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PREPARATION OF PHYTOPHARMACEUTICALS FOR THE MANAGEMENT OF DISORDERS

PREPARATION OF PHYTOPHARMACEUTICALS FOR THE MANAGEMENT OF DISORDERS The Development of Nutraceuticals and Traditional Medicine Edited by

CHUKWUEBUKA EGBUNA, MICCON, AMRSC Department of Biochemistry, Faculty of Natural Sciences, Chukwuemeka Odumegwu Ojukwu University, Uli, Anambra State, Nigeria Nutritional Biochemistry and Toxicology Unit, World Bank Africa Centre of Excellence, Centre for Public Health and Toxicological Research (ACE-PUTOR), University of Port Harcourt, Port Harcourt, Rivers State, Nigeria

ABHAY PRAKASH MISHRA, Ph.D. (PHARMACY) Academic Researcher, Department of Pharmaceutical Chemistry, Hemvati Nandan Bahuguna Garhwal University, Srinagar, Uttarakhand, India

MEGH R. GOYAL, PhD, P.E. Retd. Distinguished Professor, Univ of Puerto Rico-Mayaguez Campus. Senior Editor in Chief for Apple Academic Press, Inc. Rincon, Puerto Rico, United States

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 © 2021 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. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN 978 0 12 820284 5 For information on all Academic Press publications visit our website at https://www.elsevier.com/books and journals

Publisher: Andre Gerhard Wolff Acquisitions Editor: Erin Hill Parks Editorial Project Manager: Tracy Tufaga Production Project Manager: Kiruthika Govindaraju Cover Designer: Matthew Limbert Typeset by SPi Global, India

Contributors College of Medicine and Health Science, Ambo University, Ambo, Ethiopia

Yusuf Abubakar Department of Biochemistry, Natural Product Research Laboratory, Bauchi State University, Gadau, Bauchi State, Nigeria

Jaspal Chauhan Department of Himalayan Aquatic Biodiversity, H. N. B. Garhwal University, Srinagar, Uttarakhand, India

A.A. Adam Biomedicinal Research Centre, Forestry Research Institute of Nigeria, Ibadan, Nigeria

Priyanka Dhar Department of Biotechnology, Techno India University, Salt Lake City, Kolkata, West Bengal, India

Charles Oluwaseun Adetunji Department of Microbiology, Applied Microbiology, Biotechnology and Nanotechnology Laboratory, Edo University Iyamho, Iyamho, Edo State, Nigeria

Adom Dickson Department of Educational Innovations in Science and Technology, Kwame Nkrumah University of Science and Technology, Kumasi-Accra, Ghana

Juliana Bunmi Adetunji Department of Biochemistry, Nutrition and Toxicology Research Laboratory, Osun State University, Osogbo, Osun, Nigeria

Chukwuebuka Egbuna Department of Biochemistry, Faculty of Natural Sciences, Chukwuemeka Odumegwu Ojukwu University, Uli, Anambra State; Nutritional Biochemistry and Toxicology Unit, World Bank Africa Centre of Excellence, Centre for Public Health and Toxicological Research (ACE-PUTOR), University of Port Harcourt, Port Harcourt, Rivers State, Nigeria

Khalil Ahmad Institute of Home and Food Sciences, Faculty of Life Sciences, Government College University Faisalabad, Faisalabad, Pakistan Muhammad Akram Department of Eastern Medicine, Government College University Faisalabad, Faisalabad, Pakistan

Chioma Bertha Ehis-Eriakha Environmental and Molecular Biology Laboratory, Edo University Iyamho, Iyamho, Edo State, Nigeria

Amanjot Annu Department of Pharmacology, Faculty of Medicine, Mbarara University of Science and Technology, Mbarara, Uganda Shabnam Ansari Department of Biotechnology, Faculty of Natural Sciences, Jamia Millia Islamia, New Delhi, India

Chika Precious Ekwuabu Department of Clinical Pharmacy and Pharmacy Practice, University of Benin, Benin City, Edo State, Nigeria

Godwin Anywar Department of Plant Sciences, Microbiology & Biotechnology, College of Natural Sciences, Makerere University, Kampala, Uganda

Mahitab H. El Bishbishy Department of Pharmacognosy, Faculty of Pharmacy, October University for Modern Sciences and Arts (MSA), 6th of October, Egypt

Asha Department of Botany, HNB Garhwal University, Srinagar, Uttarakhand, India

Dina M. El Kersh Department of Pharmacognosy, Faculty of Pharmacy, British University in Egypt, El Sherouk, Egypt

Tamirat Bekele Beressa Pharmbiotechnology and Traditional Medicine Center of Excellence, Mbarara University of Science and Technology, Mbarara, Uganda; Department of Pharmacy,

Abeer M. El Sayed Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo, Egypt

xii

Contributors

Benson Chukwunweike Ephraim-Emmanuel Department of Dental Health Sciences, Bayelsa State College of Health Technology, Otuogidi, Ogbia Town; Environmental Health Unit, World Bank Africa Centre of Excellence, Centre for Public Health and Toxicological Research (ACE-PUTOR), University of Port Harcourt, Port Harcourt, Rivers State, Nigeria Nebechi Jane Ezeofor Department of Food Technology, School of Applied Science and Technology, Federal Polytechnic, Oko, Anambra State, Nigeria Devaraj Ezhilarasan Department of Pharmacology, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences, Chennai, Tamil Nadu, India Shahira M. Ezzat Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo; Department of Pharmacognosy, Faculty of Pharmacy, October University for Modern Sciences and Arts (MSA), 6th of October, Egypt Muhammad Adil Farooq School of Food Science and Engineering, Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, South China University of Technology, Guangzhou, China Reena Gangwar Department of Botany and Microbiology, H.N.B. Garhwal University, Srinagar, Garhwal, Uttarakhand, India Ajay Kumar Gautam Department of Plant Sciences; Department of Biochemistry and Microbial Sciences, School of Basic and Applied Sciences, Central University of Punjab, Bathinda, Punjab, India Laurence John Francis J. Gido Research Center of the College of Medicine, Davao Medical School Foundation, Inc., Davao City, Philippines Ashutosh Gupta Department of Biochemistry, University of Allahabad, Allahabad, Uttar Pradesh, India Ena Gupta Department of Home Science, University of Allahabad, Allahabad, India

Rakesh Kumar Gupta Environmental Biotechnology and Genomics Division, CSIRNational Environmental Engineering Research Institute (NEERI), Nagpur, Maharashtra, India Poonam Gusain Uttarakhand State Council for Science & Technology, Dehradun, Uttarakhand, India Sadia Hassan Institute of Home and Food Sciences, Faculty of Life Sciences, Government College University Faisalabad, Faisalabad, Pakistan; Food Processing Centre, Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE, United States Muhammad Bilal Hussain Institute of Home and Food Sciences, Government College University Faisalabad, Faisalabad, Pakistan Muhammad Imran University Institute of Diet and Nutritional Sciences, Faculty of Allied Health Sciences, The University of Lahore, Lahore, Pakistan Farhat Jabeen Department of Zoology, Government College University Faisalabad, Faisalabad, Pakistan Jaison Jeevanandam Department of Chemical Engineering, Faculty of Engineering and Science, Curtin University, Miri, Sarawak, Malaysia Rajappa Joga ICAR Research Complex NEH Region, Umiam (Barapani), Meghalaya, India Abolanle A.A. Kayode Phytomedicine Research, Drug Discovery and Development Laboratory, Department of Biochemistry, Babcock University, Ilishan-Remo, Ogun State, Nigeria Shyam Sundar Kesh Department of Veterinary Clinical Complex (Veterinary Biochemistry), Faculty of Veterinary and Animal Sciences, West Bengal University of Animal and Fishery Sciences, Kolkata, West Bengal, India Merve Keskin Vocational School of Health Services, Bilecik Şeyh Edebali University, Bilecik, Turkey Şaban Keskin Vocational School of Health Services, Bilecik Şeyh Edebali University, Bilecik, Turkey

Contributors

Fahad Said Khan Department of Eastern Medicine, Government College University Faisalabad, Faisalabad, Pakistan Muhammad Kamran Khan Institute of Home and Food Sciences, Faculty of Life Sciences, Government College University Faisalabad, Faisalabad, Pakistan Muhammad Usman Khan Department of Energy Systems Engineering, University of Agriculture, Faisalabad, Pakistan; Bioproduct Sciences and Engineering Laboratory, BSEL, Washington State University, Richland, WA, United States Sevgi Kolaylı Department of Chemistry, Faculty of Science, Karadeniz Technical University, Trabzon, Turkey Shambhu Kumar Forest Pathology Department, KSCSTE-Kerala Forest Research Institute, Thrissur, Kerala, India Shashank Kumar Department of Biochemistry and Microbial Sciences, School of Basic and Applied Sciences, Central University of Punjab, Bathinda, Punjab, India Jaya Vikas Kurhekar Department of Microbiology, Bharati Vidyapeeth’s Dr. Patangrao Kadam Mahavidyalaya, Sangli, Maharashtra, India Prem Prakash Kushwaha Department of Biochemistry and Microbial Sciences, School of Basic and Applied Sciences, Central University of Punjab, Bathinda, Punjab, India Umme Laila Department of Eastern Medicine, Government College University Faisalabad, Faisalabad, Pakistan Fanuel Lampiao Africa Center of Excellence in Public Health and Herbal Medicine, College of Medicine, University of Malawi, Blantyre, Malawi I.O. Lawal Biomedicinal Research Centre, Forestry Research Institute of Nigeria, Ibadan, Nigeria Muhammad Faisal Manzoor School of Food Science and Engineering, South China University of Technology, Guangzhou, China

