced dissociation (CID) or infrared multiple photon dissociation (IRMPD) techniques in tandem MS, offering a helpful tool for determining the existence of CP domains [64]. The Proteomic Investigation of Secondary Metabolism (PrISM) method was established to take advantage of the large size and distinctive PPant marker ions [54,65]. In PrISM, the complex whole-cell mixture of high-molecular weight proteins is pre-fractionated by SDS-PAGE, digested, and applied to LC-MS/MS. The corresponding peptide is recognized as a component of an NC synthase when PPant

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diagnostic ions are found using tandem mass spectrometry. The gene and subsequently the BGC can be obtained using reverse genetics based on the genome sequence [63]. 5.3 Proteomics-based analysis of peptidic natural products In order to find peptidic NPs (PNPs) useful for tandem MS technology, the de novo peptide sequence elucidation is carried out in a short gun proteomics study [66,67]. Due to the frequent complexity and ambiguity of tandem MS spectra, this is not a simple task. With the goal of de novo peptide identification from tandem MS data, several new programs have been created. Programs like Deep Novo have surpassed other algorithms in terms of peptide identification rates and accuracy by incorporating artificial intelligence into the pipeline [68]; however, due to high biodiversity and complexity, de novo identification of undiscovered PNPs remains difficult. Natural product peptidogenomics (NPP), which aims to rapidly characterize ribosomally synthesized posttranslational peptides (RiPPs) and nonribosomally synthesized peptides (NRPs) and their BGCs from sequenced organisms, is one technology to solve this issue [69]. The NPP process flow begins with the analysis of peptidic compounds in a sample using tandem MS, followed by peptide enrichment and analysis. Following that, sortable sequence tags are created, and the mass shifts are considered as potential posttranslational modifications (PTMs). As a result, the complexity of tandem MS data is significantly reduced, and the peptide tags can be produced effectively for data mining.

6. Metabolomics and mass spectrometry approach in the discovery of NCs The metabolomics studies attempted to characterize the diversity of biological molecules in samples. The analysis is mainly performed using mass spectrometry (MS). The MSbased metabolomics is able to detect thousands of metabolites simultaneously from sample in minimum preparations [70e72]. The metabolomics studies are useful to determine complete metabolic pathways which could not detected by genome annotation [73], to describe the function of theoretical pathways [74], to find novel metabolic pathways [75,76], and to discover novel biomarkers related to diseases [77,78]. Cutting-edge mass spectrometry approaches such as laser ablation electrospray ionization coupled with image mass spectrometry (LAESI-IMS) are used to analyze the expressed metabolites directly from the biological sample in high throughput manner. The LASESI-IMS is advanced technique which carried out the rapid analysis of previously known and unknown samples [79]. It is in demands due to efficiency and rapid analysis even at very less sample concentration. The mass spectrometer (MSeMS) coupled to LAESI make it more relevant for the detection of NCs directly from biological samples in high throughput manner. The LASESI-IMS is capable of analyzing a variety of molecules, including phenolic, lipids, peptides, and other compounds derived

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from bacteria, fungi, and plants. The sample analysis can be done using an ambient ionization method at atmospheric pressure without any prior preparation. The dereplication is the advanced approach in metabolomics mining of novel compounds, in this process the compound is identified on the basis of its molecular mass, formula, and structural information obtained from NP databases. By utilizing molecular networking, hundreds to thousands of data sets of MS/MS recorded from a specific set of extract samples were examined to determine how the analytes related to other structurally related molecules [80]. The method provides the rapid detection of chemical compound on the basis of crude MS data obtained during screening [36]. The chemical information available from databases helps to reveal the detected compounds. By using the advanced and recent tools in bioinformatics assist to dereplication process for identification of the novel drug compound from huge crude MS-data matching with data of MS data repositories. For rapid analysis, dereplication are usually consider the cocktail of all essential methods in mining including separation methods, spectroscopic technique, and databases searching processes [81]. Molecular Networking (MN), which included the visualization and interpretation of raw mass data for further compound identification, is one of the best methods used in the dereplication of NCs [82]. This method makes it easier to profile meta-mass shift chemicals, which reveals the precise biochemical transformation and enables the identification of known chemical groups. Researchers more recently developed the algorithm tool “DEREPLICATORþ” that makes use of spectral networks for the high-throughput identification of variants of well-known NCs. The DEREPLICATORþ is currently the most popular and in-demand tool for high throughput screening of NCs including alkaloids, flavonoids, terpenes, benzenoids, and polyketides [83].

7. Conclusion and future prospects By using the modern approaches in genome mining and using bioinformatics tools, it is now possible to find out untapped microbial sources for NCs. In future, rapid developments in bioinformatics, metabolomics, genome sequencing, and synthetic biology will speed up the search for new alternative bioactive compounds, or BGCs, for the rapid drug discovery.

Acknowledgments The author SDG would like to thank Chhatrapati Shahu Maharaj Research Training and Human Development Institute (SARTHI), Pune, Government of Maharashtra for research fellowship. The author SNM sincerely acknowledges the financial support from University Grant Commission (UGC), India through Dr. D.S. Kothari Postdoctoral Fellowship (No.F.4-2/2006/BSR/BL/18-19/0416).

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[48] Tang X, Li J, Millan-Agui~ naga N, Zhang JJ, O’Neill EC, Ugalde JA, et al. Identification of thiotetronic acid antibiotic biosynthetic pathways by target-directed genome mining. ACS Chem Biol December 18, 2015;10(12):2841e9. [49] Strieth D, Lenz S, Ulber R. In vivo and in silico screening for antimicrobial compounds from cyanobacteria. Microbiology Open April 2022;11(2):e1268. [50] Ehrenreich IM, Waterbury JB, Webb EA. Distribution and diversity of natural product genes in marine and freshwater cyanobacterial cultures and genomes. Appl Environ Microbiol November 2005; 71(11):7401e13. [51] Noureen N, Cheema MT, Anwar S, Hasnain S, Sajid I. PCR-based screening approach: a rapid method to detect the biosynthetic potential of antimicrobials in actinobacterial strains. Pol J Microbiol September 1, 2020;69(2):139e49. [52] Tomm HA, Ucciferri L, Ross AC. Advances in microbial culturing conditions to activate silent biosynthetic gene clusters for novel metabolite production. J Ind Microbiol Biotechnol October 1, 2019;46(9e10):1381e400. [53] Udwary DW, Gontang EA, Jones AC, Jones CS, Schultz AW, Winter JM, et al. Significant natural product biosynthetic potential of actinorhizal symbionts of the genus Frankia, as revealed by comparative genomic and proteomic analyses. Appl Environ Microbiol June 1, 2011;77(11):3617e25. [54] Bumpus SB, Evans BS, Thomas PM, Ntai I, Kelleher NL. A proteomics approach to discovering natural products and their biosynthetic pathways. Nat Biotechnol October 2009;27(10):951e6. [55] Chapman T. Mining the proteome. Nature July 1, 2004;430(6995):109. [56] Lai JR, Koglin A, Walsh CT. Carrier protein structure and recognition in polyketide and nonribosomal peptide biosynthesis. Biochemistry December 19, 2006;45(50):14869e79. [57] a. La Clair JJ, Foley TL, Schegg TR, Regan CM, Burkart MD. Manipulation of carrier proteins in antibiotic biosynthesis. Chem Biol February 1, 2004;11(2):195e201. b. Meier JL, Mercer AC, Rivera H, Burkart MD. Synthesis and evaluation of bioorthogonal pantetheine analogues for in vivo protein modification. J Am Chem Soc September 20, 2006;128(37): 12174e84. [58] Evans MJ, Cravatt BF. Mechanism-based profiling of enzyme families. Chem Rev August 9, 2006; 106(8):3279e301. [59] Aligiannis N, Halabalaki M, Chaita E, Kouloura E, Argyropoulou A, Benaki D, et al. Heterocovariance based metabolomics as a powerful tool accelerating bioactive natural product identification. ChemistrySelect July 1, 2016;1(10):2531e5. [60] Meier JL, Niessen S, Hoover HS, Foley TL, Cravatt BF, Burkart MD. An orthogonal active site identification system (OASIS) for proteomic profiling of natural product biosynthesis. ACS Chem Biol November 20, 2009;4(11):948e57. [61] Sommerer N, Centeno D, Rossignol M. Peptide mass fingerprinting: identification of proteins by MALDI-TOF. Methods Mol Biol 2007;355:219e34. https://doi.org/10.1385/1-59745-227-0:219. [62] Ida Chiara Guerrera, Kleiner O. Application of mass spectrometry in proteomics. Biosci Rep February 4, 2005;25(1e2):71e93. https://doi.org/10.1007/s10540-005-2849-x. [63] Fischbach MA, Walsh CT. Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: logic, machinery, and mechanisms. Chem Rev August 9, 2006;106(8):3468e96. [64] Meluzzi D, Zheng WH, Hensler M, Nizet V, Dorrestein PC. Top-down mass spectrometry on lowresolution instruments: characterization of phosphopantetheinylated carrier domains in polyketide and non-ribosomal biosynthetic pathways. Bioorg Med Chem Lett May 15, 2008;18(10):3107e11. [65] Chen Y, McClure RA, Kelleher NL. Screening for expressed nonribosomal peptide synthetases and polyketide synthases using LC-MS/MS-based proteomics. In: Nonribosomal peptide and polyketide biosynthesis. New York, NY: Humana Press; 2016. p. 135e47. [66] Mohimani H, Pevzner PA. Dereplication, sequencing and identification of peptidic natural products: from genome mining to peptidogenomics to spectral networks. Nat Prod Rep 2016;33(1):73e86. [67] Ng J, Bandeira N, Liu WT, Ghassemian M, Simmons TL, Gerwick WH, et al. Dereplication and de novo sequencing of nonribosomal peptides. Nat Methods August 2009;6(8):596e9. [68] Tran NH, Zhang X, Xin L, Shan B, Li M. De novo peptide sequencing by deep learning. Proc Natl Acad Sci USA August 1, 2017;114(31):8247e52.

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CHAPTER 8

Informatics and computational methods in natural product drug discovery Heena Shoket and Monika Pandita School of Biotechnology, Shri Mata Vaishno Devi University, Katra, Jammu and Kashmir, India

1. Introduction A natural product is defined as a bioactive molecule derived from living organisms, for the development of drug development and discovery. The main source of natural products is often the isolated metabolites from plants or animals, which has been in practice for years as part of traditional medicine. Contrary to the standard small-molecule drug candidates, natural products have properties that necessitate the development of novel tools and procedures for evaluating their therapeutic potential. The scaffold variety and structural complexity of natural products are immense. With a higher molecular mass, the sp3 carbon and oxygen atoms are more in number than the nitrogen or halogen atoms, whereas donor and acceptor hydrogen bonds contribute to the complex structure. While a decreased value of octanol-water partition coefficients amplifies the hydrophilicity, higher molecular rigidity gives an advantage to natural products over synthetic compounds. As such, natural products have long been derived as key components for the development of therapeutics for various human diseases including cancer, or vesicular disorders [1]. 1.1 Category of natural products To categorize natural products, all types of chemical compounds created or recruited by living organisms that can be extracted and reused are considered. The subcategories of natural products include phytochemicals, fungal metabolites, immune systemassociated cells especially antibodies, along with toxins extracted from living organisms. Plant extracted products were primarily a source of food and related products like spices; however, their significance was highlighted by the medicinal properties many plants (medicinal) exhibited. Compared to the synthetically derived drugs, plant extracted drugs are considered safe along with their diverse chemical structure, which gives them an advantage over synthetic products [2]. Phytochemicals are plant-based chemical compounds that depict wide characteristics of natural products. They can range from supplying essential dietary elements like amino acids, antioxidants, or dietary

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supplements, to being poisonous; or in some cases, inactive in humans. Derivation of traditional medicines has been resourced from the different types of phytochemicals. The extraction of numerous phytochemicals that displayed cytotoxic activity displayed promising results during trials for therapeutic development of cancers, e.g., paclitaxel, vinblastine, docetaxeletc, etc [1]. Another category of natural products, the fungal metabolites, are functionally similar to plant metabolites. Fungi are abundantly rich in physiologically active natural components. For their variable diversity of small yet effective compounds, fungal metabolites are extracted for the preparation of a wide variety of drugs, for the treatment of variable disorders, and are a basis of variable antibiotics. The antibiotics range from anti-malarial drugs, statins, and immune suppressors among many more. Cephalosporin and penicillin are commonly used antibiotics, derived from beta lactam fungal metabolites and is used for the past several years [3,4]. Cells of the immune system, the antibodies are associated with the identification and targeting of antigens (bacteria, viruses, or other infectious pathogens). For its specificity to target diverse types of cells, their utilization in the development of therapeutics became a highlighted area of interest for variable genetic and related diseases. Treatment of diseases ranging from autoimmune disorders like rheumatoid arthritis, and nervous system-related diseases (multiple sclerosis) to diseases inflicted through antigens, e.g., viruses [1]. Immunotherapy is one such approach that uses monoclonal antibodies to identify as well as selectively target the disease-causing cells. Its application is recognized as a “vehicle” to deliver the drug to the target locations, without being discarded by the immune system and is currently being employed in cancer treatment. However, when the activity of monoclonal antibodies was studied in mouse models as well as human trials for Alzheimer’s disease treatment, the monoclonal antibodies exhibited failure in ensuring the targeted delivery of drugs [5,6]. In this regard, two leading theories have been proposed for the failure of antibodies. The first is that monoclonal antibodies fail to cross the blood-brain barrier, and the second is that early treatment is needed in case of certain degenerative diseases, for antibodies to be therapeutically effective. Furthermore, antibody therapy is linked to the activity of the drugs themselves, like in the case of the drug theralizumab, which was developed for the treatment of rheumatoid arthritis and leukemia, displayed a life-threatening reaction in healthy volunteers and thus was declared a failure. Keeping the previous failures into perspective, new treatments are being developed with higher success rates that show minimal to no risk in humans [7e9]. Another class of natural products obtained from living organisms and preferred for pharmacological development is toxins. Categorized as poisonous if toxins entry into the body is through dermal contact or inhalation, it inflicts harmful effects, e.g., toxic chemicals like mercury, isocyanates, etc [10], while the venomous toxins are the ones that enter the body through sting or get injected into the skin via animal bite e.g., Snake, bees, etc [11]. Organisms including plants, some animals, bacteria, or fungi along with some other terrestrial and aquatic organisms are known to synthesize chemical poisons

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as part of the defense strategy against predation. Natural poisons are non-natural molecules, such as proteins and carbohydrates. Some venoms are used in the preparation of chemotherapy drugs if they act selectively on cancer cells more strongly than on the unaffected, normal ones [12]. Animals produce venoms as complex combinations of chemicals, for defense or even offensive purposes. Several variable chemical compounds may be found in a single species’ venom which includes peptides, and neurotransmitters among other proteins that operate on specific molecular targets. Venoms have been fine-tuned over millions of years to fit the ecological niches of living creatures. Because of interspecies homology and their biological role in human diseases, individual venoms were studied for several therapeutic uses, including FDAapproved therapies for hypertension, diabetes, neuropathic pain, and many other diseases. Venoms, like poisons, have shown significant anti-cancer effects, and their high target specificity has made them particularly appealing for precision medicine applications, particularly for uncommon or aggressive cancer forms, for which they are isolated, improvised, and distributed in different drug libraries [13,14]. Apart from these types, some natural products show limited utilization in drug development strategies. These include structural compounds like biopolymers, or spider net forming silk (spider silk). Such components might not be useful as a drug but are useful in nanotechnology, as drug or gene vehicles, to carry the drug across specific sites or for gene integration. Using bioengineering techniques, their functional implications are redirected for better application [15,16]. Drug discovery for natural products first step for drug development and discovery is the identification of relevant chemicals that have activity in a living system and display success in the treatment of specific target diseases. Plant metabolites necessitate the collaboration of several disciplines, including pharmacology, medicine, chemistry, and pharmaceutics. Phytochemicals derived from medicinal plants are utilized as lead molecules in drug research and are then employed in the production of synthetic or semi-synthetic drugs to secure patent protection. A summary of the methods used in drug discovery of natural compounds is provided in Fig. 8.1. 1.2 Semantic methods for drug discovery While bioinformatics is used as a major tool for drug development and the discovery of novel compounds, there are other related strategies including the use of different information platforms, like ontologies and organized terms to collect details. Rule-based natural language processing, extraction of clinical data and mining of technical information, standardization of semantic data, and other activities are all part of this category. Thus, knowledge-based methods are widely used in conjunction with bioinformatics/cheminformatics methods in the context of drug development, to serve as one of the key ways of merging collective information from various research activities. Although NMR, HPLC, and mass spectrometry are helpful in the identification of plant compounds for utilization

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Figure 8.1 A summarized methodology for the utilization of natural compounds for drug discovery.

in drug discovery, online semantic tools provide additional information collected across different experiments [12]. Some of them include Semantic Medline (National Library of Medicine or NLM), National Centre for Biomedical Ontology Annotator, OMIM (gene to the drug), MetaMap, etc., which help draw inferences from the algorithmic data including drug compounds and contribute to drug discovery. The elaborate semantic web tool applications via literature mining used for drug discovery that may be applied in therapeutic applications of natural products are discussed here. 1.2.1 Literature mining Drug development is a costly and time-consuming process, and the probability of identifying drugs that can be used for the treatment of diseases is limited. So, an initial but critical part of identifying natural compounds is screening the data of drug compounds. Owing to the well-managed data of semantic tools, complimented with categorized information, inferences, and refined data structures, they are an excellent tool for generating knowledge from diverse research. One of the preferred sources regarding drug

Informatics and computational methods in natural product drug discovery

discovery is Gene Ontology, which differentiates the accessible information consortium based on molecular, cellular as well as biological characteristics [17]. Such a consortium was created toward the unification of diverse information of genes and proteins across diverse living organisms identified and detailed beforehand, which provided great help in not only generating information about a single organism but also in understanding the comparative functionality across different living organisms. One knowledge graph-based method for screening therapeutic drug compounds is SemaTyP, which not only helps in the identification of potential drugs via literature mining but also provides the mode of action of the target drug in a diseased state [18]. In regards to the advances in semantic tool development, other semantic tools including OWL2, RDF, and SPARQL facilitate a flexible transference as well as reuse of systematic data. Among these, RDF-associated BioRDF is a platform that collectively stores the research-based data. In addition, its chemical biology domain, Chem2BioRDF contains information from various chemogenomics databases (like ExCAPE-DB), so that the repository allows its users to search for the chemical and biological properties of drugs for inhibiting signaling pathway or drug response pathway analysis [19]. Likewise, many other databases were established to sort the enormous information, both literature as well as in silico data, generated through research on a global level and made available to the research community. These include PubMed for literature and UniProt/SwissProt, ChEMBL, DrugBank, etc. for analysis of chemical compounds for therapeutic applications, based on their structural and functional characterization established so far [20e22]. For PubMed articles, Medical Subject Headings or MeSH is a software program that classifies, records, and analyses biological and health-related data. The subject headings from MEDLINE/PubMed are included in MeSH [23]. MeSH is particularly used for the ability to accumulate PubMed articles using refined terminologies like “Drug discovery” or “therapeutic utilization,” which has been used to outline the natural products of plant origin as potential drug targets [24]. A small number of databases offer curated collections of articles about Natural Products. Examples include VenomKB which contains articles attributed to venom constituents, and include literature predictions outlining the components’ probable medicinal effects [1]. Another database NPASS includes references to PubMed articles describing manually curated measures of biological activity in a variety of taxa, as well as chemical properties of a larger range of NPs [25]. Others like NAPRALERT and MarinLit provide curated data about natural compounds on a subscription/payment basis. As such, many digital libraries have, therefore, developed additional tools, specific to drug discovery, for data availability related to compound drugs. Such tools store structural information about the natural compounds in both incomplete and unstructured to a more detailed and complete format.

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1.2.2 Electronic health record mining In the absence of highly controlled clinical research investigations, data from observational sources provides a way for analyzing the impact of substances on individuals in drug discovery. This method of data analysis has numerous significant advantages over clinical trials, including the avoidance of testing of potentially hazardous drugs for enrolled patients and the reduction of certain types of bias connected with screening and selection of patients. This way, compared to clinical trials, higher cohorts are generated through observational data. For its widespread use and demonstrated efficacy in many translational research activities, electronic health records or EHRs are a preferred source as they are a digital version of a patient’s data chart. EHRs contain real-time records of patients that securely avail the information to authorized users. While an EHR system does provide background information regarding a patient’s medical treatment, it extends beyond traditional clinical data collection methods and can encompass a broader perspective toward therapeutics employed in patient’s treatment [26]. The main objective of adapting the EHR system is to have all of the information about patient care, education, and practice management accessible at the point of treatment. Such an approach is required by oncologists to provide better patient support and have a reminder for the therapeutic judgments being applied in specific disease cases. This can amplify the collaborative approach between health professionals across different institutions, in providing better care to the patients, and keeping a track of the patient’s therapeutic progress. EHR data is extensive and prone to a variety of biases as well as ethical and regulatory limitations. While the text reports of practitioners are freely available through the sources, a variety of data like information regarding medication details, patient claims, their demographics, etc. are also structurally formatted for the users. Concerning drug discovery of natural products, EHR can be used either for the development of specific drugs based on the type of disorder or by identifying the genotype to phenotype relationship disease associations and accordingly generating the remodeled drugs. Alternately, analysis of the drugs for their safety and usage can also help in identifying the appropriate drug targets [27]. Specific benefits and drawbacks that NPs give as compared to non-NP medications and drug prospects might be explored from the perspective of natural products in drug development. Drug repositioning is the process of repurposing an existing drug to treat a condition other than the one for which it was originally developed. A variety of drug repositioning techniques have been implemented using EHRs. A frequently used repositioning method includes finding commonalities between diseases and then implying novel therapies based on those common factors. This is predicted by the notion that diseases with similar underlying factors would create similar EHR signals, and that such similarity can be profoundly used for similar treatments. An example of this is Tamoxifen, a drug used for the treatment of breast cancer and based on similar etiologies has been identified to be effective for the treatment of the bipolar disorder [28]. When targeting genes and gene

Informatics and computational methods in natural product drug discovery

products that are difficult to reach with small compounds, natural products like monoclonal antibodies and protein-based treatments are a preferred option, considering the facilitated targeting of specific cell types, which is particularly effective in malignancies with specific genetic signatures [29]. Additionally, GWAS and PheWAS can likely emerge for analysis and screening of natural products for target finding through clinical trials. EHR data is beneficial in the case of the previously approved drugs that can be analyzed for any harmful repercussions. Although efficient classification of experimental as well as the putative drugs can be mediated by natural language processing, it is rather simple for sanctioned drugs with implied descriptions in EHR software [30]. Elimination of medical mistakes, reduction of lost or unnecessary paperwork, and assistance for remuneration for our work are all major reasons to adopt EHRs. EHRs can also assist that the oncology community is fully contributing to the establishment of a national health care system that is evidence-based and responsive to the requirements of all stakeholders. EHR deployment should allow us and our patients to participate if the National Health Information Infrastructure is activated. Furthermore, adopting an EHR strategy is helpful in the elimination of medical errors, preventing any loss or unnecessary paperwork, and assistance with remuneration for work. In the future, EHR deployment can promote patient participation if the National Health Information Infrastructure is activated [26]. Despite its advantages, the problem in using EHRs is that there are no wellestablished standards for software tools, functionalities, or datasets for storing electronic records. While patient data is still extremely vulnerable, physicians are also concerned about the swiftness with which software products might become obsolete, as well as the profitability of software companies. They are also deterred as the exchange of details is often difficult in many EHR programs. Thus, EHRs are a beneficial tool that will contribute to discovering natural products in the coming years. 1.2.3 Association of HTS data to recognize disease treatmens Another semantic approach for drug discovery is the high-throughput screening (HTS) which has achieved broad acceptance in the biomedicine and pharmaceutical industry in recent times and has become a standard method for drug development. It entails assessing and evaluating a large number of biological effector molecules against a set of predetermined targets. Multimodal chemistry, gene as well as protein libraries are all selected with HTS experiments. The objective is to speed up the screening of vast chemical libraries for drug discovery at a rate of a few thousand compounds within some days or a week. The parallel and combinatorial methods for the synthesis of chemical compounds produce various novel components, including those applicable in metabolic, toxicological, and pharmacology. An advantage of HTS is that it has the potential to lower drug development costs. The steps followed in HTS include target discovery, preparation of reagents and compounds, assay formulation, and HTS of available libraries [31e33]. HTS is

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further categorized into two types: one is the functional type which tends to assess a compound’s ability to alter the function of a target protein and is more consistent, and the other is the non-functional type. It is mostly used to observe the binding capacity of the target compound with the select protein. An example of functional HTS includes ion currents through K-ion channels; whereas measurement of fluorescence activity in calcium signaling associated binding analysis exemplifies the non-functional HTS. The formation of linkages between sets of genes and the metabolic pathways they participate in; along with the pathways and phenotypes is requisite for successful knowledge integration of this sort. Highly annotated databases like Reactome and KEGG which store information on gene-pathway are extensively utilized by researchers. So, combining the variances in gene regulation and morphological output at the cellular and molecular level with available data on target pathways seems to be particularly intriguing. Although some impediments interrupt the application of this strategy for drug discovery in natural products, the characteristic property of natural products can be helpful in the phenotypic analysis. Using metabolomics, one can acquire knowledge of functional aspects of natural products in the source itself), whereas by phylogenomics, parallels between the previously identified genetic pathways/mechanisms in human diseases and model organisms can be evaluated, perhaps hinting which species can be preferred for extraction of drugs to treat these illnesses. An example of this can be understood by Conus geographus, a cone snail that targets its prey by releasing venom insulin into water. This release of insulin puts the prey in a state of hypoglycemic shock and led to the identification of the smallest identified insulin type termed as Con-Ins-G1. Despite lacking an analog to the standard eight-residue C-terminal region of the human insulin B chain, Con-Ins G1 exhibited binding and activation with the human insulin receptor, which made it an ideal component for therapeutic insulin [34]. So, the use of HTS as a semantic knowledge database can help characterize variable diseases in NP drug development. Other possible applications include exploring biodiversity data along with drug terminologies that can be searched through clinical datasets and published literature, which could help identify novel components for drug discoveries and further dissect the basis of theories that are yet unexplained. The new era of science demands the advancement in technology and provides the bulk amount of information in the form of “omic” data. In “biology” what is the challenging point for sciences? The exact challenge is to understand and to analyze the immensely large amount of structural and sequential data that have been generated in the biological system [35]. Bioinformatics is a remarkable revolution that helps interpret complex data into simpler forms and this approach is necessary for the development of computational biology for a better understanding of the mechanism that is unanswered through traditional biological studies [35]. Bioinformatics and proteomics have been independently developed and advocate a classical influence on available knowledge. The bioinformatics approach is crucial to understanding the complexity of biological

Informatics and computational methods in natural product drug discovery

sciences and through the development of specific programming, the unidentified aspects of data can be extracted from the available biological information through various researches. This approach involves the enhancement and application of biological and computational tools to collect, visualize and interpret biological data. These tools aid in the analysis of the distributed research findings in one place and suggests new findings using computer algorithms, for solving biological problems. This section of the review aims to overview the following steps of bioinformatics: (1) Introduction to Bioinformatics, (2) Three-dimensional structure of the protein, (3) Single nucleotide polymorphism, and (4) High-throughput virtual screening. 1.3 Importance of bioinformatics in discovery of natural products In case of NPS, scientists can employ a wide range of methodologies relating to the manufacture of substances from organisms. Phylogenetics and evolution, in particular, avail numerous pathways for drug discovery related events. Because phylogenetically close animals generally synthesize comparable proteins and metabolites, libraries of analogous compounds can be conveniently structured in organisms within the same genus; that is when one natural molecule with potential activity has an inadequate therapeutic index for human usage. Such methods, however, must be used carefully implied, as some natural compounds, e.g., venom proteins, are carefully tailored to match a specific biological niche, therefore, completely different metabolic profiles can be identified within same species when it comes to substances.

2. Evolution of bioinformatics concept: a new vision Bioinformatics is the application of informatics to a biological system. This field utilizes functional information of a target molecule (gene, proteins, enzymes, drug agents) and combines it with computational technology to merge it into a single, separate discipline. The analysis of biological systems has been carried out for decades now, and the bioinformatics approach is a boon to evaluate the applications of biological systems and their related molecules. First, computational methods are used in the development of several programs which can provide new data regarding the target molecules, through the usage of different kinds of machine learning algorithms and techniques, for information and prediction purposes. Next, the mathematical concepts of bioinformatics help to derive some principles and relate the data. An example is that of protein sequence, where the distribution of residues in the Ramachandran plot, analysis and interpretation of data, and significance of the data are generated, using various mathematical tools that work on the principles of regression or correlation techniques. Likewise, using information technology for large scale data analysis, one can develop the online resources that will enhance the application of bioinformatics to other fields like physics wherein we can analyze diverse interactive properties like electrostatic, van der Waals interaction,

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hydrophobic interactions, etc., and the mechanisms with which these interactions are tightly controlling the folding mechanism of proteins (concepts of physics). Therefore, bioinformatics requires the collaborative role of life sciences, mathematics, statistics, information technology, physics, or chemistry, for the evaluation and generation of functional information regarding biological compounds. The concept of bioinformatics has been investigated to learn its principles which come from previously evaluated studies and collective approaches through computational and experimental researches in molecular and cell biology [36]. One of the major contributions to the development of bioinformatics was that of Margaret O. Dayhoff, also known as the mother of bioinformatics (M.O.) [37]. As a biochemist, she applied the concepts of mathematics and computational sciences with biochemistry, for the development of computational biology-based databases, useful for nucleic acids and protein analysis. The development of a computational analysis database helped in the analysis of the protein sequences, generating the protein structure using the X-ray crystallography technique while many other computational tools were used for regulating peptide sequence [38,39]. Even the single letter code for amino acids was also introduced by her to simplify the complexities of nomenclature that otherwise led to heavily sized data files. Dr. Dayhoff also worked toward the development of sequence databases and cataloged all available amino acid sequences in the Atlas of Protein Sequence and Structure (M.) [40]; all of which strengthened the foundation of bioinformatics as a promising field of science. Although the early approach of bioinformatics was adequate for the data available during that period; however, with the enormous data generated in the later years through global research, it became imperative for further up-gradation of computational biology and bioinformatics with other disciplines of science [39]. While Watson and Crick (three-dimensional structure of DNA), Pauling (spiral structure of proteins using X-ray crystallography) and Ramachandran (Ramachandran plot for structural stereochemical properties of protein) cataloged the data and knowledge regarding protein structure [41]. Thus, bioinformatics is a multidisciplinary approach that determines how the computational tools organize, store, and analyzes the information associated with biological macromolecules [35]. 2.1 Three-dimensional structure of proteins Characterization, quantification, and identification of the target proteins are very important to understanding the molecular process which helps to mediate the cellular functioning [42]. The proteomic study has been expanded rapidly to understand the structural or functional interactions with other proteins and the protein dynamics in nature [43]. First of all, let us understand what exactly a structure predicts and its different parameters. What is a building block of proteins? These individual building blocks are termed amino acids which are structurally constituted of an amine group, a carboxyl

Informatics and computational methods in natural product drug discovery

group, and a side chain. A sequence of amino acids upon undergoing linear arrangement, if destined to participate in the biological activities of the cell, the protein will be referred to as a functional protein. They are mainly classified into four different groups; primary, secondary, tertiary, and quaternary structures. In addition, there are two structures in between them, super-secondary structures and the combination of different structures plus domains that can fold and perform functions. The sequence of proteins that Margaret Dayhoff collected was, later on, converted from the book into the database, known as Protein Information Resource (PIR), NCBI database, and SWISS-PROT which have a unique algorithmic system for protein sequence, created and maintained by the Swiss institute of bioinformatics. UniProt is a database that offers various functional aspects; with a lot of information regarding protein sequence and their analysis. The structural detail of protein can be retrieved from the protein data bank (PDB), where one can find all the available information regarding the structure of the protein. There are two approaches for comparative modeling of a protein; either we can take NMR directly or use X-ray crystallography structure from a protein data bank or we generate a new structure with the help of homology modeling. There is one more approach that is Ab-initio (S [44]. modeling, which uses a calculation of the most favorable confirmation, with the help of physical and chemical principles; however, homology modeling produces more accurate results [45]. Homology modeling is a well-established bioinformatics approach that can generate the three-dimensional structure of proteins with the help of other homologous or reference structure that has already been resolved [46]. The three-dimensional structure of protein helps in understanding the interactions, dynamics, function, and other parameters like drug target and functional prediction [43]. It also helps to understand the evolutionary sequences having the same folding patterns in tertiary structure [47]. Homology modeling calculates the accuracy, based on similarity and query coverage between the protein of interest and the template structure [46]. At least 30%e40% identity of structure is acceptable, but the higher the identity, the better the quality of the predicted model [46]. Following are the five basic steps for structure prediction of any protein: (1) Homologous or reference identification of structure; (2) Selection of templates; (3) alignment of reference structure with the target one; (4) model building; and (5) model evaluation [46]. The first step in homology modeling is to identify the amino acid sequence of protein structure that has already been resolved with above 30% similarity with the target sequence [46]. Using BLAST, we can perform this comparison in which identity and query coverage of reference structure should be predetermined. Upon selection of reference structure, global alignment between the protein of interest and template sequence is generated based on identity ( > 30%), the final model is selected based on model evaluation and quality of alignment. Softwares such as SWISS-MODEL and Modeller can be used for model generation. But if the identity is less than 30%, then we should prefer the

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hhpred tool. For remote protein homology detection and structure prediction based on hidden Markov models or HMM, this tool is used and is the first to implement a pairwise comparison of HMMs profiles [48]. If the similarity is more than 95%, then the homology is nearly certain in the hhpred method. Alignment firstly sets the atomic coordinates for the three-dimensional models, which contain the atoms of major and side chains of amino acid residues and software calculates the spatial constraints in a special direction [49]. Finally, the model evaluation which depends on some factors like a steric hindrance, torsion angles, peptide bond, planarity, and chirality of chains is essential [46]. The evaluated model is determined by the Ramachandran plot, which depicts the overall quality of the protein structure [47]. In addition, softwares such as Rampage, Molprobity, ProSA, and verify_3D are useful for validation of the structure generated. 2.2 Cheminformatics and natural products Chemical activity can be measured directly or indirectly (e.g., chemical constants, reactive groups, or ADME measurements) using cheminformatics approaches (e.g., structural motifs, compound class membership, or other observations of higher-order). Prospective structure mining is a directed method of predicting the therapeutic potential of query compounds by comparing the previously identified chemical properties of characterized compounds to the structures of query compounds.

3. High-throughput virtual screening Due to advancements in molecular and genomic studies, the findings of novel drug targets are expanded exponentially. A combination of computational and experimental methods leads to an escalation of optimization. The idea is to discover small drug-like molecules which are capable to manipulate the function of a target protein and is further utilized as a therapeutic drug against selected diseases. As experimental procedures need a lot of time and a huge cost to screen a drug library, the computational screening approach is a cheaper and more effective option for the accelerated identification of drug leads. High throughput virtual screening is an in-silico approach that helps to screen the library of compounds to check the binding affinity of the target receptor with the library [50]. Generally, the choice of scoring functions has three main criteria: (1) scoring function based on molecular mechanics which includes van der Waals and electrostatic interactions to find the best binding position of the protein-ligand complexes [51]; (2) observational scoring function that is completely based on the assessment of binding energy due to the presence of some energy components like hydrogen bond, hydrophobic effect, and ionic interaction; (3) a knowledge-centered scoring function, wherein the statistical assessment is done and the exact distance between protein-ligand complex is obtained

Informatics and computational methods in natural product drug discovery

[52]. As it is already known that the docking conformation ranked according to the scoring function shows the most favorable protein-ligand complex and identifies the best binding pose from the invalid ones. The docking method is carried out by either a receptor-based or ligand-based approach. By using three-a dimensional structure, the receptor-based screening method determines to find out the inherent compound which can change the function of the target receptor. With the help of databases, the target receptor is docked with compounds within active sites and the best pose is predicted. There are so many applications of a receptor-based method that has already been reported [53]. While in the case of the ligand-based method, known inhibitors are used because structural information is unavailable. This approach is identified by a variety of methods using chemical databases which includes pharmacophore, 3D shape matching, substructure, and similarity searching. Various kinds of applications of ligand-based methods have also been reported [54]. In both cases, compounds are selected based on similarity and from the library, the topranking compounds are selected for further analysis. Many databases are available for a selection of a library of compounds like PubChem and zinc database. The high throughput virtual screening helps to categorize the best binding pose for docking. The flexibility of protein structure and its accurate representation for ligand recognition is very important. Protein flexibility for the protein binding site is crucial and remains a great challenge, although several advanced methods determine the correct description of protein docility and its ligand identification [55] Since for protein flexibility, crystal structure like NMR or X-ray is provided by UniProt which makes for a good starting point. An appropriate docking tool that indicates feasible flexibility of protein conformation enhances the fidelity of prediction of protein binding affinity. There are so many web servers available for docking like PyRx; which is a free open source software. PyRx is based on SBVS and constituted with a python molecular analysis viewer for results, added with SQLite database for storage of resultant data [56]. Another one is AutoDock Vina, in which filtering of ligands based on molecular properties, visualization of ligands, and monitoring job progress and docking efficiency is immensely outperformed [57]. The application part determines that drug repositioning helps to find novel applications for existing drugs and also provides numerous other advantages like efforts and expenses with drug discovery, reducing time endeavor and failure with a new drug. Researchers have explored many strategies for repositioning such as ligand-based methodologies [58], networks, and structure-based approaches [59], and molecular docking studies [60]. Docking studies also help in predicting the potential side effects; e.g., docking-based tools anticipate the potent therapeutic compounds and the accidental and excludable communications between specific compound and human error. It can be exemplified by the hepatotoxicity inducing Acetaminophen (APAP), the overdose of which often leads to acute liver failure. For therapeutic development, this experiment

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proceeded in mice, which used docking as a tool to analyze the effect of Schisandrol B (SolB), a component of Schisandra sphenanthera (a well-known Chinese hepato-protective medicine). It was confirmed that when SolB binds with the CYP2E1 and CYP3A11 residues it leads to the inhibition of the APAP substrate NAPQI-GSH activities. Further, the study suggested that SolB has a shielding effect against the disease which is mediated by inhibiting the activation of APAP, and simultaneous regulation of the p53, PCNA, BCL-2, and p21 activity which can boost the regeneration of hepatic cells [61]. Apart from this, virtual screening and docking have led to the identification of new target drugs, which can help reduce the time and efforts of screening drugs through traditional experimental methods rather than high throughput screening. As of now, there are some ongoing docking studies where docking and experimental system are simultaneously being used to confirm the predictions. Mostly, these studies are based on new drug inhibitors for inhibition of infectious agents which include Bacillus anthracis, HIV, Mycobacterium tuberculosis, and many more. 3.1 Gene expression perturbation The advent of multi-omics approaches to identify disease mechanisms has resulted in a plethora of methods for evaluating the effect of potential medications on cells. Gene expression disturbance, as measured by RNA-sequencing and transcriptomics, has led to several novel medication discovery advances for diseases involving gene dysregulation, such as cancer and other diseases with complicated genetic etiologies ([62]. Abnormalities in gene expression, resultant of the systems-level impact of expression perturbation in the larger context of cell signaling and metabolic networks are attributed as the disease causing factors. Other additional factors include environmental influences, structural abnormalities, and other prompting factors [63,64]. Differential expression can be taken as a phenotypic signal, resulting from the underlying illness etiology. As a result, medications and drug candidates that effectively reverse such negative consequences could be used to treat these disorders. This method is especially well-suited for the drug development of NPS because a large number of compounds from all types of NPs have been carefully designed to play essential role in signaling networks or metabolic processes and are established as physiologically stable [65]. Traditional Chinese Medicine (TCM) compounds are mainly useful in this regard. In 2014, a study was conducted where researchers discovered plausible mechanisms, using which the TCM component berberine displays an anti-cancer effect, which utilized gene expression data, available in Connectivity Map (CMap) project, for berberine-perturbed human cells [66]. Analysis of the differential expression among organisms that produce the NPS is a separate but related method (rather than the organisms that NPS acts upon). Comparative profile analysis of transcriptome of different bacterial species in the genus Salinispora, various novel

Informatics and computational methods in natural product drug discovery

NPs, along with putative mechanisms were detailed, which elaborated on their functional aspects and highlighted the diversity of emergent multi-omics systems, beneficial in NP drug discovery [67]. 3.2 Data required for natural product The variety and complexity of information, essential for NP associated drug discovery research make storage, representation, and distribution of this information, difficult. As a result, various NP databases are restricted to a small number of closely related NPs, resulting in data fragmentation [68]. ConoServer [70] and ArachnoServer [69] are two known NP databases with extensive and detailed information, although each is limited to toxins synthesized by a single clade of species. One fractional resolution is to create specialized, more conventional databases to improve the categorization of NPs, as the Tox-Prot manual annotation program within UniProtKB/Swiss-Prot has done [21]. However, the larger issue of being able to use all of the critical data types which are exclusive to certain types of NPs remain unresolved. The inclusion of APIs and other tools that enable computational access is another advantage that larger database efforts have over smaller, specialized NP databases. Although many specialist databases allow for mass data downloads, these can be partial and out-of-date. Additionally, APIs can aid in the interoperability of databases as a cohesive network of specific, labeled datasets that can provide the user with semantic knowledge and solves problem of adequately significant granular characteristics, while contributing the benefits of larger data repositories. Taking these and other challenges into account, informatics researchers and data scientists have a lot of opportunities to qualitatively/quantitatively improvise, and develop interconnection of NP databases for knowledge representations. Some of the databases that are used to avail the requisiste informtion on natural productss are mentioned in Table 8.1. 3.3 A vision for natural product drug discovery in future Despite the inequities mentioned above, growing interest in bio-ontologies, integration of semantic information and data-driven methods for drug discovery suggest that change is on the way. This study identifies several concrete solutions for the research sector to overcome current difficulties and support the development of new NP drug discovery innovations. For NPS, creating public HTS data: Information regarding a vast majority of non-human species are scarcely present in public repositories, even though the biomedical field is inundated with multi-omics HTS data. Many of the strategies we’ve outlined will remain out of reach for most academics unless additional resources are dedicated to disseminating multi-omics data for species of interest in reference to NP-based drug discovery.

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Semantic Tools Databases associated with drug discovery of natural products

Literature mining

Bioinformatics Tools EHR mining/ HTS

Three dimensional structure prediction of proteins

Homology modeling

Gene ontology

GWAS

PubMed

PheWAS

Protein information database (PIR) NCBI

SemaTyP

Reactome

OWL2, BioRDF, SPARQL ChEMBL, DrugBank, MeSH VenomKB, NPASS

KEGG

Uni-prot/Swissprot Protein data bank (PDB)

High throughput screening Docking databases

Protein structure flexibility

Expression databases

Ab-initio modeling

PubChem

PyRx

Connectivity map (CMap)

Modeller

ZINC database

AutoDock vina

ConoServer

Swiss-model

ArachnoServer

Hidden markov models (HMM)

Tox-prot

New Horizons in Natural Compound Research

Table 8.1 Some databases that are currently in use for acquiring information related to natural products and drug discovery.

Informatics and computational methods in natural product drug discovery

Using clinical data: New collaborative efforts like OHDSI and eMERGE give researchers more access to genuine clinical data that can be used for both medication discovery and evaluation. As the coverage of NPS in semantic knowledge resources improves, so will the capacity to do inference on NPs using observational data.

Author contributions HS and MP conceived, wrote, and edited the content of this review.

Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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[61] Jiang Y, Fan X, Wang Y, Chen P, Zeng H, Tan H, Bi H. Schisandrol B protects against acetaminophen-induced hepatotoxicity by inhibition of CYP-mediated bioactivation and regulation of liver regeneration. Toxicol Sci 2015;143(1):107e15. [62] Sirota M, Dudley JT, Kim J, Chiang AP, Morgan AA, Sweet-Cordero A, Butte AJ. Discovery and preclinical validation of drug indications using compendia of public gene expression data. Sci Transl Med 2011;3(96). 96ra77-96ra77. [63] Cookson W, Liang L, Abecasis G, Moffatt M, Lathrop M. Mapping complex disease traits with global gene expression. Nat Rev Genet 2009;10(3):184e94. [64] Nica AC, Dermitzakis ET. Using gene expression to investigate the genetic basis of complex disorders. Hum Mol Genet 2008;17(R2):R129e34. [65] Lewis RJ, Garcia ML. Therapeutic potential of venom peptides. Nat Rev Drug Discov 2003;2(10): 790e802. [66] Lee K-H, Lo H-L, Tang W-C, Hsiao HH-y, Yang P-M. A gene expression signature-based approach reveals the mechanisms of action of the Chinese herbal medicine berberine. Sci Rep 2014;4(1):1e9. [67] Amos GC, Awakawa T, Tuttle RN, Letzel A-C, Kim MC, Kudo Y, Jensen PR. Comparative transcriptomics as a guide to natural product discovery and biosynthetic gene cluster functionality. Proc Natl Acad Sci USA 2017;114(52):E11121e30. [68] Williams A, Martin G, Rovnyak D. Modern NMR approaches to the structure elucidation of natural products: volume 1: instrumentation and software. Royal Society of Chemistry; 2015. [69] Pineda SS, Chaumeil P-A, Kunert A, Kaas Q, Thang MW, Le L, Cristofori-Armstrong B. ArachnoServer 3.0: an online resource for automated discovery, analysis and annotation of spider toxins. Bioinformatics 2018;34(6):1074e6. [70] Kaas Q, Yu R, Jin A-H, Dutertre S, Craik DJ. ConoServer: updated content, knowledge, and discovery tools in the conopeptide database. Nucleic Acids Research 2012;40:D325e30. https://doi.org/ 10.1093/nar/gkr886. 22058133.

CHAPTER 9

Compound synergy in natural crude extract: a novel concept in drug formulation Vivek T. Humne1 and Mahendra N. Lokhande2 1

Department of Chemistry, Shri R.R. Lahoti Science College, Amravati, Maharashtra, India; 2Department of Chemistry, Avvaiyar Government College for Women, Karaikal, Pondicherry, India

1. Introduction The crude extract of plant, leaf, and bark has been commonly used as remedies since the beginning of ancient history [1]. As per a survey by the Centers for Disease Control and Prevention in the year 2012, approximately 8% of the US population uses natural products for the treatment of disease. To date, the use of the medicinal plant is a broad area of research while a significant part of the world has utilized herbal natural products and supplements as the primary mode of treatment [2]. Plant-based medications are widely used in developing nations, where they offer the only source of health treatment. Essentially, the herbal crude extract is a combination of different constituents of plants which include primary and secondary metabolites. Various proteins, lipids, amino acids, and carbohydrates produced by the plants are known as primary metabolites [3]. While small molecular, bioactive compounds produced by the plants are known as secondary metabolites [4]. To identify and enrich active ingredients, isolation of phytochemical components or bioactivities consisting of fractions of crude extracts has been carried out [5]. Many pharmacological activities of bioactive constituents became weakened or dissipate after their separation and purification from the crude extracts [4]. It has been noticed that after the extraction of a pure component from crude extract, pharmacological effects decrease which is found to be the same as that of pharmacokinetic synergies of pure constituents [6]. For example, the level of exposure of pure artemisinin in the bloodstream after their oral administration was found to be more than 40 times reduced as compared with the component medicated with dried plant extract of A. annua [7]. Furthermore, artemisinin is a major component of A. annua plant extracts, and it promotes pervasive metabolism via cytochrome P450 enzymes (CYPs) (e.g., CYP2B6 and CYP3A4) [8]. Another example is arteannuin B having a supportive component of crude which suppress hepatic CYP3A4 (IC50 1.2 M) and enhanced AUC0et (2.1-fold) and peak concentration (Cmax, 1.9-fold) by oral administration of artemisinin in mice [9].

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2. What is the synergy effect? The separate constituents or components of a mono- or multi-extracts of crude mixture affected various targets. This is due to the synergistic multi-target effect and thus combined in an agonistic in a synergistic way (Fig. 9.1). In addition, synergistic effects can also occur when constituents of an extract interact with various targets of the cell, together to increase the solubility and resulting to increase the pharmacokinetic properties of more than one compound in the extract [6]. In vitro pharmacological models can be depicted as the isoboles of two different doses. The graphical representation of Fig. 9.2 shows a result of the synergistic effect of the mixture of two well-known natural products of Ginkgolides A and B. The different concentration of Ginkgolides A and B is used to measure the IC50-values for PAF-induced in vitro thrombocyte-aggregation inhibition test according to Born’s protocol. The interaction matrix is less than 1, which is corresponding to a concavely curved isobole pointing toward zero, indicating a synergistic effect. If the interaction matrix is greater than 1, the isobole indicates antagonism effect. However, no interaction was found when interaction matrix is equal to zero [10].

3. Factors responsible for the synergetic effect The following types of mechanisms are proposed on the basis of classic pharmacological, molecularebiological, and clinical studies. 3.1 Synergetic multi-target effects Many plant extracts contain rich sources of polyphenols and terpenoids as a group of constituents. If they show a sufficiently high bioavailability, these constituents could strongly increase the overall efficacy of the synergistic effect. Polyphenols have a significant

Figure 9.1 Effect of mono and multi-targets of different constituents of plant extract.

Compound synergy in natural crude extract: a novel concept in drug formulation

Figure 9.2 Isoboles of zero-intraction, synergerism, and antagonism.

potential to bind to various protein or glycoprotein structures, whereas terpenoids are lipophilic in nature. As a consequence, they can pass through the cell walls of the human body or microorganisms. In the case of a mono-extract mixture of compounds having the ability to bind only one target, thereby additive effect can be considered. Over-additive or synergistic effects can be achieved when a single element binds to many targets. For example, cannabis and tetrahydrocannabinol show antispastic effects along with other activities such as antiemetic, anti-inflammatory and analgesic. It has been noticed that the same proportion of Cannabis extract and tetrahydrocannabinol is more effective than tetrahydrocannabinol toward antispastically. An early investigation found that a tetrahydrocannabinol-free extract had no discernible antispasmodic effect. An improved synergistic effect could be attributed to the concurrent ingredients of Cannabis extract, such as cannabidiol [11]. Phytopreparation of Iberogast is another example of the multi-target principle. It is a combination of nine plant extracts that are used to treat dyspepsia and intestinal motility disorders. A clinical study has been carried out with two synthesized drugs, cisapride and metoclopramide. In comparison to the two synthetic medications, phytopreparation of Iberogast has no negative effects. Iberogast can be used for gastrointestinal motility function, gastrointestinal hypersensitivity, gastrointestinal autonomic afferent function and inflammation. Synthetic monodrugs, on the other hand, work in the same way as regular proton pump inhibitors. Nevertheless, they only treat one symptom of functional dyspepsia [12].

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3.2 Pharmacokinetic or physicochemical effects based on solubility, resorption rate, and bioavailability Polyphenols and saponins are examples; they do not have enough pharmacological effects on their own. However, the incorporation of such components may improve the solubility and rate of resorption of primary extract active constituents, hence increasing its bioavailability. Consequently, the efficiency of the extract becomes more effective than its isolated constituent. For example, because of poor bioavailability, hypericin from Hypericum perforatum has a modest antidepressant (Mono Amine Oxidaseinhibiting) action. However, when hypericin is conjugated with different metabolites of plant extract, the level of plasma for hypericin is also increased which increases the potency of the antidepressant effect [13]. 3.3 Interactions of agents with resistance bacteria The synergistic effect is also dependent on the comedication of antibiotics with drugs that can inhibit the bacterial resistance mechanism partially or entirely. There are three reasons to occur bacteria gain antibiotics: (1) to improve the active site of the target, resulting to decrease in the effectiveness of the drug’s binding site, (2) antibiotics produced by the microorganism directly destroyed or modified by enzymes, or (3) the role of efflux of antibiotics of the cell to hindrance the antibiotic effect to penetrate into the bacteria cell and extrude the accumulated drug out of the bacteria cell after penetration [14]. For example, baicalein is a class of flavonoids that is isolated from the roots of Scutellaria baicalensis and Scutellaria lateriflora. Baicalein possesses weak anti-Methicillin resistant Staphylococcus aureus (MRSA) activity similar to other flavones. The comedication study of antibiotics such as tetracycline and b-lactams with baicalein is carried out on MRSA strains. Baicalein has no effect on the MRSA strains resistance similar to other antibacterial drugs such as erythromycin, kanamycin, chloramphenicol, and ofloxacin. It concluded that baicalein affects the resistance cell of both tetracycline and b-lactams in MRSA. Therefore, it showed a synergy effect with b-lactams against MRSA [15]. In a different study, Nandre et al. found that flavonoids like chrysin, galangin, and phenethyl caffeate that were isolated from Indian propolis samples worked synergistically on multidrug resistant bacterial isolates, whereas a single or individual flavonoid had very little efficacy [16].

4. Synergistic effect on diabetes Diabetes mellitus is a type of chronic metabolic disease which belongs to the class of hyperglycemia, which destroys the multiple cells of the body systems. There are two kinds of diabetes systems: type-1 and type-2 diabetes mellitus. Type-1 diabetes mellitus is due to the impaired production of insulin; therefore, it is also known as insulin-dependent diabetes mellitus. While type-2 diabetes mellitus is due to the inability of cells toward

Compound synergy in natural crude extract: a novel concept in drug formulation

insulin (insulin resistance); therefore it is also known as non-insulin-dependent diabetes mellitus. Momordica charantia is also called bitter melons which treat diabetes mellitus. Polypeptide-p was isolated from M. charantia and found effective as a hypoglycemic agent [17]. A novel functioned glucose homeostasis has been identified as MCIR-binding protein that is bound to the various sites of the insulin protein on insulin recepter [18]. The 9-cis, 11-trans, 13-trans-conjugated-linolenic acid (9c, 11t, and 13t-CLN) was isolated from the n-hexane fraction of M. charantia. It induced acyl CoA oxidase activity in a peroxisome proliferator-responsive murine hepatoma cell line (H4IIEC3) indicating that 9c, 11t, 13t-CLN was able to act on a natural peroxisome proliferator activated receptors signaling pathway [19]. Charantin, momordenol, and momordicilin are important potent drugs having insulin-like chemical frameworks and properties [20]. Momordicine II and Kuguaglycoside G also stimulated insulin secretion [21]. Several mechanisms are reported for the treatment of diabetes such as stimulation of insulin, a decrease in hepatic glucose and increase in the peripheral uptake of glucose. The combination of both metformin 1 (Table 9.1) and M. charantia are having the ability to decrease body weight during treatment and also significantly decrease blood glucose level [22]. The combination of glibenclamide 2 (Table 9.1) and Aloe Vera juice has been investigated for diabetic activity. The glibenclamide 2 possesses no effect on blood sugar levels. However, the combination of Aloe Vera gel, and glibenclamide 2 showed antihyperglycemic activity and also decreases triglyceride levels, which are usually found high in diabetic patients [23]. The synergistic effects of nateglinide 3 (a nonsulfonylurea D-phenylalanine derivative) and meals on insulin secretion have been studied for type 2 diabetes. Table 9.1 Synergy effect of different constituents with herbal extract. Compound

Structure

1

Herbal extarct

Activity

Refs

M. charantia

Antidiabetic

[21]

Aloe vera

Antidiabetic

[22]

Meals on insulin secretion

Antidiabetic

[23]

Metformin 2

Glibenclamide 3

Nateglinide Continued

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Table 9.1 Synergy effect of different constituents with herbal extract.dcont’d Compound

Structure

4

Herbal extarct

Activity

Refs

Methicillin resistant strains of Staphylococcus aureus

Antimicrobial

[28]

Vangueria madagascariensis piper betle L. extracts

Antimicrobial

[29]

Betulinic acid 5

Chloramphenicol

In Table 9.1, Nateglinide 3 is the derivative of an amino acid that exhibited in a novel chemical class of medications for the treatment of type 2 diabetes that differs pharmacologically and therapeutically from currently available drug. Nateglinide 3 binds and inhibits the b-K1 cell’s ATP channel, resulting in membrane depolarization and an influx of extracellular calcium, which leads to insulin production. It was noticed that increase in insulin secretion was noticed when nateglinide 3 was taken before a meal as compared to nateglinide 3. Nateglinide 3 substantially lowers plasma glucose levels. Nateglinide 3 has a synergistic effect on insulin secretion when given before meals, revealing that it is more effective when glucose concentrations are at their optimum level [24].

5. Synergistic effect on antimicrobial activity Medicinal plants or their extracts have long been used as a medication to treat a myriad of infections. Antimicrobial properties have been identified in a variety of plants and plant extracts. Investigations of the antibacterial properties of natural compounds have been a central topic for medication discovery around the world during the last few decades. Several herb-to-drug interactions can occur when plant treatments are used with pharmaceutical drugs, with possible outcomes including synergistic amplification of antibacterial potential and a reduction in the undesirable side effects of synthetic drugs. These combined actions have reduced the chances of medications used alone to treat a microbial ailment having lower efficacy. Synthetic antibiotics can currently be used in combination with inexpensive, practical, and nontoxic medicinal herbs as per the herb-to-drug combination technique [25]. Plant extracts are commonly known for their antibacterial properties, but in association with antibiotics can improve their efficiency. The ability of chemical components of plant extract itself explains the modification or inhibition of resistance mechanisms to become susceptible for the action of antibiotics at lower doses.

Compound synergy in natural crude extract: a novel concept in drug formulation

The efficiency of antibacterial activity of plant components depends on various factors: (1) to identify the microorganism target (viz., type, genus, species, strain), (2) to identify the plant material such as botanical source, development stage, the composition of the bioactive compounds, or method of extraction and (3) to identify the chemical properties such as hydrophilicity, lipophilicity, concentration, pH value. However, the inhibition of bacterial growth is found through several mechanisms: disruption of membrane function and structure (including the efflux system), interruption of DNA/RNA synthesis and function, interference with intermediary metabolism, and induction of coagulation of cytoplasmic constituents. The combination of different antibiotics mainly ciprofloxacin showed good synergistic interaction in the n-hexane fraction, resulting in a 32-fold reduction of minimum inhibition concentration (MIC). The mode of action of Plant Growth Regulator (PGR) ethanol and sequential fractions was also investigated, and it was revealed that adding PGR extract increases norfloxacin accumulation intracellularly [26]. The mechanism of synergistic action for antimicrobial activity has depended on the following four points: (1) modification of active sites on bacterial cell: the cell wall becomes mechanically weak without the mesh structure, affecting the bacteria cell’s integrity. Resistance develops when penicillin-binding proteins (PBP) affinity for antibiotics is lowered or when their production is reduced, (2) modification of antibiotics through inhibition of enzymes: antibiotics are inactivated by a variety of enzyme systems found in bacterial cells. It occurs due to the hydrolysis, active group replacement and oxidation-reduction process [27], (3) increase of membrane permeability: antibiotics and other chemicals are penetrated to the cell wall which is the first barrier in order to reach their targets and exhibit the inhibitory effect. Before successfully reaching the cell membrane, compounds can be carried out from the periplasmic space from the efflux pumps or inactivated by enzymes after passing the outer membrane. The increase of membrane permeability resulted in an increase in the level of the antibiotics in the bacterial cells and their better interaction with intracellular targets [28], (4) inhibition of efflux pumps: one of the resistance mechanisms is the development of efflux pumps by bacteria to remove drugs from cells. Efflux pumps are activated by ATP hydrolysis or by a change in ion concentration. Active compounds inhibited tetracycline excretion by inactivating the efflux pump [29]. Pentacyclic triterpenoids have anti-staphylococcal properties, and while individually weaker than common antibiotics produced by bacteria and fungi. Synergistically, these compounds may use different mechanisms of action or routes to achieve antimicrobial effects, as suggested by the lower MICs. The three pentacyclic triterpenoids such as a-amyrin, betulinic acid, and betulinaldehyde exhibited antimicrobial activity against the two strains of S. aureus, one of which was methicillin-sensitive and the other methicillin-resistant. The good result of synergistic combination was found with betulinic acid 4 against methicillin [30]. Chloramphenicol 5 and Ciprofloxacin has little effect on

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Acinetobacter spp. when combined with Vangueria madagascariensis (VM) extracts, potent activity was observed. The methanolic fruit extract at lower concentration of Chloramphenicol (30%) gave better synergistic effect (MIC 1/4 3.75 mg/mL) as compared to 50% (MIC 1 /4 12.5 mg/mL) [31]. Piper betle L. extracts and antibiotics combination showed additive and synergistic effects. The effective synergy has been observed in the combination against P. aeruginosa and chloramphenicol (70:30). Synergy was also noticed against S. aureus, Propionibacterium acnes, S. epidermidis, and Streptococcus pyogenes [32].

6. Synergistic effect of natural drugs on breast cancer cell Worldwide mortality by breast cancer has gained more concern, as lifestyle and stress increase day by day. Breast cancer starts in the breast of males as well as females, as it is more commonly observed in the female breast than male breast. The main affected part of the breast is glands, ducts, and fatty tissues of the breast. Depending upon the presence of a lump in different parts of the breast are given different terms, such as lobules glands which make milk and are termed as lobular cancer, milk carrying vessels to the nipple are called duct and cancer known as ductal cancer. Ductal cancer has two types: non-invasive cancer and invasive cancer. Noninvasive present in only duct while invasive spread out of the duct. Minor breast cancer was observed in the areola of a nipple and termed as Paget’s disease of the breast. The place in the breast, fats, and connective tissues are presently known as a stoma. This helps to maintain the position of lobules and ducts; at this place cancer tumor is termed as phyllodes tumor. Angiosarcoma is observed in blood vessels; this sarcoma and lymphomas are also termed as tumors. The remaining types which occurred are Medullary, Mucinous, Tubular, Metaplastic, Papillary breast cancers, but these are very rare. Some of the specific tests are used to determine the tumor and stage of propagation. These tests include (1) “Hormone receptor positive” which includes progesterone and estrogen receptor simultaneously known as PR positive and ER positive. (2) “Human epidermal growth factor receptor 2” i.e., HER2 positive indicates changing genes to protein. (3) “Triple negative” indicates the absence of ER, PR, and HER2 positive tests but still showing lumps. Breast cancer can, however, spread through either lymph or blood. If lymph vessels carrying infected cells under arms are known to be auxiliary lymph nodes, internal mammary lymph nodes, supraclavicular and infra-clavicular lymph nodes, these lymph nodes are typified as per their position. Looking at natural products and their synergistic effect, some of the existing drugs mixed with natural products and screened for their activity against infected cells, such as Paclitaxel is an anti-cancer drug, widely used in chemotherapeutic methods and shown to have a cytotoxic nature. Piperine is isolated from black and long pepper (Piper nigrum L., Piper longum L.) natural extract mixed with Paclitaxel and tested against breast cancer. The drug molecules are listed in Table 9.2. The bioactivity of the Paclitaxel increases in presence of Piperine and it is confirming the synergistic effects. In this effect the combination of both the drug were screen for

Compound synergy in natural crude extract: a novel concept in drug formulation

Table 9.2 Chemical structure of natural drugs on the breast cancer cell.

Paclitaxel

Piperine

(Curcuma longa L)

Cisplatin

the MCF-7 cells line and IC50 values found to be 0.50  0.022 mM to IC50 (0.78  0.022) and IC70 (0.95  0.032) at different concentration, while the separate paclitaxel and piperine shown 0.22  0.003 mM and 1.21  0.21 mM values respectively. The IC50 values indicate the superior effect when drug combination takes place [33]. The study of polyphenol curcumin isolated from Curcuma longa L and OMDM-2 in combination were investigated against breast cancer specifically (MCF-7) and glioblastoma (U-87) cell line. The synergetic effect was studied by a combination of curcuma extract. The method used herewith is resazurin assay; both the cells (MCF-7) and glioblastoma (U-87) were investigated individually and in combination. The OMDM-2 was tested against both cell lines and showed anti-proliferative activity in the presence and absence of curcumin and recorded its IC50 values at different concentrations. The OMDM-2

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shown IC50 values for MCF-7 Cells is 4.9 mM and glioblastoma U-87 is 2.7 mM. To study the synergetic effect different ratios of both the drugs were applied in IC50, IC60, IC70, and IC80. The OMDM-2 effect increases 3 to 13 times more in presence of curcumin in IC50 and IC70 and it was measured by IC method. At the dose ratio of 1:1 in IC75 when curcumin was added to OMDM-2, the synergetic effect decreased almost two times [34,35]. Berberine is a natural chemical compound isolated from Coptis Chinensis and Berberis aristata and shows breast cancer activity. Theophylline is also extracted from tea leaves, alone theophylline has no effect on the breast cancer cell. Normally, crude theophylline is used for respiratory related difficulties. But in combination with berberine shows activity against breast cancer cells. Moreover, the chemical structure of berberine has a quaternary nitrogen atom and due to the positive nature of nitrogen showing strong binding with the nucleic acid. This may be responsible for activity against breast cancer cell line and in a combination of theophylline as it reduces the cytotoxicity of berberine against noninfected cells. The action of berberine inhibits proliferation in MDA-MB-231cells, and this synergistic effect depends upon the dose of a combination of berberine and theophylline. The presence of theophylline is showing a synergistic effect. The amount of substance required is from 100 to 50 mM of Berberine [36]. Berberine has anti-proliferative activity against MCF-7 and MCF-7/TAM cells. The combined effect of berberine and tamoxifen was studied by increasing dose ratio at 24, 48, and 72 h for cell proliferation and apoptosis activity. The IC50 values of berberine were reported for MCF-7 and MCF-7/TAM cells of 130 and 99.7 mM. Looking at the initial results, the berberine is more receptive to MCF-7/ TAM cells than MCF-7 cells. The cell viability for infected cells by treatment with tamoxifen was found to be 0.87  0.24% for MCF-7 cells, while a combination of berberine at 20 mg shown 29  3.25% for MCF-7 and 17.87  2.05% for MCF-7/TAM cells. The combination dose at 20 mM of berberine and 1 mM of tamoxifen increases the apoptosis effect 61.8  7.47% [37]. We observed reaction and combination of berberine with several organic molecules which shows anti-proliferative and apoptosis activity against breast cancer cell MCF-7. There are several examples of inorganic compounds showing anti-breast cancer activity. In such a way when berberine combined with cisplatin shows apoptosis activity by breaking DNA molecules [38].

7. Conclusion Synergetic effect is a significant aspect to enhance the biological activities. It is noticed that combination of particular drug constitute with herbal extract has playing a crucial role in antidiabetic, antimicrobial, and anti-cancer activity. The mode of mechanism, mode of action, and effectiveness are well explained for various molecules. Finally, identification of drug constitute with proper target of the crude herbal extract decides the future database studies on bioavailability, pharmacodynamics, and mechanism of action. Consequently, it will give a good contribution in drug discovery program.

Compound synergy in natural crude extract: a novel concept in drug formulation

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[23] Bunyapraphatsara N, Yongchaiyudha S, Rungpitarangsi V, Chokechaijaroenporn O. Antidiabetic activity of Aloe vera L. juice II. Clinical trial in diabetes mellitus patients in combination with glibenclamide. Phytornedicine 1996;(3):245e8. [24] Keilson L, Mather S, Walter YH, Subramanian S, Mcleod JF. Synergistic effects of nateglinide and meal administration on insulin secretion in patients with type 2 diabetes mellitus. J Clin Endocrinol Metab 2000;(85):1081e6. [25] Borchers AT, Hackman RM, Keen CL, Stern JS, Gershwin ME. Complementary medicine: a review of immunomodulatory effects of Chinese herbal medicines. Am J Clin Nutr 1997;(66):1303e12. [26] Rafiq Z, Narasimhan S, Haridoss M, Vennila R, Vaidyanathan R. Punica granatum rind extract: antibiotic potentiator and efflux pump inhibitor of multidrug resistant Klebsiella pneumoniae clinical isolates. Asian J Pharmaceut Clin Res 2017;(10):191e5. [27] Hu Z-Q, Zhao W-H, Hara Y, Shimamura T. Epigallocatechin gallate synergy with ampicillin/sulbactam against 28 clinical isolates of methicillin-resistant Staphylococcus aureus. J Antimicrob Chemother 2001;48:361e4. [28] Araya-Cloutier C, Vincken JP, Ederen R, den Besten HMW, Gruppen H. Rapid membrane permeabilization of Listeria monocytogenes and Escherichia coli induced by antibacterial prenylated phenolic compounds from legumes. Food Chem 2018;240:147e55. [29] Mikulasova M, Chovanova R, Vaverkova S. Synergism between antibiotics and plant extracts or essential oils with efflux pump inhibitory activity in coping with multidrug-resistant staphylococci. Phytochem Rev 2016;(15):651e62. [30] Chung PY, Navaratnam Lip P, Chung Y. Synergistic antimicrobial activity between pentacyclic triterpenoids and antibiotics against Staphylococcus aureus strains. Ann Clin Microbiol Antimicrob 2011; (10):25. [31] Mahomoodally MF, Dilmohamed S. Antibacterial and antibiotic potentiating activity of Vangueria madagascariensis leaves and ripe fruit pericarp against human pathogenic clinical bacterial isolates. J Trad Combi Med 2016;(6):399e403. [32] Taukoorah U, Lall N, Mahomoodally F. Piper betle L. (betel quid) shows bacteriostatic, additive, and synergistic antimicrobial action when combined with conventional antibiotics. South Afr J Bot 2016; (105):133e40. [33] Motiwala MN, Rangari VD. Combined effect of paclitaxel and piperine on a MCF-7 breast cancer cell line in vitro: evidence of a synergistic interaction. Synergy 2015;(2):1e6. [34] Ammar RM, Ulrich-Merzenich G. Curcumin synergizes with the endocannabinoid reuptake inhibitor OMDM-2 in human MCF-7 breast cancer and U-87 glioblastoma cells. Synergy 2017;(5):7e14. [35] Wang K, Zhang C, Bao J, Jia X, Liang Y, Wang X. Synergistic chemo preventive effects of curcumin and berberine on human breast cancer cells through induction of apoptosis and autophagic cell death. Sci Rep 2016;(6):1e14. [36] Niasari F, Chadegani AR, Razmi M, Fallah S. Synergy of theophylline reduces necrotic effect of berberine, induces cell cycle arrest and PARP, HMGB1, Bcl-2 family mediated apoptosis in MDAMB-231 breast cancer cells. Biomed Pharmacother 2018;106:858e67. [37] Wen C, Wu L, Fu L, Zhang X, Zhou H. Berberine enhances the anti-tumor activity of tamoxifen in drug sensitive MCF 7 and drug resistant MCF 7/TAM cells. Mol Med Rep 2016;(14):2250e6. [38] Zhao Y, Jing Z, Li Y, Mao W. Berberine in combination with cisplatin suppresses breast cancer cell growth through induction of DNA breaks and caspase-3 dependent apoptosis. Oncol Rep 2016;36: 567e72.

CHAPTER 10

Small molecules vs biologics Preethi Poduval1, Sonia Parsekar1 and Surya Nandan Meena2 1

Department of Biotechnology, Dhempe College of Arts and Science, Miramar, Goa, India; 2Department of Chemistry, Division of Biochemistry, Savitribai Phule Pune University, Pune, Maharashtra, India

1. Introduction Researchers continuously strive attempting to ensure safe and effective cure for new emerging diseases and old uncurable diseases. In view of such attempts tremendous focus has been laid on the development of molecules for drug discovery. The two main candidates for preparation of drugs involves small molecules and biologics. Small molecules and biologics are basically disease-fighting “weapons.” A small molecule drug (SMD) is derived through chemical synthesis from natural products produced by bacteria, fungi, algae, plants, etc. The common drugs under this category are aspirin, felbamate, varenicline, procaine, diphenhydramine among many other “medicine cabinet” drugs. On the other hand, the biologics drugs derived through the complex biochemical processes to diagnose, treat, or prevent diseases. These drugs are prepared from live organisms or their by-products such as live cells, proteins, enzymes, blood, etc Humulin (5.8 kDa), the first biologics drug (BD), was available in 1982 marking a new era in the pharmaceutical sector. It was observed that the share of biologics in the pharmaceutical market increased remarkably from 16% (2006) to 25% (2016) and had won by a small margin in 2020 with 60% of the top 20 drugs sold as compared to small molecules with 40% consumption [1]. However, SMD occupies about 90% of the global sales and this is majorly because the BD cost around 22 times more than the SMD [1]. Amino acid molecules constitute the therapeutic proteins and vary in sizes ranging from small peptides of 1 kDa to less than 10 kDa and larger peptides with sizes larger than 10 kDa such as monoclonal antibodies [2]. The smallest of such molecules for example antibodies have sizes much greater than the small molecule-based drugs used to treat squamous carcinoma (auristatin) [3]. Simple chain amino acids make peptides that have a characteristic tertiary structure and larger protein complex molecules that are arranged into unique three-dimensional folding structures processing on integral biological function. The properties such as polarity, heat sensitivity, proneness to enzymatic digestion and permeability are specific to the therapeutic proteins with certain exceptions of monoclonal antibodies and vary in behavior as compared to the small molecule drugs with respect to the route of administration via systemic injection into the body as against the small molecule drugs that can be administered orally.

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Increase in the involvement and novel approaches in the study of biologics has opened many doors for further research allowing the present era to witness the rise of biologics for making drugs. The highlight of recent small molecule drugs includes anti-tumorigenic (PAWI-2) [4]; niclosamide a potential drug against COVID-19 [5]; immunosuppressant ISAtx-247, FTY-720, and Alemtuzumab and that of biologics include anti-melanoma drug candidate TVEC [6]; anti-asthmatic antibody Tezepelumab [63]; disease-modifying antirheumatic drugs against skin disease psoriasis [7], and tumor necrosis factor TNF-a against Atherosclerosis [8]. The traditional approaches have almost been completely replaced by the advanced state of the art research and development. The collaborative, interdisciplinary, and cutting-edge research has improved the entire process of drug discovery and aims to reduce the high expenses, time factor, and increase the effectiveness of the drug (Fig. 10.1). 1.1 Sources of small molecules and biologics The small molecule drug (SMD) are made from compounds found in nature. The common natural sources for SMD are plants, fungi, algae, and microbes, but most prevalent sources are plants [11]. Few examples that include medium derived from plants are from Papaver somniferum (morphine), Digitalis lanata (digoxin), and Salix spp. (aspirin). Biological are large molecules such as sugars, proteins, nucleic acids, enzymes, amino acids, and therapeutic proteins. The pharmaceutical ingredients used to design the drugs are extracted from the microbes using traditional and latest methods of culturing, storing,

Figure 10.1 Size comparison between small molecule v/s biologics.

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and extraction of biologics. Receptor modulators, enzymes, vaccines, and antibodies can be recovered from engineered microorganisms designated as MCF (Microbial Cell Factories) [12]. Ref. [13] highlighted the methodology to overexpress the genes for producing NDP (nucleotide di phosphate) sugars which ultimately led to the generation of salicylate glucoside and other glycosylated variants by the metabolic engineering approach. Escherichia coli has been popularly used as a robust microbial source for production of pharmaceutically important biologics such as antibodies, vaccines, and cytokines [14]. Fungi such as Penicillium chrysogenum were used as a cell factory biologics such as b-lactam antibiotic and secondary metabolites roquefortines, fungisporin, penitric acid, siderophores, chrysogine, chrysogenin sesquiterpene, u-hydroxyemodin, and sorbicillinoids [15]. Actinomycetes of the marine Streptomyces spp. produced various halogenated biologics, i.e., polyketides, alkaloids with promising anticancer, and antibacterial drug potential [16]. 1.2 Isolation of small molecules vs biologics 1.2.1 Isolation of small molecules Extraction is the primary step to isolate the small molecules in the form of natural products from natural crude materials. Hence, there are several efficient extraction procedures available of small molecules. According to the extraction principle, solvent extraction, distillation method, pressing, and sublimation are common in use [17]. Among all these methods, most widely used method is solvent extraction as it favors increase in yield. Small molecules are embedded well inside complex plant tissue framework, and hence extraction of these molecules from plant material is a tedious process. Different techniques of extraction are used in various solvent systems [18]. Following are the various extraction procedures categorized based on the temperatures during extraction. Table 10.1 describes the various techniques used for isolation of small molecules from their natural sources. The factors that enhance the diffusivity and solubility of small molecules, facilitates, the extraction. Extraction efficiency also depends on various other factors such as the properties of solvent used for extraction, starting material, small molecule size, the ratio of solvent-to-solid, extraction temperature and duration. The crucial step in solvent extraction is the selection of the solvent. This is done by considering the solubility, selectivity, cost, and safety of solvents. Based on the universal law of “like substances dissolves like,” polar solvents dissolve polar solutes and vice versa. Alcohols such as ethanol and methanol are universal solvents for phytochemical investigation. Better results of extraction are achieved when the particle size is fine, due to deep penetration of solvents and solute diffusion. Very fine particles result in difficulty in filtration due to solute absorption in the solid. Solubility and diffusion increase with increase in temperature but high temperature results in solvent loss due to vaporization leading to impure extracts and

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Condition

Type of method

Description

Advantages

Disadvantages

Low or room temperature methods of extraction

a. Cold extraction method

Dried, cut, crushed, or milled plant samples are soaked in solvents with shaking followed by filtration using a whatman filter paper drying of residue obtained under vacuum at room temperature and the determination of yield by calculating the difference in weight. e.g., Maceration [19] Selected enzymes such as protease, lipase, and phospholipase are used under favorable conditions, e.g. Pectinase and a-amylase are employed for extraction of essential oils [20] Fresh, wet, or dried plant parts are ground, soaked in solvents and then vigorously shaken for 5e10 min or soaked

Simple, environmentalfriendly, cost effective, and can be performed in the field [18]

It may not extract all compounds from the plant material

The method is nondegrading

The setup employed is costly. Method is too demanding in terms of nutrients required, oxygen presence, and temperature optimization

Efficient method

Mostly, the process requires a laboratory set up, and hence cannot be easily done in the field

b. Enzyme-assisted extraction (EAE)

c. Plant tissue homogenization

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Table 10.1 Various techniques used for isolation of small molecules from their natural sources.

d. Ionic liquid extraction

a. Decoction

Very high yields of extraction for organic and inorganic ligands. High quality and efficiency of extraction because of ionic interactions

Ionic liquids are costly compared to organic solvents

Cost effective, extracts more oil-soluble compounds than infusion and maceration due to high temperatures, can be used in a field or homestead set up [23, 24]

Only water soluble and thermally stable compounds can be extracted

Continued

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High temperature extraction thermally stable compounds are extracted at high temperatures [25]

for additional 24 h with shaking followed by filtration, centrifuged, and then dried or may be directly dried under vacuum and if required then redissolved in the solvent [21] Organic salts in liquid form are employed for selective interaction with polar and nonpolar compounds by hydrogen bonding, p-stacking interactions, ion exchange, and hydrophobic interactions [22] Plant parts are boiled in water, cooled, strained, and then diluted with cold water if required

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Type of method

Description

Advantages

Disadvantages

b. Soxhlet extraction

Employed when compounds of interest have limited solubility in a solvent and impurities are insoluble

One pass of hot solvent is sufficient because of solvent recycling [25]

c. Microwave assisted extraction

Sample and solvent is heated using microwave energy due to which cell walls rapture and exudates active compounds [26] Employs universal extraction systems e.g., BUCHI systems with thimbles containing test samples in solvents suspended in glass jars with temperature set just below boiling point of that solvent. After extraction, the extract is filtered and concentrated under reduced pressure [26]

Less time consuming than other conventional methods

Cannot extract thermolabile compounds due to high temperature degradation. This extraction requires a laboratory set up with soxhlet apparatus Not suitable for thermally unstable compounds. Possibility of generation of free radicals

d. Automated solvent digester extraction method

Fast and consumes less solvent

Process requires a laboratory set up, and hence cannot be easily done in the field

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Table 10.1 Various techniques used for isolation of small molecules from their natural sources.dcont’d Condition

e. Pressurized liquid extraction (PLE)

Optional temperature extraction: The methods can be used, depending on the nature of the components of interest in the samples

a. Serial exhaustive extraction

This method is ecofriendly, suitable for thermally stable compounds, and prevents degradation of oxygen and photo sensitive compounds

This method is expensive

Used for extraction of wide polarity range of compounds

Not used for thermolabile compounds [25]

Simple, easy, and fast process

High temperature makes this method not useful for thermolabile compounds Continued

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b. Infusion and digestion

Samples are put in a sample holder with solvent in an apparatus and heated upto 200
C and pressure between 35 and 200 bars resulting in low viscosity of solvents and maintaining it in liquid phase [20] Series of solvents are used for extraction from the least polar, e.g., n-hexane to the most polar, e.g., methanol for complete extraction of compounds with wide range of polarities. It can be performed at higher temperature (e.g., Soxhlet) or room temperature Useful for readily available components of the drug in crude form, e.g., tea in tea bags. Infusions are prepared freshly by

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Type of method

c. Supercritical fluid extraction method

d. Sonication

Description

maceration of the ready drug material in hot or cold water. Digestion is closely related to infusion, in which a steady gentle heat is applied during the extraction process to increase the extraction efficiency [25] Liquefied gases like CO2 is pumped through a cylindrical channel containing the sample material at around 32
C or higher and 74 bars [20]. In separating chamber, the gases are recovered for reuse and there is separation of extract from the solvent. Used in the extraction of essential oils

Advantages

Disadvantages

Supercritical fluids are non-toxic, easy to recover and nonflammable. No traces of solvent remain in the extract. At lower temperatures, high yields of thermal labile compounds like terpenes and terpenoids (B.P w150
C) are obtained

It requires an expensive set up

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Table 10.1 Various techniques used for isolation of small molecules from their natural sources.dcont’d

Ultrasound waves (20 e2000 KHz) penetrates the sample materials. Mostly performed at higher temperature [27], thereby increasing cell wall permeability. Based on viscosity, polarity, surface tension and vapor pressure, solvent is chosen which influences the cavitation phenomena [20]

This method effectively extracts components

High cost of installation and operation, alteration of some active compounds and the generation of free radicals in the extract leading to unreliable result [28]

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decomposition. There is increase in the efficiency of extraction with the longer extraction time until a certain time. After the equilibrium is reached, extraction efficiency will not depend on time. The extraction yield will be higher when the solvent-to-solid ratio is large. However, too high solvent-to-solid ratio will result into excess solvent and hence more time for concentration. Large volume of organic solvents and more extraction time is required in case of conventional extraction methods such as percolation, and reflux extraction. Greener extraction methods include the pressurized liquid extraction (PLE), microwave-assisted extraction (MAE), and super critical fluid extraction (SFC) can also be used since it consumes lower organic solvent and extraction time is shorter with high selectivity [29]. Advanced extraction methods of vinblastine (antineoplastic drug) were obtained using SeCO2 that was treated with 2% ethanol at a pressure of 300 bar and at 40
C from the source organism Catharanthus roseus. This method resulted in 92% more efficiency than traditional extraction methods. 1.2.2 Isolation of biologics There are three common procedures for isolating biologics from its natural source or crude extracts. (a) Salting out: The process of salting out involves co-precipitation with ammonium sulfate as the saturation concentration allows precipitation of most of the proteins due to high molarity [30]. Also, the method provides an advantage of retaining the native structure of proteins as salting out lacks a large heat of solution causing rapid heat dissipation and saturated solution of 4.04 M concentration as a temperature of 20
C with a density of 1.235 g/cm3 limit the interference with precipitated proteins. Such conditions so not interfere with the precipitated proteins obtained after a brief centrifugation step. The concentrated proteins are protected from denaturation in the solution state and are rather bacteriostatic [31]. (b) Isoionic precipitation Column method: The isoionic state of a protein is a salt-free state where the proteins are least soluble easily precipitated, compact, and in a least hydrated conformation. The method of column separation of proteins is favorable only when the proteins are soluble at their isoionic points. By using a deionization column or a dialysis process, the proteins are rendered isoionic to easily precipitate them. The main factors influencing the precipitation is the pH of the solution with respect to the proteins’ pI (Isoelectric point) and a low (0e0.1/0.2 M) salt concentration [31]. An option of using a mixed bed resin deionization is also viable besides adjusting the pH of the proteins so as to reach the isotonic point pH. The outcome of this method yields salts free proteins. Inorganic salts have a major effect on protein solubility even if present in minimum concentrations [32]. (c) Dialysis method: A proven effective method to yield salt free proteins is dialysis. The drawbacks of the conventional dialysis methods are swelling of the dialysis bag when the salt diffuses outwards, due to increase in osmotic effect and although deionization could be achieved by dialysis against a suitable buffer, the isoionic point is usually uncertain. To counter

Small molecules vs biologics

these drawbacks, the resin deionization method (also known as the Dinitz method) is used where in the protein is automatically adjusted to its isoionic pH and the salt is diffused outside the membrane resin in which the protein is trapped by the process of counterion exchange [32]. Once the exchange is completed, a brief centrifugation step ensures the recovery of the precipitated protein from the dialysis tubing. The only drawback of this method as against the column method of isolation of proteins is that dialysis takes more time. The proteins that are insoluble at their respective isoionic point is retained inside the dialysis bag as a precipitate whereas outside the resin exchanger bags, salts are forced out allowing the proteins to reach their isoionic pH [31]. 1.2.3 Purification techniques of small molecules and biologics Chromatography is one of the important separation techniques which is useful for the separation/purification of the small molecules in a crude mixture for their further analysis. The type of chromatographic techniques which are usually used based on characteristics of molecules and type of interaction are namely ion exchange chromatography, surface adsorption chromatography, partition chromatography, and size exclusion chromatography. Other chromatographic techniques are based on the type of stationary bed used such as column chromatography, thin layer chromatography, and paper chromatography. So, according to the principle of chromatography, molecules present in a mixture gets separated from other molecules when spotted onto the surface or applied into the solid and fluid stationary phase (stable phase) while they move with the help of a mobile phase. The efficiency of separation depends on characteristics of molecule which deals with adsorption, partition, and differences in their molar mass or affinity [33,34]. These differences result in longer stay of some molecules present in the mixture on the stationary phase and hence their slow movement in the system used in chromatographic separation, while rapid movement of other molecules with the mobile phase leading to separation [35]. In order to separate and identify small molecules such as amino acids, fatty acids, and carbohydrates, partition chromatography is very useful. However, effective separation of large molecules such as nucleic acids and proteins can be achieved by affinity chromatography (i.e., ion-exchange chromatography). Chromatography is not only a separation technique but also a method of quantitative analysis. Various chromatographic methods that have been developed are simple column chromatography, gel-permeation (molecular sieve) chromatography, ion-exchange chromatography, affinity chromatography, paper chromatography, thin-layer chromatography, liquid chromatography, gas chromatography, hydrophobic interaction chromatography, dye-ligand chromatography, pseudo affinity chromatography, capillary electrophoresis (CE), and high-performance liquid chromatography (HPLC) [36]. The HPLC analysis of small molecules is mostly performed in reversed-phase separation mode due to their polarity [37]. [a] Sodium dodecyl sulfate (SDS) gel electrophoresis: The method involves the separation of charged proteins as per their physical properties as they undergo sieving

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through the gel under the influence of an electrical current. The proteins denature due to the dual action of sodium dodecyl sulfate and b mercaptoethanol. Additionally, the SDS detergent imparts a negative charge to the protein, and because the SDS-coated proteins demonstrate consistent charge to mass ratio, the proteins are sorted according to their sizes [38]. [b] One-dimensional gel electrophoresis: The PAGE (polyacrylamide gel electrophoresis) allows the proteins to undergo separation via a molecular sieve due to the external electric field through the pores of the polyacrylamide gel. The pore size of the acrylamide gel decreases as the concentration of the gel matrix increases. The combination of the protein size, charge, shape, and the pore size determine the rate of migration of the protein in one dimension. This modification of the conventional electrophoresis method provides information not only about the molecular size of the protein but also regarding the number and size of subunits contained in the protein of interest [39]. [c] Two-dimensional gel electrophoresis: The electrophoresis involving the separation of proteins based on two forces is the two-dimensional gel electrophoresis. The first dimension involves the separation of proteins by the process of isoelectric focusing and in the second dimension, the protein is lysed by the detergent activity of SDS. This approach is considerably enhanced for resolving complicated protein mixtures and for determining the protein purity because these competing pressures cause the proteins to separate and offer information about the protein size, shape, and charge [40]. 1.3 Techniques for the characterization of small molecules and biologics Identification of the small molecules after their separation and purification process involves the use of various spectroscopic techniques in order to characterize. Each of these techniques provides vital information which is then linked with each other and with the data obtained by the other necessary techniques. The spectral data interpretation and data finding/comparing with the small molecules which are already identified and known by their spectras in spectral libraries results in identification of unknown compounds. Some of the public chemical compound repositories which can be used for this purpose are ChemSpider [41], PubChem [42], and Human Metabolome Database (HMDB); DrugBank [43]; KEGG [64]; MZedDB [44]; and ChEBI [65]. This is a web-based search of small molecules by their molecular formulae and masses in which biological activities of small molecules are already known. Following are some of the techniques used in identification of small molecules: (a) Fourier-transform infrared spectroscopy (FTIR): This spectroscopic technique is used to identify different functional groups present in the small molecule; (b) Nuclear Magnetic Resonance Spectroscopy (NMR): Valuable technique which uses magnetic fields for the identification of structural frameworks. 1H-NMR detects type and positions of protons present in the structure and the types of isotope present is determined by C-NMR; (c) Mass spectrometry (MS): Useful

Small molecules vs biologics

technique in determination of elements in natural crude extracts and other small molecules. According to stages of advancement, there are four types of mass spectroscopy. As per [45], versatile instrument is ion trap MS, whereas for general purpose it is quadruple MS. A very sensitive technique is triple quadruple MS and is used only for targeted samples. Fourier transform instruments are used nowadays for very high resolution and mass accuracy which is necessary for identification of structure. This technique gives the information pertaining to the mass of the parent small molecule as well as masses of various fragments obtained from it [46]. One of the key techniques used during the recent years for the identification of small molecules is liquid chromatography coupled with mass spectrometry (LC-MS) [47]. 1.3.1 Biologics 1.3.1.1 Reversed-phase high-performance liquid chromatography (RP-HPLC) Reverse phase-high performance liquid chromatography is a technique used for isolation of biologics such as proteins, peptides, and the molecules separate from the mobile phase to the stationary phase containing the hydrophobic immobilized ligands. The stationary phase is made of n-alkyl silica and acetonitrile comprising of trifluoroacetate (TFA) owing to its ion modifying ability, due to which it is used as an RP-HPLC gradient. Biologics such as small polypeptides and globular proteins of molecular weight 10,000, peptides, are separated and purified by RP-HPLC. Whereas for the separation of proteins at a larger scale RP-HPLC holds very limited potential as the acidic buffering action combined with the hydrophobicity posed by the n-alkyl silica leads to lesser yields and/or loss of biological functioning of the large polypeptides [48]. 1.3.1.2 Electrospray (ES) and matrix-assisted laser desorption ionization (MALDI) Electrospray (ES) and matrix-assisted laser desorption ionization (MALDI) procedures are basically employed to purify the polar and large biomolecules which are otherwise are not readily ionized. This technique is used for the purification of recombinant proteins, and study of post-translational modifications of proteins or for detection of the protein’s molecular weight. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an established technique used to characterize proteins of 2000e30,000 Da. 1.3.1.3 Liquid chromatographyemass spectrometry (LC-MS) LC-MS has been used widely for metabolomic studies due to its soft ionization, high throughput, and good coverage of metabolites. This method combines the physical separation of a mixture of proteins by liquid chromatography and analysis the mass of the proteins via mass spectrometry. Liquid samples can be easily prepared for mass spectrometry, as peptides exhibit a greater solubility as compared to proteins. In the solution, proteins are subjected to endoprotease digestion and passed across the high-pressure liquid

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chromatography column. The solution is sprayed out via a narrow nozzle located at the end of this positively charged column and is introduced inside a mass spectrometer. The charge on the sample droplets leads to the fragmentation till the single ions exist. The peptides are then broken down and the charge to mass ration is measured. The process is again repeated with another separate enzyme, and the overlapping patterns that are generated are used to construct a protein sequence [31,49]. 1.3.1.4 Tandem mass spectrometry of peptides and proteins The identification of proteins by MS have emerged substantially over the past decades and the method of peptide mass fingerprinting gained importance. The method involves the enzymatic digestion of protein following the measurement of the resulting peptide fragments with a MALDI-TOFF MS. The data generated after measuring the protein is then matched with the database of proteins that was generated in silico. A modification of this method is the shotgun proteomics that involves the enzymatic digestion, subsequent chromatographic separation and finally subjecting to tandem mass spectrometry of an entire proteome. An advantage of this modification is that thousands of proteins can be analyzed from any cell lysate [66]. The application of two separate phases: LCeMS and LCeMS/MS, is employed for protein identification. LCeMS offers superior-quality data for the peptide mass as compared to MALDI mass spectrometry [50,51]. 1.4 Mechanism of action of biologics and small molecule Due to the smaller molecular mass, small molecule drugs can penetrate easily into the cell. After entering the cell, they interact differently with various proteins. Receptors and enzymes are the most for small molecule drug target. Receptors act like sentinels for the cell [52] and convey the messages from the outside environment to the internal cells to pass the information of what to produce or what not to. The catalysts inside the cells are enzymes and their production should be sufficient to support life. Hence, the small molecule drugs trigger the important molecular pathways inside the cells that regulate the vital functions and hampers the unnecessary molecular pathways [53]. Overall, small molecule drugs prevent the occurrence of a disease and help in relieving the other symptoms of diseases. A limitations of small molecule drugs is the lack of regenerative ability. Hence, they are unable to restore the regular functions of cells, tissues, and organs [54]. Aspirin is a common example of an over-the-counter medication small molecule drug. It was first approved by FDA in 1965 as a pain reliever (analgesics) drug. Aspirin targets enzymes known as cyclooxygenase enzyme 1 (COX1) and cyclooxygenase enzyme 2 (COX2) after entering in the body. Basically, the pain and inflammation is caused by prostaglandins synthesized due to these enzymes. However, cyclooxygenase enzyme pathway is suppressed by Aspirin by targeting COX1 and COX2 enzymes which results in inhibition of production of prostaglandins which relieves pain [55].