xiii

Anand Prakash Maurya Institute of Microbiology and Infection, School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Birmingham, United Kingdom Vineet Kumar Maurya Department of Botany and Microbiology, H.N.B. Garhwal University, Srinagar, Garhwal, Uttarakhand, India Mohammad Mehdizadeh Department of Agronomy and Plant Breeding, University of Mohaghegh Ardabili, Ardabil, Iran Abhay Prakash Mishra Academic Researcher, Department of Pharmaceutical Chemistry, Hemvati Nandan Bahuguna Garhwal University, Srinagar, Uttarakhand, India Neha Mishra Sam Higginbottom University of Agriculture, Technology and Sciences, Allahabad, India Ghulam Mohiudin Department of Eastern Medicine, Government College University Faisalabad, Faisalabad, Pakistan Andrew G. Mtewa Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom; Chemistry Department, Malawi Institute of Technology, Malawi University of Science and Technology, Thyolo, Malawi; Pharmbiotechnology and Traditional Medicine Center of Excellence, Mbarara University of Science and Technology, Mbarara, Uganda Patricia A. Nalumansi Department of Science in Environmental Management, Faculty of Science and Technology, International University of East Africa, Kampala, Uganda Abha Negi Department of Botany and Microbiology, H.N.B. Garhwal University, Srinagar, Garhwal, Uttarakhand, India Kennedy J. Ngwira Institute of Molecular Science, School of Chemistry, University of the Witwatersrand, Johannesburg, South Africa Manisha Nigam Department of Biochemistry, School of Life Sciences, Hemvati Nandan Bahuguna Garhwal University, Srinagar, Uttarakhand, India

xiv

Contributors

Godswill Ntsomboh-Ntsefong Department of Plant Biology, Faculty of Science, University of Yaounde I; Institute of Agricultural Research for Development (IRAD), Yaounde, Cameroon Alloysius Chibuike Ogodo Department of Microbiology, Faculty of Pure and Applied Sciences, Federal University Wukari, Wukari, Taraba State, Nigeria Patrick E. Ogwang Pharmbiotechnology and Traditional Medicine Center of Excellence, Mbarara University of Science and Technology, Mbarara, Uganda Chinenyenwa Ohia Department of Environmental Health Sciences, Faculty of Public Health, College of Medicine, University of Ibadan, Ibadan, Nigeria Kelechi Justin Okere Department of Chemical Sciences, Faculty of Natural and Applied Sciences, Hezekiah University, Umudi, Imo State, Nigeria Eleonora Okushanova Shakarim State University of Semey, Semey, Kazakhstan Ahmed Olatunde Department of Biochemistry, Abubakar Tafawa Balewa University, Bauchi, Nigeria T.O. Omogbene Biomedicinal Research Centre, Forestry Research Institute of Nigeria, Ibadan, Nigeria Peculiar Feenna Onyekere Department of Pharmacognosy & Environmental Medicine, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria Temitope A. Oyedepo Department of Biochemistry, Adeleke University, Ede, Nigeria Santwana Palai Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science and Animal Husbandry, OUAT, Bhubaneswar, Odisha, India Sunil Pareek Department of Agriculture and Environmental Sciences, National Institute of Food Technology Entrepreneurship and Management, Kundli, Sonepat, Haryana, India Chioma Obianuju Peculiar-Onyekere Department of Pharmaceutical Microbiology & Biotechnology, University of Nigeria, Nsukka, Enugu State, Nigeria

Ranu Prasad Sam Higginbottom University of Agriculture, Technology and Sciences, Allahabad, India Alina Abdul Rahim Institute of Halal Research and Management, Universiti Sains Islam Malaysia, Nilai, Malaysia Muhammad Riaz Department of Allied Health Professional, University of Sargodha, Sargodha, Pakistan Tahreem Riaz Department of Eastern Medicine, Government College University Faisalabad, Faisalabad, Pakistan Narashans Alok Sagar Department of Agriculture and Environmental Sciences, National Institute of Food Technology Entrepreneurship and Management, Kundli, Sonepat, Haryana, India Maha M. Salama Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo; Department of Pharmacognosy, Faculty of Pharmacy, British University in Egypt, El Sherouk, Egypt Mohamed A. Salem Department of Pharmacognosy, Faculty of Pharmacy, Menoufia University, Shibin Elkom, Egypt M.S. Sandeep Veda Narayana Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, India Barbara Sawicka Department of Plant Production Technology and Commodities Sciences, Faculty of Agrobioengineering, University of Life Sciences in Lublin, Lublin, Poland Mohammad Ali Shariati Laboratory of Biocontrol and Antimicrobial Resistance, Orel State University Named After I.S. Turgenev, Oryol, Russia; Kazakh Research Institute of Processing and Food Industry (Semey Branch), Semey, Kazakhstan Nadia Sharif Pakistan

Women University, Mardan,

Singh P. Shivakumar Department of Botany, Palamuru University, Mahabubnagar, Telangana, India

Contributors

Atul Kumar Singh Department of Biochemistry and Microbial Sciences, School of Basic and Applied Sciences, Central University of Punjab, Bathinda, Punjab, India

Lisbeth Vallecilla-Yepez Department of Biological System Engineering, University of Nebraska-Lincoln, Lincoln, NE, United States

P. Shivakumar Singh Department of Botany, Palamuru University, Mahabubnagar, India

P. Seshu Vardhan School of Biotechnology, Jawaharlal Nehru Technological University, Kakinada, Andhra Pradesh, India

Priyanka Singh Centre of Food Technology, University of Allahabad, Allahabad, India

G.M. Vidyasagar Medicinal Plants and Microbiology Research Laboratory, Department of Post-Graduate Studies and Research in Botany, Gulbarga University, Kalaburagi, Karnataka, India

Dominika Skiba Department of Plant Production Technology and Commodities Sciences, Faculty of Agrobioengineering, University of Life Sciences in Lublin, Lublin, Poland A. Stephen Department of Botany, St. Joseph’s College (Autonomous), Bengaluru, India Ram Lal Thakur Department of Microbiology, SardarBhagwan Singh Post Graduate Institute of Bio-Medical Sciences and Research, Dehradun, India Habibu Tijjani Department of Biochemistry, Natural Product Research Laboratory, Bauchi State University, Gadau, Bauchi State, Nigeria Casim U. Tolo Pharmbiotechnology and Traditional Medicine Center of Excellence, Mbarara University of Science and Technology, Mbarara, Uganda Genevieve D. Tupas Department of Pharmacology, College of Medicine, Davao Medical School Foundation, Inc., Davao City, Philippines Krishnan Umachandran India

Nelcast Ltd., Chennai,

D.P. Uniyal Uttarakhand State Council for Science & Technology, Dehradun, Uttarakhand, India

Abdul Wahab Institute of Home and Food Sciences, Faculty of Life Sciences, Government College University Faisalabad, Faisalabad, Pakistan Angelo Mark P. Walag Center for Advanced Plant Science and Phytotechnologies; Department of Science Education, University of Science and Technology of Southern Philippines, Cagayan de Oro City, Philippines Anke Weisheit Pharmbiotechnology and Traditional Medicine Center of Excellence, Mbarara University of Science and Technology, Mbarara, Uganda Dinesh Kumar Yadav Department of Chemistry, Mohanlal Sukhadia University, Udaipur, Rajasthan, India Saurav Yadav Department of Biotechnology, HNB Garhwal University, Srinagar, Uttarakhand, India Rida Zainab Department of Eastern Medicine, Government College University Faisalabad, Faisalabad, Pakistan Ahmed Zayed Department of Pharmacognosy, College of Pharmacy, Tanta University, Tanta, Egypt

C H A P T E R

General principle of primary and secondary plant metabolites: Biogenesis, metabolism, and extraction Charles Oluwaseun Adetunjia, Santwana Palaib, Chika Precious Ekwuabuc, Chukwuebuka Egbunad,e, Juliana Bunmi Adetunjif, Chioma Bertha Ehis-Eriakhag, Shyam Sundar Keshh, and Andrew G. Mtewai,j,k a

Department of Microbiology, Applied Microbiology, Biotechnology and Nanotechnology Laboratory, Edo University Iyamho, Iyamho, Edo State, Nigeria bDepartment of Veterinary Pharmacology and Toxicology, College of Veterinary Science and Animal Husbandry, OUAT, Bhubaneswar, Odisha, India cDepartment of Clinical Pharmacy and Pharmacy Practice, University of Benin, Benin City, Edo State, Nigeria dDepartment of Biochemistry, Faculty of Natural Sciences, Chukwuemeka Odumegwu Ojukwu University, Uli, Anambra State, Nigeria e Nutritional Biochemistry and Toxicology Unit, World Bank Africa Centre of Excellence, Centre for Public Health and Toxicological Research (ACE-PUTOR), University of Port Harcourt, Port Harcourt, Rivers State, Nigeria fDepartment of Biochemistry, Nutrition and Toxicology Research Laboratory, Osun State University, Osogbo, Osun, Nigeria gEnvironmental and Molecular Biology Laboratory, Edo University Iyamho, Iyamho, Edo State, Nigeria hDepartment of Veterinary Clinical Complex (Veterinary Biochemistry), Faculty of Veterinary and Animal Sciences, West Bengal University of Animal and Fishery Sciences, Kolkata, West Bengal, India iDivision of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom jChemistry Department, Malawi Institute of Technology, Malawi University of Science and Technology, Thyolo, Malawi kPharmbiotechnology and Traditional Medicine Center of Excellence, Mbarara University of Science and Technology, Mbarara, Uganda

Preparation of Phytopharmaceuticals for the Management of Disorders https://doi.org/10.1016/B978-0-12-820284-5.00018-6

1. Biogenesis, metabolism, and extraction

1.1 Introduction Since the beginning of life, man has utilized plants for different purposes. The early man intensified effort in search of food to cope, which led him to distinguish between plants that serve as food from those that have medicinal value [1]. Ever since, man has depended on plants for their daily needs for food, medicine, and other things including shelter, clothing, and fertilizers. Plants synthesize hundreds of thousands of metabolites. Metabolites are products and intermediates of metabolism with numerous functions including signaling, inhibitory effects on enzymes, defense, cofactor to enzyme, and protection of the plant against several pathogens and diseases. Various plants of different species and genera have been recognized to produce both primary and secondary metabolites of different types. The advances in the application of metabolomics have helped in the profiling of numerous types of metabolites. Metabolomics in effect entails the comprehensive analysis of various metabolites present in any biological system. Also, it helps give a better knowledge of the organization and principle of cellular purposes at several levels including an insight of biological processes in an integral system [2–8].

1.2 Plant metabolites Biochemical reactions that occur in organisms are collectively referred to as metabolism. The products of these reactions, as well as their intermediates, which are usually small molecules, are called metabolites. As earlier mentioned, they are classified as either primary or secondary, based on their intrinsic functions within the organism.

1.2.1 Primary metabolites Primary metabolites are different types of organic compounds secreted by plants that are involved in the various development and growth, synthesis of hormone and protein, photosynthesis, and respiration. Primary metabolites are present in almost every part of the plant species within extensive phylogenetic clusters, which are synthesized using the same or almost biochemical pathways. Examples of primary metabolites include carbohydrates, that is, glucose, fructose, sucrose, raffinose, stachyose, mannitol, xylitol, and sorbitol; the nonstarch includes lignin; amino acids, that is, arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan, and valine; vitamin B complex, that is, thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, and folic acid; organic acids; ascorbic acid; oxalic acid; aconitic acid; and unsaturated fatty acids, that is, monounsaturated and polyunsaturated [9].

1.2.2 Secondary metabolites Plants secrete some useful biologically active chemical substances that are not involved in the development and growth of plants. These substances are called secondary metabolites and are often differentially circulated among taxonomic sets within the plant kingdom. Although the function of many of the plant secondary metabolites still remains unknown,

I. Phytopharmaceuticals and technology

1.2 Plant metabolites

there are four major groups of secondary metabolites that include the sulfur-containing compounds and alkaloids, which are almost about 12,000 compounds; phenolic and polyphenolic compounds, which are about 8000 compounds; and the terpenoids, which are about 25,000 compounds. Moreover the function of these secondary metabolites mainly is to prevent the body from diseases. Examples of secondary metabolites includes flavonoids, that is, quercetin, apigenin, luteolin, myricetin, and cyanidin; phenolic acids, that is, chlorogenic acid, caffeic acid, ferulic acid, p-coumaric acid, vanillic acid, sinapic acid, and protocatechuic acid; lignans, that is, lariciresinol, pinoresinol, secoisolariciresinol, and matairesinol; carotenoids, α-carotene, β-carotene, β-cryptoxanthin, zeaxanthin, and lycopene; tocopherols and tocotrienols, that is, α-tocopherol, β-tocopherol, γ-tocopherol, α-T3, and γ-T3; quinones, that is, phylloquinone; sterols, that is, campesterol, campestanol, stigmasterol, β-sitosterol, β-sitostanol; alkaloids, that is, α-tomatine, dehydrotomatine, α-solanine, α-chaconine, lactucin, and lactucopicrin; glucosinolates, that is, glucoiberin; and glucosinolates, that is, glucoraphanin, glucoalyssin, sinigrin, gluconapin, progoitrin, and gluconasturtiin [10]. These bioactives are capable of eliciting pharmacological/toxicological response in humans (Table 1.1) while increasing healthy nutrition as the major component of nutraceuticals and dietary supplements. They also help increase agricultural yields as growth enhancers of plants and animals as well as being used as dyes, fragrances, flavors, and cosmetics [23]. Traditionally, secondary metabolites are obtained from plant parts by extraction methods such as steam, solvent (organic/inorganic), and super critical extraction. Other modern methods exist [24]. However, this has encountered a lot of challenges, which the scientific community has constantly been working to overcome. Plant tissue and cell culture is a method in metabolic engineering that has been found as a worthy alternative to mitigating the limitations associated with the conventional methods of extraction. This involves the isolation of advantageous cells and culturing them under special organized environments. The metabolites obtained are free from contaminants; the metabolic process can be rationally controlled, labor cost is decreased, increased yield of high-value metabolites is achieved, and cells are cultured under controlled condition free from climatic variations/soil types [23]. Improved methods of biotechnology have been applied in plant cell and tissue culture to further increase yield of metabolites for commercial and industrial purposes. As seen in Table 1.1, the most prominent and bioactive principles of medicinal and toxicological significance are as follows: 1. Alkaloids: These are group of nitrogenous heterocyclic bases obtained from various parts of the plant. Alkaloids are bitter in taste. They are waste product of plant metabolism, and their names usually end with “ine.” Alkaloids are alkaline secondary metabolites with pharmacological activity. 2. Flavonoids: These are pigments found in plant more specifically in the colored areas of the plant, like petals of flower or leaf itself. 3. Anthocyanins: These are antioxidants made up of aromatic rings of the cyanidin with substituents such as hydroxyl, methyl, or glycosyl groups. They exhibit antioxidant and antiviral properties. 4. Saponins: These are group of compounds with sugar moieties bound to a sapogeninsteroid alkaloids, steroids, or triterpene resulting to structural varieties of saponins. They reduce the digestion of protein due to formation of less soluble saponin-protein complexes and stop the absorption of micronutrients. They exhibit antioxidant properties.

I. Phytopharmaceuticals and technology

1. Biogenesis, metabolism, and extraction

TABLE 1.1 Medicinal importance of secondary plant metabolites. S/no.

Phytochemicals

Examples

Medicinal uses

References

Alkaloids

Morphine, caffeine, berberin, codeine, piperidine, camptothecin, vinblastine, vincristine, serpentine, aceclidine, rutaecarpine, quinine, artemisinin

Diuretic, local anesthetic, bactericidal, anticancerous, antihypertensive, cholinomimetics spasmolytics, antiasthmatics, antimalarials

[11]

Flavonoids

Quercetin, reservaratrol kaempferol, caffeic acid, flavones, rutin, naringin, hesperidin tannic acid, gallic acid, ellagic acid

Hyperlipidemia, hyperglycemia, anticancerous, antioxidative, antiartherosclerotic

[12, 13]

Anthocyanins

3 Caffeoylquinic acid, 5 caffeoylquinic acid, polymeric procyanidins, 5 caffeoylquinic acid, procyanidins

Antioxidative, antiinfluenza, antiviral

[14, 15]

Saponins

Diosgenin and hecogenin α hederin and hederasaponin C

Nutraceuticals, functional foods, natural food, preservatives

[16, 17]

Tannins

Procyanidins, prodelphinidins, epicatechin, epigallocatechin procyanidin, or prodelphinidin

Hyperglycemia, hyperlipidemia

[18]

Phenols and phenolic acids

p Hydroxybenzoic acids, protocatechuic acids, vanillic acid, syringic acid

Antioxidant, antiulcer, antidiabetic, cardioprotective, anticancerous, antiinflamatory, neuroprotective, hepatotprotective, antiaging, antimicrobial activity

[19]

Glycosides

Amygdalin, gentiopicrin, rographolide, polygalin, cinnamyl acetate, anthraquinone

Antifungal, antibacterial, antioxidant, antidiabetic, fluid and electrolyte loss, hepatoprotective, cardioprotective

[20]

Terpenoids

Isopreniods mono and sesquiterpenes, saponins, sapogenins, sesquiterpene lactones, tetraterpene carotenoids, β sitosterol, artemisinin

Antimicrobial, antifungal, antiviral, antihyperglycemic, antiinflammatory, antioxidants, antiparasitic, immune modulatory, skin permeation enhancer

[21]

Coumarins

Umbelliferone, aesculetin, herniarin, psoralen, imperatorin

Antidiabetic, hepatoprotective, antithrombotic, antifungal, antiviral, analgesic, anticarcinogenic

[22]

5. Tannins: These are bitter astringents, condensed, or hydrolyzable, water-soluble polyphenols. They bind to protein and precipitate it as well as to other various organic compounds including amino acids and alkaloids. Due to its astringent action, it produces a tangy sensation in mouth. Condensed tannins of polyphenolic compounds exhibit powerful antioxidant activity. I. Phytopharmaceuticals and technology

1.3 Biogenesis of primary and secondary plant metabolites

6. Phenols: Phenols are associated with lignin as ester groups. They are extracted as alcoholsoluble fraction bound to simple glycosides and insoluble fractions. 7. Glycosides: Glycosides are found in all plants with large range of therapeutic value. They have glycosylated principles of medicinal and toxicological value. Cardiac glycosides derivatives of digitalis and strophanthus such as ouabain are notable inhibitor of Na +/K + -ATPase activity of biomembranes. 8. Terpenoids: Terpenoids combine in many combinations to get a wide range of isoprenoids. These are present as glycosides. 9. Coumarins: The coumarins exist in different forms, for example, as benzopyrene derivatives in many plants. They are found as aroma present in essential oil [25].

1.3 Biogenesis of primary and secondary plant metabolites Biogenesis is a term that refers to the processes involved in the synthesis of organic substances from intermediate compounds. Cell metabolism includes two distinct processes: anabolism, which involves production of complex compounds using energy and reducing power, and catabolism, which involves the degradation of complex molecules to produce energy and reducing power. These processes enable the plant cell to utilize absorbed nutrients and stay alive. Plant cell metabolism involves a complex cascade of chemical reactions called metabolic pathways. As earlier stated, same biochemical pathway may produce both primary and secondary metabolites with common intermediates, which further complicates the process. Plants undergo photosynthesis and respiration, which uses carbon dioxide (as the sole source of carbon), water, sunlight energy, and ammonia as starting materials to produce primary metabolites such as glucose, amino acids, lipids, and nucleic acids. Major metabolic pathways involved in plant metabolism include photosynthesis, Calvin’s cycle, glycolysis, citric acid (Kreb’s) cycle, pentose phosphate pathway, oxidative phosphorylation, fatty acid oxidation, and respiration (anaerobic).