Small molecules vs biologics

Biologics have a much higher specificity to drug targets as compared to small molecules. The biologics have characteristic shapes that allow ergonomic interactions with ligand gated ion channels, G protein coupled receptors and other signaling receptors. Small compounds drugs, on the other hand, may distort or switch off target specific binding due to their lack of specificity in comparison to biologics [56]. The larger biologics such as the monoclonal antibodies and antibody drug conjugates exhibit more promising ADME characteristics as compared to the small molecules. This is so because, the larger biologics have stronger affinity, better excretion from the body [10]. Vedolizumab is a biologics drug used to cure severe ulcerative colitis and Crohn’s disease. The pathway of this biologic proceeds by binding to the a4b7 integrin protein that is specifically expressed by the gastrointestinal T lymphoctes. Once the binding between the monoclonal antibody and the integrin is initiated, the mucosal adhesion molecule-1 is then expressed onto the mucosal endothelial surface cells present in the gut which attract the lymphocytes for further action against the antigens. Vedolizumab’s preference in the therapeutic process over other monoclonal antibodies is mostly due to its selectivity [57]. 1.4.1 Pharmacodynamic of small molecules and biologics Small molecule drugs and biologics interact in different way with their targets resulting in different effect on the human body. Biologics are more specific in comparison with the small molecule drugs. This property of specificity (or lack thereof) impacts the safety aspect. Small molecules are constructed in such way that it easily binds with targets like ligand-gated ion channels, receptor tyrosine kinases and G protein-coupled receptors either extracellularly or intracellularly interspersed throughout the body. Also, small molecules through physical interaction can interfere with the biological processes. There is possibility of small molecules to induce off-target effects since they lack the property of specificity as possessed by the biologics. This means that there is unwanted interaction of the small molecule drugs with cells, cellular components, and tissues. Sometimes, these interactions may be harmless, but frequently, they may lead to adverse side effects. Contrastingly, biologics exhibit high specificity while binding with the target molecules by either binding to the cell surface or intracellularly. One advantage biologic has over small molecules is that the chemical moieties of the therapeutic proteins may not cause side effects by themselves but pose some risk, as they can hinder certain vital functions of the body resulting in life threatening and rather rare events that shows a direct relation to cytokine release syndrome, immunogenicity, encephalopathy, and more. 1.4.2 Physiochemical properties of small molecules and biologics Pharmacokinetics, which studies how drugs work in the body, comprises information on how molecules are taken up, dispersed (including by transport), metabolized and excreted (ADME). After the drug is taken inside the body by any route, its efficiency

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is based on its interactions with tissues, cells, various enzymes, other proteins, etc. Some drugs are not absorbed into the systemic circulation and act locally while its necessary for absorption of most drugs into the blood for their pharmacological effects. After the absorption of drug molecules into the blood, drug concentration can be determined via blood sampling at regular intervals of time after administration. Absorption is followed by distribution of these drug molecules in various tissues and organs by the process of systemic circulation. Here, they interact with the components of cell, are metabolized and finally eliminated. Another less well-known circulatory system which is equally important but found to be more “sluggish” is lymphatic system. This circulatory system (Approximately 100e500 times slower than blood) is important in case of drugs having higher molecular weights (i.e., >10 kDa) and also for small molecules which are driven by association with macromolecular carriers (e.g., liposomes, nanoparticles, polymers) to the lymphatic system or the molecules that are connected with endogenous macromolecules (e.g., proteins and lipoproteins) once they enter inside the body. Biologics may circulate in the human body via blood or lymphatic systems and may even transcellularly migrate by connective transportation, pinocytosis, phagocytosis, and receptor mediated endocytosis. The ADME patterns displayed by the biologics are more pronounced as compared to that of small molecules due to several reasons, some of which are limited distribution volume, longer periods to reach peak concentration, and longer half-life of the larger biologics. 1.5 Pharmacokinetics of small molecules vs biologics Pharmacokinetics, which studies how drugs work in the body, comprises information on how molecules are taken up, dispersed (including by transport), metabolized and excreted (ADME). After the drug is taken inside the body by any route, its efficiency is based on its interactions with tissues, cells, various enzymes, other proteins etc. Some drugs are not absorbed into the systemic circulation and act locally while it’s necessary for absorption of most drugs into the blood for their pharmacological effects. After the absorption of drug molecules into the blood, drug concentration can be determined via blood sampling at regular intervals of time after administration. Absorption is followed by distribution of these drug molecules in various tissues and organs by the process of systemic circulation. Here, they interact with the components of cell, are metabolized, and finally eliminated. Another less well-known circulatory system which is equally important but found to be more “sluggish” is lymphatic system. This circulatory system is crucial in case of higher molecular weight drugs of less than 10 kDa and small molecules driven by the attachment with macromolecular carriers (e.g., liposomes, nanoparticles, and polymers) to the lymphatic system or the molecules that are connected with the macromolecules that are endogenous (e.g., proteins and lipoproteins) once they enter inside the body.

Small molecules vs biologics

Larger biologics have a dual mode of distribution, i.e., the blood and lymphatic systems. The movement of the complex biologics is transcellularly by means of the convective transport, endocytosis, phagocytosis, and pinocytosis. Apparently, the ADME characters displayed by the larger biologics is more distinct as compared to the small molecule drugs. Some of the distinction parameters include a longer shelf life, more time to reach peak concentration and limited distribution volumes. The binding efficiency of biologics has a direct relation with the receptor interaction, more popularly known as “target mediated drug disposition.” The therapeutic proteins are broken down to the constituent amino acids mostly by the action of protease and/or peptidase akin to the endogenous substrates. The broken down amino acids are recycled, and the remaining amino acids are either secreted into the bile juice or filtered out of the human body via the urine or feces for their elimination. In contrast, the small molecules are removed out of the body by the cytochrome p450, kidneys by the non-targeted organs. 1.5.1 Differences between small molecules and biologics The major differences between SMs and biologics are described in Table 10.2. As compared to small molecule, biologics exhibit high specificity toward the target molecules and the receptor -drug interactions are survived by the action of peptidase and protease [58]. Biologics have low toxicity since they are obtained from the living organisms. However, the immunogenicity may influence the safety and efficacy aspects of biologics. In 2015, Stratton et al. reported a significant rise in the approval of biologics-based drug for a period of over the last 30 years. The highlights of which included the monoclonal antibodies and vaccines vis-a-vis a persistent decline in the small-molecule drug counterpart. The disadvantages of biologics are because of their characteristics, mainly the complex and large nature of biologics which need high skill to analyze and generate extensive

Table 10.2 Major differences between biologics and small molecules [9,10]. Small molecules

Biologics

Entirely chemical in nature Obtained by chemical processes Completely characterizable Excretion may be renal and biliary Stable Half life is short (usually 24 h) Non-immunogenic Low molecular weight Well defined structure Completely as per ADME Dose intervals are daily

Protein or conjugate þ protein in nature Obtained by living cell cultures Not entirely characterizable Excretion is mostly by recycling by body Unstable Half-life is long (usually weeks) Immunogenic High molecular weight Complex, heterogeneous structure ADME processes are still evolving Dose intervals are intermittent

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data. Biologics need to be handled in specialized manner and exhibit lesser stability than the small molecule-based drugs. In addition, the biologics require further attention with respect to maintenance of an optimum temperature, light, and prevention of jostling. In terms of drug delivery, the biologics have a longer half-life but reach the peak concentration at a relatively slower pace as compared to the small molecules. 1.5.2 Economical sustainability of biologics and small molecule drugs There are two important criteria for any drug to be a pharmaceutical success viz. accessibility and the cost of the drug. The economics of any drug, be it small molecules or biologics, determines the saleability of the drug worldwide and may also be a limiting factor in deciding to choose an alternative by the consumers. Although biologics have been considered to be superior in matters of efficacy, they still have economic drawbacks. The new and novel treatment solution strategies involve high costs especially for biologics-based treatment. For example, the DNA barcoding technology is an expensive treatment option, due to the heavy cost of the high throughput screening which requires few hundred billions of dollars. Such massive financial burden cannot be borne by startups and small-scale pharmaceuticals [59]. The small molecules on the other hand pose economic advantages over biologics as the former have been designed with cost cutting measures as the generic pharmaceutical business developed. Competition with the giant pharmaceutical companies all over the world has further decreased the costs of small molecules drugs due to the increase in sales and use of the generic drugs by consumers. Biologics being complex molecules are more expensive in terms of production and in contrast to the small molecules, the biologics-based drugs do not have alternate versions, such as bio-similars. It is estimated that the production of biologic based drugs is approximately 12 times more than that of small molecules [60]. Additionally, the constituents used for the preparation of biologics are more complex as they comprised protein/enzymes, small peptides from microbes, plant or animal cells, where modifications involve tedious experiments and the manufacturing process may be sensitive to any minor changes. On the other hand, more stable small molecules and the manufacturing process can be subjected to changes whenever needed [61]. Furthermore, there may be a non-disclosure clause in patients who opt for biologicsbased treatment which results in a substantial decrease in competition of other drug manufacturing companies and eventually results in the increased cost of the biologicsbased drugs. Also, the tailor-made biologics cannot be mass produced in the form of a tablet or pill, unlike the small molecules [62]. The biologics-based drugs need to be administered directly to the patient leading to the limitation in the commercial viability of biologics in comparison to small molecule-based drugs.

Acknowledgment The authors would like to thank Professor Vrinda Borker for her support and guidance in writing the article. The author SNM sincerely acknowledges the financial support from University Grant Commission (UGC), India through Dr. D.S. Kothari Postdoctoral Fellowship (No.F.4e2/2006/BSR/BL/18e19/0416).

Small molecules vs biologics

References [1] Buvailo A. Will biologics surpass small molecules in the pharma race. 2018. BiopharmaTrend.com. https://www.biopharmatrend.com/post/67-will-small-molecules-sustain-pharmaceutical-racewith-biologics. [2] Di L. Strategic approaches to optimizing peptide ADME properties. AAPS J 2015;17(1):134e43. [3] Durbin KR, Nottoli MS, Jenkins GJ. Effects of microtubule-inhibiting small molecule and antibodydrug conjugate treatment on differentially sized A431 squamous carcinoma spheroids. Sci Rep 2020; 10(1):907. [4] Cheng J, Moore S, Gomez-Galeno J, Lee DH, Okolotowicz KJ, Cashman JR. A novel small molecule inhibits tumor growth and synergizes effects of enzalutamide on prostate cancer. J Pharmacol Exp Therapeut 2019;371(3):703e12. [5] Al-Kuraishy HM, Al-Gareeb AI, Alzahrani KJ, Alexiou A, Batiha GE. Niclosamide for Covid-19: bridging the gap. Mol Biol Rep 2021;48(12):2021e8195. [6] Makurvet FD. Biologics vs. small molecules: drug costs and patient access. Med Drug Discov 2021;9: 100075. [7] Felquer ML, LoGiudice L, Galimberti ML, Rosa J, Mazzuoccolo L, Soriano ER. Treating the skin with biologics in patients with psoriasis decreases the incidence of psoriatic arthritis. Ann Rheum Dis 2022;81(1):74e9. [8] Wierzbowska-Drabik K, Lesiak A, Skibi nska M, Niedzwiedz M, Kasprzak JD, Narbutt J. Psoriasis and atherosclerosisdskin, joints, and cardiovascular story of two plaques in relation to the treatment with biologics. Int J Mol Sci 2021;22(19):10402. [9] Declerck PJ. Biologicals and biosimilars: a review of the science and its implications. GaBI J 2012;1(1): 13e6. [10] Wan H. An overall comparison of small molecules and large biologics in ADME testing. ADMET DMPK 2016;4(1):1e22. [11] Bhaskar R, Xavier LS, Udayakumaran G, Kumar DS, Venkatesh R, Nagella P. Biotic elicitors: a boon for the in-vitro production of plant secondary metabolites. Plant Cell Tissue Organ Cult 2021:1e8. [12] Davy AM, Kildegaard HF, Andersen MR. Cell factory engineering. Cell Syst 2017;4(3):262e75. [13] Qi R, Pfeifer BA, Zhang G. Engineering heterologous production of salicylate glucoside and glycosylated variants. Front Microbiol 2018;9:2241. [14] Chiba CH, Knirsch MC, Azzoni AR, Moreira AR, Stephano MA. Cell-free protein synthesis: advances on production process for biopharmaceuticals and immunobiological products. Biotechniques 2021;70(2):126e33. [15] Guzman-Chavez F, Zwahlen RD, Bovenberg RA, Driessen AJ. Engineering of the filamentous fungus Penicillium chrysogenum as cell factory for natural products. Front Microbiol 2018;9:2768. [16] Wang C, Du W, Lu H, Lan J, Liang K, Cao S. A Review: halogenated compounds from marine actinomycetes. Molecules 2021;26(9):2754. [17] Rasul MG. Extraction, isolation and characterization of natural products from medicinal plants. Int J Basic Sci Appl Comput 2018;2(6). [18] Dhanani T, Shah S, Gajbhiye NA, Kumar S. Effect of extraction methods on yield, phytochemical constituents and antioxidant activity of Withania somnifera. Arab J Chem 2017;10:S1193e9. [19] Ncube NS, Afolayan AJ, Okoh AI. Assessment techniques of antimicrobial properties of natural compounds of plant origin: current methods and future trends. Afr J Biotechnol 2008;7(12). [20] De Monte C, Carradori S, Granese A, Di Pierro GB, Leonardo C, De Nunzio C. Modern extraction techniques and their impact on the pharmacological profile of Serenoa repens extracts for the treatment of lower urinary tract symptoms. BMC Urol 2014;14(1):63. [21] Das K, Tiwari RK, Shrivastava DK. Techniques for evaluation of medicinal plant products as antimicrobial agent: current methods and future trends. J Med Plants Res 2010;4(2):104e11. [22] Tang B, Bi W, Tian M, Row KH. Application of ionic liquid for extraction and separation of bioactive compounds from plants. J Chromatogr B 2012;904:1e21. [23] Azwanida NN. A review on the extraction methods use in medicinal plants, principle, strength and limitation. Med Aromatic Plants 2015;4(196):0412e2167.

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[24] Mtewa AG. Antibacterial potency stability, pH and phytochemistry of some Malawian ready-to-serve aqueous herbal formulations used against enteric diseases. Int J Herbal Med 2017;5(3):01e5. [25] Banu KS, Cathrine L. General techniques involved in phytochemical analysis. Int J Adv Res Comput Sci 2015;2(4):25e32. [26] Ingle KP, Deshmukh AG, Padole DA, Dudhare MS, Moharil MP, Khelurkar VC. Phytochemicals: extraction methods, identification and detection of bioactive compounds from plant extracts. J Pharmacogn Phytochem 2017;6(1):32e6. [27] Salehan NA, Sulaiman AZ, Ajit A. Effect of temperature and sonication on the extraction of gallic acid from Labisia Pumila (Kacip Fatimah). ARPN J Eng Appl Sci 2016;11(4):2193e8. [28] Handa SS. An overview of extraction techniques for medicinal and aromatic plants. Extra Technol Med Aromatic Plants 2008;1:21e40. [29] Feingold KR. Cholesterol lowering drugs. Endotext (Internet) March 30, 2021. [30] Balasundaram B, Sachdeva S, Bracewell DG. Dual salt precipitation for the recovery of a recombinant protein from Escherichia coli. Biotechnol Prog 2011;27(5):1306e14. [31] Nehete JY, Bhambar RS, Narkhede MR, Gawali SR. Natural proteins: sources, isolation, characterization and applications. Phcog Rev 2013;7(14):107. [32] Matulis D. Selective precipitation of proteins. Curr Protocols Protein Sci 2016;83(1):4e5. [33] Cuatrecasas P, Wilchek M, Anfinsen CB. Selective enzyme purification by affinity chromatography. Proc Natl Acad Sci USA 1968;61(2):636e43. [34] Porath J. From gel filtration to adsorptive size exclusion. J Protein Chem 1997;16(5):463e8. [35] Harris DC. Exploring chemical analysis. Macmillan; 2004. [36] Harwood LM, Moody CJ. Experimental organic chemistry. Blackwell Scientific; 1989. [37] Wang QC, Svec F, Frechet JM. Reversed-phase chromatography of small molecules and peptides on a continuous rod of macroporous poly (styrene-co-divinylbenzene). J Chromatogr A 1994;669(1e2): 230e5. [38] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(5259):680e5. [39] Brunelle JL, Green R. One-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-PAGE). Methods Enzymol 2014;541:151e9. Academic Press. [40] Rabilloud T, Chevallet M, Luche S, Lelong C. Two-dimensional gel electrophoresis in proteomics: past, present and future. J Proteonomics 2010;73(11):2064e77. [41] Williams AJ. A perspective of publicly accessible/open-access chemistry databases. Drug Discov Today 2008;13(11e12):495e501. [42] Wang Y, Xiao J, Suzek TO, Zhang J, Wang J, Bryant SH. PubChem: a public information system for analyzing bioactivities of small molecules. Nucleic Acids Res 2009;37(Suppl. l_2):W623e33. [43] Wishart DS, Knox C, Guo AC, Eisner R, Young N, Gautam B, et al. HMDB: a knowledgebase for the human metabolome. Nucleic Acids Res 2009;37(Suppl. l_1):D603e10. [44] Draper J, Enot DP, Parker D, Beckmann M, Snowdon S, Lin W, et al. Metabolite signal identification in accurate mass metabolomics data with MZedDB, an interactive m/z annotation tool utilising predicted ionisation behaviour ‘rules. BMC Bioinf 2009;10(1):1e6. [45] Wang HY, Jackson SN, Woods AS. Direct MALDI-MS analysis of cardiolipin from rat organs sections. J Am Soc Mass Spectrom 2007;18(3):567e77. [46] Kind T, Fiehn O. Advances in structure elucidation of small molecules using mass spectrometry. Bioanal Rev 2010;2(1):23e60. [47] Pitt JJ. Principles and applications of liquid chromatography-mass spectrometry in clinical biochemi stry. Clin Biochem Rev 2009;30(1):19. [48] Aguilar MI, Hearn MT. [1] High-resolution reversed-phase high-performance liquid chromatography of peptides and proteins. Methods Enzymol 1996;270:3e26. Academic Press. [49] Zhou B, Xiao JF, Tuli L, Ressom HW. LC-MS-based metabolomics. Mol Biosyst 2012;8(2):470e81. [50] Elzoghby AO, El-Fotoh WS, Elgindy NA. Casein-based formulations as promising controlled release drug delivery systems. J Contr Release 2011;153(3):206e16. [51] Elzoghby AO, Samy WM, Elgindy NA. Albumin-based nanoparticles as potential controlled release drug delivery systems. J Contr Release 2012;157(2):168e82.

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[52] Li Y, Chen J, Bolinger AA, Chen H, Liu Z, Cong Y, et al. Target-based small molecule drug discovery towards novel therapeutics for inflammatory bowel diseases. Inflamm Bowel Dis 2021;27(Suppl_2): S38e62. [53] Pinilla-Vera M, Hahn VS, Kass DA. Leveraging signaling pathways to treat heart failure with reduced ejection fraction: past, present, and future. Circ Res 2019;124(11):1618e32. [54] Sun G, Rong D, Li Z, Sun G, Wu F, Li X, et al. Role of small molecule targeted compounds in cancer: progress, opportunities, and challenges. Front Cell Dev Biol 2021;9:694363. [55] Zacharias-Millward N, Menter DG, Davis JS, Lichtenberger L, Hawke D, Hawk E, et al. Beyond COX-1: the effects of aspirin on platelet biology and potential mechanisms of chemoprevention. Cancer Metastasis Rev 2017;36(2):289e303. [56] Stater EP, Sonay AY, Hart C, Grimm J. The ancillary effects of nanoparticles and their implications for nanomedicine. Nat Nanotechnol 2021;16(11):1180e94. [57] Wyant T, Fedyk E, Abhyankar B. An overview of the mechanism of action of the monoclonal antibody vedolizumab. J Crohn’s Colitis 2016;10(12):1437e44. [58] Gul L, Modos D, Fonseca S, Madgwick M, Thomas JP, Sudhakar P, et al. Extracellular vesicles produced by the human commensal gut bacterium Bacteroides thetaiotaomicron affect host immune pathways in a cell-type specific manner that are altered in inflammatory bowel disease. J Extracell Vesicles 2022;11(1):e12189. [59] Sunkari YK, Siripuram VK, Nguyen TL, Flajolet M. High-power screening (HPS) empowered by DNA-encoded libraries. Trends Pharmacol Sci 2021;43(1):4e15. [60] Frech HE, Pauly MV, Comanor WS, Martinez JR. Costs and benefits of branded drugs: insights from cost-effectiveness research. J Benefit-Cost Anal 2022;13(2):166e81. [61] Pelz L, Göbel S, Jaen K, Reichl U, Genzel Y. Upstream processing for viral vaccinesegeneral aspects. Bioprocess Viral Vaccines 2022:79e135. CRC Press. [62] Heled Y. The case for disclosure of biologics manufacturing information. J Law Med Ethics 2019; 47(S4):54e78. [63] Ando K, Fukuda Y, Tanaka A, Sagara H. Comparative efficacy and safety of tezepelumab and other biologics in patients with inadequately controlled asthma according to thresholds of type 2 inflammatory biomarkers: a systematic review and network meta-analysis. Cells 2022;11(5):819. [64] Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, et al. KEGG for linking genomes to life and the environment. Nucleic Acids Res 2007;36(Suppl. l_1):D480e4. [65] Degtyarenko K, De Matos P, Ennis M, Hastings J, Zbinden M, McNaught A, et al. ChEBI: a database and ontology for chemical entities of biological interest. Nucleic Acids Res 2007;36(Suppl. l_1): D344e50. [66] Coon JJ, Syka JE, Shabanowitz J, Hunt DF. Tandem mass spectrometry for peptide and protein sequence analysis. Biotechniques 2005;38(4):519e23.

Further reading [1] Aguilar MI. Methods in molecular biology, HPLC of peptides and proteins, methods and protocols. 2004.

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CHAPTER 11

Introduction to enzymes and organocatalysis G.D. Ametefe1, O.O. Ajani2, E.E.J. Iweala1 and S.N. Chinedu1 1

Department of Biochemistry, Covenant University, Ota, Ogun State, Nigeria; 2Department of Chemistry, Covenant University, Ota, Ogun State, Nigeria

1. Definition and classifications of enzymes Green chemistry has gained recognition as a concept and strategy for attaining sustainable development over the last few decades [1]. The ability of chemistry to support cuttingedge technologies that reduce the use and/or production of hazardous materials is known as “green chemistry”; ‘the 12 tenets of green chemistry’ were established by Anastas and Warner (Fig. 11.1) [2]. In order to make chemistry more environmentally friendly, catalysis particularly enzyme catalysis, heterogeneous catalysis, and organocatalysisdhas been identified as the key component [4]. This is because this field of study has been shown to lessen the negative effects that chemical processes have on the environment (Clark and Rhodes, 2000). Enzymes are simply biologically derived catalysts that speed up or accelerate chemical reactions; yet, not used up in the process [5]. A catalyst is a substance known to influence the rate of reaction [6]. Catalysts are so important that industrialization and the activities occurring in the living things highly depend on them. The Haber-Bosch process for the production of nitrogen would be impaired without a catalyst, leading to a significant effect on life on earth [7,8]. Hence, the role of catalysis cannot be overestimated. However, catalysts are much more a broader term with enzymes emphasizing biologically derived proteins [9]. Enzymes are proteins, and specific in their mode of action, with evidence in the reduction of the activation energy (Ea) yet, not undergoing a permanent change in the process [9e11]. They can accelerate chemical reactions many times more than the reaction would proceed without an enzyme. Although hemoglobin is a protein, it is not an enzyme because it transports oxygen, not merely catalyzing the reaction process. Some catalytic RNA molecules are referred to as ribozymes [12]. So, enzymes not only react with the substrate but forms an enzyme-substrate complex which induces the formation of product/products in the final state. That is: E þ S#ES / E þ P where E is the enzyme, S is the substrate, ES is the enzyme-substrate complex, and P is the product [9,14].

New Horizons in Natural Compound Research ISBN 978-0-443-15232-0, https://doi.org/10.1016/B978-0-443-15232-0.00018-7

© 2023 Elsevier Inc. All rights reserved.

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Figure 11.1 The green chemistry 12 principles [2,3].

The lowering of the activation energy in an enzyme-catalyzed reaction is the basis for the preference of enzymes in reaction processes, without the enzyme altered at the end of the reaction [5]. The chemical reactions that take place in nature have a significant role in the existence of living things. Although some naturally occurring reactions may proceed without the presence of enzymes, they are enhanced by the presence of enzymes, with proof in the lowering of the activation energy of the reaction process, which significantly contributes to the maintenance of life (Fig. 11.2) [11,15]. As a result, enzymes are described as biocatalysts that quicken reactions, showing that these essential compounds are not only present in humans but also in living things like bacteria which secret enzymes in the course of feeding [13].

Introduction to enzymes and organocatalysis

Enzyme absent

Activation energy

Enzyme present

F re e e n e rg y

S

P

Reaction coordinate

Figure 11.2 Enzyme effect in lowering of the activation energy of reactions [9,13].

It is evident in the symbiotic relationship’s humans share with microbes in the gut, where the microbes understandably secrete enzymes to further degrade undegraded materials in the colon before its expulsion from the body [16e18]. It is expedient to say that enzyme action has been exploited for many years, such as in the production of alcohol, where fermentation occurs by imploring microbes like fungi. However, until reasonably recent enzyme application, the roles of enzymes have not been understood [19,20]. The understanding of the functions of an enzyme in biological reaction processes has now aided the merging of this knowledge with other fields of human endeavors such as engineering, computer science programming for the design, and construction of instruments to further optimize its production for human needs [21]. Many enzymes exist as numerous studies have engaged in this field, and more insights are still revealed for industrial applications, among other needs [22,23]. The clamor for an enzyme is mainly dependent on the ability of enzymes to be specific in their mode of action, their high precision, the need for a relatively mild environment for its action, and not forgetting its environmentally friendly characteristic (Table 11.1) [13,15]. 1.1 General mechanism of action of enzymes Enzymes are given names depending on the reactions they catalyze, making it simpler to distinguish between different enzymes. For instance, hexokinase adds a phosphate group to glucose, whereas DNA polymerase produces DNA polymers as the name suggests [24,25]. In summary, because enzymes have distinct modes of action, the following

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Table 11.1 Classification by reaction type and functions of the enzymes. Class

Reaction type

Example

Oxidoreductases

They oxidize or reduce species. Hence, they are known for the transfer of hydrogen atoms or electrons from one molecule to another

Transferases

Moving a functional group (phosphate, methyl, etc.) from one compound to another; so, the other compound gains the functional group Hydrolysis (catabolic) with the help of water. That is hydrolytically cleaving a bond with the aid of water Rearrangement of atoms within a molecule. That is to say, changes spatial orientation to make isomers Splitting chemicals into smaller parts without using water (catabolic). Hence, its nonhydrolytic cleavage/removal of compounds from the substrate Forms bond by condensation reaction by ATP cleavage. Joins substrates often by condensation reaction “addition reaction” Joining of two molecules by the formation of new bonds (anabolic)

Oxidases, dehydrogenases Molecules involved are NADH/NADPH etc Transferase, kinases, phosphatase

Hydrolases

Isomerases

Lyases

Ligases

Synthetases

Protease, lipases, amylase, pepsin Phosphohexoisomerase, fumarase Aldolases, decarboxylases

DNA ligase

DNA polymerase, DNA ligase

Robinson PK. Enzymes: principles and biotechnological applications. Essays Biochem 2015;59:1e41.

are some illustrations of each of the six classes of enzyme actions (https://www.youtube.com/watch?v ¼ AD3-v1oKjSk&t ¼ 115s https://www.youtube.com/watch?v¼AD3v1oKjSk&t¼115s retrieved August 5, 2022): 1. Transferase: removes the functional group “Y” from molecule B [26]. A þ BY / AY þ B This is evident in protein translation where amino acids bound to tRNA are transferred to the growing polypeptide chain by the enzyme peptidyl transferase. Met-Ala-Leu + Lys

Met-Ala-Leu-Lys + tRNA

tRNA

2. Ligase: it involves the joining of separate molecules. As in the joining of the molecule A to B, with ATP involved in the process. A þ B/AB

Introduction to enzymes and organocatalysis

For instance, during DNA replication, the two strands of the DNA molecules are joined together by the DNA ligase to form a single strand [27].

3. Oxidoreductase: A little different from the “others” as it does two different reactions. The reactions involve the transfer of electrons from one molecule (A) to another (B) and, from B to A, thereby making the reactions reversible. While the oxidase oxidizes or takes away electrons from a molecule, a reductase reduces or gives electrons to a molecule [28]. A þ B: #A: þB Hence, catalyzing both forward and reverse reactions. A great example of such equilibrium reactions is the catalysis of pyruvate and NADH to lactic acid and NADþ. Where electrons are either passed from NADH to pyruvate or from lactic acid to NADþ, catalyzed by the lactate dehydrogenase. The enzyme removes electrons from a molecule of lactic acid. Pyruvate þ NADH#Lactic acid þ NADþ 4. Isomerase: As the name suggests, it converts a compound to one of its isomers [29]. For instance, the known reaction involving conversion of glucose-6-phosphate to fructose-6-phosphate is catalyzed by phosphoglucose isomerase. This enzyme involves the creation of an isomer of glucose which is phosphorylated. Hence, an intramolecular group transfer. Glucose-6-Phosphate / Fructose-6-phosphate 5. Hydrolases: These enzymes use water to cleave molecules. As in the cleaving of a molecule A to B and C [30]. A þ H2 O / B þ C For instance, serine hydrolases (“protease”) hydrolysis action on peptide bonds in: Lys  Ala þ H2 O / Lys þ Ala They can be called serine proteases as they are hydrolases that use the serine residue as the crucial catalytic amino acid responsible for breaking the peptide bond. Also, hydrolases could act by transferring functional groups to water or addition of the constituent of the water to aid the splitting of the molecule A  B þ H2 O / A  H þ B  OH

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As in the transfer of water molecules to pyrophosphate to form two phosphate molecules: Pyrophosphate þ H2 O/2 phosphate molecules 6. Lyases: These enzymes are involved in the catalysis of the dissociation of a molecule [31]. Hence, the dissociation of molecule A, to molecules B and C, in the absence of water (nonhydrolytic) or oxidation and reduction as in an oxidoreductase. Hence, they generate either a double bond between two atoms or a ring structure in a molecule to work.

A-B

A=B + X-Y

X Y For instance, the cleaving of pyruvate to acetyldehyde and carbon dioxide. Pyruvate þ Hþ / Acetyldehyde þ Carbon dioxide 1.2 Properties of enzymes The i. ii. iii. iv. v. vi. vii.

following are some common properties of enzymes: Enzymes speed up the chemical reaction. Enzymes lower the activation energy of chemical reactions (Fig. 11.2). Enzymes are required in a “small” amount. Enzymes are specific for the substrate they catalyze. Temperature affects the activity of the enzyme-catalyzed reactions. pH affects the activity of the enzyme-catalyzed reactions. Enzymes catalyze reversible reactions. [13,32].

1.3 Factors that influence enzyme production process The following factors influence the enzyme production process in industries; they include: temperature, pH, inoculum size, and agitation of the fermented substrates along with other factors below influence enzyme production [33,34], they include the following: a. Temperature effect: Typically, minimal activity is obtained at low temperatures. The enzyme’s activity does, however, increase as the temperature gets closer to the optimum, with the optimum being the best activity, which in humans has been commonly known to be 37 C until the enzyme begins to lose its activity (denaturation) at temperatures above the optimum. Therefore, for optimal functioning, each enzyme has its specific temperature or range of optima temperatures (Figs. 11.3A and B).

Introduction to enzymes and organocatalysis

Optimum

Enzyme activity

T e m p e r atu r e

Figure 11.3A Sample of temperature effect on the activity of enzymes [32].

Optimum

Enzyme activity

pH

Figure 11.3B Sample of pH effect on the activity of enzymes [32].

The protein attribute of enzymes cannot be overlooked in considering temperature, as high temperature denatures enzymes, thereby negatively affecting their activity [33,35]. Studies undertaken on optimizing the effect of temperature on pectinase (pectin enzyme) production have shown that temperature affects pectinase production. As 30 C with Saccharomyces cerevisiae (ATCC 52712) and Aspergillus niger, and 37 C with Bacillus subtilis were the optimal temperatures obtained beyond which there was a decline in pectinase activity [32,33,35]. Polygalacturonase’s best activity from the mold was obtained at

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50 C and 40e60 C for yeasts in another study [36], as 25 C was reported for another strain of Saccharomyces cerevisiae by Ref. [37]. So, differences in strains of microorganisms could lead to differences in the optimum temperature for the best activity. b. pH effect: Similar to the temperature effect, enzymes have their unique optimum pH in which they function. A change in the pH from the optimum leads to a change in the chemical nature of the amino acids that make up the protein; which may result in a change in the bonds, and the tertiary structure may break down. This leads to the disruption in the active site leading to denaturation of the enzyme. pH influences the production of enzymes, with favorable pH leading to increase in activity of enzymes and the reverse for unfavorable pH with denaturation of the enzymes, as seen with a study on pectinase activity [32]. Pectinase has been reported to have been optimally secreted by Bacillus subtilis and Bacillus amyloliquefaciens SW106 at pH 6.0 [38]. So, pH influences the growth of microorganisms and aids their rate of product formation. c. Enzyme concentration: In reactions where the concentration of the enzyme is low, it leads to competition for the active sites on the enzymes which results in a low rate of reaction. However, as the enzyme concentration increases, the more available active sites lead to a faster reaction rate, with an increase beyond the optimum results in no effect as the substrate concentration becomes the limiting factor. d. Substrate concentration: When the substrate concentration is low relative to the enzyme’s active sites, it indicates that the many enzyme’s active sites are not occupied, leading to a low reaction rate. More substrate concentration added to the reaction vessel results in more enzyme-substrate complexes formed, and is associated with an increase in the reaction rate to the optimum; however, further substrate concentration leads to a decrease in activity due to saturation of the active sites on the enzyme’s active sites by the substrates. e. Effect of metal ions and inhibitors: Enzymes are known to have active sites that participate in the reaction process. However, each enzyme’s active sites are functional group/groups that aid catalysis, though the substrate changes in orientation relative to the functional group on the active site [11]. For catalytic stability in the conformation of the enzyme to be maintained, metal ions could accept or give out electrons, thereby restricting mainly the desired reaction process to take place as it limits other undesired reactions; this process keeps hold of the substrate and enzyme bond, before the product formation [39,40]. Most enzymes with pectin enzymes inclusive are known to be metallo-enzymes. Hence, they need metal ions such as Ca2þ, Mn2þ, Fe2þ, and Mg2þ, among many other ions, to increase their activity [41]. However, some elements reduce the interaction of the substrate with the enzyme’s active sites, and these are given the term inhibitors [40,42]. f. Effect of fermentation time and substrate composition on pectinase production: Hussain and colleagues (2019) reported the production of pectinase on the 4th day using

Introduction to enzymes and organocatalysis

the conventional approach of one factor at a time (OFAT). The 7th day was for endoglucanase production with the best activity using Aspergillus niger, Aspergillus flavus, and Penicillium atrovenetum. Another study showed that Aspergillus flavus fermentation in pectin (extracted from African star cherry) as substrate was used for the production of pectinase with the best activity obtained in 4 days [43]; while in another study on the optimization of the production process showed differences in the duration of fermentation for the production of the enzyme [44]. Agrowastes like the peels of pineapple and orange as well as sawdust, wheat bran, and sugar cane pulps were also used as substrates for the production of pectinase with strains of Aspergillus clavatus, Aspergillus niger, Fusarium sp., Penicillium chrysogenum, and Trichoderma sp [45]. The use of corn cob, sawdust and sugar cane pulp as well as utilizing microbes like Aspergillus niger, Penicillium chrysogenum, and Trichoderma harzianum used to produce cellulase in fermentation times of 36 h for A. niger while P. chrysogenum and T. harzianum for 12 and 60 h respectively have been reported [46]. In producing pectinase, the medium used is essential in that each type of enzyme is affected by the type of substrate and the microorganism used in the fermentation process [47,48]. For instance, polygalacturonic acid (PGA), as well as lactose, has been shown to increase the production of pectinase [49]. g. Effect of solvents on enzyme extraction: Some extraction solvents like distilled water are used in the recovery of industrial enzymes from the fermentation medium [50,51], buffer [52] and 0.1 M Na2SO4 [53] among many other extraction solvents. The rationale for the use of solvent for extraction is that it aids in the splitting of the bond between proteins and carbohydrates of such substrates, thereby releasing the “trapped enzyme” [32]. However, other studies have confirmed the use of 0.05 M acetate buffer, 0.05% of Tween 80 and Tween 40, distilled water, and glycerol (0.05%) as extraction solvents for pectinase, of which acetate buffer proved to be the best [54,55]. The use of distilled water was also reported as a solvent for enzyme extraction [50,51]; another solvent used was Na2SO4 (0.1 M) [53]. The extraction solvent for the recovery of the enzyme from the fermented substrate is essential. Where the solvent volume is too much, it gives rise to a dilute solution with reduced enzymatic activity [56]. In another report, in cases of reduced volume of buffer, decreased enzyme activity is obtained with the same report advocating for more extraction solvent volume [54]. So, there is the need to optimize the actual volume of extraction solvent necessary for the best activity of such enzyme as a reduced volume of solvent leads to the insufficiency of the solvent to permeate the solid fermented mass in cases of SSF.

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h. Effect of inoculum size on enzyme production: Studies have shown the need for inoculum concentration. Optimization of inoculum concentration is needed for the optimal yield of the enzyme produced. Ref. [57] showed that 1  107 spores/ mL led to the peak activity of xylanase, with the lowest being 1  106 spores/mL. Similar studies, too, showed peak activity of xylanase from Fusarium oxysporium at 1  107 spores/mL concentration. In contrast, reduced activity was obtained with an increase in inoculum concentration of 2  107 spore/mL [58]. This reduction with increase in inoculum is associated with competition for the substrates, hence, negatively affecting the enzyme’s activity. i. Effect of ammonium sulfate precipitation for practical application of enzyme: The effect of ammonium sulfate salt primarily precipitates the protein from the extract. The groups on the molecules of proteins with charges are stabilized in the concentration of low salts. This process increases the solubility of such proteins; hence, resulting in a phenomenon referred to as salting-in. In some situations, increased salt addition leads to insoluble protein due to a reduction in water, making the proteins precipitate, a phenomenon referred to as salting-out in response to excess salt [59,60]. A study by Ref. [61] showed that in purifying PME (pectin methyl lyase), using 20%e80% NH4(SO4)2, an increase in the salt concentration further led to an increase in enzyme activity with significant increase of 21.50 in 80% of the salt in comparison to 8.25 for 0%. In the same study, however, the soluble content of protein was reduced from 62 mg/mL to 21 mg/mL, with an 80% ammonium sulfate concentration of the protein in its crude state [61]. Dialysis with ammonium sulfate precipitation has proven to increase the purification of the crude enzyme from orange peel [62]. The peak activity with 65% ammonium sulfate precipitation has been shown with the yeast Kluyveromyces marxianus [63]. Apart from using ammonium sulfate for partial purification through precipitation of crude protein, ethanol, and ethylene glycol are reportedly used [64,65]. 1.4 Enzyme kinetics The extent to which enzymes catalyze biological reactions is explained using the phenomenon of enzyme kinetics. It describes how the concentration of substrate affects the velocity of reaction in enzyme studies [66]. Before a reaction occurs, there is a need for overcoming the energy barrier, which is termed the activation energy, as illustrated in Figs. 11.2 and 11.4, with the topmost point in Fig. 11.4 referred to as the “transition state.” As the temperature increases, the molecules gradually become activated, thereby overcoming the energy barrier [68,69]. The study of the kinetics of enzymes investigated with model substrates and assays that generate color is determined using the spectrophotometer. These assays are geared toward measuring the disappearance of a substrate and the appearance of a product [32],

Introduction to enzymes and organocatalysis

T r a n s itio n s ta te E n erg y A ctivation energ y P ro d u c ts

R e a c ta n ts

R e a c tio n p ro g r e s s

Figure 11.4 Sample of a reaction profile of an enzyme-catalyzed reaction [67]. (Modified from https:// doctorlib.info/medical/biochemistry/8.html retrieved September 05, 2022.)

thereby giving rise to readings of absorbance from which the activity of the enzyme is then mathematically calculated. The two commonly known models or plots for determining enzyme kinetics are the MichaeliseMenten and the Lineweaver Burk plots. (a) MichaeliseMenten Plot: This plot measures the kinetics of enzyme-catalyzed reaction by determining the effect of an increase in substrate concentration on the reaction velocity or rate of the reaction with the generation of a hyperbolic graph [70]. From Fig. 11.5, as the substrate concentration increases, the reaction rate of the enzyme on the substrate increases. However, beyond the maximum velocity (Vmax), adding more substrates into the medium does not correspond to an increase in the reaction rate. This phenomenon arises because enzymes become saturated with the substrates at such point, hence, suggesting too much concentration of substrates for the enzymes to handle [73,74]. In other words, at the beginning of the reaction, there is a linear relationship which is an indication of the first-order kinetics; however, when the enzymes become saturated with the substrates, the zeroth-order is established, denoting that no matter the order of increase, it does not affect the reaction rate as shown in the equation below Rate of reaction (R) ¼ K [S]0 .. Zeroth order kinetics. In the zeroth order, (R) ¼ K [S]0 Since any number to power 0 is 1, So, [S]0 ¼ 1. Hence, the zeroth-order is (R) ¼ K  1

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V

Vmax

Vm ax/2

Km

[S]

Figure 11.5 MichaeliseMenten plot [67,71,72].

Therefore, (R) ¼ K. With reference to http://pharmaquest.weebly.com/uploads/9/9/4/2/9942916/ order_of_reaction.pdf (retrieved August 07, 2022 http://pharmaquest.weebly.com/ uploads/9/9/4/2/9942916/order_of_reaction.pdf (retrieved August 07, 2022). Since the Vmax denotes the peak reaction rate, Km is simply the substrate concentration at half the maximum velocity. In further understanding the hyperbolic plot, there is the need for the knowledge of how enzymes act to give product and still not used up in the process as exemplified in https://www.cs.tufts.edu/comp/150CSB/handouts/enzymes.pdf (retrieved September 07, 2022);

where ‘E is an enzyme’, ‘A is the substrate’, ‘EA’ is the Enzyme-substrate complex, and ‘P is the product’. It can be observed that both k2 and k-1 are rate constants associated with reactions leading to the dissociation of the enzyme-substrate complex; k2 describes the rate constants related to the formation of product and the release of the enzyme. In contrast,

Introduction to enzymes and organocatalysis

k-1 describes the breaking up of the enzyme-substrate [ES] complex into enzyme and substrate. Therefore, while the k2 and k-1 denote the breaking apart of the enzymesubstrate complex, k1 shows the formation of the enzyme-substrate complex. Thus, the Km is simply the ratio of the enzyme-substrate complex breaking apart to it staying together. So; Km ¼

k2 þ k1 k1

https://www.cs.tufts.edu/comp/150CSB/handouts/enzymes.pdf (retrieved September 07, 2022) there by making Km the measure of dissociation or affinity of an enzyme for a substrate. However, the calculation of the velocity of an enzyme-catalyzed reaction is derived from the hyperbolic equation of MichaeliseMenten; v¼

Vmax ½S Km þ ½S

This hyperbolic equation of MichaeliseMenten is useful because if Km and maximum velocity (Vmax) for a particular enzyme are known, the velocity (5) is calculated at any substrate concentration ([S]) [75]. (b) LineweavereBurk plot (Double-reciprocal plot): This is an extension of the MichaeliseMenten model as it still respects the hyperbolic equation of Michaelise Menten. However, the difference is that it takes the inverse of both sides of the above hyperbolic equation; hence, the name “double-reciprocal plot” (Fig. 11.6). That is: 1 Km þ ðSÞ ¼ vO Vmax ðSÞ 1 Km ðSÞ þ ¼ VO Vmax ðSÞ Vmax ðSÞ 1 Km 1 þ ¼ VO Vmax ðSÞ Vmax [76]. From the double reciprocal plot, the y-intercept is used to calculate the Vmax by taking the inverse number on the y-intercept, the same for Km. Still, the negative value is because the x-intercept is in the negative quadrant; hence, the negative value so obtained cancels out the negative in the formula to give a positive value for Km. As the Km value is positive because, as previously mentioned, Km is the substrate concentration at half the maximum velocity. The unit for Km could be micromolar (mM), molar (M), or millimolar (mM). Simultaneously, the Vmax is the rate where denoting saturation of the active

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Figure 11.6 LineweavereBurk plot or the double reciprocal plot [14,76].

sites by the substrates, making the unit, concentration per time such as micro-molar per minute (mM/minute), molar per second (M/second), etc. The ratio of Km to Vmax is the slope (Fig. 11.6), thereby fitting into the commonly known equation of a slope where y ¼ mx þ c with “m” the slope and “c” the y-intercept. However, in comparing the MichaeliseMenten plot to the Lineweaver Burk plot, there is the challenge of approximation for the MichaeliseMenten plot, regarding the maximum velocity and Km; hence the preference for the Lineweaver Burk plot over the MichaeliseMenten plot, which is relatively more accurate [77]. 1.5 Enzyme yield The yield of an enzyme is the ratio of the enzyme activity relative to its original activity multiplied by 100%. However, to calculate yield and in turn level of enzyme purification, there is the need for information on total protein, total enzyme, and specific activities of the enzyme [78]. Though total protein shows the amount in milligram of protein present in the mixture, enzyme activity is an indicator of the ability of the enzyme in such mixture to promote the catalysis of the substrate. Specific activity is the ratio of the calculated enzyme activity to the total protein concentration mentioned earlier [32]. However, determining these parameters is not enough without calculating the level of purification, which is done by determining the ratio of the specific activity of such purification step (undertaken) to the specific activity of the original or the crude enzyme extract [78]. The yield of a crude pectin enzyme is 100%; the yield decreases with purification [78]. However, both specific activity and purification levels increase upon purification. Hence,

Introduction to enzymes and organocatalysis

this phenomenon of a decrease in the yield of enzymes further explains the concept of the use of crude extracts for the clarification of fruit juices [78e80]. Additionally, the application area of an enzyme is indicative to a large extent of the level of purification of such an enzyme. For instance, the yields of crude pectin enzyme filtrate in a study were reported to be 100% each; upon undergoing ammonium sulfate precipitation and dialysis, the yields decreased to 5.40%, 7.66%, and 5.99% for the purified pectinase from Aspergillus niger, Aspergillus fumigatus, and Aspergillus flavus, respectively [78]. Further giving credence to the need to produce pectin enzyme based on its intended application, as the intended goal with regards to application of the enzyme should inform the level of purification of such enzyme to avoid reduction in the yield. 1.6 Inorganic elements that serve as cofactors for enzymes Cofactors that are widely regarded as enhancers of enzyme activity are vital aspects of this discussion. From studies in literature, below are some of the cofactors for some enzymes: a. Cu2þ for cytochrome oxidase [81]. b. Kþ for pyruvate kinase [82]. c. Mg2þ for Hexokinase, glucose-6-phosphatase, pyruvate kinase [83]. d. Mo for Dinitrogenase [84]. e. Ni2þ for Urease [85]. f. Se for Glutathione peroxidase [86].

2. Introduction to organocatalysis Generally, reactions occur at slow rates in the absence of a catalyst. Catalysts accelerate a chemical reaction without affecting the position of the equilibrium [87]. Hence, the need for catalyst/catalysts. Both enzymes and organocatalysts are catalysts and have been acknowledged under the umbrella, “green chemistry”; as they reduce the chemical processes known to impact the environment [2,4,88]. Organocatalysis is a form of catalysis where the rate of a chemical reaction is increased by small molecule organic catalyst/catalysts [89]. In other words, Benjamin List (one of the fathers of organocatalysis) in one of his interviews defined organocatalysts as small organic (or low molecular weight) molecules acting as catalysts where metal is not part of the active principles. These catalysts function by either removing or donating electrons or protons. These types of catalysts are made up of carbon, hydrogen, sulfur, and other non-metal elements found in organic compounds. Though this reaction type requires nonmetals, the metals could be present in sub-stoichiometric amounts. Examples of organocatalysts are L-proline, quinine, DMAP, Nobin, BOPHOZ, and Macmillan’s catalyst (Fig. 11.7). Hence, organocatalysis defines Brostead acid and bases, and the Lewis acids and Lewis bases, as contained in the two-book volumes of the Science of Synthesis Asymmetric

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Figure 11.7 Examples of organocatalysts [3].

Organocatalysis 1: Lewis Base and Acids Catalysts edited by Benjamin List and the Science of Synthesis Asymmetric Organocatalysis 2: Bronstedt Base and Acids Catalysts, and Additional Topics edited by K. Maruoka. The relevance of this field is evidenced in the 2021 Noble Prize in chemistry awarded to Benjamin List and David Macmillan. Both works were vital to the “modern era” of enantioselectivity of organocatalysts; the beginning of the use of the term “organocatalysis” published in 2000 [90,91]. This field could be used as a tool for molecule building. The previous knowledge on catalysts was centered on enzymes and metals, not until these researchers introduced the third type of catalyst, the organocatalysts, which are believed to build from small organic molecules. Though metal catalysts are still in use today, the disadvantage of their sensitivity to oxygen and water, plus the tendency of some of the metal catalysts to cause harm to the environment as a result of them being heavy metals is a concern [92]. 2.1 Chiral molecules The two types of common organocatalysis available are the achiral and chiral (mirror images).

Introduction to enzymes and organocatalysis

Figure 11.8 Images depicting the chirality of organocatalysis (https://www.youtube.com/watch? v¼gevukNKvNPA&t¼545s).

They are symmetric and asymmetric organocatalysts. Most organic molecules abound which are found to have two variants, that is, a mirror image of the other; yet, have different effects on the human body. By structural mirror image or chirality, it is meant that they are like the human hands which though similar, cannot be effectively superimposed on each other (Fig. 11.8). This simple action of ‘non-superimposable’ in organic chemistry is referred to as “enantiomer.” An example is the limonene molecule, having two variants with one of the variants possessing a lemon scent and the other an orange scent. However, producing one of the variants has been made easier today. The sensitivity of enantiomers becomes vital in the synthesis of drugs as the body can perceive the functional differences in the enantiomers. The exclusion in the production of one enantiomer in favor of the other is made possible with the asymmetric organocatalysis or enantioselective organocatalysis, a methodology introduced by these 2021 Noble laureates in Chemistry. Benjamin List in his study showed proline as a catalyst and the ability of this amino acid in driving asymmetric catalysis [90]. The study was on aldol reaction. This reaction can be undertaken in the body with Aldolase A; a huge enzyme known to contain many amino acids. However, among these amino acids, only three were found by Benjamin List to be actively involved in catalysis. They are lysine, glutamic acid, and tyrosine. In further studies, he found L-proline to be capable of use via asymmetric organocatalysis for the production of the enamine intermediate. His study is of significance as it was affirmed that, proline is relatively simple and cheap with little to no recognizable adverse effect on the environment to date. David W.C. Macmillan on the other hand initially employed metal catalysis (copper) in his reaction which was known in “that era” but found to be slow. The metal-based

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catalysts were not sustainable and sensitive for the formation of a six-member ring on an industrial scale. He subsequently discovered an organic molecule, a moiety for use in carrying out the reaction with none of the above demerits. His work on the DielseAlder reaction led to the discovery of imidazolidinone which carried out the reaction in an asymmetric manner [94]. In summary, the making of a stereo center for chemical reactions favoring the production of one of the chiral molecules is illustrated in Figs. 11.9 and 11.10, as depicted by Karl Anker Jorgensen. A racemic mixture is optically inactive due to the possession of equal amounts of enantiomers as denoted in the first illustration; however, for the second illustration, if one of the faces is shielded to allow for ‘B” to react only with the right side (other catalysts also do same), then only one molecule is produced.

Figure 11.9 Enantiomers of limonene; A is R-(þ)-limonene, and B is S-()-limonene [93].

Figure 11.10 Chemical reaction showing preference for one chiral compound (https://www.youtube. com/watch?v¼gevukNKvNPA&t¼545s).

Introduction to enzymes and organocatalysis

These two discoveries opened up a new significant path for a new reaction methodology. Before their discoveries as outlined earlier, the known catalysts methodology was the popular metal and enzyme-based catalysis. 2.2 Advantages and disadvantages of organocatalysts Below are the advantages and disadvantages of organocatalysts [3]. Advantages

Disadvantages

Easy preparation or availability Easy handling; inert toward moisture and oxygen Easy scale-up No metal contamination Easy screening Useful in complex (steric) reactions

High catalyst loading Relatively new field compared to enzymes

The preference for organocatalysis over the other types of catalysis includes the following as found in literature: 1. The knowledge of organocatalysis can be used to drive myriad reactions. 2. Some pharmaceutical products such as thalidomide (Thalomid) for the reduction of morning sickness in pregnant women, was reported to result in fatal deformities in the 1960s, giving birth to children with impairment in the growth of arms and legs [95]; due to the ability of the drug to form a mirror image of each other with one of benefit and the other causing deformities to the embryos of humans through the reduction of blood supply to the arms and legs of the embryos, was a challenge. However, with asymmetric organocatalysis, such a challenge has been significantly handled, resulting in the selective production of only the useful enantiomer. 3. The intermediate products formed during reactions do not need to be isolated and purified, but allowed to progress, hence, reducing wastages or environmental pollutants that would have resulted are curtailed to the barest minimum, thanks to organocatalysis making the reaction cost-effective, hence increasing the scaling up of laboratory experiments for industrial production of products. 4. The use of organocatalysis reduces the number of reaction steps to arrive at the product desired. As in the scientific world, the lesser the number of reaction steps to arrive at the desired product and lesser concerns on the environment, the better the route to consider for the production of such product. 5. This type of catalysis makes a major contribution to green chemistry as there is significantly no need for metal-based catalysis; hence, makes organocatalysis more sustainable and environmentally friendly. In summary, the advantages of organocatalysis are in its cost-effectiveness of the natural molecule, non-toxicity and simple structure in comparison to organometallic

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catalysis, and enzyme catalysis. Hence, this is an area of great potential for industrial scaleup due to its eminent potential for efficient syntheses. Some compounds in the market where organocatalysis has been used for their production with a reduction in the number of manual operations include the following: a. Telcapant by Merck [96]. b. Tamiflu (oseltamivir) for bird flu treatment [97]. c. INCIVEK (telaprevir) for hepatitis C treatment [98]. d. Paroxetine-an antidepressant compound [99].

3. Conclusion Shortly, the replacement of metal-based catalysis with a more environmentally friendlybased methodologies for catalysis, such as organocatalysis is envisaged.

Suggestion For more on organocatalysis, read the two book volumes of the Science of Synthesis: Asymmetric Organocatalysis edited by Benjamin List and K. Maruoka.

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[39] Al-Najada AR, Al-Hindi RR, Mohamed SA. Characterization of polygalacturonases from fruit spoilage Fusarium oxysporum and Aspergillus tubingensis. Afr J Biotechnol 2012;11:8527e36. [40] Okonji RE, Itakorode BO, Ovumedia JO, Adedeji OS. Purification and biochemical characterization of pectinase produced by Aspergillus fumigatus isolated from soil of decomposing plant materials. J Appl Biol Biotechnol 2019;7(3):1e8. [41] Chen AY, Adamek RN, Dick BL, Credille CV, Morisson CN, Cohen SM. Targeting metalloenzymes for therapeutic intervention. Chem Rev 2019;119(2):1323e455. [42] Ouertani A, Neifar M, Ouertani R, Masmoudi AS, Mosbah A, Cherif A. Effectiveness of enzyme inhibitors in biomedicine and pharmacotherapy. Adv Tissue Eng Regen Med 2019;5(2):85e90. [43] Nsude CA, Ujah II, Achikanu CE, Ezenwali MO, Udaya CL, Chilaka FC. Optimization of fermentation condition for pectinase production from Aspergillus flavus, using African star cherry pectin as a carbon source. Int J Ecol Environ Sci 2018;1(1):15e9. [44] Ametefe GD, Lemo AO, Orji FA, Kolawole LA, Iweala EEJ, Chinedu SN. Comparison of optimal fungal pectinase activities using the Box-Behnken design. Trop J Nat Prod Res 2021;5(9):1656e64. [45] Okafor UA, Okochi VI, Chinedu SN, Ebuehi OAT, Onygeme-Okerenta BM. Pectinolytic activity of wild-type filamentous fungi fermented on agro-wastes. Afr J Microbiol Res 2010;4(24):2729e34. [46] Chinedu SN, Okochi VI, Omidiji O. Cellulase production by wild strains of Aspergillus niger, Penicillium chrysogenum and Trichoderma harzianum grown on waste cellulosic materials. IFE J Sci 2011;13(1): 57e62. [47] Farinas CS. Developments in solid state fermentation for the production of biomass degrading enzymes for the bioenergy sector. Renew Sustain Energy Rev 2015;52:179e88. [48] Soccol CR, da-Costa ESF, Letti LAJ, Karp SG, Woiciechowski AL, Vandenberghe LPS. Recent developments and innovations in solid state fermentation. Biotechnol Res Innov 2017;1:52e71. [49] Des RK, Sanjiv KS, Rupinder T. Enhanced production of pectinase by Bacillus sp. DT7 using solid state fermentation. Bioresour Technol 2003;88(3):251e4. [50] Patil NP, Chaudhari BL. Production and purification of pectinase by soil isolate Pencillium sp. and search for better agro-residue for its SSF. Recent Res Sci Technol 2010;2(7):36e42. [51] Silva D, Tokuioshi K, Martins ES, da-Silva R, Gomes E. Production of pectinase by solid-state fermentation with Penicillium viridicatum RFC3. Process Biochem 2005;40:2885e9. [52] Sudeep KC, Upadhyaya J, Joshi DR, Lekhak B, Chaudhary D, Pant BR, et al. Production, characterisation and industrial application of pectinase enzyme isolated from fungal strains. Fermentation 2020;6:59e69. [53] Singh SA, Platter H, Diekmann H. Exopolygalacturonase-lyase from thermophilic Bacillus sp. Enzym Microb Technol 1999;25:420e4. [54] Ahmed SA, Mostafa FA. Utilization of orange baggase and molokhia stalk for the production of pectinase enzyme. 2013. https://doi.org/10.1590/S0104-66322013000300003. . [Accessed 2 June 2014]. [55] Ibrahim D, Weloosamy H, Lim SH. Effect of agitation speed on the morphology of Aspergillus niger HFD5A-1 hyphae and its pectinase production in submerged fermentation. World J Biol Chem 2015; 6(3):265e71. [56] Aikat K, Bhattacharyya BC. Protease extraction in solid state fermentation of wheat bran by a local strain of Rhizopus oryzae and growth studies by the soft gel technique. Process Biochem 2000;35: 907e14. [57] Maria L, Garcia S, Samia MT. Optimisation of xylanase biosynthesis by Aspergillus japonicas isolated from a Caatinga area in the Brazilian state of Bahia. Afr J Biotechnol 2006;5(11):1135e41. [58] Kuhad R, Manchanda M, Singh A. Optimization of xylanase production by a hyperxylanolitic mutant strain of Fusarium oxysporum. Process Biochem 1998;33:641. [59] Fu C, Li Z, Sun Z, Xie S. A review of slating-out effect and sugaring-out effect: driving forces for novel liquid-liquid extraction of biofuels and biochemical. Front Chem Sci Eng 2020;15(4): 854e71. 15://doi.org/10.1007/s11705-020-1980-3. [60] Mehringer J, Hofman E, Touraud D, Koltzenburg S, Kellermeier M, Kunz W. Salting-in and saltingout effects of short amphiphilic molecules: a balance between specific ion and hydrophobicity. Phys Chem Phys 2021;23(2):1381e91.

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[84] Seefeldt LC, Hoffman BM, Dean DR. Mechanism of Mo-dependent nitrogenase. Annual Chem Rev 2009;78:701. [85] Zambelli B, Musiani F, Benini S, Ciurli S. Chemistry of Ni2þ in urease: sensing, trafficking, and catalysis. Acc Chem Res 2011;44(7):520e30. [86] Michiels C, Raes M, Toussaint O, Remacle J. Importance of Se-glutathione peroxidase, catalase, and Cu/Zn-SOD for cell survival against oxidative stress. Free Radical Biol Med 1994;17(3):235e48. [87] Ostwald W. Die Uberwindung des wissenschaftlichen Materialismus. In: Verhandlungen der Gesellschaft Deutscher Naturforscher und Arzte; 1895. p. 155e68. [88] Clark JH, Rhodes CN. Clean synthesis using porous inorganic solid catalysts. Cambridge, UK: RSC Clean Technology Monographs; 2000. [89] Jacobsen EN, MacMillan DWC. Organocatalysis. PNAS 2010;107(48). [90] List B, Lerner RA, Barbas- III CF. Proline-catalyzed direct asymmetric aldol reactions. J Am Chem Soc 2000;122:2395e6. [91] Jen WS, Wiener JJM, MacMillan DWC. New strategies for organic catalysis: the first enantioselective organocatalytic 1,3-dipolar cycloaddition. J Am Chem Soc 2000;122(40):9874e5. [92] Tchounwou PB, Yedjou CG, Patlolla AK, Sutton DJ. Heavy metal toxicity and the environment. Exp Suppl 2012;101:133e64. [93] Blanch GP, Nicholson GJ. Determination of enantiomeric composition of limonene and limonene1,2-epoxide in lemon peel by multidimensional gas chromatography with flame-ionization detection and selected ion monitoring mass spectrometry. J Chromatogr Sci 1998;36:37e43. [94] Ahrendt KA, Borths CJ, MacMillan DWC. New strategies for organic synthesis: the first highly enantioselective organocatalytic Diels-Alder reaction. J Am Chem Soc 2000;122:4243e4. [95] Timmermans S, Leiter V. The redemption of thalidomide: standardizing the risk of birth defects. Soc Stud Sci 2000;30(1):41e71. [96] Xu F, Zacuto M, Yoshikawa N, Desmond R, Hoerrner S, Itoh T, et al. Asymmetric synthesis of telcagepant, a CGRP receptor antagonist for the treatment of migraine. J Org Chem 2010;75(22): 7829e41. [97] Smith JR. Oseltmivir in human avian influenza infection. J Antimicrob Chemother 2010;65(2): ii25e33. [98] Wilby KJ, Partovi N, Ford JE, Greanya ED, Yoshida EM. Review of boceprevir and telaprevir for the treatment if chronic hepatitis C. Canadian J Gastroenterol Hepatol 2012;26(4):205e10. [99] Kowalska M, Nowaczyk J, Fijalkowski L, Nowaczyk A. Paroxetine- overview of the molecular mechanisms of action. Int J Mol Sci 2021;22(4):1662.

CHAPTER 12

Natural products for the prevention and management of nephrolithiasis Farah F. Al-Mamoori Department of Pharmaceutical Sciences, Faculty of Pharmacy, Zarqa University, Zarqa, Jordan

1. Introduction Over the last 25 years, the prevalence of nephrolithiasis among children and adolescents has rapidly grown. Among adults, about 50% of patients with occurrence nephrolithiasis will develop a recurrent stone within 5e10 years of the first kidney stone [1]. Long-term pharmacological treatment is required to decrease stone recurrence. The majority of medications used to treat kidney stones have been around for a long time. There hasn’t been a new medicine created since the introduction of potassium citrate treatment [2]. The belief that kidney stone illness is a self-limiting condition obstructs nephrologists and urologists in preventing frequent kidney stone disease, hence delaying the development of new medications. Furthermore, new agent testing is lacking due to a lack of succinctly assessed outcomes. This misunderstanding, combined with poor patient compliance, resulted in the pharmaceutical industry paying no attention to the development of a novel medicine due to its perceived low profitability [3]. There are four general categories of kidney stones: calcium-based, uric acid, struvite, and cystine stones. Surgical procedures like extracorporeal shock wave lithotripsy (ESWL), percutaneous lithotripsy, and trans-ureteral lithotripsy are used to treat and manage nephrolithiasis [4]. The traumatic action of shock waves, remaining stone fragments as potential nidus for future stone formation, acute renal injury, loss of renal function, and increased stone recurrence, ESWL generated severe hematuria, hypertension, pancreatitis, and infection [4]. ESWL often requires numerous treatments because of inadequate clearance and leads to surgical delays or an increased burden of expenses [5]. Currently, well-known herbal medications have a nephrolithiatic action by modifying the ionic composition of urine, for example, by lowering the amounts of calcium and oxalate ions. The majority of these treatments also include diuretic, crystallization inhibition, and antioxidant properties [6]. Exact understandings of these medicinal plants or plant remedies mechanisms of action are critical for the development of effective and safe anti-nephrolithiatic medicines [6].

New Horizons in Natural Compound Research ISBN 978-0-443-15232-0, https://doi.org/10.1016/B978-0-443-15232-0.00026-6

© 2023 Elsevier Inc. All rights reserved.

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2. Prospects for nephrolithiasis management and improvements in natural product approaches 2.1 Medicinal plants Despite the fact that natural products, mainly medicinal plants, have been used for kidney stones in numerous nations for thousands of years, there has been a growing interest in herbal and traditional medicine in recent years for the prevention and treatment of a variety of disorders. The anti-inflammatory and therapeutic properties of medicinal plants and phytochemical components were studied [7]. They utilize a variety of ways to produce their nephrolithiasis benefits, including: Assistance to spontaneously pass through stones by increasing the volume of urine and pH. • Enhanced renal function. • Reveal a significant improvement in the relieving symptoms such as pain, burning micturition and hematuria. • Balance the inhibitor and promoter of urine crystallization and affects the nucleation, growth, and aggregation of crystals. • Strengthen the antioxidant status of the renal tissue and the integrity of the cell membrane. • Antimicrobial activity [8,9]. 2.1.1 The role of plant polyphenols in nephrolithiasis The anti-nephrolithiatic activity of plant polyphenols are supposed to be due to: • Diuretic effect: Polyphenols have retained their diuretic properties. As a result of flushing out the salt deposits, diuretic activity increases the amount of fluid passing across the kidneys. The increased urine volume reduces salt saturation and consequently prevents crystal precipitation at physiological pH [10]. Gallic acid showed a significant increase in urinary excretion of chloride, potassium, and sodium [11]. Rutin showed a significant increase in urine volume and cation excretion [12]. Luteolin, at dose of 3 mg/kg, showed induction in diuretic and natriuretic effect [13]. Rutin quercitin, cyanidin, and delphinidin also showed diuretic activity [1]. • Anti-inflammatory effect: Pain and inflammation are tightly linked to arachidonic acid, prostaglandin-E2, nitric oxide (NO), COX-2, and cytokines (IL-1, IL-6, and TNF-). Nephrolithiasis symptomatic alleviation is dependent on analgesic, antispasmodic, and anti-inflammatory actions [14]. By controlling inflammatory signaling pathways and preventing the synthesis of inflammatory mediators, curcumin has anti-inflammatory actions [15]. Tumor necrosis factor (TNF-), interleukin-1 (IL-1), and interleukin-6 (IL-6) transcriptional levels were all decreased by ferulic. Importantly, ferulic acids promoted autophagy and prevented the activation of the NLRP3 inflammasome [16]. Syringic acid, vanillic acid, diosmin, p-coumaric acid, sinapic acid, catechin, and apigenin also showed anti-inflammatory activity [1].

Natural products for the prevention and management of nephrolithiasis

•

•

Antioxidants: Renal oxalate exposure promotes lipid peroxidation and the production of reactive oxygen species (ROS), as well as damage and inflammation in renal cells. The ensuing loss of membrane integrity promotes fibrosis and collagen synthesis, as well as CaOx adherence, preservation, and eventual stone formation. Many studies suggest that polyphenols’ antioxidant activity helps to avoid CaOx by reducing the damage to renal endothelial tissue caused by cytotoxic chemicals with oxidative capability [1]. Caffeic acid is a powerful DPPH scavenger, superoxide anion radical scavenger, overall reducing power, and metal chelating agent for ferrous ions [17]. p-Coumaric acid, vanillic acid luteolin, quercetin, resveratrol, cyanidin, and malvidin also showed antioxidant activity [1]. ACE inhibition: The RAAS increases NADPH oxidase in renal cells, which creates reactive oxygen species (ROS). ACE inhibition reduces the deposition of CaOx crystals and renal inflammation through lowering ROS generation. The hydroxyl groups of free phenolic compounds chelate with the active site of ACE, and the zinc atom is present, inhibiting ACE [1]. It has been demonstrated that quercetin inhibits ACE in vitro, perhaps due to its capacity to chelate metal ions like zinc [18]. Apigenin, kaempferol, luteolin, cyanidin, and sinapic acid also showed ACE inhibition activity [1] (Table 12.1).

2.1.2 Examples on anti-nephrolithiatic medicinal plants Examples on some medicinal plants have been researched for their defensive or therapeutic role in nephrolithiasis, including in vitro, in vivo, and/or clinical data. The details on the pharmacological evidence of these medicinal plants are illustrated in Table 12.2.

Table 12.1 Mechanisms of many polyphenols for the prevention of CaOx formation: diuretic, antioxidant, anti-inflammatory, and ACE inhibition. Diuretic

Anti-inflammatory

Antioxidant

ACE inhibition

Gallic acid Chlorogenic acid Genistein Rutin Quercitin Cyanidin Delphinidin Luteolin Luteolin

Curcumin Syringic acid Vanillic acid Diosmin p-Coumaric acid Sinapic acid Ferulic acid Catechin Apigenin

Caffeic acid p-coumaric acid Vanillic acid Luteolin Quercetin Resveratrol Cyanidin Malvidin

Daidzein Caffeic acid and chlorogenic acid Apigenin Kaempferol Luteolin Cyanidin Sinapic acid Quercetin

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Table 12.2 List of medicinal plants reported for nephrolithiasis activity. Plant name

Part used

Study type

Annona squamosal L.

Leaf

In vivo

Terminalia chebula R. Mallotus philippinensis Lam. Macrotyloma uniflorum lam.

Fruit Leaf

In vitro In vivo

Seed

In vivo

Arnebia euchroma royle.

Root

In vitro

Boldoa purpurascens cav.

Leaf

In vitro

Boldoa purpurascens cav.

Leaf

In vivo

Lawsonia inermis L.

Bark

In vivo

Costus igneus nak. Prunus Mahaleb L.

Leaf Root

In vitro In vivo

Nigella sativa linn.

Seed

Clinical

Results

References

• [Urine volume, pH • YUrea, creatinine, and uric acid in serum and urine • YCaOx crystallization • YCalcium, oxalate and phosphate in urine • [Urine volume and magnesium • YCalcium, oxalate, uric acid and urea in urine • [The highest dissolution of CaOx • YNucleation and aggregation of CaOx crystallization • YUric acid in urine • YUric acid and creatinine in serum • YCalcium, oxalate, phosphate in urine • [Dissolution of CaOx • YUrea, uric acid, creatinine, calcium, and phosphorus in serum • [Excretion of stone

[14]

[19] [20] [20]

[21] [22]

[22]

[20] [23] [24]

[5]

• [ Demonstrates increasing trend; Y Demonstrates decreasing trend.

2.2 Natural products 2.2.1 Fish oil The active component of fish oil is dietary eicosapentaenoic acid (EPA), an n-3 fatty acid. The same metabolic process is used to process EPA and n-6 fatty acids. As a result, raising EPA lowers n-6 fatty acid metabolites, especially PGE2. Reduced PGE2 levels also activate the nephron Naþ/Kþ/2Ca transporter, which increases renal calcium reabsorption in addition to reducing urine calcium excretion. Significant reductions in urine calcium and oxalate concentrations and increases in urinary citrate concentration have been linked to consuming 1200 mg/d of fish oil [11,25].

Natural products for the prevention and management of nephrolithiasis

2.2.2 Probiotics Investigators have successfully shown that oxalate can be utilized by some types of the endogenous digestive bacteria, potentially limiting its absorption from the intestinal lumen. Gastrointestinal problems like antibiotic-induced diarrhea have frequently been treated with probiotics that contain Lactobacilli spp [12]. O. formigenes obligate anaerobe utilizes oxalate as its only energy source. The most effective oxalate-degrading organism discovered in the human digestive tract, according to in vitro research, is O. formigenes. For instance, O. formigenes destroyed up to 98% of the available oxalate under controlled circumstances [4]. 2.2.3 Vitamins Alanine glyoxylate aminotransferase (AGT), the enzyme that catalyzes the conversion of glyoxylate to glycine, requires pyridoxine (vitamin B-6) as a cofactor. Glyoxylate may turn into oxalate when AGT is insufficient or pyridoxine levels are low. Studies have shown a negative correlation between vitamin B-6 intake and the likelihood of developing stones [26] and have shown that dietary therapy combined with pyridoxine supplementation works well for patients with hyperoxaluric stone development [9]. 2.2.4 Citrus juices Citrus fruits, such as oranges, grapefruits, and lemons, as well as the juices made from them, are essential to the modern diet and are gaining popularity as a result of current health trends. The use of these liquids may have a variety of effects on how kidney stones develop. Citrus fruit juices have a variety of potential beneficial effects. The first and most evident one is the advantages of combining citrus juice consumption with fluid intake. Citrate, one of the greatest inhibitors of urinary stone formation, is found in citrus fruit juices, which is the second factor. Citrus juices include an acid called citric acid, which is a tricarboxylic acid that primarily occurs as the salt citrate at physiological blood pH. (7.4) [25].

References [1] Ahmed S, Hasan MM, Mahmood ZA. Antiurolithiatic plants: multidimensional pharmacology. J Pharmacogn Phytochem 2016;5(2):4. [2] Sakhaee K. Pharmacology of stone disease. Adv Chron Kidney Dis 2009;16(1):30e8. [3] Sakhaee K. Recent advances in the pathophysiology of nephrolithiasis. Kidney Int 2009;75(6): 585e95. [4] Tiwari A, Soni V, Londhe V, Bhandarkar A, Bandawane D, Nipate SONALI. An overview on potent indigenous herbs for urinary tract infirmity: urolithiasis. Asian J Pharmaceut Clin Res 2012;5(1):7e12. [5] Ardakani Movaghati MR, Yousefi M, Saghebi SA, Sadeghi Vazin M, Iraji A, Mosavat SH. Efficacy of black seed (Nigella sativa L.) on kidney stone dissolution: a randomized, double-blind, placebocontrolled, clinical trial. Phytother Res 2019;33(5):1404e12. [6] Barghouthy Y, Somani BK. Role of citrus fruit juices in prevention of kidney stone disease (KSD): a narrative review. Nutrients 2021;13(11):4117.

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[7] Boeing T, da Silva LM, Mariott M, de Andrade SF, de Souza P. Diuretic and natriuretic effect of luteolin in normotensive and hypertensive rats: role of muscarinic acetylcholine receptors. Pharmacol Rep 2017;69(6):1121e4. [8] Nirumand MC, Hajialyani M, Rahimi R, Farzaei MH, Zingue S, Nabavi SM, et al. Dietary plants for the prevention and management of kidney stones: preclinical and clinical evidence and molecular mechanisms. Int J Mol Sci 2018;19(3):765. [9] Yadav RD, Jain SK, Alok S, Mahor A, Bharti JP, Jaiswal M. Herbal plants used in the treatment of urolithiasis: a review. Int J Pharmaceut Sci Res 2011;2(6):1412. [10] H€am€al€ainen M, Nieminen R, Vuorela P, Heinonen M, Moilanen E. Anti-inflammatory effects of flavonoids: genistein, kaempferol, quercetin, and daidzein inhibit STAT-1 and NF-kB activations, whereas flavone, isorhamnetin, naringenin, and pelargonidin inhibit only NF-kB activation along with their inhibitory effect on iNOS expression and NO production in activated macrophages. Mediat Inflamm 2007;2007:45673. [11] Ito H, Kotake T, Nomura K. Effect of ethyl icosapentate on urinary calcium excretion in calcium oxalate stone formers. Urol Int 1995;54:208e13. [12] Stephen I, Revathi G, Pradeepha P, Arthy M, Swathi PR, Mounnissamy VM. Diuretic activity of rutin isolated from cansjera Rheedii J. Gmelin (opiliaceae). 2018. [13] Kim CH, Chung DY, Rha KH, Lee JY, Lee SH. Effectiveness of percutaneous nephrolithotomy, retrograde intrarenal surgery, and extracorporeal shock wave lithotripsy for treatment of renal stones: a systematic review and meta-analysis. Medicina 2020;57(1):26. [14] Korah MC, Rahman J, Rajeswari R, Sherief H, Lalitha V, Sengottavelu S, et al. Evaluation of diuretic efficacy and antiurolithiatic potential of ethanolic leaf extract of Annona squamosa Linn. in experimental animal models. Indian J Pharmacol 2020;52:196e202. [15] Peng Y, Ao M, Dong B, Jiang Y, Yu L, Chen Z, et al. Anti-inflammatory effects of curcumin in the inflammatory diseases: status, limitations and countermeasures. Drug Des Dev Ther 2021;15:4503. [16] Larson AJ, Symons JD, Jalili T. Therapeutic potential of quercetin to decrease blood pressure: review of efficacy and mechanisms. Adv Nutr 2012;3(1):39e46. [17] Lieske JC. Probiotics for prevention of urinary stones. Ann Transl Med 2017;5(2). [18] Liu Y, Shi L, Qiu W, Shi Y. Ferulic acid exhibits anti-inflammatory effects by inducing autophagy and blocking NLRP3 inflammasome activation. Mole Cellular Toxicol 2022:1e11. [19] Anu V, Akhila S, Kumar IA, Antony S. In-vitro antiurolithiatic activity of macerated aqueous extract of Terminalia chebula by using titrimetry method. Int J Pharmacogn 2020;7(6):144e7. [20] Patel TB, Golwala DK, Vaidya SK. Antiurolithiatic activity of alcoholic leaf extract of Mallotus philippinensis lam. Against ethylene glycol induced urolithiasis in rats. Aegaeum J 2020;8(4):759e65. [21] Shirani M, Arjaki D, Kheiri S, Bijad E, Mohammadi S, Lorigooini Z. An in vitro screening potential traditional medicinal plants for nephrolithiasis. Clin Phytosci 2020;6(1):1e8. [22] Mosquera DMG, Ortega YH, Quero PC, Martínez RS, Pieters L. Antiurolithiatic activity of Boldoa purpurascens aqueous extract: an in vitro and in vivo study. J Ethnopharmacol 2020;253:112691. [23] Kushagra D, Rajiv S, Ruchi G, Neelesh M. In-vitro evaluation of anti-urolithic activity of leaves extract of Costus igneus. Res J Pharm Technol 2020;13(3):1289e92. [24] Akbari F, Dashti A, Vahedi L. Effect of prunus mahaleb L. Seed extract on ethylene glycol-and ammonium chloride-induced urolithiasis in BALB/c mice. Iran J Med Sci 2020;45(2):134. [25] Yasui T, Tanaka H, Fujita K, Iguchi M, Kohri K. Effects of eicosapentaenoic acid on urinary calcium excretion in calcium stone formers. Eur Urol 2001;39:580e5. [26] Mogna L, Pane M, Nicola S, et al. Screening of different probiotic strains for their in vitro ability to metabolise oxalates: any prospective use in humans? J Clin Gastroenterol 2014;48(Suppl. 1):S91e5.

CHAPTER 13

Enzymatic preparation, purification, and therapeutic applications of marine oligosaccharides Md Imran, Hetika Kotecha, Elaine Da Costa, Devika R. Jadhav and Sanjeev C. Ghadi School of Biological Sciences and Biotechnology, Goa University, Taleigao Plateau, Goa, India

1. Introduction The marine realm covers a majority of the Earth’s surface providing habitat to various microbes. These microbes are adapted to harsh conditions such as low light intensity, low nutrient levels, varying salinity, low temperatures, and high-pressure conditions for their survival [1,2]. The bacterial population mainly dominates in the marine environment as compared to other microbes as they thrive in diverse marine econiches like surface waters, ocean floors, hydrothermal vents, and coral reefs. Due to their abundance in diversities, these marine bacteria play a vital role in the carbon recycling of the marine environment [1,3]. The purified bacterial polysaccharide degrading enzymes are being used for the mass production of oligosaccharides at the laboratory or industrial scale. Various complex polysaccharides (CPs) such as agar, alginate, carrageenan, chitin, pectin, pullulan, and ulvan are abundantly found in the marine environment [4,5]. CPs are degraded by the action of carbohydrate active enzymes (CAZymes). The marine environment offers a close association between seaweeds and extracellularly produced microbial polysaccharide degrading enzymes leading to the formation of oligosaccharides as shown in Fig. 13.1 [5e8]. Various bacterial CAZymes have been studied in the marine environment for their capability to degrade multiple CPs. Flammeovirga, Microbulbifer, and Saccharophagus sp. are a few bacteria known to degrade multiple polysaccharides [9e11]. The marine bacteria produce catalytically diverse polysaccharide degrading enzymes leading to the formation of structurally diverse oligosaccharides with different degrees of polymerization (DP). Furthermore, the oligosaccharides produced by enzymatic hydrolysis are known to possess unique chemical properties such as varying DP, the flexibility of glycosidic bonds, and molecular weights that confer diverse pharmacological activities. Several researchers are conducting studies on oligosaccharides obtained from the marine environment and significant efforts have been made to develop potential drugs for various chronic diseases from marine oligosaccharides [12,13]. Increasing interest in research related to oligosaccharides has confirmed varying bioactivities such as anti-

New Horizons in Natural Compound Research ISBN 978-0-443-15232-0, https://doi.org/10.1016/B978-0-443-15232-0.00028-X

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Figure 13.1 Polysaccharides of seaweed origin and screening for polysaccharide degrading bacteria.

biofilm, antidiabetic, anticoagulation, anti-inflammatory, antitumor, antimicrobial, and antioxidant [14,15]. Oligosaccharides obtained from polysaccharide degradation by their respective CAZymes demonstrate biocompatibility with little or no toxicity [16]. At neutral pH, marine oligosaccharides with low viscosity and high solvency have increased their potential to be used as drugs for various treatments. Due to their nontoxic, nonallergic, and biodegradable properties, these oligosaccharides have enhanced scientific interest [17]. Alginate, carrageenan, chitosan, and fucoidan are widely used in biomedical and pharmaceutical industries because of their unique biological and physicochemical properties [18]. This chapter describes the production of agar, carrageenan, and alginate oligosaccharides from seaweeds/CPs using marine bacterial agarase, carrageenase, and alginate lyase, respectively. The methodology employed for purifying and separating enzymatically produced oligosaccharides is discussed. Furthermore, the structural peculiarities owing to the biological activities of enzymatically produced agar, carrageenan, and alginate oligosaccharides are also described. Additionally, applications of these oligosaccharides in biomedicine are also mentioned.

Enzymatic preparation, purification, and therapeutic applications of marine oligosaccharides

2. Marine polysaccharides and their oligosaccharides Marine organisms like bacteria, cyanobacteria, fungi, algae, and crustaceans produce a diverse range of polysaccharides. Marine oligosaccharides are produced either naturally or from chemical/enzymatic hydrolysis of marine polysaccharides [19,20]. The enzymatic polysaccharide hydrolysate produces oligosaccharides that are low molecular weight and are composed of sugar moieties in the range of 10e12 monosaccharides. Oligosaccharides are known to show exceedingly high structural diversity and DP that confer diverse biological activities to them [21]. One of the significant components of the cell walls of Rhodophyceae is agar [22]. Agar is routinely extracted from Gelidium and Gracilaria. Agar is a heterogeneous polysaccharide composed of agarose and agaropectin polymers [23]. Although typically agar consists of 70% agarose and 30% agaropectin, the proportions of agarose and agaropectin fractions vary depending on the source of agar. Agarose is a chain of repeating disaccharide agarobiose units (made up of b-D-galactose and 3,6-Anhydro-a-Lgalactose). Agar oligosaccharides are classified into two groups namely agarooligosaccharides and neogaro-oligosaccharides. This classification is based on the presence of galactose or 3,6-Anhydro-a-L-galactose at the terminal end. The agarooligosaccharides contain a galactose molecule at the terminal reducing end whereas the neoagaro-oligosaccharides have 3,6-Anhydro-a-L-galactose at their terminal end. Carrageenan is a linear, anionic, sulfated polysaccharide obtained from certain red seaweeds like Chondrus crispus and Eucheuma [24]. It comprises alternating units of b-Dgalactose and 3,6-Anhydro-a-D-galactose with a-(1,3) and b-(1,4) glycosidic linkage. Depending on the pattern of sulfation, anhydrogalactose content and substitution by glycosyl, methoxyl, and/or pyruvate groups, there are about 20 different types of carrageenan. Kappa (k), iota (i), and lambda (l) types are the most commercially exploited carrageenans, having one, two, and three sulfate ester groups respectively on the repeating disaccharide units [25]. Carrageenases are endolytic enzymes that generate even numbered oligosaccharides by cleaving the b-(1, 4) glycosidic bond. Alginate is found in brown macroalgae (Phaeophyceae) cells like Laminariales and Fucales. Alginate oligosaccharides (AOS) are produced upon the degradation of alginate. AOS are linear oligomers linked with a 1,4-Glycosidic bond with DP of two to eight monomeric molecules. This acidic polysaccharide is a polymer of a-L-guluronate (G), and its C5 epimer b-D-mannuronate (M) [26]. These monomer units can be linked to form three different types of polymer chains: poly b-D-mannuronate (polyM), poly a-L-guluronate (polyG), and heteropolymer (polyMG). Alginate polymers are known to exist as one of the above types. However, a single alginate polymer may comprise all three types of chains viz. polyG, polyM, and polyMG. Differences in the M/G ratio

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and its distribution confer varying physical properties such as the stiffness to the polysaccharide [27]. More rigidity of the G block is attributed to its axial-axial linkage compared to the M blocks that are di-equatorially linked.

3. Enzymatic preparation of bioactive oligosaccharides from complex polysaccharides 3.1 Agaro-oligosaccharides (AGOS) Agarase enzymes, responsible for agar degradation, are classified into a-Agarases (GH96 and GH117) and b-Agarases (GH16, GH50, GH86, and GH118). b-Agarases belonging to (GH 16), exhibit an endo mode of action and produce neoagarotetraose and neoagarohexaose, whereas a-Agarase (GH117) eliminates the 3,6-Anhydro-L-galactose from the nonreducing end of neoagaro-oligosaccharides [28]. AGOS prepared from b-agarase hydrolysis of the agar obtained from G. lemaneiformis in 50 mM sodium phosphate buffer at pH 6.5 was reported to have an enzyme activity of 10 U/mL [29]. This process was carried out at 55
C for 2 h. The amount of oligosaccharides thus obtained was estimated using the 3,5-Dinitrosalicylic method. A 59 kDa b-Agarase from Microbulbifer strain CMC-5 was utilized for agar hydrolysis at 50
C and pH 7.0 [30]. 0.05% of melted agarose can be used as a substrate with b-Agarase for 40
C/24 h to produce neoagaro-oligosaccharides [31]. The authors - successfully purified neoagaro-oligosaccharides by enzymatic degradation of agarose with agarase (AgaA) at 37
C for 12 h in a rotatory shaker incubator [32]. The neoagarooligosaccharides hence obtained displayed prebiotic activity without any side effects. Highly soluble oligosaccharides produced by the action of agarase on agarose by the shake flask method at 50
C for 24 h (pH 5) demostrated antioxidant activity [33]. Similarly, the enzymatic breakdown of agar by the b-agarase from various bacteria like Alteromonas sp. C-1 (30
C, pH 6.5), Vibrio sp. JT10107 (30
C, pH 8.0), Bacillus megaterium (40
C, pH 6.6) and Agarivoran albus YKW-34 (40
C, pH 8.0) have been reported [34e37]. 3.2 Carrageenan oligosaccharides (COS) Carrageenases hydrolyze internal b-(1e4) glycosidic bonds of carrageenan and are categorized as k-, i-, and l-Carrageenases based on their substrate specificity. The enzyme hydrolyses carrageenan by a double displacement reaction in the presence of carboxylic acid-containing amino acid residues [38]. The authors in Ref. [32] used carrageenan from Kappaphycus alvarezii for the production of carrageenan oligosaccharides (COS) via enzymatic degradation by carrageenase for 6 h at 28
C. The unhydrolyzed polysaccharide was eliminated by centrifugation, followed by partial purification of the supernatant using a rotary evaporator at 60
C and ethanol precipitation. The authors in Ref. [39] prepared COS using a two-step enzymatic hydrolysis approach by treating Eucheuma cottonii first with cellulase followed by

Enzymatic preparation, purification, and therapeutic applications of marine oligosaccharides

recombinant k-Carrageenase. The authors in Ref. [40] optimized the reaction (4 h/pH 7.5/50
C) between k-Carrageenan and a-Amylase to obtain COS. A study shows that k-Carrageenase immobilized on carboxyl functional magnetite nanoparticles is an effective method for the production of COS [41]. The k-Carrageenase from various marine bacteria namely Cytophaga sp. 1k-C783, Vibrio sp. CA-1004 and Bacillus subtilis, ϊ-Carrageenase from Pseudoalteromonas porphyrae and l-Carrageenase from Pseudolateromonas sp. CL-19 are reported to be optimally active at temperatures varying from 25 to 55
C and with pH of 5.6e8.0 [42e46]. 3.3 Alginate oligosaccharides (AOS) Alginate lyase degrade alginates by b-elimination to form unsaturated AOS. Alginate lyases are classified into 12 different polysaccharide lyases families (PL). They can be divided into polyM-specific lyases, polyG-specific lyases, and bifunctional lyases along with their categorization as endo- and exo-type of enzymes [47]. Sodium alginate depolymerization can be carried out using partially purified alginate lyase enzyme in phosphate citrate buffer (pH. 7.0) at 40
C followed by incubation in a boiling water bath for 15 min to stop the reaction [48]. The partial purification of the AOS thus obtained can be done using the ethanol precipitation method. AOS were generated by Ref. [49]; using alginate lyase produced by the marine bacterium Pseudomonas sp. HJZ216 against alginate dissolved in TriseHCl buffer (50 mmol L1, pH 7.0). This procedure was carried out at 30
C to the point where absorbance at 235 nm changed and then stopped reactions by heating the solution in a boiling water bath. The pH of the filtrate obtained from the degraded alginate solution was brought to 2.85 to separate AOS from the non-depolymerized substrate followed by ethanol precipitation. Bacillus subtilis KCTC 11782BP was inoculated with 3% sodium alginate extracted from Laminaria hyperborean and incubated for 14 days at 37
C [50]. The culture supernatant obtained after the incubation was centrifuged and passed through an Amicon filter for separating oligosaccharides with a molecular weight below 10 kDa.

4. Purification of oligosaccharides The oligosaccharides prepared by enzymatic reactions may give rise to contaminants like unhydrolyzed polysaccharides, monosaccharides, furfurals etc. [51]. These impurities may interfere with the biological activities of the oligosaccharides and compromise their medicinal properties. To obtain pharmaceutical-grade oligosaccharides, the resultant enzymatic product needs to be subjected to purification. A schematic representation depicting oligosaccharide extraction, purification, and its application is shown in Fig. 13.2. The hydrolysate obtained by enzymatic degradation is subjected to ethanol precipitation and membrane separation methods for partial purification [52]. The percentage of alcohol used for precipitating the unhydrolyzed polysaccharides and the required time of

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Figure 13.2 Enzymatically derived marine oligosaccharides, purification, and its therapeutic applications.

incubation are optimized as per the requirements. The authors in Ref. [53] used activated carbon along with 10% alcohol for the partial purification of oligosaccharides followed by gel filtration by Sephadex LH-20 columns. Size exclusion chromatography is frequently used for oligosaccharide purification. The authors in Ref. [54] used Sephadex G 25 (for hexose and octaose) and Sephadex G 10 (for biose and tetraose) columns to purify AGOS, followed by HPLC using isocratic mode on the C8 column with 40% MeOH as the mobile phase. Similarly, the authors in Ref. [13,55] employed Bio-Gel P-2 columns using water and 0.1 mol/L NH4HCO3 as eluents, respectively. Bio-Gel P-6 gel filtration columns are also reported for successful purification of AGOS and COS [56,57]. Ref. [31] refined the AGOS cocktail with Asahipak GS-320 HQ multimode column and deionized water as eluent. AGOS have been isolated using diethylaminoethyl (DEAE)-cellulose 52 ion exchange affinity columns by eluting it with 2 M NaCl at 10
C [58]. Anion exchange columns like AG MP-1 resin column and Q-Sepharose Fast Flow are used as purification systems by applying a gradient of acetic acid (0.5e2.0 M) or 0.2e0.8 M Sodium acetate buffers [59,60]. Ref. [61] used “click” maltose columns with three mobile phases (water, acetonitrile, ammonium formate) for oligosaccharide separation. Ref. [62] reported the use of high-performance anion exchange chromatography (HPAEC) by using Dionex CarboPacÔ PA100 and PA100 columns to obtain AGOS fractions. Along with the methods mentioned above, high-performance liquid chromatography (HPLC) has been routinely used in the carbohydrate purification step after

Enzymatic preparation, purification, and therapeutic applications of marine oligosaccharides

enzymatic hydrolysis. HPLC can be also be employed using a reversed-phase C18 column with acetonitrile and ammonium acetate as mobile phase [57]. Ref. [63] separated COS fractions by applying a NaCl gradient (0.1e2.0 M) on a POROS HQ50 column by the SAX-HPLC technique. The carbohydrates can be labeled with 3-Amino-9Ethylcarbazole (AEC) and processed using an LTQ-XL ion-trap mass spectrometer and an HPLC system [64]. Ref. [65] performed COS fractionation using medium pressure-liquid chromatography (MPLC) using HiLoadÔ SuperdexÔ and 0.1 M NH4HCO3 as an elution buffer. Ref. [66] purified alginate oligosaccharides employing reverse phase chromatography with SepPak C18 cartridges and further processed it using ultra-highperformance liquid chromatography (UHPLC) system.

5. Application of oligosaccharides in biomedicine 5.1 Agaro-oligosaccharides (AGOS) Based on molecular structure composition and linkages, different agar-derived oligosaccharides have different properties that can be exploited for various uses in biomedicine (Table 13.1) [67]. Reports suggest that AGOS derived by enzymatic methods exhibit higher antioxidant activities as compared to AGOS derived by acid hydrolysis [29]. Ref. [33] conducted studies to determine the relationship between antioxidant activity and the degree of hydrolysis. They enzymatically derived AGOS with different degrees of hydrolysis from 3.73% to 20.51% showing DPPH and ABTS radical scavenging activities. Their study suggests that the higher the DP, the increase in antioxidant activities was noted. Enzymatically derived AGOS have been reported to diminish alcohol-induced liver injury by boosting the action of hepatic alcohol dehydrogenase. This hepatoprotective effect is a result of antioxidant activity of AGOS [58]. Ref. [68] reported that AGOS obtained during 6-h hydrolysis with a higher DP demonstrated the higher antioxidant activity as compared to AGOS obtained during 12-h hydrolysis with a lower DP. Recent reports suggest that 3,6-Anhydro-Lgalactose (AHG), obtained from Rhodophyta, has skin-whitening activity. AGOS are known to possess antioxidant, anti-inflammatory, anti-tyrosinase as well as skin moisturizing effects [69]. Ref. [69] also suggested that AHG demonstrated the strongest antimelanogenic effect on murine B16 melanoma (B16F10) cells and human epidermal melanocytes. Xylitol is an anticariogenic sugar substitute, inhibiting the growth of Streptococcus mutans (at conc. 30 g/L and above) an organism responsible for dental caries. Ref. [70] studied the anticariogenic property of AHG using S. mutans. The study demonstrated that S. mutans is incapable of fermenting AHG. The inhibitory effect of AHG is much higher as compared to that of xylitol. The growth of S. mutans and its lactic acid production was found to be completely negligible at 10 g/L of AHG. Thus, AHG could be a strong potential anticariogenic agent to prevent dental caries [70].

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Table 13.1 Biomedical applications of Agar oligosaccharides. S. No.

Marine oligosaccharides

1

Agarooligosaccharides (AO) Agarooligosaccharides AOS-1, AOS-2 and AOS-3 Neoagarooligosaccharides NA2, NA4, NA6

2

3

Enzyme

Genes cloned from/Enzyme source

Microbulbifer sp. Q7

Prebiotic

[107]

Agarase

Unidentified marine bacterium

[60]

b-Agarase

Streptomyces coelicolor A3(2)

1. Antioxidant activity 2. Radical scavenging 1. Antia-Glucosidase activity 2. Anti-tyrosinase activity 1. Whitening effect toward B16F10 murine melanoma cells 2. Antioxidant activity 1. Antioxidant activity 2. Radical scavenging Prebiotic

Neoagarooligosaccharides NA4

b-Agarase

Pseudoalteromonas sp. AG4

5

Neoagarooligosaccharides (NAOS)

Agarase

Gelidium amansii

6

Neoagarooligosaccharides (NAOS) Neoagarotetraose

b-Agarase AgaA and AgaB b-Agarase AgWH50A

Neoagarooligosaccharides (NAOs) NA2 and NA4

b-Agarase

8

Refs.

Agarase

4

7

Biomedical properties

e Agarivorans gilvus WH0801

e

Inhibit inflammation in LPS-stimulated macrophages Antidiabetic (type II diabetes mellitus)

[108]

[109]

[29]

[32]

[110]

[111]

5.2 Carrageenan oligosaccharides (COS) Sulfated polysaccharides such as carrageenan have been gaining attention due to their wide applications. Apart from being used as an emulsifier and stabilizer in various foods, the oligosaccharides formed by enzymatic degradation of carrageenan have been reported

Enzymatic preparation, purification, and therapeutic applications of marine oligosaccharides

to possess various bioactivities. These include antiviral, immuno-modulatory, antimicrobial, anticoagulant, antidiabetic, antioxidant, and antitumor activities (Table 13.2). Carrageenans have been identified with high-anticoagulant activity and are used as anticoagulants in blood products [71]. COS appears to inhibit thrombin, thus preventing the formation of clots. COS with higher sulfate content depicted greater antithrombic activity. However, it was demonstrated that the position of substitution has a bigger impact on the anticoagulant activity as compared to the degree of substitution [72]. Treatment of cells with purified COS significantly decreased reactive oxygen species (ROS) levels in the cells [65]. The antioxidant activities of COS have been attributed to the DP, content of reducing sugar, substituent molecules, and the reducing terminus of COS [73]. Chemical modification of COS such as sulfation, phosphorylation, and acetylation can enhance their antioxidant activity [74]. Furthermore, COS with lower DP had higher antioxidant activity [75]. l-Carrageenan possesses better antioxidant activity than k-Carrageenan and i-Carrageenan [76]. Table 13.2 Biomedical applications of carrageenan oligosaccharides.

S. No.

Marine oligosaccharides

1

Carrageenan oligosaccharides

k-Carrageenase

2

k-Carrageenan oligosaccharides (KOS)

k-Carrageenase

3

K-Neocarrabiose, kNeocarratetraose, k-Neocarrahexaose k-Neocarrabiosesulfate, k-Neocarrahexaosesulfate and kNeocarraoctaose ‘sulfate k-Carrageenan oligosaccharides CO-1, CO-2 and CO-3

k-Carrageenase

4

5

Enzyme

Genes cloned from/ Enzyme source

Cytophaga sp. MCA2 Cellulophaga lytica strain N52

k-Carrageenase

Thalassospira sp. Fjfst332 e

k-Carrageenase

e

Biomedical properties

Refs.

Antitumor activity

[112]

Immune regulation (inhibit the viability and content of NO, TNF-a and IL-10) Antiinflammatory activity 1. Antitumor 2. Antiangiogenic activity

[113] [114]

Inhibits multiplication of influenza A virus

[65]

[52]

[115]

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COS and their different derivatives play a crucial role as anticancer agents as they have been reported to possess antiproliferative, antimetastatic, and antioxidant activity [76]. They achieve this by blocking interactions between cancer cells and the basement membrane, thereby inhibiting tumor cell proliferation and tumor cell adhesion. The COS carraheptaose simultaneously inhibits both bFGF signaling and heparanase activity thus preventing the growth of the tumor cell and its metastasis respectively [77]. COS plays a role as a free radical scavenger and suppress the development of cancer by preventing oxidative damage in healthy cells [75]. Additionally, non-sulfonated or lightly sulfonated COS depicted more antitumor activity in comparison to the highly sulfonated COS. COS is known to have therapeutic properties as they affect the immune system in both ways, by stimulating or suppressing it [78]. COS is known to cause inflammation by triggering TNF-a production in mice [79]. In vivo experiments suggest that COS can also act as inhibitors of macrophage function. k-Carrageenan hexamers regulate macrophage activity by binding to CD14 and suppressing the NF-kB inflammatory pathway [80]. Studies done on COS of variable lengths suggest that DP along with the number of sulfate groups affects their anti-inflammatory activity. Seaweed polysaccharides are a good source of anti-diabetic agents. Inhibition of a-Amylase and a-Glucosidase reduces the rate of carbohydrate absorption after food intake. The antidiabetic effect of hydrolyzed carrageenans can be attributed to their inhibitory effects on these enzymes, thus controlling blood glucose levels in patients with diabetes mellitus [81]. COS has also been incorporated into sponge dressings as an immunomodulatory biomaterial to accelerate wound healing in diabetic rats [82]. There are numerous reports of COS action against viruses such as HIV, HPV, human metapneumovirus (hMPV), and HSV-2. The antiviral activity is mainly via two mechanismsd(1) binding to viral particles and inactivating them or (2) preventing the release of viral particles from the host cell by modifying the cell membrane plasticity. The increasing molecular weight of COS enhanced their virus-killing activity [83]. Carrageenan and its corresponding oligosaccharides also act as prebiotic agents. They promote the growth of beneficial human gut microflora which generate bioactive metabolites capable of inhibiting pathogens and regulating the host immune system [84]. They have also been used for the treatment of bowel problems such as diarrhea, constipation, and dysentery. COS have potential cosmetic applications as they have been reported to be useful for skin whitening as they suppress the production of melanin by inhibiting tyrosinase [85]. COS also exhibits cholesterol-lowering effects by increasing viscosity in the gastrointestinal tract as well as binding to cholesterol-rich bile [86]. 5.3 Alginate oligosaccharides (AOS) Alginate oligosaccharides (AOS) are known for their non-immunogenicity, low toxicity, and biodegradability (Table 13.3). These properties make oligosaccharides derived from alginate an excellent biomaterial having various pharmacological applications [12].

Table 13.3 Biomedical applications of alginate oligosaccharides. Marine oligosaccharides

Enzyme

Genes cloned from/Enzyme source

1

Alginate oligosaccharide

Alginate lyase

Bacterial

2 3 4

Alginate oligosaccharide Alginate oligosaccharide Alginate oligosaccharides (AOS)

Alginate lyase Alginate lyase S Alginate lyase

e Sphingobacterium e

5

Alginate oligosaccharides

Microbulbifer sp. BY17

6

Alginate oligosaccharides

Alginate lyase, BY17PV7 Alginate lyase

7

Alginate oligosaccharides (AlgO) Alginate oligosaccharides ALO1, ALO3x Di-alginate oligosaccharides (AOS)

Alginate lyase

Microbulbifer sp. Q7

Alginate lyase

Alteromonas portus HB161718T Pseudoalteromonas carrageenovora ASY5

8 9

Alginate lyase Aly1281

Pseudoalteromonas sp. 272

Biomedical properties

Refs.

Improves immunometabolic pathways (reduction in obesity) Prebiotic Antioxidant 1. Anti-hyperglycemic activity 2. Reduced high glucoseinduced mortality, oxidative stress and apoptosis 1. Antioxidant activity 2. Radical scavenging Immunomodulatory (regulation of nitric oxide (NO), reactive oxygen species (ROS), and tumor necrosis factor (TNF)-a production) Prebiotic

[116]

[117] [92] [118]

[119] [120]

[107]

Antioxidant activity

[98]

1. Antioxidant activity 2. Radical scavenging

[121]

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S. No.

241

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Ref. [87] demonstrated that AOS possesses antitumor activity due to a significant decrease in the development of osteosarcoma. AOS prepared from alginate lyase and Vibrio sp. 510 when sulfated with formamide-chlorosulfonic acid showed a significant drop in tumor weight when compared with the control [88]. OligoG, an alginate-oligosaccharide nanomedicine obtained from hydrolysis of Laminaria hyperborean alginate, was screened against 21 multidrug-resistant clinical isolates. The study suggested that OligoG leads to an increase in the efficacy of traditional antibiotics against various important clinical isolates. OligoG also showed some anti-biofilm activity suggesting it as a novel nanomedicine target for multidrug resistant clinical pathogens [89]. Anti-biofilm activity plays a vital role in eliminating chronic bacterial infections. AOS possesses an inhibitory effect on the growth of many pathogenic bacteria [90]. AOS produced by the action of alginate lyases on alginate from Flavobacterium sp. LXA having DP of 6.8 inhibited the proliferation of Pseudomonas aeruginosa [48]. Ref. [91] performed studies on a novel oligomannuronate-chromium (III) complex known as OM2 derived from marine alginate. OM2 induces mitochondrial biogenesis and boosts lipid metabolism better than oligomannuronate (OM). Reports also suggest that OM2 has played an important role in sensitizing insulin action better than OM and can be used in treating Type II diabetes. Ref. [92] prepared AOS enzymatically from Sphingobacterium sp. which showed complete inhibition of lipid oxidation in emulsions and radical scavenging activity against superoxide radicals, ABTSþ, eOH. Enzymatically derived AOS using alginate lyase from Microbulbifer sp. ALW1 showed antioxidative properties by scavenging DPPH, ABTSþ, and eOH radicals [93]. 5.4 Other oligosaccharides Fucoidan is a highly sulfated polysaccharide found in the cell walls of some brown seaweeds. It has a complex structure normally composed of fucose, uronic acid, galactose, and xylose. Fucoidan oligosaccharides (FOS) have multiple applications as a result of their low viscous and highly soluble nature along with their exceptional bioavailability. The physiological and metabolic benefits of FOS are numerous. They have been shown to significantly reduce serum glucose, triglyceride, cholesterol, and low-density lipoprotein (LDL) levels [94]. FOS are potent free-radical scavengers and thus they exhibit antioxidant activity higher than that of fucoidan [95]. FOS also shows anticancer activity by restoring the immune system, preventing the attack of ROS and inhibiting the nutrition of cancer cells and other pathways. For instance, hydrolyzed fucoidan from Undaira pinnatifida enhances anticancer activity from 37% to 75%. Low molecular weight FOS are reported to have higher cytotoxicity in cancer cells by inhibiting cell transformation [96].

S. No.

Marine oligosaccharides

Enzyme

Genes cloned from/Enzyme source

Biomedical properties

Refs.

1

Laminaran oligosaccharides

2

Porphyran oligosaccharide (PO)

Endo-b-(1 fi 3)glucanase e

Bacillus circulans

Antiapoptotic activity

[99]

Arthrobacter sp.

1. Anti-obesity 2. Antihypercholesterolomic activity Antioxidant activity Antiviral (vesicular stomatitis virus) 1. Antioxidant activity 2. Angiotensin-converting enzyme-inhibitory activities

[122]

3 4

Porphyran oligosaccharides Ulvan F1

a-Agarase, AgaA33 Ulvan lyase

Thalassomonas sp. JAMB-A33 -

5

Ulvan oligosaccharides (UOS)

e

Pseudomonas vesicularis MA103 and Aeromonas salmonicida MAEF108

[123] [102] [124]

Enzymatic preparation, purification, and therapeutic applications of marine oligosaccharides

Table 13.4 Biomedical applications of marine oligosaccharides.

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Laminarin (also referred to as laminaran) is a CP found in brown seaweeds and is used as a carbohydrate food reserve. The biological functions of laminarin and laminarin oligosaccharides (LOS) have been widely reported and can be applied to drug development. They have prebiotic, antioxidant, and anti-inflammatory properties [97e99]. It also exhibits excellent antitumor and anticancer activities by inducing apoptosis, inhibiting angiogenesis and preventing metastasis [69,100,101]. Ulvan is a sulfated polysaccharide found in the cell walls of some green seaweeds. Various applications of ulvan lyase-produced ulvan oligosaccharides (UOS) have been reported. These include antiviral, anti-inflammatory, antioxidant, anticancer as well as immunomodulation activity [102e106] (Table 13.4).

6. Conclusion and future prospects Marine bacterial enzymes such as agarase, alginate lyase, carrageenase and chitinase have been extensively used for the production of seaweed-derived oligosaccharides. Bacterial enzymes could be exploited for the production of oligosaccharides with various degrees of polymerization that could be studied for determining potential therapeutic activities. The enzyme-derived oligosaccharides could be purified and structures determined. Understanding the basic biology of seaweed-derived oligosaccharides and -omics technology to explore the metabolic pathways of polysaccharide degradation, along with molecular investigations will enhance our understanding of the enzymatic mechanisms of seaweed oligosaccharides production leading to low-cost production technologies. The future challenge will be to develop a chemical synthesis approach to synthesize these therapeutic oligosaccharides for large-scale production and possible commercialization of seaweed-derived oligosaccharides.

Acknowledgments The authors acknowledge the Department of Biotechnology, New Delhi, India for funding project (DBT sanction no. BT/PR41474/NDB/39/760/2020). The authors are also grateful to Goa University for providing infrastructure facilities.

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Enzymatic preparation, purification, and therapeutic applications of marine oligosaccharides

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Further reading [1] Hu X, Jiang X, Aubree E, Boulenguer P, Critchley AT. Preparation and in vivo. Antitumor activity of k-Carrageenan oligosaccharides. Pharmaceut Biol 2006;44(9):646e50. [2] Huang Y, Jiang H, Mao X, Ci F. Laminarin and laminarin oligosaccharides originating from brown algae: preparation, biological activities, and potential applications. J Ocean Univ China 2021;20(3): 641e53. [3] Kim YE, Kim YJ. Effects of nanofibrous membranes containing low molecular weight b-Glucan on normal and cancer cells. J Nanosci Nanotechnol 2017;17(5):3597e605. [4] Xu XQ, Su BM, Xie JS, Li RK, Yang J, Lin J, et al. Preparation of bioactive neoagaroligosaccharides through hydrolysis of Gracilaria lemaneiformis agar: a comparative study. Food Chem 2018;240: 330e7.

CHAPTER 14

Marine microalgae: an emerging source of pharmaceuticals and bioactive compounds M.K. Anusreea, K. Manasa Leelaa, M. Sreehari, Subhisha Raj, Arathi Sreenikethanam and Amit K. Bajhaiya Algal Biotechnology Lab, Department of Microbiology, School of Life Sciences, Central University of Tamil Nadu, Thiruvarur, Tamil Nadu, India

1. Introduction Microalgae are photoautotrophic organisms with different phyla such as Cyanophyta, Chlorophyta, Rhodophyta, Haptophyta, Streptophyta, and Heterokondophyta. Primary and secondary metabolites produced in microalgae are complex organic compounds accumulated by microalgae with the help of H2O, CO2, and energy from sunlight. They are adapted to different environmental stress conditions that extend their growth ranging from freshwater to extreme salinity. They can survive on moist, black earth, and even on desert sands, extending up to clouds [1]. Their large spectrum of existence leads to varieties of chemical compounds with a myriad of properties and represents a remarkable opening to discover novel metabolites, or to produce available metabolites at lower costs [2]. Microalgae possess metabolic versatility and plasticity that are commended in physiological states. Their secondary metabolism can be effortlessly activated by externally applied stresses [3]. Microalgae are deemed to be cosmeceutical with their effortless response to stress. Cosmeceuticals are products with biologically active ingredients with medical or druglike advantages, which aim to improve the structure, morphology, and appearance of skin [4]. Polysaccharides are the active compounds in microalgae. They have potential application in the prevention of blemishes, damaged skin repairing, and inhibiting inflammation process (e.g., genus chlorella). Hence, they are used to produce thickener moisturizers and gelling agents. Numerous other bioactive substances in microalgae accelerate healing process and maintain skin moisture [5,6]. The quest for these biologically active molecules to treat metabolic disorders and immune ailments drives the way to harvest bioactive compounds from microalgae. The easiness of micro-algal cultivation benefited from using its metabolites in biological applications related to health. Microalgae are considered as sustainable source of biologically a

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© 2023 Elsevier Inc. All rights reserved.

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Figure 14.1 Flow-diagram showing various applications of microalgal biomass.

active compounds like biofuels, foods, feed, and pharmaceuticals [7] (Fig. 14.1). Microalgae have 20%e40% higher productivity when compared to oil crops. Some of them can build up to 80% dry lipid biomass weight [8,9]. Another application of biologically active molecules in microalgae is as nutraceuticals, which focus on saying “Prevention is better than cure.” Microalgal nutraceuticals include compounds like phycocyanin, astaxanthin, Beta carotene, fucoxanthin, lutein, lycopene, phycobiliproteins etc. Microalgae are creators of biomolecules that are relevant in diverse domains. Here in this chapter, we will focus on bioactive compounds and pharmaceuticals from microalgae.

2. Bioactive compounds from microalgae Microalgae are autotrophic organisms that can grow in a wide range of habitats. To survive under different conditions, different microalgae produce different types of secondary metabolites which have beneficial therapeutic activities like anti-cancerous, antimicrobial, etc. These secondary metabolites of microalgae are referred to as “HighValue Molecules” (HMV). Microalgae have the ability to co-produce other compounds like pigments, proteins, polyunsaturated fatty acids, and antioxidants and are attracting interest as therapeutic agents for a variety of health conditions as well as additional uses in the food, cosmetics, energy, and pharmaceutical industries [10].

Marine microalgae: an emerging source of pharmaceuticals and bioactive compounds

2.1 Secondary metabolites from microalgae Secondary metabolites are the organic compounds that are indirectly involved in the usual growth and development of a living organism. These organic compounds have potential biological activities that contribute to the biological systems of the organism by which they are being produced [11]. The secondary metabolites are produced by many organisms and microalgae are one of these organisms that produce these metabolites via various metabolic pathways [12]. The metabolites which microalgae produce are mentioned below: 2.1.1 Carotenoids The carotenoids produced by microalgae have major commercial importance. Different carotenoids include b-carotene, astaxanthin, lycopene, lutein, and canthaxanthin [13] (Table 14.1). Carotenoids are lipophilic and naturally pigmented in yellow, orange, or red colors. These are a major class of terpenoids that commonly share a 40 Carbon backbone structure and are classified into carotenes and xanthophylls. Xanthophylls are the oxygenated derivatives of carotenes and are hydrophilic in nature. 2.1.1.1 b-carotene b-carotene is one of the most common carotenes produced by algal strains like D salina; Haematococcus sp. b-carotene is found to have high pro-vitamin A activity, which is important for proper eyesight and to maintain a healthy immune system [20,21]. Hence, the demand for b-carotene has increased and being used in preparations of multivitamins as well. Apart from this, b-carotene has been used as food colorant in many foods and beverages. It has been employed in animal feed and has shown to decrease the risks of AMD (age-related macular degeneration). b-carotene can also be used for the treatment of disorders like asthma, cardiovascular diseases, etc. [10,20,22]. Table 14.1 Carotenoids produced by various microalgae strains. Carotenoids

Source

Therapeutic indication

References

b-carotene

D. salina

[13,14]

Astaxanthin

Zeaxanthin

H. pluvialis, C. zofigiensis, C. vulgaris D. salina, C. pyrenoidosa, C. protothecoids C. ellipsodiea

Violaxanthin Fucoxanthin

C. ellipsoidea P. tricornutum

Antioxidant, provitamin A, AMD, liver fibrosis, anti-inflammatory Antitumoral, anti-oxidant, antiinflammatory AMD, atherosclerosis, retinal neural damage Antioxidant, maculopathy, cataracts, anti-inflammatory Anti-inflammatory, anti-cancer Antioxidant, anti-inflammatory, anti-cancer

Lutein

[15] [16] [17] [18] [19]

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2.1.1.2 Astaxanthin Astaxanthin is an oxidative derivative of carotenes that is red-orange in color. Astaxanthin is produced by algal species like Haematococcus pluvialis, Chlorella zofigiensis, Chlorella vulgaris and Chlorococcum sp. It is also reported in seafoods like salmon, shrimps, trouts, lobsters, and fish eggs. Its antioxidant qualities are thought to play a major part in a variety of other features, including protection against UV-light, photo-oxidation, inflammation, cancer, ulcers, bacterial infections, age-related disorders, or the enhancement of the immune response, heart health, eye health, liver function, joint health, and prostate health [10,23]. The use of astaxanthin from H pluvalis as anti-inflammatory agent has been experimented in zebrafish [24]. 2.1.1.3 Zeaxanthin Zeaxanthin is one of the carotenoids present as yellow color in nature. There are several different groups of microalgae that contain zeaxanthin, such as Cyanobacteria, Rhodophytes, Chlorophytes (green algae), and some species of heterokonts. Apart from these microalgae like D salina, Spirulina and Chlorella sp. also produce zeaxanthin and lutein [25,26]. It is used as supplements for age-related macular degeneration and as a yellow colorant in the food sector [27]. 2.1.1.4 Lutein Lutein is a lipid-soluble carotenoid obtained by humans from food. It is produced by some species of microalgae that produce zeaxanthin like D salina, Spirulina, and Chlorella sp [10]. It has various recognized therapeutic benefits, including assisting in the prevention of macular degeneration, lowering the risk of heart stroke and heart attack, and mitigating the effects of other severe metabolic disorders [28]. The accumulation of lutein and zeaxanthin in the macula of the eye acts as blue light filter or as anti-oxidant, resulting in the protection of the eyes [28,29]. The dietary intake of lutein has found to inhibit the development of several disorders like antherosclerosis [30,16]. Apart from this, there are several studies related to therapeutic effects of lutein which exhibit the decrease in the risk of age-related macular degeneration [31,16]. 2.1.1.5 Violaxanthin Violaxanthin (5,6,50 ,60 -diepoxy-5,6,50 ,60 -tetrahydro-,-carotene-3,30 -diol) is an orangecolored natural xanthophyll pigment found in plants, macro- and microalgae. Chlorella ellipsoidea and Dunaliella tertiolecta are two microalgae from which this substance has been extracted (J [32,33]. Violaxanthin is found to be a potent anti-cancer and antiinflammatory therapeutic. The violaxanthin isolated from C. ellipsoidea has shown to exhibit anti-proliferative activity of human colon cancer cell lines, HCT-116. It also shows anti-inflammatory effects on other inflammatory diseases by suppression of NF-

Marine microalgae: an emerging source of pharmaceuticals and bioactive compounds

kB and MAPK pathways [10,33]. An experimental study has revealed that the violaxanthin of C. ellipsoidea is a favorable species for the treatment of inflammatory disorders [34]. 2.1.1.6 Fucoxanthin Fucoxanthin is a golden-brown colored xanthophyll mainly produced by marine organisms. The different microalgae that produce this carotenoid are Bacillariophytes, Bolidophytes, Chrysophytes, Silicoflagellates, Pinguiophytes, and brown macroalgae (Phaeophytes). The commercial source of fucoxanthin is diatom, Phaeodactylum tricornutum. Fucoxanthin has many health benefits, which is one of the reasons it is gaining more commercial importance. It has been found to exhibit therapeutic activities like anticancer, anti-obesity, anti-inflammatory, anti-oxidant, and prevents cerebrovascular diseases [10,33]. One of the experimental studies on fucoxanthin of P tricornutum has demonstrated the anti-diabetic activity, where it shows inhibiting activity against a-amylase and a-glucosidase in 3T3- L1 cell lines linked to type-2 diabetes [35]. 2.1.2 Polyunsaturated fatty acids (PUFA) PUFAs are mostly obtained from fish oil, but due to their unpleasant smell and taste, their use has been limited. Alternatively, microalgae have gained importance as source of PUFAs namely EPA (eicosapentanoic acids) and DHA (docosahexanoic acid). Some of the microalgae strains that are rich in fatty acids are Tetraselmis sp. and Nannochloropsis oculata [36]. EPA and DHA are the two main u-3-fatty acids present in marine oils that have a wide range of biological activities. It is well established that EPA and DHA have an impact on blood pressure, coagulation, platelet and endothelial function, and lipoprotein metabolism [33]. The clinical trials of algal DHA has shown the reduction of serum triglyceride levels and seems to be reducing blood pressure as well [37]. EPA (eicosapeantanoic acid) is a precursor of various lipid regulators involved in cellular metabolism and serves several important functions in biological membranes. The effect of u-3-fatty acids also improves other health conditions such as dyslipidemia, hypertension, diabetes mellitus, and vascular diseases. The use of EPA in animal models has shown to be beneficial for diabetic animals [38,39]. DHA (docosahexanoic acid) is a significant component of the brain, retina, and heart muscle, and it has been linked to brain and ocular development as well as excellent cardiovascular health [39]. The DHA algal oil from Schizochytrium sp is found to have a good safety profile in at lower intake [40]. Apart from this supplementation of algal DHA oils to breastfeeding mothers also helps in brain development of infants [41]. Both EPA and DHA play important roles in the treatment of diseases like Parkinson’s, Alzheimer’s, psoriasis, cancer, atherosclerosis, rheumatoid arthritis, and other inflammatory diseases [33]. Experiments in animal models have shown the beneficial effects of u3-fatty acids in progression of Parkinson’s disease, reporting that algal oils can be effective in treatment of neurodegenerative diseases [42].

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2.1.3 Proteins Microalgae are considered as the potential cellular factories for protein synthesis. Due to their high protein content, microalgae are used as feedstocks in animal and poultry and as human feed. The microalgal strains like Arthrospira, Cholorella, D. salina, and Spirulina are reported to have high protein content [10,43]. Microalgal peptides have antiinflammatory and anti-cancer activities [33,44]. Phycobiliproteins from marine cyanobacteria and red algae have also been shown to have anticancer, anti-inflammatory, immunomodulatory, antioxidant, hepatoprotective, and neuroprotective properties [45,33]. One of the recently discovered biomolecules named MAAs (mycosporine-like amino acids) that are abundantly produced by marine organisms, like microalgae have been known to exhibit anti-photoaging activity in different skin lines [46,33].

3. Applications of bioactive compounds The ease in cultivation and fastidious growth of marine microalgae makes them a good source of bioactive compounds [47]. During the extraction, the maintenance of the structural and chemical composition of these bioactive compounds is simpler. The bioactive compounds derived from the marine microalgae have a wide range of applications in various sectors including health, energy, nutrition, and environment [48]. Polysaccharides are one of the bioactive compounds derived from marine microalgae, which have various applications and utilized in different fields, e.g., as gelling and thickening agents in the food industry. It improves the texture and quality of the food materials [49]. When considering the ecological aspects, the micro-algal polysaccharides have a great role in the field of soil treatment. It will help to fertilize the soil by acting as a nutrient carrier and work as a soil conditioner. Additionally, it also helps to increase the water holding capacity of the soil [49,50]. The usage of micro-algal polysaccharides acts as a growth promoter in crops [51]. Apart from the polysaccharides, the marine microalgae are also prodigal with proteins. This high level of protein content can be hydrolyzed to get the bioactive peptides. These peptides will usually be 10e20 amino acids in length. They have a great role in the pharmaceutical and cosmetic industry. The higher protein content value in most of the species and less expensive methods for the extraction makes micro-algal peptides an alternate for the traditional dietary protein source. The bioactive peptides from marine microalgae has a better efficiency, selectivity, and tolerance which makes them ideal candidates in the field of pharmaceuticals and cosmetics [52]. These days the marine micro-algal lipids are getting more attention due to their ability to accumulate in a large quantity and the presence of therapeutically important omega-3 long-chain polyunsaturated fatty acids (u-3 PUFAs), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA). Nowadays, fish oil is used as a source of PUFAs and the marine microalgae can be a better alternative because of high PUFA levels in many of

Marine microalgae: an emerging source of pharmaceuticals and bioactive compounds

the species including Isochrysis sp., Pavlova sp., Nannochloropsis sp., Chaetoceros calcitrans, Thalassiosira sp., and Schizochytrium sp. The US Food and Drug Administration has approved the use of DHA from Crypthecodinium cohnii and from Schizochytrium sp. as a dietary supplement for humans in the year 2001 and 2015 respectively [53,52]. Microalgal bioactive pigments include phycoerythrin, phycocyanin, beta-carotene, astaxanthin, fucoxanthin, lutein, zeaxanthin, etc. They have different applications in the food industry, therapeutics, and poultry. The beta-carotene derived from the Dunaliella salina is used as a food colorant and food additive; it also acts as Vit-A precursor and possess antioxidative activity [10]. Astaxanthin derived from Haematococcus pluvialis is used in the field of aquaculture as a coloring agent for salmon meat [54]. Fucoxanthin is used in the poultry field to improve the meat color of broiler chicken [55]. Lutein, zeaxanthin, and canthaxanthin can be also utilized in poultry for improving chicken skin color, which can be an alternative for the synthetic coloring agents [56]. Phycocyanin can be extracted from Arthrospira sp., and it can be used as a natural coloring agent in food and cosmetics [57]. Phycoerythrin can be extracted from Porphyridium cruentum, and it can be used as a food marker and can be used as reagent for fluorescence immunoassays [58].

4. Microalgae as a source of pharmaceuticals Pharmaceutical applications of microalgae are dependent on antioxidant properties, anticancer activity, anti-inflammatory, and antimicrobial activities. Polyphenols, phycobiliproteins, and vitamins are water soluble antioxidants found in microalgae those that help in cancer inhibition by causing premalignant lesions regression [59]. Oxidative damage occurs when exposed to ionizing radiation which forms free radicals and active oxygen. Microalgal metabolites help in preventing oxidative damage and are effective against chronic disorders, cardiovascular diseases, and inflammations. Pholorotannins (a polyphenol found in marine algae), beta carotenes, sulfate polysaccharides, seaweeds, and filamentous green algae exhibit great antioxidant properties [60]. 4.1 Antimicrobial activities of microalgae Antimicrobial activities of microalgae are exhibited by certain compounds like terpenes, indoles, phenols, and in some microalgae, it is even due to lipid composition, for example, Chaetocerous muelleri [61,62]. Autoinhibition is observed in some microalgae like H pluvialis and S. costatum [63,64]. In the case of I. galbana, interference with chlorophyll and protein synthesis confers antimicrobial activity. In other cases, it is associated with membrane permeability changes along with dissociation of phycobilin assemblages in thylakoid membrane which results in leakage across cell wall [65]. Microalgae and its active compounds for antimicrobial activities are summarized in Table 14.2.

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Table 14.2 Antimicrobial activities of active compounds from microalgae. S. No.

Microalgae

Activity

Active compound

1. 2.

P. tricornutum H. pluvialis

Antibacterial Antibacterial

Eicosapentaenoic acid Short chain fatty acids

Antifungal

Butanoic acid and methyl lactate Pheophorbide like compounds Sulfated polysaccharides Methanolic extracts

3.

Dunaliella sp

Antiviral

4. 5.

C.autotrophica Chlorella vulgaris I. galbana

Antiviral Antifungal

6.

Antimicroalgal

Ascorbic acid free cell lysate

Target organism

References

MRSA E. coli, S. aureus Candida albicans HSV 1

[66,67] [68]

VHSV A. niger

[70] [71]

C. vulgaris

[72]

[68] [69]

4.1.1 Antibacterial activity Infections caused by bacteria have a high impact on public health and some diseases are deadly. Antibiotics acts against the disease by rupturing the cell and destroying the organism itself or controlling the growth. Resistance of pathogenic organisms to antibacterial agents has been significantly increased over the past decade. Expanding number of antibacterials offer diverse resistance mechanisms which should be closely monitored and taken care off [73]. Microalgal cell-free extracts are now tested as additives to avoid the use of artificial antibiotics for food and feed composition and are also administered as sub-therapeutic doses during the period of animal breeding [74]. First antibacterial compound isolated form microalgae Chlorella is chlorellin which are inhibitory to gram-negative and gram-positive bacteria [75]. Organic extracts of microalgae (Scenedesmus costatum) with fatty acids longer than 10 carbon atoms indicated antibacterial activity against aquaculture bacteria. The ability of fatty acids in microalgae to intrude bacteria depends on its chain length and degree of unsaturation [62]. 4.1.2 Antiviral activity Viral cells are reproduced in different stages like adsorption or invasion of cells stage, multiple cell synthesis stage, or maturity and release stage. Antiviral action can take place in any stage of viral growth. Microalgae Dunaliella species has anti-HSV factor which interfere with stage 1 of viral growth [76]. Stage 1 of some enveloped viruses like viral hemorrhagic septicemia virus and African swine fever virus are affected by sulfated exopolysaccharides from marine algae [77]. Encephalomyocarditis virus is widespread

Marine microalgae: an emerging source of pharmaceuticals and bioactive compounds

in subclinical level which when present as acute form leads to sudden death of piglets and reproductive failure in adult animals. Microalgae, Gyrodinium impudicum, has homopolysaccharide of galactose with uronic acid and sulfate groups which exhibit an impressive activity against encephalomyocarditis virus [78]. 4.1.3 Antifungal activity Fungal diseases and fungi were not significant until 1970s. Unique and prudent antifungals encompassing algae came into the role when deadly fungal infections got expanded. Katircioglu et al. studied the development of infections of three different yeasts using 10 microalgal strains from the freshwater reservoir in turkey for antifungal properties of microalgae and concludes that Oscillatoria and Chlorella species were attaining the best antifungal activity [79]. Antifungal activity of pressurized liquid extracts of H. pluvialis was argued on butanoic acid and methyl lactate which was effective against C. albicans [80,81]. Surveys of C. pyrenoidosa and S. quadricauda against eight different fungi (A. niger, A. flavus, P. herquei, F. moniliforme, Helminthosporium sp., A. brassicae, S. cerevisiae and C. albicans) shows that antifungal activity of these microalgae is extended to all the fungi studied [82]. 4.1.3.1 Algal drugs: applications and future Photosynthetic chloroplast in algae helps to make targeted and complex anticancer drugs. Ability of algae to fold proteins into three-dimensional structures is used to produce human antibodies. Therapeutic drugs like human vascular endothelial growth factor against pulmonary emphysema are successfully produced by algae [83]. A compound named cryptophycin is a significant component in anti-cancer drugs, which is isolated from blue green algae. Saxitoxin and polyketide are alkaloidal neurotoxins in algae which possess anti-inflammatory and anti-cancer properties. Medicinal properties of algae are illustrated in Table 14.3 [84,85,86,87,88]. The future of algae derived drugs counts on high value oils, cosmetics, colorants, waste water treatment, food supplements, personalized drugs, fertilizers, and forensic medicines. Table 14.3 Medicinal properties of selected Algal strains. Algae

Medicinal uses

Enteromorpha Acetabularia Laminaria Sargassum

Hemorrhoids, parasitic disease, coughing, bronchitis Edema, urinary disease Urinary diseases, thyroid problems Cervical lymphadenitis, edema, inflammation, induce urination Blood sugar lowering

Grateloupia

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5. Enhancement of algal metabolites Algae being photosynthetic, they are grown in controlled light conditions in vitro. In order to enhance the secondary metabolites of the microalgae, different light sources with different intensities can be used to improve secondary metabolite production [89]. Apart from this, application of different stress conditions helps improve the secondary metabolite production. Various stress conditions are present like salinity, pH, temperature, etc. Application of salinity stress has resulted in increase of secondary metabolites in many algal members [90]. Another way of enhancing secondary metabolites in microalgae is the genetic engineering, where new strains can be developed to increase the production of these metabolites. Apart from traditional gene transfer techniques, advances in microalgal research and engineering development are under study [91].

6. Conclusion Marine microalgae produce various primary and secondary metabolites. Marine microalgae like D. salina, Haematococcus sp., Arthrospira, etc. are some of the species employed to isolate these bioactive compounds. The metabolites produced by the microalgae include carotenoids, PUFAs, proteins, and polysaccharides. These metabolites have several applications in improving the human lifestyle as these are used in the treatment for inflammation, infections, and cancers. The increase in demand for food and nutrients has pushed the emergence of the use of microalgae in various sectors ranging from food to pharmaceuticals and cosmetics. Although the discovery for the use of the metabolites has been put forward, but essential developments still need to be done in order to utilize these metabolites efficiently. The advancements in the field of biotechnology will help in dealing with these challenges to develop and isolate novel bioactive molecules from microalgae. The exploration of various microalgal species would also provide promising results in the biomedical sector.

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CHAPTER 15

Natural compounds in chemopreventive foods for prevention and management of non-communicable diseases Asmita N. Bambole, Surya Nandan Meena, Vinod S. Nandre and Kisan M. Kodam Department of Chemistry, Division of Biochemistry, Savitribai Phule Pune University, Pune, Maharashtra, India

Abbreviations AD Alzheimer’s disease CAT Catalase ECP Electron transport chain GPx Glutathione peroxidase GSH Reduced glutathione GR Glutathione reductase GST Glutathione S-transferases IPF Idiopathic pulmonary fibrosis NADPH Nicotinamide adenine dinucleotide phosphate NO Nitric oxide NSCLC Human non-small cell lung cancer PD Parkinson’s disease RNS Reactive nitrogen species ROS Reactive oxygen species SOD Superoxide dismutase WHO World Health Organization

1. Introduction Non-communicable diseases (NCDs), also known as civilization diseases, include cancer, cardiovascular disease (CVD), chronic respiratory diseases, and type 2 diabetes. According to a World Health Organization (WHO) report of 2014, NCDs account for more than half of all premature deaths worldwide. Over the past few decades, NCDs have surpassed infectious diseases as the primary cause of death in the majority of nations. Every year, 41 million people die due to NCDs, resulting for 74% of all deaths worldwide. Above 15 million people die due to NCDs between 30 and 69 of age, every year. CVDs kill the most individuals each year (17.9 million), followed by New Horizons in Natural Compound Research ISBN 978-0-443-15232-0, https://doi.org/10.1016/B978-0-443-15232-0.00008-4

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cancer (9.3 million), respiratory illness (4.1 million), and both type of diabetes (1.5 million). Over 85% of all premature NCDs deaths are caused by these diseases [1]. According to WHO, type 2 diabetes (2%), chronic respiratory illnesses (5%), cancer (27%), and cardiovascular diseases (46%) account for the majority of mortality from civilization diseases [2]. NCDs, usually referred to as chronic illnesses, are characterized by a protracted course and are brought on by a combination of genetic, physiological, environmental, and behavioral variables. Chronic illnesses, often known as NCDs, are long-lasting conditions brought on by a mix of genetic, biochemical, environmental, and lifestyle factors. NCDs now account for seven of the world’s top 10 causes of mortality, rise from 4 of the 10 in 2000, according to WHO’s 2019 Global Health Estimates. About 60%e90% mortality of COVID-19 was due to either one or more NCDs [3]. In recent study of Italy revealed that 96% of COVID-19 death had NCDs among which 69% had hypertension, 31% type 2 diabetes, 28% heart diseases, 16% chronic obstructive pulmonary diseases, and 16% cancer [4]. According to the WHO NCDs progress monitor 2022, 88% of fatalities in the United States of America are caused by NCDs, resulting in a total of 2,600,000 deaths in the population. The 90% of deaths occur due to NCDs in Canada, resulting in a total of 249,800 NCD deaths. In Niger, however, only 30% of deaths from NCDs occur, with a total of 55,600 NCD deaths. In Central African Republic, 32% of deaths occurred due to NCDs, resulting in a total of 18,100 NCD deaths. This data reveals that the rate of mortality from NCDs is higher in industrialized countries than in developing countries. In China, the most populous country, 90% (i.e., 9,058,000) of deaths occur due to NCDs. In India, the overall number of NCD deaths is 66% (i.e., 6,047,000 in number) of total deaths [5]. The data presented above show that the NCD fatalities does not depend on country’s population. Indeed, the mortality rate from NCDs is higher in industrialized countries than in poor countries. As a result, poor countries that continue to rely on natural and unprocessed foods are less likely to be afflicted by NCDs. NCDs largely onset by aspects such as lifestyle, diet, and physical activity. The sluggish lifestyle of urban countries and minimum physical activity affects immensely in digestion. The new craze for junk food or processed food is major reason for health issues in urban countries. These foods are not only high in bad fat (e.g., fried and canned food, pizza, burger, street food, etc.) but also rich in salt, high calories, flavored, zero nutritional value which give rise to high blood pressure, obesity, CVD, digestive issues etc. 0.1 billion people are obese in America. Therefore, urban countries are mostly affected by NCDs than poor counties.

2. Cause of civilization diseases Reactive oxygen species (ROS) are produced in the body’s normal physiological conditions by various mechanisms of the cell. These ROS produced in cell are then scavenged by endogenous antioxidants. This balanced process maintains the normal cellular

Natural compounds in chemopreventive foods for prevention and management of non-communicable diseases

condition of the cell. ROS are byproducts of regular cellular metabolism and are likely to function as a second messenger. Despite having significant physiological roles, ROS have the potential to do significant harm. The relative rates of ROS generation and clearance control the balance between physiological functioning and damage. ROS maintain the cellular “redox homeostasis” under physiological settings to protect cells from oxidative stress via several redox-regulatory processes. Overproduction of ROS, by many reasons such as internal (excessive activation of nicotinamide adenine dinucleotide phosphate (NADPH) by cytokines or the electron transport chain (ETC) in mitochondria, xanthine oxidase, obesity, increased Na level) or external (UV, diet, environment, lifestyle, smoking), results in oxidative stress [6]. Oxidative stress results from an imbalance between the biological system’s capacity to detoxify these reactive products and the synthesis and accumulation of ROS in cells. Endogenous antioxidant defenses are compromised by the generation of excessive ROS [7]. Therefore, an increased quantity of ROS may result in oxidative tissue damage and pro-inflammatory gene up regulation, which in turn may result in inflammation. The outcomes of studies into the factors that add to the development of civilization diseases consistently point to oxidative stress as a common occurrence [8e10]. Reactive species include reactive nitrogen species (RNS), ROS and reactive sulfur species (RSS). Proteins, lipids and nucleic acids (RNA and DNA) are the most typical targets of these reactive species [11,12]. DNA damage from oxidation is one of the causes that contributes to the onset of cancer [13e15]. According to recent studies, oxidative stress is either the major or secondary cause of many CVDs [16]. Oxidative stress has been linked to a number of neurological conditions, including Parkinson’s disease (PD), Alzheimer’s disease (AD), sclerosis, depression, and impaired memory [17e19]. Nephritis, kidney failure, proteinuria, and uremia are all associated to oxidative stress [20,21]. Idiopathic pulmonary fibrosis (IPF), a progressive non-inflammatory lung disease, is characterized by oxidative damage [22]. Diabetes and its consequences are mostly developed by oxidative stress [23]. The relation between balance of ROS and antioxidants is shown in Fig. 15.1.

3. How oxidative stress cause NCDs and antioxidants can prevent NCDs ROS, oxidative stress, NCDs, and antioxidants are all linked. Increased production of ROS prompts oxidative stress, which leads to NCDs, whereas antioxidants can reduce oxidative stress by scavenging ROS and so prevent or reverse NCD conditions. A free radical molecule has an unpaired electron in its outer shell, which makes it unstable and highly reactive. A free radical will attempt to take an electron from its nearby molecule in order to stabilize itself. However, if it is successful in grabbing another electron, its victim also becomes a free radical. This unstable molecule will then attempt to steal an electron as well. The result is referred to as a free radical cascade. The process by which

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Figure 15.1 Relation between ROS and antioxidants. A balance between ROS levels and the antioxidant (AO) defense is maintained under normal physiological circumstances (A). This equilibrium can be changed in favor of antioxidant defense (B) or ROS (C), reversion of the imbalanced state resulting into lower severity of NCDs and other diseases (D).

a free radical molecule attacks another molecule in order to take its electron is known as “oxidation” Antioxidants neutralize free radicals by donating an electron and therefore putting an end to the electron-stealing chain process without becoming itself destabilized. Therefore, antioxidants are sometimes referred to as “free radical scavengers” or “self-sacrificing soldiers” and help prevent oxidative stress. Different antioxidants have different mechanisms of neutralizing damage. The mechanism of antioxidant is shown in Fig. 15.2. 3.1 Cancer According to figures from the WHO fact sheet on cancer 2022, approximately 400,000 children develop cancer every year. Breast, lung, colon, rectum, and prostate cancers were the most prevalent types of cancer that caused mortality in 2020. The use of cigarettes, having a high BMI, drinking alcohol, eating less fruits and vegetables, and not exercising account for about one-third of cancer fatalities [24]. Oxidative stress damage DNA which may result into mutations in tumor suppressor genes. Series of mutations in tumor suppressor genes can lead to cancer. Modulation in gene expression of downstream affect DNA repair pathways and endogenous antioxidants. In cancer, ROS increase p53, tumor growth, invasiveness, and metastasis. Chronic inflammation in oxidative stress leads to malignancy [25]. The oncogenic phenotype

Natural compounds in chemopreventive foods for prevention and management of non-communicable diseases

Figure 15.2 Function of antioxidants. Free radical snatches electron from stable molecule making it unstable which then acts as free radical. Antioxidant stops this cascade by donating its electron to unstable free radical and making it stable.

of cancer cells is induced by ROS which acts as secondary messengers in intracellular signaling cascades [15]. ROS along with base mutation can also cause DNA crosslinking, strand cleavage, and chromosomal damage [26,27]. Antioxidants like vitamin C, E, retinoids, and selenium are additional micronutrients that play essential roles in metabolic activities. They have immuno-modulatory and apoptosis-inducing capabilities, as well as the ability to govern cell proliferation and differentiation. Antioxidants such as caffeine, phenolic compounds, trigonelline, diterpenes, and melanoidins in coffee elevates the intracellular activity of cytochrome P450 and enzymes such as glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferases (GST), reduced glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT). According to the Huffington Post, glutathione (GSH) is the “mother of all antioxidants.” Antioxidants in coffee downregulate the messenger RNA (mRNA) expression of endogenous antioxidant enzymes [28]. These antioxidants prevent the prostate cancer. Vitamin E decreases the viability of breast cancer cells [29]. Antioxidants prevent the generation of ROS or intercept that are already generated. Antioxidants prevent or reduces the epidermal toxicity and prevent the skin cancer [30]. In vivo study on human non-small cell lung cancer (NSCLC) showed that lycopene (carotenoid) from watermelon has anticancer activity against lung cancer [31]. In vivo research showed that phenolic compounds including epigallocatechin gallate and resveratrol had antitumor activity against T24 cells.

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3.2 Diabetes Diabetes mellitus is a degenerative non-communicable disease. Oxidative stress causes pancreatic b-cell damage which causes faulty gene expression and secretion of insulin [32]. Diabetes mellitus both type 1 and type 2 causes oxidative damage and deficit the antioxidant defense [33]. Diabetes is also a rooting cause of other NCDs such as CVD and hypertension. Statistical meta-analyses indicate that antioxidants have potential to treat three complications of diabetes mellitus [34]. Endothelial dysfunction or vascular dysfunction is a typical of diabetes complications. In the etiology of diabetes and its consequences, oxidative stress plays a critical part. Undoubtedly, a redox imbalance has been connected to both insulin resistance and b-cell degeneration, two key events in the pathogenesis of type 2 diabetes. Oxidative stress in diabetic vascular disease that is overproduction of ROS contributes to endothelial dysfunction. As a result, the bioactivity of nitric oxide (NO) is lowered, inhibits platelet functioning, restrict leukocyte adhesion, and lessens anti-atherosclerotic mechanism [35]. According to previously covered in clinical experiments, resveratrol (RSV) is an effective antioxidant molecule for treating metabolic diseases like diabetes, dyslipidemia, and obesity. RSV is a naturally occurring polyphenol with antioxidant properties that can help mitochondrial dysfunction. RSV induces NO expression, activity, and bioavailability. By changing the NADPH pathway, curcumin can halt the death of Langerhans islet cells. In vitro study showed that quercetin and anthocyanin help prevent diabetes. In curing of type 2 diabetes, antioxidant compounds activate the AMP-activated protein kinase (AMPK) pathways, suppress the expression of COX2 related genes that release pro-inflammatory mediators, improve glucose tolerance and insulin responsiveness, decrease the number of inflammatory cells, and lower the levels of cytokines. The primary isothiocyanate in Moringa oleifera, moringa isothiocyanate (MIC1), inhibits transforming growth factor beta 1 (TGF-1), and activates Nrf2-ARE at levels comparable to sulforaphane (SFN). It also suppresses pro-inflammatory cytokines, reduces ROS, and activates Nrf2-ARE. Additionally, it has been observed that simmondsin and puerarin lower oxidative stress by triggering the Nrf2 pathway [36]. Lipoic acid, vitamin C, and N-acetylcysteine can prevent diabetes related complications. Vitamin D in type 2 diabetes decreases inflammatory markers like IL-6 and TNF-a. 3.3 Hypertension Animal and mechanistic investigations have confirmed a strong causative relationship between ROS, redox signaling, and hypertension [37]. Oxidative stress in brain RVLM (rostral ventrolateral medulla) causes hypertension [38]. Antioxidants can lower blood pressure by reducing the oxidation stress [39] and evidently antioxidant vitamin C reduces ROS in the RVLM area that results in lowering the blood pressure [40]. In addition to being a major risk factor for chronic renal disease, hypertension is also a cause of myocardial injury, cardiac arrest, stroke, peripheral arterial disease, and arterial disease. Diabetes and dyslipidemia are two metabolic disorders that are frequently

Natural compounds in chemopreventive foods for prevention and management of non-communicable diseases

linked to hypertension. NADPH oxidase is the initial source of ROS. Vitamins E and C scavenge the ROS and downregulates the NADPH oxidase, reduction in systolic blood pressure. Polyphenols promote and improve endothelial NO synthase (eNOS) production, inhibit ROS-producing enzymes including NADPH and xanthine oxidases, and raise glutathione levels. Carotenoids (lycopene, b-carotene and vitamin E) in tomato prevent atherosclerosis, lower blood pressure and help prevent hypertension. Sesamin and sesaminol in sesame reduces oxidative stress and also increases GPx, SOD, and CAT activities [41]. 3.4 Cardiovascular diseases (CVD) CVD include heart attacks and stroke. Though previously assumed to be caused by arterial cholesterol accumulation, cardiovascular disease is involved in cascade of inflammatory events. Among many factors, oxidative stress is the chief risk factor of CVD. Production of ROS and RNS by cells like endothelial cells, vascular smooth muscle cells, and monocytes or macrophages leads to oxidative stress which damage the endothelial cells. Endothelial damage is the starting step in CVD [42]. Mitochondrial ROS are crucial in numerous signaling pathways that lead to CVD [43]. Epidemiologic studies exposed that dietary vitamins E and C decrease the risk of CVD. Therefore, antioxidant vitamin C have capability to scavenge lipid radicals and inhibit the oxidation of low-density lipoprotein (LDL). Vitamin E prevents CVD, especially coronary heart disease and atherosclerosis [44,45]. According to epidemiological research, increased consumption of fruits containing anthocyanin and flavonoids reduces the risk of both oncological and cardiovascular disorders [46,47]. Low production of NO causes CVD and antioxidants which restore production of NO can prevent CVD. 3.5 Lung diseases Lung diseases or pulmonary diseases related with oxidative stress include sarcoidosis, hyperoxia, chronic beryllium disease (CBD), chronic obstructive pulmonary disease (COPD), chronic respiratory diseases, and asthma. Overproduction of ROS also induces bronchial hyperreactivity, which is a hallmark of asthma [48]. Increased ROS production can cause direct oxidant damage and epithelial cell loss [49]. Reactive species hydrogen peroxide (H2O2) constricts airway smooth muscle [50]. The etiology of different lung illnesses such as asthma, COPD, acute lung injury (ALI), pulmonary fibrosis, and lung cancer is influenced by oxidative stress. COPD and other inflammatory illnesses can lead to lung cancer [51]. The intracellular antioxidant enzymes in lung maintain a normal redox state. Endogenous antioxidants include GSH, catalase, SOD, GPx, transferrin, vitamin E and C, ferritin, ceruloplasmin, serum proteins, and bilirubin [52e55]. One of the studies showed that fruits and dietary antioxidant intake in can prevent asthma and lung disease development [56]. Vitamin C is more effective at reducing nitrogen dioxide (NO2) oxidation, whereas vitamin E is more efficient at preventing ozone (O3).

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Flavonoids such as isoorientin, vitexin, and isovitexin show activity in the prevention of pulmonary disorders [57]. Catechins, flavonols, and flavones prevent asthma. Oxidative stress affects the endothelium cells which helps in progression of COVID-19 infection. Also, any viral infection including COVID-19 leads to ROS generation. In both circumstances, antioxidants can play important role of prevention as well as treatment of oxidative stress in COVID-19. Recent experimental studies show that the people got affected with COVID-19 contain low concentration of antioxidants such as vitamins, minerals and endogenous antioxidants. COVID-19 infection causes CVD by activating the NADPH oxidase-2 (NOX-2) and increasing oxidative stress. Curcumin can bind to SARS-CoV-2 target receptor and prevent COVID-19 infection. COVID-19 primarily affects respiratory system. Polyphenols, hydroxytyrosol, quercetin, carotenoids, vitamins C and D antioxidants which reduces NOX-2, oxidative stress and inflammation play role in prevention of COVID-19 [58]. Vitamin C was found beneficial in treatment of COVID-19 infection as well as CVD. 3.6 Neurological diseases or neurodegenerative disorder (NDDs) Oxidative stress causes a rise in ROS and the progression of neurodegenerative disorder (NDDs) [59,60]. Oxidative stress causes extracellular amyloid-b (Ab) plague [61], phosphorylation of s protein, and subsequently the development of the neurofibrillary tangles (NFTs). Ab plague are responsible for AD. Oxidative stress causes autophagy dysfunction which leads to PD. The etiology of Huntington’s disease (HD) is heavily influenced by neuronal autophagy dysfunction. Amyotrophic lateral sclerosis, ataxia, spiral ganglion neuron degeneration, and cerebral ischemia are some of the major NDDs caused by autophagy dysfunction [62]. H2O2 induces production of ROS, interleukin [IL]-1b and inflammatory factor (tumor necrosis factor [TNF]-a). Quercetin reduces oxidative stress in a-Amyloid-induced models of AD through activating the Nrf2 signaling pathway [63]. It improves the mitochondrial activity in living cells [64]. By stimulating Nrf2-mediated anti-oxidative pathways, calycosin from kudzu roots protects against brain injury [65]. The antioxidant thymoquinone (TQ) guards against oxidative damage in brain cells. TQ’s anti-inflammatory and antioxidant qualities shield brain cells from inflammation and oxidative stress. The fact that TQ demonstrates anti-neurotoxin properties suggests that it may have a role in preventing NDDs including AD and PD. TQ’s ability to block certain inflammatory mediators and Toll-like receptors (TLRs), which reduces neurotoxicity and inflammation [66]. For the treatment of neurological illnesses by preventing lipid peroxidation, a variety of antioxidants, such as pyrrolopyrimidine which can cross the blood-brain barrier (BBB), are often recommended. Coenzyme Q10, pyrrolopyrimidine, and deferoxamine prevent PD [67]. Selenium activates the GPx and help prevent NDDs [68]. Fig. 15.3 shows that how various factors can cause the generation of ROS and oxidative stress.

Natural compounds in chemopreventive foods for prevention and management of non-communicable diseases

Figure 15.3 The flow chart of how various factors can cause the generation of ROS and oxidative stress which give rise to various cellular disfunctions causing the non-communicable diseases.

4. What are chemopreventive foods? The chemoprevention term was first coined by Sporn, which means suppressing inhibiting, or reversing carcinogenic progression and development by the use of natural or synthetic agents [69]. Chemoprevention is no longer limited with cancer prevention but has grown to include all civilization illnesses. Several epidemiologic studies show that diet rich in vegetables and fruits can reduce the occurrence of all civilization diseases.

5. Antioxidants in chemopreventive foods to prevent or control civilization diseases The role of oxidative stress in the emergence of civilizational diseases has now been scientifically confirmed and acknowledged [70]. Dietary antioxidants have been shown to be able to counterbalance both exogenous and endogenous free radicals, which prevents the oxidation of lipids. For example, vitamin C can shield a1-proteinase inhibitors from oxidative damage [71]. Therefore, an imbalance between the production of ROS and the antioxidant defense mechanisms leads to oxidative stress. Both enzymatic and nonenzymatic antioxidant mechanisms can eliminate ROS. As a result, suggestions to increase dietary antioxidant consumption as a chemo preventive strategy, whether via organic plant-based foods or dietary supplements, became a hot issue in human nutrition. Several studies show that chemo preventive diets serve a major role in avoiding or reducing civilization diseases, according to the literature [72]. For the treatment of oxidative stress, antioxidants are regarded as the first-line therapy [73]. According to the American Institute for Cancer Research, a diet high in proteins and fats may raise the risk of cancer, but fruits and vegetables include cancer-preventive

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chemicals [74,75]. The intake of these vegetables has an inverse relationship with the incidence of colon, pancreatic [76], lung [77], breast [78], stomach [79], and ovarian [80] cancer. Healthy diet is the key to prevent NCDs and improve healthy life. Replace trans fats, saturated fat, refined carbohydrate, dietary cholesterol, sodium to polysaturated fats, dietary omega-3, dietary fibers, phytonutrients, and antioxidants. Antioxidants are very crucial components in cells which can reduces or balances the oxidative stress by specifically quench free radicals, by chelating the redox metals, have a positive impact on gene expression [15]. Antioxidants repair other antioxidants, restoring them to their pre-damage state. This mechanism is named as “antioxidant network” Antioxidants can be enzymatic or non-enzymatic. Enzymatic antioxidants involve SOD, CAT, and GPx [81]. And non-enzymatic antioxidants involve vitamins C and E, carotenoids, natural flavonoids, thiol antioxidants (glutathione, thioredoxin, and lipoic acid), hormones, and other compounds [82]. Antioxidants can be divided into three groups based on their function or mode of action, first enhances the function of endogenous antioxidant enzymes such as SOD, CAT, and GPx, which accelerates the inactivation or conversion of free radicals; second, nonenzymatic scavengers of free radicals, and third upregulates the other important mechanisms. Vitamin E or a-tocopherol (a membrane-bound antioxidant) which terminates the reaction of lipid peroxidase by scavenging lipid peroxyl radicals [83]. Vitamin C can also directly scavenge oxygen and hydroxyl radical [84,85]. Other non-enzymatic antioxidants which can scavenge different radicals are b-carotene (scavenges oxygen anions and peroxyl radicals), bilirubin (lipid radical), uric acid (hydroxyl radical, oxygen, and peroxyl radical), glucose (hydroxyl radical), taurine (hypochlorous acid), albumin (transition metal binding), and cysteine and cysteamine (donates sulfhydryl groups) [86]. Flavonoids can prevent many diseases caused due to oxidative stress [57]. Curcumin downregulates numerous inflammatory cytokines (IL-8, IL-6, and TNFa). The M. Oleifera (family: M. Oleiferaceae), popularly known as “Miracle Tree” or “Tree of Life,” is a medicinal plant with a numerous health beneficials [87]. This plant contains several antioxidants such as vitamins, carotenoids, phytosterols, polyphenols, and tocopherols, glucosinolates, polyunsaturated fatty acids, and minerals. The literature states that both in vivo and in vitro investigations on this plant have revealed more than 20 distinct pharmacological properties [88]. 5.1 Food and vegetables for controlling cancer Phytochemicals in plants, vitamin E, C, and A, anthocyanidins in berries, epigallacatechin-3-gallate in green tea, beta-carotene, lycopene in tomato can lower the risk of developing pancreatic cancer. Tomatoes, watermelon, and papaya are the best source of Lycopene. Selenium can prevent cancer and sources of selenium are Brazil nuts,

Natural compounds in chemopreventive foods for prevention and management of non-communicable diseases

walnuts, saltwater fish, beef, and poultry. Diets high in fiber and whole grains have been associated with a lower risk of colorectal cancer. Several observational studies propose that increase in omega-3 fatty acids consumption is essential for lowering your risk of breast and bladder cancer [89,90]. The prevention of cancer and chronic illnesses linked to renal cell carcinoma is advised by fiber-rich diets high in vegetables and fruits [91]. Anthocyanins in Black Raspberry and Ellagitannins and Urolithins in Pomegranate prevent the colon cancer risk [92,93]. Chlorogenic acids (group of polyphenols) in coffee avert free radical damage and control inflammation [94]. Broccoli, cabbage, cauliflower, turnips etc. are good sources of dithiolthiones and isothiocyanates which has anticancer activity. Isoflavones in soyabean are anticarcinogenic. Lignans in rye and flaxseed help in prevention of breast cancer. Green leafy vegetables rich in leutin are also anticarcinogenic. The different type of antioxidant found in different food and vegetables are shown in Fig. 15.4. 5.2 Food and vegetables for controlling lung disease or cardiovascular diseases (CVD) Consuming foods high in vitamin C [95], minerals, beta-carotene [96], dietary fiber [97], and other antioxidants can help prevent the development of COPD. Apples high in flavonoids protect against asthma and COPD. An American Society for

Figure 15.4 Types and classes of antioxidants found in natural food source.

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Preventive Cardiology Clinical Practice stated with evidence that the diet best for preventing atherosclerotic cardiovascular disease (ASCVD) includes fatty fish, fruits, vegetables, legumes, nuts, seeds, and plant protein. The main role of antioxidant vitamin E or a-tocopherol is to prevent lipid oxidation. Main dietary sources of a-tocopherol are vegetable oils and nuts. Daily intake of 12 mg vitamin E is recommended to improve CVD health. Along with vitamins A, E, and C and beta-carotene are also involved in CVD prevention. Lycopene is found effective in prevention of atherosclerosis and CVD. Edible mushrooms are rich in phenolic content which influences many metabolic markers such as total LDL, cholesterol, fasting triacylglycerol, homocysteine, BP, homeostatic function and oxidative and inflammatory damage, which potentially lessen the risk of CVD. 5.3 Food and vegetables for controlling diabetes Obesity and type 2 diabetes (T2D) are both risk factors in the development and progression of CVD. Therefore, a primary public health priority is the incorporation of efficient and maintainable dietary advice for the control and treatment of obesity and diabetes. Tulsi, turmeric, onions, ginger, green tea, lemon, and other foods can help prevent diabetes directly or indirectly. Flaxseeds have antioxidant a-linolenic acid which have hypolipidemic and hypoglycemic properties. Allium cepa Linn (onion) and ginger control blood sugar level. Catechin in green tea control diabetes. Turmeric control blood sugar level and enhances endogenous antioxidant enzyme GPx. Blackberries, red grapes, apricots, eggplant, coffee, and green tea are all rich in polyphenols which help treating diabetes. 5.4 Food and vegetables for controlling neurological disorders (NDDs) Fruits and vegetables rich in anthocyanin [98], Quercetin [99] antioxidants evidently help reduce occurrence of neurological diseases. Antioxidants in honey can prevent various neurological disorders such as AD, Huntington’s disease, PD, and multiple sclerosis, etc. Honey contains pigenin, benzoic acid, caffeic acid, catechin, chlorogenic acid, chrysin, cinnamic acid, coumaric acid, ellagic acid, ferulic acids, galangin, gallic acid, hesperetin, kaempferol, luteolin, quercetin, naringenin, and syringic acid which help prevent NDDs [100]. By boosting the expression of peroxisome proliferator-activated receptors, the punicic acid in pomegranates reduces oxidative damage and inflammation and prevents NDDs [101]. 5.5 Food and vegetables for controlling hypertension National Institutes of Health evidently reduces the BP among patients with as well as without hypertension. Eating a low sodium DASH (dietary approaches to stop hypertension) dietary pattern, rich in potassium from fruits and vegetables, is suggested for the prevention and treatment of hypertension [102]. Garlic helps increase in NO. Study on rats showed that increase in NO in the RVLM reduces BP, heart rate, and commencement of the sympathetic nervous system (SNS) [38]. Relaxation and vasodilation of smooth muscles are caused by NO production. Oats have soluble fibers that help lower hypertension

Natural compounds in chemopreventive foods for prevention and management of non-communicable diseases

[103]. In a dose-dependent way, tea lowers both systolic and diastolic blood pressure [104]. Carrot, tomato, pomegranate, and soybean help treat hypertension. Flaxseed oil rich in a-linolenic acid which help in hypertension prevention and reduce total cholesterol [105]. In dose dependent manner, Eugenol in crude extract of Basil have property to reduce systolic, diastolic, and BP [106]. Table 15.1 is depicting the various antioxidants and their sources.

6. Challenges or loopholes in chemoprevention strategy Various recent clinical trial and experiments shows that fruit and vegetable intake can help improve NCDs. But in one of the studies, it shows that improvement in BP by consuming fruits in hypertensive patients was dose dependent. That means, antioxidants which evidently can help prevent NCDs but temporally or dose dependent. For example, green tea intake can prevent from CVD, but results were varying [154]. Also, there is no enough data which can clearly indicate the effect antioxidants in both the genders is same. Epidemiological studies have stated that dietary intake of foods rich in vitamin E, vitamin C, and b-carotene have been inversely associated with the frequency of coronary heart disease. High doses of Vitamin C can lead to kidney stone. Zinc is an important trace element which acts as antioxidants but its high concentration can lead to lungs diseases [155]. Therefore, severe clinical trials, particularly among high-risk groups, are needed before natural source antioxidants can be recommended routinely to patients. There aren’t enough of certain fruits and vegetables plants in some areas where they may help reduce civilization disease. The limited form of one dish with several therapeutic characteristics; nonetheless, other forms in dietary food must be explored so that people might accept them in their everyday lives. Food scientists must work on a variety of features of chemo preventive foods, including the transfer of new plant species from one location to another, as well as their production to assure easy access to locals. It is necessary to investigate the many dishes of a given fruit or vegetable plants so that they may be consumed at any time of day, such as breakfast, lunch, or supper. The goal of this project is to bring a popular, palatable cuisine dish that has outstanding scientifically confirmed chemo preventive effects. In order to manage NCDs, it is necessary to identify, test for, and treat these conditions as well as give those who require it access to quality healthcare. Advancing in better management of NCDs is critical. WHO have started an NCD management intrusions crucial for achieving the global target of 25% of reduction in premature death from NCDs by the year 2025. Some initiatives of Government of India including Poshan Abhiyan, Fit India, Eat Right India and National Mental Health Program, National Program on prevention and control of CVDs, diabetes, Cancer, stroke (NPCDCS), National Multisectoral Action Plan etc. and many more management strategies have been incorporated throughout the countries. WHO constantly been informing the severeness of

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Table 15.1 List of antioxidants, their natural sources and targeted NCD. S. no.

Antioxidants

Flavonoids

1.

Anthocyanins

2.

Quercetin

3.

Epicatechingallate

4.

Catechins

Natural source (foods and vegetables)

NCDs target

References

Cranberry, apple, onion, citrus fruit, tea, red wine, chocolate, honey Eggplant, cabbage, plums, grapes, berries (raspberry, strawberry), pomegranate Splitgill mushroom, tomatoes, onions, potatoes and broccoli, apple, tea, honey Cranberries, strawberries, kiwi, cherries, peach, apple, avocado Hazelnut, pistachios, almond Tea, green tea Apples, apricot, cherries, peaches, chocolate, red wine, grapes, tea, honey

Diabetes, asthma

[107]

Diabetes, cancer, NDDs

[36]

Gastric cancer, AD, HD, diabetes

[63,108]

Cancer (Bladder, Blood, brain, liver, pancreatic, prostate), diabetes

[31], [109e111], [112,113]

Diabetes, CVDs, COPD, NDDs

[114,115]

Cancer (brain, breast, gastric, leukemia, lung esophagus, pancreas, prostate, skin), NDDs (AD, HD, stroke) Cancer, inflammation, CVDs, AD

[116e119], [120,121]

Non-flavonoids

5.

Resveratrol

Grapes, blue berries, cranberries, Peanuts, pistachios, dark chocolate Red wine

6.

Gallic acid

Strawberries, grapes, banana, apple, avocado, guava, mango, pomegranate Walnut, cashew, hazelnut Honey, green tea, red wine

[110]

Natural compounds in chemopreventive foods for prevention and management of non-communicable diseases

Table 15.1 List of antioxidants, their natural sources and targeted NCD.dcont’d Natural source (foods and vegetables)

S. no.

Antioxidants

7.

Caffeic acid

8.

Gingerol

9.

Curcumin (polyphenol)

Turmeric (curcuma longa)

10.

Lignans

11.

Luteolin

12.

Isoflavonoids

13.

Calycosin (isoflavone)

Blueberries, broccoli, brussel sprouts, cabbage, sesame seeds, flax seed, bran, whole grains, lentils, soybean, cashew nuts, cherries Celery, palsy, onion leaves, broccoli, cabbage, carrots, apple skin, chilli (capsicum annuum) Soybeans, tofu, lentils, peas, beans, potatoes, pistachios, resins, currants milk Kudzu root, purple clover, red clover

Fruits, tea, coffee, oil, spices, and vegetables Ginger (zingiber officinale)

NCDs target

References

Cancer, NDDs, AD

[65]

Cancer (Bladder, Blood, breast, cervical, pancreatic, oral), lower BP Cancer (leukemia, blood, neck, skin, colon, duodenum, forestomach), nasopharyngeal carcinoma, NDDs (AD, vascular dementia, brain injury, stroke), diabetes Cancer (breast), CVD, hypertension

[122e125]

Cancer (colon), hypertension, AD, NDDs

[132,133]

CVDs

[134]

NDDs

[65]

[126e129], [130]

[131]

Continued

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Table 15.1 List of antioxidants, their natural sources and targeted NCD.dcont’d S. no.

Antioxidants

Natural source (foods and vegetables)

NCDs target

References

Cancer (pancreas)

[70]

Cancer (cervical, breast), leukemia, CVD, diabetes

[135]

Cancer (breast), CVD, diabetes, NDDs

[29]

Diabetes, CVD, AD, PD

[136]

Cancer, CVD

[137]

Diabetes, lung cancer

[155]

AD, NDDs

[138]

Vitamins and minerals

14.

Vitamin A

15.

Vitamin C

16.

Vitamin E

17.

Manganese

19.

Selenium

20.

Zinc

21.

Copper

Sweet potato, carrot, egg yolk, milk, liver, cheese Strawberries, orange, blackcurrants, kiwifruit, mangoes, broccoli, spinach, capsicum, ginger, pepper Tomatoes, avocado, nuts, seeds, whole grains, vegetable oils (wheatgerm oil) Soyabean, legumes, rice, Leafy vegetables, pineapple, seafood, lean meat, milk, nuts, tea, coffee Pork, beef, turkey, chicken, fish, shellfish, eggs, whole grains, milk, yogurt, spinach, green peas, beans, potatoes, mushrooms Seafood, lean meat, milk and nuts, nuts, sesame seeds, soya been, pumpkin Seafood, lean meat, milk, nuts

Natural compounds in chemopreventive foods for prevention and management of non-communicable diseases

Table 15.1 List of antioxidants, their natural sources and targeted NCD.dcont’d S. no.

Antioxidants

22.

Allicin

Natural source (foods and vegetables)

NCDs target

References

Allium sativum (garlic), onion

Cancer (cervical, ovarian, pancreatic), diabetes

[139e141]

Grapes, peas, kale, lettuce, broccoli, spinach pistachios, eggs, corn Oranges, papaya, persimmons, mangoes red capsicum, pumpkin Saffron crocus

Cancer, NDDs, lung diseases

[142,143]

Obesity, CVDs

[144]

Cancer (liver)

[145,146]

Nigella sativa (black cumin) Tomatoes, pink grapefruit, watermelon Yellow, green vegetables, spinach, pumpkins, mangoes, apricots, carrots, parsley, oranges Broccoli, cabbage, cauliflower Thyme and oregano, herbs, berries, flax seed, tea Holy basil, clove, nutmeg, cinnamon, bay leaf

NDDs

[66]

Cancer (lungs, prostate), CVD

[31,73]

Cancer (pancreatic, lung), CVD, diabetes

[147]

Prostate cancer, NDDs Diabetes, colorectal cancer, CVD

[148]

Cancer (breast, liver)

[149,150]

Carotenoids

23.

Lutein

24.

Cryptoxanthins

25.

27.

Saffron (crocin, crocetin, safranal, and kaempferol) Thymoquinone (TQ) Lycopene

28.

Beta-carotene

29.

Indoles

30.

Polyphenols

31.

Eugenol

26.

[98,119]

Continued

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Table 15.1 List of antioxidants, their natural sources and targeted NCD.dcont’d S. no.

Antioxidants

32.

zoo chemicals (omega-3 fatty acid, fatty acids, linoleic acid)

33.

Alpha linoleic acid

34.

Lectin

35.

Punicic acid

Natural source (foods and vegetables)

Animal based foods (red meat, offal and fish), plants (dates, pomegranate) Flaxseed, walnut Red meat, dairy product, sunflower oil Beans, soyabean, peanut, tomatoes Pomegranate (punica granatum)

NCDs target

References

Breast cancer, CVDs

[151]

Diabetes, CVD

[152]

Nasopharyngeal carcinoma NDDs (AD, PD, HD), cancer

[153] [103]

NCDs and been consistently updating report of number of deaths and cases, successful implementation of preventive measures in all countries. Still there is need to understand the severeness of NCDs in many countries.

7. Conclusion and future perspective Antioxidants healings include use of natural antioxidant from natural source and activation of cellular signaling pathways to activate the endogenous antioxidant cascade, are promising strategies to overcome oxidative damage associated with NCDs. Therefore, NCDs prevention and control program must incorporate policies in which people are encouraged to maintain healthy living choices, increase health literacy. They should be provided with the health services focused on early detection and prevention of NCDs and other risk factors. There is a need of strategies to improve the sluggish lifestyle and physical activity. Also there is a need to improve the dietary habits and proper nutritional knowledge of food. It will require a complex series of legislative actions, awareness campaigns, clinical facilities, and adequate supply of medicines. NCDs are predominant global public health challenge of the 21st century. Prevention of premature death due to NCDs should be the main goals of health policies. At global level, many preventive and awareness policies can be implemented, but primary prevention of NCDs will start at individual level by maintaining healthy lifestyle.

Natural compounds in chemopreventive foods for prevention and management of non-communicable diseases

Acknowledgment AB sincerely acknowledges the financial support from University Grant Commission (UGC), India through UGC NET JRF-2019 fellowship (No. 836, (CSIR-UGC, JUNE 2019). The author SNM sincerely acknowledges the financial support from University Grant Commission (UGC), India through Dr. D.S. Kothari Postdoctoral Fellowship (No.F.4e2/2006/BSR/BL/18e19/0416).

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CHAPTER 16

Insect metabolome: New paradigm of novel metabolites discovery and its potential applications Yogita P. Patil1, 2, a, Sharada D. Mohite1, 2, a, Ashok P. Giri1, 2 and Rakesh S. Joshi1, 2 1

Biochemical Sciences Division, CSIR-National Chemical Laboratory, Pune, Maharashtra, India; 2Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India

1. Introduction Megadiverse insect communities contribute significantly to wealthy biodiversity. To date, more than 1 million species of insects have been reported. These insects show diverse behavior in terms of feeding, mating, and social structure; also, they have highly variable physiology and metabolisms [1,2]. Based on alterations in molecular, biological, and physiological divergence, insects show a wide array of metabolic diversity. As metabolites are final effector molecules, they show modulation of the immune response, feeding, courtship, communication, and other specialized responses. Metabolite abundance or occurrence is more dynamic than any other molecular mechanism. Due to its short life cycle, metamorphosis, and specialized physiology, insect metabolism is very dynamic, making it challenging to study and characterize specific metabolites [3]. Metabolites are important molecules that are generated by biochemical reactions that undergo chemical conversions during metabolism and are easier to be in tune with the phenotype [4]. Functional metabolism can explain links between a metabolic body and many aspects of life, including behavior, response to heat stress, dietary changes, health, and safety of an organism [5]. Multiple chromatographic and analytical techniques help to identify metabolite’s differential chemical and physical properties. They serve as a superior platform for qualitative and quantitative analysis of metabolites than other techniques [6,7]. This technique can detect, identify, and compare metabolites between the insect developmental stages, body organs, and tissues. In addition, the life stages of insects, from eggs to all instars of larvae, pupae, and adults of various insects used as a human diet for thousands of years, whether as a common food or in times of famine, as an ingredient, medicine, or ritual practice and even as a novelty dish. They are of great medical importance in humans and in veterinary due to they play a very important role as vectors for the transmission and infection and necrosis in the tissues; their activities can affect agriculture as they act as plant pollinators, pests, and parasites. Also, they’re used in medical a

Both authors have contributed equally.

New Horizons in Natural Compound Research ISBN 978-0-443-15232-0, https://doi.org/10.1016/B978-0-443-15232-0.00007-2

© 2023 Elsevier Inc. All rights reserved.

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treatments for several diseases as maggot therapy, which has gained important value with effectiveness significantly [8]. These various functions and applications of insects are attributed to a diverse range of insect metabolites. Insect metabolites possess tremendous potential and functions, so they need to be detected, identified, and characterized further for human welfare.

2. Specialized methods for insect metabolome analysis Metabolomics includes a set of high-throughput techniques that have new insights into biological processes by detecting and identifying a subset of molecules. This makes it possible to detect the whole metabolism of an organism in an exceedingly specific state with a metabolic phenotype [6]. The metabolic dynamics of insects go hand in hand with insect development and insect immunity, which involve the up-and-downregulation of biochemical factors, including small molecules. There are combined techniques for metabolite detection such as gas chromatography (GC), high-performance liquid chromatography (HPLC), ultra-performance liquid chromatography (UPLC) (Modified version of HPLC), mass spectrometry with nuclear magnetic resonance (NMR), and also capillary electrophoresis [9e11]. These specific metabolite detection methods are used to target the diverse nature of the molecules. GC-MS technique can measure as small as molecules of a mass around 500 Da, which need a transition of molecules between the liquid and gaseous phase; many polar compounds need to be chemically altered or derivatized, which possess the intermediate metabolism and must be formed into the more volatile compound. GC-MS can be used for the testing of low-polar metabolites like silicane derivatives, esters, fragrances, and volatiles. GC-MS can be used to analyze metabolites from termites from the pinewood (PW) and Mikania micrantha leave (ML) diet. Based on diet, upregulated key metabolites, including isomaltose, melibiose, palatinose, lactose, sorbitol, d-arabitol, myo-inositol, pelargonic acid, palmitic acid, 4-hydroxyphenyl acetic acid, and downregulated 2-oxoglutarate, tyrosine, kynurenine, 5-aminovaleric acid, maltose, 5-hydroxytryptophan in termites fed an ML diet [12]. Further, in Bombyx mori, the ingestion of sodium fluoride (NaF) can lead to an increase in the activity of metabolic pathways in fluorine-sensitive strains such as purine metabolism, glutathione metabolism, and oxidative phosphorylation. Among them, fumaric acid, phosphate, and guanine are upregulated metabolites, whereas other metabolites such as xanthosine, glycine, and L-cysteine showed differential downregulation. The fluoride tolerance mechanism of NaF of both the susceptible and tolerant strains was attributed to upregulation of hypoxanthine and 30 -adenylic acid, whereas downregulated metabolites are pyruvate, guanine, allantoic acid, xanthine, and N-acetyl-L-glutamic acid [13]. LC-MS is another suitable technique for detecting several highly polar metabolites like organic amines, nucleosides, nucleotides, and ionic compounds. LC-MS studies detect the

Insect metabolome: New paradigm of novel metabolites discovery and its potential applications

difference between treated insects having less biomass up to the power, to differentiate fruit flies (Drosophila melanogaster) from subspecies or mutant kinds of these species with different variables [14,15]. Many studies combine GC-MS and LC-MS to support an in-depth study of metabolism in organisms. Some specific compounds with different properties, like alcohols, amino acids, fatty acids, and organic acids, can be detected in GC-MS and LC-MS [13,16]. During the insecticidal detoxification metabolites analysis study, Anopheles sinensis mosquitoes were exposed to deltamethrin insecticide. Deltamethrin Susceptible (DS) and Resistant (DR) strains were collected and studied by LC-MS/MS method for differential metabolite analysis. The detected metabolites, deoxyguanosine, D-myo-inositol-3-phosphate, glycerophosphocholine, N-acetyl-alpha-Dglucosamine1-phosphate, DL-methionine-sulfoxide, aspartyl-glutamine, and organooxygen compounds, purine nucleic acids, and glycerophospholipids along with 2-formaminobenzoylacetate, isoleucyl-glutamate, leucyl-leucine, and 11-deoxy-PGF2a, showed upregulation and downregulation in DR and DS, respectively [17]. The identified metabolites possibly perform a critical involvement in the metabolic detoxification of deltamethrin. In Chilo suppressalis, on the third day of parasitism, metabolite analysis by using UPLC-MS/MS revealed the temporal metabolites, alanine, arginine, serine, and tyrosine are significantly increased in the hemolymph. On the other hand, it was found that 5-hydroxylysine, lysine, glutamic acid, and methionine levels dropped down [18]. Rapid, quantitative, and highly reproducible metabolome measurements are also possible using NMR. H1 NMR-based analysis of insect metabolism is mainly focused on the unique features of insects and depends on the hemolymph composition of larvae, pupae, and adults. 1H NMR-based metabolomics in honey bees (Apis mellifera) reveals that the glyphosate herbicide mediated metabolite alteration in the glyphosate-treated honey bees exposed to short- and long-term duration. The few essential amino acids, isoleucine, leucine, lysine, and valine, were downregulated by the second day of exposure. While prolonged exposure of ten days, other essential amino acids such as methionine, threonine, and histidine, besides non-essential amino acids proline, glutamine, and sucrose, were downregulated; these changes imparted the sedentary behavior [19]. In another study, 1H NMR-based metabolomics was used to detect citrate in honey bees’ whole body, 10-HDA in bee heads (mandibular glands), while GABA, arginine, and proline in the bee brain [20]. NMR, however, requires a larger sample mass and is less sensitive compared to MS-based studies. Lately, mass spectrometry imaging (MSI), a molecular technology with which we can visualize metabolites, has received plenty of attention. MSI is employed as a technique to show the tissue-wise distribution of macromolecules to small metabolites in insects and enables the assessment and identification of molecules present on the surface of insect tissues. Matrix Assisted Laser Desorption Ionization (MALDI-MSI) has been used to study the biologically complex material from various insects, including D melanogaster, A. mellifera, Cataglyphis nodus, An. stephensi, and Periplaneta americana. The MSI system is further connected with an electrical beam laser and

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Atmospheric-Pressure (AP-MALDI) [21,22]. MALDI-MSI can characterize various small molecules’ spatial and temporal distribution (e.g., lipids) and macromolecules (e.g., peptides and proteins). In soybeaneaphid interaction, aphids usually secrete honeydew, a complex mixture of oligosaccharides, composed of raffinose and stachyose while feeding. This is visualized with high-resolution MALDI-MSI around the feeding sites of aphids. Also, in bees, the localization along with the concentration of age-dependent neuropeptides (AmAST-1 and AmTRP-5) in the brain regulated their task inside the hive and outdoors. This neuropeptide distribution can be easily located by the MALDI-MSI. In the visualization of glucosinolate sinalbin of rapid sequestration in hemolymph upon ingestion by larvae of Athalia rosae, MALDI-MSI was used to give an idea of activation by plant myrosinases during digestion and to avoid any further toxicity [23]. Furthermore, high-resolution AP-MALDI-MSI is used to characterize the spatio-temporal distribution of endogenous metabolites in pomace flies and locate, pheromones, lipids, and a few more metabolites in males and females [21].

3. Insect metabolome: diversity and spatio-temporal dynamics Behavioral and physiological alterations can modulate the spatio-temporal distribution of metabolites. For instance, all sugars and alcohols are significantly reduced during the wandering phase compared to the feeding phase. Metabolites such as trehalose (a vital energy supplier) in insects and polyunsaturated fatty acids were decreased within the brain during the wandering phase, and it concluded that the wandering phase brain requires more energy than the feeding stage [10]. The many findings revealed that the infections altered the metabolite flux in insects. In the case of fungal infection in silkworm (B. mori), the level of metabolites, including amino acids, lipids, eicosanoids, and carbohydrates is substantially altered, which imparts the adverse effect on insects like nutrient deprivation, inhibition of host immunities [24]. Furthermore, based on diet (artificial feed and mulberry leaves), the metabolome for cocoon formation was detected and quantified in silkworms. The metabolites from both groups of insects were compared within the hemolymph, midgut, and posterior sericterium of the artificial feed and mulberry leaves, respectively. Among them, the three main pathways affected are cysteine and methionine metabolism, arginine biosynthesis, and proline metabolism. These pathway metabolites improve the cocoon yield performance [7]. Six major metabolites were detected; among them, homo-citrulline, glycitein, valylthreonine, propyl gallate, and 3-amino 2,3-dihydrobenzoic acid were positively correlated with whole cocoon yield, but inversely correlated with cocoon shell weight and cacoon shell ratio. This inverse correlation pattern was also observed with 3dimethylallyl-4-hydroxyphenylpyruvate levels in the hemolymph [7]. Additionally, the neuroactive molecules adrenaline, adenosine, and serotonin were upregulated with the downregulation of acetylcholine, g-aminobutyric acid, norepinephrine, and taurine

Insect metabolome: New paradigm of novel metabolites discovery and its potential applications

in the artificial feed group [25]. Insect larvae, including beetles and butterflies, have a precise mechanism for storing unexpected amounts of lactate. Majority of oxalate from rumex leaves is converted to lactate in Gastrophysa atrocyanea. This finding suggests that beetles and butterflies accumulate lactate within the larvae and will be used as an energy source for the insect during development [26]. In insects, most biological activities, such as growth, development, reproduction, and diapause, are regulated and controlled by the brain via the modulation of endocrine glands by employing neuroactive molecules dopamine (DA), tyramine (TA), and octopamine (OA). DA and TA have a higher concentration in the brain, finally inducing foraging behavior. In addition, OA concentration increases in a stressed condition. The glutamic acid concentration in the brain significantly decreased at the wandering stage. On the other hand, the wandering stage stimulates the prothoracic glands, the prothoracicotropic hormone secreted from the brain. It elevates the 20-hydroxyecdysone concentration in the hemolymph [27,28]. It can be inferred that the insect shows the spatio-temporal distribution of various metabolites under several conditions by imparting their unique nature.