1.3.1 Biogenesis of carbohydrates Plants are capable of synthesizing carbohydrate from carbon dioxide (CO2) and water (H2O) in the presence of sunlight (energy) via photosynthesis [26]. The assimilation of carbon from CO2 to produce carbohydrate is a cyclic process that involves various intermediates and enzymatic catalysts and is referred to as the Calvin’s cycle. The light energy from sunlight is captured and converted to chemical energy, adenosine triphosphate (ATP) and nicotinamide dehydrogenase (NADH) via ATP synthase, to initiate subsequent reactions. Another enzyme, ribulose-1,5-diphosphate carboxylase oxygenase (RuBisCO), then catalyzes the reduction of CO2 and its subsequent covalent bonding to a 5-carbon compound, ribulose 1,5 bisphosphate to form an unstable 6-carbon compound, which splits into two molecules of 3-phosphoglycerate. Then, 3-phosphoglycerate kinase catalyzes the introduction of a phosphoryl group to yield 1,3-bisphosphoglycerate, which is acted upon by glyceraldehyde 3-phosphate dehydrogenase to form glyceraldehyde 3-phosphate. Next, triosephosphate isomerase catalyzes the conversion of glyceraldehyde 3-phosphate to dihydroxyacetone phosphate, most of which is used I. Phytopharmaceuticals and technology

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to reproduce ribulose 1,5 bisphosphate (as starting material to continue the cycle), and the rest carbohydrate. Two molecules of dihydroxyacetone phosphate combine to produce fructose 1,6-bisphosphate, which is further converted by fructose 1,6-bisphosphatase to fructose 6-phosphate and complex carbohydrates like starch.

1.3.2 Biogenesis of lipids Fatty acids are biosynthesized in seed plastids from acetyl CoA, produced from pyruvate, the end product of glycolysis [27], and then transferred to the ER for lipid storage. Enzymes act on glucose in glycolysis pathway to produce little amount of ATP and NADH, as well as intermediates (glycerates and pyruvates), with the most important being pyruvate. Pyruvate is infused into Kreb’s cycle (also called citric acid or tricarboxylic cycle) and undergoes oxidation to produce greater amount of energy (ATP) and several biosynthetic intermediates, one of which being acetyl CoA, the precursor of fatty acids. Plant studies have shown that plant lipids are made up of a triacylglycerol (TAG) core, surrounded by proteins [28], which eventually bud off as lipid bodies. This is formed at specific sites in the endoplasmic reticulum [29, 30], where substantial amounts of lipids are stored in plant seeds as TAG, although not exclusively, as lipids have also been found in other types of cells within the plant [28]. It has been proven that lipid biogenesis in plants is one of the various functions that occurs within the endoplasmic reticulum [31–33]. Certain regions in the ER stores diacylglycerol acyltransferase (DGAT), which is the precursor enzyme for TAG biosynthesis, and proteins such as oleosin, which is the major structural protein of lipid bodies formed in seeds [28, 34–38], others being caleosin and steroleosin. There is a possibility that the mechanism of lipid formation differs in other cell types (nonseed) or varying species and may be specific to them [39, 40].

1.3.3 Biogenesis of volatile oils Volatile oils (also known as essential oils) are odoriferous liquids produced by diverse (several hundreds) odor-active chemicals in plants. Characteristically, they evaporate quickly when exposed and are produced in various plant parts. They are products of metabolism, with primary metabolites as the building blocks, and as such are yields of the basic processes such as glycolysis, Kreb’s cycle, fatty acid oxidation, and pentose phosphate pathway. Precursors include fatty acids, amino acids, terpenoids, phenolics, glucosinolates [41], and carbohydrates [27]. They may be classified as primary or secondary metabolites depending on the point of formation in the metabolic pathway [42]. 1.3.3.1 Volatile oil derivation by carbohydrate pathway Carbohydrate-derived essential oils are uncommon, and two types have been reported: terpenoid compounds and fluranones [42]. Terpenes represent a group of organic compounds synthesized from active isoprene C5 units dimethylallyl diphosphate (DMPP) and isopentenyl diphosphate (IPP). They are classified as hemiterpenes, monoterpenes, sesquiterpenes, diterpenes, and triterpenes having 1, 2, 3, 4, and 6 isoprene units respectively; however, monoterpenes and sesquiterpenes make up most terpenoid volatile oils [42]. Terpenes are generated from mevalonic acid (MVA) or an alternative methylerythritol phosphate (MEP) pathway.

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1.3 Biogenesis of primary and secondary plant metabolites

They exist majorly as hydrocarbons and their oxidized forms as aldehydes, ketones, phenols, alcohols, oxides, peroxides, and ester. Both MVA and MEP pathways have acetyl-CoA and pyruvate, respectively, as the starting material to produce isoprene units, which combines in head-to-tail fashion to produce terpenes. Multiple reactions including hydrolysis, ionization, isomerization, cyclisation, and oxidoreduction occur, and large group of enzymes, generally referred to as “terpene synthases,” act on the pathways, to yield the different classes of terpenes. According to Reineccius [43], oxygenated monoterpenes account for the volatile oils in many citrus fruit species. The most prominent examples of furanone group are 4-hydroxy2,5-dimethyl-3(2H)-furanone, HDMF known as furaneol, and 2,5dimethyl-4-methoxy-3(2H)-furanone, DMMF known as methoxyfuraneol. They are considered key odorants in the plants where they occur such as pineapple and strawberry fruits. The furanones are generated via multiple pathways. D-fructose 6-diphosphate has been identified as the biosynthetic precursor, which undergoes a sequence of reactions and enzymatic action to yield the intermediate 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone (HMMF), and then the product, furaneol and methoxyfuraneol. Furaneol is also generated in heated foods during Millard reaction as is observed to give an attractive look [27, 42]. 1.3.3.2 Volatile oil derivation by fatty acid pathway Fatty acids that are derived from acetyl CoA, produced from pyruvate, the end product of glycolysis, act as nonvolatile precursors in the biosynthesis of some volatile oils. Fatty acids are stored in plant ER as TAG and released by lipases to produce volatiles such as alcohols, lactones, esters, aldehydes, ketones, and acids through three major pathways, namely, alpha-oxidation, beta-oxidation, and lipoxygenase (LOX) pathway. The LOX pathway occurs majorly in disrupted plant cells associated with wounding or damage as oxygen is introduced. Alpha- and beta-oxidation occurs in intact plants. They cause the degradation of fatty acids to produce short- to medium-chain fatty acids, which undergo series of enzymatic reactions to yield diverse odoriferous compounds [27, 42]. 1.3.3.3 Volatile oil derivation by amino acid pathway Volatile oils can also be formed directly from the amino acid pathway [27], and this depends on the availability of free amino acids, which in turn depends on degree of ripeness and environmental conditions during growth [43, 44]. Several amino acids such as methionine, cysteine, alanine, phenylalanine, leucine, isoleucine, and aspartic acid are involved as precursors. Importantly, branched-chain amino acids produce branched-chain volatile oils such as methyl 2-methylbutanoate in prickly pear, isoamyl acetate in banana, and 2-methyl butyl acetate in apples [27]. The biosynthesis of these volatiles proceeds from amino acid pathway through diverse reactions and enzyme action. Initially, amino acid undergoes deamination by aminotransferase to yield alpha-ketoacid, which loses one carbon atom by decarboxylase enzyme to form an aldehyde. Series of subsequent reactions follows to produce corresponding volatile oils [42]. 1.3.3.4 Glucosinolate-derived flavors Glucosinolates are naturally occurring compounds found majorly among plants of the Brassicaceae family like cabbage, broccoli, mustard, and horseradish, majorly for the purpose of repelling insects, and it is also responsible for the sulfury or sharp flavor characteristic to

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these plants [27, 42]. In humans the hydrolyzed products of glucosinolate have been reported to possess anticancer properties [45, 46]. In intact plants, glucosinolate and thioglucosinolate (also known as myrosinase) exist in the vacuoles and cytoplasm, respectively, and are thus separated physically. Once cells disrupt (damage), myrosinase acts to hydrolyze glucosinolate to yield the corresponding nitriles, thiocyanates, and isothiocyanates and by-products of glucose and sulfates [42].

1.3.4 Biogenesis of resins Plant resins are secondary metabolites of complex mixtures, which include volatile and nonvolatile terpenoid and/or phenolic compounds. They often exist in combination with essential oils (oleoresins), gums (gum resins), oil and gum (oleo-gum resins) sugars (as glycosides), and benzoic/cinnamic acid (balsams). They not only are formed and stored in specified structures internally or externally on the tree surface but also may be found in other plant cells and can be produced spontaneously at the site of injury to the plant. They occur mainly as exudates from the tree trunk and are generally insoluble in water but soluble in alcohol, oil, chloroform, and ether. Terpenoids and phenolic resins have been identified with the former consisting majorly of the internally formed resins while the latter largely constitute resins formed on the surface. Terpenes as mentioned in the metabolism of volatile oils are made up of isoprene C5 unit building blocks and are classified according to the number of isoprene units they contain. Unlike volatile oils, most resins consist of di- and triterpenes, rarely both; however, they occur in coexistence with other classes of terpenes such as mono- and sesquiterpenes (volatile oils) and tetra- and polyterpenes (gums, rubber, and gutta-percha). Phenolic resin metabolism involves multiple pathways. The shikimic acid pathway yields the aromatic amino acid, phenylalanine, which is converted to cinnamic acid (phenylpropane) by phenylalanine ammonia-lyase (PAL). Cinnamic acid is a C9 compound occurring as a benzene ring with C3 side chain. It serves as the substrate from which other phenolic compounds are derived via cleavage of the side chain or substitution. Malonyl acid pathway from acetyl CoA also produces aromatic benzene ring that combines with the benzene ring and C3 side chain from the shikimic acid pathway, to yield flavonoid, components of phenolic resins. Many plant resins have been observed to elicit pharmacological effects; hence, they find application in medicine and pharmacy.

1.4 Medicinal potentials of plant secondary metabolites 1.4.1 Anticancer effects Many plant metabolites including isothiocyanate [45–47], luteolin [48], resveratrol [49], genistein [50], vitamin A derivatives [51], curcumin [52], green tea extract [53], soybean extract [54], and lycopene [55] have been investigated and found to possess antitumor properties. Nutraceuticals have gained increasing interest due to minimal side effects and general acceptance and hence have been applied as preferred alternative in the prevention of cancer. Most plant extracts have been studied for prevention rather than treatment of cancer resulting in minimal efficacy and adoption in practice. An investigation carried out showed that

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1.4 Medicinal potentials of plant secondary metabolites

combination of anticancer plant extracts, produced a more effective result when compared with the efficacy of individual compounds.

1.4.2 Antiinflammatory activity Inflammation is a natural physiological response to cell injury, disease, or other forms of disturbance in animals. While it may be protective in certain cases, it can trigger a cascade of events resulting in a continuum of disorders, acute and chronic pain/diseases such as rheumatoid arthritis, allergies, hyperventricular dystrophy, atherosclerosis, osteoarthritis, inflammatory bowel disease, cancer, and coronary heart failure. Inflammation is associated with redness, heat, swelling, pain, and loss of function. During the process the vessels are dilated, which allows increased blood flow and mobilization of plasma proteins, leucocytes, and fluid to the affected site. Inflammation is the result of a number of biological pathways including the synthesis of plasma proteins, which act as chemical mediators for inflammation such as prostaglandins, cytokines, leukotrienes, vasoactive amines, and nitric oxide [56]. At the onset, cyclooxygenase and lipoxygenase together with their by-products, prostaglandin (PGE2 and PGH2), leukotrienes (LTC4 and LTB4), and cytokines (IL-1 and TNF-a) are produced by enzymatic action in response to a trigger (cell injury, disease, allergen, and so on). These by-products are referred to as proinflammatory mediators and make up the principal mediators of inflammation. Currently, antiinflammatory chemical agents such as aspirin and nonsteroidal antiinflammatory drugs (NSAIDs) exist but are however known to cause side effects such as suppression of the immune system and gastrointestinal bleeding [57]. For this reason, alternative therapy is being sought. Several plants have been investigated and discovered to have capacity to interrupt the inflammatory process at various points and hence possess substantial antiinflammatory properties (Table 1.2), some of which have been applied in therapy.

1.4.3 Antimicrobial effect of compound present in plant metabolites Chang et al. [107] investigated the antimicrobial activity of essential oils obtained from Cinnamomum osmophloeum in an in vitro trial. The result obtained shows that the tested essential oil exhibited an antimicrobial effect against methicillin resistant Staphylococcus aureus, Enterococcus faecalis, Vibrio parahaemolyticus, and Escherichia coli. The high antibacterial activity might be linked to the presence of cinnamaldehyde as the active agent in the essential oil of Cinnamomum osmophloeum. Moreover, some other essential oil with high antimicrobial activity includes oregano oil, which inhibit Escherichia coli O157:H7 [108], peppermint and spearmint oil against pathogenic bacterial [109], essential oil from herbs most especially from Lamiaceae and Compositae family [110], essential oil against Helicobacter pylori [109]; and essential oil against oral pathogens [111]. The composition and the rich nature of plant extract have influenced and enhanced their antimicrobial activity against pathogenic microorganisms. Some of the factor responsible for their antimicrobial activity might be linked to the presence of various compounds with low molecular weight [112]. The nature of chemical structure, the position, and number of substitutions, for example, in the benzene ring of phenolic acids and saturated side chain length are factors to consider. It has been observed that some of these compounds have the capability

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TABLE 1.2 Plants with antiinflammatory properties. Plant species

Family name

References

Annona squamosa

Annonaceae

[58]

Clerodendrum phlomidis

Lamiaceae

[59]

Erycibe obtusifolia Benth

Convulvulaceae

[60]

Garcinia mangostana

Clusiaceae

[61]

Gaultheria yunnanensis

Ericaceae

[62]

Glycyrrhiza uralensis

Fabaceae

[63]

Gymnaster koraiensis

Apiaceae

[64]

Magnolia officinalis

Magnoliaceae

[65]

Malus domestica

Rosaceae

[66]

Saussurea heteromalla

Asteraceae

[67]

Stephanotis mucronata

Apocynaceae

[68]

Terminalia chebula

Combretaceae

[64]

Withania somnifera

Solanaceae

[69]

Aquilaria agallocha

Thymelaeaceae

[70]

Arachis hypogaea

Fabaceae

[71]

Chromolaena odorata

Asteraceae

[72]

Citrus unshiu

Rutaceae

[73]

Curcuma longa L.

Zingiberaceae

[74]

Mangifera indica

Anacardiaceae

[75]

Panax ginseng

Araliaceae

[76]

Punica granatum L.

Punicaceae

[77]

Juniperus cedrus

Cupressaceae

[78]

Vitis vinifera L.

Vitaceae

[79]

Zanthoxylum piperitum

Rutaceae

[80]

Andrographis paniculata

Acanthaceae

[81]

Belamcanda chinensis

Iridaceae

[82]

Cimicifuga racemosa L.

Ranunculaceae

[83]

Forsythia koreana

Oleaceae

[84]

Salvia miltiorrhiza

Lamiaceae

[85 87]

Schisandra chinensis

Schisandraceae

[88]

Schisandra sphenanthera

Schisandraceae

[89]

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1.4 Medicinal potentials of plant secondary metabolites

TABLE 1.2 Plants with antiinflammatory properties

cont’d

Plant species

Family name

References

Eriobotrya japonica

Rosaceae

[90]

Evernia prunastri

Parmeliaceae

[91]

Lonicera japonica

Caprifoliacea

[92]

Psoralea corylifolia

Fabaceae

[93]

Sarcandra glabra

Chloranthaceae

[94]

Scutellaria baicalensis

Lamiaceae

[95]

Vitex rotundifolia

Lamiaceae

[88]

Artemisia argyi

Asteraceae

[96]

Caesalpinia sappan

Fabaceae

[97, 98]

Chloranthus henryi

Chloranthaceae

[99]

Cratoxylum formosum

Hypericaceae

[100]

Ligusticum chuanxiong

Umbelliferae

[101]

Olea europaea

Oleaceae

[102]

Amorphophallus konjac

Araceae

[103]

Commiphora mukul

Burseraceae

[104]

Commiphora incisa

Burseraceae

[105]

Dumortiera hirsuta Nees

Marchantiaceae

[106]

to destroy the proton motive force, cytoplasmic membrane, and the active transport available in these pathogenic microorganisms [113, 114]. Also, some other compounds present in plant metabolites are organic acid, which form part of plant extract. It has been observed that in the nondissociated nature, they have the potential to migrate through the bacterial cell wall and move into the cell where they dissociate and bring down the cytoplasmic pH and destabilize the normal physiology of pH-sensitive bacteria. Moreover, because the organic acids are present in anionic form, they cannot penetrate the cell membrane, and therefore they accumulate within the cell surface, which later increase the level of osmotic pressure and interrupt some of the metabolic process. Volatile oil has been observed to possess antimicrobial action [115, 116]. It has been reported that for plant extract to be effective against their target microorganism, they have to be in contact with the cell, but essential oil are more volatile. It should be noted that hydrophilic compounds are less volatile, but there is a need for the equilibrium achieved between volatile compounds available in the headspace [117]. Moreover the presence of lipophilic molecules in the essential oil present in the lipid bilayer can affect the lipid-protein interaction [118] and enhance the increase in the rate of membrane permeability, which later leads to functional and structural impairment of the targeted microbial cells. This might also

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lead to seepage of intracellular components and damage of microbial enzyme systems, which could result to autolysis of the affected microbial cell [112, 119]. On the whole, lipophilic compounds have the potential to interreact with the hydrophobic parts of cell proteins [120]. This might enhanced the hydrophobicity nature of the essential or volatile oil to overcome the compound accumulation, which might led to destruction of cell membrane [117, 121].

1.5 Processing, extraction, and isolation of plant metabolites The processes involved in the extraction, isolation, and identification of plant secondary metabolites were summarized.

1.5.1 Selection of specific species of plant For the investigation of plants for medicinal effects, a common methodology is undertaken. Whole plant or its parts are randomly collected and taken for investigation in which a common chemical test is carried out to identify the phytochemicals present. The selection of plant is done on the basis of being traditionally used, and the approach is termed ethnobotanical bioprospecting approach. The plants used traditionally have generally got biologically active compounds with medicinal properties. The selection of plants can also be done on the basis of chemotaxonomical approach where taxonomically related plant is established with structurally similar biomolecules. Plants are also selected on the basis of local field observation with immunity against bacteria, fungi, or virus as they synthesize defensive natural products with bactericidal, fungicidal, or virucidal effects. Plant selection also utilizes a combination of these three, that is, ethnopharmacological, taxonomic, and random approaches with a database of relevant information of a particular plant. The database prioritizes the plant species for extraction and screening of medicinal activity. This is the accepted approach by pharmacological companies.

1.5.2 Collection of plant material and its identification Either whole or parts of a plant are collected. The portions where metabolites are stored in the plant are selected. It is better to select aboveground parts like leaf, stem, flower, fruit, seed, and bark and underground parts like tuber, bulb, and root separately. Various factors like age of the plant, environmental condition of light, rainfall, and humidity should also be taken into consideration for perfect selection of active plant material. Accurate identification of the plant for isolation of active principles is a must. The plant must be authenticated by a botanist or a plant taxonomist. The plant name, portion of the plant collected, place, and date of collection should be noted and be deposited in the herbarium for future reference.

1.5.3 Drying and grinding of plant material After collecting the plant, it is usually dried on trays at room temperature with proper ventilation. Ideally, dry conditions prevent the growth of microbial organisms, which lead to

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1.5 Processing, extraction, and isolation of plant metabolites

fermentation or deterioration of metabolites of the plant. The plant materials should be ground into fine powder to give a uniform sample with increased surface area. The plant material can be cut into more small pieces for easy grinding. Mechanical grinding can be done with the help of grinders, mixture, hammer, or cutting mills. However, sometimes the process results in clogging of sieve due to fat and volatile oils present in plant. Plants can be cut into small pieces to facilitate uniform and faster drying. Plants should not be dried in direct sunlight to minimize formation of artifacts by unwanted reactions. Simple air drying, microwave drying, oven drying, and freeze-drying of plants samples are the preferred methods of drying. Air drying usually takes long say 3–7 days to months depending on the types of sample. As high temperature is not involved, heat-sensitive compound is not affected. The only drawback is the longer time and contamination. Microwave drying utilizes electromagnetic radiation with electrical and magnetic fields. The method will decrease the drying time but will also lead to deterioration of the phytochemicals. Oven drying is the easiest method using rapid heat energy to decrease the moisture content along with preserving phytochemicals. This method nowadays is common because it is faster and minimizes the chemical reactions in the plant material. After drying the samples, they are to be kept in cool and dry place. Longtime storage of samples is not advisable as it leads to degradation of plant metabolites. In freeze-drying, the sample is frozen overnight at 80°C to 20°C before lyophilization to avoid the frozen liquid in the sample from melting. It is a complex and expensive method [122].