4. Uniqueness of insect metabolome Insects share many similar metabolite profiles with plants and other organisms. Comparatively, insects produce many specialized and unique metabolites, which regulate insect survival differently. In insects, trehalose is the major hemolymph sugar. Trehalose serves multifaceted roles in insect development, regulates insect nutritional behavior, and uptake nutrients such as carbohydrates, proteins, and micronutrients. It also acts as a source of energy and sometimes facilitates the feedback mechanism during overwintering, and diapause of insects. Many studies proposed that the trehalose level in hemolymph imitates the dietary status of the insect [29]. During gluconeogenesis in Manduca sexta, nutrient levels were linked to hemolymph trehalose levels, and the carbon-to-nitrogen ratio from carbohydrate absorption affected the growth [30]. Ghost moth’s (Hepialus humuli) samples from fat bodies and hemolymph were analyzed for heat exposure duration. Ghost moth excessive heat exposure has induced differential metabolic profiles in the hemolymph and fat body. Trehalose and lactate indicate that they are the most eminent during heat tolerance. When the insect was exposed to 27 C, trehalose levels in hemolymph and fat body dramatically decreased, whereas massively accumulation of lactate was observed. Even so, the intermediates of lipid catabolism, mainly monoacylglycerols and free fatty acids (FFAs) were elevated in the hemolymph [31]. Furthermore, the invertebrate-specific neurotransmitters, OA and TA, play a salient neuromodulatory role in modulating insect behaviors, such as locomotion and grooming; conditional courtship in fruit flies; dance and sting behavior in honeybees; rhythmic behaviors and sensitization and dishabituation of sensory input in locusts; discrimination of nestmates from non-nestmates and invasive

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insects in honeybees and fire ants; feeding regulation of blowflies, cockroaches, and honeybees; age-wise and work division as foraging preference and labor in honeybees; visual responses with learning and memory processes in locust and honeybees, fruit flies, and crickets and many others [27,32]. The lipid b-oxidation key molecule, carnitine, and its acyl derivatives were known as differential metabolites that show a connection with respective time courses of phase transition in gregarious locusts to solitarious and vice versa. Locusts phases in the solitarious and gregarious groups, both have distinct hemolymph metabolic profiles. Differential metabolites were identified, and many of them are majorly involved in lipid metabolism, and the concentration of this significantly differed in the phases [33]. Beetle and butterfly’s metabolites, such as lactate and amino acids, along with that oxalate downregulated during feeding. The metabolites correlated during feeding were citrate, arginine, and asparagine during diet-based behavior. In comparison, metabolism was negatively dominated by fumarate, glutamate, aspartate malate, and phosphorylated compounds such as ribulose-1,5-bisphosphate and glucose-6-phosphate [26]. The special glands in the lepidopteron insects are Filippi’s gland, also called the Lyonet’s gland. Filippi’s glands contain 59 differential metabolites, including fatty acids, amino acids, and sugars. Homocysteine, D-glucose, allose, putrescine, D-mannose, N6-acetyl-Llysine, glutaric acid, B- alanine, L-lysine, pipecoli acid, L-phenylalanine, uridine, L-Histidine, L-glycine, vitamin B5, oxalic acid, cholesterol, linoelaidic acid, docosanamide, spermine, b-sitosterol, L-cystathionine, a-linolenic acid, arachidic acid, behenic acid, Lasparagine, N-dodecane, and D-arabinose [34]. FFAs are essential factors during microbial infection which determine resistance or susceptibility toward infection; they are present in cuticle compounds and an eicosanoids synthesis precursor. These FFAs play key regulators in insect cellular and humoral immunity. FFAs provide energy and supply material during metamorphosis and pupae formation. Depending on the insect’s developmental stage, the cuticular lipid pattern changes and forms an exoskeleton. Research on Sarcophaga bullata (Diptera: Sarcophagidae) and D. melanogaster has shown that FFAsconstitutes all cuticular lipids in newly emerged flies and in seven-day-old flies, concentration is increased. Unhardened cuticle majorly transports the FFAs, and this process is essentially over timedependent. Also, Drosophila females show increased age-dependent lipid and fatty acid metabolite content, which correlates with starvation resistance [35e37]. In addition, the ratio of saturated to unsaturated FFAs play a very critical role in insect survival in cold conditions, which is vital for insects’ survival tactics in different living in climate zones [36]. Parallel to this, the arrest during the developmental process in insects is known as diapause. Diapausing insects can withstand environmental stress by suppressing metabolic activity and increasing adversity resistance. Helicoverpa armigera arrests in diapause at the pupal stage, characterized by the accumulation of specialized metabolites like g-Aminobutyric acid in diapause-destined larvae. In addition to this, there are other metabolites present at differential levels in diapause-destined larvae like glycerol, fructose, glucose,

Insect metabolome: New paradigm of novel metabolites discovery and its potential applications

glycine, pyruvate, ribitol, and urea, phosphoric acid, 3-hydroxy-3-methylglutaric acid, phosphorylethanolamine, ornithine, and ribonic acid. Furthermore, Nacetylglutamate, glucose, N-acetylglucosamine (GlcNAc), and gluconic acid are found in the higher concentration during the later-stage larvae and prepupal stage. Metabolites like ribitol and glycerol are key molecules in cryoprotectants found in insects [38]. Accumulation of such metabolites has been observed in the diapausing Sarcophaga crassipalpis, flesh fly, and, C. suppressalis, the rice stem borer. Also, shows an increase in urea levels in hemolymph of diapause-destined larvae in the middle stage of the late instar larvae. While the precursor of urea, ornithine, was found to be decreased. Also, in diapausedestined larvae,GlcNAc levels were found to be upregulated in the late-stage instar. GlcNAc is the major and monomeric unit of chitin. The cuticle structural components implemented waterproofing to promote survival and enhance the diapause preparation phase [38]. Furthermore, it was observed by transcriptomic and metabolomic studies that in the process of cold tolerance, glutathione, and proline metabolism play very important roles in D. melanogaster [39]. Cannibalistic feeding behaviour is one phenomena where insects are prone to feed on body parts of smaller and/or weaker individual or whole individual of the same species. This cannibalism phenomenon is termed “quasi-vitamins” or “conditionally essential” nutrients. The absence of artificial diets imparts a reduction in insect growth rate. Lepidopteran insects like H. armigera transported choline into the hemolymph and later converted it into phospholipids then in the fat body. The choline phosphate and its ester derivatives are the most abundant choline metabolite in the B. mori and in other lepidopteran insects’ hemolymph. Concentration of these metabolites is elevated as the larval behavior shifts toward cannibalism [40]. GC/MS-based metabolomics approaches in A. gambiae females have shown an elevated accumulation of certain amino acids, organic acids, and other metabolites post 24 h blood meal [41]. These above discussed metabolites are originated and associated with the differential interactions between insects and other components like the microbiome, plant, and other insects.

5. Insect-associated metabolome The insect produces various metabolites, including amino acids, proteins, and lipids, to achieve normal growth, development, and response to stimuli. The role of specialized metabolites is studied in D. melanogaster, Paederus riparius, Prorhinotermes simplex, Graphosoma lineatum, and B. mori. More than 60 metabolites were reported in the hemolymph of the silkworm. These metabolites include quinate, lactate, acetate, pyruvate, valine, alanine, glycine, glucose, trehalose, glycogen, GlcNAc, putrescine, dopamine, trimethylamine, citrate, 2-ketoglutarate, succinate, malate, betaine, ascorbate, nicotinamide, choline, purines, and pyrimidines. The level of these metabolites varies across the developmental stages and stress responses. For example, branched-chain amino acids level was

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low in hemolymph on the 3rd day of the 5th larval instar. In contrast, the citrate level was higher relative to the 3rd day of the 3rd larval instar and prepupa stage. Trehalose level was higher on the 3rd day of the 3rd larval instar and the 3rd day of the 5th larval instar. Other metabolites such as GlcNAc, methionine-sulfoxide, mesaconate, tyrosine-o-b-glucuronide, and hydroxykynurenine were observed only in hemolymph at a pre-pupal stage but not observed in other stages [42]. Neuropeptides are involved in various physiological and developmental processes of insects. Fourteen neuropeptides were reported in the coronal brain part of D. melanogaster [21]. Neuropeptides like allatostatins and tachykinin-like peptides are responsible for odor perception and locomotion. Baratin is a nonamidated neuro stimulating neuropeptide found in the cockroach brain. It plays a role in modulating motor patterns in the abdominal ganglia [43]. Insects also produce defense compounds to protect themselves from natural enemies. For example, the Rove beetle (P. riparius) has defensive molecules such as pederin, pseudopederin, and pederon [44]. Similarly, the venom of honeybees contains three allergens and two toxins [45]. In addition to this, (e)-1-nitropentadec-1-ene and (e)-hex-2-enal are essential defensive compounds found in a P. simplex, a Cuban subterranean termite and in Graphosoma lineatum, stink bug respectively. This compound is most abundantly present in openings and reservoirs of glands of termites and stink bugs [46]. Insects are preferred for drug testing instead of mice, rabbit, and monkeys because of their high fecundity, easy handling, low maintenance cost, and fewer ethical regulations. Locust has emerged as a new model system for drug metabolism experiments because of its anatomical similarity with mammals. The effect of terfenadine treatment (an antihistamine drug) on the distribution of secondary metabolites in locust has been studied by Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) imaging [47]. In this study, terfenadine was observed in the stomach and intestinal walls, whereas the smaller amounts of other metabolites such as fexofenadine, terfenadine glucoside, and terfenadine phosphate were observed only in an unexcreted fecal matter stored in the lower part of the intestine. So, no colocation of the terfenadine drug and its respective metabolites was observed, indicating quick defecation of metabolites through fecal matter [47]. Similarly, the temporal distribution of the drug midazolam and its glucose conjugates were studied in locust. Midazolam was observed in fecal matter abundantly after 30 min upon its treatment, whereas its glucose conjugate, i.e., midazolam glucoside, was observed mainly in gastric caeca (part of midgut), intestine, and fecal matter after 2 h of treatment. This result showed that locust removes the toxic effects of midazolam by its conjugation with glucose to remove the toxic [47]. 5.1 Plant-insect interaction metabolome The plant produces specialized metabolites to protect itself from herbivores in response to insect feeding. The insect also produces metabolites in response to plant defense. It is essential to study metabolite changes in insects and plants to shed light on

Insect metabolome: New paradigm of novel metabolites discovery and its potential applications

understanding the association between plant resistance and the adaptability of insects. Interaction of host-insect leads to changes in the metabolite profile of both. In one of the studies involving aphid (Aphis glycines) and soybean (Glycine max), defense-related compounds were accumulated [48]. The compounds accumulated in aphids-infested soybean leaf include phosphorylcholine, pipecolic acid, salicylic acid, formononetin, dihydroxyflavone, and amino acid [48]. Similarly, in another study, isoflavones were accumulated explicitly in the mesophyll cells or epidermis. These compounds were absent in the vasculature, suggesting the contribution of isoflavones in non-phloem defense response [49]. Recently, advanced mass spectroscopy-based techniques were used to study the fate of plant-insect interaction at the metabolite level. Initially, glucosinolates accumulated in sinalbin and then circulated in the hemolymph, and in the end, they accumulated around the malpighian tubules [50]. Similarly, metabolites produced by Dactylis glomerate (a grass) were traced in Chorthippus dorsatus (grasshoppers) by using Laser Desorption/Ionization (LDI). Metabolites such as quinic acid, apigenin, and luteolin were accumulated in wounded grass leaves until the healing process was completed. Quinic acid was detected in all the digestion steps in the gut and feces of grasshoppers [51]. NMR is also used to study metabolite changes in plant-insect interaction. Metabolite analysis in plant exhibit different responses to specialist and generalist insects. The level of glucose, ferulic acid, gluconapin and alanine, and sinapoyl malate increased in leaves of Brassica rapa L upon attack by generalist insect Spodoptera exigua. In contrast, gluconapin, glucose, feruloyl malate, sinapoyl malate, and threonine levels were increased in response to the specialist insect Plutella xylostella L [52]. Similarly, other biotic interactions alter insect metabolome in response to interaction. 5.2 Insect gut microbiome metabolome Insects not only cause the loss of crops but also helps with pollination and associated microbes to improve their impact on crops. Insects and their associated gut microbes are critical units of natural and human-impacted ecosystems involved in biogeochemical cycling. For example, insect microbes help to decompose the biomass of plants and contribute substantially to the carbon cycle [53]. Further, they also help in nitrogen fixation and enhances the nitrogen cycle [54]. Microbial communities are mainly found in the gut and show a mutualistic relationship with their hosts. The involvement of gut microbiota in insect function is extremely applicable to medicine, agriculture, and ecology [55]. Beneficial microbes are associated with herbivores and pollinators help them to digest recalcitrant food compounds and efficient nutrient uptake from poor diets. They also protect insects’ from predators, parasites, and pathogens. These useful microbes are involved in intra- and inter-specific communication and help insects’ in mating and reproduction [55]. Microbes associated with insects’ gut use specific secondary metabolites for interaction with their host [56]. Photorhabdus and Xenorhabdus are symbiont of

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nematodes, Steinernema (Rhabditita: Steinernematidae) and Hetero-rhabditis (Rhabditita: Heterorhabditidae), respectively, that feed on the diverse host range. These two symbionts produce abundant secondary metabolites to help their survival [57,58]. For example, Xenorhabdus species produce a series of rhabdopeptides that governs nematode virulence against the host insect [59]. Similarly, X. nematophila produces cell wall-localized phenoloxidase inhibitor rhabduscin that inhibits the immune response of insects [56,60]. Some secondary metabolites produced by Photorhabdus are capable of governing multiple symbiotic interactions. P. luminescens produces a molecule called stilbenes, that acts as a phenoloxidase inhibitor of insects’ virulence factors [61]. Moreover, it can function as a specific signal for the nematode’s development. P. luminescens produces another molecule called pyrone that triggers the insects’ virulence and clumping of the host cell [56,57]. Folate-producing bacteria secreted 5-formyl-tetrahydrofolate in the gut of lower termites. The spirochete Treponema primitia utilizes this metabolite as a co-factor to produce acetic acid, a source of energy for the insect host [62]. Numerous candidate cross-feeding bacteria coexist in the honey bee gut, such as Bifidobacterium spp., Gilliamella apicola, and Snodgrassella alvi. These bacteria improve the digestion of the pollen diet and utilize bacterial by-products of other co-occurring taxa [60,63]. Bacteria from Acetobacteraceae family, Lactobacillales order, and yeast from Saccharomycetales are the major microbes found in the Drosophila gut microbiome [64,65]. Interactions of bacteria and yeasts may help insects in their life cycle and events. Drosophila female flies prefer diets having SaccharomyceseAcetobacter co-culture for feeding and laying more eggs than monocultures. In case of using only Acetobacter strain in diet, acetic acid was not produced. While, four microbial metabolites, namely acetic acid, acetoin, ethanol, and lactic acid, were produced upon the interaction between AcetobactereLactobacillus and Acetobactere Saccharomyces interactions [64]. These metabolites are mediators of the microbe and microbe-host interactions. They have a vital role in the regulation of Drosophila metabolism, immunity, and behavior [66e70]. Table 16.1 shows the major characteristics of the insect metabolites along with their applications.

6. Potential application of insect metabolites 6.1 Therapeutics application Reports suggest that products from insects or insect-based extracts have several therapeutic applications in human health [83]. Insects protect themselves by producing specialized metabolites as a result of self-defense. These chemicals are screened for various therapeutic activities like antioxidant, antibacterial, and anticancer. In the traditional medicine system, ant, wasp, and bee products, such as honey and venom, were used to treat wounds, arthritis, rheumatism, ulcers, inflammation, infections, pain, cancer, and allergic reactions [71]. Traditionally honey has been included in various pharmaceutical preparations and used to improve human health [84]. Beehive propolis is a resinous, waxy substance

Table 16.1 Characteristic insect metabolites and their applications. Class

Insect

Application/Activity

References

1.

Venom

Peptide mixture

Wasp and ant

[71]

2.

Honey

Peptide mixture

Bee

3. 4.

Beehive propolis Apalbumins

Polyphenols Protein

Bee Bee

5.

Honey bee based products

Peptides, polyphenols

Bee

6.

Melittin

Peptide

Bee

PLA2 and apamine

Protein and polypeptide

Bee

Wounds, arthritis, rheumatism, ulcers, inflammation, infections, pain, cancer, and allergies Wounds, arthritis, rheumatism, ulcers, inflammation, infections, pain, cancer, and allergies Lip balms and tonics Strengthen the human body, improve appetite, prevent aging of skin and leukemia, and treat other cancers Diarrhea, tuberculosis, impotency, asthma, exophthalmic goiter, and mouth galls Inhibits binding of NF-kB with DNA. Also used for the treatment of rheumatoid arthritis and multiple sclerosis. Anticancerous activity against renal, lung, liver, prostate, bladder, mammary cancer cells, and leukemia cells Anticancerous activity by reactivating p53 tumor suppressor

7.

[71]

[72] [72]

[73]

[73e77]

[78]

303

Continued

Insect metabolome: New paradigm of novel metabolites discovery and its potential applications

Metabolite/Product

304

8. 9.

Metabolite/Product

Class

Insect

Application/Activity

Allantoin T cantharidin

Monocarboxylic acid Terpenoid

Blowfly (calliphoridae) Blister beetle, Cantharis vesicatoria.

Wound treatment Used as a diuretic and to recover from epilepsy, asthma, rabies, and sterility Components of medicine used against malaria Chronic diarrhea and profuse menstruation Antibacterial

10.

Eggs

Red ant (Solenopsis invicta)

11.

Cocoon extract

Mulberry silkworm

12.

Proteins

13.

Pierisin, cecropins, defensins Pierisin and cercopin

14. 15.

Sericin, sericin powder Silk

Proteins Protein fiber

Proteins

Pieris rapae, P. brassicae, and P. napi Pieris rapae, P. brassicae, and P. napi Silkworm Silkworm

Against human gastric cancer, mammalian lymphoma, and leukemia cells Wound suturing. Relief of spasms and flatulence and also for the treatment of vascular impotency

References

[79]

[80] [80] [81] [81]

[31] [82]

New Horizons in Natural Compound Research

Table 16.1 Characteristic insect metabolites and their applications.dcont’d

Insect metabolome: New paradigm of novel metabolites discovery and its potential applications

produced by bees upon mixing saliva with beeswax and compounds from various plants. It is used in lip balms and tonics. In contrast, albumin in royal jelly strengthens the human body, improves appetite, prevents skin aging and leukemia, and treats other cancers. The honey bee is considered a medicinal insect because 80% of its weight is used as medicine. In comparison, 10% is used in traditional ayurvedic and pharmaceutical formulations. The therapy where honey bee products are used for treatment is called Apitherapy. Honey bee products have been used as conventional medicine to cure several diseases such as diarrhea, tuberculosis, impotency, asthma, exophthalmic goiter, and mouth galls [84]. The venom of the honey bee is a combination of multiple vital compounds. Bee venom contains enzymes such as phospholipase A2 (PLA2) and hyaluronidase, peptides such as melittin, apamin, mast cell degranulating peptide (MCD), and adolapin, and biogenic amines such as histamine, serotonin, and catecholamine, have many pharmaceutical properties [71,73]. Melittin is a potent peptide found in bee venom that has inhibitory activity on the DNA binding action of NF-kB. This key transcriptional factor regulates the expression of the gene responsible for inflammation by inhibiting phosphorylation of IkB, so it reduces swelling and pain. Melittin is also used to treat people with rheumatoid arthritis and multiple sclerosis [77,84]. PLA2 and apamine from the venom of bees also showed anticancerous properties. PLA2 acts mutually with the phospholipid, phosphatidylinositol-(3,4)-bisphosphate of the cell membrane, inhibits tumor cell growth and potent cell lysis. Apamine also acts as a potential anticancerous agent. It resuscitates the p53 tumor suppressor pathway and triggers the rapid elimination of tumors [78]. Bee venom also has anticancerous properties as melittin acts on various cancerous cells such as renal, lung, liver, prostate, bladder, mammary cancer cells, and leukemia cells [85,86]. There might be many such insects that contain medicinally essential compounds. Allantoin, secreted by blowfly (Calliphoridae) larvae, has a curative effect on treating wounds [84]. Cantharidin, approved medicine, is produced from the blister beetle, Cantharis vesicatoria. Cantharidin was used as a water pill to recover from epilepsy, asthma, rabies, and sterility [79]. The red ant (Solenopsis invicta) eggs are used as one of the components of medicine used against malaria. Mulberry silkworm cocoon extract is supposed to check chronic diarrhea and profuse menstruation [84]. In China, Japan, Korea, India, and Thailand, pupae of silkworms are usually used as medicine. The waste liquor containing sericin produced during degumming silk fiber is a used crude material for producing sericin powder. This powder has various medicinal applications [84]. In Chinese medicine, silk relieves spasms and flatulence and treats vascular impotency [82]. Also, the larvae of different species, which include Lucilia sericata (Diptera: Calliphoridae) or Protophormia terraenovae (Diptera: Calliphoridae), also are used as maggot remedy withinside the remedy gangrene and wounds [87]. Apart from these extensive therapeutic uses, insect metabolites are also used for other applications. The insect metabolome applications are shown in Fig. 16.1.

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Figure 16.1 Insect metabolite applicationsdinsect metabolites identified and isolated by various high-throughput methods have diverse therapeutic, nutraceutical, and biotechnological applications.

6.2 Nutraceutical application Insect consumption is historically practiced in 113 nations across the globe. More than a thousand insect species are recognized to be suitable for human eating. The most regularly ingested species are beetles, caterpillars, bees, wasps, ants, grasshoppers, locusts, crickets, bugs, termites, dragonflies, flies, cicadas, leafhoppers, and different species. Asia, Africa, and Latin America countries are the highest consumer of insects as feed. Insects have excessive material of mono- and polyunsaturated fatty acids; and are rich sources of iron, magnesium, copper, manganese, selenium, zinc, phosphorus, and nutrients like riboflavin, B, biotin, and B complex in an exceedingly few cases. Edible bugs comprise an oversized quantity of fibers, which can help the digestive process. In a few butterfly caterpillars, retinol and b-carotene were present. Fat-soluble vitamins became determined with inside the larvae of the crimson palm weevil Rhynchophorus ferruginous [88]. Larvae with a more substantial weight contained an improved concentration of saturated fatty acids and a decreased percentage of unsaturated fatty acids, with the incorporation of eicosapentaenoic (EPA) and omega-3 (DHA) [89,90]. There are few traditional practices where insects fed medicinal herbs are used as feedstock or fortified feeding for holistic health benefits.

Insect metabolome: New paradigm of novel metabolites discovery and its potential applications

6.3 Industrial application Potential applications and products originating from the insect hold high promises in the biotechnology industry. Carminic acid is the primary pigment that will be extracted from the Dactylopius coccus. It is predominantly utilized in cosmetics, meals, pharmaceutical programs, and fabric and plastic programs [91]. Kermes bugs (Kermes vermillion, Kermes palestinensis) are scale bugs from the Mediterranean, which may be parasitic on numerous species of dryland oak shrubs utilized to produce red-colored dye kermesic acid. A topnotch crimson dye is extracted from the shell of the female bugs. Their use has been considered the very fact that historic instances, while this color cited because the ‘King’s crimson’ and precious as a painter’s pigment. The dyes are similar in color, highsatisfactory and intensity; however, cochineal dye is 10 or 12 times as powerful because of the kermes dye. Cochineal comes from the scale insect, which produces carminic acid. Carmine dye is crafted from carminic acid extracted from the female beetle eggs. This crimson dye is employed to produce scarlet, orange, and sun sunglasses of crimson. Also, it is used in cosmetics and food as a colorant. The lac bugs produce a crimson dye a great deal, just like the cochineal and kermes bugs; however, it is also recognized for its manufacturing of a glassy resin processed to produce shellac. Also, scale bugs, the Laccifer lacca or Kerria lacca, secrete a resin to defend themselves among hatching and maturing into adults. They’re determined in massive colonies on various bushes in a geographic area [92,93]. Beeswax considers the most versatile and widely used material in the industry. Wax is composed of a variety of complex metabolites such as hydrocarbons, mainly heptacosane, nonacosane, hentriacontane, pentacosane, and tricosane; free fatty acids, with a free fatty alcohol. For centuries, beeswax was widely used as the most suitable material for candles, polishing woods and floors, also in general-purpose varnishes. Furthermore, it has been used in the packaging and processing of preserving foods, confectionery, and cigarette filters. Beewaxes impart waterproof properties to textiles and papers. Emulsions for leather goods were softened by beeswax. It is used in slow-release pyrethrum pesticides pellets in the pesticide industry. Also, small amounts of beeswax are incorporated in various inks, pens, markers, and even carbon paper often contain. Beeswax facilitates ingestion, so it has also found use in cosmetics and medicinal pills as a coating agent. Other products in which beeswax is a regularly used ingredient are grafting wax, crayons, sealing wax, protective car polishes, and thread for sewing canvas sails and leather shoes [94]. Resilin is a rubber-like protein that attributes elastic properties used to repair arteries. Sericin is the main component of silk proteins, appears vital and elastic, and has been used as biomaterial [95]. 6.4 Other applications In addition to these applications, the flies from the Families Calliphoridae (blowflies), Sacrophagidae (flesh flies), and Muscidae (residence flies) are also used for

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entomotoxicology [96,97]. Some insects including D. melanogaster (Diptera: Drosophilidae), also Galleria mellonella (Lepidoptera: Pyralidae), A. mellifera (Hymenoptera: Apidae), and B. mori (Lepidoptera: Bombycidae), are taken into consideration as model organisms in clinical and veterinary research [1,98,99]. Fatty acid content of black soldier fly larvae Hermetia illucens (Diptera: Stratiomyidae) is utilized in aquaculture as they feed on several natural substances and reassert each protein and fat [100,101]. These applications highlight the importance of insect metabolites but need to shed light on neglected insect metabolites and their applications.

7. Conclusion and future prospects Insects are diversified organisms present on earth. Insect metabolites help in regulating and modulating insects’ every activity. Here, we discussed specialized methods used for the insect’s metabolite detection. There are analytical techniques like LC-MS, UPLC, GC-MS, NMR, capillary electrophoresis, and MALDI-MSI can be used to identify, quantify the metabolites with high polarity and high formula weight, and more than other platforms. Insects’ unique metabolites show tremendous spatiotemporal effects on insect behavior. In addition, metabolites are used in gangrene and wound treatments, such as maggot therapy. They are also used in human welfare as dietary supplements and industrial applications. As during nutraceuticals and industrial aspects, feeding future populations of humans and animals will require the development of beetles, caterpillars, grasshoppers, locusts, bees, wasps, bugs, termites, dragonflies, flies, ants, crickets, cicadas, leafhoppers, and many more species as alternative sources of protein, vitamins like riboflavin, B, biotin, and B complex and minerals like copper, iron, phosphorus, selenium, magnesium, manganese, and zinc. Consuming insect metabolites has several advantages over animal products for direct human consumption by employing therapeutic and nutraceutical products. Recent research and development show edible insects and their respective metabolites can be promising to add on benefits to some extent in alternation for the meat products, through direct consumption or for secondary utilization in the feedstock. Another industrial usage of insect metabolites carminic acid, kermesic acid, crimson dye includes dye-making, and metabolites complex present in bee wax used in various insulators and coating, resilin and sericin metabolites elasticity to repair arteries and to make biomaterials. Nevertheless, to fully comprehend the possibility that insects and their metabolites have to achieve and discover the beneficial facts, a tremendous amount of effort still needs to be made. Additionally, insect rearing activity should be promoted as a socially inclusive activity and encouragement should be done. This kind of activity requires less technical support and knowledge with less capital investment. Since it can be done in minimal space, it can be done by underprivileged members. As the prices of traditional animal and plant products increase, insects may become the most suitable and beneficial alternative, a

Insect metabolome: New paradigm of novel metabolites discovery and its potential applications

cheaper, and more valuable resource. Technological innovation, changes in the feeding preferences of consumers, insect-circumscribed food, and sustainable food production are needed to promote insects as feed. Insects can partly replace compound feed’s increasingly expensive dietary ingredients in the aquaculture industries, livestock, and poultry. As the insect is one of the unavoidable constituent of human diet, potential needs to be re-evaluated by including their metabolites. The immense insect biomass is needed to replace current human and animal dietary ingredients such as meal and oil from fish in aquaculture industries and soybean, so automated and quantitative mass rearing imparts the increase in the stable production reliable, and innocuous harvests need to be industrialized. The challenge for this insect production, the industry will be to ensure the profitable, dependable production of insect biomass of high and consistent beneficial superiority. However, many of the insect’s metabolites are neglected and are yet to be discovered. It is necessary to tap insect diversity to explore more metabolites and investigate their possible use in therapeutic, agricultural, or industrial use. These may contribute to human well-being.

Acknowledgment YPP and SDM would like to thank University Grant Commission for the fellowship. RSJ and APG acknowledge the Council of Scientific and Industrial Research (CSIR), India, and CSIR-National Chemical Laboratory, Pune, India, for financial support under project codes MLP36626 and MLP101526.

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[88] Kourimska L, Adamkova A. Nutritional and sensory quality of edible insects. NFS J 2016;4:22e6. https://doi.org/10.1016/j.nfs.2016.07.001. [89] Ewald N, Vidakovic A, Langeland M, Kiessling A, Sampels S, Lalander C. Fatty acid composition of black soldier fly larvae (Hermetia illucens)dpossibilities and limitations for modification through diet. Waste Manag 2020;102:40e7. https://doi.org/10.1016/j.wasman.2019.10.014. [90] Oonincx DGAB, Laurent S, Veenenbos ME, van Loon JJA. Dietary enrichment of edible insects with omega 3 fatty acids. Insect Sci 2020;27:500e9. https://doi.org/10.1111/1744-7917.12669. [91] Borges M, Tejera RL, Díaz L, Esparza P, Iban ~ez E. Natural dyes extraction from cochineal (Dactylopius coccus). New extraction methods. Food Chem 2012;132:1855e60. https://doi.org/10.1016/ j.foodchem.2011.12.018. [92] Pigments | Causes of Color (WWW Document), n.d. URL http://www.webexhibits.org/ causesofcolor/7.html (accessed 5.29.22). [93] Cooksey CJ. The red insect dyes: carminic, kermesic and laccaic acids and their derivatives. Biotech Histochem 2019;94:100e7. https://doi.org/10.1080/10520295.2018.1511065. [94] Fratini F, Cilia G, Turchi B, Felicioli A. Beeswax: a minireview of its antimicrobial activity and its application in medicine. Asian Pac J Trop Med 2016;9:839e43. https://doi.org/10.1016/ j.apjtm.2016.07.003. [95] Takahashi M, Tsujimoto K, Yamada H, Takagi H, Nakamori S. The silk protein, sericin, protects against cell death caused by acute serum deprivation in insect cell culture. Biotechnol Lett 2003; 25:1805e9. https://doi.org/10.1023/A:1026284620236. [96] Byrd J, Sutton L. Forensic entomology for the investigator. WIREs Forensic Sci 2020;2:e1370. https://doi.org/10.1002/wfs2.1370. [97] Hodecek J. Revisiting the concept of entomotoxicology. Forensic Sci Int Synergy 2020;2:282e6. https://doi.org/10.1016/j.fsisyn.2020.09.003. [98] Menzel R. The honeybee as a model for understanding the basis of cognition. Nat Rev Neurosci 2012;13:758e68. https://doi.org/10.1038/nrn3357. [99] Wojda I, Staniec B, Sułek M, Kordaczuk J. The greater wax moth Galleria mellonella: biology and use in immune studies. Pathog Dis 2020;78. https://doi.org/10.1093/femspd/ftaa057. [100] Diener S, Gutierrez FR, Zurbr€ ugg C, Tockner K. Are larvae of the black soldier flye hermetia illucens e a financially viable option for organic waste management in Costa Rica?vol 7; 2009. [101] Wang Y-S, Shelomi M. Review of black soldier fly (Hermetia illucens) as animal feed and human food. Foods 2017;6:91. https://doi.org/10.3390/foods6100091.

Further reading [1] Despland E, Noseworthy M. How well do specialist feeders regulate nutrient intake? Evidence from a gregarious tree-feeding caterpillar. J Exp Biol 2006;209:1301e9. https://doi.org/10.1242/jeb.02130. [2] Huis A van. Edible insects: future prospects for food and feed security, FAO forestry paper. Rome: Food and Agriculture Organization of the United Nations; 2013.

CHAPTER 17

New targets for old drugs: drug repurposing approach for accelerating the drug discovery engine with minimum financial inputs Navanath M. Kumbhar1, M.A. Aparna1, Snehal K. Nimal1, Pallavi Shewale1, Sagar Barale2 and Rajesh Gacche1 1

Department of Biotechnology, Savitribai Phule Pune University, Pune, Maharashtra, India; 2Department of Microbiology, Shivaji University, Kolhapur, Maharashtra, India

1. Introduction to drug repurposing: beyond the “old wine in new bottle” Drug repositioning (DR) is the process of finding new clinical indications for existing drugs [1,2]. In the current “target rich-lead poor” scenario of drug discovery, the repurposing or repositioning is gaining increasing interest over the traditional de novo drug discovery, which is more time-consuming (13e15 years) and expensive (US$ 2-3 billion) [1]. At present, there are over 20,000 drugs prescribed by medical practitioners and approved by the FDA (www.fda.gov). The majority of FDA-approved drugs have been tailored against a specific target, but more than one molecular target can potentially be affected by most small inhibitor molecules [1,2]. Owing to the non-affordable prices of anticancer drugs, the slow pace of anti-cancer drug discovery and development, and evolving drug resistance, there is a need to develop novel drug candidates against deadly human diseases through a strategic drug development process. The DR tackles two main problems in the pharmaceutical industry such as the cost and time required to develop new drugs (Fig. 17.1). The safety and efficacy of FDA-approved drugs have been already accessed; therefore, repurposing accelerates the discovery and drug development process [3]. Furthermore, the risk of failure in clinical trials and the timeframe from discovery to approval of drugs decides the overall costs of drugs from bench to bedside and which is lower in the case of repurposed drugs as compared to new or previously untested drugs [1,2]. Therefore, repurposing is a suitable strategy to discover new indications for existing drugs to treat various human diseases within a short timeframe and cost-effective manner. The DR is being appreciated and becoming an increasingly attractive and popular approach for the design, discovery, and development of new drugs. Of note, such repurposed drugs are likely to capture a significant portion of the pharmaceutical market in a short period. Therefore, several pharmaceutical companies are using the repurposing New Horizons in Natural Compound Research ISBN 978-0-443-15232-0, https://doi.org/10.1016/B978-0-443-15232-0.00021-7

© 2023 Elsevier Inc. All rights reserved.

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approach to discover new indications for old drugs, and their clinical trials to repurpose against new diseases [4]. However, some researchers have made the statement “drug repurposing is a well-intentioned but misguided approach,” based on arguments such as the repurposed drugs are lacking single-agent activity in treating cancer, the evidence which supports the DR is obtained from retrospective observational studies, and the substantial costs of testing of repurposed drugs. These are valid concerns about some of the repurposed drugs in oncology, but it does not apply to the drugs which are repurposed against other human diseases. Furthermore, DR is a two-edged sword that reduces the cost and time of finding new drugs by shortening the drug development process by 3e5 years and increasing the success rates from 10% to 25% as compared to the conventional de novo drug development process. Therefore, the repositioned drugs entering in the regulatory approval pipeline are increasing every year and could account for about 30% of total approved drugs [1,2]. Here, we are summarizing the repurposed drugs against various human diseases such as cancer, cardiovascular disorders, neurodegenerative disorders, diabetes, and viral and microbial infections. Thalidomide, aspirin, itraconazole, metformin, sildenafil, azidothymidine, imatinib, and nelfinavir are the successfully repurposed drugs widely used for their new indications [1].

Figure 17.1 Timeline between classical de novo drug development and drug repurposing.

New targets for old drugs: drug repurposing approach for accelerating the drug discovery engine

2. Experimental and computational approaches for drug repurposing In the era of big-data (omics) analysis, the DR is evolving through the use of multiparametric techniques including experimental and computational strategies that allow the rapid screening of old drugs against new biological targets from various human diseases/disorders [5]. Experimental strategies like cellular thermostability assays, binding assays, single-cell sequencing, and microarray gene expression analysis are enabling successful repositioning. Furthermore, computational techniques such as genome-wide association studies, transcriptional signature-based, 3D-QSAR pharmacophore modeling, structure/ligand-based screening, molecular docking, MD simulations, machine/deep learning, biological network analysis, and data-mining-based screening are playing a key role in the repositioning of drugs for cancer, cardiovascular, diabetes, neurodegenerative disorders, microbial infections, and other rare diseases [6,7]. Indepth computational analysis followed by experimental validations predicts the drugdisease interactive response. The biomarker-guided strategy was developed in the past decade for repurposing drugs against breast cancer with an increasing approval rate. With high prediction accuracy, network-based techniques identify novel drug-disease or drug-target interactions; nevertheless, they have several drawbacks, such as the inability to recognize overlapping clusters [8,9]. The drug-centric method anticipates the relational link between the new indications for known medicine and their different novel target proteins. Molecular docking helps to screen a library of compounds against an archive of protein targets. Pharmacophore modeling predicts the pharmacophoric features of drugs and is used to screen against various commercial databases. In addition, the protein-ligand interaction-based 3D-pharmacophoric mapping describes the pharmacophoric features that are participated in stabilizing the protein-ligand interactions which help in drug-centric repositioning [10]. In addition, artificial intelligence (AI), deep neural networks (ANN), and graph representation learning are machine learning-based approaches that are playing a significant role in the repurposing of drugs against human diseases. The AI was widely used for accelerating drug repurposing for emerging human diseases such as COVID-19, Alzheimer’s disease, Cancer, and viral infections [11,12]. The list of databases, software, and servers that are useful in the repurposing of drugs against various human diseases has mentioned in Table 17.1 [13].

3. Repurposing the drugs for effective cancer management Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells in the body [14]. The 18.07 million cases of cancer are diagnosed and 9.55 million cancer deaths are reported around the world [14]. More than 70% of cancer deaths occur in low and middle-income countries, and become the second major cause of

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Table 17.1 List of databases, servers, and software useful for drug repurposing. Database/Server

Method

Website

DrugBank ChemSpider

Compound database Compound database

PubChem

Compound database

DPubChem

Machine learning/QSAR

ZINC Therapeutic target database PyRx AutoDock

Compound database Provide therapeutic drug targets SBVS SBVS

https://go.drugbank.com/ http://www.chemspider. com/ https://pubchem.ncbi.nlm. nih.gov/ https://openebench.bsc.es/ tool/dpubchem https://zinc.docking.org/ http://db.idrblab.net/ttd/

GOLD

SBVS

SwissDock ChemDes

SBVS Molecular descriptor and fingerprint computation

AlzPlatform (chemogenomics Database) LightBBB server

TargetHunter, HTDocking, and BBB predictor Blood brain permeability prediction Pharmacokinetics prediction Ligand similarity tool based on hierarchical clustering Predict molecular descriptors and fingerprint for small molecules 3D shape pharmacophore generation and ligand similarity search Small molecules toxicities prediction Molecular and bioactivity prediction of molecule Pharmacokinetics and toxicity profile of small molecule

SwissADME ChemMine ChemDes

BRUSELAS

ProTox II Molinspiration cheminformatics admetSAR

https://pyrx.sourceforge.io/ http://autodock.scripps. edu/ https://www.ccdc.cam.ac. uk/solutions/csddiscovery/components/ gold/ http://www.swissdock.ch/ http://www.scbdd.com/ mopac-optimization/ optimize/ https://www.cbligand.org/ AD/mainpage.php http://ssbio.cau.ac.kr/ software/bbb http://www.swissadme.ch/ https://chemminetools.ucr. edu/ http://www.scbdd.com/ mopac-optimization/ optimize/ https://bio-hpc.ucam.edu/ Bruselas/web/Tutorial/ Tutorial.php https://tox-new.charite.de/ protox_II/ https://www. molinspiration.com/ http://lmmd.ecust.edu.cn/ admetsar1

New targets for old drugs: drug repurposing approach for accelerating the drug discovery engine

Table 17.1 List of databases, servers, and software useful for drug repurposing.dcont’d Database/Server

Method

Website

T.E.S.T

Toxicity estimation

PharmaGist

Ligand base pharmacophore generator Pharmacophore base ligand similarity search Pharmacophore base drug target identification

https://www.epa.gov/ chemical-research/ toxicity-estimationsoftware-tool-test https://bioinfo3d.cs.tau.ac. il/PharmaGist/php.php http://zincpharmer.csb.pitt. edu/ http://www.lilab-ecust.cn/ pharmmapper/

ZINCPharmer PharmMapper

high morbidity and mortality worldwide, followed by cardiovascular disorders [14]. Although there are many potent anticancer drugs available in the market; the increased chemoresistance of cancer cells, the long and expensive discovery of new drugs, and their failures in clinical trials represent the “repositioning” as a powerful tool to overcome these issues [1e4,9]. Drug repositioning hence paves the way as an affordable, systematic, and effective strategy to find novel drug action against cancer, uniquely combined with current systems biology [15]. Approved toxicity and safety utilities of one compound against one disease can easily be redirected toward another disease which will help to reduce the timeframe required to validation of the drugs in clinical trials. Below, we have discussed the repurposing of FDA-approved non-cancer small molecules against the various cancer subtypes. 3.1 Breast cancer Breast cancer is characterized by the spread of abnormal cells in the breast [14]. Breast cancer is contributed to 23% of total cancer cases and 2.09 million deaths worldwide and becoming the second major cause of high female mortality [16]. Out of the total, 43.6% of breast cancer cases are reported in Asian countries, and it is common in Indian women [16]. Around 162,468 new breast cancer cases and 87,090 deaths were reported in India in 2018 [17,18]. Based on the expressions of progesterone receptor (PR), estrogen receptor (ER), and human epidermal growth factor receptor 2 (HER2), breast cancer can be sub-grouped into four types such as (1) ER/PRþ, HER2þ; (2) ER/PR-, HER2þ; (3) ER/PRþ, HER2-; and (4) ER/PR-, HER2-. The ER/PR-, HER2- (triple negative breast cancer, TNBC) is a highly heterogeneous most aggressive cancer which accounts for 10%e20% of breast carcinoma [19e21]. TNBC is mainly attributed to a poor prognosis that exhibits resistance to the current targeted and hormonal therapies [22]. This results in the highest rate of metastatic disease and the poorest overall survival rate [23]. Furthermore, the emerging drug resistance and severe side effects impose limitations on the present chemotherapy regime for cancer treatment. Therefore, there is an urgent need to develop new anti-cancer agents that combat drug resistance and avoid side effects.

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Also, the success rate of the conventional drug discovery process is very low in TNBC. Therefore, researchers have tried to repurpose the various FDA-approved drugs against TNBC using various computational and experimental approaches. Several non-cancer small molecules have been repurposed against the TNBC. The non-steroidal anti-androgen drugs (Bicalutamide, Seviteronel, Orteronel, and Enzalutamide) initially were developed for treating prostate cancer [24,25]. Now, these are repurposed against the AR-positive TNBC. The Bicalutamide inhibited the Wnt/catenin signaling pathway by downregulating the c-Myc and inducing apoptosis that reduces cell proliferation and colony formation in MDA-MB-231 [25,26]. Furthermore, the selective/non-selective b-blockers (atenolol, metoprolol, and propranolol) arrested the cell cycle at G0/G1 and S phases and inhibited cell proliferation by reducing angiogenesis and metastasis. Seviteronel reduced cell growth and tumor size in TNBC (MDAMB-453) cells and patient-derived xenografts (PDX), respectively [27]. The enobosarm and ostarine inhibited the cell proliferation in TNBC cells [25]. Bazedoxifene is approved to treat osteoporosis in postmenopausal women. Now, it is decreasing the cell viability, migration, and colony formation, and increasing the apoptosis in MDA-MB-231 cells [28]. Flubendazole is approved to treat intestinal parasites and is repurposed against breast cancer [29]. Niclosamide inhibited cell and tumor growth in TNBC [30]. Other drug candidates are also in pipeline to repurpose against the TNBC. 3.2 Colorectal cancer Colorectal cancer (CRC) is the third most common cancer and the second leading cause of high mortality worldwide, contributing 10% of the global cancer burden [31,32]. More than 1.9 million new cases of CRC are diagnosed and 935,000 deaths were reported around the world in 2020 [32]. Systemic metastasis and postoperative recurrences result in a low survival rate (11%) of CRC [33]. In CRC, the efficacy of the current treatment modalities is limited by several factors. For example, the adverse side effects following treatment with chemotherapy drugs are diverse and vary significantly in terms of disease severity. Although the response rate of conventional systemic chemotherapies can achieve up to 50%, CRC drug resistance has been reported to develop in almost all patients with CRC and thereby limiting the therapeutic index of anti-CRC agents and finally resulting in failure chemotherapy regime [34]. Therefore, the entire situation warrants the development of novel, effective, safe, and cost-affordable anti-CRC drugs which makes repurposing a suitable strategy to discover new drugs. Nebivolol, a hypertension drug, is repurposed against CRC by suppressing the growth of colon cancer cells [35e37]. Aspirin decreased cancer metastasis and immune invasion in CRC. The anti-retroviral drugs (Tenofovir, Zidovudine, Efavirenz, Indinavir, Saquinavir, Ritonavir, and Raltegravir) arrest the cell cycle, suppress the growth, invasion, and angiogenesis, and induce the apoptosis in CRC. Similarly, the FDAapproved antimicrobial drugs (Clarithromycin, Azithromycin, Gemifloxacin, Artesunate,

New targets for old drugs: drug repurposing approach for accelerating the drug discovery engine

and Mefloquine) have been repurposed against the CRC [35]. These drugs suppressed the proliferation and invasion ability of CRC, decreased angiogenesis, and induced apoptosis. Furthermore, Valproate, Fluoxetine, Sirolimus, and butyrate reduced cell viability and angiogenesis by inducing apoptosis in CRC [35]. Celecoxib (antiinflammatory) has significantly increased the therapeutic response in chemo-refractory CRC cells which makes CRC more sensitive to 5-FU and irinotecan in in vivo/ in vitro studies. Metformin inhibited the DNA replication of MCM2 and PCNA proteins in 5-FU-resistant colon cell lines and demonstrated a synergistic effect with chemotherapy against CRC [35,36]. 3.3 Glioblastoma Glioblastoma multiforme (GBM) is the most malignant astrocytic and grade IV tumor subtype of brain cancer according to the World Health Organization (https://www. abta.org). GBM is classified based on its source of origin in the cell and can be astrocytoma, oligodendrogliomas, ependymomas, and mixed gliomas. According to WHO, the distribution of primary glioma’s is GBM (51.2%), gliomas (11.1%), ependymomas (5.8%), oligodendroglioma’s (8.4%), pilocytic astrocytoma’s (5.8%), diffuse astrocytoma’s (1.5%), anaplastic (7.5%), and other subtypes (8.7%). GBM is a most malignant and commonly occurring brain tumor in adults, comprising 60% of all CNS tumors [38]. The characteristic features of GBM are cellular polymorphism, necrosis, angiogenesis, quick mitotic activity, diminished apoptosis, and genetically heterogeneous tumor [39,40]. The high doses of radiation and overexpression of ovarian steroid hormones are the main causes of GBM formation [41]. In GBM, the increased tumor size produces pressure in the skull which ultimately leads to memory loss, headache, and focal neurological deficits [41]. However, the exact molecular mechanism of GBM initiation cannot be pulled down as there is an amalgam of oncogenic events leading to this dreadful disease [42]. Currently, temozolomide (TMZ) is the most commonly used drug for chemotherapy treatment. However, patients with GBM develop resistance against TMZ due to the over-expression of O6-methylguanine methyltransferase (MGMT) [43]. Therefore, the MGMT is considered a promising target for the drug discovery process against the GBM. The repurposed drugs that are in clinical trials for the treatment of GBM are stated below. The Memantine drug was initially used to treat Alzheimer’s disease, now it is repurposed to treat GBM. It promotes cell death, induces morphological changes in cancer cells, and inhibits cell proliferation and migration in GBM [44]. The anti-epilepsy drug (Levetiracetam) inhibits cell growth and proliferation and increased the autophagy in GBM which makes GBM more sensitive to TMZ and radiotherapy. Levetiracetam downregulated the expressions of HDACs and MGMT protein while increasing the expression of P53 in GBM [45]. Furthermore, Sertraline (anti-depression drug) demonstrated anti-tumor activity against glioma by suppressing the AKT activity [46].

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Fluoxetine is a selective serotonin reuptake inhibitor (SSRI) repurposed to treat gliomas, and it also increases the efficacy of TMZ and ionizing radiation [47]. The anti-diabetic “Metformin” drug inhibits the chloride intracellular channel-1 (CLIC1) which arrests the cell cycle in glioma stem cells (GSC) [48]. Similarly, Repaglinide and Ciglitazone (anti-diabetic drugs) downregulated the Bcl-2, Beclin-1, and programmed deathligand 1 factor in GBM and increases ROS production which ultimately leads to cell death [49,50]. Furthermore, the antiviral drugs (Ritonavir, Lopinavir, and Nelfinavir) displayed anticancer activity by decreasing the expression of Matrix metalloproteinases (MMPs) in astrocytes and microglia, and also by inhibiting the PI3K/AKT/MTOR signal transduction pathways in GBM [51,52]. Itraconazole (antifungal drug) has arrested the GBM tumor growth by inhibiting the AKT1-MTOR pathway and by inducing autophagy [53]. Furthermore, the anti-malarial drugs (Chloroquine, Mefloquine, and hydroxychloroquine) were reported to induce autophagy, arrest cell cycle at G2/M phases, inhibit MMP-2/TGF activity, and increase the expression of P21 and P53 in GBM. Mebendazole, an anti-parasitic drug, increases the overall survival rates of mice with glioma [44,54]. The cardiovascular drugs (Captopril and Losartan) reduce cell proliferation and angiogenesis by inhibiting the MMP-2 and by decreasing the levels of proangiogenic factors (VEGF, PDGF, and FGF) [55,56]. The use of repurposed drugs for the treatment of GBM will provide cost-effective treatment in middle-income countries. 3.4 Lung cancer Lung cancer is an uncontrolled proliferation of cells in the lungs. There are two major types of lung cancer such as non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) based on their types of aggravates. The NSCLC is caused among 8 of every 10 people suffering from lung cancer due to the accumulation of damaged cells. Lung cancer is a leading cause of cancer death with an estimated 2.2 million new cases and 1.8 million fatalities in 2020 [57]. The transcription factors Ras, Raf, MEK, and ERK are involved in cell proliferation and apoptosis in lung cancer. Lung cancer acquired resistance toward the presently prescribed anticancer drugs and also showed adverse side effects which worsens the treatment. These challenging circumstances in treatment scenarios demand the need to modernize research to develop promising anti-cancer therapeutics. Therefore, DR will provide a suitable approach to designing new and potent drugs against lung cancer. Disulfiram, an anti-alcoholism drug, has been repurposed against lung cancer. Disulfiram inhibits the proliferation of cancer stem cells (CSCs) which leads to lung cancer cells being more sensitive to chemotherapeutic treatment [58]. Nelfinavir (HIV-1 protease inhibitor) inhibits the PI3K/AKT signaling pathway which makes tumor cells sensitive to ionizing radiations [59]. The heat shock protein-90 inhibitor (Ganetespib) exhibited promising single-agent efficacy with an effect of 50% in patients with ALK-rearranged illness [60]. The tyrosine kinase (Dasatinib) and Src (Osimertinib) inhibitors have shown

New targets for old drugs: drug repurposing approach for accelerating the drug discovery engine

a synergistic effect on Cripto-1 overexpressing lung tumors [61]. The combination of Verapamil with Docetaxel/Vincristine induced death in lung cancer cells through the autophagy process [62]. The anti-malarial drugs (artemisinin and dihydroartemisinin) induced apoptosis in lung cancer cells through cell cycle arrest at the G1 phase [63]. Non-steroidal and anti-inflammatory drugs namely Ibuprofen and Cisplatin involved in the elevation of mitochondrial apoptosis in A549 lung cancer cells [64]. Metformin and Nivolumab are known anti-diabetic and immunotherapeutic drugs that trigger apoptosis and down-regulate the c-FLIPL expression in lung cancer [65]. A known antibiotic (minocycline) reduced adverse side effects in NSCLC patients when treated with chemoradiation. Itraconazole, an antifungal drug, showed dose-dependent early antivascular, metabolic, and anti-cancer effects in NSCLC patients. In this way, repurposing will ease the process of lung cancer treatment by providing better therapeutic options. 3.5 Pancreatic cancer Pancreatic cancer (PC) is one of the most harmful cancer, worldwide. It does not display symptoms in patients until the tumor is unresectable and advanced. The present treatment is not satisfactory and hence there is an urgent need to develop more potent drugs with an efficient strategy [66]. Due to the poorest prognosis and less availability of therapeutic options the 5 year survival rate of PC is only 5%, in the last 20 years. Additionally, infections from H. pylori, hepatitis B, and human immunodeficiency virus increased the risk of pancreatic cancer [67]. Surgical resection is the only treatment presently available for pancreatic ductal adenocarcinoma. However, tumor reoccurrence was observed after the surgical treatment in 80% of patients [68]. In the last decade, the combination of gemcitabine and nab-paclitaxel was approved for the treatment of metastatic PDAC. The combination of 5-fluorouracil and leucovorin was approved for metastatic PDAC [67]. Here, we highlighted the few preclinical studies that help to understand the potential of selected repurposed drugs against PDAC by targeting the pancreatic cancer cells or cells from the TME of PDAC. The Carglumic acid that was originally used for treating Hyperammonemia is currently repositioned for the treatment of PDAC by inducing apoptosis. The role of Metformin in the inhibition of mTOR, STAT3, and TGF-b1/ Smad2/3 signaling pathways has been elucidated in PC. The potent antibiotic agents (Azithromycin, doxycycline, tigecycline, and pyrvinium) have induced apoptosis, arrested the cell cycle, and impaired mitochondrial biogenesis and oxidative phosphorylation in PC cells. Ritonavir (anti-HIV drug) induced apoptosis and cell cycle arrest through the inhibition of E2F-1 and AKT pathways. Similarly, Itraconazole an antifungal drug down-regulated the TGF-b/SMAD2/3 signaling pathways, increasing ROS production and mitochondrial membrane depolarization in PC cells and mouse model studies. Antipsychotic drugs (Penfluridol, Pimozide, Trifluoperazine, and Olanzapine) inhibited the activity of protein phosphatase 2A (PP2A), SRC, AKT, and p70S6k targets which decrease the survival of CSCs and induce cell death through apoptosis in PC cells.

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Disulfiram (anti-alcohol addiction drug) inhibited the NF-kB signaling pathway and down-regulated the stemness-related genes in PC cells. Similarly, many other drugs are in clinical trials which are tested against the PC. Although additional studies are required to ensure the safety and effectiveness of many of these repurposed candidates, this possibility offers new hope for pancreatic cancer treatment [69]. 3.6 Leukemia Leukemia is characterized by the uncontrolled growth of blood cells, usually white blood cells in the bone marrow. The WBCs are a basic component involved in the immune system of the body. The leukemia cells proliferate more and substitute the normal blood cells during infection. According to the American Cancer Society, about 60,650 patients are diagnosed with leukemia in 2022 in the USA (https://www.cancer.org/). There are five subtypes of leukemia including acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), and hairy cell leukemia (HCL) (https://www.cancer.org/). The leukemia treatment is depending on the types and stages of cancer. Targeted therapy, chemotherapy, and radiation therapy are some of the recommended therapeutic approaches being used either alone or in combination. Due to the prolonged side effects, and low success rate in leukemia treatment, the repurposing approach will be suitable for designing new anti-leukemia drugs within a short period. The anthelmintic drugs (Mebendazole and Pyrvinium) repurposed against the MLLrearranged AML by targeting the c-MYB and b-catenin proteins that are involved in the initiation, self-renewal, and maintenance of AML [70]. The verteporfin, a drug approved for the photodynamic therapy of macular degeneration, was found to suppress the growth and disease development of AML cells in the in vitro study [71]. Fidaxomicin (antibiotic) alone and in combination with doxorubicin were inhibiting the cell growth of MLL-rearranged HSC cells and reduces the tumor in the NSG xenograft model [72]. The antipsychotic drug (thioridazine) showed cell cycle arrest and reduced tumor growth by inducing apoptosis in MLL-AF6 cells [73]. The antimalarial drug (artesunate) has shown cytotoxic potential toward the MLL-rearranged AML cells expressing the MLL-AF9 in in vitro and in vivo studies [74]. Further, the ABT-737 (BCL-family inhibitor) and Purvalanol A (CDK2 inhibitor) demonstrated therapeutic potential against MLL-rearranged leukemia [75]. These drugs have shown promising effects against Leukemia and hence can be used for the treatment of leukemia by reducing the time and cost of the drug discovery process.

4. Repurposing for cardiovascular diseases Cardiovascular diseases (CVD) are a group of disorders related to the heart and blood vessels and is one of the prime reason for deaths in the UK [76]. In atherosclerosis, plaques

New targets for old drugs: drug repurposing approach for accelerating the drug discovery engine

accumulate in the artery walls which produce difficulties in blood flow, resulting in CVD. Cholesterol is the main dietary risk factor for developing cardiovascular diseases [77]. The cardiovascular diseases are coronary heart disease (CHD), cerebrovascular disease, peripheral artery disease, and aortic atherosclerosis. Myocardial infarction and heart failure can occur as a result of myocardial ischemia that leads to CHD. One-third to 50% of all cases of cardiovascular disease are caused by CHD [78]. The repurposing approach will help to treat cardiovascular diseases more effectively by combating severe side effects of currently prescribed drugs. Aspirin (antipyretic drug) is repurposed against cardiovascular diseases and approved by FDA. Furthermore, the drugs (Atomoxetine and Dapoxetine) used for the treatment of Parkinson’s and anti-depression are repurposed against cardiovascular diseases. Fingolimod is originally used for overcoming transplantation rejection in the case of multiple sclerosis; now it is used to treat cardiovascular diseases. Methotrexate has initially been used to treat several malignancies and autoimmune conditions including psoriasis and rheumatoid arthritis. Now, it is displaying cardio-protection effects through its antiinflammatory and immunomodulatory properties by inhibition of aminoimidazole carboxamide, ribonucleotide transformylase, and dihydrofolate reductase activities. Metformin has been repurposed against CVD that reduces major risk factors of cardiovascular disease such as blood lipids, body weight, and blood pressure [79,80]. The donepezil, a cholinesterase inhibitor (AChI), has been approved for the treatment of mild-to-moderate Alzheimer’s disease (AD) which lowers cardiovascular mortality [81]. Allopurinol, a drug used for the treatment of gout disease, has also been found to reduce oxidative stress and improve morbidity and mortality in congestive heart failure patients [82]. Anticancer drugs (Rituximab and Raloxifene) are repurposed for the treatment of CVD. The antiHIV drug (zidovudine) is now used to treat CVD [83]. Similarly, many more molecules are in clinical trials against CVD which are discovered through a drug repurposing approach. Therefore, repurposing becomes a fascinating method for finding safe and effective medications to treat cardiovascular diseases and disorders.

5. Repurposing for neurodegenerative disorders Neurodegenerative disorders (NDs) are increasing at a frightening rate, worldwide. NDs are the most complex type of disease associated with a medical illness that affects the brain and nervous system. About 35.6 million people are suffering from dementia and 6.2 million cases are registered for Alzheimer’s disease (AD) [84]. The most studied neurodegenerative disorders are Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis (MS), while Huntington’s disease, Friedreich’s ataxia, Wolfram syndrome, and amyotrophic lateral sclerosis are the rare NDs [85]. The dysfunctioning and loss of specific neurons lead to more progressive, chronic, and life-threatening NDs [86]. Due to the complex mechanism and severity of NDs, pharma companies are facing continuous failures in

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the development of promising drugs in the first and second phases of clinical trials [87]. This can be overcome by using a drug repurposing approach. DR provides an efficient way to treat NDs by offering approved toxicology and pharmacokinetics studies of candidate drugs. Anticancer, anti-diabetic, and anti-microbial drugs have shown promising effects against NDs. 5.1 Drugs repurposed as neurodegenerative disorders play a neuroprotective role 5.1.1 Alzheimer’s disease In Alzheimer’s, the accumulation of amyloid-b (Ab) plaques and Tau protein leads to gradual loss of memory and cognition function by increasing the age of patients [88]. Anticancer drugs (Bexarotene, Carmustine, Nilotinib, Tamibarotene, and Thalidomide) are playing a crucial role in managing Alzheimer’s disease by reducing Ab deposition [89e91]. Bexarotene also reduces the huntingtin levels which promotes microglial phagocytosis and improves motor functions through P53/P73 pathways [89]. Similarly, Nilotinib decreases the a-syn, parkin solubility, and restores dopamine levels via JAKSTAT, MAPK, and PI3K-Akt pathways. Tamibarotene reduces the level of inflammatory cytokines and chemokines [90]. Thalidomide suppresses the expression of the Beta secretase 1(BACE-1) enzyme [92]. Likewise, Paclitaxel showed a neuroprotective effect in AD by inhibiting the phosphorylation of tau protein [93]. Furthermore, Dictolisib, a mTor/PI3K inhibitor, reduces memory impairment in AD [94]. Furthermore, the inhibition of microgliosis-dependent amyloid formation was observed after the treatment of Dasatinib by modulating the JAK-STAT, MAPK, and PI3K-Akt pathways [95]. The acetylcholinesterase inhibitor (Sunitinib and Pazopanib) inhibits angiogenesis and NO formation and reduces tau phosphorylation [96,97]. In addition, Metformin exhibits anti-inflammatory actions to improve neuropathological conditions by decreasing impaired cognition in AD [98]. Interestingly, metformin specifically reduces Ab-plaques by activating the microglial cells and astrocytes in the hippocampus and cortex region of APP/PS1 mice models [99]. 5.1.2 Parkinson’s disease Parkinson’s disease (PD) affects the central nervous system and impairs movement which causes rigidity and tremor. Some PD patients also exhibit cognitive dysfunctions during the illness [93]. Unfortunately, PD has no cure up till now. However, the drugs that are available at present can only delay the progression of this disease [100]. The main pathological feature of PD is the accumulation of a-synuclein and the formation of Lewy bodies in various parts of the brain which leads to the loss of dopaminergic neurons. The role of a-synuclein accumulation has been reported in increased oxidative stress conditions in PD. Thus, a-synuclein could act as a significant therapeutic target [101]. The anticancer drugs (Nilotinib, Dabrafenib, and Sarcatinib) act as neuroprotective

New targets for old drugs: drug repurposing approach for accelerating the drug discovery engine

agents by inhibiting the accumulation of a-synuclein, activating extracellular signaling ERK, and restoring the synapse loss through JAK-STAT, MAPK, and PI3K-Akt pathways in PD [102,103]. Similarly, metformin increases the expression of peroxisome proliferator-activated receptor gamma coactivator-1a and anti-oxidative defense genes in mitochondria ultimately producing a neuroprotective effect in PD [104]. 5.1.3 Multiple sclerosis Multiple sclerosis (MS) is an autoimmune disorder. It is associated with demethylation which leads to delayed remyelination of axons and irreversible neurological loss [105]. In addition, aging also promotes the delayed remyelination of axons due to the slow differentiation of oligodendrocytes progenitor cells (OPCs) to oligodendrocytes which are responsible for the formation of myelin sheath in CNS [106,107]. The anticancer drugs (Alemtuzumab, Cladribine, Cyclo-phosphamide, Mitoxantrone, Methotrexate, and Rituximab) are repurposed against MS. Alemtuzumab and Rituximab are monoclonal antibodies which target CD52 and CD20 in MS, respectively [108,109]. Mitoxantrone and Cyclo-phosphamide reduced the progression which relapses the MS. Therefore, these drugs are acts as neuroprotective agents [110,111]. 5.1.4 Amyotrophic lateral sclerosis Amyotrophic lateral sclerosis (ALS) is characterized by the degeneration of motor neurons, shortening of muscle size, and decreased movement. It is also known as Lou Gehrig’s or Charcot disease. The neuropathological hallmark of ALS is the accumulation of ubiquitinated proteins in the cytosol of motor neurons. Several studies have been performed to repurpose the drugs against ALS [112]. The anticancer drugs (5Fluorouracil, Imatinib, Masitinib, and Thalidomide) are repurposed against ALS. These drugs showed a neuroprotective role toward ALS by regulating the inflammatory action of mast cells, Schwan cells, macrophages, and neutrophils [113]. Imatinib and bosutinib, a tyrosine kinase receptor inhibitor increased the survival rates of ALS mouse models which having SOD1 mutation [112,114]. 5.1.5 Huntington’s disease Huntington’s disease (HD) is associated with rigidity, dystonia, and weakness in muscles followed by dementia and loss of cognition [115]. HD is a dominant autosomal disease caused by unstable 36e70 CAG trinucleotide repeat in exon-1 on the Huntington gene (HTT) [116]. Currently, there is no treatment available to treat HD. However, some drug targets have been identified which provide relief from the multiple manifestations of HD [117]. Tetrabenazine, an antipsychotic drug is repurposed to cure HD patients by inhibiting the vesicular monoamine transporter 2 (VMAT2) and preventing the dopamine release from synaptic vesicles [117]. Cysteamine bitartrate, an FDA-approved drug used for the management of nephropathic cystinosis is now safe for HD patients.

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Further, lithium citrate an inhibitor of inositol monophosphatase and glycogen synthase kinase-3 has demonstrated an improvement in motor phenotype in HD preclinical models [118,119]. 5.1.6 Wolfram syndrome Wolfram syndrome (WS) is a rare and life-threatening disorder [120]. The WS occurs due to mutations in the WFS1 and CISD2 genes. The WS is characterized by nonautoimmune juvenile diabetes mellitus, diabetes insipidus, and endoplasmic reticulum (ER) stress which leads to the loss of pancreatic b cells and neurons [121]. Therefore, targeting ER stress by developing medicines would help to treat WS [122]. Unfortunately, no approved drugs are available to treat WS. The dantrolene sodium was used to treat malignant hyperthermia and muscle spasms now repurposed against the WS. Dantrolene inhibits ryanodine receptors on ER and suppresses b-cell expression which reduces neuronal death by preventing calcium leakage from ER [123]. The anti-epilepsy drug (sodium valproate) is repurposed against WS [124]. The sodium valproate increases the mRNA expression of WFS1 and enhances the dissociation of WFS1 from GRP94 in neuronal cells, and reduces ER stress [125]. 5.1.7 Friedreich’s ataxia Friedreich’s ataxia (FRDA) is an autosomal recessive NDs mostly occurring in the Caucasian population and rare in far sub-Saharan and far east [126]. The FRDA is caused by the gradual degeneration of dorsal root ganglia neurons followed by degeneration of the spinocerebellar tract and neuronal loss [127,128]. The homozygous trinucleotide GAA repeat expansions in the frataxin gene (FXN) are also a major cause of FRDA [129]. The clinical symptoms of FRDA are progressive limb incoordination, defective vision, speech and hearing, weakness in muscles, and scoliosis. Initially, deferiprone was used for the treatment of Thalassemia syndrome; now it is repurposed for FRDA. Deferiprone works as an iron chelating agent, thus reducing the iron overload of mitochondria in FDRA models. Deferiprone moderately improves cardiomyopathies in FDRA models [130]. Idebenone an anti-neuropathy drug inhibited apoptosis in frataxin-deficient cells and improves cardiac health in mouse models. Furthermore, omaveloxolone, an anticancer drug, shows improvement in neurological functions in FDRA models [131]. Vatiquinone is used to treat mitochondrial disease with refractory epilepsy now being repurposed against FRDA by managing oxidative stress levels which serves neuroprotection [132]. Very limited drugs are available for the treatment of NDs. Therefore, drug repurposing would provide a suitable therapeutic approach to treating NDs. Moreover, extensive experimental and in silico studies are needed to understand the mechanism of repurposed drugs to save time and money.

New targets for old drugs: drug repurposing approach for accelerating the drug discovery engine

6. Drug repurposing in diabetes Diabetes is a hyperglycemic condition in which the blood glucose level is higher than 7.0 mM after a standard glucose tolerance test. The continuous increase in blood glucose levels accompanies several major health complications including cardiovascular diseases, retinopathy, nephropathy, and neuropathy [133]. According to the International Diabetes Federation, nearly 380 million individuals are affected with diabetes, and the estimated cases will be 592 million by 2035. Several factors like urbanization, a sedentary lifestyle, and increasing obesity are responsible for the epidemic nature of diabetes. The destruction of insulin-producing b-cells causes type 1 diabetes, an autoimmune disease. Great challenges are ahead to overcome and control the diabetes in coming decades [134]. Therefore, new therapies like a b-cell replacement, repositioning of drugs, and regenerative potential of commercially available drugs should be tested using bioinformatics analysis and a systems biology approach [135]. Type 2 diabetes (T2DM) is a chronic disease caused by abnormal secretion of insulin by pancreatic b-cells. Also, a sedentary lifestyle and too much calorie intake lead to T2DM [136]. Gestational diabetes takes hold of nearly 1 in 12 pregnancies in the United States and is increasing in developing countries [137]. The targeting of insulin is one of the important strategies to treat type 2 diabetes. Four main classes of oral anti-diabetic agents are available. The sulphonylureas and rapid-acting secretagogues help in increasing pancreatic insulin secretion. While biguanides reduce hepatic glucose, production and inhibitors of a-glucosidase delay the digestion and absorption of intestinal carbohydrates or improve insulin action [138]. Metformin and thiazolidinediones are currently being used for the treatment of diabetes. Furthermore, several molecules are in clinical trials which specifically target glucose metabolism and insulin secretion. However, presently available anti-diabetic drugs are producing adverse side effects such as intestinal discomfort in a major population, lactic acidosis, and undesirable weight gain which is also linked to cardiovascular and hepatic disorders [139]. Diabetic kidney disease is a serious complication associated with diabetes and endstage kidney disease. Diabetic kidney disease is continuously increasing because of the growing epidemic of diabetes and obesity. The anti-inflammatory drug (pentoxifylline) is currently used for the treatment of diabetic kidney patients where it showed antiinflammatory and anti-fibrotic effects [133]. The epalrestat, an aldose reductase inhibitor, is repurposed for the treatment of the congenital disorder of glycosylation (PMM2CDG). The niclosamide ethanolamine, an anti-parasitic drug, is repurposed against diabetes by increasing the whole-body energy expenditure and improving the lipid metabolism in the T2D model [140]. The anti-inflammatory drug (Salicylate) is repurposed as an anti-diabetic agent which positively supports glycemic control and inflammation [141]. The anti-malarial and anti-rheumatoid drug (hydroxychloroquine) showed anti-diabetic effects in many clinical studies. Similarly, the asthma drug (amlexanox)

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showed beneficial effects on the T2D rodent model [133]. Diacerein is an antiinterleukin-1b drug used for the treatment of osteoarthritis and also demonstrated beneficial effects on insulin sensitivity and control of metabolic activities [142]. Furthermore, anti-hepatitis (matrine) and anti-cardiac ischemia-reperfusion injury (BGP-15) drugs are repurposed against diabetes by reducing adiposity, ameliorating hepatic steatosis, improving glucose tolerance, and increasing insulin action [143]. The drugs (cholic acid, ursodeoxycholic acid, and tauroursodeoxycholic acid) used for the treatment of primary biliary cirrhosis or cholestasis are now repurposed as potent anti-diabetic agents by improving diabetic conditions through changes in energy expenditure, and alleviation of endoplasmic reticulum (ER) stress [144]. A second-generation bile acid sequestrant (Colesevelam) lowers the level of circulating lipids and modulates the metabolism of bile acid which improves the glycemic control in T2D [145]. The MLR-1023 was originally developed for the treatment of gastric ulcers. Now it is enhancing the signal transduction of insulin and improves insulin action and metabolism of glucose in in-vivo and clinical trial Phase II studies [146]. The anti-glaucoma drug (methazolamide) is repurposed against T2D using a gene expression signature that improves glucose tolerance and reduces glycemia [147]. Furthermore, the combined analysis of genome-wide association studies (GWASs), proteomics, and metabolomics data analysis were used to repurpose the diflunisal, nabumetone, niflumic acid, valdecoxib, phenoxybenzamine, and idazoxan against diabetes (Table 17.2). Table 17.2 Drug repurposed for the treatment of T2D. Drug name

Original use

Proposed mode of action

NRTI Telmisartan Doxepine

Anti-retroviral drugs Treatment of high blood pressure Antidepressant

Niclosamide ethanolamine Salicylate Amlexanox Matrine Hydroxychloroquine Methazolamide

Anthelmintic drug Anti-inflammation Anti-inflammation Hepatitis B Malaria, rheumatoid arthritis Glaucoma

Diacerein

Osteoarthritis

Berberine

Infectious diarrhea

Inhibit reverse transcription Angiotensin II receptor blocker (ARB) Increases concentration of neurotransmitters serotonin (5-HT) and norepinephrine (NE) in the brain Mitochondrial uncoupler NFkB Anti-inflammatory HSP90/HSP72 Anti-inflammatory Suppresses hepatic glucose production Improves ER stress and reverses inflammation Activates AMPK by inhibiting mitochondrial complex 1

New targets for old drugs: drug repurposing approach for accelerating the drug discovery engine

Table 17.2 Drug repurposed for the treatment of T2D.dcont’d Drug name

Original use

Proposed mode of action

Tauroursodeoxycholic acid Bile acids

Cholestasis Primary biliary cirrhosis

Bile acid sequestrants MLR-1023

Hypercholesterolemia Gastric ulcers

Triterpenoids BGP-15

Anti-tumor and antioxidant Ischemia-reperfusion

Clobetasol Phenoxybenzamine

Inflammation and itching Hypertension, hypoplastic left heart syndrome Chronic glaucoma Inflammatory diseases Pain

Alleviates ER stress Increases energy expenditure, agonism of FXR and TGR5 Small heterodimer partner Activation of lyn tyrosine kinase AMPK, FoxO1 PARP inhibitor/HSP72 inducer Phospholipase A2 Alpha-2A adrenergic receptor Cholinesterase Nitric oxide synthase Prostaglandin G/H synthase 2 NMDA receptor Serotonin-1A Management of prediabetes and type 2 diabetes mellitus Treatment for diabetic neuropathy Increases the success rate of blood sugar control Treatment of type 2 diabetes mellitus Can be used as a treatment for type 1 diabetes mellitus Effective in treating or preventing GDM

Echothiophate iodide Hydrocortisone Carprofen D-cycloserine

Buspirone Colesevelam

Bacterial infections Anxiety disorder Hyperlipidemia

Pregabalin

Epilepsy

Alpha 1 (a1)-adrenoceptor antagonist Bromocriptine

Benign prostate hyperplasia

Cyclooxygenase-2 inhibitor Calcium channel blockers

Parkinson’s disease Non-steroidal antiinflammatory drug Anti-hypertensive drug

7. Repurposing for viral diseases Multiple human infections including COVID-19, SARS, HIV, and many others, and cancer are caused by different types of viruses. The viruses are also infected to animals and plants. Even though there are thousands of viruses that can harm humans, most of them have no known cures [148]. There are 2.80  1022 virus-like particles per biome associated with humans. In the COVID-19 pandemic, the drug repositioning approach played a crucial role in designing and developing novel drugs against COVID-19. A new tactic for accelerating the approval of efficient and secure therapies for screening orphan

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disorders is the repurposing of currently available medications. Similarly, repositioning has gained significant importance in the treatment of common as well as rare viral infections. The typical strike rate for creating a new medicinal compound is 2.0% [149]. Novel target validation strategies, including CRISPR/Cas9 gene editing, and new experimental organoid models are helping to find new antiviral medicines and decipher the molecular mechanisms underlying viral disease. The in-silico screenings, mining of databases with transcriptomic profiles, and screening of bioactive small-molecule collections help to repurpose the drugs by identifying new biological targets and pathways that combat viral infections. 7.1 Drug repurposing in retrovirus The WHO has proclaimed the latest outbreak of Coronavirus illness (COVID-19), which started in Eastern Asia before spreading everywhere in the world. In the initial stage of viral spread, there was no vaccination available for the novel virus SARSCoV2 (2019-nCoV) [150]. Additionally, current antiviral medications have failed, leading to an extremely high fatality rate. Therefore, researchers across the world were attempting to find new drug candidates that can help to control COVID-19 infections and get control over them. Repurposing was the major approach used to find new drugs with anti-viral activity. The FDA-approved and experimental antiviral medications that have been utilized in the past to treat SARS and MERS are being studied for COVID-19. The drugs (remdesivir, teicoplanin, favipiravir) which are used to treat SARS-CoV, MERS, and influenza are repurposed against COVID-19. These drugs showed effective control over COVID-19 with high safety [150]. Furthermore, lopinavir, ritonavir, arbidol, and niclosamide have shown potent activity against COVID-19. Further, the anti-malarial medication chloroquine has been used as a top contender for the treatment of COVID-19 [151]. Ivermectin, an FDA-approved anti-parasitic, was tested for its anti-viral efficacy in the in vitro study [152]. An antirheumatoid drug (baricitinib) is in its clinical trial phase III which is repurposed against COVID-19 [153]. Digoxin is a cardiac glycoside that has been used to treat a variety of heart diseases including paroxysmal atrial tachycardia, atrial fibrillation, and atrial flutter [154]. Recent studies conducted on digoxin revealed the potential of digoxin and other cardiotonic steroids against the treatment of the virus. The digoxin was involved in decreasing the production of the HIV-1 regulatory protein Rev by generating overs splicing in the HIV-1 RNA which blocks the synthesis of structural proteins required for the formation of new virion assembly [155]. The cardiac glycosides including procillaridin A, bufalin, covallatoxin, and digitoxin inhibited the Hepatitis B virus (HBV) in cell culture studies [156]. The broad spectrum antiviral action of Sunitinib (chemotherapeutic drug) was reported, where it showed inhibition of protein kinase R (PKR) and 20 -50 -oligoadenylate synthetase (OAS)/RNase L systems which are required for the replication of the virus

New targets for old drugs: drug repurposing approach for accelerating the drug discovery engine

[157,158]. The antimalarial drugs (chloroquine and hydroxychloroquine) showed antiviral activity by disturbing the acidic pH condition that is essential for virus replication [148,159]. Cyclosporin-A has displayed antiviral properties against the human papillomavirus (HPV) by binding to cellular cyclophilins which inhibits binding to viral proteins [160]. Remdesivir was initially prescribed for the treatment of Filoviridae members, now it has been repurposed against COVID-19 [161]. The drugs (camostat and nafamostat) used to treat chronic pancreatitis are repurposed against SARS-CoV/-2 and HCoVNL63 infections by inhibiting the activity of Serine 2 transmembrane protease. Mycophenolic acid was initially prescribed as an immunosuppressant for organ rejection and the treatment of Crohn’s disease. Now, it is repurposed against several viral infections such as dengue virus, hepatitis C virus, SARS-CoV-2, HIV, and Zika virus [162]. The anti-parasitic agent (nitazoxanide) has been tested against coronaviruses, influenza, and hepatitis C virus [163e165]. In this way, the drug repurposing approach can provide a more suitable strategy to design and discover drugs against deadly human viral infections.

8. Repurposing for microbial diseases Human diseases from microbial infections are increasing around the globe. Since the premodern era, society has been devastatingly suffering from microbial infections including diseases like cholera, tetanus, tuberculosis, polio, and many others [166]. Although, the advancement in medical facilities, improved sanitation and the development of vaccines are helping to reduce the mortality rate during the 1980s. The incremental shift in morbidity has been observed due to the currently occurring COVID-19. However, the human populations are very susceptible to new microbial infections due to genetic mutations which lead to acquired drug resistance, use of imprecise immunosuppressants, broadrange antibiotics, diabetic complications, and other viral-mediated infections which weaken immunity [167]. Antimicrobial resistance (AMR) is a major challenge to public health which makes treatment more difficult. Around 700,000 death cases have been reported due to AMR across the world and it also adversely affects the global economy [168]. In the last decade, almost all the common pathogens have acquired resistance against the first line of drugs. Whereas, some of the bacterial strains have shown resistance toward the second and third-line drugs. However, acquiring multidrug resistance is a major threat and is responsible for the severity of infections and increase in disease morbidity. The commonly known multi-drugs resistant bacteria are Klebsiella pneumonia, Escherichia coli, cephalosporin-resistant E. coli Vancomycin-resistant enterococci, penicillin- and macrolide-resistant cotrimoxazole-resistant Streptococcus pneumonia [167]. Further, another group of Gram-positive and Gram-negative drug-resistant bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas

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aeruginosa, and Enterobacter species) is often abbreviated as “ESKAPE” [169]. This group of bacteria is known to cause serious health infections and very efficiently escapes from the effect of antimicrobial drugs [170]. The excessive use of antibiotics in public health, industrial agriculture, and veterinary medicine resulted in acquired drug resistance in pathogens [171]. This produces complications in the treatment of diseases caused by drug-resistant pathogens such as viruses, fungi, and bacteria [172]. Unfortunately, during preclinical and clinical trials most bacteria acquire resistance to antimicrobial drugs [173]. Therefore, there is a need to discover efficient novel antimicrobial drugs which combat drug resistance and provide an efficient way to treat human infections more efficiently. The process of drug development can be accelerated by drug repurposing. The wide variety of non-antimicrobial drugs significantly demonstrates anti-microbial activity, thus successfully repurposing against microbial infections [174,175]. Examples of drugs studied for repurposing and their supposed antimicrobial mode of action are listed below [166] (Table 17.3). The schematic representation of repurposed drugs against various human diseases is given in Fig. 17.2.

Table 17.3 Repurposed drugs against Microbial infections. Original indication

Drug

Tested bacteria

Mode of action

Refs.

Anti-depressants

Fluoxetine

S. aureus, MRSA, S. epidermidis, E. faecalis, vancomycinresistant enterococci (VRE), B. cereus, M. luteus, E. coli, A. baumannii, K. pneumoniae, P. aeruginosa S. aureus, MRSA, S. epidermidis, E. faecalis, VRE, A. baumannii

Apoptosis, DNA fragmentation, chromosomal condensation, inhibits efflux pump, disrupts of basic metabolic processes

[176]

Damages membrane, disrupts the basic metabolic processes Inhibits efflux pump ion,

[177]

Fluvoxamine

Sertraline

S. aureus, E. coli, P. aeruginosa, H. pylori

[178,179]

New targets for old drugs: drug repurposing approach for accelerating the drug discovery engine

Table 17.3 Repurposed drugs against Microbial infections.dcont’d Original indication

Drug

Tested bacteria

Mode of action

Refs.

Local anaesthetics

Lidocaine

S. aureus, S. epidermidis, S. viridans, S. pyogenes, E. faecalis, E. coli, P. aeruginosa, K. pneumoniae, N. gonorrhoeae

Disrupts cell membrane, alters cellular respiration and DNA synthesis, inhibits membranebound enzymatic activities

[180]

Bupivacaine

S. aureus, S. epidermidis, S. pyogenes, E. faecalis, S. pneumoniae, E. coli S. aureus, E. faecium, Enterobacter spp., P. aeruginosa, K. pneumoniae, A. baumannii, M. tuberculosis S. aureus, MRSA, E. faecium, E. coli, P. aeruginosa, A. baumannii S. aureus, S. epidermidis, Streptococcus mutans, E. coli, A. baumannii, P. aeruginosa

Anticancer

Gallium nitrate

Tamoxifen

5-Fluorouracil

Imatinib

M. tuberculosis

Methotrexate

S. aureus, P. aeruginosa

Streptozotocin

S. aureus

[180]

Interferes with irondependent metabolism

[181 e183]

Interacts with bacterial membrane

[184]

Inhibits quorum sensing and biofilm formation, blocks synthesis of DNA and RNA Inhibits ablfamily TKs Abl1/Abl2 Inhibits nucleotide biosynthesis Interferes with DNA replication

[185,186]

[187]

[188]

[189]

Continued

335

336

New Horizons in Natural Compound Research

Table 17.3 Repurposed drugs against Microbial infections.dcont’d Original indication

Drug

Tested bacteria

Mode of action

Refs.

Antineoplastics/ treatment of osteoporosis

Raloxifene

P. aeruginosa

[190]

Zoledronic acid

C. pneumoniae

Antipyretics

Paracetamol

Anti-helmintics

Niclosamide

S. aureus, S. epidermidis, E. coli, P. aeruginosa, A. baumannii, P. gingivalis, T. denticola, T. forsythia S. aureus, MRSA, vancomycinresistant S. aureus, (VRSA), linezolid-, daptomycinresistant S. aureus, P. aeruginosa, K. pneumoniae, A. baumannii P. aeruginosa, K. pneumoniae, A. baumannii, C. difficile

Inhibits quorum sensing, and decreases virulence Inhibit proliferation of infected SaOS-2 cell line and facilitates immune response toward infection Reduces biofilm formation

Inhibits virulence and quorum sensing, increases membrane permeability

[194,195]

Increases membrane permeability and disrupts cell membrane

[196]

Rafoxanide

Closantel Ivermectin

C. difficile S. aureus, P. aeruginosa, K. pneumoniae, A. baumannii, M. tuberculosis, M. ulcerans

[191]

[192,193]

[197] [198]

New targets for old drugs: drug repurposing approach for accelerating the drug discovery engine

Table 17.3 Repurposed drugs against Microbial infections.dcont’d Original indication

Drug

Tested bacteria

Mode of action

Refs.

Anti-protozoics

Pentamidine

P. aeruginosa, K. pneumoniae, A. baumannii

Increasing membrane permeability, cell membrane disruption

[199]

Robenidine

P. aeruginosa, K. pneumoniae, A. baumannii P. aeruginosa

Anti-diabetes

Metformin

Mucolytic agents

Mesna

S. aureus, S. epidermidis, E. faecalis, E. coli, P. aeruginosa, K. pneumoniae, H. pylori

Pan-assay interference compounds

Curcumin

S. aureus, P. aeruginosa

Epigallocatechin 3 gallate

S. epidermidis, S. pneumoniae, B. subtilis, B. cereus, E. coli, P. aeruginosa

Simvastatin

S. aureus, MRSA, S. epidermidis, S. pneumoniae, S. pyogenes, Enterococcus sp., E. coli, P. aeruginosa, K. pneumoniae, M. catarrhalis, S. marcescens, H. influenzae, M. tuberculosis S. aureus, S. pneumonia, E. coli S. aureus, S. epidermidis, S. pneumonia, S.

Antihyperlipidemics

Pravastatin

Rosuvastatin

[200]

Inhibits quorum sensing Inhibits biofilm formation by reducing the expression of extracellular polysaccharide and protein genes Damages bacterial cell membrane Inhibits DNA gyrase, inhibits biofilm development and quorum sensing Reduces formation of mature biofilms, decreases EPS production cholesterol synthesis

[201] [202]

[203]

[204]

[205,206]

Reduces in cell viability

[207]

Disrupts bacterial membrane, inhibits

[208,209]

Continued

337

338

New Horizons in Natural Compound Research

Table 17.3 Repurposed drugs against Microbial infections.dcont’d Original indication

Drug

Tested bacteria

Vasodilators

Bepridil

pyogenes, E. coli, H. influenzae, K. pneumoniae, A. baumannii L. monocytogenes

Antipsychotics/ antihistaminics

Promethazine

S. aureus

Trimeprazine

S. aureus, MRSA, E. coli, A. baumannii

Mode of action

Refs.

protein synthesis

Reduces of intracellular infection by inhibiting escape from vacuole phagocytes Increases bacterial membranes permeability Interacts with bacterial membrane

[210]

[211,212]

[213]

Figure 17.2 Repurposed drugs with their original indications and new disease indications.

New targets for old drugs: drug repurposing approach for accelerating the drug discovery engine

9. Conclusion Drug repurposing is a boon for drug development to treat various human diseases and disorders. It is indeed a promising strategy to develop anti-cancer, anti-viral, anti-diabetic, cardiovascular, neurodegenerative, and antimicrobial drugs in a short time at low cost leading to the discovery of innovative drugs. It also minimizes the risk factor which is usually involved in the preparation of new drugs. A combination of original and repurposed drugs also shows an additive advantage over single-drug treatment as such combinations reduce the dose and enhance the drug efficacy. Furthermore, the repurposed pharmaceuticals can join clinical trials or be used for compassionate use right away, notably in the case of human diseases lacking a specific treatment, in addition to the clear economic advantage resulting from such a strategy in the medication development process. The time required for the drug discovery and development process will be reduced due to approved and accessibility of in vitro and in vivo studies, complete chemical characterization, toxicity profile analysis, drugs production, formulation development, and pharmacokinetic profiles of FDA-approved medications. However, there are certain limitations to these strategies. Very few drugs have been approved by the authority, for which quick resistance could also be developed. Drug repurposing requires strong valedictory data from preclinical trials. Also, repurposed drugs develop resistance. These drugs have a chance of influencing the human microbiome which could make new complications also. These drugs might show a difference in in vitro activity than in vivo activity. Thus, the mechanism of these new repurposed drug molecules has to be completely studied before application.

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CHAPTER 18

Modern role of essential oils in drug discovery and medicinal products Varsha Kumari1, Priyanka Kumawat1, Surya Nandan Meena2, Shyam Singh Rajput1, Ramesh Saini3, Sharda Choudhary4, Bhuri Singh5, S.B. Yeri6, D.K. Gothwal1, Radheshyam Sharma7, Poonam Kumari1 and Sarfraz Ahmad1 1

S.K.N. Agriculture University, Jobner, Rajasthan, India; 2Department of Chemistry, Division of Biochemistry, Savitribai Phule Pune University, Pune, Maharashtra, India; 3Deaprtment of Crop Science, Konkuk University, Seoul, Korea; 4National Research Centre on Seed Spices, Ajmer, Rajasthan, India; 5Agriculture University, Kota, Kota, Rajasthan, India; 6University of Agricultural Sciences, Raichur, Karnataka, India; 7Jawaharlal Nehru Krishi Vishwa Vidhalya, Jabalpur, Madhya Pradesh, India

1. Introduction Essential oils (EOs) are obscured liquid compound constituents of volatile plant secondary metabolites. They are present in plant naturally and obtained through wet or steam distillation from medicinal plants. Essential oils are named according to their source plant as for example ginger essential oils from ginger, lemon essential oils from lemon, almond essential oils from almond, lavender essential oils from lavender, nutmeg essential oil extracted from nutmeg etc. [1]. Essential oils are mainly composed of terpenes accompanied with alcohols, ethers, carbohydrates, monoaldehydes, and ketones which cause essence and aroma in medicinal flora. EOs have been used in giving gratifying taste to processed food items and also utilized in cosmetics, perfumes, and beverage items. EOs from medicinal plant contain monoterpenes, monoterpenes hydrocarbons, thymol etc. responsible for antioxidant, antibacterial, antiviral, anti-inflammatory, and insecticidal properties. EOs are very sensitive and volatile in properties, so they need particular care while handling and packing as compared to vegetable oils [2,3]. Extraction of EOs from raw base can be categorized into two classes: conventional and advanced. Conventional method includes hydrodistillation, entrainment by water steam, organic solvent extraction, and cold pressing. Because of elevated thermolability, prolong extraction duration and poor quality of extracts, conventional methods are not very profitable [4]. In contrast to the classical method, advanced ones have various advantages because of solvents utilization and reduction in extraction time. Advanced/innovative methods include supercritical fluid extraction, subcritical extraction liquids, ultrasound-assisted extraction, microwave-assisted extraction, and instant controlled pressure drop [5,6] (Figs. 18.1 and 18.2). Fast growth of knowledge of isolation, purification, processing of essential oils are crucial in determining its role in drug formulation procedure. It has significant effects New Horizons in Natural Compound Research ISBN 978-0-443-15232-0, https://doi.org/10.1016/B978-0-443-15232-0.00004-7

© 2023 Elsevier Inc. All rights reserved.

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Figure 18.1 Flow diagram depicting methods of extraction of essential oils.

Figure 18.2 Diagram depicting medicinal plants as a source of essential oils.

Modern role of essential oils in drug discovery and medicinal products

in area of pharmacy and aromatherapy [7,8]. In this chapter, we emphasized on importance of essential oils and their role in the development of novel antimicrobials, antibiotical drugs, and medicinal products.

2. Methods of extraction of essential oils (EOs) Among the conventional methods of essential oil extraction, hydrodistillation is ancient and most simple method where floral parts of plants are boiled in the water and vapors are condensed to collect essence [9]. In entrainment by water steam method, same principle is followed where flowers or plant parts are not directly contacted with water [10]. In organic solvent extraction method, flower or plant part are kept within organic solvent mixture and mashed inside it; pressure is applied to take out solvent in order to concentrate extracts. In cold pressing, oil sacs are ruptured in course of extraction process and centrifuged at fast speed to liberate and collect fragile and vaporous oil [11]. In innovative method, supercritical fluid extraction is very usual where CO2 is taken as solvent. This method consists of supercritical phase and depression phase. In supercritical phase, CO2 is passed to plant parts by pressure and in depression phase compressed CO2 is loaded which leads to separation of essential oils [12]. In subcritical extraction liquids both water and CO2 is utilized to reduce heat at subcritical phase. Ultrasound is used to extract essential oils from plant part through vibratory action of cell membrane after submerging in solvent and water in ultrasound-assisted extraction method [13]. Microwave-assisted extraction is one of powerful method of oil extraction which requires less time and produce high quality of extract. In instant controlled pressure drop method, steam is enforced at particular temperature and pressure through plants under vacuum followed by passing through normal atmospheric pressure leads to extraction of oils [14,15]. Merits and demerits of numerous essential oils extraction methods are explained in Tables 18.1 and 18.2.