1.5.4 Extraction protocol Extraction is the process by which we separate the bioactive principle from the plant using solvents like diethyl acetate, ethanol, chloroform, acetone, and so on through standardized procedures to get the active ingredient, which can provide medicinal effect. The solvent is chosen based on the solubility and polarity of the bioactive principle to be extracted. The most common solvents of use are water, organic and inorganic solvents. The extraction procedure is chosen based on the nature of the source of plant material and the bioactive compound to be isolated. The soluble extract is separated from insoluble residue. The most common extraction techniques used by researchers in the field of phytopharmacology are as follows: Maceration: By this technique, the plant material is soaked in closed container containing a solvent for 4–5 days at room temperature with intermittent agitations. This process softens and breaks the plant cell wall leading to release of the phytochemicals. After the intended days, the mixture is filtered. The type of compound extracted is determined by the solvent used. Infusion and decoction: Infusion and decoction have the same principle as maceration. The sample is boiled for short time to extract volatile ingredients in infusion and to get mineral salts, bitter samples from hard materials like root, bark, wood, and seed in decoction. Percolation: Percolation is slowly passing a liquid through filter. The equipment percolator is used for percolation where boiling water and acid are added with shaking till extraction is done.

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Soxhlet extraction/hot continuous extraction: The sample is finely grounded and placed in thimble or a porous bag made up of filter paper in the Soxhlet apparatus. The solvent is heated by heating mantle in the solvent chamber, which vaporizes in thimble and then condenses in the condenser and drips back to the solvent chamber. It has the drawbacks of exposure to flammable solvents, toxicity, costly, and nonenvironmental friendly. Microwave-assisted extraction: Here, microwave energy is utilized for the separation of bioactive principles from the sample to the solvent. This method favors polar solvents with high dielectric constant more than nonpolar solvents. This method needs less time and volume of solvent. The drawback is thermal degradation. Ultrasound-assisted extraction or sonication extraction: Ultrasound-assisted extraction (UAE) uses ultrasound of 20–2000 kHz. The ultrasound enhances the contact surface of solvents and samples. It increases the permeability and disrupts plant cell wall, thus facilitating extraction. The procedure has advantages of being simple and of low costly. Accelerated solvent extraction: Accelerated solvent extraction (ASE) is an efficient method that uses minimal amount of solvent. The plant sample is packed with an inert material in the extraction cell. This helps to prevent sample aggregation leading to further blocking of system. The method has advantages of temperature and pressure control and requires less time. Supercritical fluid extraction: Supercritical fluid or dense gas is a substance that has characteristics of both gas and liquid at its critical point, for example, CO2 that becomes supercritical fluid at above 31.1°C and 7380 kPa. It is a better solvent for nonpolar analytes. The addition of ethanol or methanol makes it a solvent for polar compounds too. The advantages are low cost and have low toxicity of CO2. Also the strength is easily alterable by change of temperature and pressure. The methods employing solvent for extraction are highly influenced by the type of solvent used. However, the amount of solvent used for extraction causes no difference in effect. All the processes done for extraction or preextractions are very much important in studying the medicinal effect of those plants. The steps like sample preparation by grinding and drying also affect the level of phytochemicals in final extractions. All the methods have their own benefits and drawbacks [123].

1.5.5 Identification and characterization of bioactive principles Plant samples usually contain various bioactive principles whose isolation, identification, and characterization need recent analytical protocol and instruments. Mostly, thin-layer chromatography (TLC), column chromatography, flash chromatography, Sephadex chromatography, and high-performance liquid chromatography (HPLC) are used for isolation. Also, nonchromatographic techniques like immunoassay using monoclonal antibodies (MAbs) and phytochemical screening assay are also remarkable. The bioactive fractions separated can be further identified or characterized by Fourier transform infrared spectroscopy (FTIR) and gas chromatography-mass spectrometry (GC-MS). Hyphenated techniques like LC/UV, LC/MS, and LC/NMR can be used to differentiate between new and already established bioactive principles from medicinal plants. Also, spectroscopic techniques like UV/VIS and IR spectrophotometry, carbon and proton NMR, MS,

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References

and X-ray diffraction are helpful for structural elucidation of bioactive principles. Furthermore, chemical reactions involving hydrolysis and hydration reaction are done to confirm structural or functional relationships.

1.6 Quality and safety Pure compounds isolated from medicinal plants are usually vetted in the laboratories through standard protocols. To establish the effects of medicinal plants, the ethnobotanical claims and pharmacological, observational, and clinical studies are taken into account. The dose of bioactive compound varies. The changes coming to these compounds over time may also be a drawback of its efficacy. So based on this, the United States and Europe saw the need to regulate the market and license these products. A minimum level of quality phytoingredients is needed in the product for appropriate response. Standards for active ingredients of medicinal products may be found in monographs and/or pharmacopeias. Standardized products provide a better sense of security for using herbal drugs. At the international level, WHO developed a strategy to review traditional medicines by developing monographs for herbal ingredients. Chemical compounds in herbs have properties that add to the pharmacological activity of the extract [124–130]. However, adulteration of herbal products can be intentional and sometimes accidental. In some cases, herbal remedies have been found to contain heavy metal residues, which can lead to harmful effects when consumed. Also, microbial contamination is of great concern. So it is needful that all herbal products go through some quality control processes before being allowed to be released into the market.

1.7 Conclusion This chapter provides detailed information on the different types of primary and secondary metabolites and their application for the treatment of diseases. The advances in plant metabolomics have facilitated the detection of some other new metabolites that might be useful in the production of useful drugs for the management of diseases. However, there is need to intensify efforts on the application of plant metabolomics in synergy with other functional genomics, proteomics, and bioinformatics for better results.

References [1] N. Kumar, Z.A. Wani, S. Dhyani, Ethnobotanical study of the plants used by the local people of Gulmarg and its allied areas, Jammu and Kashmir, India, Int. J. Curr. Res. Biosci. Plant Biol. 2 (9) (2015) 16 23. [2] L.W. Sumner, P. Mendes, R.A. Dixon, Plant metabolomics: large scale phytochemistry in the functional geno mics era. Phytochemistry 62 (2003) 817 836, https://doi.org/10.1016/S0031 9422 (02) 00708 2. [3] A.R. Fernie, The future of metabolic phytochemistry: larger numbers of metabolites, higher resolution, greater understanding. Phytochemistry 68 (2007) 2861 2880, https://doi.org/10.1016/j.phytochem.2007.07.010. [4] R. Verpoorte, Y.H. Choi, H.K. Kim, NMR based metabolomics at work in phytochemistry. Phytochemistry 6 (2007) 3 14, https://doi.org/10.1007/s11101 006 9031 3.