3. Medicinal plants as a source of essential oils Ocimum bassilicum: It is bushy erect shrub with less height having a strong aroma. Leaves are used for extraction of essential oils. Methyl eugenol (80.00%) and a-cubebene (7.00%) are major component [16]. It inhibits antibacterial activity for most of species of bacteria, viz., E. coli, Salmonella, Staphylococcus, Shigella, Pseudomonas etc. Cinnamomum spp.: They belong to hard trees with very excellent sources of essential oils. They are commonly called as cinnamon, a type of spice used to add flavor in the food items. Cinnamon essential oil is popular oil drained from bark of tree of cinnamon. Cinnam-aldehydes and cinnamic acids are major components of cinnamon essential oils. This oil is gaining popularity as having antibacterial, antifungal and antimicrobial activity [17] (Table 18.2).

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Table 18.1 Merits and demerits of numerous essential oils extraction methods. Extraction methods

Merits

Demerits

Hydrodistillation

Simple

Entrainment by water steam Organic solvent extraction

Short extraction time No chemical deterioration Less expenditure

Chemical degradation due to lengthy extraction duration Essence degradation Residues of organic solvents in oil Glucosinolate contamination

Superior grade of extract Less costly More efficient Fast and eco-friendly Less energy utilization

Costly Low quantity of terpenes Free radicals production Utilization of toxic compounds Not any as most potent method

Conventional methods

Cold pressing Innovative methods

Supercritical fluid extraction Subcritical extraction liquids Ultrasound-assisted extraction Microwave-assisted extraction Instant controlled pressure drop

Table 18.2 List of medicinal plants with major constituent of essential oils and control. Medicinal plant

Ocimum bassilicum

Major constituent of essential oils

Eucalyptus globules

Methyl eugenol, a-cubebene Cinnam-aldehydes, cinnamic acids Cineole, terpinene

Citrus limon

D-limonene,

Pelargonium graveolens

Eugenol, geraniol, geranic acid

Lavandula officinalis Mentha piperita

Camphor, terpinenol, linalool, cineole Menthol, carvacrol, menthone

Rosmarinus officinalis

Tannins, resinic acid

Cinnamomum spp

monoterpenes

Treatment/cure

References

Antibacterial activity

[16]

Antibacterial, antifungal, and antimicrobial activity Antioxidant, antiinflammatory, antiproliferative, and antibacterial activities Detox activity, antiseptic, and antiallergic activity Antimicrobial, anticancer, antidiabetic, and antibacterial properties Aromatherapy

[17]

Nerve stimulant activity, anti-inflammation, antiinfection, antimicrobial, and antiseptic properties Control of hysteria, paralysis, menstrual cramps, cardiovascular disease

[18]

[19] [20]

[21] [22]

[23]

Modern role of essential oils in drug discovery and medicinal products

Table 18.2 List of medicinal plants with major constituent of essential oils and control.dcont’d Medicinal plant

Major constituent of essential oils

Melaleuca alternifolia

Terpinenol, monoterpenes

Salvia sclarea Anthemis nobilis

Linalool, linalyl, terpineol Angelic acid, tiglic acid

Cananga odorata

Geranyl, benzyl acetate, eugenol

Aspilia africana

Germacrene, a-pinene

Lipia multiflora

Monoterpenoids, thymol

Spondias mombin

Octadecane, hexatriacontane

Treatment/cure

References

Antibacterial, antiinflammatory, antiviral, and antiseptic activity Antimicrobial property Relaxes menstrual pain, wounds, inflammation, tensions, muscle cramps, antianxiety, joint pain etc. Reduces heart rate, fast breathing, depression, anxiety, late menopause effect Control of wounds, inflammation and infection inhibition Antifungal, antiviral, antibacterial activity Anti-inflammatory and antibacterial property

[24]

[25] [26]

[27]

[28]

[7] [29]

Eucalyptus globules: It is long plant with height of approximately 250 feet. They are deep rooted evergreen plants with profuse water requirement. Essential oils are mainly consist of cineole and terpinene. Eucalyptus oil from leaves of plants has demonstrated antioxidant, anti-inflammatory, antiproliferative, and antibacterial activities. Oils have been utilized for various purposes as cold and cough treatment, cosmetics, immunity booster, asthma, cure of joint pain, skin infections etc. [18,30,31] (Fig. 18.2). Citrus limon: Rutaceae is the family of Lemon which bears abundant flavored fruit throughout the year. It is bushy shrub with medium height. Essential oils are rich in monoterpenes and D-limonene up to 92% of total. Odor of the oil is determined by oxygenated compound aldehyde citral. Essential oils from lemon are called as lemon essential oil which are utilized to control pain associated with labor, nausea, vomiting, boost immumity, refresh dull skin, improve digestion, detox activity, antiseptic and antiallergic actions etc. [19,32]. Pelargonium graveolens: Geranium is a perennial shrub of hairy nature with less height. Eugenol, geraniol, and geranic acid are major composition of essential oils. Herbal perfumes, soaps, and cosmetic products are constituted by geranium oils. This oil is being source of antimicrobial, anticancer, antidiabetic, and antibacterial actions [20,33,34].

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Lavandula officinalis: Lavender has beautiful flower of family Lamiaceae. Essential oil contains camphor, terpinenol, linalool and cineole. Lavender oil shows its antibacterial and antifungal ability. Lavender oil is potentially utilized in aromatherapy for control of skin problems, abrasions patterns, burning symptoms, stresses, and headaches [21]. Mentha piperita: Peppermint is creeper plant with dark green leaves which exist in Lamiaceae family. It carries sweet smell with menthol. Essential oils are extracted from leaves and flower of peppermint composed of menthol, carvacrol, and menthone. Peppermint oil is very ancient medicine which has nerve stimulant activity, antiinflammation, anti-infection, antimicrobial, and antiseptic properties. It also soothes pain, headache, sinus, and lung infection [22,35]. Rosmarinus officinalis: Rosemary carries light blue flower which exist in family of Lamiaceae utilized for its medicinal quality. Rosemary is enriched with fragile oil, tannins, and resinic acid. Rosemary oil has intense control of hysteria, paralysis, menstrual cramps, and cardiovascular diseases [23] (Table 18.2). Melaleuca alternifolia: It is commonly called as Tea tree which is a shrub belongs to Myrtaceae family bears yellow flower and feathery leaves of muddy regions. Terpinenol and monoterpenes are major components of Tea tree oil which gives pleasant essence to it. The tea tree oil has antibacterial, anti-inflammatory, antiviral, and antiseptic activity. Oils are also being utilized for control of cough, asthmaticitis, flu, tuberculosis, cold etc. [24,36e38]. Salvia sclarea: Clary sage bears purple hairy leaves belongs to Lamiaceae family. They are of perennial in nature where tinted large leaves are source of essential oils. Clary sage essential oils are majorly constituent of linalool, linalyl, and terpineol. Its oil aids for treatment of women as muscle cramp during the menstrual cycle, uterus complications, cortisol levels, skin related problems, and antimicrobial property [23,25,39,40]. Anthemis nobilis: Roman chamomile is called as prized herbs from ancient times due to its potentiality to sooth emotions. It carries golden flowers with white petals belongings to the Asteraceae family. Chamomile essential oils are rich in angelic and tiglic acid. It has role in relieving brain and body to keep sleep. It relaxes menstrual pain, wounds, inflammation, tensions, muscle cramps, antianxiety, and joint pain [26,41]. Cananga odorata: Ylangeylang is short tree belongs to Annonaceae family with clean sweet smell. Ylangeylang essential oils are composed mainly of geranyl and benzyl acetate, eugenol etc. Oil reduces heart rate, fast breathing, depression, anxiety, late menopause effect [27,42] (Fig. 18.2). Aspilia africana: It is ancient and essential reservoir of essential oils belongs to Asteraceae family. Aspilia essential oils are made up of germacrene and a-pinene having antibacterial property for numerous species of bacteria including Escherichia and Pseudomonas. Oils are worked as medicine in wounds, control inflammation, infection inhibition etc. [28,43]. Lipia multiflora: It is aromatic plant belongs to Verbenaceae family having shrub in nature. Lipia essential oils contain high level of oxygenated monoterpenoids and thymol majorly. Oils have antifungal, antiviral and antibacterial activity [7].

Modern role of essential oils in drug discovery and medicinal products

Spondias mombin: They are from Anacardiacae family. Spondias essential oils are constituted by octadecane and hexatriacontane used as anti-inflammatory and antibacterial drug [29]. Origanum vulgare: It is plant of family Lamiaceae which is perennial in growth habit. Leaves of this herb can add aroma to the food items. It is used as medicine in conventional and recent pharma industry [44,45].

4. Essential oils as a source of medicine and drug discovery Antibacterial: Essential oils from many species of herbs have antibacterial potential for numerous Gram-negative and Gram-positive bacteria. For example, eucalyptus oil and tea tree oil have bactericidal property for several genera of Staphylococcus species. Similarly, Basil oil bears antibacterial activity against Pseudomonas, Streptococcus and Aeromonas species [46,47]. Anti-malaria: Bark of Cinchona pubescens and Quillaja saponaria have been utilized as component of vaccine adjuvant for malaria disease [48]. Anti-inflammatory: Inflammations are caused by histamine reactions results to sprain, blemishes, regional swelling etc. Essential oils have pain relieving action compared to allopathic drug without side effects. Tea tree oil suppresses inflammation and pain after 15 min past histamine diphosphate stimulation [38]. Anti-oxidant: Free radicals create oxidative stresses which are main regions of stress and anxiety. Antioxidants suppress the oxidative stresses and anxiety. Oregano and Clary sage oils prevent olive oil from oxidation reaction and tested positive for their antioxidation potential [49]. Antiviral: Essential oils of Melaleuca species possess antiviral property. They reduce viral infection up to 99% [50]. Antifungal: Tea tree oil possesses antifungal property against broad spectrum of fungus as Aspergillus spp. and dermatophytic fungi [36]. Anti-lice: Tea tree oil also bears anti-lice action for hair lice due to presence of promising anti-cholinesterase reaction [51]. Anti-dandruff: Tea tree oils are used in shampoo to control infestation of dandruff [52]. Insecticidal activity: Essential oils belongs to Nepeta parnassica bears insect repellent property [53]. Anti-tumor: Essential oils from tea tree oil also reduce tumor growth during cancer in human being [54]. Hormone stimulation: Geranium essential oil contains geranial and geraniol leads to induction of estrogen hormone [55].

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5. Conclusions Essential oils are expression of pericarp from herbal and medicinal plants extracted by steam distillation. They are mainly composed of terpenes and mono-terpenes like secondary metabolites, the key reasons of essence in medicinal plants. They are nonmedicine agent which has symbiotic effect with allopathic drug to control various diseases considering safety issues and quality concerns. Essential oils have multivariate applications in pharmaceuticals, drug discovery, and aromatherapy as a natural gift of nature to human beings which was known for millennium but extensively utilized in recent years. The information provided in this chapter related to various types, extraction methods, source, and application of essential oils which could be further dissected finely to uncover more of its implication in future.

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[16] Ozcan M, Chalchat J. Essential oil composition of Ocimum basilicum L and Ocimum minimum L in Turkey. Czech J Food Sci 2002;20(6):223e8. [17] Ahmadi SGS, Farahpour MR, Hamishehkar H. Topical application of Cinnamon verum essential oil accelerates infected wound healing process by increasing tissue antioxidant capacity and keratin biosynthesis. Kaohsiung J Med Sci 2019;35:686e94. [18] Aazza S, Lyoussi B, Megıas C, Cortes-Giraldo I, Vioque J, Figueiredo AC, et al. Anti-oxidant, antiinflammatory and antiproliferative activities of Moroccan commercial essential oils. Nat Prod Commun 2014;9(4):587e94. [19] Namazi M, Amir A, Akbari S, Mojab F, Talebi A, Alavi Majd H, et al. Aromatherapy with citrus aurantium oil and anxietyduring the first stage of labor. Iran Red Crescent Med J 2014;16(6):e18371. [20] Ghannadi A, Bagherinejad M, Abedi D, Jalali M, Absalan B, Sadeghi N. Antibacterial activity and composition of essential oils from Pelargonium graveolens L’Her and Vitex agnus-castus L. Iran J Microbiol 2012;4(4):171e6. [21] Kim S, Kim HJ, Yeo JS, Hong SJ, Lee JM, Jeon Y. The effect of lavender oil on stress, bispectral index values, and needle insertion pain in volunteers. J Alternative Compl Med 2011;17:823e6. [22] Ravid U, Putievsky E, Katzir I. Enantiomeric distribution of piperitone in essential oils of some mentha spp., Calamintha incana (sm.) heldr. and Artemisia indaica L. Flavour Fragrance J 1994;9:85e7. [23] Svoboda KP, Deans SG. A study of the variability of rosemary and sage and their volatile oils in British market: their antioxidative properties. Flavour Fragrance J 1992;7:81e7. [24] Hammer KA, Carson CF, Riley TV. In vitro activity of Melaleuca alternifolia (tea tree) oil against dermatophytes and other filamentous fungi. J Antimicrob Chemother 2002;50(2):195e9. [25] Sienkiewicz M, Głowacka A, Poznanska-Kurowska K, Kaszuba A, Urbaniak A, Kowalczyk E. The effect of clary sage oil on staphylococci responsible for wound infections. Postepy Dermatologii Alergologii 2015;32(1):21e6. [26] Srivastava JK, Shankar E, Gupta S. Chamomile: a herbal medicine of the past with bright future. Mol Med Rep 2010;3(6):895e901. [27] Gnatta JR, Piason PP, Lopes Cde L, Rogenski NM, Silva MJ. Aromatherapy with ylang ylang for anxiety and self-esteem: a pilot study. Rev Esc Enferm USP 2014;48(3):492e9 [Portuguese]. [28] Ogunwande IA, Eresanya O, Avoseh NO, et al. Chemical composition of essential oils from Nigerian plants. Chem Sin 2012;3(1):279e86. [29] Ayoka AO, Akomolafe RO, Akinsomisoye OS, et al. Medicinal and economic value of spondias mombin. Afr J Biomed Res 2008;11:129e36. [30] Sadlon AE, Lamson DW. Immune modifying and antimicrobial effects of eucalyptus oil and simple inhalation devices. Alternative Med Rev 2010;15:33e47. [31] Mulyaningsih S, Sporer F, Reichling J, Wink M. Antibacterial activity of essential oils from and of related components against multi-resistant bacterial pathogens. Pharmaceut Biol 2011;49(9):893e9. [32] Yavari Kia P, Safajou F, Shahnazi M, Nazemiyeh H. The effect of lemon inhalation aromatherapy on nausea and vomiting of pregnancy: a double-blinded, randomized, controlled clinical trial. Iran Red Crescent Med J 2014;16(3):14360. [33] Ben Hsouna A, Hamdi N. Phytochemical composition and antimicrobial activities of the essential oils and organic extracts from Pelargonium graveolens growing in Tunisia. Lipids Health Dis 2012;11:167. [34] Boukhris M, Bouaziz M, Feki I, Jemai H, El Feki A, Sayadi S. Hypoglycemic and antioxidant effects of leaf essential oil of Pelargonium graveolens L’Her. in alloxan induced diabetic rats. Lipids Health Dis 2012;11:81. [35] Tassou CC, Drosinos EH, Nychas GJ. Effects of essential oil from mint (Mentha piperita) on Salmonella enteritidis and Listeria monocytogenes in model food system at 4 degrees and 10 degrees C. J Appl Bacteriol 1995;78(6):593e600. [36] Hammer KA, Carson CF, Riley TV. Antifungal activity of the components of Melaleuca alternifolia (tea tree) oil. J Appl Microbiol 2003a;95:853e60. [37] Hammer KA, Dry L, Johnson M, Michalak EM, Carson CF, Riley TV. Susceptibility of oral bacteria to Melaleuca alternifolia (tea tree) oil in vitro. Oral Microbiol Immunol 2003b;18(6):389e92. [38] Koh KJ, Pearce AL, Marshman G, Finlay-Jones JJ, Hart PH. Tea tree oil reduces histamine-induced skin inflammation. Br J Dermatol 2002;147:1212e7.

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[39] Baratta MT, Dorman HJD, Dean SG, Brondi DM, Ruberto G. Chemical composition, antimicrobial and antioxidant activity of laurel, sage, reosemary, oregano and coriander essential oils. J Essent Oil Res 1998;10(6):618e27. [40] Lee KB, Cho E, Kang YS. Changes in 5-hydroxytryptamine and cortisol plasma levels in menopausal women after inhalation of clary sage oil. Phytother Res 2014;28(11):1599e605. [41] Setzer WN. Essential oils and anxiolytic aromatherapy. Nat Prod Commun 2009;4(9):1305e16. [42] Hongratanaworakit T, Buchbauer G. Relaxing effect of ylang ylang oil on hup;mans after transdermal absorption. Phytother Res 2006;20:758e63. [43] Lu SM, Chen JX, Chen ZL. A study on the in vitro bacteriostatic action of eugenol. Shi Pin Ke Xue 2008;29(9):122e4. [44] Russo M, Galletti GC, Bocchini P, Carnacini A. Essential oil chemical composition of wild populations of Italian oregano spice (Origanum vulgare ssp. hirtum (link) Ietswaart): a preliminary evaluation of their use in chemotaxonomy by cluster analysis. 1. Inflorescences. J Agric Food Chem 1998;46(9): 3741e6. [45] De Martino L, De Feo V, Formisano C, Mignola E, Senatore F. Chemical composition and antimicrobial activity of the essential oils from three chemotypes of Origanum vulgare L. ssp. hirtum (Link) ietswaart growing wild in Campania (Southern Italy). Molecules 2009;14(8):2735e46. [46] Wan J, Wilcock A, Coventry MJ. The effect of essential oils of basil on the growth of Aeromonas hydrophila and Pseudomonas fluorescens. J Appl Microbiol 1998;84:152e8. [47] Takarada K, Kimizuka R, Takahashi N, Honma K, Okuda K, Kato T. A comparison of the antibacterial efficacies of essential oils against oral pathogens. Oral Microbiol Immunol 2004;19:61e4. [48] Rossi P, Cappelli A, Marinelli O, Valzano M, Pavoni L, Bonacucina G, et al. Mosquitocidal and antiinflammatory properties of the essential oils obtained from monoecious, male, and female inflorescences of hemp (Cannabis sativa L.) and their encapsulation in nanoemulsions. Molecules 2020;25: 3451. [49] Amorati R, Foti MC, Valgimigli L. Antioxidant activity of essential oils. J Agric Food Chem 2013; 61(46):10835e47. [50] Deans SG, Ritchie G. Antibacterial properties of plant essential oils. Int J Food Microbiol 1987;5: 165e80. [51] Mills C, Cleary BJ, Gilmer JF, Walsh JJ. Inhibition of acetylcholinesterase by tea tree oil. J Pharm Pharmacol 2004;56:375e9. [52] Satchell AC, Saurajen A, Bell C, Barnetson RS. Treatment of dandruff with 5% tea tree oil shampoo. J Am Acad Dermatol 2002;47:852e5. [53] Gkinis G, Tzakou O, Iliopoulou D, Roussis V. Chemical composition and biological activity of Nepeta parnassica oils and isolated nepetalactones. Z Naturforsch C Biosci 2003;58:681e6. [54] Calcabrini A, Stringaro A, Toccacieli L, Meschini S, Marra M, Colone M, et al. Terpinen-4-ol, the main component of Melaleuca alternifolia (tea tree) oil inhibits the in vitro growth of human melanoma cells. J Invest Dermatol 2004;122(2):349e60. [55] Howes MJ, Houghton PJ, Barlow DJ, Pocock VJ, Milligan SR. Assessment of estrogenic activity in some common essential oil constituents. J Pharm Pharmacol 2002;54(11):1521e8.

CHAPTER 19

Cyclodextrins (CDs) derived from natural source as an essential component in biopharmaceutics Bharat Shinde1, Priyanka Khot2, Dadasaheb Patil1, Pooja Doshi2, Manish Gautam1 and Sunil Gairola1 1

Serum Institute of India Pvt. Ltd, Pune, Maharashtra, India; 2Department of Chemistry, Division of Biochemistry, Savitribai Phule Pune University, Pune, Maharashtra, India

1. Introduction 1.1 History From 1891 to 1911, the primary good-sized duration within the records of cyclodextrins covers their discovery by way of Villiers, in addition to their characterization and chemistry by means of Schardinger, the residences of cyclodextrin had been now not properly comprehended. It far believed that Schardinger performed a significant position in laying a foundation for cyclodextrin chemistry; however, Freudenberg and Meyer-Delius, French and Rundle, D. F. Cramer, Thoma and Stewart, Irie, Uekama and Otagiri, Szejtli, Duchene Kurkov, and Loftson have made historical landmarks in the exploration of cyclodextrin from 1911 to 2013. The term “cyclodextrin” was first coined by F. Cramer [1]. They revealed the cyclic structure of cyclodextrin with its central cavity over the next decade. Cyclodextrins (CDs) are non-reducing cyclic oligosaccharides composed of six, seven, eight, or more glucose units linked by -(1, 4) glycosidic linkages. Schardinger dextrins, cyclomaltoses, and cycloamyloses are other names too for these. Three major cyclodextrins are naturally present and well-known: a-cyclodextrins, b-cyclodextrins, and g-cyclodextrins. Cyclodextrins are synthesized from starch by cyclodextrin glycosyl transferases through enzymatic degradation (CGTase). CDs are soluble in water because of the particular steric arrangement of their glucose units and can feature, as crucial complexation sellers for a broad range of more lipophilic molecules, such as herbal compounds and plant bioactive, with increasing use in all commercial sectors involved. There will be two major research fields in the history of CDs. On the one hand, the complexation property of CDs, as well as their role as host molecules in improving the properties of guest molecules, is of particular interest. This conventional research area, as previously stated, commencing the 1950s, has become well-established and continues to be ongoing. With the millennium’s turn, a new branch of CD research, namely modified CDs, emerged. Due to the initial incredible unique structure of cyclodextrins and the advantages of polymeric backbones, over 11,000 cyclodextrin New Horizons in Natural Compound Research ISBN 978-0-443-15232-0, https://doi.org/10.1016/B978-0-443-15232-0.00005-9

© 2023 Elsevier Inc. All rights reserved.

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derivatives have seemed to be known as CD derivatives and cyclodextrin polymers (CDPs) have been developed as biotech drug delivery systems [2]. Due to their high efficacy, biological drugs such as genes, proteins, and monoclonal antibodies are a wide range of applications including solubilizers, stabilizers, growth promoters, viral gene delivery agents, artificial chaperones, process aids, adjuvants, and cryo-preservatives in the vaccine industry. As a result, many biomaterials for biotech drug delivery have been developed in recent decades. Despite their high efficacy, stability, and low immunogenicity, cyclodextrins are one of the most suitable materials. When cyclodextrins were derivatized by replacing hydroxyl groups with hydrophobic moieties, the modified CDs retained the same properties as the parent cyclodextrins. 1.2 Natural production of CDs Cyclodextrins are cyclic oligosaccharides formed as a result of starch hydrolysis by cyclodextrin glucanotransferase (cyclizing1, 4-a-D-glucan: 1,4-a-D-glucopyranosyltransferase; CGTase; EC 2.4.1.19). This multiple-function enzyme belongs to glycosyl hydrolase family 13 of a-amylase superfamily, capable of catalyzing reversible intra- and intermolecular transglycosylation reactions, as well as hydrolysis of starch, CDs, and other related compounds (Fig. 19.1) [3]. Nevertheless, the successful production of CDs

Figure 19.1 Schematic illustration of the reaction mechanism of CGTase, where R1, R2, and R3 represent different glucose units and n is the number of glucose residues (n  6). (Reproduced from Qi MQ, Zimmermann W. Cyclodextrin glucanotransferase: from gene to applications. Appl Microbiol Biotechnol 2005;66:475e485.)

Cyclodextrins (CDs) derived from natural source as an essential component in biopharmaceutics

depends significantly on the conditions of the hydrolytic process as well as the properties of the enzyme. It should be noted that the CGTase produces all three a, b, and g types of CDs but in various percentages. An enzyme having the capacity to produce predominantly one type of cyclodextrin can reduce the cost of purification of CDs and may have industrial importance. However, b cyclodextrin is the major product of many bacteria and are less reported for a and g cyclodextrins. To date, CGTase has been found in a variety of microorganisms. Besides Bacillus, the groups of bacteria, being the largest producers of CGTase, other genera are also reported for CGTase production using various substrates [4-6]. Apart from this, anaerobes like Thermoanaerobacterium thermosulfurigenes [7], Anaerobranca gottschalkii [8], and Haloferax mediterranei, a halophilic archaeon [9] have been reported. There seems to have been a surge of interest in robust enzymes in recent years because a large number of industrial processes are carried out under exceptionally harsh environmental conditions. Furthermore, it is important to take into account that new CGTase producers will explore (1) the selection of the most efficient strains, (2) the determination of the conditions of the enzyme synthesis in various microorganisms, and (3) designing methods that simultaneously leads to targeted high-yield CD production [10]. 1.3 Chemical and physical aspects of CDs Typically, cyclodextrins are of three main types: a-cyclodextrins, b-cyclodextrins, and gcyclodextrins that comprise 6, 7, and 8 glucose monomers, respectively. Cyclodextrins exhibit complexation behavior and catalytic properties, which they use as enzyme mimics. CDs are chiral, crystalline in nature, non-hygroscopic, and available as a homologues series, which is comprised of a torus-shaped ring structure with a hydrophobic cavity inside and hydrophilic exterior. A hydrophobic cavity in cyclodextrin enables them to interact with guest molecules that are nonpolar or less polar (than water). It is important to note that the selectivity of guest molecules varies depending on the size of the cavity in which cyclodextrins are housed [11]. By encapsulating guest molecules within its hollow, truncated, cone-like structure, cyclodextrin can change its chemical and physical characteristics. Self-associating properties of cyclodextrin make them beneficial for aggregate formation even at the nanoscale [12]. Comparatively to other linear dextrins, cyclodextrins are sparingly soluble in aqueous solutions because of the strong relative bond between molecules in the crystalline state. The probability of solubility of CDs increases with an increase in temperature. Apparently, the strain in rings, orientation, and hydroxyl groups of adjacent glucose units are also other factors that affect the solubility of cyclodextrins. Therefore, g-CDs are more soluble than a- and b-CDs, while b-CDs are poorly soluble among these [11,13]. The hydroxyl groups in cyclodextrin can be substituted with hydrophobic moieties in order to increase their aqueous solubility. Therefore, at present, chemically or

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enzymatically modified cyclodextrin derivatives such as hydroxyl propyl b-CD (HPb-CD), hydroxyl ethyl-b-CD (HE-b-CD), 2,6-dimethyl b-CD (DIM-b-CD), 2,3,6trimethyl b-CD (TRIM-b-CD), cyclodextrin cross-linked with epichlorohydrin (EPC-b-CD), etc have been shown functionality in various applications [14]. Nonetheless, modified CDs retain the same functional characteristics as the parent cyclodextrins. 1.4 Cyclodextrins inclusion complex formation Cyclodextrins have unique geometry and special property interact with various active pharmaceutical ingredients used in the treatment of viral and bacterial diseases. In an aqueous solution, the slightly non-polar cyclodextrin cavity “host” is occupied by thermodynamically unfavored (polar-nonpolar) water molecules, which can be easily reinstated by less polar appropriate “guest” molecules (Fig. 19 2). The hydroxyl groups on the outer exterior of CD molecules can associate with other molecules by hydrogen bonds, allowing CDs to form water-soluble complexes with lipophilic water-insoluble compounds, similar to non-cyclic oligosaccharides and polysaccharides. The lipophilic component of CDs creates a microcavity (inner diameter 5.3e8.3 Ao) into which appropriate-size apolar molecules or molecular fragments of vaccine active ingredients can fit to form inclusion complexes [15]. These CD complexes engage in interactions with membrane surfaces. The majority of the vaccine manufacturing process (predominantly the fermentation process) involves enzyme-

Figure 19.2 Schematic illustration of the host (CD) and guest inclusion complex formation with different stoichiometry [1].

Cyclodextrins (CDs) derived from natural source as an essential component in biopharmaceutics

catalyzed substrate conversion in an aqueous phase. CDs and their derivatives are known to improve the aqueous solubility of a hydrophobic complex substrate [16]. The solubilization effect reduces the toxic effect of substrates or inhibitors on enzymes or microbial cells, thereby increasing bioavailability. CDs, in many cases, act as artificial enzymes or reaction accelerators, altering the reaction pathway. The increased hydrophilicity of guest molecules reduces their volatility and negative effect. As a result, in the new millennium, CDs used in the production of combination vaccines must be designed on the fly to improve bioavailability, preserve antigenicity or immunogenicity, and lessen adverse effects.

2. Application of cyclodextrins in biopharmaceuticals A major role for cyclodextrins in the pharmaceutical industry can be attributed to the continued search for novel formulations and drug delivery systems that are costeffective, bioavailable, and non-toxic (Fig. 19.3). A substantial number of pharmaceutical products contain cyclodextrin, as drug delivery vehicles, excipients, and other additives [17,18]. In addition to improving drug solubility, CDs reduce unpleasant odors and enhance the release of lipophilic drugs. Pharmaceutical CD research has been fundamentally transformed by these characteristics, providing a viable, novel, and promising formulation tool [12,19]. Cyclodextrins are remarkable macrocyclic molecules that hugely impact our daily lives. They have a unique structure that allows the encapsulation of other compounds into them and forms host-guest inclusion complexes. This can result in structural changes in the physicochemical properties of the guest molecules. Cyclodextrins and their derivatives are currently gaining prominence in nanobiotechnology due to their ability to generate novel nanomaterials [20]. The modern approach of CD-based nanomaterials with supramolecular architecture, such as nanosponges, nanoparticles, and nano vesicles, has been used [21]. These CD-containing nanomaterials have pharmaceutical, nanomedicine, and biomedical applications. They are also used for controlled drug delivery and sensing [19]. Hundreds of studies have been conducted on cyclodextrins and their derivatives due to their bioadaptability and multifunctional properties in oral, nasal, ocular, transdermal, and dermal administration routes. Cyclodextrins when complexed with drugs led to improve their functionality by heightening bioavailability, bioadaptability, absorption, stability enhancer, reducing local irritancy, and toxicity. Along with active ingredients, cyclodextrins also form complexes with various proteins and peptides [22], oligonucleotides, and oligosaccharides [23], because CDs can enhance cellular uptake by interacting with cell membranes. Recently, clinical trials revealed that CD derivatives (anionic cyclodextrins with sulfate substituents) disrupt the virus life cycle (Dengue virus and Japanese encephalitis virus) by lowering the expression of the protein, NS1, a non-structural protein required for

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Figure 19.3 Schematic representation of the application of cyclodextrins in biopharmaceuticals.

viral replication [24-27]. The majority of pathologies are caused by one or more proteins malfunctioning. Establishing a link between a specific illness, involved protein(s), and the encoding gene(s) is a valuable tool for knowing the molecular causes of disease and designing effective drugs. In this context, one of the more interesting approaches would be to use a nucleic acid drug to supplement or alter genes within the patient’s cells, allowing the functional protein to be produced endogenously. In comparison to viral vectors, artificial (non-viral) carriers have materialized as a safer and more promising alternative for gene delivery. The vast majority of them are cationic lipids or cationic polymers. It seems that green and secure delivery systems are able to reversibly complexing the relevant nucleic acid and enhance cellular membrane crossing, endosome escaping, cytoplasm trafficking, and nuclear membrane pore passing is crucial in gene therapy. Carbohydrates have proven to be especially useful in this regard, regulating nucleic acid condensation,

Cyclodextrins (CDs) derived from natural source as an essential component in biopharmaceutics

improving DNA-vector assembly bioavailability and biocompatibility, and protecting aggregates from nonspecific interactions. Due to the fact that polyanionic biomolecules are nucleic acids, a combination of cationic CD shaping and ligand appending is conceivably used to rationally design site-specific gene delivery systems. Polycationic CDs (pCDs) formed nano-complexes (CDplexes) with plasmid DNA (pDNA), allowing for remarkable transfection [28]. The development of a supramolecular bridge between nucleic acids and proteins ought to pave the way for a promising future in gene therapy. Cyclodextrin scaffolds have been reported to be used in the sulfonation of competent drug candidates used in HIV therapy and malaria treatment. It has been found that sulfonated CDs exhibited a role in decreasing the infectivity of HIV and ceasing viral replication in vitro [29-32]. In another application, CDs can act as a cholesterol sequester [33-35], causing deformation in the viral membranes of human metapneumovirus [36], parainfluenza virus type 3 [37], coronavirus infectious bronchitis virus [38], herpes simplex virus 1 [39] and, less commonly, Newcastle disease virus [40]. The majority of CD-complexed processes involved an enzyme-catalyzed substrate transformation in aqueous media. This increases the solubility of complexed substrate in aqueous media while decreasing toxicity. Product-inhibited fermentation yield can be increased and metabolized by microbial cells at higher concentrations. In the presence of a suitable CD derivative, i.e., DIMEB 2, 6 dimethyl-beta cyclodextrins are used as supplement aids, and lipid-like inhibitor substances are complex in the production of acellular pertussis vaccines. Heptakis is a DIMEB (2, 6-di-O-methyl)-cyclodextrin methylated-CD derivative that has historically been used as a Bordetella pertussis culture growth stimulant in the production of acellular pertussis vaccine. As a result, Bordetella pertussis spread, and the production of pertussis toxin (PT) increased up to 100-fold [41]. Since the recent outbreak of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), scientists are working around the clock for safe and most effective drugs to reduce SARS-CoV-2 symptoms and hazards. Due to the adverse effects or ineffectiveness, many repurposed medicines and combinations of antiviral therapies have already gone into the background. Nevertheless, b-cyclodextrin derivatives were recommended as a host molecule for some miraculously effective anti-COVID-19 pharmacon, as solubilizers of remdesivir (SB-b-CD) and lopinavir-ritonavir combination (HP-b-CD), and as a taste masker for oseltamivir (b-CD). Interestingly, bCD-chlorhexidine-containing mouthwashes have been envisioned as protective against COVID-19 [42]. Recently, 2-(hydroxyl) propyl-b-CD was used as a cryopreservative in SARS-CoV-2, preventing cold-induced virus particle damage [43]. Because CDs cause less allergic reactions than alum, they are proven the best choice of adjuvant in the human vaccine manufacturing process. CDs induce a synergic immune response by interacting with immunoglobulins and producing Th2 cells in the influenza vaccine developed by Daiichi Sankya, a Japanese pharmaceutical company [44]. Cyclodextrin aids in the stimulation of immune cell responses, making it suitable for use as a

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veterinary vaccine adjuvant [43]. Recent research revealed that g CD metal-organic framework (g CD-MOF) was modified with Span-85 fabricated as an animal vaccine adjuvant. This finding suggested the potential of SP-g CD-MOF as vaccine adjuvants and paved the idea for the development of novel adjuvants in antigen delivery [45].

3. Regulatory aspects of CDs Natural CDs and chemically modified CDs are being investigated for a variety of biotechnology applications. Many CD-containing biotechnology-derived preparations are primarily administered orally, nasally, or parenterally. Cyclodextrin’s ability to form complexes with a variety of compounds aids in the modification of solubility, inhibition, stability, and biocompatibility. The extent of CD-related adverse effects (e.g., hemolysis, nephrotoxicity) on humans is highly dependent on the route of administration. Regulatory agencies are concerned about the use of CDs in preliminary preparation because it is an excipient and has been linked to toxicity issues; however, there are almost no restrictions on administering native CDs orally. The European Medicines Agency does not recommend native CDs for parenteral applications. CDs can be used as an auxiliary substance in drug formulation in Germany and Denmark [46]. France and other European countries such as the Netherlands, Benelux, and Spain approved CD as a flavor carrier in 1986 [47,48]. The Japanese and Dutch health ministries declared the CDs to be enzymatically modified starch, allowing them to be used in food and other products. According to regulators, CDs appear to be excipients rather than drug substances. For CDs in food products, the FAO/WHO expert committee on food additives has established an Allowable daily intake of 5 mg/kg/day [49,50]. Native CDs were also added to the FDA’s list of Generally Recognized as Safe ingredients [51]. The promising data obtained in influenza, herpes simplex, and dengue virus research could be new spectrum steps in developing a vaccine. The various CD applications are currently undergoing phase I and II clinical studies, demonstrating how cyclodextrins are steadily making a strong foothold in medicinal applications, in biomedical engineering. Cyclodextrins are becoming an important part of the biotechnology options in many products in terms of quality, safety, and efficacy.

4. Conclusion Cyclodextrins are exceptional molecules, which have diverse effects on cell membranes and biological barriers; however, these effects may have appeared in their host-guest interactions. CDs can be suitably employed in biopharma formulations containing antifungal activity chemical auxiliary substances, excipients, and supplementary aids in mechanisms for the improvement of solubility, bioavailability, absorption, synergistic, and the building block of many drug delivery systems. Despite their nontoxic nature,

Cyclodextrins (CDs) derived from natural source as an essential component in biopharmaceutics

the new CDs are promising candidates for antiviral use, but the research is still limited to in vitro studies, so more research is needed to grasp their practical application. The adaptability of cyclodextrins and modified CDs in a variety of applications ranging from cosmetics to food to biomedicine. The usage of CDs for the removal of cholesterol and different lipids, which builds up wonderful biocompatibility, interactions with biomolecules, and the ability of functionalization to gain derivatives so that it will especially engage with a specific bimolecular goal, provide them a vast medicinal capability and plenty of greater programs expected to emerge soon.

References 1. Crini G. Review: a history of cyclodextrins. Chem Rev 2014;114:10940e75. 2. Rezanka M. Synthesis of substituted cyclodextrins. Environ Chem Lett 2018;17:49e63. 3. Qi MQ, Zimmermann W. Cyclodextrin glucanotransferase: from gene to applications. Appl Microbiol Biotechnol 2005;66:475e85. 4. Mori S, Hirose S, Oya T, Kitahata S. Purification and properties of cyclodextrin glucanotransferase from Brevibacteriumsp. No. 9605. Biosci Biotech Biochem 1994;58:1968e72. 5. Rajput KN, Patel KC, Trivedi UB. A novel cyclodextrin glucanotransferase from an alkaliphile Microbacterium terrae KNR 9: purification and properties. 3 Biotech 2016;6:1e11. 6. Sin K, Nakamura A, Kobayashi K, Masaki H, Uozumi T. Cloning and sequencing of a cyclodextrin glucanotransferase gene from Bacillus ohbensis and its expression in. Escherichia coli Appl Microbiol Biotechnol 1991;35:600e5. 7. Wind LED, Liebl W, Buitelaar RM, Penninga D, Spreinat A, Dijkhuizen L, Bahl H. Cyclodextrin formation by the thermostable a-amylase of Thermoanaerobacteriumthermosulfurigenes EM1 and reclassification of the enzyme as a cyclodextrin glycosyltransferase. Appl Environ Microbiol 1995;61:1257e65. 8. Thiemann V, Dönges C, Prowe SG, Sterner R, Antranikian G. Characterization of a thermoalkali-stable cyclodextrin glycosyltransferase from the anaerobic thermoalkaliphilic bacterium. Anaerobrancagottschalkii Arch Microbiol 2004;182:226e35. 9. Bautista V, Esclapez J, P
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19. Bilensoy E, Hincal A. Recent advances and future directions in amphiphilic cyclodextrin nanoparticles. Expert Opin Drug Deliv 2009;6:1161e73. 20. Arima H, Motoyama K, Higashi T. Potential use of cyclodextrins as drug carriers and active pharmaceutical ingredients. Chem Pharmac Bulle 2017;65(4):341e8. 21. Tejashri G, Amrita B, Darshana J. Cyclodextrin based nanosponges for pharmaceutical use: a review. Acta Pharm 2013;63:335e58. 22. Irie T, Uekama K. Cyclodextrins in peptide and protein delivery. Adv Drug Deliv Rev 1999;36: 101e23. 23. Szejtli J. Introduction and general overview of cyclodextrin chemistry. Chem Rev 1998;98:1743e53. 24. Alcala AC, Hern
andez-Bravo R, Medina F, Coll DS, Zambrano JL, del Angel RM, Ludert JE. The dengue virus non-structural protein 1 (NS1) is secreted from infected mosquito cells via a non-classical caveolin-1-dependent pathway. J Gen Virol 2017;98:2088e99. 25. Carro AC, Damonte EB. Requirement of cholesterol in the viral envelope for dengue virus infection. Vir Res 2013;174:78e87. 26. Jones ST, Cagno V, Janecek M, Ortiz D, Gasilova N, Piret J, Gasbarri M, Constant DA, Han Y, Vukovic L, Kr
al P, Kaiser L, Huang S, Constant S, Kirkegaard K, Boivin G, Stellacci F, Tapparel C. Modified cyclodextrins as broad-spectrum antivirals. Sci Adv 2020;6. 27. Puerta-Guardo H, Mosso C, Medina F, Liprandi F, Ludert JE, del Angel RM. Antibody-dependent enhancement of dengue virus infection in U937 cells requires cholesterol-rich membrane microdomains. J Gen Virol 2010;91:394e403. 28. Fernandez JMG, Benito JM, Mellet CO. Cyclodextrin-scaffolded glycotransporters for gene delivery. Pure Appl Chem 2013;85(9):1825e45. 29. Ambrose Z, Compton L, Michael Piatak Jr M, Lu D, Alvord WG, Lubomirski MS, Hildreth JEK, Lifson JD, Miller CJ, KewalRamani VN. Incomplete protection against simian immunodeficiency virus vaginal transmission in rhesus macaques by a topical antiviral agent revealed by repeat challenges. J Virol 2008;82:6591e9. 30. Mori H, Otake T, Oishi I, Kurimura T. Characterization of human immunodeficiency virus type 1 resistant to modified cyclodextrin sulphate (mCDS71) in vitro. Antivir Chem Chemother 1999;10: 15e21. 31. Moriya T, Kurita H, Matsumoto K, Otake T, Mori H, Morimoto M, Ueba N, Kunita N. Potent inhibitory effect of a series of modified cyclodextrin sulfates (mcds) on the replication of HIV-1 in Vitro. J Med Chem 1991;34:2301e4. 32. Moriya T, Sato K, Kurita H, Matsumoto K, Otake T, Mori H, Morimoto M, Ueba N, Kunita N. A new candidate for an anti-HIV-1 agent: modified cyclodextrin sulfate (mCDS71). J Med Chem 1993; 36:1674e7. 33. Castagne D, Fillet M, Delattre L, Evrard B, Nusgens B, Piel B. Study of the cholesterol extraction capacity of b-cyclodextrin and its derivatives, relationships with their effects on endothelial cell viability and on membrane models. J Incl Phenom Macrocycl Chem 2009;63:225e31. 34. Lee CJ, Lin HR, Liao CL, Lin YL. Cholesterol effectively blocks entry of flavivirus. J Virol 2008;82: 6470e80. 35. Sun X, Whittaker GR. Role for influenza virus envelope cholesterol in virus entry and infection. J Virol 2003;77:12543e51. 36. Chen SH, He H, Yang H, Tan B, Liu EM, Zhao XD, Zhao Y. The role of lipid rafts in cell entry of human metapneumovirus. J Med Virol 2019;91:949e57. 37. Tang QP, Liu PF, Chen MZ, Qin YL. Virion-associated cholesterol regulates the infection of human parainfluenza virus type 3. Viruses 2019;11(5):438. 38. Guo HC, Huang M, Yuan Q, Wei YQ, Gao Y, Mao LJ, Gu LJ, Tang YW, Zhong YX, Liu DX, et al. The important role of lipid raft-mediated attachment in the infection of cultured cells by coronavirus infectious bronchitis virus Beaudette strain. Plos ONE 2017;12. article e0170123. 39. Wudiri GA, Schneider SM, Nicola AV. Herpes simplex virus 1 envelope cholesterol facilitates membrane fusion. Front Microbiol 2017;8:2383. 40. Martin JJ, Holguera J, Sanchez-Felipe L, Villar E, Munoz-Barroso I. Cholesterol dependence of newcastle disease virus entry. Biochim Biophys Acta Biomembr 2012;1818:753e61.

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41. Imaizumi A, Suzuki Y, Ono S, Sato H, Sato Y. Heptakis (2,6-Dimethyl), beta cyclodextrin: a novel growth stimulant for Bordetella pertussis phase I. J Clin Microbiol 1983;17:781e6. No. 5. 42. Jicsinszky L, Martina K, Cravotto G. Cyclodextrins in the antiviral therapy. J Drug Deliv Sci Technol 2021;64:102589. 43. Braga SS, Barbosa JS, Santos NE, EI-Saleh F, Paz FAA. Cyclodextrins in antiviral therapeutics and vaccines. Pharmaceutics 2021;13(409):1e24. 44. Onishi M, Ozasa K, Kobiyama K, Ohata K, Kitano M, Taniguchi K, Homma T, Kobayashi M, Sato A, Katakai Y, et al. Hydroxypropyl-b-cyclodextrin spikes local inflammation that induces Th2 cell and T follicular helper cell responses to the coadministered antigen. J Immunol 2015;194:2673e82. 45. Li C, Chen C, Wei Y, Tan M, Zhai S, Zhao J, Wang L, Dai T. Cyclodextrin metal-organic framework as vaccine adjuvants enhances immune responses. Drug Deliv 2021;28(1):2594e602. 46. European Medicines Agency. Background review for cyclodextrins used as excipients. London, UK: EMA; 2014. 47. Astray G, Mejuto JC, Simal-Gandara J. Latest developments in the application of cyclodextrin hostguest complexes in beverage technology processes. Food Hydrocoll 2020:106e105882. 48. Fenyvesi E, Vikmon M, Szente L. Cyclodextrins in food technology and human nutrition: benefits and limitations. Crit Rev Food Sci Nutr 2016;56(12):1981e2004. 49. Abbott PJ. JEFCA 55th meeting. Safety evaluation of certain food additives and contaminants (gcyclodextrin). WHO Food Addit Ser 2000:44e969. 50. Kroes R, Verger P, Larsen JC. Safety evaluation of certain food additives (a-cyclodextrin-addendum). WHO Food Addit Series 2006;54:3e15. 51. Pollit FD. Safety evaluation of certain food additives (b-cyclodextrin). WHO Food Addit Ser 1996;35: 257e68.

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CHAPTER 20

Natural compound-based scaffold to design in vitro disease systems Chirag Varshney1, Brijesh Kumar2 and Swapnil C. Kamble1 1

Department of Technology, Savitribai Phule Pune University, Pune, Maharashtra, India; 2School of Bio-Medical Engineering, Indian Institute of Technology, Banaras Hindu University, Varanasi, India

Abbreviations 2D Two-dimensional 3D Three-dimensional CA Chitosan-alginate Coll Collagen fibres-I CSCs Cancer stem cells CTNT Chitosan-titanium oxide nanotube ECM Extracellular matrix GAF Gelatin-alginate-fibrinogen HANF Hybrid aligned electrospun nanofiber HPKs Human primary keratinocytes MgHA Mg-doped hydroxyapatite PAA Polyacrylamide PEC Polyelectrolyte complex PLA Polylactic acid PLA-HA Polylactic acid -calcium-deficient hydroxyapatite-coated TCP Tissue culture plastic TMM Tiger milk mushroom XGH Xanthan gum hydrogels YLD Yearly lived with disability

1. Introduction The number of individuals with communicable and non-communicable diseases have increased tremendously in the last few decades. It is estimated that only 1 out of 20 individuals is disease-free, i.e., 95% world population is currently dealing with health problems and one-third of the total population has an average of five disease conditions simultaneously [1]. In 2019, 241 billion people worldwide had conditions that could benefit from rehabilitation, accounting for 310 million [235e392] all-age Yearly Lived with Disability (YLD). From 1990 to 2019, the percentage of persons affected with disability and contributing to YLDs has increased from 21.2% to 63% [2]. These analyses point to the inevitability of intense investigation on them. Change in lifestyle is the major

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underlying reason for the increase of these diseases. Cardiovascular disorders, diabetes, cancer, and hypertension are a few examples of major health concerns within noncommunicable diseases. Loss of function of an organ, and tissue degradation becomes the limiting factor in achieving a healthy and medication-free life. The best possible intervention in such cases is an organ or tissue transplantation. However, transplantation may cause side effects like kidney damage, excessive weight gain, dizziness, daily pricking of the skin, lifelong dependency on immuno-suppressors, chances of graft rejections, etc. [3]. To mitigate these challenges, the development of artificial tissue through tissue engineering potentiates the reduction of the risk-to-benefit ratio. Tissue engineering employs cell culturing techniques that play a vital role in preparing disease models. The tissue culturing approach aid researchers to study the cell’s biology, tissue morphology, mechanisms of disease development, drug action, and protein production [4]. Various representations are being used to mimic the disease state. The widely used imitation disease models are two-dimensional (2D) and three-dimensional (3D) cell culture based systems, disease animal systems, and computational models. Each model and its design have been suited to discover and understand the underlying pathways, and eventually the development of suitable drugs. The in vitro models are popular due to their easy handling over animal systems, and a certain degree of resemblance to the tissue of origin. Rodents and primates have been the most common and closest animal models. However, not all diseases are expressed in any of the animals and their upkeep is expensive. The computational models have gained popularity with databases becoming available readily. None of these can completely replicate the disease state and are interdependent for validation. Requirement of a system that incorporates as many features as possible of natural tissue remains. In vitro scaffold based tissue culture presents a possible solution to these limitations. Here we have briefly referred to in vitro models to garner interest in the need for 3D systems. We have emphasized on natural compounds broadly under the domains of sugars, proteins, and Extracellular matrix (ECM) derived that have been evaluated for the development of 3D scaffolds. We conclude with the urgent research questions that may be taken up by avid researchers.

2. 2D cell culture system The 2D system is based on growing cells either on the flat Petri plates or in a culture flask [5]. The adherent monolayer cells derived from epithelial, mesenchymal, or endothelial origin are allowed to adhere, grow, and proliferate to mimic the surface-dependent nature present in the body. In contrast, suspension cultures are derived from the cells of the circulatory systems. Both types of cell culture practices are simple and inexpensive than animal models [4]. Numerous pathways, cellular functionality, mutational studies, and expression profiling have been elucidated through them [4]. The significance is

Natural compound-based scaffold to design in vitro disease systems

exemplified in in vitro cancer biology research wherein numerous cell lines are developed and studied. The success of tumor-derived cell line has catapulted cancer discovery research to new heights. Unfortunately, the involvement of adherent cells in the 2D comes with numerous disadvantages. Some of them are listed below. 1. One of the characteristic features of a typical tumor microenvironment is presence of different non-tumor cells in it. The non-tumor cells include immune cells, fibroblasts, and healthy cells (neighboring cells) that produce cytokines, ECM, and growth factors responsible for tumor cell survival, local invasion, and metastatic dissemination [6,7]. In 2D system, lack of interaction with these cells is responsible for altered gene and protein expression, cell differentiation and proliferation, responses to stimuli, drug metabolism, and other functions [8,9]. 2. The 2D cultured cells do not mimic the natural structure of the source tissue. This phenotypic change is accompanied by altered gene expression to accommodate the new environment. As a result, most of the cell lines developed are of cancer origin and not many noncancerous cell types have been successfully replicated in the in vitro system [10]. 3. Certain cells lose their polarity in 2D environment which alters their response to various phenomena like apoptosis and cell proliferation [4]. 4. Cells derived from solid tissue form a monolayer in 2D system with an unlimited supply of nutrients such as oxygen, metabolites, signal molecule, etc. that favors the most-proliferative cell population over slow-cycling cells, leading to the development of a homogenous clone [11]. This aspect prevents the identification of slow-cycling populations that are constituents of the tumor. Cancer cell-line monolayers lack this very heterogenicity among the cancer cell types. 5. Typically, tumor cells adhere to their substrate. However, loss of anchorage dependence may occur in epithelial-to-mesenchymal transition [12]. Upon transformation toward 2D monolayer formation, some cells may spontaneously lose anchor dependency. Hence certain types of cells may be lost during cell passaging. 6. The extent of ECM secretion is variable in monolayers vis-
a-vis natural tissue [13]. The ECM gives certain cues that guide cancer cells and cancer stem cells toward the cycling phase [13,14]. Hence, the role of ECM on tumor progression is less detailed. In addition, ECM secretary cells like fibroblasts and tumor-associated macrophages are absent in monolayer. 2D cell culturing model fails to replicate the tumor microenvironment whereas 3D models have the potential to replicate the microenvironment, which can have a significant impact on results [15].

3. 3D system Here cells are grown either in anchor-dependent or anchor-independent ways to attain 3D structure. The 3D entity usually mimics the microenvironment of the donor

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tissue, as the cells secret ECM and other growth-regulated molecules [16]. As such, they can be used as a specific disease model with high accuracy over cell monolayers [17]. Based on the method of preparation, 3D systems are of three types (Table 20.1).

4. Scaffold for 3D cell culturing The scaffolds are used as a base on which cells can grow into a 3D entity that mimics the properties and functions of their parent tissue. The scaffold material may be synthetic, natural, or mixed. The scaffold can disintegrate into by-products once the cells have colonized it and begun forming their own ECM [17]. Permeability, surface chemistry, porosity, mechanical properties, biodegradation, and ability to release biomolecules, all contribute to mimicking the cellular niche [18,19]. The scaffold matrix has a close chemical and physical interaction with the cells, influencing their ability to proliferate, differentiate and secrete extracellular components [20]. As a result, the matrix used is significantly influenced by the cell type used and the nature of the research [21,22].

5. Types of scaffolds 5.1 Synthetic scaffolds Synthetic scaffolds are artificially made from organic, inorganic materials or synthetic polymers [17] (Table 20.2). They lack biocompatibility with cells, signal molecules, growth factors, hormones, or cellular receptors. Encapsulation of signal molecules, growth factors, hormones, or cellular receptors is associated with the surface of synthetic scaffolds for better cell proliferation and differentiation [20,23,24]. Excellent physical strength, well-defined components, and low risk of pathogen transmission make artificial scaffolds of high relevance in bone and cartilage engineering [18,24,25]. One such example is the use of polylactic acid (PLA). It provides the necessary mechanical support but lacks the bioactivity required to induce cell proliferation. To tackle this problem, polyethyleneimine and citric acid were chemically attached to the alkalitreated PLA scaffolds [26]. These polymer-grafted scaffolds are soaked in the simulated body fluid to develop calcium-deficient hydroxyapatite-coated scaffolds (PLA-HA). These PLA-HA scaffolds show 50% more cell proliferation than PLA scaffolds. 5.2 Natural scaffolds Unlike synthetic scaffolds, natural scaffolds easily achieve resemblance to cellular niches without incorporating encapsulated signaling moieties and growth factors into the matrix [30]. They are biocompatible and have a great ability to degrade after the completion of task without causing any harmful effects to the system like toxicity or inflammation [23].

Table 20.1 Three-dimensional cell models based on the method of preparations [4]. Features of system

Benefits

Limitations

Suspension culture on nonadherent plates

1. Single donor cells are inoculated on nonadherent plates containing nutrient medium. 2. 3D cells can be observed after 3e4 days. 1. Donor cells are grown in a medium containing gelatin substances like agarose or matrigel. 2. The 3D structure can be observed after a week of culturing.

1. Simple and speedy procedure. 2. Extraction of cells is easy for further usage.

1. The non-adherent coating on plates varies as per the cell lines. 2. Aggregates formation due to the movement of the cells. 1. Time-consuming and inconvenient due to the preparation of doable layered agar. 2. Low reproducibility of the result. 3. Cannot be used for direct immunofluorescence analysis. 4. Gelatin material like matrigel influences the morphology of cells. 1. Variations observed in the cell behavior due to the scaling of scaffolds and topography of cell distribution. 2. Scaffolds material may alter the cells’ growth and adherent properties. 3. Tissue observation and cell extraction are restricted to few analyses (like immunohistochemistry).

Culture in concentrated medium or gel-like substances

Culture on scaffold

1. Cells are grown in a medium containing biodegradable material called scaffolds such as silk, collagen, etc. to which cells migrate and fill the spaces among fibrous structures.

1. Easy extraction procedure. 2. Interaction of cells with the local environment.

1. Highly compatible with other commercially available tests like DNA and RNA extraction kits. 2. Easy to prepare for immunohistochemical analysis.

Natural compound-based scaffold to design in vitro disease systems

Types of the 3-D model

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Table 20.2 Features of different types of artificial scaffolds. Material

Advantage

Disadvantages

Example

References

Metals

1. Biocompatible 2. Excellent mechanical properties

Stainless steel, cobaltbased alloys, and titanium alloys are the most used metallic scaffolds used in dental and orthopedic surgeries

[27,28]

Ceramics

1. Osteoconductive and osteoinductive properties (bioactive ceramics) 2. The composition can be similar to the human bone in mineral content 1. Good tunability of physical properties 2. Low immune response 3. Low production cost 4. High reproducibility 5. Defined purity and composition

1. Poor biodegradability 2. Oxidation and aggregation issues 3. May require to be combined with polymer 4. Secondary release of metal ions may cause toxicity 1. Significantly brittle 2. May display inappropriate degradation/ resorption rates

Calcium phosphates, hydroxyapatite, and tricalcium phosphate are widely used in bone and tissue engineering

[28,29]

1. Often hydrophobic 2. Lack of cellular recognition patterns 3. Poor biocompatibility 4. Risk of biodegradation side effects (inflammation, toxicity, etc.)

Poly(a-ester), polyurethanes, polyacetals, poly(ester amide), polyanhydrides, polyphosphazenes, and pseudo poly(amino acids) are the most prominent classes of synthetic polymers with utility in tissue engineering

[27e29]

Polymers

All these properties of natural scaffolds put them on a pedestal over the artificial scaffolds. However, the unknown composition of natural scaffolds creates batch-to-batch variations, difficulty in getting regular size and shape, and potential immunogenicity issues [17].

Natural compound-based scaffold to design in vitro disease systems

Sugars, amino acids, ECM-derived materials, and acellular matrix are widely used precursors for scaffolds that are produced by living organisms like bacteria, yeast, plants, and animals [30e32] (Fig. 20.1). 5.2.1 Sugars Sugars are long-chain polymeric carbohydrates that are made up of small monomeric units called monosaccharides bound together by glycosidic linkages. Physical qualities of sugars such as solubility, flow behavior, gelling potential, and/or surface and interfacial properties are determined by differences in monosaccharide composition, linkage types and patterns, chain morphologies, and molecular weight which make it a potential candidate for the natural scaffolds. Sugars can be derived from plants, animals, and

Figure 20.1 Typical approaches for the development of in vitro 3D systems using natural scaffolds. Biomaterial-rich organs or tissues are isolated from the living organism that are processed to get purified precursors. These precursors are fabricated into a 3D scaffold using various techniques and then supplemented with suitable cells and growth factors. This functional in vitro scaffold-based 3D model can be used for disease studies, drug interaction, tissue engineering and regenerative medicines (created with BioRender.com).

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microorganisms. They have several physiological roles and could have a variety of applications in tissue engineering and regenerative medicine [30] (Table 20.3). 5.2.2 Proteins Proteins are made up of monomeric amino acids linked by peptide bonds. Proteins or peptides can also be used for the synthesis of scaffolds for 3D cell cultures. Typical examples of proteins that can be used for scaffold development include collagen, gelatin, gellan, and fibrin. 1. Collagen is the one of most abundant proteins present in human and is responsible for maintaining the structural integrity of various tissues [40]. Collagen, one of the primary components of ECM, has inherent biocompatibility, low antigenicity, and high tunability which allow the formation of porous scaffolds easily, but they lack mechanical strength [41e45]. Inoculated cells can easily attach to the scaffolds using their integrins which activate the cell signaling pathways that increase the cell proliferation in the 3D model [17]. Cell secreted enzymes can disintegrate this collagen protein into smaller and non-toxic by-products, reducing the risk of post-infection toxicity [46]. All these qualities of collagen make it a promising candidate for the synthesis of scaffolds. The primary production of collagen is done from collagen-rich tissues namely skin, tendon, intestine, and bladder [17]. The origin of collagen (from animal sources) makes it a tricky entity to use due to associated ethical issues and it does not match with the approach of animal-free biomaterial research. It led to the emergence of human recombinant collagen that is produced using tobacco plants [47]. 2. Gelatin, a polymer derived from type-I collagen that has undergone irreversible enzymatic hydrolysis, has gained interest in tissue engineering in the last few years because of its biocompatibility and biodegradation profile, and low antigenicity [48]. On the downside, low mechanical strength, and its extraction from animal sources such as cows, pigs, fish, and insects deviate from 3R (replacement, refinement, and reduction) principle of ethical and animal testing [17,48,49]. 3. Fibrin aggregates at the site of injury in vertebrates and provides a structure for the cell to adhere while the neighboring cells are secreting components of ECM required to heal the injury [50,51]. The ECM secretion induced by fibrin along with inbuilt biocompatibility, fast degradation, and high affinity toward cells is exploited in 3D based scaffolds system [52]. The scaffolds constructed using fibrin fibers depict the highest cell growth and closely mimic the ECM when supplemented with growth factors and endogenous molecules [52,53]. The major drawbacks of fibrin are its low mechanical strength and rapid degradation rate which can be overcome by adding another polymer during scaffold preparation [53,54]. 4. Recently soy protein from soya beans has been reported to form porous scaffolds [55]. Soy scaffolds are biocompatible and generate non-toxic by-products after degradation but have low molecular weight and rapid enzymatic degradation which are mostly

Table 20.3 Widely used sugars as a scaffold in tissue engineering and regeneration medicines. Subunits

Source

Advantages

Disadvantages

References

Chitosan

N-acetyl Dglucosamine and Dglucosamine

Insects, crustaceans, and fungi

Weak physical strength

[33]

Alginate

b-D-mannuronic acid and a-L-guluronic acid

Seaweed and bacteria

Lack of cell recognition pattern resulting in lower cell adhesion

[34e36]

Hyaluronic acid

N-acetylglucosamine and D-glucuronic Acid

Epithelial, connective, and nerve tissues of vertebrates and microbes

Poor mechanical properties

[37,38]

Cellulose

b-D-glucose

Bacteria, tunicates, and plants

1. Good biocompatibility 2. Biodegradable 3. Mucoadhesive 4. Non-toxic and nonantigenic 5. Antibacterial properties 6. High adsorption properties 1. Low production cost 2. Hydrophobic 3. Good biocompatibility 4. Biodegradable 5. Non-toxic 6. Non-allergenic 1. Component of ECM 2. Good biocompatibility 3. Proper viscosity and elasticity 4. Biodegradable 5. Low immunogenicity 1. Excellent biocompatibility and mechanical strength 2. Practical optical transparency 3. Inexpensive

Low degradation rate in in vivo

[29,39]

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Polysaccharides

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overcome by mixing with synthetic polymers [55,56]. The use of soy protein scaffolds is in an early stage and is looked forward with bright future as it could reduce the burden on animal-sourced scaffold materials for research purposes. 5.2.3 ECM-derived Few of the scaffold materials can be directly extracted from ECM. Matrigel is an example of such a matrix that has been extracted from the ECM components of the Englebreth-Holm-Swarm mouse tumor [57]. As Matrigel is extracted from natural ECM, it can accurately mimic the microenvironment which is required for cells to form a 3D cell system. The main concern with Matrigel is its origin from tumor cells which causes a lot of variation and uncertainty in the concentration and composition of components [17,57]. 5.3 Hydrogels Hydrogels are 3D network structures composed of hydrophilic polymers consisting of natural, artificial, or a mixture of both, cross-linked either through covalent bonds or inter- or intra-molecular forces of attraction [58]. Hydrogels have an excellent water holding capacity that enables high nutrient intake, oxygen, and even growth factors from the medium leading to cell proliferation in the system [59]. Like natural scaffolds, hydrogels are biocompatible and have excellent tunability which makes them widely used in tissue engineering [60]. However, they do have significant drawbacks. An uneven gradient of soluble substances can affect normal cell differentiation as endogenous factors are not incorporated directly into the matrix. The unique architecture of hydrogel makes cell imaging and analysis more difficult than other scaffold types. Immunohistochemistry is difficult to perform on some cells [61]. Different hydrogels can be prepared by altering the integration methods of macromolecules. (1) MgHA/ColI hybrid scaffolddA biomimetic hybrid composite scaffold made by the direct nucleation of Mg-doped hydroxyapatite (MgHA) on self-assembling collagen fibres-I (MgHA/Coll) was made by Bassi et al. [62]. These hybrid composite scaffolds when supplemented with human osteosarcoma SAOS-2 cells and MG63 cells formed a 3D system that mimicked the heterogenicity of the osteosarcoma stem cell niche [62]. It was observed that the cancer stem cells (CSCs) maintained greater stemness traits over monolayers when cultivated in both MgHA/ Coll and HA biomaterials. The suggested biomimetic scaffolds, which mimic the stem cell niche composition and architecture of genuine tissue, have the potential to replace current in vitro screening methods [62]. (2) Silk Fibroin-XGH scaffolddSilk fibroin and xanthan gum hydrogels (XGH) were fabricated using ultrasonication that generates b-sheets by Byram et al. [63]. The b-sheet formed by cross-linkage enhanced the mechanical properties of hydrogels

Natural compound-based scaffold to design in vitro disease systems

(3)

(4)

(5)

(6)

as required for connective tissue regeneration. The produced scaffolds had altered architecture, greater porosity and interconnected networks. Silk-XGH scaffolds supported murine fibroblast cell line, L929 cell viability and increased cellular proliferation even after 120 h [63]. Hybrid aligned electrospun nanofiber (HANF) scaffoldsdGellan is a polysaccharide with excellent biocompatibility and tunable mechanical properties. It lacks specific cell adhesion sites which can be rectified by fabricating it with another scaffold material like gelatin [64]. HANF scaffold comprised of polyvinyl alcohol, gelatin, and gellan that were bonded in a hydrogel matrix. This hydrogel can imitate the natural physical and chemical properties of neuronal ECM. Quercetin was employed for testing the HANF scaffold’s drug delivery capability. It was found that the quercetin was released in two phases, one within 24 h (immediate release) releasing 60% of the total drug followed by sustained release that extended 5 days [64]. Therefore, these HANF scaffolds can be used in neural tissue engineering and drug delivery. Col1-PAA scaffolddCollagen-I coated hydrogels constructed of polyacrylamide gels (PAA) with variable substrate elasticity by adjusting cross-linking density to approximate skin elasticity were prepared by Mogha et al. [65]. In comparison to conventional tissue culture plastic (TCP) dishes, the use of Collagen-I coated hydrogel accelerated proliferation and generated more human primary keratinocytes (HPKs) [65]. The effect of elasticity on HPKs migration was investigated after prolonged passaging on TCP and PAA gels. Intriguingly, when rigid TCP control and 10 and 20 kPa gels were compared for HPKs at early passages, there were minimal variations in cell migratory behavior. HPKs produced from gels moved more quickly in wound closure at late passage than the control. The cells from 10 kPa gels migrated more rapidly than cells from 20 kPa gels when comparing the migratory behavior of cells in both gels in the presence of exogenous epidermal growth factor [65]. Ca-alginate scaffolddTo create bone-like tissue precisely imitating in vivo settings, a 3D porous Ca-alginate scaffold cell culture system was designed in conjunction with a functionally closed process bioreactor [66]. The Ca-alginate-based closed bioreactor was inoculated with human osteoblasts. Ca-alginate scaffolds were found to promote human osteoblasts growth and differentiation, as well as bone-related gene expression upregulation. However, after 7 days, the cells stopped proliferation and the doubling time was lowered to 132 h from 125 h in the presence of osteogenic signals [66]. CTNT-TMM scaffolddDirect-blending and freeze-drying procedures were used to create chitosan-titanium oxide nanotube (CTNT) scaffolds fortified with tiger milk mushroom (TMM-CTNTs) [67]. Varying varieties of these scaffolds were built using different TMM concentrations ranging from 0% to 5% by weight.

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(7)

(8)

(9)

(10)

MG63 cells were seeded into these TMM-CTNTs scaffolds [67]. Supplementation of TMM in CTNTs scaffolds increased the porosity, mechanical strength, and biocompatibility qualities of scaffolds, allowing artificial 3D templates for bone regeneration to meet the physical requirements. The cell proliferation assay test demonstrated that the addition of TMM slowly enhanced cell growth, extending the length of proliferation. On the 7th day of incubation, scaffolds with 5 wt.% TMM were shown to be the best scaffolds when compared to other scaffolds [67]. Soy protein-Chitin scaffoldsdThe sponge-like scaffolds were constructed using soy protein and b-chitin derived from a squid pen. To investigate the biocompatibility and cytotoxicity profile of soy-chitin scaffolds, researchers inoculated them with L929 fibroblasts and human mesenchymal stem cells [68]. Increased collagen fiber deposition and increased neovascularization capacity showed great biocompatibility in in vivo [68]. Furthermore, these scaffolds promoted cell adhesion, proliferation, and a high load capacity. Finally, when these stem cells were 3D-cultured in these soy-chitin scaffolds, the released vascular enothelial growth factors were four fold higher than the 2D plates [68]. Chitosan-alginate scaffoldsdChitosan-alginate (CA) scaffolds were created by first generating a polyelectrolyte complex (PEC) between chitosan and alginate molecules, followed by freeze-casting and lyophilizing the CA PEC solution. TRAMPC2 mouse prostate cancer, MDA-MB-231 human breast cancer, SK-Hep 1 human hepatocellular carcinoma, and U-87 MG human glioblastoma cell lines were seeded in 3D porous CA scaffolds to investigate the proliferation, morphology, and gene expressions of cells cultured in CA scaffolds as compared to 2D controls. In comparison to monolayers, 3D CA scaffold cultures revealed a lower proliferation rate, development of tumor spheroids, and enhanced expression of CSCrelated mark genes (CD133 and NANOG) [69]. This research shows that the enrichment of cancer stem-like cells in CA scaffolds is a resilient process that is not limited to certain cancer types. Decellularized apple hypanthium scaffoldsdIn vitro cultivation of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts, and human HeLa epithelial cells can be performed using 3D cellulose scaffolds made by decellularizing apple hypanthium tissue [70]. The cells proliferated and increased in number by three to four fold in all cases. C2C12 and HeLa cells had roughly twice the number of NIH3T3 cells. Even after 12 weeks of continuous in vitro cultivation, a large percentage of cells remained viable [70]. Many apoptotic cells were found near the cellulose scaffold’s interior. The possible reason could be a lack of media turnover, which results in a shortage of nutrients and oxygen, which is a frequent issue observed in the 3D system. GAF scaffoldsdA hydrogel made of gelatin, alginate, and fibrinogen was constructed using 3D bioprinting, to support glioma stem cells. A survival rate of

Natural compound-based scaffold to design in vitro disease systems

86.92% was attained by glioma stem cells in gelatin-alginate-fibrinogen (GAF) scaffolds, which proliferated rapidly after bioprinting [71]. The 3D glioma cell system displayed differentiation potential (glial fibrillary acidic protein and -tubulin III) as well as maintaining their inherent properties as CSCs over the in vitro culture time. When compared to a 2D monolayer system, a 3D printed glioma cell system demonstrated greater drug resistance to temozolomide at doses of 400e1600 g/ ml [71]. Chaicharoenaudomrung N. et al. [72] have elaborated and compared chitosanalginate scaffold, Chitosan-alginate-fibrinogen scaffolds, fibrin hydrogel scaffold, collagen-based hydrogel scaffold and GFR Matrigel hydrogel scaffold against the human umbilical cord mesenchymal stem cells, HeLa cells, dental pulp stem cells, prostate cell lines (PC3 and LNCaP) and human embryonic stem cells (HUES66), C57 respectively for the various medical applications.

6. Conclusion Out of various available model systems, 3D systems are widely used and studied system because of their biomimetic nature. This system gives the nearest true microenvironment that is needed for studying disease condition and drug development. The usage of scaffolds in a 3D system has shown to be a novel way of simulating the microenvironment of respective tissue. Natural compounds are the most favorable material in terms of the preparation of scaffolds. Though natural compound-based scaffold may lack mechanical strength and tunability, they are highly biocompatible, non-allergenic to the system and show higher cell proliferation in the 3D system. These issues can be easily overcome by doping natural scaffolds with another biomaterial either natural or artificial to form a hydrogel. These hydrogels show excellent biocompatibility along with higher mechanical strength and tunability. The use of such biomaterial-based scaffolds derived from various sources is the best option for researchers to study the diseases and find the treatment of disease.

7. Future perspective The utilization of a natural material-derived scaffold for the 3D system is the optimum alternative for researchers. The choice of a suitable scaffold is directly dependent upon the application of 3D scaffolding. Developing variety of scaffolds for diverse cell lines makes it a laborious and time-consuming process. It would be a good strategy to reduce these scaffolds to a select handful that can support different cell lines. It has been noted that in most situations, researchers were only able to maintain cell lines in 3D systems for a short period. Therefore, there is a need for intensive study to find a way to increase the vitality of cell lines in 3D cell culture scaffolds for a longer duration. Additionally, the

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lack of nourishment intake causes a necrotic cell mass formation inside the core of the scaffold. These necrotic cell masses interfere with health of neighboring cells, altering the study results. Hence, it is necessary to construct a 3D system in such a way that necrotic cell development within the scaffolds can be minimized. The primary goal of using a 3D cell culture method is to create an organ or tissue that performs similarly to its parent tissue. Therefore, it is also necessary to consider whether the 3D cell culture system can persist in the human body for the time needed to display the intended function. We hope that these thoughts will intrigue the readers mind and give direction to future research.

Acknowledgment The research in the SCK laboratory is supported by internal funding from the Department of Technology, Savitribai Phule Pune University, Pune, India.

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[38] Knopf-Marques H, Pravda M, Wolfova L, Velebny V, Schaaf P, Vrana NE, et al. Hyaluronic acid and its derivatives in coating and delivery systems: applications in tissue engineering, regenerative medicine and immunomodulation. Adv Healthc Mater November 1, 2016;5(22):2841e55. [39] Luo H, Cha R, Li J, Hao W, Zhang Y, Zhou F. Advances in tissue engineering of nanocellulose-based scaffolds: a review. Carbohydr Polym November 15, 2019;224:115144. [40] Fratzl P, Misof K, Zizak I, Rapp G, Amenitsch H, Bernstorff S. Fibrillar structure and mechanical properties of collagen. J Struct Biol January 1, 1998;122(1e2):119e22. [41] del Mercato LL, Passione LG, Izzo D, Rinaldi R, Sannino A, Gervaso F. Design and characterization of microcapsules-integrated collagen matrixes as multifunctional three-dimensional scaffolds for soft tissue engineering. J Mech Behav Biomed Mater September 1, 2016;62:209e21. [42] Donnaloja F, Jacchetti E, Soncini M, Raimondi MT. Natural and synthetic polymers for bone scaffolds optimization. Polymers April 14, 2020;12(4):905. [43] Fertala A, Shah MD, Hoffman RA, Arnold W v. Designing recombinant collagens for biomedical applications. Curr Tissue Eng 2016;5(2):73e84. [44] Ferreira AM, Gentile P, Chiono V, Ciardelli G. Collagen for bone tissue regeneration. Acta Biomater September 1, 2012;8(9):3191e200. [45] Zhang D, Wu X, Chen J, Lin K. The development of collagen based composite scaffolds for bone regeneration. Bioact Mater March 1, 2018;3(1):129e38. [46] Kutschka I, Chen IY, Kofidis T, Arai T, von Degenfeld G, Sheikh AY, et al. Collagen matrices enhance survival of transplanted cardiomyoblasts and contribute to functional improvement of ischemic rat hearts. Circulation July 4, 2006;114(Suppl. 1):167e73. [47] Shoseyov O, Posen Y, Grynspan F. Human collagen produced in plants. Bioengineered August 9, 2013;5(1):49e52. [48] Bello AB, Kim D, Kim D, Park H, Lee SH. Engineering and functionalization of gelatin biomaterials: from cell culture to medical applications. Tissue Eng B Rev April 16, 2020;26(2):164e80. [49] Echave MC, Burgo LS, Pedraz JL, Orive G. Gelatin as biomaterial for tissue engineering. Curr Pharmaceut Des May 12, 2017;23(24):3567e84. [50] Breen A, O’Brien T, Pandit A. Fibrin as a delivery system for therapeutic drugs and biomolecules. Tissue Eng B Rev April 9, 2009;15(2):201e14. [51] Shiu HT, Goss B, Lutton C, Crawford R, Xiao Y. formation of blood clot on biomaterial implants influences bone healing. Tissue Eng B Rev July 17, 2014;20(6):697e712. [52] Barsotti MC, Felice F, Balbarini A, di Stefano R. Fibrin as a scaffold for cardiac tissue engineering. Biotechnol Appl Biochem September 1, 2011;58(5):301e10. [53] Barsotti MC, Magera A, Armani C, Chiellini F, Felice F, Dinucci D, et al. Fibrin acts as biomimetic niche inducing both differentiation and stem cell marker expression of early human endothelial progenitor cells. Cell Prolif February 2011;44(1):33e48. [54] Noori A, Ashrafi SJ, Vaez-Ghaemi R, Hatamian-Zaremi A, Webster TJ. A review of fibrin and fibrin composites for bone tissue engineering. Int J Nanomed July 12, 2017;12:4937e61. [55] Chien KB, Shah RN. Novel soy protein scaffolds for tissue regeneration: material characterization and interaction with human mesenchymal stem cells. Acta Biomater February 1, 2012;8(2):694e703. [56] Ahn S, Chantre CO, Gannon AR, JU L, Campbell PH, Grevesse T, et al. Soy protein/cellulose nanofiber scaffolds mimicking skin extracellular matrix for enhanced wound healing. Adv Healthc Mater May 1, 2018;7(9):1701175. [57] Benton G, Arnaoutova I, George J, Kleinman HK, Koblinski J. Matrigel: from discovery and ECM mimicry to assays and models for cancer research. Adv Drug Deliv Rev December 15, 2014; 79e80:3e18. [58] El-Sherbiny IM, Yacoub MH. Hydrogel scaffolds for tissue engineering: progress and challenges. Glob Cardiol Sci Pract September 2013;2013(3):316. [59] van Vlierberghe S, Dubruel P, Schacht E. Biopolymer-based hydrogels as scaffolds for tissue engineering applications: a review. Biomacromolecules May 9, 2011;12(5):1387e408. [60] Shin H, Quinten Ruhe P, Mikos AG, Jansen JA. In vivo bone and soft tissue response to injectable, biodegradable oligo(poly(ethylene glycol) fumarate) hydrogels. Biomaterials August 1, 2003;24(19): 3201e11.

Natural compound-based scaffold to design in vitro disease systems

[61] Tibbitt MW, Anseth KS. Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnol Bioeng July 1, 2009;103(4):655e63. [62] Bassi G, Panseri S, Dozio SM, Sandri M, Campodoni E, Dapporto M, et al. Scaffold-based 3D cellular models mimicking the heterogeneity of osteosarcoma stem cell niche. Sci Rep December 18, 2020; 10(1):1e12. [63] Byram PK, Sunka KC, Barik A, Kaushal M, Dhara S, Chakravorty N. Biomimetic silk fibroin and xanthan gum blended hydrogels for connective tissue regeneration. Int J Biol Macromol December 15, 2020;165:874e82. [64] Vashisth P, Kar N, Gupta D, Bellare JR. Three dimensional quercetin-functionalized patterned scaffold: development, characterization, and in vitro assessment for neural tissue engineering. ACS Omega 2020;5(35):22325e34. [65] Mogha P, Srivastava A, Kumar S, Das S, Kureel S, Dwivedi A, et al. Hydrogel scaffold with substrate elasticity mimicking physiological-niche promotes proliferation of functional keratinocytes. RSC Adv April 1, 2019;9(18):10174e83. [66] Chen CY, Ke CJ, Yen KC, Hsieh HC, Sun JS, Lin FH. 3D porous calcium-alginate scaffolds cell culture system improved human osteoblast cell clusters for cell therapy. Theranostics 2015;5(6):643e55. [67] Rosli N, Loh HS, Chiang CL, Lim SS. Fabrication and characterization of chitosan-titanium oxide nanotubes scaffolds reinforced with tiger milk mushroom. IOP Conf Ser Mater Sci Eng October 1, 2021;1195(1):012021. [68] las Heras K, Santos-Vizcaino E, Garrido T, Borja Gutierrez F, Aguirre JJ, de la Caba K, et al. Soy protein and chitin sponge-like scaffolds: from natural by-products to cell delivery systems for biomedical applications. Green Chem June 8, 2020;22(11):3445e60. [69] Florczyk SJ, Kievit FM, Wang K, Erickson AE, Ellenbogen RG, Zhang M. 3D porous chitosanalginate scaffolds promote proliferation and enrichment of cancer stem-like cells. J Mater Chem B, Mater Biol Med October 10, 2016;4(38):6326. [70] Modulevsky DJ, Lefebvre C, Haase K, Al-Rekabi Z, Pelling AE. Apple derived cellulose scaffolds for 3D mammalian cell culture. PLoS One May 19, 2014;9(5):97835. [71] Dai X, Ma C, Lan Q, Xu T. 3D bioprinted glioma stem cells for brain tumor model and applications of drug susceptibility. Biofabrication October 11, 2016;8(4):045005. [72] Chaicharoenaudomrung N, Kunhorm P, Noisa P. Three-dimensional cell culture systems as an in vitro platform for cancer and stem cell modeling. World J Stem Cell December 12, 2019;11(12): 1065e83.

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CHAPTER 21

Natural compounds as pesticides, emerging trends, prospects, and challenges Puja Gupta1, Mohd Shahnawaz2, 3, Vasudeo Zambare4, 5, Naresh Kumar6 and Amanpreet Thakur6 1

Department of Life Sciences, RIMT University, Mandi Gobindgarh, Punjab, India; 2Department of Botany, Government College for Women, Jammu, Jammu and Kashmir, India; 3Biofuel’s Institute, School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, China; 4R&D Department, Om Biotechnologies, Nashik, Maharashtra, India; 5 University Teknologi Malaysia, Skudai, Johor, Malaysia; 6RIMT University, Mandi Gobindgarh, Punjab, India

1. Introduction The population rate is increasing day by day. Food production needs to be enhanced to cover the expanding requirements. Regrettably, the majority of the crops are destroyed each year in the field or during storage by pests [1,2]. The chemical compounds known as pesticides are employed to restrict or destroy any pests including insects, rodents, and fungi [3e5]. Synthetic pesticides are mostly preferred because of their quick effects to save the crops. Numerous issues are noticed due to the usage of these synthetic pesticides, e.g., pest resistance and uncleanness of the vital resources on our planet earth, e.g., water, air, and soil [6,7]. Most modern pesticides (synthetic) remain in the soil for decades. The insects develop resistance to these chemicals due to their extended perseverance in the biosphere [8,9]. Chlordecone (CLD), an organochlorine pesticide, was extensively applied throughout the French West Indies (FWI) during the period 1972 to 1993 [10]. It strongly binds to organic mattererich soils due to high hydrophobicity and steric barrier. Moreover, it is neither volatile nor biodegradable. Additionally, it has been shown that CLD retains in soil for a long molecule which leads to bioaccumulation in living forms. It was later banned in France in 1993. This chemical exists in the soils and waters of the FWI despite the prohibition on its use in the early 1990s [11]. Various neonicotinoid insecticides (such as imidacloprid and thiacloprid) were widely used from 1999 onward. But these were later on restricted in Europe in 2013 due to persistence in soil [12]. Neonicotinoids have been also prohibited in several US states out of concern for pollinators and bees [13,14]. The three primary neonicotinoids, namely, clothianidin, imidacloprid, and thiamethoxam, have been officially outlawed by the EU for all outdoor usage [15]. Several US states have restricted neonicotinoids out of concern for pollinators and bees (2013). There are several other examples where these synthetic pesticides have contaminated the biosphere and caused potential health hazards to New Horizons in Natural Compound Research ISBN 978-0-443-15232-0, https://doi.org/10.1016/B978-0-443-15232-0.00022-9

© 2023 Elsevier Inc. All rights reserved.

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consumers [16]. The bio-based natural pesticides known as biopesticides could be a better substitute for synthetic ones to overcome the toxic effects in the soil and water. Biopesticides are decomposable, environmentally friendly, and less toxic than synthetic pesticides. Such pesticides enhance the productivity of crops and sustainably overcome the food crisis while defending consumer health. Natural compounds used as natural pesticides could be of microbial, plant, or biochemical origin. Mostly microbes and plants produce these natural chemicals to defend against insects and pathogenic organisms. Biopesticides are described and controlled by US Environmental Protection Agency (EPA) [17]. It may be noted that all herbal biochemicals are not regulated as biopesticides. The registrant must demonstrate to the EPA that the substance (biopesticide) acts on pests with a nonpoisoning effect. The Biochemical Classification Committee formed by Biopesticide and Pollution Prevention Division must receive a petition. Herbal materials but with a poisonous effect are controlled as chemicals. For example, avermectin, pyrethrins, and spinosyns act as poison on the nervous system of insects. In this context, biopesticides that meet the required standards encourage sustainable production and become the key to get rid of pest problems when they are biodegradable, cheap, ecofriendly, precise, and less dangerous for human health. In this chapter, we intend to provide information on different sources that could serve as biopesticides. The mode of action of these biopesticides on pests will also be discussed. Furthermore, we will also cover the challenges faced by biopesticides along with future prospective.

2. Different sources for biopesticides 2.1 Microbial origin The microbial biopesticides comprise naturally occurring or genetically modified fungi, bacteria, protozoans, etc. 2.1.1 Entomopathogenic fungi The fungi that target insects are called entomopathogenic fungi. These fungi and their by-products are applied as biopesticides. These are vital natural regulators of insect populations and are used as mycoinsecticides to get rid of various pests (insects) in agriculture. These natural pesticides (entomopathogenic fungi) are either spread as spores or mycelium that later germinates. Entomopathogenic fungi have a great potential as control agents, as they constitute a group with over 750 species that, when dispersed in the environment, provoke fungal infections in insect populations. These fungi begin their infective process when spores are retained on the integument surface, where the formation of the germinative tube initiates; the fungi start to excrete enzymes such as proteases, chitinases, quitobiases, upases, and lipoxygenases. These enzymes degrade the insect’s cuticle and help in the process of penetration by mechanical pressure that is initiated by the apresorium, a specialized structure formed in the germinative tube. Once inside the insect, the

Natural compounds as pesticides, emerging trends, prospects, and challenges

fungi develop as hyphal bodies that disseminate through the haemocele and invade diverse muscle tissues, fatty bodies, Malpighian tubules, mitochondria, and hemocytes, leading to death of the insect 3e14 days after infection. Once the insect dies and many of the nutrients are exhausted, fungi start micellar growth and invade all the organs of the host. Finally, hyphae penetrate the cuticle from the interior of the insect and emerge at the surface, where they initiate spore formation under appropriate environmental conditions. Fungi spores rest on the integument (cuticle) of insects or wounds then form the germ tube and finally form hyphae by exploiting the nutrients available in the hemocoel to proliferate and attack diverse tissues and organelles leading to the death of the insect [18]. Beauveria bassiana is a widely studied biopesticide. It invades the insect hosts B. bassiana and produces certain toxins, e.g., beauvericin, bassianolide, oosporein, and tenellin. These toxins aid B. bassiana to capture and destroy the hosts [19]. Boverin produced by B. bassiana with certain minimal concentrations of trichlorophon helped in controlling outbreaks of codling moth (Cydia pomonella) [20]. Alike toxins were used to regulate the Colorado potato beetle (Leptinotarsa decemlineata) [21]. There are abundant examples where the blastospores of fungus are used to kill insects, e.g., Metarhizium anisopliae are documented to be lethal to Aedes aegypti and Aedes albopictus mosquitoes [22]. Hirsutella thompsonii is targeted against phytophagous mites. Verticillium lecanii is targeted against Aphid (homopteran) and scale insects. Nomuraea rileyi is targeted against Achaea janata and Spodoptera litura [23]. 2.1.2 Bacteria Bacterial biopesticides are one of the low-priced biopesticides. These are mostly specific to certain species of beetles, butterflies, flies, mosquitoes, and moths that need to be ingested properly to closely interact with the target pest. Bacterial pathogens used as bacterial pesticides generally belong to the genus Bacillus. It includes rod-shaped, sporeforming bacteria and occurs frequently in soils. Genus Bacillus can thrive the harsh conditions of temperatures, pH, oligotrophic nutrients, or scarcity of water. Differentiation of vegetative Bacillus subtilis into heat-resistant spores is initiated by the activation of the key transcription regulator Spo0A through the phosphorelay. Subsequent events depend on the cell compartmentespecific action of a series of RNA polymerase s factors. Analysis of genes in the Spo0A regulon has helped delineate the mechanisms of axial chromatin formation and asymmetric division. There have been considerable advances in our understanding of critical controls that act to regulate the phosphorelay and to activate the s factors. Sporulation is another important characteristic of the genus Bacillus that makes this genus appropriate for application as a biopesticide [24]. B. subtilis is a wellstudied example. The US Food and Drug Administration has given it the designation of “generally recognized as safe” (GRAS) and thus known as nonpathogenic. Bacillus thuringiensis (Bt) is a worldwide marketed bacterial insecticide known to control primarily caterpillars of the Lepidopterans, larvae of mosquitoes, and blackflies (Simuliidae). It is a

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soil bacterium that forms diverse types of crystal proteins (cry proteins) internally at the time of sporulation. The commercially available Bt preparations are powders that comprise a combination of dried spores and toxic crystals. The powder is sprayed on leaves and the insect (larvae) eats it. Insect-resistant crops like cotton, maize, rice, and potato have been produced using Bt insecticidal toxins. Paenibacillus popilliae is used to get rid of Japanese beetle [25]. Bacillus sphaericus is yet another bacillus that is used worldwide against Diptera [26]. It is also used to control Anopheles and Culex populations [27,28]. 2.1.3 Viral pesticides Viruses attack above 1100 insect species representing 13 varied orders [27]. Entomogenous viruses are categorized as inclusion viruses (IVs) and noninclusion viruses (NIVs) based on the formation and absence of inclusion bodies inside the host cells. The IVs are characterized by polyhedron viruses (PVs) and granulosis virus. The former produce polyhedral bodies and the latter produce granular bodies [29]. Most of the commercial viral biopesticides belong to the baculovirus family (Baculoviridae) [30]. Baculoviruses are rod shaped, have an outer protein coat, and carry DNA. These viruses are transmittable by mouth. Once these viruses are consumed by an insect, the protein covering is dissolved by the high pH inside the insect’s midgut and the viral components are released. These viral components adhere to the epithelial lining of the midgut, proliferate swiftly, and ultimately destroy the host. Baculoviruses are much more advantageous as a biopesticide owing to the fact these are proved to be harmless for vertebrates as they cannot replicate in vertebrates [31]. As per literature, more than 7000 baculoviruses are known to infect insects belonging to the class Lepidopterans, Hymenopterans, and Dipterans [32,33]. Elcar is the first wide-range baculovirus preparation. It was introduced by Sandoz Inc. in 1975. This baculoviral preparation attacks several species of genera Helicoverpa and Heliothis which attacks beans, maize, soybean, sorghum, and tomato [16,34,35]. Later on, the production of this viral preparation was discontinued in 1982 and it was registered again with another label GemStar [36]. Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) is another baculovirus that is used as a biopesticide [37]. It is used against the velvet bean caterpillar in soybean. This viral preparation is widely used in Brazil and much beneficial economically, ecologically, and socially. The viral preparation of baculovirus has certain drawbacks also, e.g., these are costlier than chemical agents, are specific for a particular host, and do not have a broad spectrum application. Moreover, the viral preparations are unstable under the UV so these are encapsulated to provide stability and protect baculovirus against harmful UV radiation [38e41]. Baculovirus, Cydia pomonella GV (CpGV), used for killing codling moth pests was efficaciously microencapsulated with Titanium dioxide. The resulted formulation was biologically viable with advanced UV protection [42]. Although there are no updates about the efficiency of this formulation on crops, the mentioned technology has been approved commercially yet. This might be owing to

Natural compounds as pesticides, emerging trends, prospects, and challenges

expensiveness, phytotoxicity, storage incompatibility, and blocking of spray filters, as observed with other particulate additives. Baculovirus products as a biopesticide gain a market of approximately 2.8 billion dollars a year [41]. The number of baculovirusbased registered pesticides is increasing slowly and steadily [43]. 2.1.4 Protozoa Protozoan attacks a varied range of insects wherein many of the hosts (insects) are killed during the infection process. Protozoans need a live host for proliferation. Few protozoans even need an intermediate host. The entomopathogenic protozoans that invade insects are generally referred to as microsporidians [19]. Microsporidians are largely host specific and slow in action but produce acute infections thereby disabling the host. Protozoans produce spores during the infectious sporulation stage. The insect ingests the spore, and it enters the midgut. The spore germinates in the midgut, increases its number, and invades more host cells. Microsporidian infection takes a few hours to weeks to proliferate within the host and cause disease. These microsporidians are used as microbial insecticides due to their capability to harm the hosts. Nosema bombycis, the natural pathogen of the silkworm, is the first reported microsporidium [11,44]. Pei and coworkers (2021) also studied the infection of N. bombycis in Spodoptera litura and Helicoverpa armigera. N. bombycis transmits vertically in S. litura and H. armigera concluding that it could be a potential biological pesticide for regulating the pest population [45]. P
ebrine is widespread and has a significant negative economic impact in countries that produce silk, including China [13]. Microsporidian Nosema pyrausta targets European corn borer besides several other insect species thus acting as a vital biocontrol [46e48]. Microsporidian Nosema locustae infects crickets and grasshoppers so applied with an insect-attractant bait. It has been registered, established commercially, and marketed under several labels [49]. This product is used for the extended accomplishment of pests due to its slow mode of action. Microsporidian Vairimorpha necatrix infects various caterpillar pests, e.g., corn earworm, cabbage looper, European corn borer, fall webworm, etc. It is much more infectious than other microsporidium species. The infective host (insects) does not survive beyond 6 days of infection [50]. 2.1.5 Nematodes Nematodes that infect insects or related arthropods are named entomogenous nematodes [51]. These nematodes are nonsegmented soft-bodied roundworms that could be strict or facultative parasites of insects. Species of Heterorhabditis heliothidis, Steinernema carpocapsae, S. feltiae, S. riobravis, and S. scapterisci are frequently used in insecticidal preparations. These pathogens are naturally prevalent in soil and search for their host by responding to chemical cues [25,52]. Various species of Steinernema, e.g., S. carpocapsae, S. feltiae, S. glaseri, S. kraussei, S. riobrave, and S. scapterisci, are pathogenic and attack various insects [53]. The genus Photorhabdus and Xenorhabdus bacteria are associated symbiotically with

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the genus Heterorhabditis and Steinernema, respectively [54]. The young nematode drives through four juvenile phases from offspring to the adulthood stage. The third juvenile phase (J3) is the infective juvenile stage (IJ). It is the only free-living stage of entomogenous nematodes. The nematode in this juvenile stage enters the hemocoel of the host insect through the spiracles, mouth, anus, or, in certain species, the intersegmental membranes of the cuticle [55]. The species of the genus Steinernema enter the body of the host (insect) through the mouth, anus, or spiracles (breathing pores). A similar route is followed by the juveniles of Heterorhabditis. These nematodes complete their life cycle inside the infected host (insect) in a moist and warm environment. The infected host does not survive beyond 24e48 h. The nematodes do not leave the host tissue even after the host dies and continue to feed, mature, and breed [56]. Heterorhabditidae and Steinernematidae families have been employed successfully in pest management programs as biological pesticides [57,58]. Entomopathogenic nematodes are nontoxic to people and highly host specific and can be easily managed. This feature makes them better fit into integrated pest management (IPM) [58]. The US EPA has exempted entomopathogenic nematodes from pesticide registration. There are no reports of problems due to insect resistance yet. 2.1.6 Advantages and disadvantages of microbial pesticides in general Advantages: The microbial pesticides are generally harmless and nonpathogenic to humans and other animals. Most microbial pesticides can be employed in conjunction with synthetic insecticides when necessary. Microbial pesticides can be applied on crops that are almost ready to be harvested due to no associated threats. They promote the growth of beneficial soil microflora, thereby promoting plant growth and hence boosting crop output [59]. Disadvantages: Microbial insecticides are highly species specific. Each treatment may only be able to control a proportion of the pests (insects) existing in that place. If there are additional pest species, they will not be killed and remain there to cause damage even after treatment. Microbial insecticides lose their potency when exposed to heat, desiccation, or UV radiation [1]. Few microbial pesticides require specific formulation and storage methods. The market for some of the microbial insecticides may also be constrained as they target particular pests only. 2.2 Biochemical pesticides Biochemical pesticides are natural chemicals with pest-repelling properties. These follow nontoxic mechanisms in contrast to the conventional pesticides that execute or restrict the pest. It includes pheromones (semiochemicals), natural insect growth regulators (IGRs), plant extracts/oils like eucalyptus oil, lemongrass oil, and rosemary oil. The aroma of plant extract charms the pests and they get trapped. Besides, biochemical pesticides inhibit mating like sex pheromones. Some plants also have specialized gland cells to produce essential oils. These oils act as insect repellants, e.g., peppermint oil controls

Natural compounds as pesticides, emerging trends, prospects, and challenges

household pests like ants and cockroaches; citrus oil controls flea, aphids, and mites; and lavender oil repels insects. EPA has constituted a distinct board to regulate whether a constituent fits into the standards for classification as a biochemical pesticide [60]. Carvone is found naturally in the essential oil of Carum carvi. It is a harmless biopesticide with the registered trademark TALENT. It prevents potatoes from sprouting at the time of storage and guards against bacterial rot without posing a threat to mammals. It enhances the shelf life of stored fruits and vegetables [61]. 2.2.1 Insect pheromones Insect pheromones are the chemicals secreted by insects to control other insects in IPM programs. These chemicals are not exactly pesticides (insecticides) as they do not destroy insects directly but indirectly affect their olfactory system [51]. Pheromones have abundant benefits in terms of pest control, e.g., cheap and easy to use, specific, and highly reactive [62,63]. Monitoring insect pests with pheromone traps the right timings for the application of insecticides [64,65]. Besides, these chemicals also prevent the rate of mating, thus decreasing insect progeny. Checkmate and Isomate are the few insect pheromones used against fruit pests [28]. Insect pheromones are extremely unique for a particular species. Aggregation pheromones are the type of insect pheromones produced mainly by social insects, e.g., ants, bees, and wood-invading beetles to communicate regarding good food sources [47]. Alarm pheromones are another type of insect pheromone produced and released by insects to inform other members of the same species of the forthcoming danger. Plant volatiles are also identified as an essential part of the pheromone system, particularly in the case of coleopteran species. The various combinations of pheromones and plant volatiles are used, and insects respond differently to pheromones alone and pheromones in combinations [25]. Besides pheromones and plant volatiles, semiochemicals are also used to regulate pests [66]. Semiochemicals are signaling chemicals released from one organism that induces a change in functional response interor intraspecies. Hence these are modes of interactions between plant and insect or insect to insect in IPM strategies [67]. 2.2.2 Plant-based extracts and essential oils Plant-based extracts and essential oils have become appealing substitutes for synthetic insecticides for the management of insect pests over the past few years. These natural insecticides comprise a range of bioactive chemicals that have wide effects on insects, e.g., they could behave as attractants, antifeedants, repellents, and respiratory inhibitors; prevent insects from recognizing their hosts’ plants; hinder oviposition; and prevent the formation of insect progeny by killing either eggs or larvae [68]. Neem and lemongrass oil are among such natural insecticides often used in herbal markets globally. A combination of neem oil and Beauveria bassiana proved to be effective against vegetable-sucking pests [55]. It is crucial to establish the dose of azadirachtin in neem oil to prevent the eradication of nontarget organisms [69].

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2.2.3 Insect growth regulators IGRs do not allow the insect to complete their developmental life cycle thereby executing them. Additionally, these compounds are many targets specific without causing much harm to nontarget organisms [70]. IGRs are categorized as chitin synthesis inhibitors (CSIs) and blockers that restrict insect hormones (such as ecdysteroids and juvenile hormone mimics) [71]. IGRs can manage a wide variety of insects, including fleas, cockroaches, and mosquitoes. They inhibit young insects from reproducing, hatching eggs, and molting. These IGRs if used in combination with other pesticides have the potential to destroy adult insects [72]. 2.3 Plant origin Plants belonging to family Asteraceae, Convolvulaceae, Euphorbiaceae, Labiatae, Lauraceae, Fabaceae, Meliaceae, Piperaceae, and Solanaceae are known to have insecticidal properties [73]. More than 2000 plants showed insecticidal property against different insects [74]. These biopesticides of plant origin could be in the form of powder, solid, or liquid extracts. Extracts or derivatives of several plant families show strong insecticidal activity. The secondary metabolites of plants are the reason for the strong insecticidal activity. The therapeutic uses and pharmacological effects of the secondary metabolites from the plants have long been recognized [75e77]. These chemicals derived from plants may be constitutive or inducible which are activated upon reciprocation to insect activity. The commonly used natural pesticides of plant origin are Anabasine, Azadirachtin, Nicotine, Neem, Ryania, and Sabadilla. Few of these are discussed in Section 3.3. Secondary metabolites are also modified postextraction to improve their insecticidal properties. Alkaloids are crucial secondary metabolites in plants that function as an insecticide. More than 10,000 alkaloids have been discovered in over 30 plants. Hexane extracts of the aerial parts of Kunzea ambigua and K. baxteri (Myrtaceae) show insecticidal activity that is comparable to natural pyrethrum extract [78]. The roots of Aglaia duperreana, Aglaia oligophylla, and pyrrolizidine alkaloids from Heliotropium megalanthum has been studied for the insecticidal property on Spodoptera littoralis [79]. The alkaloid N-isobutyl-2,6,8decatrienamide (spilanthol) isolated from Spilanthes acmella is used to get rid of cabbage moth (Plutella xylostella) [73,80].

3. Mode of action of different biopesticides In this section, the mode of action of different types of biopesticides [81] will be discussed in the text and tabulated form in Table 21.1. 3.1 Biopesticides of microbial origin (product based) Microbial biopesticides usually act at the translation level, thereby inhibiting protein synthesis, e.g., blasticidin prevents peptide bond formation and inhibits the termination step

Table 21.1 Different types of biopesticides based on their source and mode of action. Sr. No.

1

Types of biopesticide

Biopesticides of microbial origin (product based)

Entomopathogenic fungi

Mode of action

Reference

Mycoinsecticides Beauveria bassiana

Targets insects. Control the population of Cydia pomonella (codling moth), Leptinotarsa decemlineata (Colorado potato beetle). It is lethal to Aedes aegypti and Aedes albopictus mosquitoes. It works against phytophagous mites. It targets aphid (homopteran) and scale insects. It targets achaea and Spodoptera litura. It is known to control primarily caterpillars of the lepidopterans, larvae of mosquitoes, and blackflies (simuliid). It is used to get rid of Japanese beetle. It is used against Diptera and controls Anopheles and Culex populations.

[82,83] [19e21]

Metarhizium anisopliae

Hirsutella thompsonii Verticillium lecanii

Nomuraea rileyi Bacterial biopesticides

Bacillus thuringiensis (Bt)

Paenibacillus popilliae Bacillus sphaericus

[37]

[19,84] [85]

[23] [24]

[25] [26e28]

Continued

Natural compounds as pesticides, emerging trends, prospects, and challenges

Name

399

400

Sr. No.

Types of biopesticide

Viral pesticides

Entomopathogenic protozoans

Name

Mode of action

Reference

Baculovirus and GemStarÔ

It attacks several species of genera Helicoverpa and Heliothis which attacks beans, maize, soybean, sorghum, and tomato. It is used against the velvet bean caterpillar in soybean. It is used for killing codling moth pests. A potential biological pesticide for regulating the pest population (Spodoptera litura and Helicoverpa armigera). It targets the corn borer.

[16,34 e36]

Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) Baculoviral, Cydia pomonella GV (CpGV) Nosema bombycis

Microsporidian Nosema pyrausta Microsporidian Nosema locustae

Microsporidian Vairimorpha necatrix

Infects crickets and grasshoppers. Also, applied with insectattractant bait. It infects various caterpillar pests, e.g., corn earworm, cabbage looper, European corn borer, fall webworm, etc.

[86]

[42] [45]

[46e48] [49]

[87]

New Horizons in Natural Compound Research

Table 21.1 Different types of biopesticides based on their source and mode of action.dcont’d

Entomogenous nematodes

Plant origin biopesticide

Alkaloid N-isobutyl2,6,8-decatrienamide (spilanthol) isolated from Spilanthes acmella Hexane extracts of the aerial parts of Kunzea ambigua and K. baxteri (Myrtaceae) The roots of Aglaia duperreana, Aglaia oligophylla, and pyrrolizidine alkaloids from Heliotropium megalanthum

[53]

These species have been employed successfully in pest management programs. It is used to get rid of cabbage moth (Plutella xylostella).

[57,58]

The extract has an insecticidal activity that is comparable to natural pyrethrum extract. It kills Spodoptera littoralis.

[78]

[73,80]

[79]

Continued

Natural compounds as pesticides, emerging trends, prospects, and challenges

2

Steinernema carpocapsae, Steinernema feltiae, Steinernema glaseri, Steinernema kraussei, Steinernema riobrave, and Steinernema scapterisci Heterorhabditidae and Steinernematidae

It is much more infectious than other microsporidium species. The infective host (insects) does not survive beyond 6 days of infection. It attacks various insects and controls their populations.

401

402

Sr. No.

3.

Types of biopesticide

Biochemical pesticides

Insect pheromones

Plant-based extracts and essential oils

Name

Mode of action

Reference

Checkmate and Isomate

These are used against fruit pests. Used in managing pests.

[15]

Semiochemicals (signaling chemicals) Neem and lemongrass oil

A combination of neem oil and Beauveria bassiana Insect growth regulators (IGRs)

IGRs

IGRs along with other pesticides Chitin synthesis inhibitors (CSIs) and blockers

These are natural insecticides often used in herbal markets globally. This combination proved to be effective against vegetable-sucking pests. IGRs can manage a wide variety of insects, including fleas, cockroaches, and mosquitoes. This combination has the potential to destroy adult insects. They restrict insect hormones (such as ecdysteroids and juvenile hormone mimics) and restrict the growth of the pests.

[81] [55]

[55]

[88]

[72]

[71]

New Horizons in Natural Compound Research

Table 21.1 Different types of biopesticides based on their source and mode of action.dcont’d

Natural compounds as pesticides, emerging trends, prospects, and challenges

of translation [89,90]. Kasugamycin inhibits translation at the step of the initiation complex [91]. Streptomycin and mildiomycin block the activity of peptidyl transferase [8,92e94]. Polyoxins inhibit chitin synthase activity [95,96]. Validamycin inhibits trehalase and prevents the generation of glucose [97,98]. Few insecticides, e.g., avermectins and emamectin, release gamma-aminobutyric acid (GABA) blocking electrical nerve conduction [8,94]. The blockage of the nervous system by GABA has been discussed elsewhere. 3.1.1 Important microbial biopesticides and their mode of action 3.1.1.1 Bacillus thuringiensis It produces crystalline proteins. It is encoded by the cry genes. The cry proteins are represented by a single family with 50 subgroups [99]. Sporulation leads to the formation of proteinaceous protoxin crystals. Proteases are produced by the host and the ingested crystals break inside the stomach by the conversion of protoxin into an active toxin known as endotoxin. Bt endotoxins are inactive in vertebrates, especially in humans as they require an alkaline environment to be functional [100]. There are abundant strains of Bt with a variety of Cry proteins. Approximately 60 different Cry proteins have been identified [101]. 3.1.1.2 Streptomyces (avermectins) Avermectins are macrocyclic lactones that originated from the actinomycetes Streptomyces. These biopesticides attack glutamate-gated channels which occur in invertebrate nerve and muscle cells. This cause enhanced porousness of chloride ions to the cell membrane leading to nerve hyperexcitation, causing tremors, uncoordinated movement, paralysis, and eventually death [102,103]. Avermectins play depolarizing role rather than hyperpolarizing the glutamate-gated chloride channel [104,105]. However, both lead to the deactivation of the channel due to the changes in the chloride levels. These biopesticides are effective against the endogenous parasites of domestic animals [106,107]. 3.2 Biochemical pesticides Pheromones are among the biochemical pesticides. Pheromones are absorbed by the antennae of the perceiving insect and then diffuse into the sensilla through small holes in the cuticle [108]. Once inside, these are transferred through the hydrophilic sensillum to the chemosensory membranes by pheromone-binding proteins (PBPs). A particular receptor protein then binds with the pheromone or pheromoneePBP complex to convert the chemical signal into an enhanced electric signal via a second messenger system linked to neural circuitry [50]. 3.3 Plant bioinsecticides These biopesticides are of plant origin. Plants release many compounds to deal with pathogenic microorganisms. These compounds are alkaloids, nitrogenated compounds,

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phenolics, steroids, and terpenoids. These function by either interrupting respiratory enzymes, inhibiting IGRs, or binding to sodium or calcium channels [109]. 3.3.1 Important plant biopesticides and their mode of action The commercial biopesticides that have been widely used are discussed in the following sections in detail along with their mode of action. 3.3.1.1 Pyrethrin Pyrethrin occurs naturally in chrysanthemum flowers. It is regarded as a biopesticide when it is not coupled with any synthetic adjuvants. It is among the ancient domestic insecticide that is still applied. It provides quick results. Pyrethroids act on sodium channels causing a delay in the closing of sodium channels which leads to excessive neuroexcitation, eventually resulting in uncontrolled coordinated movement, paralysis, and death [102,110e112]. Pyrethrin has an insecticidal effect against a variety of insects and mites, including flies, fleas, beetles, and spider mites. It does this by interfering with the sodium and potassium ion exchange mechanism [103]. Pyrethrin needs to be frequently applied as it degrades quickly under sunlight and moisture. Besides, it exhibits low mammalian toxicity [80]. These cause toxicity in mammals when inhaled due to direct entry into the bloodstream. High doses could lead to nervous disturbances. Crude pyrethrum dust can irritate the skin or trigger allergic responses when inhaled repeatedly. Products with refined pyrethrin do not include the allergens that trigger severe reactions [103]. 3.3.1.2 Ryanodine Ryanodine is a lethal alkaloid isolated from Ryania speciosa Vahl. It is known to have insecticidal activity for a long. It stimulates the calcium channels in the sarcoplasmic reticulum of the skeletal muscle. The active calcium channels of the protein filaments release excessive calcium ions, thereby causing uncontrolled contraction of the insect’s skeletal muscles to paralyze [113,114]. Ryanodine is much effective against caterpillars (codling moth, corn earworm). However, it successfully eliminates a broad range of insects and mites that also comprises aphids, lace bugs, potato beetle, and squash bug. Avermectins (derived from Streptomyces) affect the GABA-gated chloride channel in the nervous system [67]. The registration for the usage of ryanodine as a botanical insecticide was terminated due to low selectivity between insect and mammalian ryanodine receptors [102]. 3.3.1.3 Altriset Altriset (anthranilic Diamide compounds) are now used actively as these have the same insecticidal mechanism as the natural product ryanodine but the former has clear selectivity for insect ryanodine receptors [103]. Calteryx, the active ingredient in Altriset, is

Natural compounds as pesticides, emerging trends, prospects, and challenges

a synthetic compound that affects the ryanodine receptors in the insect muscle fiber. However, agricultural pests have developed resistance to diamide, and this has emerged as a serious issue now [115]. 3.3.1.4 Nicotine, neonicotinoids, and spinosyns Nicotine, neonicotinoids, and spinosyns imitate acetylcholine and activate acetylcholine receptors. An influx of sodium ions occurs, thereby generating the action potentials. Normally (under normal physiological conditions), the enzyme acetylcholinesterase hydrolyzes the neurotransmitter to terminate the synaptic action of acetylcholine. Imitated acetylcholine is not hydrolyzed by AChE, so due to excessive stimulation of cholinergic synapses, convulsion and paralysis occur that eventually leading to the death of the insect [116]. Nicotine kills soft-bodied insects and mites, including aphids, thrips, leafhoppers, and spider mites successfully. However, many caterpillars are resistant to nicotine. Neonicotinoids that are used in agriculture are imidacloprid, acetamiprid, and thiamethoxam. Both nicotine and neonicotinoids are much more toxic to invertebrates, like insects than to mammals. These compounds have far more affinity to the insect nAChR than vertebrates in contrast to the original nicotine. Neonicotinoid insecticides are much more popular as biopesticides due to their water solubility. They can be sprayed on the soil and absorbed by plants. Neonicotinoids when ingested by the insect lead to death shortly. 3.3.1.5 Azadirachtin Azadirachta indica (neem tree) is a source of Azadirachtin, a contact poison. It inhibits the central nervous system of the insect through inhibition of synaptic transmission [117]. It has an inhibitory effect on a broad range of insects, e.g., aphids, caterpillars, mealybugs, and stored grain pests [118]. As a repellent, neem does not allow insects to feed. Insects stop feeding either due to the existence of restrictive taste factors or after ingesting the food due to the functional effects of the restrictive substance [70]. As an antifeedant, azadirachtin inhibits peristalsis and stimulation of deterrent cells, prevents sugar cells from acting as an antifeedant, and decreases enzyme production [119]. As a growth regulator, neem interferes with chitin synthesis, thereby affecting normal development [71]. The effects of neem vary for different insect species. Azadirachtin shows the growth regulatory effect on effects on larval insects. It reduces the level of ecdysteroid and juvenile hormones in the insect that in turn reduces the production of the other vital hormones which are required for the molting, growth, and development of the insect [71,120]. Azadirachtin is known to inhibit or block cell division [121,122].

4. Challenges These bio-based pesticides face few challenges. Most of the biopesticides are not quick in action and act more like insect inhibitors. These get damaged by UV light very rapidly

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and thus have short residual action [7,69,112]. It is worth mentioning that plant pesticides are not always less toxic than synthetic ones e.g., nicotine is of plant origin (tobacco leaves) but it is more injurious than most synthetic pesticides. Although it is agreeable that few natural pesticides are much safe and more ecofriendly than synthetic pesticides. Many times, commercially available biopesticides are generally found contaminated and have low microbial count than mentioned [108,123], hence show poor field performance. Therefore, all the steps need to be monitored to maintain high standards so that product quality is not compromised. The end users, i.e., farmers, are reluctant to use biopesticides in place of chemical pesticides due to their high cost, delayed results, inconsistent performance on fields, and shorter shelf life. These challenges and constraints demand advanced strategies and technological revolution that rule out the shortcomings of commercial biopesticides. Another hurdle related to the promotion of biopesticides is the lack of documentation and proper profiling that reflects the weakness of the supporting policy network. Lack of profile not only piles up the cost of registration but also slows down the process. There is also a need to set up a regulatory framework for biopesticides that could be accepted globally. A common regulatory system will promote the transportation of these products across national boundaries and makes the registration process simpler [108]. The pest develops resistance to the biopesticides when they are used repeatedly. Therefore, rather than using the same biopesticides, emphasis should also be put on discovering and creating more effective microbial strains and their products [124]. Understanding the mechanisms operated in insects for detecting the presence of semiochemicals by the receptor proteins and exploring and studying the genomes of pests and their natural enemies at the molecular level could be helpful to make out how pests gain resistance [23]. It might result in the creation of next-generation biopesticides that are more effective [30]. Formulation of microbial consortia and their products, e.g., essential oils, extracts, secondary metabolites with much longer shelf life, ecofriendly, and better efficacy to control pests, would serve as next-generation biopesticides [125]. The use of carriers and adjuvants provides stability to the product with longer shelf life, protection from harsh environmental conditions making transportation, and application feasible and also improving efficiency [108]. Formulated biopesticides BioShield that constitute bacterial cells of Serratia entomophila besides Zeolite and biopolymers have a shelf life of more than 6 months [59]. All the steps from formation of biopesticides to delivery, e.g., mass production of microbial culture, formulation of product, and packaging and application to fields, should be done with care to maintain the quality of the product.

5. Prospects Biopesticides are considered a safe and effective substitute for chemical pest control. It is need of the hour that both the public and private sectors must work in collaboration for

Natural compounds as pesticides, emerging trends, prospects, and challenges

the growth of this alternative ecofriendly option. The research-oriented universities need to be exploited to encourage research on other possible products as a source of bioinsecticides. Financial support needs to be provided by different organizations to build the necessary infrastructure and conduct the necessary research and development of new biopesticides. On-farm training must be organized by the Governmental, NGO, and educational institutes wherein the end users, i.e., farmers, must be able to demonstrate the profits of using biopesticides effectively. Various programs should be initiated that boost growing of plants with pesticide potentials on large scale. The profile of biopesticides needs to be raised among policymakers and the general public. This can be done by spreading awareness and understanding of their mode of action, effect, and regulatory issues [108]. This will aid in marketing the usage of biopesticides and expedite the registration procedure. Farmers should be given access to biopesticides either for free with the purchase of agricultural products or as a Government subsidy in order to increase their usage of them. For the creation of biopesticides, it is essential to have a thorough grasp of the various living microorganisms and botanical materials that are used. This could be improved by appropriate research and development at the university and corporate levels. The effectiveness of biopesticides could be improved by using molecular techniques, such as genetic engineering [126]. Biofungicide, Serenade, produced from B. subtilis, is available commercially. It regulates many fungal infections [127]. The finding of the most efficient compound or combination of compounds associated with the pesticide activity of each relevant species may result through bioassay-guided fractionation or the bioassays assessment of numerous plant extracts and their fractions. The isolated molecule might serve as a model for creating derivative pesticides, which might be more potent. It is important to note that occasionally the chemical does not exhibit any activity when it is isolated. Only a few plant species exhibit activity as a result of the synergistic effects of chemical mixtures. Although biopesticides have a target-specific quality that makes them safer and more environmentally friendly, their demand and use in agriculture would increase if they had a wider activity spectrum against more pests from a commercial point of view for selling.

6. Conclusions Synthetic pesticides have many negative effects on the environment and human health. So, people have shifted toward biopesticides these days. The market for biopesticides is growing steadily due to increased yield, quality, biodegradable, and ecofriendly characteristics in comparison to synthetic pesticides. Biopesticides can be developed from different sources, e.g., plants, microbes, and biochemicals. Different biopesticides have varied modes of action. Researchers all around the globe are trying to develop new and efficient biopesticides. A partnership between enterprises and research institutes

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could help make much development in the field of biopesticides, thereby substituting chemical pesticides entirely. The end users, i.e., farmers, need to be made aware of the usage and benefits of biopesticides [128e143].

Acknowledgments PG is thankful to the Department of Science and Technology (DST) for the award of Women Scientist-A (SR/WOS-A/LS-136/2017) and RIMT University, Mandi Govindgarh, Punjab, for financial support.

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[65] Peng C-L, Gu P, Li J, Chen Q-Y, Feng C-H, Luo H-H, et al. Identification and field bioassay of the sex pheromone of trichophysetis cretacea (Lepidoptera: crambidae). J Econ Entomol 2012;105: 1566e72. [66] Mweresa CK, Mukabana WR, van Loon JJA, Dicke M, Takken W. Use of semiochemicals for surveillance and control of hematophagous insects. Chemoecology 2020;30:277e86. [67] Lahm GP, Cordova D, Barry JD, Pahutski TF, Smith BK, Long JK, et al. 4-Azolylphenyl isoxazoline insecticides acting at the GABA gated chloride channel. Bioorg Med Chem Lett 2013;23:3001e6. [68] Sukh D, Opender K. Insecticides of natural origin. Routledge; 2017. [69] Khater HF. Prospects of botanical biopesticides in insect pest management. Pharmacologia 2012;3: 641e56. [70] Lokanadhan S, Muthukrishnan P, Jeyaraman S. Neem products and their agricultural applications. J Biopestic 2012;5:72e6. [71] Sarwar M. Experimental induction of insect growth regulators in controls of insect vectors as well as crops and stored products pests. Sci Arena Publ Spec J Agric Sci 2020;6:32e41. Available onlinewww.sciarena.com. [72] Hazarika LK, Bhuyan M, Hazarika BN. Insect pests of tea and their management. Annu Rev Entomol 2009;54:267e84. [73] Fowsiya J, Madhumitha G. A review of bioinsecticidal activity and mode of action of plant derived alkaloids. Res J Pharm Technol 2020;13:963. [74] Tembo Y, Mkindi AG, Mkenda PA, Mpumi N, Mwanauta R, Stevenson PC, et al. Pesticidal plant extracts improve yield and reduce insect pests on legume crops without harming beneficial arthropods. Front Plant Sci 2018;9:1425. [75] Croteau R, Kutchan TM, Lewis NG. Natural products (Secondary metabolites). In: Biochemistry and molecular biology of plants; 2000. p. 155e67. [76] Shakya. Medicinal plants: future source of new drugs. Int J Herb Med 2016;4:59e64. [77] Wink M. Modes of action of herbal medicines and plant secondary metabolites. Medicines 2015;2: 251e86. [78] Khambay BPS, Beddie DG, Simmonds MSJ. An insecticidal mixture of tetramethylcyclohexenedione isomers from Kunzea ambigua and Kunzea baxterii. Phytochemistry 2002;59:69e71. [79] Jeyasankar A, Raja N, Ignacimuthu S. Insecticidal compound isolated from syzygium lineare wall. (Myrtaceae) against Spodoptera litura (Lepidoptera: noctuidae). Saudi J Biol Sci 2011;18:329e32. [80] Sharma A, Kumar V, Rattan RS, Kumar N, Singh B. Insecticidal toxicity of spilanthol from Spilanthes acmella. Am J Plant Sci 2012;3:1568e72. [81] Abd El-Azi SE. Control strategies of stored product pests. J Entomol 2011;8:101e22. [82] Diaz M, Rossini C. Bioactive natural products from sapindaceae deterrent and toxic metabolites against insects. In: Insecticidesdpest engineering. InTech; 2012. p. 287e308. [83] Díaz MP, Macías AF, Navarro SR, De La Torre M. Mechanism of action of entomopathogenic fungi. Interciencia 2006;31. [84] Peng CYS, Zhou X, Kaya HK. Virulence and site of infection of the fungus, Hirsutella thompsonii, to the honey bee ectoparasitic mite, Varroa destructor. J Invertebr Pathol 2002;81:185e95. [85] Upadhyay V, Rai D, Rana M, Mehra P, Pandey AK. Verticillium lecani (Zimm.): a potential entomopathogenic fungus. Int J Agric Environ Biotechnol 2014;7:719. [86] Brito AF de, Braconi CT, Weidmann M, Dilcher M, Alves JMP, Gruber A, et al. The pangenome of the Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV). Genome Biol Evol 2016;8: 94e108. [87] Ansari MS, Ahmad S, Ahmad N, Ahmad T, Hasan F. Microbial insecticides: food security and human health. In: Management of microbial resources in the environment. Dordrecht: Springer Netherlands; 2013. p. 341e60. [88] Masih SC, Ahmad BR. Insect growth regulators for insect pest control. Int J Curr Microbiol Appl Sci 2019;8:208e18. [89] Parker KM, Barrag
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CHAPTER 22

Natural compounds as insecticidesda novel understanding Gulzar A. Rather1, Madhu Raina2 and Sakshi Saini2 1

Agricultural Research Organization, Rishon, Israel; 2Department of Botany, University of Jammu, Jammu, Jammu and Kashmir, India

1. Introduction Pharmacology, the branch of medicine dealing with the origin, composition, and possible effects of drugs, generally takes into account the therapeutic use of drugs in different processes of living matter. Today advanced research has geared up in the area of medical science. Notwithstanding these significant advancements, many diseases including fatal cancer, diabetes mellitus, chronic kidney diseases, acute myocardial infarction, coronary thrombosis etc. remain reasonably priced and without efficient and secure therapy. Moreover, confrontation and detrimental side effects of the medicines used in the clinic [1,2] have become burning issues leading to an unremitting hunt for novel compounds with advanced and effective mode of action. Consequently, development of advanced medication from natural resources remains on cards [3e9]. In fact, more than 50% of such natural product-based new drugs have been instigated for approximately 33 years in the market from 1981 [10]. Of these drugs, claim for natural antimicrobial agents and anticancer drugs is awfully a vigorous research aspect [11e13] since cancer and infectious diseases are a noteworthy root of transience across the globe [14]. Plants act as the repository for myriad compounds with potential effects especially on the health of living matter. These natural compounds demonstrate a great assortment of chemical structures owing to the extensive and discerning process of species [15]. They represent the active pharmaceutical agent, exhibit greater scientific and industrial values [7,9,16] and have constantly used in traditional medicine [17e19], thus adding to improve the human health [20]. Owing to their positive outcome on health system, demand of these plant products and their derived molecules is increasing at an alarming rate. The attributes paraded by natural compounds relentlessly persuade methodical exploration in facets that escorts momentous advances in detecting novel compounds, appraisal of the genetic activity flaunted, perception of how a biological effect is caused and in all cases with advantageous upshots for humanity. Speedy progress in the drug discovery have opened new window to determine the pharmacological use of plant products vis-a-vis the benefits of natural compounds.

New Horizons in Natural Compound Research ISBN 978-0-443-15232-0, https://doi.org/10.1016/B978-0-443-15232-0.00002-3

© 2023 Elsevier Inc. All rights reserved.

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In recent times, plants have increasingly found a particular position in curing different diseases including the ones related to cardiac dysfunctioning, glucose metabolism, chronic kidney disease, hypertension etc. Indigenous knowledge is endowed with unique traditional information existing within locals and developed around the specific conditions by the populace native to a particular geographic area. Presently, people of different communities in developing countries depend on plants for traditional medicine to cure different diseases without any prior knowledge of the effective ingredient present in it. Although a lot of progress has been made in therapeutics, there are still some ailments that either remain without practical treatment or are drastically susceptible to the side effect of drugs. Thus, a small endeavor to perk up human health is a new path to follow. In this connection, biomolecules obtained naturally from plants embody an infinite reserve that can be explored. Irrefutably, different medicinal properties of plants are yet to be unraveled. Traditionally, natural products have played a pivotal role in therapeutic areas, chiefly infectious diseases [21,22]. These compounds offer unique characteristics in evaluating conventional synthetic molecules, which bestow both recompenses as well as confronts for the drug discovery process. They are privileged structures since they are typified by boundless staging diversity, biological activities, and morphological convolutions. Characterized by higher molecular mass, presence of abundant oxygen atoms, less nitrogen and Group VIIA elements in natural products are likely to impart superior molecular stiffness to them [21,23e26]. Indubitably these superlative compounds contribute in the realistic design of novel drugs by emerging as new chemical outfits with remedial latency [6,9]. These structurally optimized natural compounds hand out several biological functions typically interaction with other organisms thereby explaining their extreme significance for contagious ailments [27]. Also, being enriched with bioactive compounds these occupy a wider chemical space and provide important imminent for their worth and security [28]. Furthermore, such compounds find a wide application in cosmetic products as one of the active ingredients [29,30] and in the food industry owing to their valuable stabilizing properties [31,32]. This book chapter is anticipated to significantly add to our knowledge on their insecticidal properties. The substances that function as pest-controlling agents are known as insecticides or pesticides. People have extracted compounds from plants to use them as natural insecticides for thousands of years. In plant kingdom, different plant species offer diverse chemical compound as secondary metabolites with differential activities. Family Asteraceae, for instance, is known to encompass different genera of perennial grasses like Pyrethrum, Tanacetum with natural insecticidal properties. These plants are known to contain esters of cyclic ketalcohols, i.e., pyrethrins. Similarly, Chrysanthemum is considered to be a rich source of 2, 2-dimethyl-cyclopropanecarboxylic acids [33e36]. While esters of Chrysanthemum are designated as pyrethrine I (PYI), those of pyrethrinic acid are referred to as pyrethrine II (PYII) [37e41] (Fig. 22.1). In general pyrethrine composition varies from plant to plant. Based on the variety, it can hold as many as six esters in different fractions. These are cinerin I and II, pyrethrin I and II, jasmolin I and II. Of these, pyrethrine

Natural compounds as insecticidesda novel understanding

Figure 22.1 Some of the esters of potent insecticide pyrethrine with chemical structures along with 3D structure produced by using online ChemSpider software. (A) Pyrethrin I, pyrethrin II; (B) Cinerin I, cinerin II and (C) jasmolin I, jasmolin II.

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I, II forms the basic component for insecticides in plant reproductive structures and manage insect pest. This compound is non-volatile at normal temperature, virtually hydrophobic but gets mixed well in many organic solvents like benzene, petroleum ether, acetone etc. It is pertinent to mention here that under aerobic conditions, pyrethrins are more prone to oxidative degradation than the other two (cinerins and jasmolines). Thus, utmost care is to be taken while selecting techniques to segregate, amass and use it for the purpose of insecticides [42]. Natural insecticides are generally harvested via two steps; maceration wherein plant tissue is first soaked and then separated in an active constituent, and distillation during which evaporation and condensation processes follow to yield the specific active compounds. Extraction of pyrethrine is usually done either in hexane or petroleum ether, or by mixing both. The precedence in selecting these solvents is related to their low boiling point so that pyrethrine can be obtained in almost pure form at a lower temperature. Literature puts on record some novel methods to isolate pyrethrine from plant materials important for the production of environmentally safe natural insecticides. The advantage of these natural insecticides is their short persistence in the environment owing to their rapid degradation. Similarly, it has also been shown that extracts from Dalmatian chamomile have quite efficient insecticidal property against aphids responsible for pest disease in cereal crops. Such extracts constitute an eco-friendly natural insecticide as well as biologically active additives to commercial insecticidal preparations. Despite having tremendous applications in therapeutics, some natural compounds act severely and have proven to be with lethal effects on various non-target organisms, including humans. Nicotine, for example, one among the most widely used insecticides several years ago, was completely banned in Washington State after 2008 due to its high mammalian and aquatic vertebrate toxicity. Likewise, Rotenone lost its registration in agricultural practices in Washington since 2009 and Oregon since 2010 due to its toxicity to fish population. Presently, there are only three rotenone products registered for use in the Pacific Northwest, though they have fish toxicity [43]. Similarly, in Indian agriculture natural insecticides are being used for more than a century to trim down deadly diseases caused by different pests and vectors [44]. Compared to synthetic insecticides, the naturally obtained compounds used for pest control are more advantageous due to following reasons: a. Low toxicity: The natural pesticides contribute minimal or no health issue and ecological pollution since they exhibit comparatively low mammalian toxicity. b. Low pest resistance: If the products are used in their native form, there is no use to develop pest resistance. However, only synthetic pyrethroids have been reported as effective against pests. c. No side-effects: Natural compounds as pesticides are least harmful toward non-targeting organisms. d. No impact on plant life cycle: NPs have undesirable effect on plant growth as well as seed viability.

Natural compounds as insecticidesda novel understanding

e. Easy availability: Compared to the synthetic insecticides, the naturally obtained ones are cheap and accessible. Various plants with natural ability to act as insecticides like neem, bel, senwar, pyrethrum, tobacco, karanj, mahua, sweet flag, etc. are not only the potential pesticides but have been actively used under Integrated Pest management wherein different crops are not only produced but protected by combining different management strategies to practice healthy cropping [44].

2. Natural plant compounds as insecticides In the present times, natural compounds have obtained a remarkable position as insecticides in different fields like agriculture, forestry, medicine, aquaculture, food industry, processing, transportation, and storage of wood and other biological products. Insecticides are important since they provide protection to crops by restricting growth and multiplication of disease causing pathogens and thus preserve the food material [44]. 2.1 Rotenone Rotenone, a natural compound derived from extracts of tropical legumes Derris and Lonchocarpus, acts as an insecticide. The main active principle, the isoflavonoid rotenone is comparatively less toxic to mammals due to poor absorption and rapid metabolism, but is highly toxic to insects and fish, due to its rapid uptake and inhibition of respiratory electron transport at site. Rotenone has been available widely as a plant extract due to its reasonable cost and is used as a dust on horticultural and ornamental crops. However, as mentioned earlier, its use has been phased out recently in different countries including US and Canada during customary reassessment [45]. 2.2 Sabadilla Sabadilla, an insecticide obtained from seed of neotropical lily Schoenocaulon officinale which contains veratridine alkaloids, demonstrates a neurotoxic mechanism of action. The seed extract of the plant has low mammalian toxicity and is a useful insecticide against a number of agricultural insects such as Lepidopterans, leafhoppers, and thrips [45]. 2.3 Ryania Ryania is an extract obtained from a South American shrub Ryania sp. Chemically, extract is diterpene alkaloid ryanodine with insecticidal properties against horticultural and ornamental crop pests. It imparts toxicity by blocking Ca2þ ion channels [45]. 2.4 Pyrethrum Pyrethrum, also known as pyrethrins, is extracted from seed of African daisy, Chrysanthemum pyrethrum and has been used as an insecticide for past 10 decades. These plants are generally grown in Kenya [43]. The compound is an oleoresin extracted with organic

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solvents and is thus one among the most important traditional botanical insecticides. Chemically pyrethrum is IPP (Isopentyl pyrophosphate) and DMAP (4Dimethylaminopyridine) derived monoterpene. The compound is least toxic to mammals but significantly noxious to fish and different aquatic invertebrates. Despite all these facts, the market for natural pyrethrum is consistently diminishing during recent decades while that of synthetic pyrethroids is increasing at an alarming rate. This owes to the property of the latter being more stable and relatively effective than the former [45]. Moreover, pyrethrum products contain piperonyl butoxide or piperonyl cyclonene, an activator with least toxicity that considerably augments its efficacy and trims down its expenditure. Even some of these products are also applied in organic farming [43]. Pyrethrum, in general, is effective against a wide range of soft-bodied garden pests such as unarmored scale insects, and insects with fringed wings. However, it is unable to keep a check on mites. Pyrethrins behave as neurotoxins that attack and impair the functioning of central nervous system of an insect. This leads to the recurring and comprehensive nerve firings. In certain cases, neurons may have an impervious effect as well [43]. While it is lethal to pest population, human beings as well as domestic animals are least affected due to its intake because pyrethrins are acted upon by acidic pH in the stomach of mammals without any difficulty. Nevertheless, it becomes fatal if applied significantly than mentioned on the label: “Do not spray pyrethrins around ponds or other bodies of water, as they can kill fish.” Pyrethrum is a wide-ranging insecticide that is noxious to favorable insects and can even paralyze them. However, it is also susceptible to sunlight and can even degrade within hours. In order to acquire ample supervision of various pests, pyrethrum needs to be put in a regular application. 2.5 Neem Neem (Azadirachta indica), a herbal plant, covers the tropical regions of the world [44]. It is indigenous to India where it acts an ultimate source of numerous products. The bark and seeds of the tree are known to exhibit insecticidal properties with main insecticidal extract being azadirachtin. The compound acts as an insect repellant, an anti-feedant (interferes with feeding), and growth regulator (interferes with molting and growth). The oil or soap prepared from neem exhibit the same poisonous properties as other soaps and oils. In certain cases, neem also behaves like systemic insecticide. This is due to the fact that when different parts of neem are mixed with the soil, its active ingredients are leached out, specifically absorbed by the plant and finally transported to the growing apices. Neem as an insecticide is quite successful against different caterpillars, flies, white flies, and even moderately effective against aphids. It is also believed to be non-hazardous to vertebrate animals and has been shown to austerely influence numerous favorable insects such as bees, spiders, and ladybugs [43]. 2.6 Horticultural oil Horticultural oils have been used to control insect population since 1763 and are still providing services at present times. Although synthetic oils are available in the market

Natural compounds as insecticidesda novel understanding

that are generally petroleum-based, yet plant-based oils thought to be satisfactory in organic farming are accessible as well. Horticultural oils directly act upon metabolism of the insect. They interrupt feeding and egg laying properties of the insect. Once oil is coated over the entire body of the pest, it seals its body pores so that there is no gaseous exchange and the pest dies of suffocation. Horticultural oils when used under controlled manner become least toxic to plants. Nevertheless, whether such oils become effective or pose any threat to the plant depends upon its application timing, temperature conditions, type of oil used and above all the plant species on which it is being applied. Browning or burning of the leaves is the major indication of its phytotoxicity. 2.7 Dormant and summer oils Term “dormant oil” refers to a well refined plant product that is applied to the plant during dormant season usually before transition to flowering. Now, it is synonymously used as horticultural oil. On the other hand, oil applied on plants during its reproductive phase is the summer oil or foliar oil. Dormant and summer horticultural oils keep a check on any kind of growth of pest from egg to adult stages. Such pests usually include overwintering leaf-rollers, aphids etc. Dormant oils are quite efficient in administering overwintering eggs and cushioned insects and can be exercised in the beginning of spring before active plant growth instigates. Dormant oil is frequently made functional on woody trees and shrubs in dormant or delayed-dormant stages. This activity typically avoids harsh foliage flaming. The efficacy of either type of oils commonly lowers down during freezing weather so they are not applied at sub-zero temperature. Summer oils can be applied to woody plants during the flowering period. Although several horticultural oils can be applied in any season their concentration is generally kept high during winter. Since these oil products contain emulsifying agents, they are administered to plant in diluted form in both seasons. Since pests seldom develop confrontation to oil sprays such products cause little or no harm to most beneficial insects. Also when such oils are employed acceptably, they are not detrimental to human well-being [43]. 2.8 Natural soap Natural soaps are derived both from plants (coconut, olive, palm, cotton) as well as animal fat (whale oil, fish oil, or lard) and have been used since 1700s to control growth and multiplication of various insects like aphids, etc. Soaps are commonly fatty acids that can disband the protective layers of the insect cuticle, making them to desiccate. Insecticidal soaps are considered to be non-toxic to humans and several other valuable insects, but unambiguously slaughter certain pests. Some soaps are also accepted for use in organic agriculture [43]. 2.9 Insecticidal soaps Insecticidal soaps are effective for managing soft-bodied insects like aphids, scales, whitefly, mealybugs, thrips, and spider mites. The soap must contact the insect’s outer skeleton to be effective. Leaf-feeding insects are often found on the abaxial surface of

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leaves, so care is to be taken to fully cover plant foliage. Insecticidal soaps are usually diluted with water before applying. Results from the application of soap are usually seen in 1e3 days. Multiple applications are often needed to be effective. In no case, household soaps should replace insecticidal soaps for disinfection purpose. Household soaps differ significantly from the insecticidal ones in terms of composition, purity, and effectiveness, and thus have the potential to harm crops to a greater extent [43]. 2.10 Resin Wood resins are known to contain an array of terpenoids. This has been exemplified by palaeotropical plant family Dipterocarpaceae [44]. Tree species from the family are known to be immune from attack by pests. One of its largest genera Shorea robusta (sal tree) exhibits high resistance against two termite species viz. Microcerotermes beesoni and Heterotermes indicola. Particle boards and other furniture items which are made from sal wood also become protected from Cryptotermes cynocephalus [46]. Owing to such resistant properties of dipterocarp woods, there is a significant increase in the mortality rates of insects feeding on them. An investigation spread across 3 months on sal species revealed 86%e99% mortality of termites feeding on this plant. This is unlike to non-dipterocarp Dyera costulata, where only 13% death rate was recorded [46]. 2.11 Soapnuts-based pesticides Soapnuts (Sapindus mukorosse) are fruits of a tropical tree usually available in India, Nepal and Bangladesh. Morphologically, these trees are of two types: big (S. mukorosse which are usually cultivated in north India and Nepal) and small (S. trifoliatus) cultivated in south India. The soapnut shell contains “saponins” which works as a naturally available soap when making contact with water. This saponin which has highly cleaning capability repels fungus and bacteria. The soapnut dry fruit contains 11.5% saponin, 10% carbohydrate and the seeds contain 45.4% oil and 31% protein with cleansing activity without any damaging effect. Soapnuts are completely biodegradable [47].

3. Conclusion An enormous amount of work has gone into creating synthetic pesticides for the management and control of numerous dangerous insect species. However, one of the most alluring sources of risk-free substitutes for synthetic insecticides continues to be natural items. Insect management and contact-based control solutions have successfully exploited naturally occurring substances as active components. There are also several known fumigants and repellents. However, these substances might only make up a small portion of the natural compounds that exist. To commercialize the found active chemicals, further, thorough, and in-depth study on this subject is anticipated, as also increased cooperation with the insecticide business.

Natural compounds as insecticidesda novel understanding

3.1 Future prospect The term “botanical insecticides” refers to natural compounds with insecticidal properties. For as long as agriculture has been, these substances have been employed to safeguard crops. Even though they have been around for more than a century, synthetic insecticides have sadly taken their position in the current day. Because of their rapid action, low cost, ease of application, and efficiency against a variety of harmful species, synthetic insecticides have been widely used in pest management in modern agricultural systems. Nevertheless, it is now evident, after decades of use, that they have negative side effects, including toxicity to humans and animals, environmental contamination, and toxicity to insects. Because of this, people are becoming more interested in safer pest management options. Discovering natural pesticides from secondary metabolite-rich plant species should be a top goal. Additionally, the scientific community’s attention is urgently needed in this area of plant-based natural pesticides. Therefore, the research needs to be done thoroughly and plant species with known insecticidal qualities need to be discovered and encouraged for further investigation.

Acknowledgment We are really grateful for the insightful recommendations provided by Dr. Afaq Tantray of the Chemistry Department of Savitribai Phule Pune University.

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[37] Reverchon E, Osseo LS, Donsi G. Modeling of supercritical fluid extraction from herbaceous matrices. Ind Eng Chem Res 1993;32(11):2721e6. [38] Naviglio D. Naviglio’s principle and presentation of an innovative solid-liquid extraction technology: extractor Naviglio(R). Anal Lett 2003;36(8):1647e59. [39] Reverchon E, Marco I De. Supercritical fluid extraction and fractionation of natural matter. J Supercrit Fluids 2006;38(2):146e66. [40] Rahimi E, Prado JM, Zahedi G, Meireles MAA. Chamomile extraction with supercritical carbon dioxide: mathematical modeling and optimization. J Supercrit Fluids 2011;56:80e8. [41] Huang Z, Shi XH, Jiang WJ. Theoretical models for supercritical fluid extraction. J Chromatogr A 2012;1250:2e26. [42] Chandre F, Darriet F, Manga L, Akogbeto M, Faye O, Mouchet J, et al. Status of pyrethroid resistance in Anopheles gambiae sensu lato. Bull World Health Organ 1999;77:230e4. [43] Murray T, Miles C, Daniels C. Natural insecticides, A pacific northwest extension publication. PNW 2013;649:10. [44] Mishra P, Tripathi A, Dikshit A, Pandey A. Insecticides derived from natural products: diversity and potential applications. In: Book: natural bioactive products in sustainable agriculture. Springer Nature Singapore Pvt. Ltd; 2020. https://doi.org/10.1007/978-981-15-3024-1_6. [45] Arnason JT, Sims SR, Scott IM. Natural products from plants as insecticides. Phytochem PharmacoNat Prod Plants Insecticides 2012:1e8. [46] Moi LC. A new laboratory method for testing the resistance of particle to the drywood termite Cryptotermes cynocephalus. Malaysian For 1980;43:350e5. [47] Chhetri AB, Islam MP, Islam R. Development of natural pesticides from fruits and plant extracts. JNSST 2008;2(3):335e46.

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CHAPTER 23

Nanoformulations of natural compounds for herbicide and agri-food application Rajashri Satvekar1, Yogita Chavan2, Akshyakumar Sahoo1 and Vinod S. Nandre3 1

Department of Technology, Shivaji University, Kolhapur, Maharashtra, India; 2MIT-School of Food Technology, MIT-ADT University, Pune, Maharashtra, India; 3Department of Chemistry, Division of Biochemistry, Savitribai Phule Pune University, Pune, Maharashtra, India

1. Introduction Nanotechnology had drawn a lot of attention due to its extensive uses in several areas including medicine, medical diagnosis, agriculture, food, and energy. Small size to large surface area ratio of nanoparticles could be used in a variety of ways in the agriculture and food industries to promote sustainable and reliable practices. Nanotechnologies provide a number of benefits because of the unique functional characteristics of nanomaterials, although perhaps the most significant benefit is that their tiny size increases the charge density and reactivity of the materials. More strength, high heat resistance, low melting point, efficient electron transfer rate, and other magnetic characteristics are also associated with high surface area to volume ratio, which leads to higher activity of the surface atoms [1]. Existing technologies offer agrochemicals which typically include antimicrobials, fertilizers, fungicides, herbicides, insecticides, and pesticides. They provide important benefits like safe food supply; however, they also impose threats to the environment. It is crucial to accelerate innovations in science and technologies to offer transformative solutions to efficiently addressing the numerous challenges facing sustainable agriculture and food systems. Pioneering discoveries in nanotechnology have revealed many promises, and researchers envision that nanotechnology will lead to revolutionary advances in agri-food applications [2]: ➢ Genetic and cellular reengineering of microorganism, animals, and crops. ➢ Developing competent, perceptive, and self-replicating production technology. ➢ Developing systems and diagnostic tools for monitoring, tracking, and recognizing. ➢ Creating novel nanoformulation and transform crops, and foodstuffs. Natural compounds included in food as dietary constituents have preventive ability to ward off conditions such as diabetes, cancer, and heart diseases. However, because of their poor absorption, i.e., low bioavailability, their medicinal use is less noteworthy. Therefore, to overcome this, natural compound based nano-formulations are being considered and studied in recent times. The epithelial system directly absorbs nano-formulation of New Horizons in Natural Compound Research ISBN 978-0-443-15232-0, https://doi.org/10.1016/B978-0-443-15232-0.00014-X

© 2023 Elsevier Inc. All rights reserved.

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natural compounds, enhancing distribution to the target cell. A significant step toward the attempt to maximize the medicinal qualities of natural compounds would be the incorporation of phytochemicals and other nanoparticles. The bioavailability of natural compounds can be greatly increased by nanoparticles both in vitro and in vivo, according to recent research [3]. The utilization of nano-materials in farming to increase crop and livestock productivity is one use of nanotechnology that has great potential to revolutionize agriculture and food production. Application for nanotechnology may be found in animal feed, food additives, food processing, and food contact materials [4]. A wide range of nano-materials has been used in agri-food application such as processing, packaging, and food supplements. There is huge diversity of nano-materials such as nano-emulsions, nanocoatings, nano-composite, and nanoparticles which are used to develop nanopesticides, nanoinsecticide, nanoherbicide and nanofertilizer. Agriculture and food industry rely on nanotechnology, which helps to enhance the human health ensuring food quality and safety; at the same time, they also improve the crop yield [5]. To manipulate crop production using nano-formulation, nanotechnology provides novel set of materials including ceramics, emulsions, lipids, liposome, metal oxides, plant extracts, polymers and silicates. Agrochemicals and other macromolecules necessary for plant growth and stress tolerance are delivered via smart delivery system, which is made possible by nano-materials. With the help of nano-formulation, these agrochemicals improve water and nutrient conditions to boost the yield [6]. Additionally, natural polymers offer sustainable approaches to develop nano-delivery system for application in food industry such as food fortification, food preservation, food security, and safety. Natural polymers are also safer for human and animal consumption. Nanoformulations like nanoparticles, nano-composites, nano-capsules, nano-emulsions, nanos-pheres, and nano-gel are synthesized by using different techniques such as coacervation, precipitation, emulsification, electro-spraying, solvent evaporation, spray drying, sol-gel, and biological synthesis to incorporate bioactive substances such as antimicrobials, antioxidants, neutraceuticals, and flavors [7]. Natural compounds like phytochemicals exhibit low solubility, stability, hydrophobicity, and biodegradability that limit their full-scale application of antimicrobial activity. Liposomes, dendrimers, nano-emulsions, micelles, and polymers are used as nanoformulations to improve the antimicrobial effectiveness of phytochemicals. Moreover, nano-formulations can sustain the release of phytochemicals for minimal dose needs through limited distribution [8]. Illustration of natural compound nano-formulation and their agri-food application are shown in Fig. 23.1. Natural compounds (e.g., curcumin, essential oils, and resveratrol) are the most efficient antimicrobial agent showing very good activity against Gram-positive bacteria including Bacillus cereus, Listeria monocytogenes, Micrococcus luteus, and Staphylococcus aureus. Several plant extracts such as Chimaphila umbellate, Hypericum roeperianum, and Oxalis corniculate are extensively documented for their antibacterial action against Gram-negative

Nanoformulations of natural compounds for herbicide and agri-food application

Figure 23.1 Nanoformulation of natural compound for sustainable agri-food application.

strains like Escherichia coli and Pseudomonas aeruginosa. Most of naturally occurring antimicrobial compounds that target bacteria impair membrane permeability leading to membrane rupture and cell lyses. However, sometimes the mechanism may be indirect, i.e., stimulating the host immune system otherwise inhibiting adhesion to the host cell [9]. Extraction of natural compounds from plant is a crucial step for their use as antimicrobial and antioxidant bioactivity for formulating healthy food products. For polyphenols, alkaloids, tannins, pigments, etc., accurate extraction techniques should be followed to preserve the effectiveness of natural compound, for example, solvent extraction techniques, ultrasonication techniques, microwaves assisted techniques, and supercritical extraction techniques. Studies have been published on the extraction of polyphenols and alkaloids from areca nuts using solvent extraction methods and ultrasound-assisted extraction methods [10,11].

2. Nanotechnologies in agriculture Nanomaterials not only improve the plant growth but also play an important role in food process industry. Nano-biosensor has a wide range of applications in agricultural field for detecting chemical residues and diseases that are associated with the specific crops and for protecting them from undesirable changes. Another tool is nano-devices, exclusively used for genetically modified plants, postharvest management, animal breeding, poultry

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production, and nano-formulations of agrochemicals for applying herbicides and fertilizers for crop enhancement. It is reported that nano-formulations increase the solubility of low soluble bioactive compounds. It helps to release component in a slow manner, and prevent premature degradation [12]. For example, gold nanoparticles increase the yield of seeds in Arabidopsis. Cellulose nanocrystals boost seed germination; this may be due to the high water holding capacity. It has been demonstrated that adding TiO2 nanoparticles can improve plant growth. As an alternative to synthetic polymers, natural biodegradable polymers should ideally be used in the development of continuous release systems. The bioavailability of natural compound or bioactive substance is increased and high local concentration of these compounds in target cells attributed to nanoformulations that can prevent their degradation. The benefits of using nanoformulations in agriculture applications are outlined in Fig. 23.2. Due to their biodegradability, biocompatibility, and low toxicity, natural polysaccharides like alginate and chitosan have been widely researched for the fabrication of sustained release systems for agriculture. Chitosan is utilized in agriculture for its antimicrobial properties, the control of biotic and abiotic stress, to improvement of plants systemic resistance to pathogens, the stimulation of certain soil microorganisms, and the regulation the crop growth and yield [13]. 2.1 Nanoherbicides Weed management is very big crises in agriculture due to ineffectiveness of herbicides for the simultaneous elimination of many weeds species and the emergence of herbicide resistance. Herbicides are frequently sprayed on leaves, which only partially eliminate perennial weeds such as Cynodan dactylon, Solanum elaeagnifolium, and Cyperus sp. However, these conventional herbicides interfere with cell division, hinder lipid biosynthesis, and destroy the structure and functionality of the plant-specific chloroplast [14]. Figure 23.2 Benefits of using nanoformulation of natural compounds in agriculture.

Nanoformulations of natural compounds for herbicide and agri-food application

Because roots do not have cuticles as leaves do, applying encapsulated herbicide through root absorption is a simpler method than applying it via foliar absorption. Even though there are significant obstacles to herbicide absorption through roots, herbicides with liphophilic composition can be absorbed with ease as it is having liphophilic structure. According to Chinnamuthu et al. (2007), a specific nanoencapsulated herbicide molecule targeted for a particular receptor in the roots of target weeds enters the system and is transferred to parts that inhibit glycolysis of food reserve in the root system causing the specific weed plant to starve for food and die. Nanoencapsulated herbicide will be precisely spread in rain-fed farming after receiving the sufficient moisture level [15]. Herbicides cause weed to develop resistance to them when they are applied regularly. Smallest amount of agrochemicals must be used in sustainable agriculture to safeguard the environment and biodiversity. Without leaving any active ingredient in the environment, nanotechnology may provide biodegradable nanomaterials and precise delivery methods enabling nanoherbicide to be applied to weeds in an eco-friendly manner. Future nanoherbicides have been suggested to be carboxy methyl cellulose nanoparticles [16]. The development of target-specific herbicide using a natural compound encapsulated with nanomaterials is designed for a particular receptor inside the roots of target weeds, which leads to entry into the weed root system and the translocation of components that inhibit glycolysis [17]. This “nanobioherbicide” is an extremely unique natural compound-based herbicide system that may be served as a model for the invention and development of bioactive agents using systematic inquisitiveness and being alert for exceptional results, for various weed species. A controlled release-nanoformulation is considered to release herbicide at certain rate and concentration on the target plant within the optimum range for a predetermined period of time. It decreases the adverse impacts of the herbicides on the environment because there is less leaching, evaporation, and degradation. The large surface area of nanomaterials result in a little amount of herbicide being required, thus increasing concentration in a smaller dose with lower cost by use of nanoformulations of natural herbicides. The vulnerability of the herbicide to environmental conditions like desiccation, heat, and UV radiation encountered during storage, delivery, and application is significantly reduced by the inherent small particle size [18]. Over the past few years, development of nanoherbicide has significantly increased. However, the present knowledge on the mechanistic impact of nanoherbicide is currently inadequate. In order to assess their effects and forecast future possible safety uses, it is crucial to comprehend how nanoherbicide interacts with plant. Montcharles S. Pontes et al. using experimental and theoretical methods investigated the inhibitory mechanisms of a paraquat nanoherbicide on photosystem I in leaves of spinach. They found that the nanoherbicide had distinct electro-activity behavior which may account for its greater herbicidal activity on plants [19].

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Mitotic index estimation using onion cells identifies the herbicide genotoxicity. For the living organism, the mitotic index must not drop below 22%. The chromosome aberration indexes have also been calculated using the onion cells for the different treatments. Diyanat et al. (2019) showed that commercial pretilachlor increases this mitotic index as compared with the nanoencapsulated herbicide. An increase of chromosomal abnormalities is caused by an increase in the concentration of the herbicide. The lower chromosomal abnormalities were observed with lesser concentration of pretilachlor nanoformulation. It was suggested that the active ingredient attachment to the carrier system would reduce the DNA damage in the onion cells [20]. 2.2 Nanopesticides Pesticides are substances that are utilized to control pests, weeds, herbivores, and pathogens. To combat biotic stress agents, different chemicals used including antimicrobials, bactericides, fungicides, herbicides, insecticides, molluscicides, nematicides, and rodenticides. In addition, pesticides such as disinfectants, sanitizers, and repellants have a number of environmental concerns. It is alarming that 95% of herbicides and 98% of insecticides are sprayed on crops that end up in areas where they are not intended, including non-target species, water, air, and soil. This results in a loss of biodiversity, soil contamination as well as water and air pollution [21]. Excessive usage of pesticides causes pesticide resistance, prompts research into new potent pesticides that are safe for human and environment. Nanotechnology limits the amount of active compounds and significantly improves efficiency to protect the environment and human health by the controlled release of pesticide. 2.3 Nanoinsecticides Nanoinsecticides are chemical or biological compounds that control insects more efficiently over the bulk insecticides. Nanomaterial-based insecticides have a number of advantages which include controlled release that enhances effectiveness of natural and chemical insecticides, less environmental contamination from rapid application time, safe and simple handling, and lower toxicity to non-target organisms. By enclosing active ingredient in a thin-walled sac of nanometer size, “nano-encapsulation” can be employed to improve the insecticidal assessment [22]. Polymer-based nanoformulations such as alginates, chitosan, starch, and polyesters (e.g., poly-ε-caprolactone and polyethylene glycol) have all been used to encapsulate of majority of the insecticides [23]. Insecticides have been encapsulated in a variety of nanoformulations comprising nanofires, nanogels, nanosphere, micelles, and nanocapsule. Kang et al. (2012) described the insecticidal efficacy of liposome-based formulations [24], and the chitosan coated nanoliposomes exhibited a sustained slower release of the entrapped core material (etofenprox and alpha-cypermethrin) because of thicker coating layer [25]. Priya Saini et al. (2014) synthesized pyridalyl nanosuspension utilizing sodium alginate which was more effective against Helicoverpa armigera as stomach toxin [26]. Furthermore, it has been discovered that

Nanoformulations of natural compounds for herbicide and agri-food application

several inorganic nanomaterials including as Ag, Al2O3, Fe (0), TiO2, ZnO, and SiO2 are good insecticides [27]. 2.4 Nanobionics Nanobionics is the application of nanoformulation to enhance functions of plant cell by examining the electronic interactions in biological systems. A novel field of nanobioengineering has emerged as result of the interaction between plant cell organelles and nanoparticles [28]. For their absorption, accumulation, translocation, and beneficial impacts on growth and development in crop, a range of metal and carbon-based nanoformulation are being used. It has also been shown that in soybean, spinach, and peanut employing metal-based nano-materials has improved physiological parameters such as photosynthetic activity and nitrogen metabolism [29]. Numerous studies have demonstrated improved photosynthesis when nanomaterials like single-walled carbon nanotubes (SWNTs) are used. Studies of SWNT-chloroplast assemblies conducted in vivo revealed a mechanism that resulted in increased photo-absorption and higher rate of leaf electron transport. Plasmon resonance of metal nanoparticles can boost the absorption of solar energy and lead to enhance carbon fixation [30]. It is important to detect the deleterious effect of nano-materials and take precautions against any negative effects before they are widely used. 2.5 Nano-fertilizer Surface-coated nano-materials or nano-coated fertilizer particles cling to the substances more firmly to the plant due to their greater surface tension than conventional surfaces. Additionally, nano-coatings offer surface protection for larger particles. A nano-capsule is made with the shell that contains natural or bioactive compound, such as a chemical or biological agent for the protection of plant against pests and diseases. The shell consists of variety of substances, such as polymers, lipids, viral capsids, or nanoclays [31,32]. 2.6 Nano-biosensor Nano-biosensor is a device that depends on the catalysis reaction between the immobilized bio-recognition element and the targeted analyte that generates electrons and affects the electrical properties of the solution. The unique physical, chemical, and biological property with large surface area to volume ratio of nanoparticles imparts catalytic property for nano-biosensor application. Additionally, many types of nano-biosensor such as immuno-sensors, enzymatic nanobiosensors, and DNA nanobiosensors are constructed depending on the immobilized bio-recognition element [33]. For the nano-biosensor development, a variety of natural polymers such as dextran [34], chitosan [35], and DNA [36] are used. The development of nanobiosensors is an emergent area, considering the demand of rapid, simple, selective, and inexpensive methods to monitor soil conditions, and crop growth in the field [37]. Nanomaterials

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can be designed to generate a chemical or electrical signal when an analyte such a bacteria is present. By giving farmers accurate information related to their fields and assisting them in making better decision, smart nanobiosensors may be used in precision farming to increase agriculture production. Nanomaterials fate in the gastrointestinal tract is being argued extensively. However, the transport of nanomaterials through the plant system seems to be a lack of information in the understanding. Investigating this could provide better insights into these systems [38]. Some of the adverse effects were reported in plants when nanoparticles of silver, aluminum, titanium, cerium, and zinc were used on different plant varieties [39]. Public awareness and acceptance toward natural product nanoformulation tend to be more positive, attributed to the natural ingredients such as polymers used, which are generally recognized as safe recognized by US Food and Drug Administration [40]. Hence, using natural compound as nanoformulation for nutrients, antimicrobial agent, herbicide, pesticide, insecticide, and fertilizer could be an encouraging step in bringing these products to the market faster for the sustainable agri-food application.

3. Nanotechnologies in food science Food production, processing, preservation, storage, packaging, and distribution are all areas in which nanotechnology is used in the food industry. Healthier, safer, and highquality foods are created by utilizing a variety of nanoparticles for potential production, processing, and packaging. It is essential to address food wastage, food spoilage, food security, and food-borne diseases in addition to increasing production rate. Nanotechnology provides enhanced security by using nanobiosensors for detection of every pathogen or containments in food to overcome food-borne diseases [41]. Food packaging made possible by nanotechnology offers an upgrading over conventional packaging while simultaneously its functional components like antimicrobial activities extends shelf life to the food products. It is also involved in the detection of food toxins and dietary contaminants, together with color formation and flavor production. Nano-enabled smart and intelligent systems provide localization, sensing, and reporting of food items with improved efficiency and security [42]. Additionally, nano-enabled delivery techniques enhance the food component’s neutraceutical values. Different bioactive components in food and nanomaterials for nanoformulation synthesis are summarized in Fig. 23 3. 3.1 Food processing The postharvest food processing has a lot of room for improvement attributable to nanotechnology. The word “nanofood” describes food that has been created using nanotechnology in production, processing, packaging, and security of food by improving bioavailability, taste, texture, and consistency, hiding the unpleasant taste or odors, and, modifying the possible cluster formation, particle size, and surface charge [43].

Nanoformulations of natural compounds for herbicide and agri-food application

Figure 23.3 Nanoformulation of natural compound for food applications.

Bakery products, confectionery items, cheese, fruits, meat, vegetables, and fast food can all employ edible thin nano-coatings ( w 5 nm), which act as moisture and gas barriers. Additionally, they offer antioxidants and anti-browning ingredients, color, flavor, enzymes, and longer shelf life to the synthesized products. There are several bakery items available coated with edible antibacterial nano-coatings [44,45]. Nanofilters have been used to eliminate color from beetroot juice while maintaining the flavor for the red wine, as well as to eliminate lactose from milk so that it may be replaced with other sugars, made appropriate for lactose-intolerant people. It aids nonboiling removal of bacterial species from milk or water. Beer and milk may be filtered for purification using nano-materials that were utilized to create nano-sieves [46]. Food companies for instance H. J. Heinz, Nestle, Unilever, and Hershey are currently researching nanofoods and food packaging material [47]. For example, nanofood ice cream from Nestle utilizes nano-emulsions to facilitate low fat content. 3.2 Food packaging Food packaging shields to protect food from temperature changes, microbial invasion, external physical attack, and vibration by providing physical, chemical, and microbiological protection. There are numerous food packaging technologies are used to ensure food integrity and safety for consumption. There are mainly two categories food packagings:

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edible and non-edible. Edible packaging means material utilized for food packaging is suitable for eating while non-edible packaging material means it is not appropriate for consuming. The edible thin coating extends food shelf life and preserves food quality by avoiding deterioration of food. For example, de Moura et al. (2011) synthesized chitosan-carboxymethyl cellulose thin film that can be consumed as nano-enabled edible coating [48]. The packaging industry has recently paying attention on various types of nanoformulations since to their widespread availability, low cost, simplicity of processing, and superior performance. Arora and Padua (2010) summarized the nanostructures as most promising nanoscale fillers including carbon nanotubes, graphene nanosheets, graphite nanoplates, montmorillonite, and kaolinite clays. Besides, it was proven that using it improves the thermal stability and gas barrier qualities [49]. Due to their antimicrobial activity, nanoparticles are employed for packaging in food industry. It serves like carriers for antimicrobial polypeptides and avoids microbial deterioration. Packaging material prepared with a coating of starch colloids and filled through the antimicrobial agent. Due to the controlled discharge of antimicrobial agent from the packaging, it works as a barrier to microbes [50]. To lengthen food’s shelf life even after the packaging, nano-materials are exploiting as carriers to add anti-browning agents, antioxidants, enzymes, flavors, and bioactive compounds [51,52]. The microbial growth observes notably on the perishable muscle-based food surface, specifically those made of meat, chicken, and fish. As a result, using antimicrobial packaging is more effective in controlling the microbial growth than just using antimicrobials as food additives. Additionally, antimicrobial packaging works well to interact with both the environment and the food product [53]. Nanomaterials like Ag, SiO2, and TiO2 are added into food packaging material to achieve hygroscopic and antimicrobial properties [54,55]. 3.3 Food preservation In the history of mankind, the preservation of food was a common practice with use of natural products related with microorganisms [56]. The utilization of natural compounds as food preservatives is attractive alternative for chemical additives as it is adopted confidently by consumers. Curcumin is the most potent and slightest steady natural compound of turmeric (Curcuma longa). Sari et al. (2015) demonstrated that upon nanoencapsulation curcumin have decreased antioxidant activity and retain stability in pasteurization at various ionic strengths [57]. Alginate-essential oil nanoemulsions were synthesized by Cofelice et al. (2019) and employed as edible coatings to assist increase the shelf-life of coated fruits [58]. 3.4 Food safety and security Ensuring safe and high quality food products is tough challenge in the food industry. Novel pathogens associated with food intake clearly revealed the level of microbial invasion as a severe issue for food safety and security. Along with that microorganisms have

Nanoformulations of natural compounds for herbicide and agri-food application

the capability for adaptation and change with varying methods of food processing, preservation, and packaging leads to new food safety hazards. More attention is spent on methodologies and approaches employed for food authentication. For food authentication and food monitoring, conventional methods like chemical experiments and spectroscopy have significant limitations. It is imperative to create new techniques to replace traditional methods in order to achieve the objectives of consistency and cost effectiveness and it is fulfill with nanobiosensor [59]. These are used to quantify the components of food that are currently available, detect pathogens in food or processing plants, and notify distributors and consumers of the safety status of the food [60,61]. 3.5 Nanonutraceuticals Nanonutraceuticals show potential as a new perspective on food that focuses on the bioactive substances found in foods and a knowledge of their mechanisms of action. In food, nanoformulations are used to more efficient delivery of bioactive substances with improved bioavailability, in that way increasing the health benefits. An attractive research area in food industry is encapsulation of neutraceuticals into biodegradable, eco-friendly nanocarriers to boost their absorption and pharmacological properties [62]. These foodderived natural compounds are supposed to be examined to preserve exceptional properties such as bioavailability, safety, and efficiency at the nanoscale. It is important to carry out clinical and nutritional study to analyze probable unintended consequences [63,64]. Emerging diseases and microbes that can acclimatize to new food processing, packaging, storage, and preservation conditions must be taken into consideration when appropriately evaluating novel technologies for potential safety hazards. Many natural antimicrobials have been effectively evaluated; even though some have the advantages in food, some may have harmful effect owing to its adverse interactions and inactivation in the food matrix [65]. Recent reports on nanoformulation of natural compounds in agri-food application are summarized in Table 23.1.

4. Summary and future prospect Optimizing the productivity of sustainable agricultural and food industry is crucial, especially in developing countries with high populations. The foundation for the production of innovative nanoformulations should be viewed as the raw materials and bioactive compounds from agriculture and food industry. In this situation, countries whose economies are heavily dependent on agriculture must use nanotechnologies to increase agricultural productivity in a biodegradable manner even in hostile environments. The adoption of nanoformulations of natural compound would engage in recreation to feeding the rising population while conserving diminishing natural resources by minimizing hazardous effect and sustainable production techniques.

437

Table 23.1 Agri-food application of natural compound based nano formulations. Natural compound

Nanomaterial

Application

Impact on

References

438

S.No.

I. Natural compound nanoformulation for agriculture application

Neem oil (Azadirachta indica)

Nanoemulsion

Larvicidal agent

2 3

Carboxymethyl cellulose a-Pinene, linalool, and silica nanoparticles

Nanocapsules Nanocomposite

Herbicide Anti-feedant

4

Citrus peel essential oil

Nanoinsecticide

5

Zataria multiflora essential oil

Polyethylene glycol (PEG) nanoparticles Nanoencapsulation

6

Aril (Myristica fragrans), seeds (Zanthoxylum alatum), and fruits (Bunium persicum) essential oil

Chitosan nanogel

Antifungal activity

Nanopesticide

Culex quinquefasciatus Potential carrier Tobacco cutworm (S. litura F) and castor semilooper, (A. janata L) Tomato borer (Tuta absoluta) Botrytis cinerea, the causal agent of gray mold disease Callosobruchus chinensis and Aspergillus flavus

[66] [16] [67]

[68] [69]

[70]

II. Natural compound nanoformulation for food application

7

Curcumin

Nanostructure lipid carrier

Antioxidant activity Antioxidant

Curcumin activity during storage Bioavailability

8

Vitamin C and folic acid

9

Chitosan and alginate layered curcumin

Microencapsulation in chitosancoated liposome Multilayer nanoemulsions

10

Vitamin C

Iron nanoparticles (Fe3O4)

Biocompatility

11

Starch dextrin

b-carotene nano-composites

12

Resveratrol

Lecithin-based nanoemulsions

Solubility and stability Bioavailability

[71]

Bioaccessibility

Functional foods development for combating obesity. Nano iron fortified biscuits as food supplement Functional food

[73]

Oral administration

[76]

[72]

[74]

[75]

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1

Nanoformulations of natural compounds for herbicide and agri-food application

Nanoformulations of natural compounds are becoming more admired in the forefront in agri-food field. In global markets, commercial nanoformulation products of pesticides are coming along with number of research articles. These products have altered solubility, improved stability; enhanced permeability, and precise surfaces adherence, targeting, and controlled release. Nanotechnologies have been used to create stable nanoformulations with long-lasting effects in order to overcome disadvantages of the natural compounds such as their relatively brief period of action low stability and permeability. However, albeit nanoformulations increase the bioavailability of specific bioactive substances, but all nanomaterials exploited in food application must be used deliberately and merely after comprehensive examination of cytotoxicity due to probable toxic effect. “Nanoformulations of natural compounds” might encompass nanomaterials made of organic and inorganic material produced with small amount of natural compound with a precise nano-enabled structure. Nanoformulated compounds have the prospective to execute the superior of agri-food inputs as well as to reduce the drawbacks of modern agri-food applications. The market breakthrough of a nanoformulation ought to not be entirely based on a size threshold (above or below 100 nm), other than relatively based on a scientific evaluation of latest risks and benefits of individual ingredients and the whole formulation fate in the environment. Future scenarios for the developing nanoformulation of natural product can be illustrated in Fig. 23.4. In the literature, impacts of nanoformulations on the outcome and

Figure 23.4 The future prospects for nanoformulations of natural compounds.

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special effects of natural compounds have been described however there is insufficient understanding of the mechanism. Explaining and investigating the consequences in requisites of environmental aspects need a proper analytical technique, and different experimental protocols which are typically used for traditional agrochemicals [77]. In agricultural practices, initial developments carried with current methods lead to new combination of contaminants. On the other hand, nanotechnology could give solutions to diminish contamination caused by agrochemicals. This can only be achieved by academic researchers, industrial synthesis, and regulatory agencies. Alternatively, new scientific tools should be developed to evaluate risks and benefits, to assess environmental impact and fully exploit nanoformulation of natural compound for sustainable agrifood application.

Declaration of competing interest Authors state that they have no conflict of interest.

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CHAPTER 24

Natural compounds for bioremediation and biodegradation of pesticides Mudasir Ahmad Dar1, a, Mohd Shahnawaz1, 2, a, Khalid Hussain3, a, Puja Gupta4, a, Mohd Yaseen Sirwal5, a, Beenish Sadaqat1, a, Sehrish Gazal6, a, Romana Akhtar7, a, Sarita Parihar8, a, Daochen Zhu1, a, Charles Oluwaseun Adetunji9, a, Tahira Fardos12, Jyoti Parihar10, Osemwegie Osarenkhoe Omorefosa11, Rongrong Xie1 and Jianzhong Sun1 1

Biofuel’s Institute, School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, China; 2Department of Botany, University of Ladakh, Kargil Campus, Khumbathang, Kargil, Ladakh UT, India; 3Department of Botany, University of Kashmir, Srinagar, Jammu and Kashmir, India; 4Department of Life Sciences, RIMT University, Mandi Gobindgarh, Punjab, India; 5Department of Chemistry, Government Degree College Kishtwar, Kishtwar, Jammu and Kashmir, India; 6Department of Environmental Science, Government Degree College Kishtwar, Kishtwar, Jammu and Kashmir, India; 7Department of Zoology, Government Degree College Kishtwar, Kishtwar, Jammu and Kashmir, India; 8 Department of Economics, Government Degree College Kishtwar, Kishtwar, Jammu and Kashmir, India; 9Department of Microbiology, Edo State University Uzaiure, Edo State, Iyamho, Nigeria; 10Department of Pedagogy of Bioscience, Government College of Education, Jammu, Jammu and Kashmir, India; 11Department of Food Science and Microbiology, Landmark University, Omuaran, Kwara, Nigeria; 12Department of Botany, Government College for Women, Jammu, Jammu and Kashmir, India

1. Introduction There is no clear understanding of how nature is counteracting the overload effects of the ecotoxigenic/genotoxic products that are generated and released on the planet by the innumerable activities of a rising global population nor the interplay of climate factors or changes in mediating phenomenal natural processes for fostering balance in different ecosystems. Technology, agriculture, industrialization, global energy demands, civilization, and other human insatiable exploitation of the earth’s resources for their existential gains have come at a cost to health, environment, ecosystem life-support, and biodiversity. Hence, the earlier reliance by humanity on chemically synthesized utilities in food processing, manufacturing, domestic care products, agriculture, and energy prospecting has confounded and exacerbated pollution-related problems in the developed and developing nations of the world. Even though the world is experiencing an exponential increase in biocidal applications, greenhouse emissions, climate change issues, food insecurity, wars, and public health outbreaks, particularly those related to pesticide applications, studies are also rapidly unrelenting in searching ecofriendly solutions to the menacing effects of pollution overload. A few biological techniques such as bioremediation, biodegradation, biotransformation, genetic engineering, transcriptomics, and biodetoxification that mimicked the natural ecophysiological responses of mostly a

These authors contributed equally to this work and are all first authors.

New Horizons in Natural Compound Research ISBN 978-0-443-15232-0, https://doi.org/10.1016/B978-0-443-15232-0.00015-1

© 2023 Elsevier Inc. All rights reserved.

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microorganisms to the intolerable presence of pollutants have been advanced in the course of time to tackle the persistence of toxigenic pollutants in the environment [1,2]. Many pollutants contaminating the environment may linger for a brief to an extended period, eventually permeates the global food webs through the trophic levels to bioaccumulate in living things (bacteria, algae, fungi, plants, animals, and humans), and initiate numerous adaptive responses from organisms that become exposed to them [3,4]. The fate of these toxigenic pollutants varies in nature according to their chemical, structural, and physical properties, and sensitivity to climate factors as well as organismic reactions [5]. Consequently, pesticides constitute one of the most menacing categories of pollutants that threaten human health by causing numerous chronic illnesses related to cardiopulmonary, infertility, neurological, dermatological, congenital, and immunological complications. They are chemicals of synthetic or semisynthetic origins used separately or as mixture to repel, prevent, mitigate, or kill any invasive pests or applied as disinfectants. Any substance or mixture of chemicals, considered as a plant regulator, desiccant, defoliant, and even nitrogen stabilizers can all pass as pesticides. In modern times, pesticides (biopesticides) may also include biological agents like the virus, bacteria, fungi, antimicrobial, and disinfectants that can incapacitate, deter, or kill pests. In addition, the ubiquitous, volatile, aerodynamic, and recalcitrant nature of pesticides along with their latent or asymptomatic health effects contributed significantly to the recent elevated attention devoted to their removal from a different environment and the increasing number of biogenic resources screened for pesticide-remediating potentials. Depending on the nature of the target pests, a pesticide may be an herbicide, insecticide, fungicide, algicide, rodenticide, nematicide, avicide, molluscicide, bactericide, and lampricide. Hence, the growing perception that pesticides are synonymous with plant protection and yield improvement in crop production. Consequently, agriculture was recognized to be the largest generator of pollutant pesticides ahead of the pesticide industries and other non-agricultural sources with an estimated 2.5 million metric tons amounting to 5.6 billion pounds applied annually worldwide. This is complicated by the growing urgency, as validated by the United Nations Sustainable Goal on zero hunger, to feed the over 7.8 billion inhabitants on earth and their applications are projected to generate 3.5 million metric tons by 2020 [6,7]. This has shifted emphasis to pesticide application risks evaluation and the emergence of numerous knowledge-based pesticide risk assessment tools that are designed to regulate pesticide applications, support the farm users as well as result in the ban of some classes of pesticides tagged the dirty dozen [8]. Notable among the pesticides banned in the USA, UK, India, Brazil, and other nations are benomyl, carbaryl, diazinon, fenarimol, fenthion, ethyl mercury, alachlor, dichlorvos, and neonicotinoid pesticides. Similarly, the authors in Ref. [6] noticed that herbicides constitute over 48% of the total global pesticide application followed by insecticides with China, the USA, and Argentina being the lead pesticide-consuming nations on the international

Natural compounds for bioremediation and biodegradation of pesticides

level while Congo, Sudan, and Cameroon dominate pesticide usage in the African continent [9]. Even though many organisms naturally evolved, due to decades of exposure, mechanisms to survive the persistence of pesticides in the environment, the slow individual or population, or community response(s) is disproportionately inconsistent with their rate of release and magnification. Suffices to say numerous natural compounds, organisms, and biotechnologies have been innovated or engineered for expedient accelerated pesticide-degradation efficiency and turnover. Due to the unabating health and environmental issues conceptually posed by the conventional use of chemical and physical degradation or removal of pesticides from the air, water, and soils, biotechnological-based approaches such as biodegradation, bioremediation, bioengineering, and sensor technology have been the preferred alternative for this purpose [10]. The authors in Ref. [11] associated these activities more with microorganisms which confirm why most studies involving pesticide-biodegraders use naturally occurring microorganisms. Although the use of organisms with the capacity to degrade xenobiotic and pesticide compounds is considered green, less intuitive attention was given to exploring the use of their metabolic product, other derived compounds (exopolysaccharides, enzymes, peptides, hydrolysates, etc.), and the augmentation of these natural compounds with non-biogenic compounds for bioremediation of pesticide-contaminated systems [12]. They present a safer, energy-efficient, sustainable, cost-effective, and eco-friendly approach to remediate pesticides contaminated systems. Biocatalytic, biometabolic, transfigurative, bioaccumulation, biostimulation, and removal activities of numerous microorganisms or their natural compounds against pesticides (biopesticides) in nature have been enhanced significantly for spatiotemporal pesticide-decontamination [1,13]. These in situ and ex situ enhancement methods are usually unconventional and accomplished through local weather manipulation, enrichment, augmentation, or supplementation techniques using compost, nutrients, or other enhancing compounds [14], hurdle technology (combining more than one pesticide-degrader or biological and nonbiological methods for the decontamination of a contaminated system), omics technologies [15e19], genetic manipulation [20], nanotechnology, and introduction of a culture pesticide-degrading organisms or their byproducts/derivatives to the contaminated system [5,21e24]. Despite these, data on the genes potentiating, or responsible for pesticide degradation, detoxification, or removal and their transgenic characteristics remains scanty. Furthermore, the understanding of the impact of interspecific and intraspecific interaction on optimizing, catalyzing, and time quotient of pesticide degradation is critical to improving the application of cometabolism or hurdle technologies in remediating pesticides-contaminated systems while associated time-bound risks to native species posed by introduced cultures of non-native pesticide-biodegraders under permissive outdoor climates are unclear. Therefore, the knowledge of pesticide bioremediation or biodegradation pathway, particularly when it involves natural compounds, and the impact of the process on the eco-diversity

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dynamics of such a system is far from comprehensive nor was where the process intersects machine learning conceptualized. Therefore, the present chapter aims to overview pesticide bioremediation under the following lines: (i) to introduce pesticides and their impacts on sustainability; (ii) to discuss the concerns related to pesticide pollution; (iii) to highlight pesticide toxicity to soil and water ecosystems; (iv) to enlist different types and methods of pesticides bioremediation; (v) to report the enzymes involved in pesticide bioremediation, and (vi) to suggest the future prospective of pesticide bioremediation.

2. Pesticides and their impacts on sustainability Pesticides are chemical compounds that are used for controlling or abatement of the pests in agriculture or households. According to the United Nations Organization for Food and Agriculture (FAO), a pesticide is defined as any chemical or combination of chemicals intended for preventing, or eliminating, any pest, including disease vectors (both human and animal health concerns), unwanted plant or animal species, that cause harm or otherwise impede the production, processing, storage, transportation, or marketing of food, agricultural or forestry products, or animal feedstuffs. Based on the target organism, pesticides are classified as nematicides, fungicides, insecticides, rodenticides, herbicides, and plant growth regulators. Ideally, a pesticide is desired to kill the target pest only, while showing no effect on other animals including humans. However, this optimal situation and pesticide specificity are very rare and impossible to achieve. The continuous and excessive use of pesticides is wreaking havoc on all forms of life including humans [25]. The extensive and improper usage of pesticides in agriculture has resulted in a variety of environmental issues and public health concerns. The majority of farmers use pesticides that are either “moderately” or “highly” toxic. Being masses, most (60%) of the farmers get information about the pesticides from the retail shop owners without themselves thoroughly following the product manual. However, a study examining the link between pesticide usage and the emergence of sickness symptoms among farmers exposed to harmful pesticides in various parts of the world has reported substantial influences on farmers’ health [25]. Since India is an agriculture-based country where 57% of the population is dealing or dependent on agriculture. In Asia, India is the largest producer of synthetic pesticides, with an approximate production of over 90,000 tons2 per year. However, India ranks 12th in the use of pesticides worldwide. The farmers are particularly vulnerable to pesticide exposure, which is common in agricultural settings [26e28]. Pesticide poisoning is 13 times more common in developing countries than the highly industrialized and developed nations that consume 85% of total pesticide production. The majority of pesticiderelated poisoning in developing countries is due to a lack of training and body protection, besides the absence of spray regulation [29]. Farmers who are continuously exposed to

Natural compounds for bioremediation and biodegradation of pesticides

such toxic chemicals may develop chronic diseases such as diabetes, hypertension, and other conditions of which they are unaware until being examined [30].

3. Concerns related to pesticide pollution Water being one of the natural resources comprises the basic element of all living creatures. Surface water has been a big concern in many countries around the world over past few years due to its contamination. Contamination of the aquatic environment due to pesticides occurs in several ways. They include spray drift, runoff, and leaching [31]. Pesticides when get transferred from agricultural source to aquatic ecosystems may prove to be harmful [32]. Fishes are directly affected by pesticides. Smaller fishes are more impacted than larger ones by pesticides [33]. Pesticides can poison fish indirectly by reducing their food supplies (algae and plankton), alter their food preferences, and degrading the quality of their aquatic environment [34]. Some pesticides may reduce the number of primary producers, which would in turn reduce the number of primary and secondary consumers [35]. Some organochlorine herbicides harm the main consumers, including zooplankton [36]. Additionally, some pesticides might have a negative impact on tiny crustaceans [37]. 3.1 Impact of pesticides on human health It has been observed that only 0.1% of the pesticide administered interacts with the targeted organism whereas the major portion of the pesticide lead to pollute the soil, air, and water causing substantial nontarget ramifications throughout the ecosystem [38]. Moreover, the cumulative properties of certain pesticides help them to migrate within the food chain and accumulate in the tissues of many living organisms [39]. One of the typical examples of the impact of pesticide pollution on human health is served by the Aral Sea area. United Nations Environment Protection Agency (UNEP) has incorporated various effects of pesticides that include “the range of oncological, hematological morbidity and pulmonary dysfunction, in addition to immune system deficiencies and inborn deformities” [40]. Employees working in the farm have high risks due to skin contact, intoxication, and inhalation while treating pesticides to crops. There are two major impacts of degradation of pesticide runoff in water on human health. The first includes the ingestion of shellfish and fishes that are contaminated by pesticides. This can affect the economies based on the fish that reside at the bottom of the main agricultural submerged areas. Another issue is the drinking water contaminated with pesticides. Inside human body, fat tissue “lipids” is known to be the main receptor for many pesticides and hence these pesticides get accumulated in the adipose organs. For example, DDT being lipophilic is accumulated in human adipose tissues when consumed through edible fish products [41]. Once accumulated in the body, pesticides pose diverse effects on the metabolism of the animals. Although many of these impacts are long-term and frequently go unnoticed by

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normal observers, there are serious concerns about their impact on the entire food chain. Examples of some physiological effects include suppression of the immune system, infertility, endocrine system disturbance, tumors, cancers, teratogenic effects, transgenerational effects like DNA damage, and ultimately poor health of fishes as evidenced by a reduced count of red-to-white blood cell ratio, severe mud on fish gills and crusts, etc. 3.2 Pesticide toxicity to soil and water ecosystems The ecological impacts of pesticides vary from habitat to habitat. Diverse impacts on aquatic ecosystems by several pesticides have been noticed. Pesticides can travel along with different components of the biosphere like air [42], land [43], water [44], and can get absorbed by plants [45] and animals [46]. Pesticides acquire varying levels of toxicity and soil endurance which is highly dependent on the type of soil and the implementation method [47]. Earthworms, which account for more than 80% of terrestrial invertebrates, significantly improve soil fertility by degrading soil organic matter into humus [48] and are thus designated as an important measure of soil quality [49]. Earthworms help to improve and preserve soil structure by establishing passages in the soil which allow gaseous exchange and facilitate drainage. However, varied agricultural operations and excessive use of pesticides pose a great danger to the earthworm diversity and density [50]. Earthworms exposed to numerous pesticides have been found to show cuticle rupture, coelomic fluid leakage, swelling, and blanching of the body and eventually, the body tissues of the earthworms get softened. In certain species of earthworm few pesticides and fungicides when administered together have shown to cause neurotoxic effects [51]. Millions of microorganisms are present in the soils that have been sequestering carbon for billions of years. These microorganisms through mycorrhizal fungi form symbiotic relationships with plants thereby making networks that help plants to access nutrients from the soil in exchange for a consistent supply of carbon from the air, which the plant photosynthesizes. However, incorrect application practices and excessive pesticide use can harm the soil and damage the soil ecosystem [52]. Pesticides have the potential to change microbial diversity, growth, and biomass in the soil, disturbing the soil ecosystem and resulting in decreased soil fertility [53]. Certain herbicides like metsulfuron methyl, chlorsulfuron, and thifensulfuron methyl have been found to inhibit the growth of certain microbial strains [54] which carry out important ecological processes, and hence alternation in the growth and development of these bacteria will ultimately hamper the soil fertility (Fig. 24.1). The competition between certain groups of microorganisms in the soil can be greatly reduced after the administration of pesticides, which can inhibit the growth of certain microorganisms while accelerating the growth of others [55]. In a study application of endosulfan to the soil resulted in a 76% rise in bacterial biomass and a 47% reduction in fungal biomass [56]. Pesticides also can activate and deactivate any particular microorganisms or enzymes, thereby influencing principal biochemical reactions occurring

Natural compounds for bioremediation and biodegradation of pesticides

Figure 24.1 Effects of the pesticides on microbial diversity in soils and agriculture. (Adopted from Meena RS, Kumar S, et al. Impact of agrochemicals on soil microbiota and management: a review. Land 2020;9:34. https://doi.org/10.3390/land9020034.)