I. Phytopharmaceuticals and technology

1. Biogenesis, metabolism, and extraction

[5] O. Fiehn, Metabolomics the link between genotypes and phenotypes. Plant Mol. Biol. 48 (2002) 155 171, https://doi.org/10.1023/A:1013713905833. [6] H.K. Kim, Y.H. Choi, R. Verpoorte, NMR based plant metabolomics: where do we stand, where do we go?. Trends Biotechnol. 29 (2011) (2011) 267 275, https://doi.org/10.1016/j.tibtech.2011.02.001. [7] J.W. Allwood, D.I. Ellis, R. Goodacre, Metabolomic technologies and their application to the study of plants and plant host interactions, Physiol. Plant. 132 (2008) 117 135. [8] R. Verpoorte, Y.H. Choi, N.R. Mustafa, H.K. Kim, Metabolomics: back to basics. Phytochem. Rev. 7 (2008) 525 537, https://doi.org/10.1007/s11101 008 9091 7. [9] A. Crozier, M. Clifford, H. Ashihara, Plant Secondary Metabolites: Occurrence Structure and Role in the Hu man Diet, Blackwell Publishing, Oxford, UK, 2006, 384. [10] G. Goldberg, Plants: Diet and Health. The Report of a British Nutrition Foundation Task Force, Blackwell Pub lishing, Oxford, UK, 2003 347 p. [11] M.F. Roberts (Ed.), Alkaloids: Biochemistry, Ecology, and Medicinal Applications, Springer Science & Business Media, New York, 2013, p. 64. [12] M.N. Sarian, Q.U. Ahmed, M. So’ad, S. Zaiton, A.M. Alhassan, S. Murugesu, J. Latip, Antioxidant and antidiabetic effects of flavonoids: a structure activity relationship based study. Biomed. Res. Int. (2017) https://doi.org/10.1155/2017/8386065. [13] S. Salvamani, B. Gunasekaran, N.A. Shaharuddin, S.A. Ahmad, M.Y. Shukor, Antiartherosclerotic effects of plant flavonoids. Biomed. Res. Int. (2014). https://doi.org/10.1155/2014/480258. [14] Y. Hamauzu, H. Yasui, T. Inno, C. Kume, M. Omanyuda, Phenolic profile, antioxidant property, and anti influenza viral activity of Chinese quince (Pseudocydonia sinensis Schneid.), quince (Cydonia oblonga Mill.), and apple (Malus domestica Mill.) fruits, J. Agric. Food Chem. 53 (4) (2005) 928 934. [15] T. Jurikova, S. Skrovankova, J. Mlcek, S. Balla, L. Snopek, Bioactive compounds, antioxidant activity, and bi ological effects of European cranberry (Vaccinium oxycoccos), Molecules 24 (1) (2019) 24. [16] K.W. Chan, S. Iqbal, N.M. Khong, D.J. Ooi, M. Ismail, Antioxidant activity of phenolics saponins rich fraction prepared from defatted kenaf seed meal, LWT Food Sci. Technol. 56 (1) (2014) 181 186. [17] D. Singh, P.K. Chaudhuri, Structural characteristics, bioavailability and cardioprotective potential of saponins, Integr. Med. Res. 7 (1) (2018) 33 43. [18] R. de la Iglesia, F.I. Milagro, J. Campión, N. Boque, J.A. Martı́nez, Healthy properties of proanthocyanidins, Biofactors 36 (3) (2010) 159 168. [19] V. Saibabu, Z. Fatima, L.A. Khan, S. Hameed, Therapeutic potential of dietary phenolic acids. Adv. Pharm. Sci. 2015 (2015) 1 11, https://doi.org/10.1155/2015/823539. [20] V. Kren, L. Martı́nková, Glycosides in medicine: the role of glycosidic residue in biological activity, Curr. Med. Chem. 8 (11) (2001) 1303 1328. [21] P.P. Brahmkshatriya, P.S. Brahmkshatriya, Terpenes: chemistry, biological role, and therapeutic applications. in: K.G. Ramawat, J.M. M erillon (Eds.), Natural Products Phytochemistry, Botany and Metabolism of Alkaloids, Phenolics and Terpenes, Springer Verlag, Heidelberg, 2013, , pp. 2665 2691, https://doi.org/10.1007/978 3 642 22144 6 120. [22] V. Patel, R. Patel, The active constituents of herbs and their plant chemistry, extraction and identification methods, J. Chem. Pharm. Res. 8 (4) (2016) 1423 1443. [23] S.N. Vijaya, P. Udayasri, Y. Aswanikumar, B.B. Ravi, K.Y. Phani, V.M. Vijay, Advancements in the production of secondary metabolites, J. Nat. Prod. 3 (2010) 112 123. [24] S. Sasidharan, Y. Chen, D. Saravanan, K.M. Sundram, L.Y. Latha, Extraction, isolation and characterization of bioactive compounds from plants’ extracts, Afr. J. Tradit. Complement. Altern. Med. 8 (1) (2011) 1 10. [25] L. Yang, C. Yang, C. Li, Q. Zhao, L. Liu, X. Fang, X.Y. Chen, Recent advances in biosynthesis of bioactive com pounds in traditional Chinese medicinal plants, Sci. Bull. 61 (1) (2016) 3 17. [26] O.K. Atkin, A.H. Millar, P. Gardestrom, D.A. Day, Photosynthesis, carbohydrate metabolism and respiration in leaves of higher plants, in: R.C. Leegood, T.D. Sharkey, S. von Caemmerer (Eds.), Photosynthesis: Physiology and Metabolism, Kluwer Academic Publishers, Netherlands, 2000, pp. 153 175. [27] M. Wust, Biosynthesis of plant derived odorants, in: A. Buttner (Ed.)Springer Handbook of Odor, SpringerCham, Switzerland, 2017, pp. 13 34. [28] D. Chapman Kent, J.M. Dyer, R.T. Mullen, Biogenesis and functions of lipid droplets in plants, J. Lipid Res. 53 (2012) 215 226.

I. Phytopharmaceuticals and technology

References

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I. Phytopharmaceuticals and technology

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Päivä oli viimeinen niistä neljästä, jotka Kotkanpää oli Eusebiolle myöntänyt. Uskollisena vihalleen intiaanisoturi, joka oli päättänyt kostaa, oli kutsunut koolle suuret päälliköt hankkiakseen luvan panna toimeen pirullisen suunnitelmansa. Toistamme tässä vielä kerran: intiaanit eivät ole julmia senvuoksi, että se heitä huvittaisi. Välttämättömyys on heidän ensimmäinen lakinsa. Koskaan he eivät tuomitse vankiaan, varsinkaan naista, kidutettavaksi, ellei heimokunnan etu sitä vaadi. Heti kun päälliköt olivat kokoontuneet neuvottelutulen ympärille, astui piipunkantaja kehän keskelle. Pitäen sytytettyä piippua kädessään hän kumarsi neljään ilmansuuntaan mutisten lyhyen rukouksen, sitten hän ojensi piipun vanhimmalle johtajalle pitäen yhä sen pesää kädessään. Kun kaikki päälliköt toinen toisensa jälkeen olivat polttaneet, tyhjensi piipunkantaja siitä tuhan tuleen lausuen: "Suuren comanchikansan päälliköt, antakoon Natosh — Jumala — teille viisautta. Toimikaa niin, että mikä päätöksenne lieneekin, se on yhtäpitävä oikeuden kanssa." Kumarrettuaan hän kunnioittavasti vetäytyi pois. Syntyi hetken hiljaisuus. Jokainen punnitsi tarkasti vastalausuttuja sanoja. Vihdoin nousi vanhin päällikkö puhumaan. Hän oli kunnianarvoisa vanhus, jonka ruumista peittivät lukemattomat arvet ja joka heimolaistensa keskuudessa nautti suurta mainetta viisautensa vuoksi.

Hänen nimensä oli Eshis — Aurinko. "Pojallani Kotkanpäällä", lausui hän, "on tärkeä ilmoitus tehtävänä päällikköjen neuvostolle. Hän puhukoon, korvamme ovat avoinna. Kotkanpää on yhtä viisas kuin uljas soturi. Kuulemme kunnioittaen hänen sanojaan." "Kiitos", vastasi soturi, "isäni on itse viisaus, Natoshilla ei ole mitään salattavaa häneltä." Päälliköt kumarsivat. Kotkanpää jatkoi: "Valkonaamat, ikuiset ahdistajamme, ajavat meitä takaa ja hätyyttelevät meitä väsymättömästi, pakoittaen meidät heille luovuttamaan paraimmat metsästysalueemme yhden toisensa jälkeen ja pakenemaan arkojen kuusipeurojen tavalla metsien perukoihin. Useat heistä rohkenevat saapua preirieille asti jotka ovat meidän tyyssijojamme, pyydystääkseen majavia ja metsästääkseen hirviä ja bisoneja, jotka ovat meidän omaisuuttamme. Nämä kunnottomat ihmiset, kansojensa hylkyainekset, varastavat ja ryöstävät meiltä voidessaan sen tehdä joutumatta rangaistuiksi. Onko oikein, että valituksitta kärsimme heidän ryöstöään? Sallisimmeko heidän leikata kurkkumme poikki kuin arat ashahat, yrittämättä nousta kostamaan? Eikö preirielaki sano: silmä silmästä, hammas hampaasta? Mitä isäni vastaa, mitä veljeni sanovat, eikö se ole oikein?" "Kosto on luvallinen", sanoi Aurinko, "se on heikomman ja sorretun vanhentumaton oikeus. Sen tulee kuitenkin olla kärsityn vääryyden mukainen."

"Hyvä! Isäni on puhunut kuin viisas vanhus. Mitä siitä veljeni ajattelevat?" "Aurinko ei voi puhua väärin. Kaikki, mitä hän sanoo, on oikein", vastasivat päälliköt. "Onko veljelläni valittamista jotakuta vastaan?" kysyi vanhus. "Kyllä", jatkoi Kotkanpää. "Muuan valkoihoinen erämies on häväissyt minua, useita kertoja hyökännyt leirini kimppuun, väijyksissä oltuaan tappanut useita nuorista miehistäni, minua itseänikin haavoittanut, kuten voitte huomata, sillä haava ei vielä ole mennyt umpeen. Tämä mies on lyhyesti sanoen comanchien julmin vihollinen, joita hän vainoo kuin metsänpetoja nauttiakseen heidän kidutuksistaan ja kuunnellakseen heidän kuolinhuutojaan." Nämä viekkaasti lausutut sanat kuullessaan koko seura vapisi raivosta. Huomaten, että hänen puheensa miellytti kuulijoita, tuo kavala päällikkö jatkoi paljastamatta lainkaan iloa, jota hän sisimmässään tunsi: "Olisin voinut, jos asia olisi koskenut vain henkilökohtaisesti minua", sanoi hän, "antaa anteeksi nämä vääryydet, niin loukkaavia kuin ne ovatkin olleet, mutta nyt on kysymyksessä yhteinen vihollinen, mies, joka on vannonut tuhoavansa kansakuntamme. Niin haikea kuin onkin välttämättömyys, joka tässä sattuu, en saa empiä iskemästä häntä siihen kohtaan, mikä hänelle on kaikkein kallein. Hänen äitinsä on käsissäni, mutta olen epäröinyt häntä kidutuksin uhrata. En ole antanut vihan hallita itseäni, olen vain tahtonut olla oikeudenmukainen, ja vaikka minun olisi ollut niin helppo surmata tuo nainen, niin olen pitänyt parempana, että te itse, kunnioitettavat heimomme päälliköt, suostuen antaisitte siihen määräyksen. Olen

tehnyt vielä enemmän: koska minua kammottaa hyödyttömästi vuodattaa verta ja rangaista syytöntä syyllisen puolesta, olen myöntänyt tälle naiselle neljä päivää odotusaikaa, jotta hänen pojallaan olisi tilaisuus pelastaa äitinsä tarjoutumalla itse kärsimään kidutukset hänen sijastaan. Muuan valkoihoinen vankini on lähtenyt häntä etsimään, mutta sillä miehellä on kaniinin sydän, hänellä on rohkeutta murhata vain aseettomia vihollisiaan, hän ei ole tullut eikä tule!… Tänä aamuna, auringon noustessa, menee umpeen myöntämäni odotusaika. Missä on tämä mies? Hän ei ole ilmestynyt!… Olenko menetellyt oikein, vai onko minua moitittava, onko tämä nainen kiinnitettävä kidutuspaaluun, jotta valkoihoiset rosvot kauhistuneina hänen rangaistuksestaan huomaisivat, että comanchit ovat peloittavia sotureita, jotka eivät koskaan jätä häväistystä rankaisematta? Olen puhunut. Olenko puhunut oikein, mahtavat miehet?" Pidettyään tämän pitkän syytöspuheen Kotkanpää istuutui jälleen ja pannen käsivarret ristiin rinnalleen painunein päin odotti päällikköjen ratkaisua. Syntyi jokseenkin kauan kestävä äänettömyys puheen jälkeen. Vihdoin Aurinko nousi puhumaan. "Veljeni on puhunut oikein", sanoi hän, "hänen sanansa ovat miehen sanoja, joka ei anna tunteen hallita itseään. Kaikki, mitä hän on sanonut, on oikein. Valkoihoiset, julmat vihollisemme, varustautuvat tuhoamaan meitä. Vaikka meidän onkin vaikeata rangaista naista, on se nyt välttämätöntä." "Se on välttämätöntä!" kertasivat päälliköt kumartaen.