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in soil like fixation of nitrogen, and formation of nitrite and nitrate [55,57]. Pesticides affect the decomposition rate of organic matter which is a pivotal soil property for ascertaining the microbial cell quality and soil productivity. In a study, when four different types of herbicides (atrazine, primeextra, paraquat, and glyphosate) were applied to the soil, a remarkable decrease in soil organic matter was observed initially which later was found to increase from the second to the sixth week of continuous herbicide application [58]. Pesticides also pose a great threat or can disrupt the enzymatic activity and various metabolic processes undergoing in soil [59]. Soil consists of both free and mobilized proteins like extracellular and intracellular enzymes [60], which can act as a measure of soil biological equilibrium, soil fertility, and quality and may indicate a change in the biological structure of the soil due to any type of pollution [61]. The soil enzymatic pool also acts as a catalyst for degradation of synthetics and various other natural substances [62], and hence, monitoring enzymatic activity can determine pesticide’s impact on various soil biological functions [63]. Organochlorine pesticides, some fungicides, herbicides, and molluscicides are found to be highly toxic to birds causing mortality and affecting their behavioral pattern and reproductive potential [64]. Every year on farmlands an estimated number of about 672 million birds come in direct contact with pesticides and about 10% of birds that are subjected to acute toxicity get killed [65]. Chemicals like carbamides, organophosphates, and organochlorines have been found to show a decrease in the raptorial bird population by affecting their feeding and reproduction behavior [66]. Biomagnification is another threat to the ecosystem posed by pesticides. The persistent nature and nonbiodegradability of the pesticides allow them to get concentrated in the tissues of organisms in the food chain at each successive trophic level [67,68]. Some pesticides like organochlorines have higher biomagnifications potential and hence have higher persistence than other chemicals [69]. A decline in the population of birds like herons, gulls, terns, etc. was observed as a result of eggshell thinning which was attributed to DDE (Grasman et al., 1998). A similar pattern of biomagnification like PCBs is also shown by organochlorine insecticides, such as DDT, DDD, and dieldrin with varied residual levels [70]. Similar to soil ecosystems, the impact of pesticides on the aquatic environment is diverse. The concentration of pesticides in water bodies poses a significant threat not only to humans but also to a number of biological communities. Pesticides can enter water bodies in several ways, like accidental leakage, effluents from industries, surface runoff, and runoff from pesticide-treated soils and through various industrial operations [71]. Pesticides are often transported from fields to various water reservoirs through runoff or drainage caused by rain or irrigation [72]. Globally, pesticides are known to cause greater fish mortality rates and extensive research has been carried out depicting the influence of pesticides on the dwindling fish populations [73]. Pesticides have been found to damage different body tissues and vital organs of carps having the potential to harm the

Natural compounds for bioremediation and biodegradation of pesticides

consumers as well [74]. Toxic chemicals and pesticides are found to damage olfaction in fishes which is crucial for conveying essential information for facilitating activities like mating, foraging, kinship, escaping predators, migrations, and behavioral patterns in various species of fishes [75]. Amphibians which are at much greater risk from environmental contaminants due to their permeable skin, rudimentary immune system, and dual aquatic-terrestrial cycle also get affected by pesticides [76]. Pesticides can alter the growth pattern of fungi, zooplankton, and phytoplankton while negatively affecting the amphibian population. In another study frogs (Rana pipiens) when exposed to pesticides in an agricultural area were found to be smaller in size and weighed less and were found more vulnerable to diseases and infections [77].

4. Bioremediation of pesticides The physicochemical properties of pesticides along with a diversified environmental setting significantly determine the possibility of the pesticide reaching its environmental fate. Few pesticides are found to degrade quickly in soil, while others may stay for a longer duration. After the release of pesticide in the environment, it can be broken down by photolysis, hydrolysis, oxidation, and reduction or gets metabolized by the microbiological activity of various groups of microorganisms, plants, and certain animals (Fig. 24.2). However, the microbial degradation of pesticides also known as bioremediation seems more promising and sustainable as the compounds are completely mineralized by the microbial activities. Bioremediation involves the cleaning up of the contaminated environments through microorganisms where they transform the pollutants into harmless products by mineralization, generating carbon (IV) oxide, water, and microbial biomass [78e80]. Bioremediation requires a mix of the correct temperature, nutrition, and feeds, and cleaning up toxins without all of these components can take a long time. Sometimes unfavorable conditions for bioremediation can be improved by adding molasses, vegetable oil, or simple air to the environment. The duration of the bioremediation process is determined by several factors such as the size of the polluted region, the concentration of pollutants, temperature, soil density, and so on. 4.1 Salient features of bioremediation Bioremediation is a biological process that uses living organisms, usually microorganisms (bacteria and fungi) and plants, to degrade, remove, alter, immobilize, and detoxify waste products and pollutants from soil or water [81]. Microorganisms secrete a militia of enzymes and cofactors that act as natural biocatalysts and facilitate the progress of biochemical pathways involved in the degradation of pesticides [82]. Some plant species are also able to degrade pollutants and can remove contaminants from the soil or water by absorbing them via the roots and then accumulating them in the leaves [83]. Therefore,

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Figure 24.2 Environmental fate of pesticides. (Adopted from Malhotra H, Kaur S, et al. Conserved metabolic and evolutionary themes in microbial degradation of carbamate pesticides. Front Microbiol 2021;12:1e31.)

this process is often considered as a cost-effective and sustainable method for environmental clean-up applications [84]. Being a natural and ecofriendly e process based on natural attenuation, bioremediation is highly appreciated than chemicals and physical technologies [85]. The salient features of bioremediation are as follows: 1. The majority of bioremediation treatment methods eliminate pollutants present in the soil. 2. Bioremediation procedures are intended to either destroy or reduce the harmful organic compounds into less toxic and simpler molecules. 3. Bioremediation major relies on indigenous microorganisms, such as bacteria and fungus. Occasionally, some plant species are used to improve biodegradation and stabilize the soils. 4. In certain cases, the supply of nutrients or electron acceptors (such as ozone or hydrogen peroxide) to promote the development and reproduction of local species is necessary. 4.2 Types of bioremediation Based on the location and treatment, bioremediation strategies are broadly classified as (a) in situ bioremediation, and (b) ex situ bioremediation technologies.

Natural compounds for bioremediation and biodegradation of pesticides

4.2.1 In situ bioremediation technologies These technologies involve at site application of biological organisms (bacteria, fungi, and plants) as a decontamination method to treat hazardous chemicals present in the subsurface with negligible disruption. As it does not require any excavation, in situ bioremediation is cheap as compared to its counterpart, ex situ bioremediation; however, the design and onsite installation of sophisticated equipments during bioremediation needs improvements. These technologies include biosparging, bioventing, biostimulation, bioaugmentation, biopiling, phytoremediation, composting methods, and some liquid delivery systems. The presence of moisture content, pH, electron acceptor, nutrient availability, and temperature are crucial environmental factors effective for in situ bioremediation [86]. While some of these procedures (biosparging, bioventing, and phytoremediation) could be improved, others function without any enhancements. Techniques for in situ bioremediation have proved effective for heavy metals clean up, hydrocarbons, dyes, and chlorinated solvents [87e90]. 4.2.1.1 Biosparging In this technique, air is injected into the soil subsurface at the saturated zone for stimulation of microbial activities via upward movement where volatile organic compounds move to the unsaturated zones to promote pollutant removal from contamination sites. Soil permeability and pollutant biodegradability are the two determinants of effective biosparging [86]. The operation of biosparing is similar to in situ air sparging (IAS) that involves high airflow rates for pollutant volatilization. In contrast, biosparging promotes biodegradation. Since small-diameter air injection points are inexpensive, they offer considerable flexibility and easiness for designing the system. The aquifers polluted with diesel and kerosene cleared with biosparging [91]. 4.2.1.2 Bioventing Bioventing involves controlled airflow stimulation by supplying oxygen to an unsaturated zone to boost the activity of indigenous species. By including nutrients and moisture, bioremediation is improved to enhance microbial degradation of contaminants into harmless products [86]. This method is applied to clean up areas where light petroleum products have been spilled [92]. The movement of volatile compounds to the soil vapor phase caused by the high airflow rate necessitates off-gas treatment of the resultant gases before discharge into the air [93]. 4.2.1.3 Biostimulation Crude oil contamination creates an imbalance among carbon, nitrogen, and phosphorus content at the polluted site, and its biodegradation in the soil can be limited by many factors, including nutrients, pH, temperature, moisture, oxygen, soil properties, and contaminant presence [94,95]. To mitigate this problem, the addition of one or more

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limiting nutrients or biosurfactants is made to the system to accelerate the degradation rate by indigenous microorganisms capable of bioremediation which is called biostimulation. This technique may be applied to open large water bodies and closed systems of soil and storage tanks by including several types of limiting nutrients and electron acceptors. The limited nutrients include phosphorus, nitrogen, oxygen, or carbon (e.g., in the form of molasses), which are available in low quantities [96]. Nitrogen and phosphorus-enriched fertilizers are commonly used in bioremediation of polluted soils [97]. The various feasible environmental factors are used to speed up microbial growth and the metabolism rate [98,99]. The most important strategic path is the presence of pollutants that act as stimulant or inducers by turning on the particular operons responsible for bioremediation [100]. Since nutrients are the fundamental building blocks of life and enable microbes to produce the basic requirements such as energy, cell biomass, and enzymes. The addition of oxygen, nutrients, and electron donors/acceptors significantly increase the population as well as activity of microorganisms involved in bioremediation [101]. The main advantage of biostimulation is that bioremediation will be carried out by an already present indigenous group of organisms that are well suited and well distributed within the subsurface. The delivery of the additives to be readily available to subsurface microorganisms makes the technique of biostimulation challenging because it is based on the local geology of the subsurface and its tight, impermeable lithology makes it difficult to spread additives throughout the affected area. The even distribution of additives in the subsurface is also prevented by the fractures and creates preferential pathways in the subsurface which additives preferentially follow. The addition of nutrient supplements may also promote the growth of heterotrophic microbes which are not innate degraders of pollutants, thereby creating a cut-throat competition between the indigenous microorganisms [102]. 4.2.1.4 Bioaugmentation Bioaugmentation involves the selection and introduction of an indigenous microbial consortium for the treatment of contaminated soil or water [103]. This technology is most commonly used in municipal wastewater treatment to restart activated sludge bioreactors and the cultures available contain research-based consortium of microbial cultures, containing all necessary microorganisms to ensure that the in situ microbes can completely degrade the pollutants [104]. Bioaugmentation has been proposed as a potential strategy to remediate oil-contaminated sites. The rationale for the addition of lab-cultured or genetically engineered microbes is the lacunae or inefficiency of the indigenous microorganisms to degrade pollutants [105]. In this process, microorganisms are collected from contaminated sites, cultured in vitro, engineered through genetic techniques, and then applied to the contaminated environments, where they act as bioremediators to quickly and totally eliminate complex pollutants. The use of genetically modified organisms can increase the degradative efficiency of various environmental

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pollutants as these organisms have diverse metabolic profiles to change into less and harmless end products [106,107]. The genetically modified microorganisms have shown promising results for bioremediation of several chemical and physical pollutants [108,109]. The primary factor in the speed of decontamination is that seeding may reduce the lag period to start the bioremediation process [110]. To make the approach successful at the site, the seed microbes must be able to degrade pollutants, maintain genetic stability and viability, survive in exotic environments, effectively compete with indigenous microorganisms and move efficiently through the pores of the sediment to the contaminants [111,112]. 4.2.1.5 Biopiling Biopile technology involves the mixing of additives with excavated soil and then put on the treatment area, under forced aeration. Typically, treatment lasts 3e6 months [113]. The contaminants are converted into carbon dioxide and water. The fundamental components of the biopile treatment include aeration system, treatment bed, irrigation/ nutrient supplementation system, and leachate collection equipment. To facilitate and enhance biodegradation, temperature, nutrients, oxygen, and pH are all provided in a controlled manner. For the air and nutrient transfer, the irrigation/nutrient system is buried beneath the soil. Soil piles can reach a height of 20 feet and can be covered with plastic to control runoff, evaporation, and volatilization, and to encourage solar heating. If soil contains volatile organic compounds, the air leaving the soil may be treated to eliminate or destroy the VOCs before they are released into the atmosphere. 4.2.1.6 Phytoremediation Phytoremediation uses plants to absorb and eliminate toxic elements from the environment or to reduce the bioavailability of those contaminants in the soil [114]. Plants perform these activities through their root systems, which can absorb ionic substances from the soil, even in lower quantities. Plants extend their root systems into the soil matrix and create rhizosphere ecosystems, which stabilize soil fertility and allow for the reclamation of contaminated soil [115]. To date, several plant species have been identified that have a natural capacity to remove organic and elemental contaminants from contaminated environments (Table 24.1). There are several benefits to using phytoremediation: (i) economic viability because phytoremediation is an autotrophic system powered by solar energy; it is easy to manage and has low installation and maintenance costs; (ii) environment and eco-friendly; it reduces exposure of contaminants to the environment; (iii) applicability; it can be applied over a large-scale field and can be disposed of easily; (iv) it prevents erosion and metal leaching; (v) it can increase soil fertility via organic matter [116]. The various subprocesses of phytoremediation are phytoextraction, phytofiltration, phytostabilization, phytovolatilization, and phytodegradation.

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Table 24.1 Plants with phytoremediation potential. S. no.

Plant

Nature of pollutant

References

1 2

Ludwigia octovalvis Aegiceras corniculatum

[117] [118]

3 4 5

Spartina maritime Arundo donax Eichhorina crassipes

Gasoline Brominated diphenyl ethers (BDE-47) As, Cu, Pb, Zn Cd and Zn Heavy metals (Fe, Zn, Cd, Cu, B, and Cr) PAHs Pb Anthracene and fluoranthene Diesel Polychlorinated biphenyls Ni TPH Agþ Pb

6 7 8 9 10 11 12 13 14

Phragmites australis Plectranthus amboinicus Luffa acutangula Dracaena reflexa Sparganium sp. Amaranthus paniculatus Rizophora mangle Arabidopsis thaliana Carex pendula

[119] [120] [121] [122] [123] [124] [125] [126] [127] [128] [129] [130]

PAH, polyaromatic hydrocarbons; TPH, total petroleum hydrocarbon.

4.2.1.6.1 Phytoextraction Phytoextraction is the process by which plant roots translocate and accumulate contaminants from soil or water into above-ground biomass, or shoots, which are then harvested [131]. Because harvesting root biomass is typically impractical, pollutant transfer to shoots is a vital biochemical activity desired for good phytoextraction [132]. Plants that collect substantial quantities of contaminants during their lifetimes can be used in continuous phytoextraction [133]. Phytoextraction involves four main steps: mobilization of the pollutant in the rhizosphere, pollutant uptake by roots, translocation to aerial tissues, and finally pollutant sequestration [134]. The soil depth available for the growth of plant roots, season, and climatic conditions are the vital parameters for efficient phytoextraction [135]. Furthermore, the efficiency of phytoextraction can be improved with additives such as citric acid, ethylenediaminetetraacetic acid, nitrilotriacetic acid, aminopolycarboxylic acids, and ethylenediaminedisuccinic acid [136]. Phytoextraction is primarily used to remove inorganic pollutants from polluted media because it is highly cost-effective; less disruptive to the soil and environment; requires no disposal sites, excavation, and transportation of contaminated media; and enjoys high public acceptance [136]. 4.2.1.6.2 Phytofiltration Rhizofiltration, or phytofiltration, is the process of absorbing contaminants into the root zone or adsorbing them onto plant roots from a solution [137]. Due to the fact that some plants may have several phytochelatins to boost the binding capacity of pollutants like metal ions, its mechanism is connected to the creation of

Natural compounds for bioremediation and biodegradation of pesticides

specific chemicals inside the roots, which result in the adsorption of pollutants [138]. Rhizofiltration is directly related to effluents, polluted streams, or groundwater systems. A thorough knowledge of the pollutants, their interactions and impact on nutrients is necessary for rhizofiltration to be successful. An ideal plant for rhizofiltration must possess rapidly growing plant roots and be able to remove pollutants continuously from solutions [139]. Terrestrial plants are frequently used in phytofiltration due to their vast root systems and fibrous roots, which enable them to pull pollutants from the groundwater as well as the rhizosphere [134,137]. 4.2.1.6.3 Phytostabilization Phytostabilization is another method that prevents mobility and entry of pesticides into the food chain or leaching into groundwater through different mechanisms, such as root adsorption or the formation of insoluble compounds in the root zone [133,137]. The ultimate goal of phytostabilization is to stabilize pollutants rather than remove them, so reducing their impact on human health as well as environment with a hope that the plants would act similarly to soil amendments [140]. However, phytostabilization cannot be considered a long-term solution for pesticide degradation because it only lowers the contamination of neighboring media/areas, while retaining the concentration of pollutants [82]. Nonetheless, phytostabilization offers many advantages over other remediation procedures, since the process mostly maintains toxins in the roots with limited translocation to the shoots, reducing the need to treat the aerial portions. When quick immobilization is required to protect ground and surface waters, it can be used as a very promising concept [141]. Phytostabilization has been recognized as one of the promising strategies of phytoremediation because it stabilizes lead, chromium, and mercury in the soil and prevents the interaction of these metals with soil biota [136]. 4.2.1.6.4 Phytovolatilization Phytovolatilization uses plants to absorb pollutants, convert them into volatile compounds, and then release those compoundsdeither in their original form or with slight modificationsdinto the atmosphere as a result of the plant’s metabolic and transpiration forces [137]. Transpiration of water from leaf surfaces occurs via stomata. Some plant species with deep roots frequently have the capacity to absorb and breakdown pollutants by producing particular enzymes or genes [131]. Pollutants are absorbed from the soil or water during phytovolatilization and transformed into less harmful vapors that are later discharged into the atmosphere by plant transpiration [82]. Till now only a few species of plants have been recognized that can naturally convert metals into volatile compounds. Therefore, the phytovolatilization technology is enhanced via the genetic engineering of plants to volatilize metals [82]. However, its use is constrained by the fact that the process merely transfers the contaminant from one environmental compartment (soil/water) to another (atmosphere), from which it is likely to precipitate with rainfall and then return to the ecosystem. Consequently, the pollutants are not completely removed from the environment [134].

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4.2.1.6.5 Phytodegradation According to Ref. [141]; phytodegradation, also known as phytotransformation, is the process of capturing pollutants and nutrients from the water, sediment, or soil and then chemically modifying them through plant metabolism. This frequently leads to contaminant inactivation, degradation, or immobilization in both plant roots and/or shoots. Some plants can use their metabolic processes or enzymes to convert the ingested toxins into less hazardous molecules [131]. Thus, phytodegradation is a metabolic process used by plants to detoxify and break down pollutants inside the plant cells [136]. 4.2.2 Ex situ bioremediation The ex situ bioremediation procedures include the biological treatment of contaminated media at a site other than the original one. Excavated soil is deposited in a lined aboveground treatment area and is then aerated to increase the rate of pollutant degradation by microorganisms. Bioreactors, biofilters, land farming, and composting techniques are some of the examples of ex situ bioremediation. 4.2.2.1 Bioreactors The bioreactor approach is used to treat polluted soils in both the solid and liquid (slurry) phases. It uses microorganisms to remove contaminants from wastewater or pumped groundwater. Apart from the exposure of medium to bioremediation, the idea is principally the same as biodegradation [142]. Slurry bioreactors are among the most effective ex situ methods utilized in the treatment of polluted soils by refractory contaminants under controlled environmental conditions. They are also among the most highly constructed bioremediation systems. Slurry bioreactors have been used extensively in several bioremediation applications and feasibility studies as full-scale equipment [143]. 4.2.2.2 Composting Composting, one of the widely used ex situ bioremediation strategies, is highly preferred for the removal of hydrocarbons from soil. Recently, it has been effectively used to treat soils that have been polluted with hydrocarbons [144]. Composting also involves the microbial breakdown of organic wastes into harmless, and stable byproducts. It is often used to treat agricultural, municipal, and other solid wastes, as well as sewage sludge into beneficial soil additives like hummus. 4.2.2.3 Landfarming Land treatment, sometimes known as land farming or land application [145], has been commercially applied at a broad scale with reasonable success. Although it is a century-old technique, it is still being applied in the oil industry [146]. Due to the low equipment requirements, landfarming is particularly beneficial for distant locations

Natural compounds for bioremediation and biodegradation of pesticides

and remote areas. It typically employs passive aeration by tilling or ploughing the polluted soil while exposing the pollutants to the sun for photocatalytic mechanisms [147]. 4.2.2.4 Biofilters This method works by immobilizing microorganisms on a solid substrate to breakdown pollutants found in air emissions. Air pollutants that are passed through biofilters are successively destroyed because they become adsorbed onto microbial biofilms. Sulfur gases, hydrogen sulfide, ethyl benzene, dimethyl sulfides, nitrous oxide, etc. are all removed from the air by using biofilters.

5. Types of pesticide bioremediion based on the type of microbes/ enzymes 5.1 Fungal biodegradation of pesticides Fungi are of paramount importance in biogeochemical cycles for mineralizing various metals and chemicals. Additionally, they degrade a variety of environmental xenobiotics, including pesticides [148]. The structure of sprayed pesticides and other resistant substances is frequently altered by fungi in the environment, which releases certain biotransformation products that are absorbed and assimilated by other microbes living in soils [149]. Table 24.2 enlists some of the potential fungal strains used for the biodegradation of pesticides. 5.2 Bacterial degradation of pesticides Bacteria have been extensively used for the bioremediation of pesticides in the environment due to their fast growth rate, low-cost, ease to handle, and manipulation through genetic engineering. Since pesticides demonstrate mild to serious effects on the surrounding organisms, including bacteria, several potential pesticide-degrading microorganisms have been isolated from different environments [25]. In soils, pesticides are metabolized and transformed by many species of bacteria. Some bacterial cultures can even metabolize highly persistent chemicals as energy source and nutrients or co-metabolism with other substrates for their development. Co-metabolism involves the chemical conversion and breakdown of most resistant substances by bacterial strains whose growth conditions are unfavorable. The biotransformation capacity of microorganisms to degrade pesticides can be applied in sustainable agriculture while reducing the deleterious impacts of these chemicals on farmers’ health. Pesticides are more efficiently biodegraded by consortia of microbes than single isolates. Mineralization of pesticides by microorganisms is currently the best possible and widely accepted approach for sustainable agriculture. However, the level of bacterial metabolism, which is positively reducing pollutants in the environment, determines the persistence of pesticides in the soil. Despite the biodegradation of most pesticides by some potential bacteria or microbial consortia, certain

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Table 24.2 Fungi as bioresource for bioremediation of pesticides. S. No.

Fungal strain

Pesticide

References

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Geastrum Fusarium solani F. verticillioides Penicillium oxalicum Aspergillus niger Pleurotus ostreatusto Verticillium sp. Ganoderma sp. Mucor racemosus Pseudomonas aeuroginosa Aspergillus vesicolor Trichoderma harzianum A.niger Lentinus subnudus Phlebia acanthocystis P.acanthocystis Bjerkandera adusta Pseudotrametes gibbosa Trametes vesicolor Anthrocophyllum discolor

Lindane Lindane Lindane Methamidophos Endosulfan DDT Chlorpyrifos Chlorpyrifos Dieldrin Cypermethrin Triclosan Pentachlorophenol b-Cypermethrin Atrazine Aldrin Heptachlor Parathion Pyrene Imiprothrin þ Cypermethrin Pentachlorophenol

[150] [151] [152] [153] [154] [155] [156] [157] [158] [159] [160] [161] [162] [163] [164] [165] [166] [167] [168] [169]

agrochemicals are recalcitrant and thereby persist in the environment. Consequently, several regulations are devised by the national environmental protection agency (NEA) that prohibits the overuse of such persistent chemicals in agriculture. Moreover, formulation of the species-specific and biodegradable pesticides is paramount. This approach may significantly reduce the pesticide residues in soil and water. Additionally, the farmers need to be educated about the proper usage of pesticides and their toxicity. To date, numerous pesticide-degrading microbial species have been isolated from various environments. Actinobacteria, Ascomycota, Bacteroidetes, Basidiomycota, Cyanobacteria, Chlorophyta, Proteobacteria, and Firmicutes have all been discovered to be the finest sources for the breakdown of Carbamate, Organochlorine, Organophosphate, and Pyrethroids pesticides (Table 24.3). Many species of bacteria such as Bacillus [170,171], Burkholderia [172], Methylobacterium [173], Micrococcus [174], Paenibacillus [175], and Pseudomonas [176] are known to degrade extremely toxic pesticides (Phorate), highly (Carbofuran), or moderately hazardous (Endosulphan), besides some low toxicity chemicals (Malathion). The precise impact of pesticides on agriculture and the reactions of pesticide-degrading bacteria (PDB) on crop yield are still elusive. Hence, the bioremediation of pesticides through bacteria requires knowledge of the microbial dynamics, their effects on the ecosystem, and consequent changes in the natural environments.

Table 24.3 List of the potential bacteria, their sources, and degradation of different pesticides. Source

NCBI accession number

Cupriavidussp. DT-1

Sludge

JQ750642

Diaphorobacter polyhydroxybutyrativorans PDB6 Achromobacter spanius PDB2

Soil

NA

Soil

NA

Bacillus cereus

Agricultural soil

MH341691

Bacillus sp. Mes11 Anabaena sp. Nostoc sp. Pseudomonas sp. Serratia marcescens Ochrobactrum sp. Pseudomonas sp. Psychrobactersp. Castellaniellasp. Variovoraxsp. Pseudoxanthomonas japonensis Burkholderia sp. Enterobacter aerogenes S. marcescens S. liquefaciens P. frederiksbergensis B. gladioli B. pumilus

Soil Water Soil Soil Soil Sewage sludge Sewage sludge Sewage sludge Sewage sludge Sewage sludge wheat fields Sludge Sewage sludge Soil Soil Soil Soil Soil

EU864320 NA NA NA NA DQ976365 DQ976368 DQ976366 DQ976371 DQ976367 KF279695 AY550913 NA NA NA NA NA NA

Pesticide degraded

References

Chlorpyrifos, 3,5,6-trichloro2-pyridinol (TCP) Carbofuran, Emamectin Benzoate, and Thiamethoxam Carbofuran, Emamectin Benzoate, and Thiamethoxam cypermethrin, imidacloprid, fipronil, sulfosulfuron Mesotrione Fenamiphos Fenamiphos Diazinon Diazinon Igepal CO-210 Igepal CO-210 Igepal CO-210 Igepal CO-210 Igepal CO-210 Isoproturon Fenitrothion Bifenthrin Diazinon Malathion Malathion Dimetoate Dimetoate

[193] [194]

[194]

[195] [189] [187] [187] [190] [190] [185] [185] [185] [185] [185] [196] [197] [198] [188] [188] [188] [188] [188] Continued

Natural compounds for bioremediation and biodegradation of pesticides

Bacteria

463

464

Bacteria

Source

NCBI accession number

Pesticide degraded

References

Acinetobacter radioresistens Rhodococcus koreensis S1-1 Stenotrophomonas sp. Ochrobactrum sp. Providencia stuartii MS09 Micrococcus sp. P.diminuta, P. aeruginosa, P. putida Achromobacter xylosoxidans C8B Enterobacter sp. Sphingobiumsp. YBL2 Achromobacter sp. Ralstoniasp. DI-3 Arthrobacter sp. Sphingobium sp. Paenibacillus polymyxa

Soil Soil Soil Soil Soil Diuron storage Soil

NA AB823727 NA AY661464 NA GQ284587 NA

Chlorpyrifos Endosulfan DDT Methyl parathion Chlorpyrifos Diuron Methyl parathion

[188] [199] [200] [201] [202] [182] [203]

Soil Soil Soil Soil Agricultural soil Soil Sewage sludge Microbial culture collection center Soil Microbial culture collection center Soil Soil Soil Water Soil Soil Coral Soil Soil

HQ426648 AJ639856 EU159275 NA KM051530 AY913770 NA NA

Endosulfan Chlorpyrifos isoproturon Glyphosate Diazinon Endosulfan Methyl parathion Cyanophos and malathion

[204] [205] [206] [191] [207] [192] [186] [175]

NA NA

Chlorpyrifos Cyanophos and malathion

[153] [175]

HQ260890 KC577853 NA HQ164550 NA NA NA NA NA

Carbofuran OP1 quinalphos I8 Carbofuran Chlorpyrifos Endrin, aldrin, DDT Chlorpyrifos Chlorpyrifos Chlorpyrifos Chlorpyrifos

[208] [209] [170] [210] [174] [211] [212] [171] [213]

B. licheniformis ZHU-1 Azospirillum lipoferum Bosea sp. HC6 B. thuringiensis B. thuringiensis B. subtilis Y242 Bacillus sp. 4585 B. pumilus C2A1 B. firmus by 6 B.cereus DH Mesorhizobium sp. HN3

New Horizons in Natural Compound Research

Table 24.3 List of the potential bacteria, their sources, and degradation of different pesticides.dcont’d

Rhizosphere wastewater sludge o Agricultural soil Soil

JQ514559 HM146925 KX518600 KP890249

Chlorpyrifos Acephate N-methylcarbamate Triazole

[214] [173] [215] [216]

Soil Agricultural soil Soil

KY646471 HQ260907 NA

Chlorpyrifos Carbofuran Carbofuran

[176] [208] [217]

Soil Soil waste water

LC099979 KX530865 NA

Propiconazole 2,4-Dichlorophenoxyacetic Methyl parathion

[172] [218] [219]

Agricultural soil Soil Soil Soil Soil Soil Soil Soil Soil

ATCC13883T FN600408 e FN600409 FN600411 KY646473 KY646475 AB571066 HQ426648 NA

Carbofuran phenol Oxamyl Oxamyl Oxamyl Chlorpyrifos Chlorpyrifos Diazinon Endosulfan sulfate Carbofuran

[220] [221] [221] [221] [176] [176] [222] [204] [217]

Soil

NA

DDT

[223]

Soil and sewage

NA

Hexachlorocyclohexane

[224]

Soil

NA

Triazine

[225]

DDT, Dithiothreitol; NA, not available; OP, Organophosphate; TCP, Trichloro propane.

Natural compounds for bioremediation and biodegradation of pesticides

Ochrobactrum sp.JAS2 Methylobacteriumsp.YAL-2 Pseudaminobactersp. SP1a Shinella sp. NJUST26 1H1,2,4Rhizobium sp. 4H1-M1 Sphingomonas sp. CN1 SphingomonaspaucimobilisS8M3-23 Burkholderia sp. BBK_9 Cupriavidus gilardii T-1 Acinetobacter radioresistens USTB-04 K. pneumoniae 13883T P. extremaustralis OXA17 P. monteilii OXA18 P. jinjuensis OXA20 Pseudomonas sp. 4H1-M3 Xanthomonas sp. 4R3-M1 S. marcescens DI101 A. xylosoxidans C8B Yersinia pseudotuberculosis S1M1-15 Pseudomonas sp., Neisseria sp., Moraxella sp., Acinetobacter sp. Pseudomonas sp., Burkholderia sp., Flavobacterium sp., Vibrio sp. Rhodococcus sp.

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The conventional methods like enrichment, isolation, purification, and screening of pesticide-degrading bacteria may be accomplished in different growth media. For the identification of distinct culturable microbial communities capable of degrading pesticides, different particular growing media and enrichment procedures can be employed. These ubiquitous and selective media may be used to isolate pesticide-degrading microorganisms from a variety of environments including extreme and cryptic habitats such as insect gut systems [177e181]. One gram of the soil contaminated with organophosphorus, carbamates, organochlorine, or pyrethroids could be supplemented with particular growth media and incubated for 3e5 days at desired temperatures while agitating at 150e200 rpm. For further purification, several techniques like serial dilution could be used for the isolation of particular bacteria. Afterward, individual colonies could be picked up, then purified to get contamination-free bacterial culture, which can be further subjected to different biochemical tests and characterization for pesticide degradation. Recently many studies on the isolation, identification, and characterization of pesticide-degrading microorganisms have been published. Pesticide-degrading microorganisms have been identified from a variety of ecosystems, including diuron storage [182], marine sponge [183], sewage sludge [184] [185,186], soil [187,188], water [187], etc. Earlier, the authors in Ref. [174] identified and characterized dieldrindegrading microbial populations that showed high catabolic activity for aldrin, endrin, benzenehexachloride (gamma isomers), DDT, and Baygon. From the screening tests, the authors successfully 13 pesticide degradation isolates that can degrade aldrin specifically, whereas all other bacteria could mineralize DDT and endrin. Similarly, the chlorinated hydrocarbon-based insecticides have been extensively used in the agriculture during the previous two decades. Consequently, these pesticides and their metabolites have substantially accumulated in the soil. However, it is astonishing that the microbial degradation of these compounds by bacteria is also reported in the literature. The bacterial species that are able to degrade these chemicals include Arthrobacter sp. 2787, Bacillus sp. 4585, Micrococcus sp. 204, Pseudomonas sp. 27 [182,188e192]. The authors in Ref. [205] used several molecular and biochemical techniques to characterize six chlorpyrifos degrading bacterial isolates including Enterobacter asburiae from the Enterobacteriaceae family. When the bacteria were tested for chlorpyrifos breakdown under different growth conditions, they were found to show potential degradation of the pesticide using it as carbon resource. The molecular analysis of the bacterium revealed that pesticide degradation is linked with chromosomes and polygenes. When compared to non-inoculated soil samples, soils supplemented with chlorpyrifos degrading bacteria enhanced bacterial proliferation as well as the rate of pesticide decomposition. These observations highlighted the utility and application of the bacterium for cleaning polluted and pesticide-contaminated environments. The authors in Ref. [190] isolated three bacterial strains, namely Serratia marcescens, Serratia liquefaciens, and Pseudomonas sp., that can degrade diazinon in soils. The bacteria were isolated on methylotrophic specific mineral

Natural compounds for bioremediation and biodegradation of pesticides

salt medium (MSM) by using pesticide diazinon (50 mg/L) as substrate. The bacteria degraded 80%e92% of the pesticides within 14 days of initial treatment showing predominant microbial activities. Therefore, these robust bacterial strains can be applied in diazinon-rich soils for microbial remediation. The authors in Ref. [203] isolated 21 bacterial isolates that showed the degradation of methyl parathion. The isolates, Pseudomonas putida (EMP12a), Pseudomonas diminuta (EMP11c), and Pseudomonas aeruginosa (EMP12b), demonstrated maximum degradation of the compound. In a similar study, Rhodococcus koreensis strain S1-1 has been isolated from the endosulfan enriched soil [199]. The bacterium showed significant degradation of the endosulfan from 12.25 to 2.11 M in just 14 days. The authors in Ref. [196] also described an isoproturon (IPU) degrading bacterium Pseudoxanthomonas japonensis from the wheat fields that exhibited better activities at pH 7.0 and 30 C. Whether under in vivo or in vitro conditions, the biodegradation of pesticides is catalyzed by enzymes majorly from the microbial world. Biodegradation involves alteration in the structure of chemical substances, which changes the specific properties of the compound. Biodegradation of chemicals causes the elimination of unwanted qualities, as well as their oxidation and reduction. Microbes are known to reduce and eliminate dangerous pesticides to a greater extent in the natural environment. The degradation of pesticides is a complicated process that involves a series of chemical reactions. The biodegradation pathways of many pesticides are still elusive and therefore require more research to fully comprehend the process and the role of enzymes. Actinomycetes are Gram-positive bacteria with high GC contents that can degrade various synthetic pesticides having unique chemical structures such as triazinones, striazines, acetanilides, organochlorines, organophosphates, sulfonylureas, carbamates, and organophosphonates. The efficient degradation of xenobiotic pesticides requires a consortium of bacteria. In a consortium, the pesticides are typically co-metabolized by the microbes frequently. Although there is a plethora of information available about pesticide breakdown by Gram-negative bacteria, the data related to pesticide degradation by actinomycetes is scanty. Actinomycetes can also be used in bioremediation and transgenic pesticide-resistant crop development. The group actinobacteria include the members like Arthrobacter, Microbacterium, Nocardioides, Micrococcus, Streptomyces, and Rhodococcus, which can degrade different pesticides efficiently. The Streptomyces sp. M7, isolated from organochlorine-contaminated sediment, is a typical example of lindane-degrading actinomycete [226]. The authors in Ref. [182] determined that Gram-positive bacterium, Micrococcus sp. PS-1 can thrive and tolerate 250 ppm concentration of phenylurea herbicide diuron using it as a source of carbon. Bacteria belonging to the genera of Pseudomonas, Neisseria, Moraxella, and Acinetobacter isolated from the Yaqui Valley in Sonora, Mexico, have been found to biodegrade DDT completely [223].

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5.3 Enzymes involved in pesticide bioremediation All the biological processes that degrade pesticides involve biocatalysts known as enzymes. The enzyme-mediated bioremediation is innovative method to remove pesticides from polluted sites and raw materials. The enzymes involved in the mineralization of pesticides have been traditionally classified on the basis of biotransformation phase (Table 24.4). The enzyme-mediated biotransformation of pesticides takes place in three phases. Phase I enzymes include the proteins that modify the functional groups of the compound. Phase II enzymes usually catalyze transfer reactions of the entire biomolecule while phase III involves translocation mechanisms causing assimilation of the pesticides. Biotransformation enzymes are divided into four categories such as oxidoreductases, hydrolases, transferases, and translocases. Monooxygenases, dioxygenases, peroxidases, and oxidases are the most common among oxidoreductases. Hitherto, numerous enzymes derived from bacteria, fungi, insects, plants, and animals have been reported to degrade pesticides. The major classes of enzymes that can degrade pesticide are described in the following section. 5.3.1 Laccases Laccases (E.C. 1.10.3.2) belong to the multicopper polyphenol oxidases thatoxidize, a variety of aromatic amines and phenolic compounds using oxygen as the terminal electron acceptor [254]. These enzymes have lately been the center of attention for the remediation of many pesticides. The major sources of these enzymes are plants, bacteria, fungi, and insects. However, microbial laccases in particular from wood-decaying fungi have received much attention because of their broad substrate specificity, and powerful antioxidant capacity [254]. Laccases catalyze the oxidation of hydrogen donating substrate with simultaneous reduction of oxygen to a water molecule [255]. The authors in Ref. [256] utilized laccase from white rot fungi to degrade dichlorodiphenyltrichloroethane (DDT) in contaminated soil samples. After 5, 10, 15, and 25 days of incubation, the concentration of compound in soil samples decreased by 21%e32%, 29%e45%, 35%e51% and 36%e51%, respectively. These enzymes have also been reported to degrade chlorophane (CP) and dichlorophen 2,20 -methylene bis(4-chlorophenol) (DCP) pesticides in aqueous environments. The authors in Ref. [227] used husks of pearl millet (PM) and finger millet (FM), for the production of laccase from Bacillus sp. which was partially purified and used for the degradation of different pesticides like Tricel and Phoskill. After incubation at 40 C and pH 7 for 5 days, the Tricel and Phoskill were degraded by 71.8  3.5 and 77.3  3.4%, respectively. Similarly, the authors in Ref. [228] used agro-waste (potato peel), for the production of thermo-alkali stable laccase (S2LAC) from Pseudomonas sp. S2. The partially purified enzyme had a molecular weight of 38 kDa and depicted a maximum specific activity of 1089.70  16.8 U m/g at 80 C and pH, 9.0. The S2LAC was able to degrade a variety of organophosphorus pesticides including dichlorophens, monocrotophos, chlorpyrifos, and profenovos to

Table 24.4 An overview of the enzymes, and their sources involved in pesticide degradation. Enzyme category

Laccases

Source

Bacteria

Fungi

Bacteria

Fungi Insects

Klebsiella sp. ZD112 Sphingobium sp. strain JZ-1 R. palustris PSB-S Ochrobactrum anthropi YZ-1 Nocardioides sp. strain SG-4G Pantoea ananatis Sd1 Aspergillus niger ZD1 Helicoverpa armigera

Accession no

Pesticide degraded

References

NA

Tricel, phoskill Dichlorophos, Chlorpyrifos Aromatic pesticides

[227] [228] [216]

Isoproturon Chlorpyrifos, profenofos, thiophanate methyl

[229] [230]

NA S2LAC pLACGy

AKN79754

NA NA

NA MH395741

TsL NA

KY234237 NA

NA lacc6, lacc9, lacc10 EstP

NA KX815352, KX815353, KX815354 AY995176

PytH

Dichlorodiphenyltrichloroethane

[231] [232]

Isoproturon chlorophenols, nitrophenols

[233] [234]

[235]

FJ688006

Pyrethroid and organophosphorus pesticides Pyrethroid pesticides

[236]

Est3385 PytZ

KU377526 JQO45333

Pyrethroid pesticides Pyrethroid pesticides

[237] [238]

mheI

GQ454795

Carbendazim

[239]

PaCes7

NA

Carbaryl pesticide

[234]

NA

NA

Pyrethroid pesticides

[240]

CarE 001G

KT345938

b-cypermethrin, l-cyhalothrin, and fenvalerate

[241]

469

Continued

Natural compounds for bioremediation and biodegradation of pesticides

Esterases

Bacillus sp Pseudomonas sp. S2 Geobacillus yumthangensis Trametes versicolor Tricholoma giganteum AGDR1 Trametes sp. F1635 White-rot fungi (polyporus) Trametes versicolor Pleurotus ostreatus HAUCC 162

Enzyme name

470

Enzyme category

Phosphatases

Glutathione Stransferases

Source

Bacteria

Bacteria

Insects

Cytochrome P450

Bacteria

Insects

Lactobacillus sakei WCP904 Agrobacterium radiobacter Bacillus thuringiensis MB497 Lactobacillus casei. 355 B. amyloliquefaciens YP6 Azohydromonas australica Spirulina platensis Klebsiella jilinsis2N3 Acidovorax sp. KKS102 Bombyx mori Helicoverpa armigera Sphingomonas bisphenolicum Rhodopseudomonas palustris Locusta migratoria

Enzyme name

Accession no

Pesticide degraded

References

opdD

NA

chlorpyrifos

[242]

opdA

NA

Organophosphate pesticides

[243]

NA

NA

[244]

NA

NA

Chlorpyrifos, triazophos, dimethoate Organophosphorus pesticides

AP3

NA

A. australica OPH NA Kj-gst

AJ605330.1.

[245]

NA NA

chlorpyrifos, dichlorvos, phoxim, and triazophos Methyl parathion, paraoxon, dichlorvos, and chlorpyrifos Chlorpyrifos Chlorimuron-ethyl

[246]

[248] [249]

BphK-KKS

NA

Polychlorobiphenyl

[250]

NA HaGST-8

NA KY780632

[251] [252]

P450bisd

NA

1-chloro-2,4-dinitrobenzene Organochloride, carbamate, and neonicotinoid Bisphenol A

CYP201A2

NA

Tributyl phosphate

[237]

YP9AQ1 and CYP9A3

JN984870 and KT716446

Pyrethroid

[250]

[247]

[253]

New Horizons in Natural Compound Research

Table 24.4 An overview of the enzymes, and their sources involved in pesticide degradation.dcont’d

Natural compounds for bioremediation and biodegradation of pesticides

different extents ranging from 45.99  0.3%, to 81.84  0.6%. In another study, the authors in Ref. [230] purified laccase from tricholomagiganteum AGDR1, which was then used to degrade different pesticides among which chlorpyrifos, profenofos, and thiophanate methyl were degraded up to 29%, 7%, and 72 % respectively within 15 h. Laccases are also found as effective decomposers of CP and DCP in the aqueous system. The action of laccases on pesticides can be expanded by using mediators. The mediators can enhance the activity of laccases using one of the following mechanisms: (1) By acting as electron transfer agents between enzyme and pesticide, where the mediator is believed to be first oxidized followed by the dissemination of oxidized form from the catalytic site of the enzyme and then oxidizes the substrate which is otherwise inaccessible to the enzyme due to large size of the substrate molecule. (2) The mediator inflates the oxidizing capability toward oxidation of higher-redox potential nonphenolic subunits in the substrate by introducing alternative reaction pathways for oxidation [229]. Recently, the authors in Ref. [257] stated efficiency of laccase to remove four different pesticides viz., oxybenzone, pentachlorophenol, atrazine, and naproxen from contaminated environmental samples after supplementation with 2,20 -azino-bis 3-ethylbenzothiazoline-6sulfonic acid (ABTS), 1-hydroxybenzotriazole (HBT), N-hydroxyphthalimide (HPI), 2,2,6,6 tetramethylpiperidinyloxyl (TEMPO), violuric acid (VA), syringaldehyde (SA), and vanillin (VAN). Among the tested mediators, HBT, ABTS, and VA resulted in the efficient removal of tested pesticides. The authors in Ref. [233] also used Trametes versicolor laccase for the removal of isoproturon in the presence of different mediators like acetosyringone, HBT, SA, ABTS, VA, and VAN. The isoproturon was degraded almost completely within 24 h using 0.3 U/mL laccase and 1 mM HBT. However, in the absence of mediator, isoproturon was hardly degraded. In another study, the organophosphorus pesticide O-ethyl S-[N, N-diisopropylaminoethyl] methylphosphonothiolate (VX), O-ethyl-S-[2-(diisopropylamino)ethyl]phenylphosphonothioate (PhX) and RVX were effectively degraded using purified T. versicolor laccase in the presence of ABTS [258]. Recently, the authors in Ref. [259] designed an optimized laccasemediator system for the degradation of six different pesticides, i.e., bentazone, carbofuran, clomazone, diuron, pyraclostrobin, and tebuconazole using laccase from T. versicolor. In this context, seven different redox-mediating compounds were tested, including caffeic acid, ABTS, p-coumaric acid, gallic acid, chlorogenic acid, ferulic acid, protocatechuic acid, and vanillin. Among the tested laccase-mediator systems, the laccase-vanillin system showed the highest degradation efficiency of 77% for all the pesticides. All these studies demonstrate that laccase-mediator systems have obviously better degradation capability compared to when laccase enzyme is used alone for degradation. However, the use of synthetic mediator-based enzyme systems is hampered by higher process costs and release of toxic molecules in the environment. Therefore, the use of natural mediators must be encouraged, the phenolic compounds generated during lignin degradation could provide efficient and green alternatives in this regard [260].

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5.3.2 Esterases Esterases (EC 3.1.1.1) are a large group of enzymes belonging to the hydrolasesuperfamily [261]. Esterases catalyze the cleavage and formation of ester bonds and have been stated to degrade the ester bonds containing pesticides like organophosphate, carbamate, and pyrethroid. Esterases belong to a/b superfamily of proteins [261]. The catalytic site of these enzymes contains acatalytic triade, serine, histidine, and glutamine, which confer catalytic activity during pesticide degradation. The first step of the catalytic mechanism involves the acylation of serine residue which results in the synthesis of acylated intimation and an alcohol molecule (from carboxyl ester) [262]. The reaction intermediate is hydrolyzed by the external water molecule which acts as a nucleophile to release the product from the enzyme. Pesticide degrading esterases have been reported from several microorganisms, plants, and animals. Most of the microbial esterases have been isolated from bacteria while only a few exist in fungi. The first microbial esterase was isolated from Bacillus cereus SM3 [263]. Since then, pesticide degrading esterases have been reported in a wide variety of microbes like Aspergillus niger ZD 11 [240], Sphingobium sp. JZ-1 [236], Klebsiella sp. ZD112 [216], Rhodopseudomonas palustris PSB-S [237], and Pseudomonas synxantha PS1 EstPS, [264]. Aspergillus niger ZD11 esterase was purified with approximately 41.5-fold homogeneity, a molecular weight of 56 kDa, and pI. 5.4. At optimum conditions of pH 6.5 and temperature of 45 C, the purified enzyme successfully hydrolyzed various pesticides sharing similar structures [240]. Besides microbes, insects have gained much attention recently for the isolation of enzymes capable of degrading pesticides. The unregulated use and mismanagement of pesticides has resulted in the development of resistance in some insects toward pesticides. The pest Helicoverpa armigera wreaks havoc on agricultural crops due to its resistance development against many pesticides in particular to pyrethroids [179,181,265]. The authors in Ref. [266] revealed that pyrethroids could be catabolized into low molecular weight compounds using homogenate from the midgut of H. armigera. Recently, the authors in Ref. [241] expressed the carboxylesterase gene from H. armigera in E. coli, and the protein (CarE 001G) was purified. The purified CarE 001G could degrade b-cypermethrin, l-cyhalothrin, and fenvalerate, displaying specific activities of 1.7, 1.4, and 0.5 nM/min/mg protein, respectively. Using transcriptomic and genomic studies, approximately 39 carboxylesterase genes have been recognized in the house fly, Musca domestica, and 11 of these genes have been identified to be upregulated during pyrethroid degradation [267e271]. However, these genes have not been expressed and are yet to be purified. In addition, numerous insects have developed resistance against pyrethroid pesticides such as Culexpipiens, C. quinquefaciatus [272], and Locusta migratoria [273]. The main reason behind pyrethroid resistance in these insects is the upregulation and overexpression of pyrethroid hydrolase genes. Some of these genes have been expressed in E. coli where corresponding proteins have been purified and utilized for pesticide degradation.

Natural compounds for bioremediation and biodegradation of pesticides

Carboxylesterases have been reported from animals also including humans that help in the detoxification of different exogenous and endogenous chemicals [274]. In humans, carboxylesterases are involved in xenobiotic degradation and maintenance of human health. Two predominant carboxylesterases viz., CES1 and CES2 are found in humans to carry that degrade different pesticides [274]. The authors in Ref. [275] evaluated the potential carboxylesterase hCE-1 and hCE-2 from the human liver for pyrethroids degradation and found that permethrin and cypermethrin were degraded effectively however, the rate of degradation for trans-isomers was faster compared to the corresponding sis-isomers. 5.3.3 Phosphatases Phosphatases (EC 3.1.3.1) are another group of enzymes that belong to the hydrolase superfamily. These enzymes have recurrently been used to eliminate phosphate groups from numerous organic compounds such as organophosphate pesticides (OP), alkaloids, proteins, and nucleotides [246]. Phosphatases like organophosphorus hydrolase (OPH), organophosphate acid anhydrolases (OPAA), and methyl parathion hydrolase (MPH) have mostly been isolated from microorganisms including bacteria which have demonstrated very good activity for OP pesticide degradation. OPH is one of the first phosphatase enzymes that was isolated from Enterobacter sp. and used for the hydrolysis of different organophosphorus compounds by breaking potassium-sulfate and potassium-oxygen bonds [276]. The OPH-like enzyme named OpdA has been isolated from Agrobacterium radiobacter which was also found effective for the hydrolysis of OP pesticides [243]. The enzymes like OPH and OpdA share the same secondary structures; however, their substrate specificities are different because of the difference in their catalytic sites [244]. Recently, the authors in Ref. [244] investigated Bacillus thuringiensis MB497 for the production of intra/extra-cellular phosphatases. The bacterial strain was found to produce a substantial amount of extracellular phosphatase. The enzyme showed 81%e94.6% degradation of three OP pesticides viz., Chlorpyrifos, Triazophos, and Dimethoate when the pesticides were used at 50 mg/L concentration for 30 min of incubation. 5.3.4 Glutathione S-transferases (GSTs) Glutathione S-transferases (EC 2.5.1.18) comprise a large family of enzymes that can hydrolyze a variety of xenobiotic compounds including pesticides. These enzymes are found in almost all living organisms including plants, animals, bacteria, and fungi [277]. Depending on the location in the cell, GTs can be categorized into three different types viz., cytosolic, mitochondrial, and microsomal GSTs, whereas bacteria contain an additional class called as bacteria specific fosfomycin-resistance protein [277]. These enzymes carry out detoxification via conjugation of hydrophobic components present in the pesticide with the glutathione group of the enzyme. The recombinant GSTs from E. coli [278], Klebsiella jilinsis 2N3 [249], Acidovoraxsp. KKS102 [250], and Bombyx mori

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[251] have been successfully used for the detoxification of OP, demonstrating their potential for bioremediation. The long-time exposure of pesticides has also led to resistance in these pests which is thought to be because of the synthesis of different enzymes including GST in these insects. Numerous GSTs have been reported from insects having significant detoxification capacity for OP including H. armigera [253], Plutella xylostella, and Galleria mellonella [118]. 5.3.5 Cytochrome P450 (CYP) The CYP-450 superfamily of proteins includes heme-containing monooxygenases that can catalyze oxidative/reductive or oxidative hydrolysis of several xenobiotic compounds. These enzymes are found in viruses, bacteria, fungi, and animals [235]. The fungi Trichoderma has been extensively used for the degradation of xenobiotic compounds including xenobiotic pesticides. The bioremediation potential of the fungus, Trichoderma sp. to degrade xenobioticsis primarily attributed to the CYP-450 proteins. Recently, the authors in Ref. [279] cloned 39 cytochrome P450 encoding genes from T. atroviride T23 genome and among these genes, 21 were reported to be involved in the xenobiotic compound degradation.

6. Conclusions and future perspectives The present study highlights the innovation of diverse natural products, particularly from microbial and animals resources as agents of pesticide degradation and bioremediation with an emphasis on enzymes that remove/reduce the obnoxious effect of prevailing pesticides in the environment. Unlike the conventional methods, the biological methods produce little to no residues, especially under laboratory conditions. Although a plethora of studies have emphasized microbial degradation of pesticides with many innovations, only a few studies have passed practical application in natural environments. This may be due to the rapidly fluctuating ecological variables in the pesticide-contaminated environment, and the poor understanding of the cryptic interacting influence of the biotic communities on the degradation pathway or process in the natural environment. The logical possibilities of unanticipated variable reactions (synergistic, suppressive, displacement, disruptive, etc.), between cell receptors and ligands, introduced and native compounds, and pesticide and extracellular polymeric substances reactions including enzymes may have also accounted for the field or large-scale applications of these methods. Even though there is a dearth of information correlating naturally occurring genetic transformation processes with pesticide biodegradation, it may be assumed to interfere with the application of bioremediation and biodegradation in the open environment. Consequently, more studies are therefore required to increase understanding of natural variables that affect the application of these methods to pesticide removal in the environment. With the advent of new technologies, a continuous bioprospection of potential microbes, and integration of engineering,

Natural compounds for bioremediation and biodegradation of pesticides

bioinformatics, and machine learning approaches with affordable cost implications might enhance the versatility, amenability, and efficiency of the practical application of bioremediation via bacterial agents in the next few decades. However, complementing this intuitive prospect with the emergence of enforceable policies, legislation, and regular pesticide risk assessment protocols by the relevant regulatory agencies may guarantee a safer environment that is less pervaded by a spectrum of chemically variable pesticides. This may dramatically lower the negative impacts of pesticide application or the need for conventional (physical, chemical, photoelectrolysis), and biological methods without threat to food security. It could equally be the game changer in the global effort to mediate climate change problems. Additionally, there is an urgent need to educate farmers about the toxicity of synthetics and avoiding its serious implications. Lastly, to reduce human exposure to pesticides, the development and dissemination of health education based on research, knowledge, skills, and practices are immensely important for the betterment of community health and overall sustainability.

Acknowledgments MAD acknowledges the grants received from Jiangsu University, China (5363000606), National Natural Science Foundation of China (31900367, 31772529), and Academic Program Development of Jiangsu Higher Education Institutions (PAPD 4013000011). MS appreciates Jiangsu University for Associate Professorship under the Invited Talent Program. The financial assistance provided to KH as a Research Associate by Department of Biotechnology (DBT), GoI is also acknowledged. PG is thankful to the Department of Science and Technology (DST) for the award of Women Scientist-A (SR/WOS-A/LS-136/2017) and the Department of Life Sciences, RIMT University, Mandi Govindgarh, Punjab for financial support. ZD is highly indebted to the National Key R& D Program of China (Grant No.2018YFE0107100), and the Primary Research & Development Plan of Jiangsu Province for financial support. The funders had no role in study design, data collection, and analyses, decision to publish, or preparation of the manuscript.

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[231] Pandey G, Dorrian S, et al. Cloning and biochemical characterization of a novel carbendazim (methyl-1 H-benzimidazol-2-ylcarbamate)-hydrolyzing esterase from the newly isolated Nocardioides sp. strain SG-4G and its potential for use in enzymatic bioremediation. Appl Environ Microbiol 2010;76(9):2940e5. [232] Zhao YC, Yi X, et al. Fundamental study of degradation of dichlorodiphenyltrichloroethane in soil by laccase from white rot fungi. Int J Environ Sci Technol 2010;7(2):359e66. [233] Zeng S, Qin X, et al. Degradation of the herbicide isoproturon by laccase-mediator systems. Biochem Eng J 2017;119:92e100. [234] Zhuo R, Yu H, et al. Heterologous expression and characterization of three laccases obtained from Pleurotusostreatus HAUCC 162 for removal of environmental pollutants. J Hazard Mater 2018;344: 499e510. [235] Wu PC, Liu YH, et al. Molecular cloning, purification, and biochemical characterization of a novel pyrethroid-hydrolyzing esterase from Klebsiella sp. strain ZD112. J Agric Food Chem 2006;54: 836e42. [236] Wang BZ, Guo P, et al. Cloning of a novel pyrethroid-hydrolyzing carboxylesterase gene from Sphingobium sp. strain JZ-1 and characterization of the gene product. Appl Environ Microbiol 2009;75(17):5496e500. [237] Luo X, Zhang D, et al. Cloning and characterization of a pyrethroid pesticide decomposing esterase gene, Est3385, from Rhodopseudomonas palustris PSB-S. Sci Rep 2018;8:1e8. [238] Haque M, Hong SY, et al. Cloning of an organophosphorus hydrolase (opdD) gene of Lactobacillus sakei WCP904 isolated from chlorpyrifos-impregnated kimchi and hydrolysis activities of its gene product for organophosphorus pesticides. Appl Biol Chem 2018;61(6):643e51. [239] Sharma N, Leung IK. Novel thermophilic bacterial laccase for the degradation of aromatic organic pollutants. Front Chem 2021;9. [240] Liang WQ, Wang ZY, et al. Purification and characterization of a novel pyrethroid hydrolase from Aspergillus niger ZD11. J Agric Food Chem 2005;53(19):7415e20. [241] Bai LS, Zhao CX, et al. Identification and biochemical characterization of carboxylesterase 001G associated with insecticide detoxification in Helicoverpa armigera. Pestic Biochem Physiol 2019;157: 69e79. [242] Wang SN, Chen QJ, et al. An extracellular yellow laccase from white rot fungus Trametes sp. F1635 and its mediator systems for dye decolorization. Biochimie 2018;148:46e54. [243] Selleck C, Guddat LW, et al. High resolution crystal structure of a fluoride-inhibited organophosphate-degrading metallohydrolase. J Inorg Biochem 2017;177:287e90. [244] Ambreen S, Yasmin A, et al. Isolation and characterization of organophosphorus phosphatases from Bacillus thuringiensis MB497 capable of degrading Chlorpyrifos, Triazophos and Dimethoate. Heliyon 2020;6:e04221. [245] Yao Q, Huang M, et al. Identification and characterization of a novel bacterial carbohydrate esterase from the bacterium Pantoeaananatis Sd-1 with potential for degradation of lignocellulose and pesticides. Biotechnol Lett 2020;42(8):1479e88. [246] Chu YH, Yu XX, et al. Purification and characterization of alkaline phosphatase from lactic acid bacteria. RSC Adv 2019;9:354e60. [247] Meng D, Jiang W, et al. An alkaline phosphatase from Bacillus amyloliquefaciens YP6 of new application in biodegradation of five broad-spectrum organophosphorus pesticides. J Environ Sci Health, Part B 2019;54(4):336e43. [248] Zhao S, Xu W, et al. In-depth biochemical identification of a novel methyl parathion hydrolase from Azohydromonasaustralica and its high effectiveness in the degradation of various organophosphorus pesticides. Bioresour Technol 2021;323:124641. [249] Zhang S, Zhang C, et al. Glutathione-S-transferase (GST) catalyzes the degradation of Chlorimuronethyl by Klebsiella jilinsis 2N3. Sci Total Environ 2020;729:139075. [250] Shehu D, Zazali A. Dechlorination of polychlorobiphenyl degradation metabolites by a recombinant glutathione S-transferase from Acidovoraxsp. KKS 102. FEBS Open bio 2019;93:408e19. [251] Yamamoto K, Zhang PB, et al. Identification of a sigma-class glutathione-S-transferase from the silkworm, Bombyx mori. J Appl Entomol 2006;130:515e22.

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[252] Thengodkar RRM, Sivakami S. Degradation of chlorpyrifos by an alkaline phosphatase from the cyanobacterium Spirulina platensis. Biodegradation 2010;21(4):637e44. [253] Labade CP, Jadhav AR, et al. Role of induced glutathione-S-transferase from Helicoverpa armigera (Lepidoptera: noctuidae) HaGST-8 in detoxification of pesticides. Ecotoxicol Environ Saf 2018; 147:612e21. [254] Morsi R, Bilal M, et al. Laccases and peroxidases: the smart, greener and futuristic biocatalytic tools to mitigate recalcitrant emerging pollutants. Sci Total Environ 2020;714:136572. [255] Unuofin JO, Okoh AI, et al. Aptitude of oxidative enzymes for treatment of wastewater pollutants: a laccase perspective. Molecules 2019;24(11):2064. [256] Zhao RB, Bao HY, et al. Isolation and characterization of Penicillium oxalicum ZHJ6 for biodegradation of methamidophos. Agric Sci China 2010;9:695e703. [257] Ashe B, Nguyen LN, et al. Impacts of redox-mediator type on trace organic contaminants degradation by laccase: degradation efficiency, laccase stability and effluent toxicity. Int Biodeterior Biodegrad 2016;113:169e76. [258] Trovaslet-Leroy M, Jolivalt C, et al. Application of laccase-mediator system (LMS) for the degradation of organophosphorus compounds. Chem Biol Interact 2010;187(1e3):393e6. [259] Kupski L, Salcedo GM, et al. Optimization of a laccase-mediator system with natural redoxmediating compounds for pesticide removal. Environ Sci Pollut Control Ser 2019;26:5131e9. [260] Bilal M, Iqbal HM, et al. Persistence of pesticides-based contaminants in the environment and their effective degradation using laccase-assisted biocatalytic systems. Sci Total Environ 2019;695:133896. [261] Bhatt P, Zhou X, et al. Characterization of the role of esterases in the biodegradation of organophosphate, carbamate, and pyrethroid pesticides. J Hazard Mater 2021;411:125026. [262] Wang D, Zou L, et al. Human carboxylesterases: a comprehensive review. Acta Pharm Sin B 2018; 8(5):699e712. [263] Maloney SE, Maule A, et al. Purification and preliminary characterization of permethrinase from a pyrethroid-transforming strain of Bacillus cereus. Appl Environ Microbiol 1993;59:2007e13. [264] Cai X, Wang W, et al. Autotransporter domain-dependent enzymatic analysis of a novel extremely thermostable carboxylesterase with high biodegradability towards pyrethroid pesticides. Sci Rep 2017;7:1e11. [265] Walsh TK, Joussen N, et al. Multiple recombination events between two cytochrome P450 loci contribute to global pyrethroid resistance in Helicoverpa armigera. PLoS One 2018;13:e0197760. [266] Ai G, et al. HPLC assay for characterizing a-cyano-3-phenoxybenzyl pyrethroids hydrolytic metabolism by Helicoverpa armigera (H€ ubner) based on the quantitative analysis of 3-phenoxybenzoic acid. J Agric Food Chem 2010;58:694e701. [267] Chintalchere JM, DarM A, et al. Evaluation of some essential oils against the larvae of house fly, Musca domestica by using residual film method. Advances in Biotechnology and microbiology9 2019:1e9. https://doi.org/10.19080/AIBM.2018.09.555752. [268] Chintalchere JM, Dar MA, et al. Impact of essential oils on Musca domestica larvae: oxidative stress and antioxidant responses. Int J Trop Insect Sci 2020:821e30. https://doi.org/10.1007/s42690-02000272-y. [269] Chintalchere JM, Dar MA, et al. Biocontrol efficacy of bay essential oil against house fly, Musca domestica (Diptera: muscidae). The Journal of Basic and Applied Zoology81 2020;6. https://doi.org/10.1186/ s41936-020-0138-7. [270] Chintalchere JM, Dar MA, et al. Bioefficacy of lemongrass and tea tree essential oils against house fly. Musca domestica. Proc Nat Acad Sci 2021;B91:307e18. https://doi.org/10.1007/s40011-02001220-z. [271] Feng X, Li M, et al. Carboxylesterase genes in pyrethroid resistant house flies, Musca domestica. Insect Biochem Mol Biol 2018;92:30e9. [272] Scott JG, Yoshimizu MH, et al. Pyrethroid resistance in Culex pipiens mosquitoes. Pestic Biochem Physiol 2015;120:68e76. [273] Zhu W, Yu R, et al. Identification and characterization of two CYP9A genes associated with pyrethroid detoxification in Locusta migratoria. Pestic Biochem Physiol 2016;132:65e71.

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CHAPTER 25

Role of natural compounds in metal removing strategies Harshada Sowani1, Mithil Mahale1, Vinod S. Nandre1, Surya Nandan Meena1, Kisan M. Kodam1, Mohan Kulkarni1 and Smita Zinjarde2 1

Department of Chemistry, Division of Biochemistry, Savitribai Phule Pune University, Pune, Maharashtra, India; Department of Biotechnology (with jointly merged Institute of Bioinformatics and Biotechnology), Savitribai Phule Pune University, Pune, Maharashtra, India 2

1. Introduction The term “heavy metal” refers to the group of elements present in the earth’s crust with a density greater than 5 g/cm3 and atomic weight varies from 63.5 to 200.6 g/mol [1]. Broadly, they can be categorized as essential and nonessential heavy metals. Essential heavy metals such as zinc (Zn), cobalt (Co), selenium (Se), molybdenum (Mo), vanadium (V), manganese (Mn), nickel (Ni), strontium (Sr), copper (Cu), and iron (Fe) are known as micronutrients which are required in trace amounts by plants, animals (including human beings) and microorganisms for their metabolic activities. However, if the concentration of essential heavy metals crosses threshold limits, they become deleterious. Nonessential heavy metals like aluminum (Al), arsenic (As), cadmium (Cd), lead (Pb), mercury (Hg), chromium (Cr), antimony (Sb), and tin (Sn) are very harmful because they react with biological macromolecules and form highly stable, biotoxic complexes that are difficult to dissociate [2,3]. Few metals such as Cd, Hg, Cr, Ni, Cu, and V generate reactive free radicles that cause DNA damage, lipid peroxidation, and protein sulfhydryl group depletion. 1.1 Entry of heavy metals into the environment Both natural events (earth’s crust and rock weathering, leaching, volcanic eruptions, geothermal activities, sand storms, wind-blown dust particles) and various anthropogenic activities add significant quantities of heavy metals to the soil, water, and air. Combustion of fossil fuels, burning of agricultural wastes, industry, or factory chimney emissions called stack emissions release various pollutants (hydrocarbons, heavy metals) and gaseous (carbon monoxide, carbon dioxide, sulfur oxides, nitrogen oxides, methane). Whereas an accidental release of toxic vapors or gases from different chemical storage reservoirs from industrial plants add pollutants in the air are called fugitive emissions, also increased heavy metal concentration in the air. Fugitive emissions release lower contents of contaminants than stack emissions, and their distribution is site-specific. On the contrary, stack emission contaminants get distributed over a large area by natural air New Horizons in Natural Compound Research ISBN 978-0-443-15232-0, https://doi.org/10.1016/B978-0-443-15232-0.00016-3

© 2023 Elsevier Inc. All rights reserved.

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currents. High-temperature processing volatilizes Cd, As, and Pb-like metals to their respective oxides. Moreover, if reducing atmospheric conditions are not maintained, fine particulates are generated and deposited on the surface of water or land. Tetraethyl lead, a fuel additive in petrol, emits Pb in the air after combustion. Hazardous heavy metals, namely Cd, Pb, Zn, and other metals, are prevalent in the soil near ore mining areas and in places where smelting is carried out. In the agriculture and horticulture sector, inputs of insecticides, fungicides, and pesticides add significant quantities of heavy metals (As, Cu, Hg, Mn, Zn, or Pb) to the soil. Applying biosolids viz. domestic sewage sludge, livestock manures, composts, and fertilizers to the land leads to the accretion of As, Se, Cd, Mo, Cr, Sb, Cu, Hg, Ni, Pb, and Zn in soil. Sewage disposals, as well as sewage treatment plants, bring heavy metals to the environment [4,5]. Metals including Hg, Pb, Cd, Ni, and Cr expelled by pharmaceutical industries get mixed with surface and ground water and have adverse effects. Textile processing units release Cd, Zn, Hg, Zn, and traces of several heavy metals. While inorganic pigment manufacturing units release chromium and cadmium sulfide, printing circuit board manufacturing produces heavy metal wastes with Ni, Pb, and Sn. Wood preservation treatments that use chromated copper-arsenate generate arsenic comprising wastes. The waste water discharge from steel industries consists of traces of Zn, Cu, Cd, Ni, and Sn used for iron finishing or coating purposes. Electroplating and decorative plating practices generate Cd, Cu, Cr, Ni, Zn, Pb, V, platinum (Pt), Silver (Ag), and Gold (Au). The petroleum refining process uses conversion catalysts and contaminates the environment with Cr, Ni, and V. Developing processes in photography add Ag and ferrocyanides in high concentrations. The aforementioned industrial operations release significant quantities of heavy metals; hence pretreatment of contaminated wastes and wastewaters is needed before their release into the environment. Heavy metals are persistent pollutants. Their accumulation in water bodies and soil adversely affects the flora and fauna that resides therein [6,7]. Heavy metals affect soil fertility and crop production. Heavy metals enter human bodies through contaminated water and food, and their associated toxicity causes serious health issues [8]. Conventional remediation strategies (physicochemical and chemical) can be applied to mitigate heavy metal concentrations and limit their toxicity. For heavy metal contaminated soils, generally, soil replacement, surface capping, thermal desorption, chemical leaching or soil washing, solidification and soil flushing, chemical fixation, electrokinetics, and vitrification are recommended as remediation strategies. For mitigating heavy metal contaminated wastewater, adsorption, chemical precipitation, electrodialysis, electrochemical treatments, floatation, flocculation, ion-exchange, membrane filtration, microencapsulation, nano-filtration, photocatalysis, polymers, reverse osmosis, and ultrafiltration can be used. However, certain protocols are time-consuming, expensive, difficult to implement on a large scale, and generate secondary toxic pollutants. To overcome

Role of natural compounds in metal removing strategies

these problems, several researchers have suggested bioremediation as a promising, ecofriendly, cost-effective, sustainable, and safe strategy [9]. Phytoremediation is an in situ remediation approach that utilizes plants and their associated microbial flora, together with the amendment of soil and agronomic practices for the removal of contaminants [10]; whereas a process that utilizes fungi, bacteria, and yeast-like microbial flora to abate the concentration of pollutants including heavy metals in the soil or water bodies by means of absorption, oxidation-reduction or precipitation is called microbial bioremediation (Fig. 25.1). Microorganisms interact with heavy metals through different mechanisms: (i) Bioaccumulation (when transporter proteins transport heavy metal ions into the cytoplasm or intracellular spaces where they are sequestered via proteins or peptide ligands to form insoluble particulates); (ii) Biosorption (nonspecific interaction between the metal ion and biomolecules present on the surface of living or nonliving microbial biomass). (iii) Biomineralization (compounds of microbial origin arrest metal mobilization and cause their precipitation); (iv) Biotransformation (microorganisms via their enzymatic machinery modify metals via oxidation-reduction, alkylation and methylation reactions and limit heavy metals mobilization by pigments, biosurfactants, and siderophores); and (v) Bioleaching (where bacteria and fungi solubilize metal sulfides and oxides from ores

Figure 25.1 Conventional and nonconventional strategies used in the remediation of heavy metals.

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deposits that can be purified by adsorption, ion-exchange, membrane separation, or selective precipitation methods). Besides this, several adsorbents such as biochar from biowaste, biopolymer-based material, industrial wastes, and agricultural residues play a crucial role in the remediation of water contaminated with heavy metals. The adsorption process has higher metal removal capacity, requires relatively less energy, and does not generate secondary pollutants. This chapter deals with the role of certain natural compounds associated with or produced by microorganisms (secondary metabolites) and plants (biopolymers and agriculture wastes) in excluding heavy metals from contaminated environments.

2. Natural compounds used in heavy metals removal 2.1 Biosurfactant-based removal of heavy metals Biosurfactants are bioactive compounds derived from certain plants and microorganisms. They lower interfacial and surface tension forming micellar structures that enhance the bioavailability and mobility of heavy metals in the soil. A complex of biosurfactant and heavy metal ions can be formed through counterion binding or electrostatic interactions. Biosurfactant-based soil remediation is achieved either by soil flushing (in situ) or soil washing (ex situ) protocols where rhamnolipids, lipopeptides, sophorolipids, surfactin, saponin, and tannins can be used. Soil flushing is an eco-friendly, inexpensive remedial strategy where biosurfactants are either injected or recirculated in polluted soils. Electrostatic bonding between cationic heavy metals and anionic biosurfactants results in the formation of complexes that can be readily removed from the soil matrix by precipitation and water flushing. The treated soil, as well as the biosurfactant, can be reused further. A report by Wang and Mulligan [11] demonstrated As, Cu, Pb, and Zn elimination from polluted mine tailings using 0.1% rhamnolipid (JBR425) flushing solution (pH 11). As a result, 148 mg/kg of As, 74 mg/kg of Cu, 2379 mg/kg of Pb, and 259 mg/kg of Zn removal were achieved after 70-pore-volume of flushing [11]. Rhamnolipids are anionic biosurfactants used to remove cationic heavy metal ions from the soil and sediments [12,13]. In another report related to soil flushing, the rhamnolipid solution was used to eliminate Cu(II) ions adsorbed on the sand surface. The elimination proficiency improved from 5% to 12%, as rhamnolipid concentration was raised from 40 to 200 mg/L. Thus, it was noticed that the elimination of metal relied on the biosurfactant concentration used [14]. Soils and sediments differ in their chemical composition. Sediments are rich in organic matter and clay content; hence, the remediation strategy for contaminated soil and sediment varies. For sediments, washing and solidification are the most suitable protocols. One such study on heavy metal contaminated sediment was carried out by Mulligan et al. [15]. Elimination of Cu (110 mg/kg) and Zn (3300 mg/kg) from contaminated sediment was studied by using three different biosurfactants, namely rhamnolipid,

Role of natural compounds in metal removing strategies

surfactin, and sophorolipid derived from Pseudomonas aeruginosa (ATCC 9027), Bacillus subtilis (ATCC 21332) and Torulopsis bombicola (ATCC 22214), respectively. A single wash with rhamnolipid (0.5%) solution eliminated 65% and 18% of Cu and Zn, respectively. Sophorolipid (4%) solution abolished 25% Cu and 60% Zn, whereas 2% surfactin solution removed 15% Cu and 6% Zn. Metal removal ability of surfactants was determined by zeta potential and ultrafiltration technique, and it was concluded that biosurfactant-metal ion complexation was involved here. Another study was conducted to remove copper, zinc, and nickel from contaminated sediment. Sediment washing was performed using 0.5% of commercial rhamnolipid JBR215 solution. As a result, 37%, 13%, and 27% of Cu, Zn, and Ni removal were noted. Copper removal was enhanced up to four times by sodium hydroxide (1%) addition than rhamnolipid (0.5%) used alone [12]. Di-rhamnolipid from P. aeruginosa BS2 was used to remove heavy metals (Cd and Pb) from the soil contaminated artificially. After 36 h of leaching, 92% of Cd and 88% of Pb were removed. Di-rhamnolipid selectively favored mobilization of Cd more than Pb. This study demonstrated that biosurfactants offered strong candidature in metal bioremediation [13]. Later, the same di-rhamnolipid was used as a washing agent in the removal of Cd (430 ppm), Cr (940 ppm), Cu (480ppm), Ni (880ppm), and Pb (900 ppm) from multimetal contaminated soil. This study showed that removal of Cr, Pb, and Cu from heavy metal spiked soil was 13, 9, and 14-fold, respectively, while Cd and Ni removal was 25-fold higher [16]. A model of a co-contaminated system was developed to study the reduction of Cd toxicity and naphthalene biodegradation by Burkholderia sp. isolated from soil. Cadmium toxicity elimination was achieved by adding 10-fold greater contents of rhamnolipid than cadmium (890 mM), and cadmium toxicity reduction was observed by rhamnolipid addition at equimolar concentrations [17]. Remediation of soil co-contaminated with heavy metal cadmium and a polycyclic aromatic hydrocarbon phenanthrene was attained by biosurfactant enriched soil washing strategy. Rhamnolipid solution (0.5%) removed 72.4% of cadmium and 87.8% of phenanthrene from cocontaminated soil. Both the studies found that rhamnolipids enhanced the solubility of naphthalene and phenanthrene and formed complexes with cadmium ions [18]. The synergistic effect of two biosurfactants on metal removal has also been studied [19]. Washing solutions were prepared using different concentrations of rhamnolipid and saponin and used for the washing of V, Ni, and Cr contaminated soil. As a result, 87% of Ni, 71% of Cr, and 70% of V removal was achieved through biosurfactant synergy at pH 5. The use of biosurfactants in a synergistic manner appears to be a good alternative to chemical surfactants for the removal of heavy metals [19]. Rhamnolipid biosurfactant solution (0.8 g/L, pH 11) was used to remove Ni, Cr, and Cd from crude oil contaminated soil. As a result, 43.05% of nickel, 34.73% of chromium, and 52.81% of cadmium removal from the soil was achieved [20]. Soil contaminated with As, Cd and Zn was treated by P. aeruginosa LFM 634 produced rhamnolipid solution. Soil

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washing and chemical precipitation resulted in the 90%, 80%, and 53% removal of Cd, Zn, and As, respectively. Basak and Das [21] have demonstrated a strategy for removing Zn(II) from electroplating wastewater using sophorolipid produced by Cryptococcus sp. VITGBN2. Immobilized sophorolipid-sodium alginate beads were packed in a column, bed height was kept at 12 cm, and the flow rate was maintained at 1 mL/min. As a result, 94.34% Zn(II) removal was observed. Sophorolipid from Candida bombicola was used to remove heavy metals from soil contaminated with hydrocarbons. About 23.11% of Cr, 15.71% of Cd, 9.93% of Pb, 7.29% of Zn, and 4.96% of Cu were removed from the soil after the treatment with sophorolipids [22]. Later, arsenic speciation from mining tailing samples after treatment with sophorolipids was studied [23]. It was found that 1% sophorolipid solution released As(III) and As(V) from the mine tailing samples. In another study, sophorolipid obtained from Starmerella bombicola CGMCC 1576 was used for metal contaminated soil washing. This resulted in the removal of 83.6% of Cd and 44.8% of Pb [24]. The biosurfactant formed from Candida tropicalis resulted in the removal of Zn and Cu from 30% to 80%, and Pb removal was 15%. With packed column experiments, Zn and Cu removal ability ranged from 45% to 65% [25]. Bacillus subtilis A21 produced lipopeptide biosurfactants surfactin and fengycin. Soil washing with this lipopeptide solution successfully removed 64.5% of petroleum hydrocarbons and 44.2%, 35.4%, 40.3%, 32.2%, 26.2%, and 32.07% of cadmium, cobalt, lead, nickel, copper, and zinc, respectively [26]. The soil isolate Bacillus cereus NWUAB01 produced lipopeptides capable of forming complexes with metal ions. This biosurfactant removed Pb (69%), Cd (54%), and Cr (43%) from the soil [27]. The anionic lipopeptide obtained from Bacillus sp. exhibited great affinity toward Pb, Hg, Mn, and Cd-like cationic heavy metals. The addition of this lipopeptide to heavy metals resulted in the formation of a white color precipitate (metal-biosurfactant complex). The biosurfactant removed 75.5% Hg, 97.73% Pb, 89.5% Mn, and 99.93% Cd, respectively, from the stock of (1000 ppm) respective metal solutions. Moreover, the lipopeptides solution also washed off the heavy metal traces present on vegetable (cabbage, carrot, and lettuce) surfaces. This study used lipopeptides of strength 2X CMC [28]. Surfactin produced from Bacillus sp. HIP3 showed heavy metal chelation. Cadmium, chromium, zinc, lead, and copper at a concentration of 100 ppm each, were treated with surfactin (10 mg/mL) solution for 24h at room temperature. This resulted in 0.7%, 1.68%, 2.91%, 12.71%, and 13.57% removal of Cd, Cr, Zn, Pb, and Cu, respectively [29]. Metal desorption has been reported in Cu(II) and Ni(II)-spiked kaolin clay by a plant-based saponin biosurfactant that formed complexes with metals. Maximum desorption was noticed at 2 g/L of saponin concentration. A single wash with saponin solution removed 83% of Cu and 85% of Ni from kaolin spiked with 0.45 mg/kg of Cu and 0.14 mg/kg of Ni [30].