"Menkää", jatkoi Aurinko, "suorittakaa alkuvalmistukset, antakaa toimitukselle sovitusuhrin leima, ei koston. Kaikkien ihmisten täytyy nähdä, että comanchit eivät kiduta naista huvin vuoksi, vaan että he osaavat rangaista syyllisiä. Olen puhunut." Päälliköt nousivat ja tervehdittyään kunnioittavasti vanhusta poistuivat. Kotkanpää oli saavuttanut tarkoituksensa. Hän saisi kostaa, ottamatta päälleen vastuuta teosta, jonka koko inhottavuuden hän oli ymmärtänyt, mutta johon hän oli taitavasti osannut saada osallisiksi heimonsa päälliköt muka oikeuden perusteella, vaikka hän siitä sisimmässään välitti perin vähän. Valmistukset rangaistusta varten suoritettiin kiireisesti. Naiset veistelivät saarnesta ohuita tikkuja, jotka oli pistettävä tuomitun kynsien alle, toiset keräsivät seljapuun ydintä valmistaakseen rikitettyjä sytykkeitä, kun taas nuorimmat lähtivät metsään tuodakseen sylimäärin tuoreita puita, joilla tuomittu oli hiljalleen poltettava, niin että hän samalla tukehtuisi savuun. Sillä aikaa olivat miehet tyvestä kuorineet paljaaksi erään kidutuspaaluksi sopivan puun. Sitten he olivat sivelleet sen hirvenihralla, johon oli sekoitettu punamultaa. Juurelle he olivat kasanneet polttopuut, ja kun tämä oli tehty, oli taikuri saapunut salaperäisin sanoin vihkimään puun, niin että se vihdoin oli kaikin puolin kunnossa käytettäväksi tarkoitukseensa. Kun nämä valmistukset oli suoritettu, tuotiin tuomittu paalun luo ja pantiin sitomatta istumaan puukasalle. Sitten alkoi päänahkatanssi.

Onneton nainen oli näköjään rauhallinen. Hän oli päättänyt uhrata henkensä. Ei mikään hänen ympärillään voinut häntä enää horjuttaa. Hänen silmänsä hehkuivat kuin kuumeessa ja olivat täynnä kyyneliä tuijottaessaan tuohon joukkoon, joka ympäröi häntä kiljuen kuin villipedot. Hänen sielunsa toimi kuitenkin yhtä pirteänä ja selvänä kuin hänen parhaina päivinään. Äitiparkaa vaivasi pelko ahdistaen hänen sydäntään ja pannen hänet kärsimään tuskaa, johon verrattuna intiaanien hänelle aikoma oli pientä. Hän vapisi ajatellessaan, että hänen poikansa, saatuaan tietää kauhean kohtalon, rientäisi paikalle ja hänet pelastaakseen antautuisi julmien vihollistensa käsiin. Kuunnellen tarkasti pienintäkin melua, hän oli joka hetki kuulevinaan avuksi rientävän poikansa kiireiset askeleet. Hänen sydämensä vavahteli pelosta. Hän rukoili Jumalaa sielunsa sisimmässä, että hän itse saisi kuolla rakkaan lapsensa, sijasta. Päänahkatanssi jatkui kiihkeänä hänen ympärillään. Joukoittain sotureja, suurikokoisia, kauniita, ylellisesti koristettuja miehiä, joilla kaikilla oli kuitenkin kasvot värjätty mustiksi, pyöri parittain paalun ympärillä, johtajina seitsemän rummuilla ja chichikueilla varustettua soittajaa, joiden kasvot oli maalattu mustia ja punaisia juovia täyteen ja joiden päässä oli tarhapöllön siipiä, ulottuen pitkälle alas selkään. Miehillä oli mustat sulat ja punainen puku ja käsissään pyssyjä ja sotanuijia, joiden päätä he tanssiessa pitivät alaspäin. He olivat laajana puoliympyränä paalun ympärillä. Vastapäätä heitä tanssivat naiset täydentäen kehän.

Kotkanpää, joka johti sotureja, piti kädessään pitkää sauvaa. Kepin päähän oli kiinnitetty ihmisen päänahka. Sen yläpuolella oli oljilla täytetty harakka siivet levällään, vähän alempana samassa kepissä oli toinen päänahka, ilveksennahka ja sulkia. Kun tanssi oli tovin jatkunut, asettuivat soittajat tuomitun viereen ja aikaansaivat korvia huumaavan melun laulaen, lyöden kaikin voimin rumpujaan ja ravistellen chichikueitaan. Tanssi jatkui jokseenkin kauan kauhean ulvonnan säestyksellä, joka oli omiaan saattamaan pelosta hulluksi tuon onnettoman, sillä se ennusti kaikkein hirveintä kidutusta. Vihdoin Kotkanpää kosketti tuomittua kevyesti sauvallaan. Tämän merkin johdosta melu lakkasi kuin taikaiskusta, rivit hajaantuivat, ja kukin otti aseensa. Kidutus alkoi!

XX Kidutus Heti kun päänahkatanssi oli loppunut, marssivat heimokunnan parhaat soturit rivissä paalun eteen aseet käsissään, naisten, varsinkin vanhempien, syöksyessä vankipoloisen kimppuun herjaten ja survoen häntä, kiskoen hiuksista ja lyöden häntä, mutta pienimmälläkään liikkeellä tämä ei tehnyt vastarintaa, vielä vähemmin hän koetti vapautua rääkkäyksestä. Tuo onneton nainen toivoi vain yhtä, nimittäin kidutuksen alkamista. Hän oli kuumeisen kärsimättömänä seurannut päänahkatanssin odottamattomia vaiheita, kovin peläten näkevänsä rakkaan poikansa ilmestyvän ja asettuvan hänen ja pyövelien väliin. Muinaisajan marttyyrien lailla hän valitti itsekseen, että intiaanit hukkasivat kallista aikaa hyödyttömiin juhlallisuuksiinsa. Jos hänellä olisi riittänyt voimia, olisi hän heitä siitä nuhdellut ja pilkannut heidän hitautensa ja sen epäröimisen tähden, jota he näyttivät osoittavan häntä uhratessaan.

Totuus oli se, että comancheja väkisinkin arvelutti, vaikka toimitus näyttikin oikealta, kiduttaa turvatonta naista, joka oli jo iäkäs eikä koskaan ollut heille tehnyt mitään pahaa, ei välittömästi eikä välillisesti. Itse Kotkanpääkin tunsi vihastaan huolimatta kuin jotakin salaista tunnonvaivaa rikoksesta, jota oli tekemässä. Siksi hän ei kiirehtinyt lopullisten valmistusten suoritusta, vaan toimitti niitä laimeasti. Häntä kalvoi inho, jota hän ei kyennyt karkoittamaan. Rohkeille miehille, jotka ovat tottuneet mitä suurimpiin vaaroihin, on kunniatonta kiduttaa sellaista heikkoa olentoa kuin naista, jolla ei ole muuta puolustuskeinoa kuin kyynelensä. Jos olisi ollut kysymyksessä mies, olisi päätös kiinnittää hänet kidutuspaaluun ollut heimokunnassa yksimielinen. Intiaanivangit pitävät kidutusta tavallisesti pilkkanaan ja solvaavat pyöveleitään. Kuolinlauluissaan he soimaavat voittajiaan, näiden pelkurimaisuutta ja taitamattomuutta uhrejaan kiduttaessaan. He luettelevat omia yleviä tekojaan ja laskevat vihollisiaan, joilta ovat nylkeneet päänahan, ennenkuin itse ovat sortuneet. Purevilla sanoillaan ja halveksivalla käyttäytymisellään he kiihoittavat raivoon pyövelinsä, yllyttävät näiden vihaa ja siten osaksi oikeuttavat näiden julmuuden. Mutta kun on uhrattava nainen, heikko ja nöyrä, joka esiintyy kuin karitsa teurastuslaitoksessa ja joka jo on puolikuollut, niin mitä mielenkiintoa voisi tuollainen toimitus herättää? Ei ollut odotettavana mitään kunniaa, vaan päinvastoin yleistä inhoa.

Comanchit ymmärsivät tämän. Siitä johtui heidän inhonsa ja epäröintinsä. Siitä oli kuitenkin tehtävä loppu. Kotkanpää lähestyi senvuoksi vankia ja vapauttaen hänet kiukkuisista akoista, jotka häntä kiusasivat, lausui synkällä äänellä: "Nainen, tahdon pitää lupaukseni. Poikasi ei ole tullut, joten sinun täytyy kuolla." "Kiitos", vastasi puhuteltu murtuneella äänellä, nojautuen paalua vastaan, ettei olisi kaatunut. Intiaanipäällikkö katsoi häneen ymmärtämättä. "Etkö pelkää kuolemaa?" kysyi hän. "En", vastasi toinen suunnaten häneen enkelimäisen lempeän katseen, "se on minulle tervetullut. Elämäni on ollut vain pitkä sarja tuskaa. Kuolema on minulle siunaus." "Mutta poikasi?" "Poikani pelastuu, jos minä kuolen, sen olet vannonut isäsi luitten kautta." "Sen olen vannonut." "Anna minun siis kuolla." "Ovatko heimosi naiset siis intiaaninaisten kaltaisia, jotka vapisematta kestävät kidutusta?" kysyi päällikkö hämmästyneenä. "Kyllä!" vastasi nainen liikuttuneena. "Kaikki äidit sitä halveksivat, kun on kysymyksessä heidän lastensa pelastus."