Role of natural compounds in metal removing strategies

2.2 Exopolysaccharides (EPSs) based removal of heavy metals EPSs are polymers secreted by certain microorganisms that mainly contain carbohydrates. Besides this, small quantities of proteins, lipids, pyruvic acid, uronic acid, nucleic acid, and inorganic molecules like phosphate or sulfate are also present in EPSs. Based on composition, they can be categorized as homo and heteropolysaccharides. Several research reports have demonstrated the role of bacterial EPSs in heavy metal biosorption, where negatively charged groups of EPSs interact with positively charged heavy metals or through ion-exchange mechanisms [31]. Hence, EPS acts as a competent biosorbent or chelating agent in heavy metal remediation. There is a report on the halophilic bacterium Salipiger mucosus, isolated from the Mediterranean seaboard, producing EPS that could chelate 15.7 mg copper/g EPS, 43.5 mg lead/g EPS, and 8.7 mg of cobalt/g EPS, respectively [32]. Another study evaluated the chelating activity of EPS produced by the halophilic bacterium Halomonas almeriensis. Under optimized conditions, this anionic EPS could chelate 10, 19.2, and 24.5 mg of cobalt, copper, and lead, respectively (per g of EPS) [33]. An extremophilic bacterium, Acidithiobacillus thiooxidans secreted EPS that facilitated heavy metal bioleaching, and EPS derived from psychrophiles (microorganisms which can survive at low temperature or cold environment) was effective in heavy metal removal [34]. Liu et al. [35] studied the efficiency of exopolysaccharides, extracted from waste sludge to remove heavy metals (Cu, Zn, Cr, Cd, Co, Ni and chromate ions) at concentrations of 10e100 ppm. According to the study, although 85%e95% of zinc, copper, chromium and cadmium were eliminated, removal of cobalt, nickel and chromate ions was comparatively lower (69%, 37%, and 26%, respectively). EPSs from nitrogen fixing bacteria are also reported to mediate biosorption of heavy metals. For instance, Rhizobium radiobacter F2 produced EPS that was tested for its capacity to eliminate Pb(II) and Zn(II) from aqueous solutions. Maximum biosorption of Pb(II) was detected at pH 5.0 and for Zn(II), it was at pH 6.0. Desorption of both the heavy metals was carried out by hydrochloric acid. After desorption 80% biosorption capacity of EPS was retained [36]. Sinorhizobium meliloti is another nitrogen fixing bacterium that produced two types EPSs namely, succinoglycan and galactoglucan which conferred resistance toward As and Hg ions [37]. There are several reports on the sequestration of heavy metals by cyanobacteria. EPSs secreted by cyanobacteria are known to be efficient chelating agents in remediating heavy metal polluted environments. EPS from Anabaena spiroides demonstrated an affinity for Mn2þ ions, and complexation brought about the removal of 8.52 mg per gram of EPS [38]. Other cyanobacteria, namely, Gloeocapsa gelatinosa and Calothrix marchica synthesized capsular polysaccharides (CPS) that could remove around 82e86 mg of Pb(II) ions per gram of CPS [39]. CPS obtained from Nostoc and Cyanospira capsulata could

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remove copper ions more efficiently than nickel and zinc ions. EPSs from two other cyanobacteria, Gloeocapsa calcarea and Nostoc punctiforme, enabled sequestration of chromium. Biosorption conditions were optimized by applying a Langmuir and Freundlich model, pH was altered from 2 to 6, and chromium concentrations were varied from 5 to 20 mg/L. Higher biosorption occurred at a Cr concentration of 20 mg/L under extreme acidic (pH 2) conditions [40]. Another study was conducted to assess the chromium adsorption ability of a polysaccharide secreted by cyanobacterium Lyngbya putealis, HH-15. Parameters including pH, temperature, and metal concentration were optimized by Box-Behnken design using response surface modeling. The peak adsorption capacity of EPS was attained by increasing its concentration from 45 to 157 mg/g and Cr (VI) concentration from 10 to 30 mg/L [41]. Hydroxyl and carboxyl groups associated with EPS of Cyanothece CCY 0110 showed the highest affinity for copper, cadmium, and lead. After acid pretreatment, the EPS demonstrated enhanced affinity (86%, 43%, and 22%, respectively) toward Cu2þ, Cd2þ, and Pb2þ ions. On the contrary, pretreatment with base improved removal of Cu2þ and Pb2þ (318% and 92%, respectively) and lowered (26%) removal of Cd2þ [42]. EPSs produced by thermophilic bacteria are rich in neutral carbohydrates and uronic acid. Their role in bioleaching and metal ion immobilization is noteworthy [43]. Thermophilic Pseudomonas sp. W6 secreted EPS that was capable of Pb biosorption at 1 mM concentrations. Moreover, this EPS could also remove Pb (65%) from synthetic water [44]. 2.3 Biopolymers-based removal of heavy metals Biopolymers are polymeric compounds produced by or derived from plants and microorganisms. Alginate, chitosan, cellulose, hemicellulose, lignin, and starch are some examples of carbohydrate-based biopolymers. These biopolymers are biodegradable, biocompatible, and cost-effective. The inclusion of chemically reactive groups (acetamide, amino, carboxymethyl, and hydroxyl) in polymer chains confers unique structure, stability, and selectivity on the biopolymers the properties that are relevant for their role as sorbents [45]. Biopolymers can be used to formulate nanocomposites and adsorbents that are important in the treatment of heavy metal contaminated wastewaters. Metal uptake efficiency and mechanical strength of biopolymer chain can be enhanced by grafting or blending with different nano-biomaterials. These biopolymer-based adsorbents are reusable and can be recycled [46]. In this section, we will discuss heavy metal removal using chemically modified biopolymers. 2.3.1 Alginate-based materials used in heavy metals removal Alginate is a copolymer of mannuronic and guluronic acid obtained from algae and some bacteria [47]. Alginate finds its applications in treating wastewaters [48]. Heavy metal

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removal using different alginate-based adsorbents, for example, alginate beads, alginate gel-coated adsorbent, and alginate capsules, have been evaluated. In one study, the alginate capsules showed high Pb(II) uptake capacity, i.e., 1560 mg/g of dry sodium alginate. Most of the alginate-coated adsorbents exhibit great potential for metal removal from contaminated water. Metal desorption from gel-coated adsorbent can be carried out by nitric acid, which could be reused further without any loss in metal uptake capacity [49]. Another study investigated the sorption capacity of calcium alginate beads for removing Cd and Pb by varying different kinetic and thermodynamic parameters. It was found that with an increase in temperature, sorption capacity increased (27.4 and 150.4 mg/g for cadmium and lead, respectively, at 25 C). The kinetics and isotherm experimental data of biosorption were best fitted in pseudo-second-order kinetics and the Langmuir-Freundlich model. Thermodynamic studies recommended the spontaneous and endothermic nature of the process [50]. Later studies demonstrated the use of calcium alginate beads in the removal of Zn(II), Pb(II), and Ni(II) metal ions from aqueous solutions. Metal ion removal was dependent on pH and adsorbent dose. The maximum removal was observed at pH-5 when the adsorbent dose concentration was 8 g/100 mL. The kinetic data of biosorption was assessed by pseudo-first and secondorder while the Weber and Morris model did the metal ions diffusion interpretation [51]. A kinetic (pseudo-first and second-order, Vermuelen) and equilibrium study using Langmuir and Freundlich models was conducted to estimate the adsorption capability of an activated carbon-calcium alginate composite for removal of Pb (II) ions under acidic (pH 5) conditions using NaCl as a medium solution. The amount of Pb (II) ion removal was determined by differential pulse anodic stripping voltammetry. It was found that calcium alginate enhanced the active carbon adsorption capacity of composites [52]. Novel hydroxyapatite-alginate-gelatin-nanocomposites (HA-Alg-Gel nanocomposite) were prepared to study Pb2þ and Cd2þ sorption capacity. The sorption kinetic data was best fitted into the pseudo-second-order equation. The equilibrium removal capacity of nanocomposites was calculated as 550 mg/g for Pb2þ and 361 mg/g for Cd2þ, slightly lower than the removal capacity of HA-Alg-Gel composite (616 for Pb2þ and 388 mg/g Cd2þ). In binary metal ion systems, the adsorbent removal capacity for a particular metal ion is slowed down if there is another ion in the medium [53]. In another study, nanocomposite beads prepared from zeolite, polyvinyl alcohol, and sodium alginate were used to remove heavy metals (Pb2þ, Cd2þ, Sr2þ, Cu2þ, Zn2þ, Ni2þ, Mn2þ, Fe3þ, and Al3þ) from contaminated wastewaters. Langmuir adsorption isotherm and adsorption kinetics (pseudo-first-order model) were studied. Thermodynamics studies of these selected heavy metals showed that the adsorption was endothermic. At pH 6, the removal efficiencies of Pb2þ (99.5%), Cd2þ (99.2%), Sr2þ (98.8%), Cu2þ (97.2%), Zn2þ(95.6%), Ni2þ (93.1%), Mn2þ (92.4%) and Li2þ (74.5%) while they were 96.5% and 94.9% for Fe3þ and Al3þ, respectively, at pH-5 [54]. Nanofibers containing polyvinyl alcohol and sodium alginate were prepared and used as adsorbents to

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remove cadmium metal ions from the aqueous solution. Factors influencing the cadmium adsorption process, including temperature, pH, and metal concentration, were optimized, and the maximum equilibrium adsorption was 67.05 mg/g. Thermodynamics data showed that the adsorption of cadmium was endothermic and spontaneous [55]. 2.3.2 Lignin-based adsorbents and heavy metal removal Lignin is a biopolymer found abundantly in nature with diverse functional groups (hydroxyl, carboxyl, phenolic, to mention a few) that provide adsorption sites for positively charged heavy metal ions. Adsorption efficiency of lignin can be improved by modification of functional groups or chemical grafting reactions [56]. Secondary amines were grafted onto alkaline lignin and evaluated for their adsorption capability for Pb(II) ions. Grafted lignin showed 4.2 times more rapid adsorption of Pb(II) than nonmodified alkaline lignin [57]. In one study, aminated epoxy groups were grafted onto bagasse soda lignin. This modified lignin could remove 72.5 and 55.4 mg/g Pb(II) and Cu(II), respectively. Spectroscopic studies indicated that amine and hydroxyl groups present on the modified lignin surface mainly play an essential role in heavy metal adsorption [58]. In another study, nitrogen functionalized corncob-based biochar showed 85.65 mg/g adsorption capacity for Cd(II) ions under neutral pH conditions [59]. Here, Cd(II) ions were removed by ion-exchange and adsorption-complexation mechanisms. The use of lignin-based biochar is another good option for removing heavy metals. The efficacy of biochar mainly depends on the properties of biomaterials used in its synthesis. Lignin-containing titanate nanotubes were capable of adsorbing Pb2þ, Cu2þ, and Cd2þ in a pH range of 2e7. Adsorption was best at pH 6 being 677.6 mg/g for Pb2þ, 258.2 mg/g for Cu2þ and 308.5 mg/g for Cd2þ [60]. The adsorption capacity of lignin-based biochar for Pb(II) was 1003 mg/g, and 519 mg/g when the raw material was used [61]. Another study evaluated the heavy metal adsorption capability of corncob-based acrylonitrile treated biochar (pyrolysed at 350 C). The resultant biochar showed an adsorption capacity of 85.65 mg/g for Cd [59]. Mixed metal-biochar composites were manufactured using lignin and red mud by pyrolysis. The addition of alumina-silicate, Fe (metallic), and carbon matrix to composites increased the adsorption and chemical reduction capacities for Pb(II), Ni(II), As(V), and Cr(VI) [62]. The thiol group containing lignin-based adsorbent exhibited 72.4 mg/g of adsorption capacity for Cd(II) and selectivity (8.6-fold) than the nonmodified lignin. Adsorption kinetics and isotherm experimental data fitted into pseudo-second-order and Langmuir isotherm model, respectively, suggested a chemical sorption process occurred between the adsorbent and heavy metal ions [63]. Carboxy-methylated formic lignin was applied for Pb2þ ions removal. At pH 5, the adsorption capacity for Cd2þ ions was assessed 67.7 mg/g, which was enhanced at pH 6.0 up to 107.5 mg/g [64]. In one study, lignin extracted from residues of paper industries and waste streams was implemented for Pb(II), Cu(II), Zn(II), Cd(II), and Ni(II)

Role of natural compounds in metal removing strategies

adsorption. The carboxylic and phenolic groups of extracted lignin were responsible for heavy metals adsorption. Both of these groups showed the highest affinity toward Pb(II) and the lowest affinity for Ni(II) ions [65].
Sciban et al. [66] studied Cu(II), Cd(II), Zn(II), and Cr(VI) ion adsorption from water by kraft lignin. The highest affinity was observed for Cr(VI), followed by Cd(II), Cu(II), and Zn(II). These results suggest that the adsorption capacity could change due to ion species interference. 2.3.3 Chitosan-based adsorbents and heavy metal removal Chitosan is a polymer made up of amino polysaccharides. It is present in crustaceans and insects. The degree of acetylation and molecular weight determines the functional properties of chitosan. This polymer exhibits a key role in the divalent heavy metal ions adsorption. Chemical modification of chitosan can be achieved by cross-linking, grafting, ion templating, and combining with other adsorbents. Modified chitosan is generally used in heavy metal remediation. Previously, studies have demonstrated that hexavalent chromium ions were eliminated from wastewater using novel chitosan composites, showing 153.85 mg/g of adsorption capacity [67]. There is a report on the successful removal of Cr(VI) ions from aqueous solution using diethylenetriaminepentacetic acid linked chitosan [68]. Chitosan, graphene oxide, and 1,2-cyclohexylenedinitrilo tetraacetic acid nanocomposite showed 166.98 mg/g adsorption capacity for Cr (VI) ions present in an aqueous solution. Graphene makes available more free groups for heavy metals adsorption from aqueous solutions [69]. The quaternized chitosan microspheres were developed and applied to remove Cr(VI) ions from wastewater. Under acidic conditions (pH 5), 97.34% of chromium was removed [70]. Chitosan crosslinked polyvinyl alcohol, and zeolite composite nanofibers showed adsorption capacity of 0.17, 0.11, and 0.03 mmol/g for Cr(VI), Ni(II), and Fe(III), respectively. The addition of polyvinyl alcohol offers excellent reusability of the nanofibers [71]. Chitosan nanofibers cross-linked with polyacrylic acid sodium showed the capability of Cr(VI) ions removal [72]. Recently, cellulose and chitosan were modified chemically, and their adsorption capacity for Cr, Pb, and Cd from aqueous solution was determined. At pH 4, the removal of Cr, Pb, and Cd was calculated as 56%, 85%, and 94%, respectively, when the metal concentration (60 ppm) and adsorbent dosage (1.0 g/L) were used. With doubled metal concentration (120 ppm) and similar adsorbent dosage (1.0 g/L), the adsorption capacity of composite for Cr, Pb, and Cd was noted at 55, 80, and 91 mg/g, respectively [73]. Chitosan-polyvinyl alcohol hydrogel having nanosized particles were prepared without any cross-linking agent and investigated its adsorption capacity for manganese [Mn (II)] ions present in the aqueous solution. Mn(II) has been considered a key pollutant as it changes water properties. Adsorption kinetics and equilibrium data were respectively fitted in the pseudo-second-order kinetic model and Freundlich isotherm model. Thermodynamic studies indicated that Mn(II) ion adsorption onto PVA/CS was an

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endothermic and spontaneous process [74]. The adsorption capacity of Chitosan-PVA magnetic composites for the exclusion of Cu(II) ions from wastewaters was 143.0 mg/g [75]. Rosli and co-workers [76] recently prepared 1-allyl-3-methylimidazolium chloride functionalized chitosan-PVA blend nanofiber membranes using gluteraldehyde as a cross-linking agent. The synthesized nanofibers’ adsorption capacity for Pb(II), Mn(II), Cu(II), and Pb(II) ions was 166.34 mg/g. In an earlier report, chitosan cross-linked with epichlorohydrin exhibited an adsorption capacity of 35.40 mg/g for Cu(II), 13.05 mg/g for Pb(II), and 10.21 mg/g for Zn(II) ions [77]. In later studies, cross-linked metal-imprinted microparticles were manufactured from chitosan, metal templates, and an epichlorohydrin cross-linking agent. Then, comparative biosorption capacities of templated and non-templated microparticles for Cu(II), Zn(II), Ni(II), and Pb(II) ions were calculated. The sorption capacities of templated microparticles for Cu(II), Zn(II), Ni(II), and Pb(II) ions were 74%, 46%, 57%, and 43%, respectively. Whereas adsorption capacity of non-templated microparticles was lower, i.e., 25%, 13%, 41%, and 12% for Cu(II), Zn(II), Ni(II), and Pb(II) ions, respectively [78]. Chitosan/hydroxyapatite composite membrane was set for Pb(II), Co(II), and Ni(II) ions removal from aqueous solutions. The composite membrane showed 296.7, 213.8, and 180.2 mg/g of adsorption capability for Pb(II), Ni(II), and Co(II), respectively [79]. Innovative, benign chitosan-alginate nanoparticles were synthesized and applied successfully in the removal of Hg ions present in water. Moreover, these nanoparticles were capable of interacting with positive as well as negatively charged metal ions. Similarly, alginate-coated chitosan nanoparticles removed 94.48% of nickel ions under acidic conditions (at pH 3) from industrial effluents [80]. 2.3.4 Cellulose-based adsorbent for heavy metals removal Cellulose is a biopolymer abundantly present in different plant resources. Modified forms of cellulose are beneficial for pollutant remediation purposes. A variety of nanocomposites, nanofibers, nanocrystals, hydrogel, beads, and membranes made up of cellulose with some physical and chemical modification are reported to have increased reactivity and binding sites for heavy metals. There is a report on carboxymethyl cellulose-coated polyacrylamide composites, those favored Pb2þ, Cu2þ, and Cd2þ ion adsorption. The kinetics of adsorption reaction fit well with the pseudo-second-order kinetic and Langmuir adsorption models [81]. The magnetic carboxymethyl chitosan nanocomposites achieved rapid removal of Zn2þ, Cu2þ, and Pb2þ ions present in the aqueous solution. These nanocomposites had improved binding sites for metals than those present in pure chitosan [82]. From pineapple leaves, cellulose fibers were prepared and modified with carboxymethyl groups and ethylenediaminetetraacetic acid (EDTA) to generate cell-EDTA and cell-carboxymethyl matrices, respectively. These modified forms were used to remove Pb2þ and Cd2þ ions from water. Adsorption capacity of cell-EDTA was 41.2 mg/g for Pb2þ and 33.2 mg/g for Cd2þ. Cell-CM showed 63.4 and

Role of natural compounds in metal removing strategies

23.0 mg/g adsorption capacity for Pb2þ and Cd2þ, respectively. The experimental kinetics data fitted in both the pseudo-first and pseudo-second-order model [83]. Nanocomposites comprising carboxymethyl cellulose and nanoscale zerovalent iron were used as an adsorbent in removing Pb2þ ions [84]. At pH 6.0, the nanocomposite displayed an adsorption capability of 1237.32 mg/g, greater than nanoscale zerovalent iron (838.84 mg/g). Hydrogel beads were also prepared from carboxymethyl cellulose using epichlorohydrin as a linking agent. The adsorption capacity of hydrogel for Cu(II), Ni(II), and Pb(II) ions was 6.49, 4.06, and 5.15 mmol/g for, respectively [85]. In another study, epichlorohydrin cross-linked carboxymethyl cellulose fibers were used to remove Cd2þ present in water, for which the adsorption capacity was calculated as 150.60 mg/g [86]. 2.4 Starch-based composites, adsorbent heavy metals removal Starch is a natural polysaccharide that requires some chemical modification to acquire heavy metal removing abilities. Modified starch is an inexpensive option for removing heavy metals from wastewaters. An earlier report on corn starch (modified by crosslinking with phosphorus oxychloride and carboxymethylation) showed effective removal of Cu, Pb, Cd, and Hg ions from water [87]. In another report, co-polymers were prepared by grafting dimethylaminoethyl methacrylate onto starch. The adsorption capacity of co-polymers displayed 2.09 and 2.12 mmol/g of dry weight for Pb(II) and Cu(II) ions, respectively [88]. Xu et al. [89] evaluated Cr(VI) ion removal present in wastewater using starch modified with quaternary ammonium (cationic) and a carboxymethyl (anionic) group cross-linking. In another investigation, modified corn starch was prepared by esterification with maleic acid and itaconic acid and evaluated as biosorbents for removing Ni2þ, Zn2þ, Cd2þ, and Pb2þ ions. While native corn starch did not show selectivity for the cations, itaconate esterified starch was highly selective and aided the removal of higher contents of Pb2þ [90]. 2.5 Agriculture wastes-based removal of heavy metals A variety of horticulture and agriculture wastes have been receiving attention as tools for heavy metal remediation. Included are rice husk, wheat bran, maize corn cob, fruit peels (lemon, lime, orange, apple, pomegranate, banana, pomelo, pistachio), tea waste, coffee beans, peat bagasse, shells of groundnut, hazelnut, walnut, coconut, grapes and cotton stalks, cotton seed, and soybean hulls [91]. These are cost-effective, eco-friendly resources and can be used in their natural or modified forms. The agricultural waste consists of lignin, hemicellulose, starch, simple sugars, proteins, lipids, and water embodied with different functional groups like phenols, ketones, acids, and aldehydes that are involved in bonding with metal ions. In one such report, Mallampati and co-workers [92] demonstrated the efficiency of fruit peels (avocado, hamimelon, and dragon) in removing Pb2þ and Ni2þ from water. Electron microscopy study of fruit peel was done to determine its

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morphology, and the functional (hydroxyl, carboxyl, and amine) groups were determined by spectroscopy. The experimental data fitted in the Langmuir isotherm model suggests electrostatic interactions between adsorbent (fruit peels) and heavy metals. The adsorption capacity for lead and nickel with avocado peels was 4.93 and 9.82 mg/g; dragon fruit peels was 4.60 and 7.59 mg/g, and with hamimelon peels, this was 7.89 and 9.45 mg/g, respectively. These adsorbents exhibit reusability. Chen et al. [93] demonstrated the adsorption of cadmium ions using banana, litchi, orange, and pomegranate peels. The highest metal removing capacity was of litchi peel, followed by orange and pomegranate peel. Banana peel showed the least metal removal capacity. These peels can be reused for up to 14 cycles; thereafter, the adsorption capacity is reduced by 6.5%, 7.6%, 8.4%, and 11% for litchi peel, orange peel, pomegranate peel, and banana peel, respectively. The adsorption capacity of orange peel for Cd(II), Ni(II), Zn(II), and Co(II) ions was detected as 93.72%, 80.11%, 87.23%, and 81.06%, respectively, when 0.025 g/L of adsorbent dose and 0.001 mg/L metal initial concentration was used. Later on, Feng and Guo [94] also used orange peels as adsorbent (dose of 0.01 g/L) to obtain 93.7%, 99.4%, and 86.6% removal of Cu(II), Pb(II), and Zn(II), respectively, using each metal at a concentration of 50 mg/L. Previous studies showed that orange peel adsorbent removed Ni(II) from waste water. Recovery of adsorbed Ni(II) using 0.05M HCl through column operation was 95.8% which is higher than the 76% recovery observed via batch processes [95]. The use of raw pomegranate peel, pomegranate peel activated carbon (native form), and pomegranate peel activated carbon (chemically modified form) accomplished potential removal of Pb(II) and Cu(II) from aqueous solutions [96]. In another study, activated carbon obtained from pomegranate husk was explored to remove hexavalent chromium ions from wastewaters. The experimental data fitted the pseudo-second-order kinetic model. The equilibrium studies showed adsorption activity of 35.2 mg/g for Cr(VI). This activated carbon was used to eliminate chromium ions from seawater (natural and synthetic) and wastewaters [97]. In another study, pomegranate peel wastes were used to eliminate nickel from water. Biosorption kinetics could be described as the pseudosecond-order, and sorption capacity for nickel elimination was calculated as 52 mg/g of peel. Thermodynamics data specified that the process was endothermic and spontaneous [98]. Moghadam and co-workers [99] demonstrated the removal of Fe(II) from the aqueous solution using pomegranate peel carbon biosorbents. The maximum removal calculated was 18.5 mg/g of peel carbon. The experiment data fitted in the first-order kinetic and the Langmuir model. When grape stalk wastes were used as sorbents, rapid uptake of Cu(II) and Ni(II) present in aqueous solutions was noted in the pH range between 5.5 and 6.0. Metal sorption on grape stalks released potassium, magnesium, and calcium, suggesting that ion exchange was mainly responsible for Cu(II) and Ni(II) ion removal [100]. Modified carrot residues assisted biosorption of Ni(II), Cu(II), Co(II), and Mn(II) ions in the batch system

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and brought about 70% removal in less than 1 h. The sorption data fitted in the Langmuir model with biosorption of Cu > Ni > Co > Mn [101]. The green tomato husk was modified by formaldehyde and used for Fe3þ and Mn2þ removal from single and binary aqueous systems. Efficient metal removal was observed from single systems. Ion-exchange and complexation mechanism was involved in Mn2þ sorption, whereas precipitation and ion exchange occurred during Fe3þ sorption [102]. Rice husk is a key by-product of rice mills. It is comprised of cellulose (35%), hemicellulose (25%), and lignin (20%). In addition, proteins, and ash, including silica, were also present in a small amount. Functional groups associated with rice husk (silanol, carbonyl, carboxyl, hydroxyl, aldehyde, and ketone group) act as ion-exchangers for removing heavy metals. There is a report on the successful removal of Cd(II) from water and wastewater using rice husk. The uptake of Cd(II) was observed to be pH dependent, and followed the pseudo-second-order of kinetic model. Sorption data was best fitted in the Langmuir model [103]. A biomatrix was obtained from partial alkali digestion of a lignocellulose waste, rice husk, and was used to remove Zn, Mn, Co, Ni, Hg, Cu, Pb, and Cd ions present in the single solution and mixed solution. Adsorption capacity followed Langmuir and Freundlich isotherms. Studies revealed that calcium and magnesium present on biomatrix and carboxyl and hydroxyl groups of lignocellulose are the primary sites for ion exchange with cationic metals. The biomatrix was biodegradable, ecofriendly, and brought about a reduction of chromium [(VI) to (III)] at pH 2 [104]. Heavy metals, namely Cr [(III) and (VI)], can be removed using sawdust from Cedrus deodera. It is a very simple, inexpensive method. Here Cr(III) ions were selectively retained at the pH ranging from 3 to 4, while Cr(VI) ions were retained at pH 1. Elution was carried out using HCl and NaOH. This speciation method was successfully employed for the remediation of environmental and industrial water samples containing chromium ions [105]. Dehydrated wheat bran was used for the adsorption of Cu(II) ions, following the Langmuir isotherm. It showed 51.5 mg/g adsorption capacity for Cu(II) ions [106]. Peanut hulls and their pellets were used for uptake of copper ions present in aqueous solutions. The experiments were carried out in batch and fixed bed systems. About 75% of copper removal occurred within 20 min. As the timespan increased up to 50 min, 92% removal of Cu was observed. Bench-scale column studies showed that (modified) pelletized peanut hulls’ uptake ability was more remarkable than (unmodified) peanut hulls [107]. Walnut sawdust was evaluated for its ability to adsorb lead, cadmium, and nickel from aqueous solutions. The adsorbent showed higher selectivity for Pb(II) than Cd(II) and Ni(II). The kinetics of metal ion adsorption kinetics was described by pseudo-first-order, second-order reaction, and temperature by the Freundlich and Langmuir isotherm model. It was found that walnut sawdust could be the best option as an adsorbent for metal ions elimination from aqueous solutions [108]. Babel and Kurniawan [109] used coconut shell charcoal (CSC) and commercial activated carbon (CAC) for chromium (VI) ions elimination from synthetic electroplating

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wastewater. A coconut shell was used to prepare granular adsorbents. On nitric acid oxidation, Cr adsorption capacities of CSC and CAC were 10.88 and 15.47 mg/g, respectively, while sulfuric acid oxidation resulted in less adsorption, i.e., 4.05 and 8.94 mg/g of CSC and CAC, respectively. These results suggested that a potent oxidizing agent can modify the shell and create more adsorption sites on the surface. A report on paper mulberry (Broussonetia papyrifera) leaves used in the heavy metal removal present in aqueous solution [110]. The paper mulberry leaf powder could adsorb 43.40, 30.65, and 43.9 mg/g of Cu(II), Cd(II), and Pb(II), respectively, from an aqueous solution containing heavy metal at a concentration of 500 mg/L. The experimental data describes the Langmuir isotherm and pseudo-second-order of kinetics. The intra-particle diffusion mechanism was involved in the heavy metal adsorption. Tea wastes generated by tea-manufacturing processes were used as adsorbents and applied for heavy metal removal in an aqueous solution. Çay et al. [111] explored fibrous Turkish tea waste for the removal of copper and cadmium. The experiment was launched in a single as well as in a binary aqueous system. The adsorption capacities of Cu(II) and Cd(II) per gram of tea waste were 8.64  0.51 and 11.29  0.48 mg for a single aqueous system, whereas, for a binary system, it was 6.65  0.31 and 2.59  0.28 mg, respectively. The experimental data fitted in the Freundlich isotherm mode. In another study, tea factory waste (TFW) was used to evaluate the uptake of hexavalent chromium Cr(VI) ions present in an aqueous solution. Different parameters, namely temperature, pH, metal ion concentration, agitation rate, and adsorbent, were optimized for the study. The maximum adsorption rate was noted at pH 2. The adsorption data followed the Langmuir model, and 54.65 mg/g of Cr (VI) ions could be adsorbed on TFW at 60 C, indicating the adsorption process to be endothermic and spontaneous [112]. In another study, tea wastes were able to remove 64% of Cu(II) ions and 92% of Pb(II) ions present in aqueous solutions [113].

3. Conclusions Using natural compounds in heavy metal remediation appears to be a lucrative alternative to existing conventional protocols. Although natural compounds derived from microbial or plant origins are nontoxic, biodegradable, readily available, and inexpensive, they have not been commercialized yet in a significant manner. Next, questions regarding the safety of a few chemically modified bio-compounds discussed in this chapter also arise. Such nonconventional remediation strategies should be thoroughly investigated by following a multidisciplinary approach so that innocuous practices for heavy metal removal can be developed on a large scale. Future research should focus on filling knowledge gaps to develop effective processes for remediation of metal-polluted environment in a sustainable and eco-friendly manner.

Role of natural compounds in metal removing strategies

3.1 Future prospects In this chapter, we discussed the use of natural compounds for heavy metal remediation. In most studies related to contaminated water, heavy metal remediation was achieved by biosorption strategy. Still, its mechanism has not been clearly understood. Hence, there is a scope for further mechanistic studies. Also, remediation work at the field level should be carried out successfully using a multidisciplinary approach.

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[18] Zhang X, Wang S, Zhao S. Improved remediation of co-contaminated soils by heavy metals and PAHs with biosurfactant-enhanced soil washing. Sci Rep March 8, 2022;12(1):3801. PMID: 35260619; PMCID: PMC8904480. [19] Khiyavi AD, Hajimohammadi R, Amani H, Soltani H. Synergistic effect of rhamnolipid and saponin biosurfactants on removal of heavy metals from oil contaminated soils. Tenside Surfactants Deterg 2020;57(2):109e14. [20] Kholghi N, Amani H, Malekmahmoodi S, Amiri A. Investigation on heavy metal removal from a crude oil contaminated soil using rhamnolipid biosurfactant as a new eco-friendly method. Tenside Surfactants Detergents 2020;57(6):515e20. [21] Basak G, Das N. Characterization of sophorolipid biosurfactants produced by Crytococcus sp. VITGBN2 and its application on Zn (II) removal from electroplating. wastewater. J Environ Biol 2014;35:1087e94. [22] Ahuekwe EF, Okoli BE, Stanley HO, Kinigoma B. Evaluation of hydrocarbon emulsification and heavy metal detoxification potentials of sophorolipid biosurfactants produced from waste substrates using yeast and mushroom. In: African Health, Safety, Security, Environment and Scocial Responsibility Conference and Exhibition, Accra, Ghana. Society of Petroleum Engineers; 2016. p. 1e16. [23] Arab F, Mulligan CN. An eco-friendly method for heavy metal removal from mine tailings. Environ Sci Pollut Res 2018;25:16202e16. [24] Qi X, Xu X, Zhong C, Jiang T, Wei W, Song X. Removal of cadmium and lead from contaminated soils using sophorolipids from fermentation culture of Starmerella bombicola CGMCC1576 fermentation. Int J Environ Res Publ Health 2018;15:2334. [25] Da Rocha Junior, Meira RB, Almeida DG, et al. Application of a low-cost biosurfactant in heavy metal remediation processes. Biodegradation 2019;30:215e33. [26] Singh AK, Cameotra SS. Efficiency of lipopeptide biosurfactants in removal of petroleum hydrocarbons and heavy metals from contaminated soil. Environ Sci Pollut Res Int October 2013;20(10): 7367e76. Epub 2013 May 17. PMID: 23681773. [27] Ayangbenro AS, Babalola OO. Genomic analysis of Bacillus cereus NWUAB01 and its heavy metal removal from polluted soils. Sci Rep 2020;10:19660. [28] Ravindran A, Sajayan A, Priyadharshini GB, Selvin J, Kiran GSS. Revealing the efficacy of thermostable biosurfactant in heavy metal bioremediation and surface treatment in vegetables. Front Microbiol 2020;11:222. [29] Badrul Hisham NHM, Ibrahim MF, Ramli N, Abd Aziz S. Production of biosurfactant produced from used cooking oil by Bacillus sp. HIP3 for heavy metals removal. Molecules July 18, 2019; 24(14):2617. PMID: 31323813; PMCID: PMC6681096. [30] Chen AH, Liu SC, Chen CY, Chen CY. Comparative adsorption of Cu(II), Zn(II) and Pb(II) ions in aqueous solution on the crosslinked chitosan with epichlorohydrin. J Hazard Mater 2008;154(1e3): 184e91. [31] Joulak I, Finore I, Poli A, Abid Y, Bkhairia I, Nicolaus B, et al. Hetero-exopolysaccharide from the extremely halophilic Halomonas smyrnensis K2: production, characterization and functional properties in vitro. 3 Biotech September 2020;10(9):395. Epub 2020 Aug 17. PMID: 32832343; PMCID: PMC7431504. [32] Llamas I, Mata JA, Tallon R, Bressollier P, Urdaci MC, Quesada E, et al. Characterization of the exopolysaccharide produced by Salipiger mucosus A3, a halophilic species belonging to the Alphaproteobacteria, isolated on the Spanish Mediterranean seaboard. Mar Drugs July 30, 2010;8(8):2240e51. [33] Llamas I, Amjres H, Mata JA, Quesada E, Bejar V. The potential biotechnological applications of the exopolysaccharide produced by the halophilic bacterium Halomonas almeriensis. Molecules June 12, 2012;17(6):7103e20. [34] Diaz M, Castro M, Copaja S, Guiliani N. Biofilm Formation by the Acidophile Bacterium Acidithiobacillus thiooxidans Involves c-di-GMP Pathway and Pel exopolysaccharide. Genes (Basel) 2018;9(2): 113. [35] Liu Y, Lam MC, Fang HH. Adsorption of heavy metals by EPS of activated sludge. Water Sci Technol 2001;43(6):59e66. PMID: 11381973. [36] Wang L, Yang J, Chen Z, Liu X, Ma F. Biosorption of Pb(II) and Zn(II) by extracellular polymeric substance (Eps) of rhizobium radiobacter: equilibrium, kinetics and reuse studies. Arch Environ Protect 2013;39:129e40.

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CHAPTER 26

Policies, regulatory requirements, and risks in natural product research Priyanka Khot1, Swapnil Jadhav2, Dinesh Chaudhari3 and Kisan M. Kodam1 1

Department of Chemistry, Division of Biochemistry, Savitribai Phule Pune University, Pune, Maharashtra, India; 2Subhadra Educational Society, Pune, Maharashtra, India; 3The College of Military Engineering, Pune, Maharashtra, India

1. Introduction The patent law describes, if any substance becomes patent, it should be an invention, novel, and none anywhere used and it should be used for pharmaceutical and industrial purposes. Rule 27(a) of the European Patent Convention states that biotechnological discoveries may be patent eligible if they deal with “biological matter which is sequestered from its natural habitat or created by technical methods even if it existed in nature.” For example, a patent might be approved under European law for a specific extract that is isolated from a naturally occurring plant in a form that does not exist in nature as such [1]. Natural products are absolutely connected with nature, and for patent criteria, it considers the special rule or exceptions about the novelty and utility, but they are isolated from the environment. Due to widespread preconceptions regarding natural products, there are many false beliefs when it comes to patenting them. Here are some crucial details that can help us determine whether or not an invention incorporating a natural product is patentable. There are many other thresholds that must be passed for the patent to be approved. The process is nearly the same as Indian law and any other country’s municipal law regarding the registration of the patient regarding any content. The first requirement for patent eligibility is whether the claims in the patent application fall into one of the four legal categories of manufacture, process, machine, or composition of matter. They ought to fit into one of these groups, as do the majority of patent requests for natural products. The second threshold is whether the application’s allegations fall under one of the four judicial exceptions listed above. Natural rules, natural occurrences, and abstract concepts are among these exclusions and the claims should not be subject to these exclusions for innovations to be patentable. For those unfamiliar with the term “claims,” it’s the section in patent applications where the inventor states exactly what he’s applying for a patent. The remainder of the application (referred to as the “specification”) along with the “figures” and the “abstract” provide supporting material for the claims made in the application. It’s crucial to keep in mind that a patent is largely about the claimed subject matter, which is the thing that innovators want to prevent others from using. In this situation, the claimed subject matter is a natural product. In the end, claims determine New Horizons in Natural Compound Research ISBN 978-0-443-15232-0, https://doi.org/10.1016/B978-0-443-15232-0.00006-0

© 2023 Elsevier Inc. All rights reserved.

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whether a patent is eligible. As we know that the object behind this is not only to register the patent and to get monetary benefits but its need to protect our unique ideas and intellectual hard work on the regional and international levels. Natural phenomena/laws, abstract ideas, and mathematical calculations are not patentable. The above-mentioned exceptions are wider and aim to guarantee that vital tools of bare scientific and technical work are not being patented. Such claims in the patent applications should be directed to one of the court exceptions, and such threshold to be passed is whether the claim as a whole creates substantially more than the exception alone, in other words, whether or not an inventive concept is present. Like this, patentability valuation of natural contents or products is done by comparing what is included in the claims to their naturally occurring components to regulate whether or not they have significantly different properties than their natural components. Significantly diverse qualities are regarded as a sign that inventive notions are included. 1.1 Natural products and intellectual property Newly invented natural products play a significant part in pharmaceutical innovations. They are active components in many medicines. For example, many molecules directly derived from natural products are used to treat [2]. Inventors and companies regarding the natural product or any other product must prefer the regions in which they aspire to patent protection. Every patent office typically levies fees for the filing, processing, and maintenance of patents after they have been granted, in addition to the prescribed periodic fees. Dealing with various national legal systems can be expensive on various levels due to the wide variations in laws and related practices, and applicants will typically be required to pay for the services of an authorized patent agent in each country [3]. In the United States, normally a new patent’s validity is 20 years from the filing date or in rare circumstances, from the filing date of a previously connected application, provided that maintenance fees are paid. Only in the United States, its territories, and its possessions are US patent grants valid. There may be possibilities for patent term extensions or changes in certain instances [4]. Genetic resources are unable to protect explicitly as intellectual property since they are neither created by humans nor acknowledged as IPs. However, whether or not they are rooted in traditional knowledge, inventions that use genetic resources related to them may be patentable. In some jurisdictions of countries, plant breeders’ rights or plant patents may cover modified plants with increased starch production capabilities. For example, there are dozens of potato varieties protected by plant breeders’ rights, including “True Blue.” “Lady Liberty,” “Elmo,” and inventive methods for using starch in food production may also be patentable. In 2014, Nihon Shokuhin Kako Co. was granted a patent for swelling-resistant starch used to improve the texture of food [5]. Patents provide the toughest intellectual property protections, but it is also difficult to obtain protection for natural or herbal medicines. Generally, patent law provides the patentee

Policies, regulatory requirements, and risks in natural product research

exclusive rights to exclude the public at large from the production, usage, sales, and import of patented novelties and inventions [5]. Natural products which can be patentable encompass: (1) Novel isolation process of natural products from their surroundings. For example, an Indian patent for the process of isolation of azadirachtin from the seeds of the neem plant. (2) Characterization of a new product either by its structure or by other physical parameters. (3) A new application of an isolated product is provided unless such knowledge or invention does not exist anywhere. For example, a Japanese patent for the use of turmeric as a stabilizer for an antifungal agent. According to the Patents (Amendment) Act, 2002 (No. 38 of 2002), section 5 includes process patents on microbiological, biochemical, and biotechnological processes. Thus, genetic engineering procedures, pharmaceutical industry processes utilizing microorganisms, and similar processes are all patentable. Plants, animals, or any component of them, including seed types and fundamentally biotic processes for growing or reproducing plants and animals, are all listed as nonpatentable elements in section 3 of the patent law. Patents can be claimed for microorganisms if they are not simply discoveries of existing organisms. Moreover, methods for making plants disease free or increasing their economic value can be patentable. The obligation for the deposition of the biological matter specified in the specification with authorized institutions as issued in the Indian Gazette is covered under Section 10 [6]. 1.1.1 Natural products are not patent eligible unless The claims relate to their composition, identical to their natural counterparts, and even if the inventor succeeded in synthesizing them. This is so that the composition is constant regardless of whether the inventor isolates or synthesizes the substance. The claims apply to combinations of two or more natural or synthetic goods that simply carry out their own functions without modifying the characteristics of either one or the other or yielding any unexpected consequences. In other words, they do not have significantly different properties than their natural counterparts. The claims are directed to the product produced by the process, but the product is no different from its natural counterpart. Claims are focused on natural processes such as pathways, signaling, metabolic pathways, and natural correlations like insulin-sugar. 1.1.2 Natural products are patent eligible if Claims allude to a formulation that has been altered by changing a product’s structure that does not exist naturally. Therefore, it differs from its natural counterpart. The claims are directed on how to make, how to produce, and how to synthesize from composition methods or manufacturers. Protection is being sought for the novel procedure that has been devised to synthesize the naturally occurring product, rather than the product itself. The potential of traditional knowledge has drawn the attention of numerous researchers, pharmaceutical businesses, and organizations. Additionally, rules vary significantly

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between nations, but broadly speaking, in order to secure protection, an inventor or company must submit an application to a patent office explaining the invention in detail enough to permit use or reproduction by a person with ordinary technical expertise. Drawings, blueprints, or diagrams are frequently included in these descriptions.

2. Protection of new plant variety India, one of the founding members of WTO, is obligated to carry out the TradeRelated Aspects of Intellectual Property (TRIPs) agreement which typically offers baseline standards of protection for intellectual property rights in all member governments [7]. The Plant Variety Protection and Farmers Rights Act of 2001 aims to reward traditional, rural, and tribal communities for their significant contribution to the nation’s agro-biodiversity, recognize farmers’ roles as cultivators and conservers, and promote investment in research and development for the establishment of new plant varieties in order to aid the advancement of the seed manufacturing industry. In order to protect new varieties, the Plant Variety Protection and Farmers Rights Act 2001 was passed in India. The act officially took effect on October 30, 2005. At the outset, 12 crop species including, wheat, sorghum, maize, rice, green gram, chickpea, black gram, kidney bean, lentil, pearl millet, etc. have been recognized for regulation. The sui generis system has been embraced by India as a substitute for patents to protect novel varieties. The administrative ministry of the Department of Agriculture and Cooperation is handling its registration and other affairs [8]. The PGRFA Treaty has also been approved by India. The three interrelated objectives of benefit sharing, sustainable use, and conservation are provided by this treaty, which broadly follows the Biodiversity Convention’s ideology. The interaction between intellectual property rights and environmental management is addressed by the Biodiversity Convention and the PGRFA Treaty. The TRIPs Agreement deals with issues concerning intellectual property rights but overlooks issues pertaining to traditional knowledge management or environmental conservation [7].

3. Natural products and international protection The Geographical Indication assures consumers that a produced product in a specific location has special characteristics constrained to that location. It cannot change ownership and may be utilized by all producers in the relevant territory who produce goods with particular qualities associated with that region. International law has evolved to strengthen and augment national and regional protections, just like other types of IP. The Paris Convention of 1883 introduced the first international standard for indications of source and appellations of origin. Recent amendments to the agreement on TRIPs added certain new clauses to stop the abuse of Geographic Information Systems. The WIPO also oversees the management of the International Lisbon System. The Geneva

Policies, regulatory requirements, and risks in natural product research

Act of the Lisbon Agreement on Appellations of Origin and Geographical Indications, enacted in 2015, expanded the system to make it possible to register other geographical indications globally as well. Previously, this only applied to appellations of origin. 3.1 Convention on Biological Diversity and Nagoya Protocol The Convention on Biological Diversity (CBD) is an international agreement established to encourage the conservation and unbiased sharing of the welfare attributed to the use of genetic resources as well as the sustainable development of biodiversity. 196 countries have ratified the CBD, including India. Its encompassing goal is to promote behaviors that will result in a sustainable future. The CBD protects genetic resources, species, and ecosystems at all levels of biodiversity [9]. Accordingly, we must comply with protocols and international treaties in order to ensure our product is protected at an international level. The Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization to CBD, the supplement agreement, was adopted on 29 Oct. 2010, in Nagoya, Japan, and entered into force on October 12, 2014. It offers a global framework for executing and advancing the CBD’s third goal. The Nagoya Protocol includes essential aspects for access to genetic resources, the equitable distribution of benefits derived from their use, and compliance with biodiversity conservation and sustainable usage. Furthermore, the Nagoya Protocol defines genetic resource utilization in a novel and innovative way. Utilizing genetic resources, as defined in Article 2 of the Protocol, includes using biotechnology, as defined in that article, to perform research and development on the genetic and/or biochemical composition of genetic resources [10]. 3.2 World Intellectual Property Organization In order to protect inherited or conventional knowledge, traditional cultural expressions (folklore), and genetic resources effectively, the members of WIPO founded the Intergovernmental Committee (IGC) on IP and inherited or conventional knowledge, folklore, and genetic resources, in 2000 and agreed to construct one or more international legal frameworks to do so in 2009. Such a document might take the form of a formal treaty obliging countries that ratify it or a recommendation to WIPO members [11]. The WIPO is a United Nations customized body formed in 1967 with the aim of fostering invention and creativity for the social, economic, and cultural advancement of all nations through the establishment of an equitable and operative global IP system. The organization strengthens the preservation of folklore, traditional knowledge, genetic resources, and IPRs [12]. The WIPO advocates for the global harmonization of intellectual property norms. So as to deal with emerging IP affairs, WIPO offers a global policy platform that brings together governments, civil society, and commercial organizations. The two fundamental aims of WIPO are to assure administrative cooperation among IPO and to provide worldwide IP protection [11,12].

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3.3 Patent Cooperation Treaty (PCT) By submitting an international patent request, the PCT allows contemporaneous protection of patents for an invention in many different nations. Anyone who is a citizen or resident of a PCT contracting state can submit such an application. It may typically be filed with the national patent office of the contracting state in which the applicant is a national or resident, or, at the applicant’s discretion, with the International Bureau of WIPO in Geneva [13]. 3.4 World Trade Organization and TRIPS The World Trade Organization (WTO) is the foremost body responsible for establishing global trade regulations. A substantial international treaty known as Trade-Related Aspects of Intellectual Property Rights (TRAIPR) was founded in order to furnish a uniform criterion for the protection and enforcement of IPRs among WTO member states. The TRIPS Agreement, which is governed by the WTO, was drafted at the Uruguay Round of the General Agreement on Tariffs and Trade (GATT) in 1989 and 1990 which pertains to trade-in services. The three pillars of the WTO have been referred to as these three treaties [14]. The TRIPS Agreement strives to establish prerequisites for the protection of intellectual property. The TRIPS, which integrates a considerable amount of legislative law from preceding international agreements like the Berne Convention and Paris Convention, is thought to be the most crucial international pact on IP [15].

4. Natural resources and associated traditional knowledge The term “traditional knowledge” tends to refer to information that is held by native people in societies and in one or more formats but is not confined to, literature, handicrafts, designs, medicines, folk culture, dance, folk remedies, medicines, biodiversity, and folk knowledge [16]. A community’s knowledge, skills, and practices are generated, maintained, and passed down through generations; they frequently contribute to the cultural or spiritual heritage of that community [17]. Traditional knowledge (TK) is an essential component of most local communities’ identity. Traditional Ecological, Agricultural, and Medicinal Knowledge (TEK, TAK, and TMK) are three categories under which the phrase “traditional knowledge” can be classified. Aboriginal knowledge is a significant proportion of traditional knowledge, that communities, peoples, and nations retain and use. Traditional Medical Knowledge (TMK) is used by indigenous people, primarily in rural areas, to sustain their health system [16,18,19]. In India, traditional knowledge is protected by the Patent Act of 1970. The WIPO is working to develop a draught of the transnational legal framework for the protection of TK that permits access to those outside the nation or community of its traditional holders on a global level through IGCdGenetic Resources, Traditional Knowledge and Folklore (GRTF) [20].

Policies, regulatory requirements, and risks in natural product research

5. Bioprospecting and biopiracy The profound information from aboriginal people and native communities is frequently exploited by biopharmaceutical companies engaged in bioprospecting for the development of new drugs. Bioprospecting is the methodical pursuit for and expansion of novel sources of molecules, genes, microorganisms, and other valuable natural products. It promotes the discovery of economically valuable genetic and biochemical resources in nature. Its goal is to discover ways to get the most out of natural reserves. The majority of medicinal plants were discovered through the bioprospecting process [12]. Recently, the dispute over intellectual property has received much international attention concerning intellectual property, resources, traditional knowledge, and legacy. International experts are inquisitive about using biopiracy to illustrate particular instances of biased or inappropriate IP claims over traditional knowledge and biological resources [20]. Pat Mooney coined the term “biopiracy” to describe a practice in which aboriginal knowledge of the environment, which is instigated by indigenous people, is used for profitable purposes by others without consent or compensation to the indigenous people themselves. For instance, bioprospecting can be viewed as nothing more than biopiracy when inherited knowledge of therapeutic floras is later patented by pharmaceutical industries without appreciating the fact that the knowledge is neither novel nor original to the patentee. The indigenous community is thus denied the opportunity to commercially exploit the technology they independently developed [21]. Biopiracy denotes the process of patenting living resources or traditional knowledge and practices in order to impose intellectual property restrictions on their utilization. It refers to the appropriation of farming and indigenous community’s knowledge and genetic resources by individuals or institutions seeking to monopolize these resources and knowledge by obtaining patents or other forms of intellectual property. It considers intellectual property to be a predator of farming communities and indigenous peoples’ rights and knowledge patents attributed to nanotechnology and synthetic biology, which produce natural products with high commercial value, is now being used to extend intellectual property claims to elements of the periodic table and critical metabolic pathways responsible for cell functioning. The well-known examples of biopiracy that the world witnessed and became aware of it. These examples turned into milestones to prevent biopiracy in the future. Lately, turmeric, Curcuma longa, is a tropical herb native to eastern India. The bitter flavor and dark color of turmeric powder are distinctive. It is used for medicinal purposes, as a food ingredient, dye, and for a litmus test. A United State patent on turmeric, particularly for wound healing applications, was first awarded to the University of Mississippi Medical Centre in May 1995. Then the Council of Scientific and Industrial Research (CSIR), India, lodged a complaint after 2 years. The CSIR contended that since turmeric has

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been used to treat wounds and rashes in India for several thousand years, the patent on its therapeutic application is not a novel invention. The proof of documented ancient knowledge in old Sanskrit manuscripts as well as an article issued in the journals of the Indian Medical Association in 1953, confirmed the CSIR’s assertion. The patent’s validity was investigated by the USPTO. Despite the patent holders’ appeal, the USPTO endorsed the CSIR objection in 1997 and revoked the patent for a lack of novelty [22]. Not to mention another example of biopiracy that sensitizes the world. The Azadirachta indica (common name, Neem) is a tropical evergreen plant native to India and other southeast countries. The leaves, bark, and seeds are comprised of ingredients that have been shown to have antimicrobial, antiviral, antipyretic, antiulcer, and antiinflammatory activities. After discovering the tree’s benefits in India, US timber importer Robert Harson started importing neem seeds to the headquarters of his company in 1971 and conducted performance and safety tests. Three years later, he sold his invention to the US Department of Agricultural and a multinational chemical corporation, WR Grace and Co. In 1992, the company secured its right to the formula that used the emulsion from neem trees, and seeds to make a powerful fungicide. In applying for the patent, the company had argued that it had used an extract of the trees, and seed to make a new fungicide, but the Indians claim that its patent was not sufficiently novel, as Indian farmers have used this as a fungicide for decades. The Indians and European Union’s green party members opposed the patent because they believed that doing so would violate the rights of underprivileged farmers in developing nations. The neem patent was the first to raise concerns about biopiracy pertaining to European and US patents. The Indian scientists contended that the Indians had long known about the medicinal properties of neem. The US patent office’s decision to award the patent to WR Grace was overturned by the European Patent Office (EPO), which accepted the justifications put forth by Indian scientists [23]. The foundation for research in science, technology, and the environment put forth a 4-years effort that paid off with the victory.

6. Conclusion Natural products are rapidly achieving extensive popularity worldwide as synthetic treatments are being relinquished to clasp natural alternatives. Interestingly, the pharmaceutical industries of natural products are making an effort to acquire a strong market grasp for their products. The contention for IP protection has thus become intense with the growing market for natural products. Consequently, companies are in search of loopholes in current laws or non-IP approaches which will lead them to monopolize the market. In such cases, intellectual property protection plays a vital role as a fundamental imperative for socioeconomic advancement. So, there should have a benchmark for products to protect the superiority of intellectual property protection. Because developing countries like India, the pivot of traditional knowledge and distinctive endowment, have the

Policies, regulatory requirements, and risks in natural product research

remarkable uncharted potential for the utilization of traditional knowledge. Thus, it is essential to deal with bioprospecting along with disputes of biopiracy at the global level using existing national and international legislation. Finally, it is the utmost responsibility of respective governments or organizations to encourage all forms of innovations for human welfare within IPR frameworks.

References [1] Nicolle F. Patenting natural products (part 1): newly isolated material; the approach under European law to patenting newly isolated natural material. 2020. [2] Newman D, Cragg G. Natural products as sources of new drugs over 30 years from 1981 to 2014. J Nat Prod 2016;79(3):629e61. [3] Courage N. Getting maximum IP from R&Ddnatural products. B and P; 2020. [4] Lecture Notes on III B. Tech II semester (JNTUH-R13), Smartzworld.com. p. 4. [5] Chan AW, Lin JC. Intellectual property protection of natural products. Asia Pacific Biotech News 2004;540(10):8. [6] The Gazette of India extraordinary PART IIdsection 1. The Patents (Amendment) Act, 2002, No. 38 of 2002. [7] Cullet P, Kolluru R. Plant variety protection and farmers’ rights: towards a broader understanding. In: 24 Delhi Law Review 2002; 2003. p. 41. [8] Savale SK, Savale VK. Intellectual property rights (IPR). World J Pharm Pharmaceut Sci 2018;5(6): 2529e59. [9] CBD. Biodiversity convention. 2012. https://www.cbd.int/intro/. [10] CBD. The Nagoya protocol on access and benefit-sharing. 2022. https://www.cbd.int/abs/. [11] WIPO. Traditional knowledge and intellectual propertydbackground brief. 2022. https://www. wipo.int/pressroom/en/briefs/tk_ip.html. [12] Javed G, Priya R, Deepa VK. Protection of traditional health knowledge: international negotiations, national priorities, and knowledge commons. Soc Cult South Asia 2020;6(1):98e120. [13] WIPO. Patent cooperation treaty (PCT). 2019. https://www.wipo.int/treaties/en/registration/pct/ summary_pct.html. [14] Pandit D, Deb PK, Tekade RK. Patents and other intellectual property rights in drug delivery. Dosage Form Design Parameters 2018;2:705e30. [15] Lai JC. Indigenous cultural heritage and intellectual property rights: learning from New Zealand experiences. Cham: Springer; 2014. [16] WIPO. Protecting India’s traditional knowledge. WIPO magazine. Available from: http://www. wipo.int/wipo_magazine/en/2011/03/article_0002.html, ; 2011. [17] WIPO. Traditional knowledge. 2000. https://www.wipo.int/tk/en/tk/. [18] Ahoyo CC, Houehanou TD, Yaoitcha AS, Prinz K, Glele KR, Sinsin BA. Traditional medicinal knowledge of woody species across climatic zones in Benin (West Africa). J Ethnopharmacol 2021:265. [19] Bhat SR. Innovation and intellectual property rights law: an overview of the Indian law. IIMB Manag Rev 2018;30(1):51e61. [20] Bency BT, Suriyaprakash TNK. Intellectual property rights: bioprospecting, biopiracy and protection of traditional knowledgedan Indian perspective. IntechOpen; 2021. [21] Robinson DF. Biopiracy and the innovations of Indigenous peoples and local communities. Indig Peoples Innov Intellect Prop Pathways Dev. 2012:77e93. [22] Jayaraman KS. US patent office withdraws patent on Indian herb. Nature 1997;389:6. [23] https://cordis.europa.eu/article/id/23505-epo-accepts-biopiracy-argument-and-revokes-patent.

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Index Note: Page numbers followed by “f ” indicate figures and “t ” indicate tables.

A Acceptor hydrogen bonds, 147 ACE inhibition, 227 Acetylcholinesterase hydrolyzes, 405 Acidic/basic properties and pH stability, 40 Acinetobacter spp, 173e174 Actinomycetes, 467 Activation energy of reactions, 203f Active sites on bacterial cell, 173 Activity-based probes, 139e140 ADME, 193e194 Adsorbent heavy metals removal, 501 Adsorption enrichment, 38 African swine fever virus, 258e259 Agaricus campestris Linnaeux, 2 Agaro-oligosaccharides (AGOS), 234, 237, 238t Agriculture wastes-based removal of heavy metals, 501e504 Agri-food application, 429f Agrobacterium radiobacter, 473 Agrochemicals, 428 AIDS, 8 Air pollutants, 461 Alanine glyoxylate aminotransferase (AGT), 229 Algae derived marine oligosaccharides (ADMO), 111e112, 117f Algal drugs, 259 Algal metabolites, 260 Algal strains, 259t Alginate-based materials used in heavy metals removal, 496e498 Alginate oligosaccharides (AOS), 233e235, 240e242, 241t Alkaloids, 87e97 Allium cepa Linn, 278 1-Allyl-3-methylimidazolium chloride, 499e500 Altriset, 404e405 Alzheimer’s disease (AD), 148, 321e322, 325e326 Amino sulphonic acid, 63 Amycolatopsis orientalis, 6 Amyotrophic lateral sclerosis (ALS), 327 Anabaena spiroides, 495e496

Anacardiacae, 357 Anaerobranca gottschalkii, 363 Anthelmintic drugs, 324 Anthemis nobilis, 356 Anthocyanin monomers from red cabbage, 42e43 Anthocyanins, 42e43 Antibacterial activity, 258 Antibiotic medicines, 122f Antibiotics through inhibition of enzymes modification of, 173 Anticancer, 76 drugs, 326 Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV), 394e395 Anti-dandruff, 357 Antifungal activity, 259 Anti-HIV, 63e64, 95 Anti-HSV, 258e259 Anti-inflammatory, 254e255, 357 drug, 329e330 effect, 226 Anti-Leishmania effects, 68 Anti-lice, 357 Anti-malaria, 357 Anti-Methicillin, 170 Antimicrobial activities of microalgae, 257e259 Anti-microbial drugs, 325e326 Antimicrobial resistance (AMR), 333 Anti-nephrolithiatic medicinal plants, 227 Antioxidant network, 275e276, 277f, 280te284t, 284 Antioxidants, 227, 271f, 357 Anti-rheumatoid drug, 329e330 Anti-tumor, 357 Antiviral activity, 258e259 Aporphine alkaloids, 92 Aquatic-terrestrial cycle, 452e453 Arabidopsis, 429e430 Arteminolides, 78 Artemisia annua, 8 Arthrocnemum glaucum, 91 Artificial scaffolds, 378t Ascomycota, 6

521

522

Index

Aspergillus niger, 207e208 Aspilia Africana, 356 Aspirin, 325 Astaxanthin, 254 Atherosclerotic cardiovascular disease (ASCVD), 277e278 Atom economy, 55te58t Atropa belladonna, 2 Autotrophic system, 457 Azadirachta indica, 405, 518 Azadirachtin, 405

B Bacillus, 363, 393e394 Bacillus amyloliquefaciens, 208 Bacillus anthracis, 159e160 Bacillus subtilis, 207e208, 235, 393e394 Bacillus thuringiensis (Bt), 393e394, 403, 473 Bacteria, 393e394 Bacterial degradation of pesticides, 461e467 Bacterial diseases, 364 Baculoviruses, 394 Ball milling, 64e67, 66f Barbier-type reaction, 62 Barbituric acid, 63 Beauveria bassiana, 392e393 Beewaxes, 307 Berberis aristata, 174e176 Betula pubescens, 6 Bexarotene, 326 Bicyclic monoterpene, 76e78 Bioactive alkaloids, 90t Bioactive compounds applications of, 256e257 from microalgae, 252e256 Bioactive polyketides, 109f Bioactive polysaccharides, 120te121t Bioassay-guided fractionation, 407 Bioaugmentation, 456e457 Bio-ccumulation, 491 Biochemical pesticides, 403 Biodegradability, 22e24 Biodegradation, 422, 474e475 Biodetoxification, 445e446 Biodiversity Convention, 514 Biofilters, 461 Biogenesis of polyphenols, 98f Bioinformatics, 154e155 concept, evolution of, 155e158

in discovery of natural products, 155 Bioleaching, 491e492 Biological-based green chemistry methods, 67e68 Biological organisms, 455 Biologics, 191e194, 195t Biologics drug (BD), 179 Biology-Oriented Synthesis (BIOS), 128 Biomagnifications, 452 Biomass, 17e18 Biomineralization, 491e492 Biopesticides, 398, 399te402t, 403e404 Biopiling, 457 “Biopiracy”, 517 Biopolymers, 496 Biopolymers-based removal of heavy metals, 496e501 Bioprospecting and biopiracy, 517e518 BioRDF, 151 Bioreactors, 460 Bioremediation, 453, 462t, 474e475 of pesticides, 453e461 salient features of, 453e454 systems, 460 types of, 454e461 Biosorption, 491 Biosparging, 455 Biostimulation, 455e456 Biosurfactant-based removal of heavy metals, 492e494 Biosurfactant-based soil remediations, 492 Biosynthetic enzymes direct proteomic analysis of, 140e141 Biosynthetic gene clusters (BGCs), 133e134, 136e137, 139 Biosynthetic pathway, 77f Biotransformation, 491e492 Bioventing, 455 Black Raspberry, 276e277 BLAST, 157e158 Blood-brain barrier (BBB), 274 Borneol-based biological active scaffolds, 126f “Botanical insecticides”, 423 Breast cancer, 319e320 Breast cancer cell, 175t Bt insecticidal toxins, 393e394

C Ca-alginate scaffold, 383 Cadmium toxicity elimination, 493

Index

Calonphyllum, 8 Cananga odorata, 356 Cancer, 270e271, 317e319 Cancer stem cells (CSCs), 322e323 Candida bombicola, 494 Cannabis extract, 168e169 CaOx formation, 227t Capsaicinoids, 105 Capsular polysaccharides (CPS), 495e496 Carbamate pesticides, 454f Carbohydrate active enzymes (CAZymes), 231 Carbohydrates, 108e119, 365e367 based on chemical structure, 110t Carbon-carbon bond formation, 62 Carbon fixation, 433 Carboxylesterases, 473 Carboxylic acid, 97e100 Carboxy-methylated formic lignin, 498e499 Carboxy methyl cellulose, 431, 500e501 Cardiovascular disease (CVD), 267e268, 273, 324e325 drug repurposing for, 324e325 Carminic acid, 307 b Carotene, 253 Carotenoids, 253e255 Carrageenan oligosaccharides (COS), 234e235, 238e240 Catalysis, 55te58t Catharanthus roseus, 188 Cationic polymers, 365e367 Cell culturing, 376 Cellular humoral immunity, 298 Cellular Thermal Shift Assay (CETSA), 134e136 Cellulose-based adsorbent for heavy metals removal, 500e501 Cellulose nanocrystals boost seed germination, 429e430 Centella asiatica, 83e85 Centers for Disease Control, 167 Cephalosporin, 147e148 CGTase, 362e363 Chaetocerous mueller, 257 Chalcones transformation using bacteria, 68 using green solvents, 69 Charcot disease, 327 Charge, 40 Chem2BioRDF, 151 ChEMBL, 134e136

Chemical analysis process, 18f Chemical process, 55te58t Chemical skeletons, 75e76 diversity of purine in nature, 97f semisynthetic or modified NPs, 119e126 structural diversity in natural products, 76e119 alkaloids, 87e97 antibiotics, 119 carbohydrates, 108e119 phenolic compounds, 97e107 polyketides, 108 terpenes, 76e87 synthetic approaches for building, 126e128 Cheminformatics and natural products, 158 Chemopreventive foods, 275 antioxidants in, 275e279 Chinese Tradition Medicine (CTM), 51 Chitosan-alginate scaffolds, 384 Chitosan-based adsorbents and heavy metal removal, 499e500 Chitosan-carboxymethyl cellulose, 435e436 Chitosan-polyvinyl alcohol hydrogel, 499e500 Chitosan-titanium oxide nanotube (CTNT), 383e384 Chloramphenicol, 173e174 Chlorella, 258 Chlorella ellipsoidea, 254e255 Chloride intracellular channel-1 (CLIC1), 321e322 Chlorogenic acids, 276e277 Chondrodendron tomentosum, 8 Chondrus crispus, 2e3 Chorthippus dorsatus, 301 Chromated copper-arsenate generate arsenic comprising wastes, 490 Chromatography, 189 Chromium, 495e496 Chronic obstructive pulmonary disease (COPD), 273e274 Chrysanthemum, 416e418 Chrysanthemum flowers, 404 Chrysanthemum pyrethrum, 419e420 Cinchona succirubra, 4 Cinnamomum spp., 353 Citrus fruit juices, 229 Citrus juices, 229 Citrus limon, 355 Claisen synthesis, 3 Classical solvent extraction, 18e19

523

524

Index

Closed vessel system, 20e22 Clostridium perfringen, 78 Coconut shell charcoal (CSC), 503e504 Col1-PAA scaffold, 383 Collision-induced dissociation (CID), 140e141 Colorectal cancer (CRC), 320e321 Combretum caffrum, 7 Commercial activated carbon (CAC), 503e504 Commiphora species, 1 Complex polysaccharides (CPs), 231 Composting, 460 Compound synergy in natural crude extract, 167e168 factors responsible for, 168e170 synergistic effect on antimicrobial activity, 172e174 on diabetes, 170e172 of natural drugs on breast cancer cell, 174e176 Compression cycles, 19e20 Condensation processes, 418 Con-Ins-G1, 154 Conus geographus, 154 Conventional method, 351 Convention on Biological Diversity (CBD), 515 Coronary heart disease (CHD), 324e325 Coumarins, 100e101 Crohn’s disease, 193 Cry proteins, 403 Cryptococcus sp, 494 Cryptotermes cynocephalus, 422 Cryptotheca crypta, 10 Crystalline proteins, 403 CTNT-TMM scaffold, 383e384 Cupressus sempervirens, 1 Curcuma longa, 517e518 Curcumin, 436 Cyanobacteria, 495e496 Cycloaddition methodologies, 62e63 Cyclodextrin polymers (CDPs), 361e362 Cyclodextrins (CDs), 361e365, 366f in biopharmaceuticals, 365e368 chemical and physical aspects of, 363e364 cyclic oligosaccharides, 362e363 inclusion complex formation, 364e365 natural production of, 362e363 regulatory aspects of, 368 Cyclooxygenase enzyme 1 (COX1), 192 Cyclooxygenase enzyme 2 (COX2), 192 Cyclosporin-A, 332e333

Cydia pomonella GV (CpGV), 394e395 Cypermethrin, 473 Cytochrome P450 (CYP), 474

D Dalmatian chamomile, 418 2D cell culture system, 374e375 Degrees of polymerization (DP), 231e232 “De-replication”, 51 “DEREPLICATOR+”, 142 Design for degradation, 55te58t Design for energy efficiency, 55te58t Designing safer chemicals, 55te58t Desorption electrospray ionization mass spectrometry (DESI-MS), 300 Diabetes, 272, 329 drug repurposing for, 329e330, 330te331t Diabetic kidney disease, 329e330 Dialysis method, 188e189 Dichlorodiphenyltrichloroethane (DDT)_, 468e471 Diels Alder cycloaddition reaction, 66e67 Dietary approaches to stop hypertension (DASH), 278e279 Digitalis lanata, 180 Digitalis purpurea, 4 Dihydropyrimidine, 69e70 Dimethylallyl pyrophosphate (DMAPP), 76 4-Dimethylaminopyridine (DMAP), 419e420 Dipolar rotation, 20e22 Dipterocarpaceae, 422 Directinfusion mass spectrometry (DIMS), 48e50 Direct proteomic analysis of biosynthetic enzymes, 140e141 Disaccharides, 111 Diterpenes, 79e83 Diuretic effect, 226 Diversity-Oriented Synthesis (DOS), 128 Diverted-total synthesis (DTS), 125e126, 128 DNA damage, 268e271 molecules, 174e176 polymerase, 203e205 Docosahexanoic acid (DHA), 255 Dormant and summer oils, 421 “Dormant oil”, 421 Doxorubicin, 7 Drosophila melanogaster, 298

Index

Drug Affinity Responsive Target Stability (DARTS), 134e136 “Drug discovery”, 151 Drug repositioning (DR), 315 Drug repurposing, 315e316, 330te331t for cardiovascular diseases, 324e325 in diabetes, 329e330, 330te331t for effective cancer management, 317e324 experimental and computational approaches for, 317 for microbial diseases, 333e334, 334te338t for neurodegenerative disorders, 325e328 in retrovirus, 332e333 for viral diseases, 331e333 Drugs derived from FOS route, 127f 3D system, 375e376 Dunaliella tertiolecta, 254e255

E Earthworms, 450 Easy availability, 419 Ebers Papyrus, 1 Eco-friendly method, 64f, 457 E. coli, 122, 333e334 Ecotoxigenic/genotoxic, 445e446 Edible mushrooms, 277e278 Effective cancer management drug repurposing for, 317e324 EHRs, 152 Eicosapentanoic acids (EPA), 20e22, 228, 255 Electronic health record mining, 152e153 Electron transport chain (ETC), 268e269 Electroplating, 490 Electrospray (ES), 191 Electrostatic bonding, 492 “Enantiomer”, 217 Encephalomyocarditis virus, 258e259 Endoplasmic reticulum (ER), 328e330 Endotoxin, 403 Englebreth-Holm-Swarm mouse tumor, 382 Enrichment and isolation, 42 Enterobacter asburiae, 466e467 Entomopathogenic fungi, 392e393 Entomopathogenic protozoans, 395 Environmental Protection Agency(EPA), 391e392 Enzyme-assisted extraction (EAE), 27, 28t, 37e38 Enzyme-catalyzed reaction, 211f Enzymes, 28t, 201, 204t, 207f

cofactors for, 215 concentration, 208 definition and classifications of, 201e215 general mechanism of action of, 203e206 kinetics, 210e214 production process, 206e210 properties of, 206 yield, 214e215 Enzyme-substrate (ES), 212e213 Escherichia coli, 181, 428e429 “ESKAPE”, 333e334 Essential oils (EOs), 351e353 medicinal plants as source of, 353e357 methods of extraction of, 353 as source of medicine and drug discovery, 357 Esterases, 472e473 Ethylenediaminetetraacetic acid (EDTA), 500e501 Eucalyptus globules, 355 Eucalyptus oil, 355 Euphorbia peplus, 7 Evaporation, 19, 418 Exopolysaccharides (EPSs), 495e496 Ex situ bioremediation, 460e461 Extracellular matrix (ECM), 374e375, 382 Extracorporeal shock wave lithotripsy (ESWL), 225 Extraction, 42, 181 methods, 37t process, 17e18 of pyrethrine, 418

F FDA, 315 Ferula ammoniacum, 100e101 Filippi’s gland, 298 Fingerprinting, 11 Fish oil, 228 Fixation of nitrogen, 450e452 Flavonoids, 102e105 compounds, 68 Fluorescence immunoassays, 257 Food and Agriculture (FAO), 448 Food and Drug Administration (FDA), 47 Food and vegetables for controlling cancer, 276e277 for controlling diabetes, 278 for controlling hypertension, 278e279

525

526

Index

Food and vegetables (Continued) for controlling lung disease or cardiovascular diseases (CVD), 277e278 for controlling neurological disorders (NDDs), 278 Food packaging, 435e436 Food preservation, 436 Food processing, 434e435 Food production, 391e392 Food safety and security, 436e437 Fourier-transform infrared spectroscopy (FTIR), 190e191 Fourier transform ion cyclotron resonance (FT-ICR), 48e50 FRDA, 328 Free fatty acids (FFAs), 297e298 “Free radical scavengers”, 269e270 French West Indies (FWI), 391e392 Freundlich isotherm model, 499e500 Friedreich’s ataxia (FRDA), 328 Fucoidan oligosaccharides (FOS), 242 Fucoxanthin, 255 Full width at half maximum (FWHM), 48e50 Function-Oriented Synthesis (FOS), 126 Fungal biodegradation of pesticides, 461 Fungal metabolites, 147e148 Fusarium oxysporium, 210

G Galactoglucan, 495 Galanthus nivalis, 8 Gallic acid, 226 Gamma-aminobutyric acid (GABA), 398e403 Ganoderma lucidum, 6 Gas chromatography (GC), 47 Gastrointestinal effect, 78 Gastrointestinal T lymphoctes, 193 Gastrophysa atrocyanea, 296e297 GC-MS, 294 Gelatin-alginate-fibrinogen (GAF), 384e385 Gelidium, 233 Gene expression perturbation, 160e161 Gene Ontology, 150e151 General Agreement on Tariffs and Trade (GATT), 516 “Generally recognized as safe” (GRAS), 393e394 Genetic resources, 512e513 Genetic Resources, Traditional Knowledge and Folklore (GRTF), 516

Genome mining, 138e139 Genome sequence of microbes, 138f Geographic Information Systems, 514e515 Geraniol-motiff as drug agents, 123f Geranyl pyrophosphate (GPP) precursor, 79f G. lemaneiformi, 234 Glioblastoma multiforme (GBM), 321e322 Glioma stem cells (GSC), 321e322 Gluconeogenesis, 297e298 Glutamate-gated chloride channel, 403 Glutamic acid, 297 Glutathione S-transferases (GSTs), 473e474 Glycoproteins, 108e110 Gracilaria, 233 Green chemistry, 55, 59f, 201, 202f, 215 future of, 58e59 general synthetic approaches to, 60e61 principles of, 55e58 Greener extraction, 24 Green synthesis of natural compounds, 55e70 ball milling, 64e67 biological-based green chemistry methods, 67e68 future of green chemistry, 58e59 general synthetic approaches to green chemistry, 60e61 grinding technique, 63e64 microorganism synthesis of natural product acutumine, 68e69 natural product psymberin using ultrasonic irradiation, 62 principles of green chemistry, 55e58 total synthesis of, 62e63 ultrasound supported green synthesis, 61e62 Grinding technique, 63e64, 65f Gyrodinium impudicum, 258e259

H H1 NMR-based, 294e296 Haber-Bosch process, 201 Haematococcus sp., 253 Hazardous heavy metals, 490 Healthy diet, 275e276 Heat reflux extraction, 19 “Heavy metal”, 489 Helicoverpa armigera, 298, 432e433, 472 Heliotropium megalanthum, 398 Hemocoagulation activity, 124

Index

Hepatotoxicity inducing Acetaminophen (APAP), 159e160 Herbicides, 430e431 Heterocyclic alkaloid, 95e97 Heterogenicity, 375 Heterotermes indicola, 422 High-performance anion exchange chromatography (HPAEC), 236 High-performance liquid chromatography (HPLC), 40e41, 189 High-resolution mass spectrometry (HR-MS), 48e50 High-speed counter-current chromatography (HSCCC), 41 High-speed vibrating mill, 63 High-throughput screening (HTS), 75, 126e128, 153e155 “HighValue Molecules” (HMV), 252 HIV, 6, 367 HIV-1 RNA, 332 HMMs, 157e158 Homalanthus nutans, 8 “Hormone receptor positive”, 174 Hormone stimulation, 357 Horticultural oil, 420e421 HPLC, 235e236 Huge libraries, 75 Human health, pesticides on, 449e450 Human Metabolome Database (HMDB), 190e191 Human microbiome, 339 Human papillomavirus (HPV), 332e333 Huntington’s disease (HD), 327e328 Hybrid aligned electrospun nanofiber (HANF), 383 Hydrogel beads, 500e501 Hydrogels, 382e385 Hydrogen peroxide (H2O2), 273e274 Hydrolases, 205 Hydrophobic immobilized ligands, 191 20-Hydroxyecdysone, 297 Hyperammonemia, 323e324 Hypericum perforatum, 170 Hypertension, 272e273

I Immune system, 449e450 Immunoglobulins, 367e368 Immunohistochemistry, 382

Immunotherapy, 148 Inclusion viruses (IVs), 394 Indian agriculture natural insecticides, 418 Indian Medical Association, 517e518 Indian propolis samples, 170 Indole (pyrrole ring fused to benzene), 87e88 Indolizidine alkaloids, 92e93 Indolizidine NPs, 94f Indolizidine rings, 69 Industrial application, 307 Inhibition of efflux pumps, 173 Innovative drugs, 339 Insect-associated metabolome, 299e302 Insect growth regulators (IGRs), 396e398 Insect gut microbiome metabolome, 301e302 Insecticidal activity, 357 Insecticidal soaps, 421e422 Insect metabolome, 293e294, 303te304t analysis, specialized methods for, 294e296 diversity and spatio-temporal dynamics, 296e297 insect-associated metabolome, 299e302 insect gut microbiome metabolome, 301e302 potential application of, 302e308 uniqueness of, 297e299 Insect pheromones, 397 In situ bioremediation technologies, 455e460 Insoluble cellular marc of natural biomass, 17e18 Integrated pest management (IPM), 395e396 Intergovernmental Committee (IGC), 515 International Lisbon System, 514e515 Intraspecific interaction, 447e448 In vitro disease systems, 373e374 2D cell culture system, 374e375 3D system, 375e376 cell culturing, 376 types of scaffolds, 376e385 Ionic conduction, 20e22 Ionic liquid-assisted extraction (ILAE), 22e24 Ionic liquids, 25t, 69e70 Isoboles of zero-intraction, 169f Isoionic precipitation Column method, 188 Isolation of small molecules from their natural sources, 182te187t Isomerase, 205 Isopentyl pyrophosphate (IPP), 419e420 Isoquinoline alkaloids, 91

527

528

Index

K Kappaphycus alvarezii, 234e235 Kasugamycin inhibits, 398e403 k-Carrageenase, 234e235 Kinetic model, 499e500 Kluyveromyces marxianus, 210 Kunzea ambiguaCarum carvi, 396e397

L Labdanoyl diterpene, 79e81 Laccases, 468e471 Lactobacilli, 229 Laminaria hyperborean, 235 Laminarin, 244 Landfarming, 460e461 Langmuir adsorption models, 500e501 Langmuir isotherm, 504 Langmuir model, 503 Lavandula officinalis, 356 “Lead-Oriented Synthesis”, 128 Less hazardous chemical synthesis, 55te58t Leukemia, 324 Ligase, 204 Lignans, 107 Lignin-based adsorbents and heavy metal removal, 498e499 Lignins, 107 Ligusticum scoticum Linnaeus, 2 LineweavereBurk plot (Double-reciprocal plot), 213, 214f Lipia multiflora, 356 Liquid chromatography (LC), 47 Liquid chromatography-mass spectrometry (LCMS), 190e192 Low pest resistance, 418 Low-pressure liquid chromatography (LPLC):, 41 Low toxicity, 418 Lung cancer, 322e323 Lung diseases, 273e274 Lutein, 254 Lyases, 206 Lymph vessels, 174

M Macrocyclic lactones, 403 Macroporous resins, 38 Macrosteles fascifron, 9e10 MALDI-MSI, 294e296

Manduca sexta, 297e298 Marine microalgae, 251e252, 260 applications of bioactive compounds, 256e257 bioactive compounds from microalgae, 252e256 enhancement of algal metabolites, 260 microalgae as source of pharmaceuticals, 257e259 Marine oligosaccharides, 231e233, 242e244, 243t application of oligosaccharides in biomedicine, 237e244 bioactive oligosaccharides from complex polysaccharides, 234e235 marine polysaccharides and their oligosaccharides, 233e234 purification of oligosaccharides, 235e237 Mass spectrometry, 48 Mass spectrometry-based metabolomics, 47e50, 49f in NP research, 50e51 bioactivity assessment, metabolism, and biomarker discovery of, 51 de-replication and drug discovery, 51 fingerprinting and profiling of, 50e51 quality control of, 51 targeted, 48 untargeted, 48e50 Mass spectrometry imaging (MSI), 294e296 Mass spectroscopy (MS), 47 Mastocarpus stellatus, 2e3 Matrix Assisted Laser Desorption Ionization (MALDI), 139e140 Matrix-assisted laser desorption ionization (MALDI), 191 Medical science, 415 Medicinal herbs, 75 Medicinal natural compounds, 2e3 Medicinal plants, 1e2, 226e227, 352f Melaleuca alternifolia, 356 Membrane-based techniques, 42 Membrane permeability, 173 Membrane separation, 39 Mentha piperita, 356 Metabolic pathways, 253 Metabolite 3-hydroxyanthranilic acid, 67e68 Metabolite abundance, 293e294 Metal desorption, 496e497 Metal ions and inhibitors, effect of, 208 Metal removing strategies, 489e492

Index

Metarhizium anisopliae, 392e393 “Metformin”, 321e322 Methicillin-sensitive, 173e174 Mg-doped hydroxyapatite (MgHA), 382 MichaeliseeMenten Plot, 211, 212f Microalgae, 258t antimicrobial activities of, 257e259 secondary metabolites from, 253e256 strains, 253t Microalgal biomass, 252f Microbe-host interactions, 301e302 Microbial Cell Factories (MCF), 180e181 Microbial diseases drug repurposing for, 333e334, 334te338t Microbial infections, 334te338t Microbial origin, 392e396 Microbial pesticides advantages and disadvantages of, 396 Microbulbifer strain, 234 Microcerotermes beesoni, 422 Microenvironment, 3, 375 Micronutrients, 489 Microorganisms, 379e380, 407, 491 Microsporidian, 395 Microwave assisted extraction (MAE), 20e22, 23t, 37e38, 353 Microwave-assisted synthesis, 61t Microwave radiation (MW), 20e22, 60 Microwave synthesis, 60e61 Mikania micrantha, 294 Minimum inhibition concentration (MIC), 173 Mitochondria, 328 Mitotic index, 432 Mmitogen-activated protein kinase(MAPK) activation, 83e85 Molecular sizeo, 40 M. Oleifera, 276 Monoacylglycerols, 297e298 Monoclonal antibodies, 179, 193 Monosaccharaides, 110e111, 379e380 Monoterpenes, 76e78 Mother of bioinformatics (M.O.), 156 M. tuberculosis, 124e125 Multicopper polyphenol oxidases thatoxidize, 468e471 Multiple chromatographic, 293e294 Multiple sclerosis (MS), 327 Murraya paniculata, 91 Mycobacterium tuberculosis, 159e160

Mycoinsecticides, 392e393 Mycophenolic acid, 333 Mycorrhizal fungi, 450e452 Mycosporine-like amino acids (MAAs), 256 Myrtenal-core structure analogues, 124f

N N-acylhydrazone, 124e125 NADH, 205 Nagoya Protocol, 515 “Nanobioherbicide”, 431 Nano-biomaterials, 496 Nanobionics, 433 Nano-biosensor, 429e430, 433e434, 436e437 Nanobiotechnology, 365 “Nano-encapsulation”, 432e433 Nano-fertilizer, 433 “Nanofood”, 434e435 “Nanoformulations of natural compounds”, 439 Nanoherbicides, 430e432 Nanoinsecticides, 432e433 Nanomaterials, 436 Nanonutraceuticals, 437 Nanopesticides, 432 Nanotechnologies, 427, 432 in agriculture, 429e434 in food science, 434e437 National Cancer Institute (NCI), 8 National environmental protection agency(NEA), 461e462 National Health Information Infrastructure, 153 National Mental Health Program, 279e284 Natural compounds (NCs), 133e134, 391e392, 428e429 biochemical pesticides, 396e398 different class of metabolites as, 3 different sources for biopesticides, 392e398 for drug discovery, 150f extraction of, 17e18 accelerated or pressurized-solvent extraction (PSE), 28e29 classical solvent extraction, 18e19 enzyme-assisted extraction (EAE), 27, 28t ionic liquid-assisted extraction (ILAE), 22e24 microwave-assisted extraction (MAE), 20e22, 23t supercritical fluid extraction (SFE), 24, 26te27t ultrasound-assisted extraction (UAE), 19e20

529

530

Index

Natural compounds (NCs) (Continued) future of, 10e11 in history, 1 as insecticides, 415e419 dormant and summer oils, 421 horticultural oil, 420e421 insecticidal soaps, 421e422 natural soap, 421 neem, 420 pyrethrum, 419e420 resin, 422 rotenone, 419 ryania, 419 sabadilla, 419 soapnuts-based pesticides, 422 medicinal natural compounds from other sources, 2e3 medicinal plants, 1e2 metabolomics and mass spectrometry approach in the discovery of, 141e142 mining for novel compounds using genomic databases, 136e139 mode of action of different biopesticides, 398e405 modern use of databases in hunting, 136 nanoformulations of, 427e429 nanotechnologies in agriculture, 429e434 nanotechnologies in food science, 434e437 plant origin, 398 proteomics approach for mining for, 139e141 traditional methods and techniques for, 134e136 vital natural compounds, 3e10 from fungi, 4e7 from marine algae, 9e10 from marine environment, 8e9 from marine sponges, 10 from plants, 7e8 Natural crude extract, compound synergy in, 167e168 factors responsible for, 168e170 synergistic effect on antimicrobial activity, 172e174 on diabetes, 170e172 of natural drugs on breast cancer cell, 174e176 Natural disaccharides, 112f Natural diversity of triterpenes, 84f Natural ecophysiological responses, 445e446 Natural poisons, 148e149

Natural products (NPs), 35, 47, 52, 75e76, 228e229, 513 acutumine, microorganism synthesis of, 68e69 drug discovery, 147e155, 161e163 bioinformatics concept, evolution of, 155e158 bioinformatics in discovery of natural products, 155 category of natural products, 147e149 high-throughput virtual screening, 158e163 semantic methods for drug discovery, 149e155 and drug discovery, 162t and intellectual property, 512e514 and international protection, 514e516 psymberin using ultrasonic irradiation, 62 purification, cutting edge approaches for, 35 anthocyanin monomers from red cabbage, 42e43 extraction, 36e38 isolation, 39e40 pre-isolation, 38e39 purification, 41e42 semisynthetic or modified, 119e126 structural diversity in, 76e119 alkaloids, 87e97 antibiotics, 119 carbohydrates, 108e119 phenolic compounds, 97e107 polyketides, 108 terpenes, 76e87 Natural pyrrole and pyrrolidine alkaloids, 88f Natural resources and associated traditional knowledge, 516 Natural scaffolds, 376e382 Natural soap, 421 Natural sources, 17 Necrotic cell mass formation, 385e386 Neem, 420 Nelumbo nucifera, 92 Nematic Protein Organization Technique (NPOT), 134e136 Nematodes, 395e396 Neoflavonoids noncommunicable diseases, 104 Neonicotinoids, 391e392, 405 Nepeta parnassica, 357 Nephrolithiasis, 225 anti-nephrolithiatic medicinal plants, 227 natural products, 228e229 role of plant polyphenols in, 226e227 Nephrolithiasis activity, 228t

Index

Neurodegenerative disorders (NDs), 325e328 drug repurposing for, 325e328 Neurological diseases or neurodegenerative disorder (NDDs), 274 New disease indications, 338f New plant variety, protection of, 514 Next-generation biopesticides, 406 Nicotine, 405 Nitrogen and phosphorus-enriched fertilizers, 456 Nitrogen fixing bacterium, 495 Nodulisporium sulviforme, 119 No impact on plant life cycle, 418 Non-communicable diseases (NCDs), 267e268, 373e374 and antioxidants, oxidative stress cause, 269e274 antioxidants in chemopreventive foods, 275e279 cause of civilization diseases, 268e269 challenges or loopholes in chemoprevention strategy, 279e284 chemopreventive foods, 275 food and vegetables for controlling cancer, 276e277 for controlling diabetes, 278 for controlling hypertension, 278e279 for controlling lung disease or cardiovascular diseases (CVD), 277e278 for controlling neurological disorders (NDDs), 278 Nonconventional remediation strategies, 504 Nonessential heavy metals, 489 Noninclusion viruses (NIVs), 394 Noninvasive, 174 Nonpolar stationary phase, 39e40 Nonribosomal peptide synthetase (NRPS), 139 Non-small cell lung cancer (NSCLC), 270e271, 322 ‘Non-superimposable’, 217 Nontoxic catalysts, 55 Nosema locustae, 395 No side-effect, 418 Novel bioactive compound, 135f Novel hydroxyapatite-alginate-gelatinnanocomposites, 497e498 Novel pathogens, 436e437 Nuclear Magnetic Resonance Spectroscopy (NMR), 11, 190e191, 301 Nucleic acid drug, 365e367 Nucleotide di phosphate (NDP), 180e181 Numerous complex natural compounds, 62

Nutraceutical application, 306 “Nutrition”, 133e134

O Ocimum bassilicum, 353 O. formigenes, 229 Oligodendrocytes progenitor cells (OPCs), 327 OligoG, 242 Oligosaccharides, 111e117, 113te114t, 235, 236f OMDM-2, 174e176 One-dimensional gel electrophoresis, 189e190 One factor at a time (OFAT), 208e209 Organic heterocycle, 89 Organocatalysis, 215e220, 216fe217f advantages and disadvantages of, 219e220 chiral molecules, 216e219 Organochlorine pesticides, 452 Origanum vulgare, 357 “Oxidation”, 269e270 Oxidoreductase, 205

P Paenibacillus popilliae, 393e394 Pancreatic cancer (PC), 323e324 Papaver somniferum, 3e4, 180 Paris Convention of 1883, 514e515 Parkinson’s disease (PD), 8, 326e327 Parmelia omphalodes, 2 Patent Act of 1970, 516 Patent Cooperation Treaty (PCT), 516 Patent law, 512e513 Patents (Amendment) Act, 2002, 513 Pathogen transmission, 376 PDAC, 323e324 Pectinase production, 208e209 Pelargonium graveolens, 355 Penicillium chrysogenum, 181 Penicillium notatum, 4e6, 119 Pentacyclic triterpenoids, 173e174 Peptidic natural products proteomics-based analysis of, 141 Permeation process, 18e19 Permethrin, 473 Pesticide-biodegraders, 447e448 Pesticide-degrading bacteria (PDB), 462e466 Pesticide-degrading microorganisms, 462e466 Pesticides, 391e392, 416e418 bacterial degradation of, 461e467 bioremediation, 447e448

531

532

Index

Pesticides (Continued) bioremediation, enzymes involved in, 468e474 bioremediation of, 453e461 degradation, 469te470t fungal biodegradation of, 461 pollution, 449e453 and their impacts on sustainability, 448e449 toxicity to soil and water ecosystems, 450e453 PGE2, 228 PGRFA Treaty, 514 Phaeodactylum tricornutum, 255 Pharmaceutical drugs, 133 Pharmaceutical ingredients, 180e181 “Pharmaceutics”, 133e134 Pharmacodynamic of small molecules and biologics, 193 Pharmacokinetics effects, 170 of small molecules vs biologics, 194e196 synergies, 167 pH effect, 208 Phenolic acids, 97e100 Phenolic compounds, 97e107 Phenotypic change, 375 Pheochromocytoma (PC12) cells, 91 Pheromone-binding proteins (PBPs), 403 Phosphatases, 473 Phosphopantetheinyl transferases (PPTase), 139e140 Photorhabdus, 301e302 Phycoerythrin, 257 Phyla Basidiomycota, 6 Physical and chemical modification, 500e501 Physicochemical properties, 453 Physiochemical properties of small molecules and biologics, 193e194 Phytochemicals, 147e148, 428 Phytodegradation, 460 Phytoextraction, 458 Phytofiltration, 458e459 Phytoremediation, 457e460, 458t Phytostabilization, 459 Phytotoxicity, 420e421 Phytovolatilization, 459 pH-zone-refining counter-current chromatograph, 41e42 Pilocarpus jaborandi, 4 Pimpinella anisun, 107 Piperidine alkaloids, 89

Piptoporus betulinus, 2 Plant-based extracts and essential oils, 397 Plant bioinsecticides, 403e405 Plant Growth Regulator (PGR), 173 Plant-insect interaction metabolome, 300e301 Plant polyphenols in nephrolithiasis, 226e227 Plant-specific chloroplast, 430 Plant Variety Protection and Farmers Rights Act 2001, 514 Plasmid DNA (pDNA), 367 Plutella xylostella, 301 Polarity, 40 Polar solvents dissolve polar solutes, 181e188 Pollution Prevention Division, 391e392 Polycavernoside-A, 9e10 Polycaverosa tsudai, 9e10 Polycyclic aromatic hydrocarbon phenanthrene, 493 Polyflavonoid tannins, 106e107 Polygalacturonase, 207e208 Polyhedron viruses (PVs), 394 Polyketides, 108 Polyketide synthases (PKSs), 139 Polylactic acid (PLA), 376 Polyphenolic amides, 105, 105f Polysaccharides, 117e119, 232f, 256, 501 Polyterpenes, 87 Polyunsaturated fatty acids (PUFA), 255 Polyvinyl alcohol, 499 Porphyra umbilicalis, 2e3 Pressurized liquid extraction (PLE), 36e37 Pressurized-solvent extraction (PSE), 28e29 Probiotics, 229 Protein data bank (PDB), 156e157 Protein kinase R (PKR), 332e333 Proteins, 256, 380e382 Proteomics-based analysis of peptidic natural products, 141 Protozoa, 395 Pseudoalteromonas porphyrae, 234e235 Pseudomonas aeruginosa, 125e126, 242, 428e429, 492e493 Pseudo-second-order of kinetics, 504 Psymberin, 62 PubMed, 151 Pulsed electric field (PEF), 37e38 Purification process, 41e43, 190e191 of natural product, 36f

Index

techniques, 41e42 Purines, 95e97 Pyrethrin, 404 Pyrethrum, 419e420 Pyrrole and pyrrolidine alkaloids, 87 Pyrrolizidine alkaloids, 95 Pyrrolizidines NPs, 95f PyRx, 159

Q

“Quasi-vitamins”, 299 Quinoline alkaloids, 90e91, 91f Quinolone alkaloid natural products, 67e68

R Raw material, 35 Reactive oxygen species (ROS), 227, 268e269, 270f, 275f Real-time pollution prevention, 55te58t “Redox homeostasis”, 268e269 Reduce derivatives, 55te58t Resin, 422 Resin deionization method, 188e189 Resistance bacteria, interactions of agents with, 170 Retrovirus drug repurposing for, 332e333 Reversed-phase high-performance liquid chromatography (RP-HPLC), 191 Rhizofiltration, 458e459 RNA, 160, 201 polymerase, 393e394 Rosmarinus officinalis, 356 Rostral ventrolateral medulla (RVLM), 272e273 Rotenone, 419 Rule 27(a) of the European Patent Convention, 511 Ryania, 419 Ryania speciosa, 404 Ryanodine, 404

S Sabadilla, 419 Saccharomicins polysaccharides, 122f Saccharomyces cerevisiae, 207e208 Saccharopolyspora erythraea, 6 Safer chemistry for accident prevention, 55te58t Safer solvents and auxiliaries, 55te58t Salinispora, 160e161

Salipiger mucosus, 495 Salixalba, 3e4 Salting out, 188 Salvia sclarea, 356 “Saponins”, 422 Saponins steroidal glycoside, 118f Sarcophaga bullata, 298 SARS-COv2, 367 Schizochytrium sp, 255 Schoenocaulon officinale, 419 S. coelicoflavus var. nankaiensis, 115 Scutellaria baicalensis, 170 Scutellaria lateriflora, 170 “Secondary metabolism”, 3 Secondary metabolites from microalgae, 253e256 Selective precipitation using supercritical/gas antisolvent (SAS/GAS)techniques, 42 Selective serotonin reuptake inhibitor (SSRI), 321e322 Selenium, 276e277 “Self-sacrificing soldiers”, 269e270 Serratia entomophila, 406 Sesquiterpenes, 78 Sesquiterpenoids’ chemical skeleton, 78 “Shunt metabolites”, 3 Silk Fibroin-XGH scaffold, 382e383 Similarity Ensemble Approach (SEA), 134e136 Single-walled carbon nanotubes (SWNTs)_, 433 Sinorhizobium meliloti, 495 Small cell lung cancer (SCLC), 322 Small molecule drug (SMD), 179e180 Small molecules vs biologics, 179e196, 180f biologics and small molecule, 192e194 isolation of, 181e190 isolation of biologics, 188e189 pharmacokinetics of, 194e196 purification techniques of, 189e190 sources of, 180e181 techniques for characterization of, 190e192 Soaking extraction, 19 Soapnuts, 422 Soapnuts-based pesticides, 422 Sodium dodecyl sulfate (SDS), 189e190 gel electrophoresis, 189e190 Sodium fluoride (NaF), 294 Soil ecosystems, 452e453 Soil microbiota, 451f Solid-liquid extraction, 29 Solid phase extraction (SPE), 39e40

533

534

Index

Solubility, 181e188 Solvent-free environment, 63 Solvent partitioning/extraction, 38 Solvents on enzyme extraction, effect of, 209 Sonochemistry, 61 Sophorolipid, 492e493 Soxhlet extraction, 19 Soy protein-Chitin scaffolds, 384 Spectrometer, 141e142 Spectrophotometer, 210e211 Spectroscopy, 10 Spinosyns, 405 Spodoptera exigua, 301 Spondias mombin, 357 Spontaneous process, 499e500 Squamous carcinoma, 179 Staphylococcus aureus, 9e10, 78, 170 Starch-based composites, 501 Steinernema, 395e396 Stilbenes, 105 Streptococcus mutans, 237 Streptomyces, 133e134, 138e139, 403 Streptomyces peucetius, 7 Streptomyces viridochromogenes, 116 Structure-activity relationship studies (SAR), 75e76 2-Styrylchromenes, 66t Subsequent analytical thin-layer, 40 Substrate concentration, 208 Succinoglycan, 495 Sugars, 110e111, 379e380 Supercritical carbon dioxide (S-CO2), 36e37 Supercritical CO2, 24 Supercritical fluid chromatography (SFC), 42 Supercritical fluid extraction (SFE), 24, 26te27t, 36e37, 353 Sweat glands, 4 Symbiotic relationship’s, 203 Synergetic multi-target effects, 168e169 Synergistic effect on antimicrobial activity, 172e174 on diabetes, 170e172 of natural drugs on breast cancer cell, 174e176 Synergy effect, 171te172t Synthetic antibiotics, 172 Synthetic compounds (SCs), 133 Synthetic pesticides, 391e392 Synthetic scaffolds, 376 Syzygium claviflorum, 6

T Tandem mass spectrometry, 48 of peptides and proteins, 192 Tannins, 106e107 Targeted MS-based metabolomics, 48 “Target mediated drug disposition”, 195 Target-Oriented Synthesis (TOS), 126e128 Taxus brevifolia, 7 Tea-manufacturing processes, 504 Temperature effect, 206 Terpenes, 76e87 Tetraterpene precursor GGPP, 86f Tetraterpenes, 85e86 Therapeutics application, 302e305 “Therapeutic utilization”, 151 Thermal Proteome Profiling (TPP), 134e136 Thermal stability, 40 Thermoanaerobacterium thermosulfurigenes, 363 Thermodynamics, 502 Three-dimensional structure of proteins, 156e158 Thymoquinone (TQ), 274 Tissue culture plastic (TCP), 383 Tissue engineering employs cell culturing techniques, 374 TNBC, 319e320 Tobacco plants, 380 Toxins, 148e149 Trade-Related Aspects of Intellectual Property (TRIPs) agreement, 514, 516 Trade-Related Aspects of Intellectual Property Rights (TRAIPR), 516 Traditional Chinese Medicine (TCM), 160e161 Traditional Medical Knowledge (TMK), 516 Traditional medicine, 147, 226 Transferase, 204 Trans-isomers, 473 “Transition state”, 210 Transpiration, 459 Treponema primitia, 301e302 Tricarboxylic acid, 229 Trichoderma, 474 “Triple negative”, 174 Tripterygium wilfordii, 7 Triterpenes, 83e85 tRNA, 204 Tropane, 95 Two-dimensional gel electrophoresis, 189e190 Type 2 diabetes (T2D), 278

Index

Tyrosine kinase, 322e323

U Ultra-high-performance liquid chromatography (UHPLC), 236e237 Ultrasonic-assisted extraction (UAE), 37e38 Ultrasound, 353 in chemical synthesis, 62 supported green synthesis, 61e62 Ultrasound-assisted extraction (UAE), 19e20, 21t Ultraviolet spectroscopy, 47 Undaira pinnatifida, 242 United Nations Environment Protection Agency (UNEP), 449e450 United Nations Organization, 448 United Nations Sustainable Goal, 446e447 Untargeted MS-based metabolomics, 48e50 Use of renewable feedstocks, 55te58t US Food and Drug Administration, 434 Usnea dillenius ex Adanson, 2 UVradiation, 396, 431

V Vangueria madagascariensis (VM), 173e174 Vatiquinone, 328 Vedolizumab, 193 Venom KB, 151 Verbenaceae, 356 Vesicular monoamine transporter 2 (VMAT2), 327e328 Violaxanthin, 254e255 Viral cells, 258e259 Viral diseases, 6

drug repurposing for, 331e333 Viral hemorrhagic septicemia virus, 258e259 Viral pesticides, 394e395 Vital natural compounds, 3e10 from fungi, 4e7 from marine algae, 9e10 from marine environment, 8e9 from marine sponges, 10 from plants, 7e8 Vitamin C, 275e276 Vitamins, 229

W Walnut sawdust, 503 Waste prevention, 55te58t Wolfram syndrome (WS), 328 World Health Organization, 321 World Intellectual Property Organization, 515 World Trade Organization, 516

X Xanthan gum hydrogels (XGH), 382e383 Xanthophylls, 253 Xenobiotic, 447e448 X-ray crystallography, 156

Y Yearly Lived with Disability (YLD), 373e374 Yeast-like microbial flora, 490e491

Z Zeaxanthin, 254 Zooplankton, 449

535

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